Retinoic acid induced differentiation of embryonal carcinoma cells

^o
RtllNOlC ACID INDOCFX DIFHiRLSTIA'IION OF EW6RYGNAI CARCINOMA
CELLS
&y
Elizabeth M.V. Jones-Vllieneuve
A thesis
presf-ntsd tc the University of Ottawa
in partial fuifillaeLt cf the
requirements for the degree of
Lector cf Philosophy
xn
Department of Blclcgy
BIBUOTHEQUET *
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"Ottawa
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/~M Elizabeth M.V. Jones-Villeneuve, Ottawa, Canada, 1983,
UMI Number: DC53313
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I authcrizt the University of Ottawa
to lend this thesis to
other li.txtutlci; cr indiviiual3 for the purpose of scholarly rts.eci.rcti.
Flizabetn M.V. Jcnes-Vilieneuve
I farther cuthcrize the Un^ver^ity
cf Ottawa to reprcauce
tnio tt€ti.i oy pLctoccpylnq or oy ether means, in total or
it part, at tte rtqaest of otner institutions cr Individuals
fcr the purpose cf ochclariy research.
EL zabeth iw.V. Jcnes-Villei eave
- li -
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tiease sign below,
and give address and date.
- lii -
ACKNOWLEDGEMENTS
I wish tc extend my sincere appreciation:
To Dr. fc.tt. McBurney, my supervisor,
helpful suggestions,
and patience
for his guidance,
throughout tne course of
this project.
To Dr. D.L Brown, Dr. P. Anderson, Dr. T.W. Moon, and Dr.
J.G.
Kaplan, my supervisory coasasittee, for their participa-
tion and neipful comments.
To Lr. J.F. Harris, Dr. V.I. Kainins, and Mr. K.A. Rogers
who collaborated with me on some aspects of this work.
To ftr. M.
Rudnlcki for his
choline acetyltransferase,
work with tetanus toxin and
during the
course of his fourth
year hcnour*3 prcject.
To Ms. B.J. Rogers and Ms. J. Little for expert technical
assistance ana unfailing qood huiour.
To »3»
B.K.S.
Featnerstone,
Edwards, Mr.
fellow stuoents,
G.D.
Paterae, ard Mr. fc.S.
for tnelr cheerful presence
and heipfux .suggestions.
- iv -
To Dr. J. Bell, Dr. J. Campione-Piccardo, and Dr. L.
Au-
jame fcr their willing participation in lengthy discussions.
To Ms. J. Craig for moral support when the end was not in
sight.
To Br. ta. Ben and Mr.
J.
Helie for their assistance in
preparing the figures.
To fis. Chantal Fregeau for translation of the abstract.
Tc the National Cancer Institute
ment of Ontario,
of Canada,
the Govern-
and the University of Ottawa for financial
support.
- v -
ABSTRACT
Murine teratocarcinomas are malignant
tumours which con-
tain a wide 3pectruai cf differeatiated cell types and a population
of eiubrycnic-llke
ste»n
cells.
termed embryonal carcinoma cells,
can
The stem
cells,
be isolated from the
tumours and grown in vitio where they asay be induced to differentiate into
a wide
variety of
therefore be used to examine
cell types.
They can
the process by which undiffer-
entiated cells become committed
to particular developmental
pathways.
In an attempt tc simplify
the differentiation pattern of
the embryonal carcinoma cells,
culture medium
served the
aDundant development
the presence
added drugs to the tissue
during the differentiation process.
embryonal carcinoma
I
I
cells w^ra
of neuron-like
aggregated and
of non-toxic concentrations of
iocum^nted this
observation with
cultures of the enbryonal carcinoma
does net
cells when
cultured in
retinolc acid.
retinoic acid
cell line,
aiff«ientiate intc neurcns
I ob-
treated
P19,
in the* absence
which
of the
druj.
The neurons ver* initially identified by their morphology
under the
light and scanning electron
identity was conflrfied by the
microscopes.
Their
presence cf neurofilaments in
- vi -
their cytoplasm
surface.
and tetanus toxin
In addition,
the activities of two
volved In neurotransmission,
acetylcholinesterase,
tures.
receptcre on
enzymes in-
choline acetyltransferase and
increased coordinately
in these cul-
Glial cells, identified by the presence of glial fi-
brillar protein
containing filaments,
and a
fibroblast-like cells were also present.
epithelial
cells were
detected
in
population of
Neither muscle nor
cultures treated
non-toxic concentrations of retinoic acid
M.
their cell
with
in excess of 10~ 7
Eibryonal caicincir»a cells, monitored by their ability tc
form colonics and by the
cell antigen,
disappeared
presence of an embryonal carcinoma
prior to the appearance
of neu-
rcns.
Neuron^ and glial
cells appeared in cultures
exposed to
retinoic acid fcr as little a3 forty-eight hours.
acid did net change the plating
did it have
was to
growth rate over
ability to respcrd to reti-
These data suggest that
induce the
a forty-
The. P19 cell population was found to be
homogeneous with respect to its
noic acii.
efficiency cf P19 cells nor
any effect on their
eight hour period.
Retinoic
development of
the erfect of the drug
neurons and
glia rather
than tc .select against ceiis differentiating along other developmental pathways.
Various r<=tincia^ were able tc
induce the development of
neurons witn a belrarcby of efficiencies similar tc that observed *n many other biological systems affected by retinoic
- vii -
acid.
Polyamlne metabolism did not appear to be involved in
the effect.
I have described a mutant clone which does not
differentiate in the presence of retinoic acid.
may
help elucidate
the chain
of events
This mutant
triggered by
the
drug.
The retinoic
acic-induced differentiation
int? neural calls
provides a model system
of P19
cells
for asking ques-
tions about the commitment of pluripctent cells tc differentiate along an embryonic cell lineage.
cells will Di useful in studying
differentiation
In addition,
these
the early events of neural
particularly since
the
neurons and
glial
ceils appear in a sequence similar to that seen in vivo.
- viii -
RESUME
Les teratocarcinorces de sourls
constitutes de plusleurs type3
sont des tumeurs aalignes
de cellules dlfferenclees et
d'une population de cellules souches. Les cellules soucbes,
appele'es cellules de carcinoies embryonnaires, peuvent etre
isolees des
tumeurs et
mi3e3 en
culture ou
elles peuvent
etre induites a se dif fe'rencier en une variete" de types cellulaires.
Elles peuvent alors
le processus par lequel
etre utillse'es pcur e'tudier
les cellules non-differencie'es sont
dirige'es vers un de'veloppement particulier.
Dans le
but de sis-pllfler
le patron
de dlffe'rentiation
des cellulesde carcinoses embryonnaires, j'ai incorpore" diverses drogues dans le milieu
de differentiation.
bryonnaire3
de culture durant le processus
Lorsque les cellules de carcinomes em-
sont sous
forme
presence de concentrations
d'aggre'gats
et cultive'es
en
non-toxiques d'acide retinoigue,
j'observe la presence de plusleurs cellules semblables a des
neurones.
Cette observation a e'te" soutenue par le fait que
les cultures de carcinomes embryonnaires de la ligne'e cellulaire P19, traitees avec l,acide re'tinoique, ne se differencient pas en neurones en l*ab3ence de cette drogue.
Les
neurones ont
morphologle a l'aide
e'te* initialment
identifiers par
de3 iBicroscopes optique et
- ix -
leur
a balayage
e'lectronlque.
presence de
Leur
neurof llaEents cytoplasmlgues et
speclfiques a la
cellulalre.
identification a e'te" confirnee
par la
de re'cepteurs
toxine te'tanlque localised a
leur surface
De plus, l^ctlvite" spe'cif lque de deux enzymes
impliques dans la neurotransmission, la choline ace'tyltransfe'rase et 1' acetylcholinesterase,
dans ces cultures.
presence de
Des cellules gliales, Identifiers par la
fllacenta conatituas de
gliales ainsi gu'une populationde
lastlque ont
a augmente" siffultanement
e'te" e'galeaent
prote'ines fibrillaires
cellules d'aspect fibrob-
observees.
type musculaire ou e'pithe'liale a
Aucune
cellule de
e'te detecte"e dans les cul-
tures traite avec un exces d'acide retinoique a 1C~ a H, concentration non-toxigue.
naires,
Les cellules de carcinoses embryon-
suivie selon leur abilite' a former des colonies et
par la presence d'un antlgene spe'cifique a ces cellules, ont
disparues suite a I'apparition des neurones.
Les neurones et
les cultures
les cellules gliales 3ont
expcsaes a l'aclde
que quarante huit heures.
apparues dans
retinoique pour
aussi peo
L'aclde re'tincique n'a pas modi-
fle' la capacite' de formation de colonies des cellules P19 et
n*a eu aucun effet sur leur taux de croissance a l'lnterieur
de la periode e~tudiee de
quarante huit heures.
La popula-
tion de cellules P19 s'est ave're'e homogene en respect de son
abilite" a repondre a l'aclde re'tinoique.
gerent que l'effet de la
Ces re'sultats sug-
droqua est d'induire le de'veloppe-
sent des neurones et des cellules gliales plutot que d'e'limlner les cellules ayant d'autres potentiel de de"veloppefflent.
- x -
Divers compose's
de neurons
re'tinoiques ont indult
avec une gamme
le de'veloppecent
d'efficacltes simllalre
a celle
observee dans d'autres systeaes blologiques affecte"s par l'aclde retinoique.
Le me"tabolisme de polyamines
pas etre impllque" dans ces effets.
J'ai
ne semble
decrlt un mutant
incapable de se differencier en presence de l'aclde retinoique.
Ce mutant peut aider a eludider la serie d'evenements
lnities par cette drogue.
La differentiation
les cellules
pour
P19 en cellules
l'etuda de
tentes vers
De plus*
induite par
l'aclde re'tincique
aeuronales procure
la differentiation
une ligne"e cellulalre
ces semes cellules
des cellules
un modele
pluripo-
embryonnaire specifique.
seront utiles pour 1'etude des
eve'nements inltiaux de differentiation
les neurones et les cellules
chez
des neurcnes puisque
gliales apparaissent selon une
sequence simllalre a celle cbservee la vivc..
- xi -
CONTENTS
ACKNOWLEDGEMENTS
. IV
ABSTRACT
.
. .
RESUME
IX
kbafiter
I.
EsL2§
INTRODUCTION
. . . . . . . .
. . . .
1
t a n y d e v e l o p went cf suuuee e ^ b r y o t
I - r a t o c a r c i n e m a s in vivo . . . . . . . . . .
t i t r > j n a l carcinoma c e i l lines. . . . . . . .
Expterimeits usxcg embryonal C d r c m o n a c t l l s
C-ll surface noieculeb
oene expretbi.cn
Leteripir a t i c n
• . . . • • . . . . • . • •
Nevir^l d i f f e r e n t i a t i o n i n t h e e m b r y o . . . .
•
. .
. .
. .
SeUEOiC
.
Ihes-i
II.
vi
JCJU
. . . . . .
prcject
.
.
.
.
.
.
.
.
.
2
7
9
15
.15
17
• • 1 7
. .
19
. 2 4
. . . . . . . . . . . . . . . . . 2 6
MATERIALS AND METHODS
.
30
Ceij. iice& and culture t e c h n i q u e s
30
tctxDDic a_id rrepardtion
. . . . . . . . . . . 3 1
bi.w«(tfi experiments . . . . . . .
. . . . . . . . 3 1
tiic:rcn microscopy
. . . . . . . . . . . . . . 3 2
l r J r f n * 3t"icxt e x e c t r u s m i c r ^ a c o p /
. . . . . .
32
b ^ a n d L g e l e c t r o n siucrcsccpy
33
i r . p a r a ^ c u of a r c i o d r i
. . . . . . . . .
. . . 3 3
lu;i une f l u o r e s c e n c e d o s a y b
. . . . . . . . . . . 3 4
Fiiair*-nt a n t i g e n s
. . . . . . . . . . . . . . 3 4
l-.tsru. tcxln . . . . . . . . . . . . . . . .
35
t i i i i j j n d l carcinoma antigen aosay
. . 3 5
Immune ilucr t ' . c ; r e d of d i oay ^ r e g a t e d c e l l i . . . 35
lit. nunc a b a c r p t i ^ n
. . . . . . . . . . . . . . 3 6
t c t i m - i t J c n cf Jtiedlaa c e l l v c l u m t
. . . 3 7
tw/ym=: a o . ~ a y -
I.-oiation
III.
ct
37
nomespon^lve
rutanrs
. . . . . . .
CBAfiACTEHIZATION OF THE CULTURE alSTEM
h-5UJt3
Dt 5^-r sspoDi-t; cnaracterl^t ics . . . . . . . .
Fiopertici cf the n e u r o n - l i k ^ cello . . . . .
-
iii
-
36
39
39
42
50
i\ r i u i e u r c r . c i c e l l - ; l.» r e t i K i c £ C J . J
t r t a t t d cuxtureb
. . . 6 1
c e l l t y t v s p r e s e n t i n u i . t r e a t e a J-19
cuituro..
. . . . . . . . 6 7
Discussion
71
IV.
MECHANISM OF ACTION OF RETINOIC ACID
76
Results
l n l u c t i o r vers.Ua s e l e c t i o n
t t t i i c i c acia analogues •
FcLyasiiitS
.••utait c e l l l . , i e i
C e l l vcluiic changes during
dif f erer.tidtlJii
i)i^cu=-3icn
V.
CONCLUSIONS
96
101
.
ki.i£JiI
A.
76
7f
.84
. . 6 6
86
106
£ag.e
RESPONSE OF P19 CELIS TO DMSO
REFERENCES
Ill
115
-
n i l
-
LIST 0? TABLES
Table 3.1
Response of different cell lines
to retinoic acid (RA)
page 40
Table 3.2
Cell types present in aggregated
P19 cultures
page 70
Table 4.1
Response of several subclones of
P19 to RA
page 78
Table 4.2
Efficiencies of some retinoids on
induction of neuronal development
page 85
-xiv-
LIST OF FIGURES
Figure 1.1
Diagrams of sections of mouse
page 4
embryos at the 4 day and 7 day stages.
Figure 1.2
Structure of all trans retinoic acid
page 27
Figure 3.1
Morphologies of the P19 cells
following various treatments
page 44
Figure 3.2
Transmission electron micrographs
of P19 cells
page 46
Figure 3.3
Relationship between retinoic acid
concentration and differentiation of
neuron-like cells
page 48
Figure 3.4
Visualization of microtubules and
neurofilaments in cells from RA
treated cultures
page 52
Figure 3.5
Tetanus toxin labels the neuronal
cells in RA treated aggregates
page 54
Figure 3.6
Acetylcholinesterase appears in
RA treated but not untreated
aggregate cultures
page 57
Figure 3.7
Choline acetyltransferase and
acetylcholinesterase activities rise
coordinately in RA treated cultures
page 59
Figure 3-8
Immunofluorescence staining of
intermediate filaments in cells
from RA treated cultures
page 63
Figure 3.9
Tetanus toxin and anti-GFP
antiserum label two different cell
populations in RA treated aggregates
of P19 cells
page 65
Figure 3.10
Immunofluorescence staining of the
intermediate filaments in the
extraembryonic endoderm-like cells
formed in the absence of RA
page 68
-xv-
LIST OF FIGURES (continued)
Figure 4.1
RA is not toxic to P19 cells
page 80
Figure 4.2
RA need not be continuously
present in aggregated cultures
page "82
Figure 4.3
RAC65 cells do not differentiate
into neurons in the presence of RA
page 89
Figure 4.4
AEC3A1-9 embryonal carcinoma cellassociated antigen disappears from
RA and DMSO treated aggregate
cultures of P19 cells but not from
similarily treated RAC65 cells
page 91
Figure 4.5
The growth rate of RAC65 cells is
not changed in the presence of
5 X 10-7 M RA
page 93
Figure 4.6
The size distribution of cells, from
4 day old RA treated and untreated
P19 aggregates
page 97
Figure 4.7
The volume of cells from RA
treated aggregates decreases
page 99
-xvi-
LIST OF ABBREVIATIONS
DMSO
dimethyl sulfoxide
EG
embryonal carcinoma
EDTA
ethylene diamine tetraacetic acid
GFP
glial fibrillar protein
PBS
phosphate buffered saline
RA
retinoic acid
-xvii-
Chapter I
INTRODUCTION
The complex
events which occur during
genesis are difficult to study
many processes take
prised of
mammalian embryo-
in the intact embryo because
place simultaneously in an
a small number of
cells.
A model
embryo comsystem which
could be easily manipulated, provide larg%* amouLts of experimental material, and u>insic the developing easbryc as closely
as possible would
ment.
simplify the study of
embryonic develop-
Murine teratocarclnoaias fulfil at least soie of these
requirements {Graham,
1977).
These malignant tumours con-
tain net only differentiated cells
from all three germ lay-
ert3 of the embryo, but also a population of undifferentiated
attbryonic-lxke cells.
grown in large
These undifferentiated
numbers as cell lines in
will differentiate
in. vitro, into
tissue culture and
a spectrum of
similar to that &een In the tumour.
troduction is
cells can be
cell types
The purpose cf this In-
tc discuss the experimental
approaches which
have teen uted to study teratocarclnomas and the undifferentiated cells derived froti them.
tains an
embryos in
outline of the
The following section con-
early development of
order to provide a
frane of reference
ensuing discussion of teratocarclnomas.
- 1 -
ncrmal mouse
for the
2
1.1
IARLY DEVELOPMENT OF MOOSE EMBRYOS
After fertilization,
the zygote divides synchronously to
form a embryo of 8 totipotent cells (TarkowsRl and Wroblewska, 1967; Kelly, 1977).
of compaction
and
whereby the cells
come into
1975).
move closer to
extensive contact
The cells
junctions and
surface.
between the inside and the outside
Anderson,
1975).
Anderson,
appearance of
later zona
This creates a
now callea a morula (Ducibella
each other
(Ducibella and
become polarized with the
intercellular tight
the outside
The embryo then undergoes a process
occludens at
permeability barrier
of the embryo,
et al,
1975;
whlcr is
Ducibella and
Gap junctions between the cells also ap-
pear at this stage (Magauson et al, 1977; Lo, 1980;
ana Johnson, 1982).
cells tegin
Goodall
At the 16 to 32 cell stage, the outside
to differentiate and
the inner cells
are dis-
placed to one end by the formation of a fluid-filled blastocoel i.n the Anterior cf the embryo.
At tha 6t cell stage, which occurs about 3 1/2 days after
fertilization,
two
trophectoderm cells
cell populations can
in a
layer around
blastocyst and the Inner cell mass
convex disc at
be distinguished;
the outside
cells (ICM)
one end of th* blastocoel.
of the
In a plano-
The polar tro-
phectoderm cells adjacent tc thrf ICM srt diploid and eventually form the
ectopiacental cone.
trophttctodermal cells
surrouad the
transfor nation into giant cells
In contrast,
blastocoel and
the mural
undergo
containing large amounts of
3
endorepiicateu DNA.
By implantation of the blastocyst into
the uterine wall at
4 1/2 days,
the cells of
differentiated into
primitive endoderm on
surface and the primitive ectoderm
phectocerftal surface
blastocoel.
with the IC*
the blastocoelic
in the Interior and tro-
(fig 1.1a).
The primitive
grows peripherally until it covers
of the
the ICM have
endoderm
the entire inner surface
That portion which
remains associated
is termed visceral feadcderm and the remainder
forsts the parietal endoderm.
teristic acrphclcgicel and
in Grahaa, 1977).
Both
cell types have charac-
biochemical properties (reviewed
After implantation,
derm, covered by visceral endoderm,
the primitive ecto-
grows downwards to form
the egg cylinder which eventually fills tae blastccoel.
7 days,
th» primitive ectoderm
is clearly divided
By
into a
dorsal extra-tabryonic region and a ventral embryonic region
(fig 1.1b).
Gastrul&t*cn
the prlttitive streak
cf the
begins at about 7
1/2 days when
stescderm appears at the
posterior end
embryonic ectoderm.lit. More
detailed reviews
of em-
bryonic development may be found in Suell and Stevens (1966)
! ' • •
and Fossant m d Papcioannou*; (1 977 ).
polar
trophectoderm
primitive ectoderm
primitive endoderm
blastocoel
mural trophectoderm
ectoplacental cone
p a r i e t a l endoderm
extra-embryonic ectoderm
visceral endoderm
embryonic e c t o d e r m
blastocoel
trophectoderm
(giant cell)
5
Figure 1.1.
day (a)
3tage,
Diagrams of oectlons of
and 7
day (b)
stages.
embryonic ectoderm and
separated by
a constriction.
the egg cylinder,
mouse embryos at the 4
At the 7
extra-embryonic ectoderm are
Visceral
enuoderffi surrounds
parAetal endoderm is located on the blas-
tocoelic surface away from the
trophectoderm has given
egg cylinder,
and the mural
rise to giant cells.
The shading
indicates the relationships between the
See text for further details.
paioanncu (1977).
day eqg cylinder
tissues in a and b.
Redrawn from Fossatt and Pa-
6
The origin of the embryonic
stood in
many organisms *here
experimental manipulation.
complex
in mammals where this
takes place
in the
fixed eabrycs,
static picture (Snell and
which lay ne misleading.
For example,
ectoderm was believed to originate
experiaental reconstituticn "of
different donors
available for
uterus,
Although much descriptive information
can be obtained by sectioning
provides only a
the embryo is
However,
determination process
much less is kncwn.
cell lineagee is well under-
this approach
Stevens,
1966),
the extra-embrycnic
from the ICM.
However,
blastocysts from genetically
and analysis of their
subseguent develop-
ment after implantation into a foster aother has denonstrated that trcphectooerm is the origin cf extra-embryonic ectoderm (Rossant and Papalcannou, 1977).
of the postimpiantaticn
Similar manipulations
embryo are not possible
can not be obtained from the utarus.
Attempts at isolating
preiaplaatation embryos and growing them
successful until the early somite
Unfortunately,
it i«
bryos.
stage (Hsu et al,
1974).
amounts of
because the embryos are
very small,
small properties
there are
in vitrc have been
diftxcult to obtain large
experimental luaterial
only a
since they
survive in
culture,
problens in obtaining synchronously
and
because
dividing em-
The dlffereatiaticn of murine teratccarcinomas pro-
vides a model
systeu which overcomes some
tages cf working with embryos.
of the disadvan-
7
1.2
IMAIQCABCIIQflAS IJ VIVO
In 1954,
whicn arise
mice.
Stevens and Little described testicular tumours
spontaneously in
about 1%
of a»ale
strain 129
These tumcurs consist of various types of differenti-
ated cells and an undifferentiated
Pierce (1975)
have defined
carcinoma (tC)
cell type.
Stevens and
the following terms:
embryonal
cells refer to the undifferentiated multipo-
tential stem cells of the tumour; teratocarcinomas are those
tutours which consist
cf EC cells and
differentiated cells
from all three germ layers of the embryo;
and teratomas are
benign tumours containing only differentiated cells.
and Dixcn (1959) and Fierce et al (1960)
Pierce
showed that the EC
cells were responsible for new
tumours when injected subcu-
taneously into a host mouse.
Kleinsmith and Fierce (1964)
showed that a single EC cell from an embryold body was capable of forming a subcutaneous tumour consisting cf many differentiated cell types
and EC cells,
thus
confirming that
the EC cells were the stem cells of tte tumour.
toneal injection
of tumour cells
led to an
Intraperi-
ascites tumour
consisting cf free floating scuctures termed emdryoid bodies
which
superficially
1960).
resembled 5-6
day
eabryos
(Stevens,
Teratocarclnowas may be propagated by either subcu-
taneous injection or as an ascites tuaiour.
Cvarian terato-
mas arise spontaneously in the LT strain of mice when ovarian
eggs
are
tarthenogeneticaliy
activated
and
become
disorganized after the blastocyst stage (Stevens end Varnum,
8
1974).
Only a small proportion
of these tumours are tran-
splantable teratocarcinomas.
Teratocarcincmas froa other Mouse strains can be produced
by
transplanting early
syngeneic mice,
embryos to
extra-uterine sites
in
usually under the testes cr kidney capsules
(Stevens, 1970b; Damjanov et al, 1971a).
Some cf these em-
bryos become disorganized and produce teratocarcincmas.
ho&t environment can determina
The
whether a teratocarcinoma or
teratoma develops; certain strains of mice such as C57B1 and
AKl are
ncnpermis-sive for
teratocarcincmas (Solter
1975) as are athymic mice (Solter and Damjanov, 1979).
bryos from
the 2 cell stage
to the eight day
stage will form teratccarcinomas
being obtained
1968;
with seven
Stevens, 1970b;
older embryos
to eight
only to
Em-
egg cylinder
with the highest freguency
day embryos
Damjanov et al,
give rise
et al,
1971b).
(Stevens,
Generally
teratomas (lies,
1977).
Transplantation of twelve to thirteen day old genital ridges
tc extrauterine
sites also laads to
teratocarcincmas (Ste-
vens, 1970a).
Spontanetus testicular
the primordial
Beals,
1964).
from both
tumours are
germ ceils of
likely derived
the fetal testis
Sher, Stevens (1967)
normal and sterile mice
from
(fierce and
grafted genital ridges
to the testes
of normal
mica, cnly the normal grafts gave teratocarcincmas. This observation supports the hypothesis of
these tumours.
It lb possible
a germ cell origin for
that grafted
esbryos alsc
9
give rise tc teratocarcinomas through parthenogenetic development of primordial germ cells.
However,
this possiblity
is not supported by the experimental evidence.
Mintz et al
(1978) demonstrated that 6 day old embryos from both sterile
and normal mice, grafted to the testes of normal mice,
gave
rise tc the same proportion of teratocarcinomas, thus eliminating the possibility that, at least in this case, the origin of the tumours was
from primordial germ cells developed
after grafting
embryos.
from eibryOi
cf the
Teratocarcinomas
derived
have both ffiale and female karyotypes
in con-
strast to those
tumours derived from primordial
which are always aale.
teratocarcinomas from
Some strains
of mice which can form
grafted embryos do
germ cell derived tutuours (Graham,
germ cells
1977).
not give
rise tc
These data sug-
gest that another pluripotent call type of the embryo,
pos-
sibly embryonic ectoderm, may give rise to teratocarcinomas.
Diwan and Stevens (1976)
carclncasas oy
have successfully obtained terato-
grafting 6
day embryonic
ectoderm into
the
testes of adult mice.
1.3
EMBRYONAL CARCINOMA CELL LINES
Lines of embryonal carcmoia celle
liitS
from teratocarcinomas
from eircryold bodies or solid
layers
of non-dividing
by
either dissociating
cells
tumours and culturing them on
feeder ceils
1970; Martin and H a n s , 1975b;
can be established J.£
(Kahan and
Ephrussl,
McBurney, 1976) or by allow-
10
ing embryoid
bodies to attacn to
tissue culture dish (Rosenthal et
the surface of
al,
1970;
Berastine et al, 1973; Lehman et al, 1974).
clusters of tc
a plastic
Evans,
1972;
in either case,
cells arise amid the differentiated cells and
ay subculturlng,
homogenous populations
cf the rapidly di-
vidiag EC cells can be obtained and cloned.
These lines of
EC cells can be naintainea in culture by freauent subculturlng tc ensure tnat they remain in exponential growth.
and Ephrussi (1967)
showed that subclones derived
Finch
fronj EC
cell3 after 25 tc 50 generations in culture could still give
rise tc teratocarcinomas with the
ated cells.
same range cf differenti-
decently, methods for generating EC cell lines
directly rrom early eabryos have been developed thus obviating the aeea for the lengthy %,U 3Liy.fi grafting procedure (Evans ana Kaufman, l^dl;
Martin,
1981;
Axelrod and Bennett,
1982).
The ceils of the ecbryc
develop in a reproducible manner
within c prtCiie organizational framework.
The differentia-
tion of EC cells is not as rigidly constrained since they dc
not develop intc actual organ structures.
However, certain
conditions are necessary tor EC ceils to differentiate maximaiLy„ia v^ico,
of ceil density.
including a requirement for a certain level
This has oeea achieved by culturing cells
in dense ftonolayers (Micoias et al, 1975), in large attached
clunpo (Mcfiurney, 1976) or as aggregates in suspension (Martin ana £VciC3,
1975a).
The outer ceils
of aggregates of
11
pluripotent EC
cells differentiate into
(Martin et al, 1977)
primitive endoderm
in a manner analogous to the formation
of embryold oodles i.n v^vg.
Baplating the aggregates leads
to aa
followed by the
outgrowth cf endoderm
many differentiated
cell types including
appearance of
neurons,
beating
aiuscle, keratinizing epithelium, cartilage, and adipose tissue (Martin
cells,
and Evans,
1975a and b ) .
Some lines
of EC
which have lost the capacity to differentiate,
to develop any encodermal layer
tail
when aggregated (Martin and
Evaas, 1975a; Martin, 1980).
Many morphological and biochemical markers have been used
to assess
the differentiation
of EC
cells lfi y.lt£o.
EC
cells have a characteristic morphology; they have sparse cytoplasms,
relatively large nuclei
spherical
mitochondria,
little
with prominent nucleoli,
endoplasmic reticulum
and
Goigi, and numerous dispersed rlbosoaes which give the cells
a uniform appearance (Pierce and Baals, 1964; Lo and Gilula,
I9d0).
EC cells
(Berstine et al,
have high levels of
1973)
alkaline phosphatase
and lactate dehydrogenase (Graham,
1977) which may be detected histochemically.
ogy to distinguish differentiated cells
fcr many ctll types,
of cell types
light and
may not be adaquate
although nerve and muscle are examples
which can be identified easily
electron microscopes.
markers have been
differentiated cell
Using morphol-
A number
used for more accurate
typet. appearing in
with both the
of biochemical
identification of
vi.tro.
Antibodies
12
used in indirect immunofluorescence
assays provide specific
markers for some cell types and allow individual cells tc be
examined.
Antibodies tc intermediate filament proteins are
an example
of antlsera
which have
been used
in this «,,way
(Jones-Villeneuve et al, 1982; Paulin et al, 1982).
ic isozymes
(muscle),
for aldolase
(nerve),
creatine
Specif-
phcsphokinase
aad phoophoglycerate mutase (muscle)
are useful,
providing tne cells differentiate fully in culture (Adamson,
1976).
Seme cssll types can be identified by a constellation
of tarkers.
For example, parietal endoderm is characterized
by production of plasminogen activator, laminin,
and colla-
gen type IV (Graham, 1977; Strickland et al, I960).
EC cells can thus differentiate
types whose appearance can be
teria in. vitro.
The Dest evidence
(1^74)
froa
resemble the ceils of
that EC cells can
blastocyst
injection
the early embryo.
differentiate normally
experiments.
Brinster
shotted that 129 derived teratocarcinoma cells trans-
ferred to
a blastocyst iiom an
mouse with agouti hair.
this observation using
ies.
monitered by biochemical cri-
The guestion then arises as to how closely
these tumour cells
comes
into many different cell
alnino mouse resulted
in a
Mintz and Illmensee (1975) extended
EC cells from ascites
eibryoid bod-
Chimeric mice resulted which had many deveiopmentally
unrelated ti^ues,
including
derived frcm the tc cells.
erated chimeric
the germ cells in
cne mouse,
Ilimensee and Mictz (1976) gen-
nice fron. single
EC cell
transfers,
thus
13
confirming that
a single EC
into many tissue types.
in tissue
cell is capable
of developing
EC cells which have been maintained
culture are also
capable of contributing
to em-
bryonic development after transfer to blastocysts (Papalcanaou et al, 1975).
Using this approach it may be possible tc
obtain strains cf mice carrying
specific mutations by using
EC ceils witn these mutetlons la blastocyst transfer experiments (Dewey et al, 1977b; Dewey and Miatz, 1980).
The con-
clusion from the blastocyst transfer experiments was that EC
cells are
capable cf
differentiating like
when they are supplied with
cues.
embryonic cells
th.3 proper set of environmental
Although EC cells are malignant, they lose this prop-
erty upon differentiation (Plsrce et al,
Graham, 1980).
1960;
Bdamson and
Thus they can be grown in tissue cultures in
large numbers due to their
tumourgenic properties but their
differentiation into mature cell types can be used as a model for normal embryonic development.
There 1c sose controversy over
which embryonic cell type
EC c e n s lost closely resemble.
ceils
can differentiate
into
It
extra-easDryonlc erdoderm
well as <2ibrycnic tissues but that
trcphbCtoociddl cells (Graham,
derived from
EC cells resemble
ICM are isolated and
cultured.
has be&n shewn that EC
they do not give rise to
1977).
The embryold bodies
the structures
termed when
These observations suggest
that EC ceils closely resemble ICM cells.
genarate pluripotent cell
as
Isolated ICM can
lines when cultured in
EC condl-
14
tioned medium (Evans and Kaufman, 1981; Martin, 1981).
ever,
since ICM cells are
ectoderm,
How-
the progenlters of the embryonic
it is possible that these pluripotent cells could
have arisen
from embryonic
accodermal cells.
Analysis of
proteins present in EC cells,
ICM cells,
ectoderm (Martin et al, 1978),
showed that EC cells share a
protein with
cells.
that is
not found
in ICM
Dewey et al (1978) have demonstrated that ICM and EC
cells differ
gels.
embryonic ectoderi
and in primitive
in protein
profiles seen
on two
dimenticnal
Evans et al (1979), also using two dimensional gels,
showed that EC cells reseaible embryonic ectoderm cf the 6 tc
7 day embryo more clcseiy than
embryo.
These observations
the ICM cells of the earlier
suggest that EC cells
equivelent of embryonic ectoderm.
ICM cells and embryonic ectoderm
to EC cello
It Is possible that both
are capable of giving rise
and that different EC cell
lines may represent
slightly different developmental stages.
ported by
the observation that
female karyotypes
and Adamson,
1978, McBurney and Strutt, 1980J.
Tbis idea is sup-
several EC cell
are at different
inactivatioii (McBurney
are the
stages of
1976,
lines with
X chromosome
Martin
et al,
15
1.4
SS2SEIMENTS S§ING EMBRYONAL £AR£J.NQHA. QELLS
The follcwiag sectica
which EC cells have beea
contains as discussion cf
ways in
used to examine developmental phe-
nomena.
1.4.1
Cell surface Sfilecules
Many
investigators have
snared by EC
ha^ been
looked
for
cells aad embryonic cells.
of particular
interest because
markers which
are
The cell surface
of its
poteatial
rola in cell-cell interactions during embryogenesis.
Immunological techniques have been used to obtain syngenic ana monocioaal antlsera against cell surface molecules of
EC cells.
Aa antiserum raised against F9 EC cells In synge-
neic mice serves
1973).
Tnl3
tc Illustrate this approach
antiserum reacts with
(Artzt et al,
*9 cells and
other EC
cell lines, sperm, and with embryos from the 2 cell stage to
the blastocyst (Jacob,
1977).
Only the embryonic ectoderm
of the o to 9 day old embryo is positive.
important in ceil- cell interactions.
F9 antibodies
(Kemier et al,
reversibly inhioit
1977),
ceil to
F9 antigen may be
Fab fragments of anti
compaction of
the morula
cell adhesion of ICM and EC
cells In culture (Nicolas et al, 1981), ana modulate gap and
tight junctions (Dunia et al,
1979),
ail without affecting
cell divisicn (Jacob, 1977).
F9 antigen is undetectable on
embryos homczygcufc for son>»s recessive genes of the T complex
(Keaiier et at, 1*76)
and it nas been suggested that the an-
16
tiserum may
recognize a
wild type
product of
(Recessive mutations In the T complex
mozygous fora
(Benaett,
at various
1975).
are lethal In the ho-
stages of
Several other
embryonic development)
antlsera have been raised
against EC calls (Stern et al, 1975;
et al, 1977a; Hebb, 1980).
this locus.
Gachelin, 1976;
Dewey
Monoclonal antibodies are a pow-
erful tool because they detect only one antigenic specificity.
Using a monoclonal antibody, Solter and Knowles (1978)
have defined a stage specific antigen (SSEA-1) which appears
on 8 cell stage embryos and is
present in highest concentra-
tions on primitive ectoderm.
The major conclusion from this
wcrk I F
that FC
cells that
cells.
cells share
are not
some antigets
present on most
Conversely,
with embryonic
adult or
other tumour
some cell surface antigens such as H-2
and beta-2 microglobulin are absent from EC cells and appear
only when
they differentiate (Jacob,
1977;
Croce
et al,
1981).
The
cartofiydrate content
differ? from
that of
of EC
cell surface
differentiated cells
molecules
as assessed
by
lectin binding (fceisner, et al, 1977; Fujinoto et al, 1982),
and fucosylglycortptide analysis (Murarmatsu
Grabel et al (1979,
on EC cells which
ably found
ceils.
1983)
et al,
1978).
detected a lectin like component
recognized oligomannosyl residues presum-
on a complementary
receptor on
neighbouring EC
A Ca++-dependent adhesion system which is shared by
early embryonic and EC ceils has also been described (Take!-
17
chi et al, 1981;
Ogou et al, 1982).
that EC cells and their
ences at the
These studies confirm
differentiated progeny show differ-
cell surface.
The challenge
is tc ascertain
what role these differences play in differentiation.
1.4.2
Gene expression
EC cells provide a system
for studying the mechanisms of
diifertntj.ai gene expression
the specific genes and gene
during development.
Although
products whicn regulate differ-
entiation are largely u&known,
several groups have examined
either endogenous genes which are
expressed only after dif-
ferentiation of the EC cells (Croce et al, 1981), cr the expression or exogenous
viral or plasmid genomes
after their
integration late the EC ceil DNA (reviewed in Levine,
also see neubner et al, 1981;
al, 19^3;
Stewart et al, 1982;
and Gautsch and Silson, 1983).
cf one of the i chromosomes
involves the turning
1982,
Niwa et
The inactivation
In-somatic feaale cells,
off of transcription of
which
almost an en-
tire chromcaG&e, may also be studied using lines cf EC cells
wxth two active
X chromosomes (McBurney and
Strutt,
1980;
Featnerstone, 1980; Paterno and McBurney, in preparation).
1.4.3fieter.iin.ation
Altnoagh there are many cell lines which differentiate in
culture along specific developmental pathways,
EC cells are
particularly valuable ' because they differentiate
into many
18
distinct ceil
types.
EC
study determination,
progeny
celis can
therefore be
used to
the process by
which a cell
and its
become committed
pathway.
to
The main obstacle
pluripotent EC cells
a particular
differentiation
in studying determination with
has been the complexity
ferentiation patterns and
of their dif-
the lack of control
perimenter had over the process.
that the ex-
One way to surmount these
problems is to »anioulate the cells so that they differentiate into only one cr a few related cells types.
Several groups
have added drugs to u differentiating cul-
tures cf EC cells in crder to achieve this goal.
Strickland
and Mahdavi (1978) reported the appearance of extra-embryonic ectoderm
in monolayer
cultures of
F9 EC
cells treated
with retinoic acid (FA). Addition of cAMP to these cultures
lea to the
development of parietal endoderm
al, 1980).
Hogan et al (1981) demonstrated that aggregation
of F9 celio during treatment with
(Stricxland et
RA led to the development
of visceral endoderm rather than parietal endoderm.
The RA
treated F9 cultures thus are potentially useful for studying
determination events
leading to
the differentiation
of EC
cells into extra-embryonic tissues.
Specrs et
al (1979)
actded hexamethylene
bis acetamide,
poiybrene, and dlrcethylacetamidd to culture^ of the pluripotent EC cell line PCC4,
and
observed trie df-rearance of e j -
ithelial-iiKe and fibrobiaat-llke cells respectively.
lln et
al (1*79)
also observed the
appearance cf
Paua flat
19
adhesive ceil
type in EC
cultures which were
treated with
hexamethyiene bis acttimide.
Growth of EC ceils la
defined medium without serum leads
to differertxatlcn of some EC cell lines into parietal endoderm (Rizzino, 1983).
Darmon at al (1981) grew pluripotent
EC cell lines in defined medium and obtained neurons and fibroblasts.
Recent hork ir cur laboratory has been concerned with the
differentiation cf EC cells into
embryonic cell types.
have reported that high doses of
RA induce several lines of
EC ceils to differentiate into
He
neurons and glia (Jones-Vil-
leneuve et ai, 1962) while dimethyl sulfoxide (DMSO) and low
dcsc3 of
RA lead to production
of nuscle (McBurney
19t>*:; Eawaras and McBurney, 1983).
ducing differentiation
thesis.
of neurons
et al,
The effect of RA in inis the
subject of
this
H oflcrt Qiscussion of the differentiation of neural
tissue ia tfie
embryo is presented in the
next section.
I
shall also summarize the Diochemical and cellular actions of
RA.
1.5
NEURAL DIFFERENTIATION IN THE EMBRJO
Th« narvuuo
bryonic
system of
ectoderm
3treak mesoderm.
after
maamals originates
the appearance
of
Neuralation, begins at 7
from the
the
em-
primitive
to 7 1/2 days in
the moucfc with tie appearance of a groove on the dorsal surface of the g&itrulating €mbryo.
Tht lateral margins turn
20
up as th^ groove widens and eventually fuse beginning at the
anterior end.
This process
forms a
aeural tube (Snell and Stevens,
stucture celled
1966).
the
Neurone and glial
cells of tne central nervous system (CNS) differentiate from
the neuroepithellum of the neural tube.
transient structure which develops on
The neural crest, a
the dorsal surface of
the neural tuba, gives rise to saost of the periperal nervous
system.
I shall ciscu^s the differentiation of the CNS.
The celis of
the CNS may be divided
into three classes;
the neurons, the glial cells, aad fibroblasts.
are classifiaa as
astrocytes,
cells and microglia.
can be
oligodendrocytes,
There are a number
usea for Identification
Glial cells
ependymal
of markers which
of the various
neural cell
typas (revi-wed by Fields, 1979 and Schachnar, 1982).
nus toxin binds specifically to
Teta-
neurons of both the central
and peripheral nervous fcy^tems. Neurcns also ccntaia three
specific intermediate filament proteins.
are divided
into protoplasmic ano fibrous
Astrocytes, which
subclasses,
are
recognized by the presence of the intermediate filament protein,
gl-idi fibrillar acidic F^otein (GFAP).
cytes synthesize CNS
myelin and are thus
prejence of galectoc*rbroside,
lin.
The ependymal cells,
Oligodendro-
identified by the
the major glycolipid of mye-
characterized by beating cilia,
line the ventricles of the brain and also the spiral cord in
a palisaae arrangement.
mesoderm,
The microglial cells, derived from
^re smell migratory ceils with phagocytic proper-
21
ties.
Fibroblasts are characterized by the presence of fl-
bronectia and also Thy-1,
a cell surface molecule also seen
on some neurone and T lymphocytes.
The very early
events in differentiation cf
glia In the CNS are still
unclear.
neurons and
This is largely due to
the complexity of the developing brain, its inaccessibility,
and a
lack of markers
for .the differentiating
cell types.
At some point, neurons and glia probably share a common precursor,
but it regains uncertain
ferentiation pathways diverqe.
at which point their difThe developmental relation-
ships between the various types of glial cells is also under
investigation.
area,
Recently,
there has
partly because monoclonal
been progress in this
antlsera recognizing anti-
gens on specific neural cell types have become available.
Some investigators have used
embryonic or neonatal brain
to generate monoclonal antlsera on
ferentiating cells contain
specific antigens characteristic
cf their developmental stage.
have isolated
Sommer and Schachner (1981)
monoclonal antlsera
oligodendrocytes
at
it is expressed on glia which
1982a).
Extensive
and upon
of
with murine
dlrferentiation.
designated CI,
the primitive; radial glial ceils
change
whlcn react
different stages
Another interesting antigen,
phological
the assumption that dif-
is detected on
of 10 day embryes.
Later
do not undergo extensive morependymai
cells
(Schnachner,
reviews on these monoclonal antlsera are
fcuad in Scnachner (1982b) and Mirsky (1982).
22
The neural tube
of the CNS can be divided
of zones (Boulder Committee,
zcne contains
1970).
mitotic cells,
into a nuaber
The inner ventricular
the subventricular
zone con-
tains dlvidmg cells which are presumably limited in differentiation potential,
mitotic cells and
the intermediate zone consists of non-
committed cells,
and the
outer marginal
zone Is comprised mainly of migrating cells and axons.
The differentiation of neurons and specific neuronal populations witnin the CNS may
be dividea into several stages;
the generation cf neuronal precursors by successive waves of
snitosis in the ventricular
zone,
their post-mitotic migra-
tica tc their final location in the developing brain,
their
aggregation with ether neurons,
and the differentiation and
aigration of processes with the
formatioa of connections to
other neurons (Cowan, 1978).
to a specific layer of the
In general, neurons belonging
brain are generated and withdraw
from nitosis at about the sams time.
this is seen
A striking example of
in the monkey visual cortex
for the inner layers form first and
where the neurons
so on with the most su-
perficially-destined neurons appearing last (Bakic, 1974).
It
was long
thought that
the ventricular
neural tube
was composed of
which first
generated neurcns and
celld
after neurogenesis
However,
was
only one primordial
the
cell type
then gave rise
complete (Jacobson,
using an iaffiuncperoxidase
electron microscopy,
zone of
tc glial
1976).
technique combined with
Ltvltt et al (1981)
have demonstrated
23
that bcth GFAP positive and
In the ventricular
GiAP negative cells are present
zone of fetal monkeys.
This work does
not iaeutlfy a common precursor for glia and neurons, but It
does show that at least some cells produced in the ventricular zone
are determined
tc be glia
wita neuronal precursors.
cells begin
It
and that
they coexist
also demonstrates that glial
to differentiate before
they cease
cell divl-
sicn.
The first differentiated glial
velopment are
the radial glial
cells observed during decells,
processes
of which
traverse tne developing brain wall from the cell body located in
the ventricular
zone.
It
ttese glial cells may function as
rcns,
has been
suggested that
a guide tc migrating neu-
thai providing a way of establishing patterns of neu-
rons within the brain (Bakic,
1*72).
after a period cf mitotic inactivity,
ate astrocytes.
Radial glial cells,
dividt and may gener-
Other astrocytes may be
derived directly
froia the GfAt positive cells of the ventricular zone.
The origin
ted.
of oligodendrocytes is also
under investiga-
Saff et al (1983) hav« recently isolated a cell type
froia neonatal rat
optic nerve which caa
differentiate into
either a fibrous & strocyt^ or an oligodendrocyte.
Thi3 cell
type is characterized by the presence cf A2B5 antigen,
ognized by a monoclonal antiserum (Elsenbarth,
chner, 19tuo).
servation*
that
1979;
recScha-
This finding is consistent with previous obA2B5
antigen
is
located
en
immature
24
astrocyte* and oligodendrocytes,
also be detected
but since this antigen can
oa some neurons it is not
a unique marker
for the precursor cell described by Raff et al (1983).
lL%LQ as <a way of sim-
Neural tissue has been studied ia
plifying the complexity encountered In
vivo.
The study of
i& lit£fi C Y isolating cells
cell lineages may be approached
at different stages of development and allowing them to form
colonies,
to
ihe cellular composition of the colonies may help
identity lanature
cells which
morphologically and may alsc allow
mature ceil types.
cannot be
distinguished
some quantitation of im-
Federoff and Doeriag (1980)
have exam-
ined the astrocyte cell lineage in this manner.
Prlxary explants of developing neural tissue allow closer
manipulat^ou of the cellular environment.
from disaggregated
Cultures derived
neural tissue are valuable
in answering
time of determination xc
specific cell
questions about the
lineages (Abney et ax, 1981),
and the importance of the mi-
croenvironient In such^processes
as the cytouifferentiation
of processes (Cowan, 1978).
1.6
R.ETINOIC A£I_D
Retinoic acid (FA) is a derivative of r e t m o l ,
noniy known da
vitamin A.
Vitamin A
more com-
and its derivatives,
termed retinoids, are ultimately derived from the plant compound, beta-carctene.
The parent structure of the retinoids
ccniists cf a tr^methylcyclohexenyl rii»g, e dimethyl substl-
25
tuted tetraeae chain,
and a polar end group which is a car-
bcxyl group In the case cf RA (fig 1.2) (Pawson, 1981).
Vitamin A
has shewn
to be essential
Since then its importance to growth,
and glycoprotein
19&0).
biosynthesis has
for life
vision,
in 1909.
reproduction,
been elucidated
(Lotan,
SA, a natural metabolite of retinol, is important to
the growth functions of vitamin A (Lotan, 1980), particularly with respect to the
dlffereatlation of epithelial tissue
and the suppression of epithelial
cancers (reviewed by Eol-
lag and Matter, 1981).
Retinoius derived froins dietary sources
are stored In the
liver as retinyl esters and secreted into the blood as retinol bound
to a retinol binding
protein which then
fonts a
complex with serum albumis for transport (Lotan, 1980).
Up-
tane into target cells is via the cell membrane, probably by
a specific membrane receptor.
proteins,
wnich are specific for retinol and retinoic acid,
are present within many cell
In the target cell retinol may
can tnea biud to the
(cbABP).
Different cytoplasmic binding
types (Chytil and Ong,
be metabolized tc RA,
1979).
which
cellular retinoic acid binding protein
It has been suggested that retinoic acid and reti-
nol 'are transported
binding proteins,
to the cell nucleus
where they may
modify gene activity in a
icnaer analogous to steroid normoaes
el, 1981).
complexed to their
(reviewed in Chader et
26
RA has other biochemical effects.
droxyl containing
tion,
combine with
traasftr tht
1977).
metabolite of
a
sugar to
Like retinol,
RA can
nucleotide
a membrane
a hy-
after phosphoryla-
sugar and
subsequently
glycoprotein (De
Luca ,
Specific cell surface glycoproteins have been shown
to altered
la retinoid treated
cells and
glycclipld bios-
ynthesisfflayalso be affected (Lotan, 1980).
On tht cellular level,
including inhibiting
RA has diverse biological effects
proliferation cf
lines (Jetten et al, 1979a;
various tumour
Lotan, 1980),
cell
antagonizing the
effects of tumour pronators (Vernsa et al, 1978;
Fish et al,
1961), reversal of karatinlzatlon in epithelial cells (Sporn
et al, 1976; fcilkoff et al, 1976), and regression of carcinogen-induced 3kir
WilKOff, 1977).
tuircurs (Lasnltzlei,
1976;
Chopra and
h& car also affect pattern formation in de-
veloping and regeneratiLg limbs (Madden, 1982; Tickle et al,
1982).
Ifle underlying aiechanlsms of
la the interaction
tceee effects may lie
of RA with its binding
prctein or could
be a result of glycoslyaticn of ceil surface proteins.
Two
excellent rtviewe of cf th.j literature concerning PA may be
found in iotas (1980) and De Luca and Shapiro (1981).
' • ' " , /
Figure 1.2. Structure of all trans retinoic acid.
.COOH
28
1.7
THESIS P£QJI£T
EC cells provide
a model systeai with which
to study the
commitment of pluripotent ceils
to ditfereatiate along par-
ticular developmental pathways.
However the differentiation
pattern of pluripoteat EC cells is
cult tc
examine the
complex and It is diffi-
events necessary
specific differentiation
pathway.
tor commitment
By adding drugs
culture aediufl. during differentiation of
hoped to reduce the spectrum of
the EC
to a
to the
cells,
I
ceil types formed by inter-
fering with some cf the determination events leading to particular developmental pathways.
Early in
the wcrk,
I ob-
served that EC cells differentiated in neurons, glial cells,
and fibroblasts in tne
of RA.
presence of non-toxic concentrations
The rebt of my
this observation.
project has consisted
cf persuing
I have collaborated with a number of peo-
ple la ord^r to bring a greater number of techniques to bear
on this prcblea.
Dr. V*I. Kalnms and Mr. K. Rogers provid-
ed 30Ee cf the antibodies fcr the intermediate filament work
and Mr.
sogers demonstrated
both the Immunofluorescent and
photographic procedures Involved
ject.
Dr.
against EC
J.H.
Harris
cells.
Mr. M.
the pro-
provided the monoclonal antiserum
ceil antigen and
volving the quantitation of
in that phase cf
performed the
experiments in-
this antigen en differentiating
Fudnicki did his fourth year honours project
under my supervision
and is responsible for
the assays for
tetaaut toxin binding and choline acetyl transferase.
29
The thesis takes the following
fori;
chapter 2 contains
details of the experimental methods which were used, chapter
3 documents tne observation obtained with RA,
describes some
and chapter 4
experiments aimed at elucidating
nism of action cf RA in this system.
the mecha-
Appendix A consists of
a paper describing the effects of DMSO in inducing P19 cells
to differentiate into
included because I
work and as
cardiac and skeletal muscle.
was Involved in the
electron microscope
a basis for discussion contained
the conclusions.
It is
in chapter 5,
Chapter II
MATERIALS AND METHODS
2.1
CELL LINES AND CULTURE TECHNIQUES.
The
cell lines
C145A12
(McBurney
and Strutt,
1979),
0C15S1 (McBurney. 1*76), and P10 (McBurney and Strutt, 1980)
are pluripotent lines cf EC cells which differentiate into a
variety of ceil types when
aggregated in vitro.
cell lines are subclones cf the P19 ceil line.
of EC cells
was isolated from a
the C3B/He strain of nice.
is a ouabain re=isteat
These
ceils are euplcid with a
a single P19 cell.
1982).
m_aj.mal tessential medium
(Glbcc Laboratories,
2.5X fetal calf
1982).
P19S18
P19S18C1A1
aad 6-thioguanine resident subclone
of P19S18 (fc£urney et al,
in alpha
The P19 line
teratocarcinoma induced in
normal male karyotype ( McBurney and Rogers,
is a cell line derived fro*
All other
All cells were cultured
(Stanners et
al,
1971)
Grand Island, fcY), supplemented with
serum and 7.5% calf
ies, Miesissauga, Ontario).
serun. (Flow Laborator-
They were maintained at 37°C in
a b% C02 atncsphere.
Dlfferentiatlor. of all the cell
follows:
ana Mg + + -
lines was carried out as
ctlli aii exponential growth were treated with Ca+ +
iree phosphate buffered saline
0.025% trypsin and InM tDTA to
(PBS)
containing
remove then from the surface
- 30 -
31
of the tissue culture dish.
10 s per
tration of
They
ml into
were plated at a concea-
a bacteriological
grade Petri
dish (Martin and Evans, 1975a) where they aggregated spontaneously.
The medium
was replaced after 3 days
and 2 days
later the aggregates were plated into tissue culture dishes.
The drugs uoed in the experiments
were added at the initia-
tion of the aggregation phase and regained In the »edium usually for the duration of
the experiment.
Aggregates were
scored for neurons at 7 to 8 days.
2.2
RETINOIC A£ID PREPARATION
RA and the
ether retinoids were prepared
tions at 1u~2 M in 95% ethanol.
as stock solu-
The stock solution was di-
luted directly Into the culture^aedium tc obtain the desired
concentration,
was removed
usually 5 X10" 7 M.
Iron the culture
In experiments where RA
mediua*,
the
aggregates were
wafcaea 3 txmes with serum-free medium before resuspension in
serum- containing medium.
13-cls-RA and the TMMP retinoids
were kind girts from Hoffman- Laroche lac. (Nutley, N.J.).
2.3
GRQHTH EXPERIMENTS
Cells were
grown for either
48 hours
presence or absence of 5 X10~ 7 M RA.
experxjueata,
Liabro
the
wells and
cells were seeded at
counted
or 8 days
in the
In the 48 bour growth
10 s per ml in
after 48 •hours
Couater (Coulter ilectrcnlcs Inc., Hiaieah,
with a
2 ml
Coulter
Florida).
For
32
the 8 day
experiments,
10 & per ml
the cells were
initially plated at
iato 2 100 aim tissue culture
dishes.
After 24
hours the cells in one dish were counted ana discarded.
At
48 hours, the cells from the remalaing dish were counted and
dishes at a coaceatration of 10 s
used tc seed 2 more 100 mm
per ml.
These dishes were counted at 3 days and 4 days re-
spectively aad
the cells from the
set up 2 more dishes.
and allowed
us to
The
4 day dish were
used to
process wa3 repeated for 8 days
keep the
cells at
optimal density
for
growth during the entire experiment.
2.4
ELECTRON MICROSCOPY
2.4.1
Transmission electrsa llcrogcofiy.
P19 cells were fixed la 4%
phate buffer,
pH
7.2,
gluteraldehyde in 0.1 M phos-
for 1.5 hours
at room temperature,
washed in the same buffer and postfixed, on ice, in 1% osmium tetroxide.
After dehydration, on ice, in increasing con-
centrations of acetone,
temperature
in 100%
the cells were brought back to room
acetone aad
infiltrated with
SPURRS.
They were then placed in Been* capsules and the resin was allowed to polymerize at 60"C
cut *ith
a glass
3taxaed fcr
knife,
7 minutes with
overnight.
collected
croscope.
on copper
uraayl acetate in
lowed by 5 minutes in lead citrate.
photographed *lth
Thin sections were
grids,
and
ethanol fol-
They were examined and
a Phillips-201 transmission
electron mi-
33
2.4.2
Sca.sniag electron mic.ros.ce£Z
The aggregates were plated
stained ia ^liu..
onto coverslips,
fixed,
and
Fixation was in 2.5% giutaraldehyde in 0.1
M sodium caccdylate buffer pH 7.3 at room temperature for 30
mla.
The cells were washed in sodium caccdylate buffer aad
postfixed in 1% csmiuir tetroxide oa
They were
dehydrated in ethanol
ice in the same buffer.
stepwise from 5%
to 1C0%.
After critical point drying, they were gold coated and examined it a AMR 100CA model scanning electroa microscope.
2.5
PREPARATION OF,ANTISERA
The aatisera tc vimetin,
glial fibrillar protein (GFAP),
and tubulin were gifts frcm Dr. V.I.
Anatomy,
University of Toronto).
vimentin (Mh 57,000),
(1979a)",
was used for the
filaments were
Electrcphoretically pure
prepared from a cytoskeletal prepara-
313 ceils according to
tioa of
Kalnins (Department of
tne aethod of Franke
immunization of rabbits.
isolated from calf
brain (Jorgensen
et al
Glial
et al,
1976) by a *i_ght irodificatioa of previously described methods (ien et al, 1976) and the filameat protiens separated by
t-AGE.
Ihe 54,GuO Mfi band was eluted from the gel,
and the
eiecropnoretically purified protein was used for the immunization of rabbits.
The preparation of the antiserum to tubulin has been prevlcasiy described (Connolly et al, 1978).
Antiserum tc ker-
atin was raided in rabbits against keratin purified from hu-
34
man stratum corneum (Sun and Green, 1978).
This was a gift
fro.ii Drs. t. fuchs and H. screen (Departmeat cf Biology, Massachusetts Institute of Technology).
lameats (Liem et al,
1978), . raised In rabbits agaiast the
160.000 Mfo component of bovine
gift fros Dr. R.
Antiserum to aeurofi-
brain neurofilaments,
was a
Llero (Department of Pharmacology, New York
School of Medicine).
2.6
IMMUNOFLUORESCENCE ASSAYS
2.6.1
filament antlgen§
Aggregates were plated directly onto coversllps, and fixation and staining was carried out in. 'Situ.
rinseu once in PBS,
nol,
pH 7.0,
and for 2 mia in
The cells were
fixed for 4 m m in 100% metha-
100% acetone,
both at -20"C.
After
wasning witn PBS, they were treated with one of the antlsera
at a dilution of 1:30 (antitubuiin,
antineurofilament,
tigxial fibrillar protein, antlviuientm)
tin).
Thi-3 was followed ny washing
treatment
with
or 1:50 (antikera-
three times ia PBS and
fluoresceln-conjugated
goat
aghast rat&it IgG (Bylano Diagnostics Div.,
ratories,
Costa Mesa,
C A ) , diluted
aa-
1:5.
IgG
raised
Travenol LaboAfter a further
thr^e vaihea in Pfio, th-a coversllps were mounted la 50% glycerol and examined
with either a Ltitz or
a Zeiss Photomi-
cro3cof.fc 2 (Carl Zeltf-, Inc., New York) equipped with epifluoresctnt optics.
35
2.6.2
Te^aaus. tgxia
The assays for tetanus toxin were carried out in collaboration with
M.
Budaicki.
After
washing in
alpha medium
(buffered with PBS 1:1), the cultures were incubated at room
tenperature for
30 min with 50
ul of tetanus
toxin (which
had been dialyzed against PBS), diluted 1:20 (Ccnnaught Research Laboratories, fclllowdale, Ontario)
1973).
After
washing,
the
coverslips were
horse anti tetanus toxia (Conaaaght)
30 min,
wasned,
(Mlrsky et al,
treated with
at a 1:50 dilution for
and exposed to rhodainine
conjugated goat
anti horse IgG (Cappel Laboratories, Cochranvllle, PA)
1:50 diiutioa for 30 min.
at a
The coverslips were thea washed
ara fixed in 5ft acetic acid ia methanol for 15 min at -20'C.
Subseguent staining with anti GFP was as described above.
2.7
EMBRYONAL CARCINOMA ANTIGEH AS§AI
2.7.1
IIIJiSSfl32Eiscen.ee of disagaESaated cells
The aggregates were dissociated in 1 mM EDTA in PBS.
cells were allowed to settle
The
onto coverslips which had been
previously coated with pcly-L-lysxnc (1 mg/ml in
fc20).
Af-
ter washing in PBS, they were treated on ice with a 1:25 dilution cf ascites flu.d from nyDidoma AEC3A1-9 (J.F.
et al,
xn
preparation)
tor 30 min.
washed and
treated wxth
fluorescein conjugated
raised against
mouse IgM (Celariane
Oatario) diluted 1:5.
Harris
The coverslips were
rabbit IgG
Laboratories,
Horaby,
The celli were txxed .[ 10C% m«thaaol
36
at -20*C,
then stained with
ethiuium bromide (1
PBb) and scored immediately.
even If they
ug/ml In
Cells w«re scored as positive
hao only one patch
of fluorescence associated
with their cell surface.
2.7.2
IIIua.Qabs.gEp.tion
Cells for the quantitative absorption analysis were fixed
at 2 X10 6
/ml in 0.1% qlutaraldehyde
room temperature.
1% (w/v).
resu^ponded m
whicn was carried out by Dr.
dixuteo sequentially
wlta 5% FCS
The cells
were washed 3
PBS and frozen until analysis
J.F.
by factors
Harris.
of 2
The cells were
in RPMI
and 0.02% azide aad absorbed with
tioa of AEC3A1-9 ascites fluid for
controlled snaking
min at
Bovine serum albumin was added to give a
final concentration of
tlmeo in Ptb,
in PBS for 15
1640 medium
a 10 - * dilu-
16h at 4°C In a humidity
chaaber. - After centrifugaticn
at CQOg
for 10 ffixn, the supernatant was tested for residual activity in
i2S
a two step binding
I-F(ab)«2 rabbit
anti mouse
concentration required
50% (D50)
assay,
to reduce
using fixed F9
cells and
Fab (12SI-fiAM).
tne AEC3A1-*
The cell
activity by
was derived from the cell titration data and nor-
malized to tne L50 value for a control Pl9i:1801A1 culture in
order to calculate
the relative amount of
present on different cell populations.
a
linear relationship
betweea
amount of antigen per ceil.
the
AEC3A1-9 antigen
This method assumes
relative DSC
and
the
37
2.6
ISTIMA2IQS Of MIB1AN £ELL VOL2ME
I estimated the median cell
butions obtained frott. a
Electronics Inc.,
Couter Counter Channalyser (Coulter
Hialeah,
calibrated with spheres
volume from the size distri-
Florida).
of knowa diameter and
was calculated tc equal 24.23 u 3 .
dian
cell voluffit
The channalyser was
was the
each channel
My estimation of the me-
caaaael midway
between the
channels containing 50% of the peak number of cells.
the distribution was soissvhat
skewed,
two
Since
this estisated value
was larger thua the peak.
2.9
ENZYME ASSAYS
The cells were removed fro*a
scraping with a
sinlzatlon.
rubber policeman or in some
cases by tryp-
The samples w«re then washed 2 tiaes ia PBS and
stored at -aO'C.
Befcre assay, all samples were resuspended
In an eguai volume of
trations were
the tissue culture dishes by
water and sonicated.
determined using
Protein concen-
Hartree's (1972)
modified
Lo*ry procedure.
Choline acetyltransferase (CAT)
nicjti
(1975).
using
a
was
radiochemical method
assayed ty M.
described
uterine, an e&ttrase mhibitor,
reaction mixture
to prevent
ture to determine the
Fonnum
was added to each
degradation of
Acetylcholinesterase was added to
by
Rud-
acetylcholine.
a duplicate reactioa mix-
activity speclficaliy attributable to
tht formati^a of acetylcholine.
38
The spectrophotometry method of Ellman et al (1961)
used to
assay acetylcholinesterase
(AchE).
The
was
activity
specifically attributable to AchE was determined by adding a
specific inhibitor of Acht, t>W 284C51 (Sigaa Chemicals,
Louis, Mo.).
St.
In some experiments, ethoprcpazine which spe-
cifically Inhibits pseudoesterases was
added to a duplicate
reaction mixture and the values thus obtained were averaged.
Both inhibitors were
10~ 2 fl at
kept in a stock solution at
5°C and used at a final concentration of 1 0 - s M.
2.10
ISOLATION OF NONRISPQNSIVE MUTANTS
Mutant ceils which did not
of RA were isolated by a
cell line by Dr. M.
were cultured in
W.
differentiate in the presence
two step procedure from the P18S18
McBurney.
Initially, P19S18 cells
meaium supplemented with 10~ 7
M RA.
The
ceils were subcultured and maintained at subccnfluent densities for
tko weeK3.
Undifferentiated cells
because of their relatively rapid growth rate.
ing at low a=jc^ity fcr a
further 10 days,
phologically undifferentiated
which was expanded
After plat-
colonies of mor-
cells were obtained,
into a cell line
PltsSl8tAC6 was subjected
were selected
and called Pl9Sl8hAC6.
to a second selection
presence of 10~ 5 M hh for 3 weeks.
one of
step in the
These cells were plated
at lo* density tor an additioaal week in the presence of the
drug aiid cue of the; colonies, P19S18PAC65,
furtcer study.
was grown up for
Chapter III
CHARACTERIZATION OF THE CULTURE SYSTEM
3.1
RESULTS
Pluripotent EC cells can often
ate into various cell types if
tured in
tissue
suspension for
culture grade
1975a and b ) .
be induced to differenti-
they are aggregated and cul-
several days
before plating
plastic surfaces
for all of my
were cultured for 5d in
re
(Martin and
experiments,
suspension.
onto
Evans,
the aggregates
They were then plated
and examined 2 to 3 days later when differentiated cells had
ffllgrated out of the aggregates.
For my
laitial experiments,
I
line C145AU (McBurney and Strutt,
was continuously
used a
1979).
present in the culture
atuidant iiun^bers of
pluripotent cell
When 10" 7 fc RA
medium,
unusually
aeuron-llke calls appeared within
of plating the aggregates.
48 h
It had been reported that RA has
no effect cu the tissue distribution which arises during the
differentiatloa
1S79b).
My
of
pluripotent
EC cells
observation on C145A12 cells
have beta i-tculicr to that cell
line.
(Jetten
may,
al,
therefore,
Thus I examined the
response cf several ether EC cell lines to RA.
- 39 -
et
TABLE 3.1
RESPONSE OF DIFFERENT CELL LINES TO RETINOIC ACID (RA)
CELL LINE
REFERENCE
#AIGGREGJIT!2S CONTAINING NEURONS
IN 10"7 M RA
WITHOUT RA
C145A12
McBurney and
Strutt, 1979
100
o2^
P19
McBurney and
Rogers, 1982
94
o*
OC15S1
McBurney, 1976
94
54
P10
McBurney and
Strutt, 1980
79
02>3
1. Three days after plating, aggregates were examined for
the presence of neuronal-like cells using phase contrast
optics. 50 aggregates were scored per measurement and
a positive was scored if the aggregate contained cells
with long processes (see figures 3.1c and d).
2. Although C125A12 and P10 aggregated cultures did not
contain neurons 3 days after plating, neurons were
routinely present 5 to 7 days after plating.
3. Some aggregates were surrounded by extra-embryonic endodermlike cells.
41
The drug hau a similar effect
tested (Table
3.1).
on the other three cell lines
Virtually
all RA
treated aggregates
contained seme cells with neuronal morphology.
gates from
some cell lines
the absence of
inducing the
the drug.
Since aggre-
formed no neuron-like
It seemed likely that
formation of these neuron-like
cells in
the RA was
cells,
rather
thaa inhibiting the development of other tissue types.
In subsequent experiments
cell line with a normal
I used P19 (fig
3.1a),
an EC
male karyotype Isolated from C3B/He
mice (McBurney and Sogers,
1982).
The RA effect could be
easily evaluated with P19 cells because no neuron-like cells
were formed in
the absence of the drug.
Figure
3.2 is an
electron micrograph of a F19 ceil showing the characteristic
Morphology of embryonal carcinoma ceils: the nucleus is relatively large
with prominent
nucleoli,
and
the cytoplasm
contains fe* organelles. tfhen aggregates of PI 9 cells were
plated m
tissue culture dishes in the absence of RA,
served
oniy undifferentiated
ceils surrounded
amount
of tissue
extraembryonic endederm
3.1b).
resembling
Coatiaued incubation of
by a
I obsmall
(fig
these cultures resulted in
the proliferation of bcth ceil types, with the appearance of
no other
differentiated cell types.
When P19
cells were
cultured as aggregates la the presence of RA, the cell types
present 2 d after plating
24 h cf plating,
a flat
were markedly different.
Within
layer of fibroblast-like cells mi-
grated out iroiit the periphery
of tne aggregate.
These fi-
42
broblast-like cells did not resemble
either EC cells or the
endoderm-llke cells seen in untreated cultures.
aad 48
h after plating,
neuron-like cells
appeared whose
processes grew rapidly
from the aggregate over
last-llke cell layer.
Phase
tron micrographs ia figs 3.1e aad
were
The processes
frequently arranged
the fibrob-
contrast B'icrographs of these
cell types are shown in figs 3.1c and d.
more detail.
Between 24
The scanning elec-
f show this morphology in
from these
in bundles.
neurcn-like cells
They had
multiple
branches with tips located on the fibroblast-like cells.
3.1.1
Dose-resEgn.se charac.te£lstlc_§
The diffentlataon of EC cells
into aeuroa-like cells was
depeadent on the coacentration of
RA present in the culture
fig 3»3 shows the response cf aggregated P19 cells
medium.
to various
FA concentrations.
than 5 XlO~e M,
At concentrations
greater
essentially all of the aggregates contained
ceils with neurcn-ilke processes by 72 h after plating.
differentiatad EC
cells could
contrast microacopy la cultures
aot be
Identified by
contaiaing neurors.
Uaphase
Cells
capable of foroiing colonies of undifferentiated cells, under
conditions ±a
which the
ceils was about 50%,
tures.
plating efficiency
of the
P19 EC
disappeared from these RA treated cul-
Experiments using an EC cell antigen confirmed this
observation (fcse secticn 4.1.4.
belcv).
Thus the drug-in-
duced appearance cf neuror;-like cells was accompanied by the
43
disappearance ot EC cells.
Between U ~ a and 5 XlC~ a M, many
aggregates tthich did not contain
aeurcn-llke cells did con-
tain fibroblast-like cells.
soue experiments,
proportion cf
In
aggregates contained
small areas
a small
of beating
muscle at RA concentrations ot 10~* to 10" a M.
fs.
Edwards ha--
cardiac and
documented the appearance
sktietal jLuscle wltb
cf both
the P19S1801A1 EC cell
f.K.S.
line (Edwards,
and McBurney, 19^3).
At 10~ 9 M, the cultures resembled un-
treated
ccntained
controls m d
only
amount*, of extra-fmbrycaic endoderm-llke
EC cells
cells.
and
SHiall
In subse-
quent experiments discussed in this thesis, I used a dose of
5 X 10~ 7 M,
a ccLcentraticn of RA with which all aggregates
ccntained neuron-like cells and few, it any, EC cells.
45
Figure 3.1.
Morphologies cf the P19 cells following various
treatments.
The undifferentiated EC cells (a) grew attached
to the surface
allowed tc
cf the tl-Lue culture dish.
aggregate for
5 1 and
platea lute tissufc culture dishas,
diff erer.tiate into
When they are
the aggregates
a sirall number of cells
the ex tra-endoderai-like ceil
cated by the arrow in b.
type indi-
If 8A is present iu the aggregated
cultures,
muren-iike and
within c d
ot pitting the aggregates (c
electron aicrographs of
are then
fibroblast-
like cells
and d ) .
such PA treated cultures
appear
Scanning
(e and f)
show networks cf processes exteiding over a monclsyer of fibroblast-like celis.
Bars: (a-d) 3.6 urn; (e and f) 11.1 urn.
47
Figure 3.2.
show that
cells.
large.
Transmission electron
they have
a morphology
The nuclear
to
n-icrcgraphs cf P19 cells
which is
cytoplasmic
typical of
ratic is
EC
relatively
A few mitochondria (m), small amounts of swollen en-
doplasmic reticulum
(er), and numerous
characterize the cytoplasm.
scattered rlbosomes
There appear
tions between the cells (arrows).
to be some junc-
Magnification is X9420.
Percent aggregates with nerve
O
I
o-
o_
%
5'
o
o" 9-J
Q
O
Q."
O
O
CD
3
CD
O
o_
1 ^
Z3
9-J
01
8
J
O
l_
(J)
O
_1
00
O
I
I
49
Figure 3.3.
belationshlp between
fereatiation of
cells,
BA concentration and dlf-
neuron-like cells.
continuously cultured
The aggregates
in the presence of
of P19
the drug,
were plated after 5 d la suspeasion and scored 2 to 3 d later.
Normally, 50 aggregates were scored for each drug con-
ceatratioa in each experiment.
obtained from
6 to
11 separata
The points indicate the mean
experiments.
The
sample
standard deviation is represeated by the vertical bars.
50
3.1.2
PraterJtleg of tjjefieurearlike, cells
The experiments in this section
taken to identify the cell types
and the next were underpresent in EA treated cul-
tures.
Microtubules form
in cells.
one of the major
cytosKeletal systems
la neurons, the microtubules are arranged la bua-
dles running down the axon parallel to Its long axis.
microtubule ouadles were
cultures using
visualized la cells In
indirect immunofluorescence
EA treated
techniques with
antibodies against purified tubulin (Connolly et al,
Figures 3.4a
and b show that
processes of
the neuron-like calls were
in a pattern
similar to that given
Such
both the cell bodies
1978).
and the
intensely stained,
by antitubulin staining
of neurons la culture (Kalnins aad Connolly, 1981).
The an-
titubulin staialng revealed varicosities on some cf the processes as indicated by the arrow
possible
to
visualize
the
in fig 3.4b.
complex
branching ard interconnections la
brcblast-ilke cells
of microtubules
patterns
It was also
cf
these cultures.
in these cultures also
in a pattern siirilar
neurite
The fi-
showed staining
tc that of
other fi-
broblasts (not shown).
Another cytoskeletal system, the 10-am iatermediate filaments,
Is comprised cf proteins specific for various tissue
types (lazarides, 1980,
termediate
1982).
filairents specific
Neurofilaments ere the infor neurons
and consist
cf
peptide subunits cf 210,000, 160,000, aud 65,0C0fcfc(Hoffman
51
ana Le-sek, 1975; Sct.laetfei, 1977; H e m tt ax, 1978).
bcaies pie^ctd
peptide »eit
dures
against the 1fcu,C0L i"! in ne urciilameat poly-
used with
to examine
&A
Staining was localized
of the
indirect lmaunoflucrescerce
treated cultures
(fig
la contrast,
ceils were unstained except for
proce-
3.4c and d ) .
along the processes and
aeuron-like cells.
specifically.
Actl-
cell bodies
the nonneuronal
nucleoli which stained non-
Neither the undifferentiated nor the extraem-
bryonic eadoderm-ilke
celis in the untreated
stained ay this antiserum,
cultures were
indicating the absence of neuro-
filaments from ttese cell types.
Ceil suriace
t-tanus toxia receptors are
for neurons (Bizzinni,
1979).
another marker
Tetanus toxin binds to neu-
rons in the central nervous system explant cultures via specific celis surface gangllosides (Diirpiel,
1977).
Nonneu-
ronal ceils rrom the ^ame cultures dc not bind tetanus toxia
(Mirsky et al, 1978).
The bmdiag of tetanus tcxln to neu-
rcns it hh tr^attd culture-^ was visualized using an indirect
i.rnancfj.acr^oCence aosay.
iig 3.5a is a pr.ase contrast ui-
crcgrcph ot a pcrt^cr. cf «
fA treated aggicgatet
Both the
neuronal ttii bodies anc their processes bcana tetanus tcxin
(f.i-j 3.bo).
Nc cth'rt cell typ^ I L thete cultures or in un-
treated cuxtare^ bound tetanus toxin.
53
Figure 3.4. Visualization ot miorotutuies and neurofilaments
lr ceii^ troai RA treatec cultures.
Inurunof lucresence stain-
ing »a 5 With ditisera tc tubulin (b)
and neurofilament prc-
tein (d). lot antituaulin antiserum stained the neuron-like
ceils, sb.cwj.ag their
branching patterns.
The flbroblast-
like cells were lightly stained by this aatlseruir*.
t^serum tc aeurofilaments -jtaln^d
their processes but
The aa-
the nturon-like cells and
did net stain the cytoplasm
ierly^ng ribroblaet-i^ke cells (d).
of the un-
The phase ccrtrast mi-
crograms ot tne same ctils are shown in panels a and c, respectively. B&r, 10 um.
55
Figure 3.5. Tetanus toxin labels the neuronal cells in retinoic acid treated
trast micrograph
aggregates. fcanel a shows
of a retinoic acid-treated
neurone ly^ng on & monolayer of fiat cell.a.
a phase conaggregate with
The surface of
the aeuronai cell body aad processes are clearly labelled in
a patchy
(b).
fashion characteristic of tetaaus
toxin labelling
(Therms is soaie randomly scattered fluorescence associ-
ated with the underlying monlayer).
Bar 10 urn.
56
Acetylcholinesterase has been freguently used as a marker
for neuronal differentiation although this enzyme is present
in some nonneuronal tissues (tfllson et al,
al, 1974; Aiamson et al, 1977).
1972;
levine et
fig 3.6 shows the result ot
one of tour experiBents in whicn the AchE activity was measured in
cells.
aggregated cultures of
both treated
and untreated
Although the absolute values of AchE activity varied
from one ex^erimeLt tc another,
and the results
the pattern was consistent
shown in fig 3.6
are representative.
treated cultures contained little activity.
treated cultures,
AchE activity peaked
Un-
However, in PA
at a tlase when neu-
rons were most nuirercus.
Chcliae acetyltransferase (CAT),
fcr the
synthesis of the
has also
been used as a
the enzyme responsible
neurotransmitter,
acetylcholine,
neuronal cell rnarKer (Pfieffer et
al, 1981) and was assayed in thus© cultures by M.
Rudalckl.
This activity was absent from the untreated cultures but did
appear in FA treattd cultures coordinately with AchE activity (fig 3.7). The decline in specific activity of these enzymes at 10 days is probably a consequence ot the proliferation of nonrjcurenal cells in these cultures.
The aoove information ca the biochemical,
immctofIncres-
cent, and anatomical aspects is contlsteat and indicates the
presence of neurons in EA treated cultures.
6H
I
>%*—
31
o o
CD
en o>
(/> c
\
MM
2H
P cn
T-
T"
0
2
4
Time (days)
6
8
58
Figure 3.6.
Acetylcholinesterase appears
not untreated aggregate cultures.
in RA treated but
The specific activity of
AchE was determined in treated (closed circles) and untreated (open circles) aggregated P19 cultures.
plated at 5
Each point
Inhibited by
strate.
d (arrow)
and neurons became abundant
represents the
dfc 284C51
Aggregates were
activity which
by 7 d.
was specifically
using acetylthiocholine
as a
sub-
The activity of acetylcholinesterase in adult mouse
brain homogenates (strain CH3/He, the genotype of P19 cells)
was 53 nmol/mia/mg protein,
about
tivity seen in EA treated cultures.
10 times the maximum ac-
Choline acetyl transferase
(pmoles/min/mg prot)
—
o
o
1
Acetylcholinesterase
(nmoles/min/mg prot.)
no
OJ
i
1
o
o
o
H
CD
(Jl-O
QL
Q
^<
(f)
- o
o-
ai
—
cn
.«- .J
o
1
—
Ul
1
l\)
o
1
60
Figure 3.7.
Choline acetyltraasferase and acetylcholinest-
erase activities
rise coordlaateiy in RA
The specltic activities
of CAT and AchE
treated cultures.
were determined in
treated (filled circles) and untreated (open circles) aggregate cultures
days (arrow)
cf P19 cells.
and
Aggregates were plated
neurons became abundant at
7 days.
at 5
(a)
Each point r«preseats an average of the specific activity of
AchE which was
specifically inhibited by B»
284C51 and the
specific activity remaining after ethopropazlne was added tc
the
reaction fixture
to
Inhibit pseudcesterases.
Aault
C3H/He mouse brain extracts contained a specific activity of
120 nuioles/ain/mg protein.
specific activity
(b)
of CAT obtained
rine, an esterase inhibiter.
Each point represents the
in the presense
of ese-
An activity of 40 cmoles/mln/
mg protein was found in adult mouse brain.
61
3.1.3
Nfifigeuional sells la re£ino.lc. acid treated cultures
The experiments described in this section were undertaken
to characterize the nonneuronai cells
present in BA treated
aggregates cr P19 cells.
As discussed above,
lament proteins provide
types.
tne tissue-specific Intermediate fia means of identifying
some tissue
Antlsera directed against vimentin, keratin and gli-
al fibrillar protein were used in immunofluorescence experiments to determine whether mesodermal-like, epithelial,
and
glial cells were present in these cultures.
Vlmentin is
an intermediate filament
protein originally
thought to be present cnly In mesodermal cells.
ever,
present m
It is, how-
many tissue culture cells of nonmesodermal
origin (Franke et al, 1978;
Franke et al, 1979b).
The fi-
broblast-like cells contain an iatermediate filament network
which stains
with antiserum to
staining patterr is
vimentin (fig
typical of that of
3.8b).
The
other viirentin-con-
talning intermediate filament systems (Franke et al, 1979b).
The keratins are a class of proteins,
rangiag ia M«r from
41,000 to 65,000, found in the iatermediate filaments of epithelial ceils (Fuchs aad Green, 197t).
agaiast keratia cid not stain
Antibodies directed
intermediate filaments in any
of the cells in RA treated cultures (fig 3.8d).
suggests that t<A treated cultures
cells.
This result
do not contain epithelial
62
Glial fibrillar protein (GFP)
is
the aajcr ceirponent of
the intcrmeolate filaments in go-iil a^trocyt^ cells (Kalnins
and Connolly, 1981).
It is very similar, it not Identical,
to glial acidic fibrillar protein, a soluble protein isolated froa gllai ceils which has been
shown to be a major com-
ponent of glial filaments (Eag at al,
1971;
1972).
fibroblast-like cells
Neither
the neurons nor the
3ta_ned witn antiserum specific for GFPter plating
hA trtated aggregates,
ccntairiiig this protein appeared at
broblast-like monolayer and the
Bignaml et al,
However, 4-5 d af-
a poculatlor
of cells
the junction cf the fl-
aggregate.
Fig 3.8f shows
the staining patterns obtained from thi3 population of glial
ceils.
Thus,
f<A treated cultures contained three distinct
ceil types ba^ed
on the antigenic characteristics
of their
intermediate filaments (Table 3.2).
HA treated aggregates wnich were exposed to tetaaus toxin
were al*o treated
3.9).
with antiserum directed agaiast
Fig 3.*d bhow^ that the aonolayer of cells uaderlylag
the neurons
in fig 3.9c is
composed of glial
neurcas did aot stain with GFP.
celi= did
not
cells.
The
ihus, neurons and glia foro>
two aitt-nct ceil populations ii
giial
GFP (fig
RA treated cultures.
labtl uniforoly
with
anti GFP
The
(fig
3.9a), prtoubiy because of the ^synchrony with which the mitct-icaiiy i«.tivt gliotlasto (GFP-)
into mcture istrocytfes (bFP+j.
terainaiiy dliferentiate
64
F:gur~ 3.o.
I Amur ofluore&cence staining of mteriredlate fi-
laments in ceils from RA treated cultures,
same fieio
ot fitrcblcst-llke
contrast (a)
a and b show the
cells photographed
by phase
and following immunofluorescence staining with
antiserum raised against vimeatin (b). The typical "basket"
pattern
of
vlmentin-contalnlng intermediate
present i? virtually all celis.
phctographea by phase contrast
uordJCtnce
staining using
filaments
is
c aad d show the same field
(c)
antiserum
Seme acnspecific perinuclear staining
and following imnuncflagainst keratin
was observed,
intermediate filaments stained with this procedure.
lial fibrillar protltn antiserum was used
(d).
but no
Antig-
in panel e and f.
A population of glial filament-containing cells appeared 4-5
d after plating ti-e aggregates. Bar, 20 um.
66
Figure 3.9.
Tetanus toxin and
different cell
populations in retinoic acid
gates cf P19 cells.
treated aggre-
All paaels show the same field of cells
after double labelling
tiserum.
anti GFP antlserua label two
with tetanus toxin aad
artl GFP an-
Paaels a aad c were photographed at a higher focal
plaae thaa b
and d.
Fanel c shows
the cells photographed
usiag rhodanine filters which allow tetanus toxin binding to
be visualized.
graph.
Panel
a Is the corresponding
phase micro-
Glial intermediate filaments are present in most of
the flat cells but the neuroa3
are clearly not stained with
the anti GFAP antiserum (d). The arrows in a and b indicate
the position of one of the neurons. Bar 50 um.
67
3.1.4
Cell ty.p.es eresent in untreated Pi9 cultures
The untreated aggregated cultures
undifferentiated EC cells
embryonic endodera.
of P19 cells contained
and cells which
These cultures
resembled extra-
were analyzed with the
anti&era described above.
Neltner glial
filaments nor
type.
fibrillar protein-containing
neurofilaments were
Fig 3.10b
shows that
like ceiib in untreated
intermediate
observed in
either cell
the extra-embryonic
cultures contained intermediate fi-
laments hhich
were stained with
al^o observed
these filaments in unditferentlated
(act shcwa).
Paul^n et al (1980)
EC cells also contain vimentin.
la many
c d l types in
endoderm-
antibody to
vimentin.
I
EC cells
have shown that PCC3/A11
Thus,
both treated and
vimeatin is preseat
untreated cultures
(Table 3.2).
The «xtra-embryonlc indoderm-like celis in untreated cultures contained bundles of
intermediate filaments that were
stainec with antiserum directed
against keratin (figs 3.10d
and f ) . Tncse filaments extended from the nuclear region to
tht periphery cf the ceil,
ending cn desmosomes which were
shared by the neighbouring cells (fig 3.101).
of cytckeratln-ccrktajaiiig
endodrrm-iikfc cells
thtoe
ctii. are
preseat la
filaments ia
the extra-embryoaic
in untreated aggregates
lilf.jr~nt from
SA treated cultures.
The presence
indicates that
the fltrcblast-like
1 he
colls
unoiiferertiated P19
cf l L 3 old uct =.ta: i With ?,atl seruui to lierat-n.
69
Figure 3.10. Immutofluoreaceace staining ot the intermediate
filaments la the extra-embryonic
in the absence of RA.
These cultures were staired with the
antibody to vimentin (b)
f).
aad the antibody to keratin (d and
Phase coatrast micrographs of the same cells are shown
in a,c and e respectively.
ment system is
cells present
cells also
The vimentin intermediate fila-
similar to that seen
in BA
la the fibrcblast-like
treated cultures.
The endoderm-like
contain an intermediate filament
by aat-bodieo to keratin.
cell border
where
system staiaed
These keratin-contaialng interme-
diate filaaeats extend rrom the
the
endoderm-like cells formed
periphery of the nucleus tc
they appear
to
end on
desmoscmes
shared by neighbouring cells (arrow, f ) . Bar, 20 urn.
TABLE 3.2
CELL TYPES PRESENT IN AGGREGATED PI9 CULTURES
Untreated
Intermediate
Filament Protein
vimentin
keratin
glial f i b r i l l a r
protein
neurofilament
protein
* n.d.
not determined
embryonal
extra-embryonic
carcinoma
endoderm
Retinoic Acid Treated
neuron
astrocyte
n.d.*
fibroblast
71
3.2
DISC.OSSIQN
The aim of this project was
dlffereatiation patterns
would
be f-csaible
to use drugs to simplify the
of EC
to examine
cells la
vitro so
the important
that it
determination
eveats leading to the differentiation of a limited number ot
cell types.
8A,
My experiments showed that, in the presence ot
EC cells differentiated into a limited spectrum of cell
types, namely neurons, glia, and fibroblast-like cells.
experiments described
In this
chapter convincingly
The
demon-
strate that neurcns and glial cells are present in P.A treated cultures of aggregated P19 EC cells.
The
neurons present
in
these
cultures were
initially
identified on the basis of taelr distinctive aorpbology.
observed with
scopes,
botn the light
and ^canning
the processes from these
and interconnected network.
As
electron micro-
aeurons formed a branched
Immuacfluoresceat staining of
these cells hith antitubulia antiserum highlighted the varlC0to4.tj.es on seme of the neuronal precedes, structures which
are often seen on isolated neurons using this technique (see
example ^.n Kainln& and Connelly, 1981).
of these celis
ments
in their
was confined by the
cytoplasm and
The neuronal nature
preseuce of neurofila-
tctaauc
toxin receptors
their cell surface, two neuron-specific cell markers.
tification cf
another way
neurotransmitters aad
ta*ea by fieifftr *t al (1981)
Iden-
associated enzymes
of characterizing neurons.
This
oa
is
approach was
who have isclatec a line of
72
EC cells which appeared to be spontaneously committed to the
formation of eholinergic neurons.
and CAT were present la
from these cells.
Elevated levels of AchE
the differentiated cultures derived
The activities of these enzymes were ele-
vated ccordinately in
EA treated cultures of
P19 cells and
were highest when the cultures contained the largest numbers
of morphologically identifiable neurons.
However,
the ac-
tivities increased many days before neurons became apparent.
Perhaps th«se enzymes are expressed very soon after neuronal
determination.
The presence of CAT
and AchE suggests that
these neurons may be cholinergic.
la addition tc neurcns,
EA treated cultures of P19 cells
also contained glial astrocytes,
identified by their stain-
ing with antibody to GEf.
In the normal embryo bcth neurons
and gila are derived from
the ectodermal germ layer.
neural ceil
rather thaa
the
adult because they lack markers of mature neural cells.
For
example,
the
types resemble
the embryonic
Both
neurons do aot
glycoprotein (see
stain with antibody
review by Fields,
1979)
and
cells do not centaia S100 proteia (Koora, 1968;
Zomzely-Neurath and Walker, 1980)
communication).
(Dr.
J.
to Thy-1
the glial
reviewed by
Bell,
persoaal
I have not been able to fully characterize
the fitrobiast-like
cells.
The presence
of vimeatin-coa-
taiaing Intermediate filaments la these cells does aot imply
that they are fflesedermaily-derived, since vimentin is present in
most celifr it tissue
culture (France et
al,
1978;
73
Franke et al, 1979b), including P19 EC cell3.
Since the fl-
broblast-iike cells did not develop into muscle, adipose, or
cartliagenous tissue, it seems likely tnat these fibroblasts
are analogous to cells of similar morphology present in cell
cultures derived from embryonic brain (Abney et al, 1981).
The extra-embryoaic endoderm-like cells
aggregated cultures of P19
filament networks,
tin.
lag
could be visualized by an-
and the other by
antiserum to vimen-
Paulln et al (1982) have reported prekeratin-containintermediate
endoderm celxs
monolayer and
and
cells contained two intermediate
one of which
tiserum to pr^ker&tin
in untreated but
filaments
in
obtained from
parietal
F9 celis
it has recently
extra-embryonic
treated with
been reported
cytokeratm-contalnlng iatermediate
RA in
that vimentin
filament
aetworks
coexist ia the
parietal endoderm cells of
et al, 1983).
I did not observe cells with trekeratin-con-
taiaiug intermediate filaments in
ceils and
EA treated aggregated P19
therefore conclude that
does net appear under these
The absence of
the embryo (laae
extra-embryonic endoderm
coaditions with this cell line.
endooen from taese cultures
the dlffereat^.atioa cf
indicates that
this tissue type It
not a prerequi-
site first step In EC cell differentiation.
More important-
ly, it deaionstrates that P19 ceils differentiate into a different set of cell types in the
in its absence.
cioion events
P19 cells.
presence of PA than they do
This suggests that EA might affect the dewhich determine the fate
of undifferentiated
74
riA
has
been
previously
ditferentiation cf EC
cells.
shown
to
induce
The ceil type(s)
the
formed by
RA-exposed tc cells seems variable depending cn the particular cell line
the drug
used and on whether the cells
as aggregates or
la monolayer cultures.
cell line F9 (Berstlne et al,
sively studied.
are exposed tc
1973),
The EC
has been nost exten-
Strickland aad Mahdavi (1978) first showed
that RA treated monolayer cultures of F9 cells differentiate
into cells
resembling extra-embryonic endoderm
cells.
If
subsequently treated with dlbutryl cAftP, these cells further
change late parietal endoderm (Sticklaad et al, 1980).
Ho-
gan et al (19o1) have shown tnat some cells in aggregates of
EA treated F9
cells develop into visceral
and Feweil (1980)
treated
endoderm.
Kuff
have observed 'neuron-like* cells
in RA
culture? of
F9 cells
but the
neuroaal nature
of
these cells has not been unequivocally established and these
cells may be
in fact have beaa the
parietal endoderm cells
described b/ Strickland et al (1980).
cells differentiated into neurons
aad treated
10~ 6
M).
foraea,
with nigh
However,
maay of
oaly small
P19 cells,
RA (greater
than
of neuroas
were
nuiraers
the aggregates coatained ao
filaments.
formed by
whea they were aggregated
conceatraticns of
seme of the noaneuroaal cells
mediate
I found that some F9
Thus,
neuroat ic our hands.
and
dia contain prekeratln interextra-embryonic
F9 cells ^.a conditions
and F9 cell
neurons,
endoderm
in which none is
only inefficiently
was
made by
developed into
75
Aggregation was first used by Martin aad Evans (1975a and
b) to induce differentiation of EC cells In culture.
et
al (1979)
treatment of
have shown
an £C
that hexamethylene
cell line results
Speers
bisacetamlde
in the
fcrmation of
differentiated cells with epithelial or fibroblast morpnologles depending cn whether or not cells are aggregated duriag
drug treatmeat.
and those
The effects of
on F9 cells reported
RA reported in this thesis
by Began et al
also dependeat oa cell agaregatloa.
tioa may reeult from
(1981)
are
The effects cf aggrega-
iaside-outside interactloas similar tc
those hypotnesizec in other mammalian developmeatal processes (Heroert and Graham, 1974).
I have
observed that the
peared in a
differeatiated cell
reproducible sequence after plating
aggregates.
Fibroblast-lxke ceils appear
followed by neurons
(2 d ) , and glial cells
types,apBA treated
initially (1 d ) ,
(4-5 d ) .
The
morphciogy of the cells and the sequence of their appearanceis identical to that seen in explants of brain from 10 d-old
rat embryco (Aoney et al, 1981; Paju et al, 1981).
This se-
quence prcbibiy reflects the rates cf neuronal and glial maturation and suggests
that *A causes a
embryonic ectoderm and neuroepithelial
dlfferentiatj.cn ot neural ceils.
in chapter 5.
plies the
One could
rapid commitmeat to
which is fclloweo by
This is discussed further
speculate that the aggregate sup-
jecsssary thr«ee dimensional environment
vivo in the neural tube.
found in
Chapter IV
MECHANISM OF ACTION OF RETINOIC ACID
4.1
RESULTS
4.1.1
Induction versus selection
There are
at least two
different models which
used tc explain the effects of
could be
RA cn P19 cells.
The first
proposes tnat RA acts by directly ineuciag P19 cells to differeatiate aleig
the developmental pathway leading
rons and glial cells.
Alternatively,
to neu-
RA could act by se-
lecting for cells capable of differentiating intc these cell
types.
The observation that neurons and glial cells do not
appear in P1^ cultures,
which have
suggests that RA sight act
not been exposed to RA,
by induction.
The following ex-
periments provide further support for the induction model.
RA did not kill EC cells.
of colonies
formed by P19
concentrations up
Fig 4.1a shows that the nuirber
cells in
to 1C~5 « was
tained in the absence of the
RA coaceatrations above
broblast-liK t, ceils,
the presence of
similar to the
drug.
RA at
number ob-
The colccies formed in
5 X10~ S M were composed
whereas colonies
formed at
cf the filower RA
concentrations were composed of cells with i-C morphology.
The growth rate of P19 celis
in medium containing RA was
measured during a 48 h drug exposure.
- 76 -
48 hours cf exposure
77
to RA is sufficient for neurons aad gl^al cells tc differeatictte la all
fig 4.1b,
aggregates exposed to RA.
RA had little effect
this time.
oa the growth
In other experiments,
growa la the presence of 5 X10~ 7
culture contained
As caa
be seen in
rate duriag
I fcuad that aggregates
M RA for 9 d in suspension
80% of the number
of cells found
treated aggregates grown for the same length of time.
in unThus,
it seems unlikely that the effects of kh can be explained by
it simply killing cells destined
to develop late other cell
types such as muscle.
The cells of
the P19 cultures may
respect to their response tc RA.
be heterogeneous with
Although uadiffereatiated
cells disappeared froa. fiA treated cultures,
selecting tor
precomaitted to
4.1.4 below).
the overgrowth
of a
form neurons and
EA might act by
subpopulatior. of
glial cells
TO test this possibility,
cells
(see section
25 F19 cells were
individually picked and plated Into separate culture dishes.
19 formed ccicnies,
clonal ceil liaes.
and 17 were suee-ssfully
15 of these
expanded into
17 ceil lines responded to
RA in a manner similar to that for the parental culture (Table 4.1).
The exceptions were
which gave rise tc a few neurons
P19S8,
a tetraplcid clcne,
even in the absence of RA,
ana P19S11, which also gave very small numbers of neuroas In
a aiBcr fraction of untreated aggregates.
within the H 9 cultures appears
to RA.
Thus,
each cell
to be capable of responding
TABLE 4.1
RESPONSE OF SEVERAL SUBCLONES OF P19 TO RETINOIC ACID (RA)
NUMBER OF AGGREGATES CONTAINING NEURONS
SUBCLONE
V/ITH RETINOIC ACID1
79/802
60/60
56/60
39/40
37/40
49/50
40/40
33/36
70/70
60/60
61/61
75/75
61/61
55/56
65/65
99/100
60/60
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
SI 1
£12
S13
S14
S15
S164
S17
r - - •> —
,-
ere "created with 5 ^- 10
WITHOUT RETINOIC ACID
0/50
0/50
0/50
0/44
0/50
0/76
ND 3
39/83
ND •
0/50
10/^2
0/50
0/41
0/18
0/70
0/50
0/46
I. r e t i n o i c a c i d .
2. I,Tu.jLe." cf a~~re.rates containing neurcns 3 fis-ys a f t e r
pdat^nr a ^ - r e g a t e s / Iiumber of agjrejaxes examined.
3 . ND = no a a x a .
4. S16 cells are referred to as P19S18 in text of thesis,
79
It PA affects determination events,
to remove the drug following
fereatlation.
tures at
ment.
it might he possible
commitment but hefore cytocif-
RA was therefore removed from aggregated cul-
various tines after
All cultures
the beginning of
were plated 5 d
the experi-
after aggregation and
i
scored 2-3 d later.
Indicate that
ensure
The
results,
a 48 h exposure
that neurons
formed
Thus, RA acts very early,
to the drug was
adeguate to
virtually all
aggregates.
in
3 to 4 days before differentiated
ceils first beccmt evident.
selection to occur and as
illustrated in fig 4.2,
There would be little time for
demonstrated abcve,
there are ao
toxic effects during this 48 hour period.
Monolayer cultures of P19 cells could be treated for 48 h
wita bk Defers aggregatica aad subsegueat culture la the absence of the drug.
abundantly from
In such experiments,
each aggregate.
were treated with RA but
neurons developed
Mien monolayer
act aggregated,
cultures
virtually all the
cells differentiated Into fibroblast-like cells anc few,
any,
neuron^ were found.
tion was
Apparently neuronal differentia-
greatly facilitated by
biga density
both t<A treatment
achieved by aggregation.
treatment are two
if
Aggregation
separate conditions which may
and the
and RA
be met si-
multaneously or PA treotment nay precede aggregation.
Relative doubling time
Relative survival
O
CT)
p
CO
<3-
o
3
O
o»
i
-
o"
O
—
o
-^
CD
ZJ
-•
O
3
9
(—-•—i
cr
q
-
i —
-+T-
1
1
I-
O
co
I
^zo
i_
81
Figure 4.1. RA is not toxic to P19 celis.
cieacy aad
growth rate of P19
tures coataiaiag RA.
The plating effl-
cells were measured
The relative survival (a)
la cul-
was calcu-
lated from experiments in which P19 cells were plated at low
density ("200 cells per 60 mm
diameter petri dish)
and the
number of colonies counted after 10 d.
The points represent
the meaa ia 3-5 separate experiments.
The meaa platiag ef-
ficleacy in the absence of PA was 41%.
The doubling time of
P19 cells was calculated after growing the P19 cells (seeded
at 10 s cells / ml) for 48 h la the presence of RA.
tio of
the doubliag
dcubling txroe
time of
cf the untreated
the RA concentration in b.
the treated
cultures is
The ra-
cultures tc
the
plotted versus
The points represent the mean of
12 or 2'4 separate determinations obtained in 3 or 6 separate
experiments.
h.
The mean doubling time of the controls was 16
Vertical bars represent the sample standard deviation.
Percent aggregates with nerve
_
ro
-& o") go
o
0
J
o-
c o
o
3
o
CD
•8
o
CO
c
CD
o
<5.
5*
o a>.
o" o
8
a
88J
0
0
I
0
I
0
I
0
I
L_
83
Figure 4.2. RA need not be coatiauously present ia aggregated cultures.
RA (5 X10~ 7 ») was added at the initiation of
aggregation and removed at various times by washing
witn
scored
normal medium.
for the
Aggregates were
presence of
Each poiat represeats the meaa
neurons 2-3
plated at
d after
3 times
5 d
and
plating.
of 2-6 separate experiments.
The sample standard deviation was
calculated for those coa-
centrations which were tested in at least 3 experimeats.
84
4.1.2
geiiQQic acid aaalQgues
RA has UJ vtrse biological ertect=
tv^as.
cn many different cell
ihc intracellular targets of BA which are important
ir thta dif f „rt.] t ic tj.on cf P19 cjilb, jay be similar to those
in other PA-st ntitivi biological
systems (although the con-
seqaerc^s of t-A action art v«ry different),
logues cf
t A have beer, usia
th c &A i;ie;uit
tan,
I9o0>.
to investigate whicr-
e.re Important for biological
A
ab.iity to incuce
Table 4.2 sbow.s
the C15
structural ana-
nuiiber of ratinclds were
Cirbcxync acid
concentrations to
attain the same
rent^Vc ctt.cenoita of
obtained in some
group of
activity (Lo-
tested for their
t-19 cells tc differentiate
that retinoids with major
parts of
into neurons.
modifications tc
bA rtqulrta
such higher
efficiency as
RA.
The
<-he analogue ire similar
to those
other biological sy^tens (Jttter
and Jet-
tei-, 19?y; Lotan, 19o0;
Lctaa •it al, 1981) aid suggest that
the intracellular rechanisss of retinoid action ir P19 ceils
art sitiiaL tc tho-= it othtr
ir.^ to the cfiABP.
systems and n<ay involve bind-
TABLE 4.2
EFFICIENCIES OF SOLIE RETINOIDS ON INDUCTION
OF NEURONAL DEVELOPMENT
RETINOID
HAIF-EFFECTIVE
DOSE' EFFICIENCY RELATIVE
b
TO ALL TR
( X 10~ U)
All trans EA
2.8
1
13-cis RA
3.8
.72
retinal
50
.06
retinol
280
.01
retinyl acetate
430
.007
2
Tii-i.i? analogue of
ethyl retinoate
Ti.I,I? analogue of
K-ethyl retinaraide
450
.006
>1000
-
1. Dose at which 5O70 of plated aggregates contained neurons
3 days after plating the aggregates.
2. TULIP = trimethylmethoxyphenol.
86
4.1.3
Efiliaiifies
kk is an anti tumour promotor.
from HA mediated suppression
key enzyme in
This activity may result
of ornithine decarboxylase,
polyamine biosynthesis (Verma et
Since polyaaines may play a
tion .'systems (Fish
et al,
1978).
role in some other differentia1981;
seemed possible that the RA effect
by a decrease in
al,
a
Scott et
al,
1982),
it
cn P19 coula be mediated
intracellular polyamiae levels.
Cultures
of aggregated P19 cells were exposed to the following drugs,
bota ia the
preseace aad absence of PA:
3
X10""6 M spermi-
3 XIO"** M alpha-methyloraithine and 10~ 7
dine, a polyamine,
M methylgiycxal-bls-(guanyi-hydrazone),
polyamine biosynthesis,
a tumour
two inhibitors
promotor,
myristate acetate and 3 XlO*"fc to dexamethasone,
our promoter.
48 a growth
These drug
tests.
the P19 cultures,
of
10~ s H phorbol
ar anti-tum-
concentrations were non-toxic in
None of taese drugs had
suggesting that
any effect on
changes in polyamine me-
tabolism ao not mediate the developmental effects iaduceo by
RA and that tumour promoters are indffectlve in altering the
response of P19 cells.
4.1.4
Mutaat cell lines
Siace the Important bioxogical
meat were not obvious,
conseguences of RA treat-
1 used a genetic approach to attempt
to determine *nich events are cruciai to the differentiation
process.
ihiJ section de^cr-Dis
the stepwise Isolation of
87
P19 derived cell lines which
rons in the presence of RA.
(see section aoove),
eace
of 10""7
do not differentiate into neuP19S18 cells, a subclone of P19
were cultured
M BA,
ana a
in the continuous pres-
partially aonrespoasive
P19S18PAC6 was isolated (fig 4.3).
clone
P19S18RAC65 (PAC65) is a
subclone of P19S18PAC6 which was isolated ia the Freseace of
1 0 _ s M EA.
RAC65 cells do BOX. dlrfereatiate into neurons at
ccncentratioais of
1 0 - 6 M (fig
SA as high as
cells are similar ia morphology to
4.3).
RAC65
P19 EC cells aad have 42
chromosomes.
No cells with
EC morphology were appareat
ia RA treated
P19 aggregates by 7-8 days after the begianlng of the experiment.
In contrast,
tures of
7-8 day
PAC65 cells coasisted
In order to document this
old identically treated culeatirely of
observatioa,
EC-like cells.
I used a moaocloaal
aatibody, AEC3A1-9, which aetects an aatigea fouad oa uaoiffereatiated EC cells but which is not present on differentiates!
cells (J.P.
Harris et
al,
AEC3A1-9 aatxger is closely related
by SSEA-1 (Solter ano Knowles, 1978).
in preparaticn).
The
to the antigen detected
As can be seen in fig
4.4b, the number of cells carrying the antigen In RA treated
P19 aggregates,
as detected by indirect immunofluorescence,
decrease! after 2
days.
day-5 aad reached a plateau of
C e n s from DWSC treated
pattern.
Tne
25% by 6-8
aggregates showed a similar
fluorescence on the
cells after 6
very weak ana guantitative ifti!iuaoabi>crptioa (Dr.
days was
J.F.
Bar-
88
ris) experiments indicated that the cells in these RA treated cultures contained
only about 1% ct
tration present cn untreated ceils
the antigen coacea-
(fig 4.4a).
The amount
of antigen on the control untreated cells decreaseo,
a muca lesser
extent.
This decrease can
the formation of extraembryonic endoderm.
but to
be attributed to
Fig.
4.4c shows
that the amouat of antigen on RA treated RAC65 cells did not
decrease during
the experiment
confirming the
that these cultures consisted of
EC cells.
cbservatxon
Experlmeats on
the growth rate cf RAC65 cells in monolayer cultures provided further support fcr thii idea.
The RAC65 cells grew con-
tinuously aao rapidly in both tne presence and absence of RA
(fig 4.5b), whereas the parental P19 cells did not grow continuously ia PA treated cultures (fig
RA,
proliferation
ceased and
the P19
ceils with a fibroblast morphology.
in RA-treated hAC65 cultures.
4.5a).
After 2 d in
cells changed
intc
No such change occured
to
c
O
100
i_
Z3
C
^
1
I
80
\1
(1>
t.
C7»
60
// 1 ••?
1 :'
/ • •'
1 •'
It
/••
200-L
,'
<^
'
1
40
o>
*-
'"
/1
///
1
to
<u
o
-*-••
V
^
^ ^
^^^^^
-r^'-i
0 I0"
•
1
9
I0"
*
jo1
'
1
l
I
'
a*'
i
'
/
S
(-il^"
1
8
''
'
**
,' '
I0"
1
7
I0"
^*«»
a
***-u
1
6
Retinoic acid concentration (M)
10ro
90
Figure 4.3. RAC65 cells ao aot differentiate into neuroas in
the presence of RA.
Aggregates of cells were cultured for 5
days in
the presence cf drug,
latsr.
Normally,
plated and scored
50 aggregates were
coaceatratioa ia each experlmeat.
2-3 days
scored for each drug
The pclats indicate the
meaa obtained from several experiments.
P19S18 (filled cir-
cles), P19S188AC6 (open circles), RAC65 (open sguares),
HY-1 (filled sguares).
aad
Relative EC antigen per cell
T
ro
.^o"
^--°"
b.
% EC antigen positive cells
o
3
ro
oi
I
l
o
CJl
o
-^
O
cn
o
i
1
JLao
••-•
SO
ro
..."
CD
.••'
-&
•
..Q
^s*
Q
o^ *
*
cn
*/
••
•
••
•••
a
03
s
9 •
•
o
1
1
o' o
I
•
•
o
PO />
:
O" •
*
9
1
o
I
i
o
1
DO©
ro
a
i
1
^
/
0)
/
03
.
1
1
oj1 *|
»
92
Figure 4.4. AEC3A1-9 embryonal carcinoma cell associated antigen disappears from PA and DMSO treated aggregate cultures
of P19
(a)
cells but not
from simllarliy treated
RAC65 cells.
The amount ot antigen per P19 cell s»as determined by an
absorption procedure
cells.
after gluteraldthyde
the
The perceat of aatigea positive cells ia P19 (b) aad
RAC65 (c)
cultures was measured using an Indirect immuncfl-
uoresceat procedure.
experiments.
Each point represeats the nean of 2-3
500 cells were scored
experiment cf panels b aad c.
cles),
fixatloa of
5x
10" 7 K RA treated
treated (open sguares).
for each pcint in each
Untreated cells (filled cir(opea circles),
and
1% DMSO
Relative cell number
94
Figure 4.5. The growth rate of RAC65 cells is not changed in
the presence cf 5X 10~ 7 M RA.
The growth rate of cells in
monolayer cultures has determined by plating cells at a conceatratioa
disnes.
cf 10 s
cells/ml lato
duplicate tissue
culture
The c^lls from the two dishes were couated after 24
aad 48 hours respectively.
the cells in the 48 hour
for 8 days,
Two aew dishes were seeded from
dish aad the process was continued
rhe points represent the meaa of 2 experiments.
P19 cells (a),
RAC65 cells (b), and HY-1
growa ia the continuous preseace
(opea circles)
(filled circles) of retiaoic acid.
as follow*: P19;
cells (c)
were
or abstace
The doubling times were
14.7h in untreated cultures, RAC65;
16.3h
in treated aad untreated cultures, and HI-1; 16.7fc in treated lad
uatreated cultures for 3
days at which time
creased to 20.1b ia treated cultures.
it in-
95
In order
to determine
dominant or recessive,
were
carried
P19S1801A1,
cut
in
whether the
RAC65 phenotype
was
cell-cell hybridization experiments
which
RAC65
a 6-thloguaaiae and
P19 cells (McBurney et al,
cells
were
fused
to
ouabain-resistent clone of
1982).
Clones of hybrid cells
vera selected in HAT (Llttlefleld, 1964) medium supplemented
by 1.5 aM ouabain.
Two clones,
HY.-1 and HY-2 (isolated by
Dr. M.s. McBurney), were examined ia datall.
The basic ob-
servatioa was
recessive be-
that the mutant
pheactype was
cause bcth hybrid lines dirfereatiated into neurons whea aggregated in
tne presence of RA
(fig 4.3), although
uadiffereatiated cells remaiaed in the aggregates.
a few
Hybrids
betweea EAC65 aad 01A1D3, which I isolated, also differentiated into neurons
sponsive tc
(data aot showa).
RA but non-responsive
thesis, 1983).
01A1D3
to DMSO
cells are re(Edwards,
MSc.
In monolayer growth experiments, the results
were not as clear-cut.
HY-1 aad HY-2 cells grew more slowly
after 3 day» in RA but
did not abruptly cease prcllferatlon
as did P19 (fig 4.5c).
Some of the hybrid cells had a cfiro-
mcsome aumbtr less thaa thy
Thus,
tne
combined parental number of 82.
iatermediate pheaotype ot
ccajegueace of
the hybrids may
th*? heterogeneity of the
te a
chromosome numbers
in the hybrids and the apparent segregation of recessive alleles.
96
4.1.5
£§1,1 vflluie cha.fig.gs duriag dif|eienti§iicn
The cells ia £19 aggregates
became smaller in EA treated
cultures as compared to those from untreated cultures.
Fig
4.6 shows a ceil size distribution obtained 4 days? after aggregation.
1 estlaated
the median cell volume
from these
acd other cultures (see Materials and Methods) and these are
plotted in fig 4.7.
cells
The 30% decrease in volume cf untreated
prcaaoiy reflects
phase cf the- cell cycle.
accumulation of
RA
cells
treated with
coatrast,
tensive
cells.
Gl
treated P19 cultures showed a
much more dramatic 75% decrease in cell volume,
the cells were aggregated.
la the
but only if
Monolayer cultures cf P19 cells
RA did not show
changes ia cell
volume.
In
RAC65 cells treated with PA did aot show this exvolume
decrease
and behaved
like
untreated
P19
Cell number per channel
O
O
o
_JL
ro_
o
o
O
a
=J
Z3
Q_
Z3
C
3
O
98
Figure 4.6. The size distribution of cells from 4 day old RA
treated (dotted line) aud untreated (solid line)
gates.
P19 aggre-
The aggregates were disassociated and the size dis-
tibation ot the cells obtained using a Coulter Counter Channalyser.
The peak channel of the dlstributioa obtaiaed from
unaggregatea P19 cells was 60.
Relative cell volume
ro
I
O-
ro •
H
3
CD
Q.
Q
(fi
a> -
00 -
w
J
*^
I
CJI
I
b> "->i co to b
I
I I I I
100
Figure 4.7.
The volume of
decreases.
Aggregated cells were
tervals.
Each
point represents
volume caicuiated from
Coulter Counter
fined as the
cells froa RA treated aggregates
dissociated at daily inthe relative
median cell
a size distribution obtained
Channalyser.
Relative cell voluae
median volume at time t divided
volume of untreated cells.
with a
is de-
by the median
Uatreated P19 celis (filled cir-
cles), 5 X10 - 7 M I A treated P19 cells (open circles),
X10~ 7 M PA treated RAC65 cells (filled sguares).
and 5
101
4.2
DISCISSION
My results argue against mo dels
which attempt to explain
the action of RA by cell selec tioa.
RA" was aot toxic to P19
cells during the first 48 aour s whea RA must be preseat.
also
platl ng
allowed unreduced
plated
into PA-containing
efficiencies
me dium.
ceils was
found to be homogea eous
spcnse to
PA treatment.
It could
The
cf EC
cells
population of
cells and that
differentiation process
but h as
its re-
be argued that
the P19
can differentiate
RA acts to
no role
differentiation cf these cells .
into extra- embryoaic
glia,
enhance the
ia directiag
However,
and muscle (McBurney et al, 19 82),
EC
with respect to
population as a whole is commi tted to making neurcns,
and fibroblast-like
RA
the
since P19 cells
endoderm-like cells
it Is iiore probable that
they comprise a pluripotent EC cell population. This is supported by experiments In which
mouse blastocysts
P19 cells were injected into
(Pcssant an d McBuraey,
ceils contributed tc
sost of the tissues
developing mouse embryo.
1982).
The P19
In the conseguent
Ia addition, other pluripotent EC
ceil lines also respond to RA, indicating that the effect is
not
peculiar to P19.
Most co nsistent with the observations
is the modaj. which propose^ th at RA acts at the level of determination to induce a set of
the absence cf
the drug.
events which do net occur in
Th us,
guences ot RA treatmeat may be
the
intracellular coase-
slmllar to the ncrmal level-
opmental signals which determi s' neuronal and glial tissues.
102
It remaias unclear how RA triggers the neural developmental pathway.
(Lotan,
RA has a number of diverse biological effects
1980).
The relative
efficiencies of PA analogues
are similar in many in y.ltr.2 systems (lotan, 1980;
al,
1980).
This heirarchy of efficiencies
Lotan et
is correlated
with the affinity ot the retinoids for the cellular retinoic
acid binding prctein (cPABP) (J^ttea aad Jettea,
taa, 1980).
in the P19 system suggests
with tha cRAfaP
celis to
Lo-
Tne effectlveaess of the various retiaoids that
I have tasted
of RA
1979;
may be a
alfferentiate into
that the reaction
first step la
naurons.
cPABP
Induciag P19
complexed RA
might then be transported to the
nucleus where it would act
directly upon the DNA.
al (1981)
demonstrated tnat
protein,
Liau et
retinol,
is transported
bound
tc the nucleus ot
where it binds specifically to
tla.
in fact,
cellular binoing
rat liver cells
sites located oa the chroma-
The otservatioas of Schiadler et al (1981) ere consis-
tent with taj.5 type cf irodel
RA ia EC ceiis.
They
for the mechanism cf action of
have Isolated RA noa-respoasive PCC4
EC cells aad have demonstrated
ty.
to its
have,
that they lack cRABP activi-
Ia soma systems RA acts by lahibitiag the Induction of
oraitnine decarboxylase,
a key
enzyme in
ynthesis (Vermd et al, 197d; Lotan, 1981;
Scott et al, 1*82).
However,
mine Dicaynthi^ls is
aot involved m
neurai developmental pathway.
polyamine biosFish et al, 1981;
our data suggest that polyathe
triggering of the
103
I could fiad
in the preseace
no evidence the RAC65
cf PA.
RAC65 calls
aoarespoaslve pheaotype for maay
cells differentiated
have maintained their
geaeraticas ia tissue cul-
ture suggesting that a stable geaetic change has occurred In
these cells.
sult of
It seems ualikely that this chaage is the re-
a biagle poiat
mutation since twc
were needed fcr its isolation.
selection steps
I have been unsuccessful in
several attempts to isolate a aoarespoaslve mutant with caly
one selection step.
3eems to
behave as
Since the Inability to
a recessive character
respoad to RA
in the
cell hy-
brids, the two-step isolatloa requiremeats probably reflects
the presence
ceils.
of two
wild-type alleles
These aoa respoasxve mutants
ia the
diploid P19
should be useful as a
basis of comparison to the responsive t-19 ceils with respect
to specific
biochemical properties.
celis in mixing experiment mignt
importaace cf
Their effect
on P19
provide iaformatloa on the
iatercellular iateracticas
duriag determiaa-
tiea.
The voiuiie ot cells ia RA treated P19 aggregates began to
decrease relative to
the control by 48 h
tica of the experimeat.
The decrease
after the iaitia-
ia cell vclume is aa
early event in the neural development of P19 cultures but it
is aot necessary tor all forms of EC differeatlatiea because
ac drot in
cell volume is se^n in
DMSO
treated aggregates
of P19 cells- destined to develop lato aonneural tissues, including muscle (Edwards, MSc. thesit, 19o3).
This ob£.erva-
104
tioa suggests that changes ia ioa flow might be importaat ia
aeural differeatlaticn.
shown to be
Decreases in cell volume have been
an early event in the commitment
ythroleukemic cells (Loritz et ai, 1977),
of Friend er-
and the spontane-
ous ditferentiatlcn of an EC ceil liae called 1009 (Pfelffer
i
et al,
1981).
The effects aoted
gates are more dramatic because the
cumulate ia the
on RA treated P19 aggrecells do not simply ac-
61 phase of the cell cycle,
25% of their original volume.
but shriak tc
RAC65 cells from RA treated
aggregates behaved like P19 calls from untreated aggregates,
showing a small decrease in
days.
volume which stabilized after 3
This observation Is consistent with the previous ob-
servatloas tnat RAC65 cells are aoa-respoaslve to BA.
Different concentrations of RA
are effective in Inducing
the development cf different cell
typaa (Edwards aad McEur-
aey,
1983)
aad structurally uarelated
drugs such as DfsSO,
butyrate, aad 6-thicguanine hava similar differentlatioa-iaduciag properties to those of
low coacentrations cf RA (Ed-
wards, MSc. thesis, 1985, Appaadix A ) .
cells tali
to respoad to aay
all of these
Irug^ may have a
The fact that RAC65
of these drugs
suggests that
common intracellular pathway
of actioa and that th<= defect(s) in SAC65 cells affects some
component cf this shared pathway.
The data in this chapter can
be used to lomulate an in-
terim working
model for
the mechanism of
this system.
in the presence of PA,
action of
RA in
P19 cells become com-
105
mitted to
differeatiating into a
type and suisequently the cell
neuroepithelial-llke cell
types of the neural develop-
mental pathway.
This may be accomplished via the intracel-
lular blndiag of
RA to its bladlag protein.
the process dees act require aggregatioa
present for 48 hours fcr
maximum effect.
the determined P19 cells must be
This steo of
and but PA must be
After this time,
aggregated la order to ex-
press the differeatiatioa program which leads to neuroas aad
glial cells.
Some
aspects of this model
further ia the next chapter.
will be discussed
Chapter V
CONCLUSIONS
The primary objective
of this project was to
find a way
of manipulating the differentiation pattera of EC cells as a
first step
ia studyiag
the eveats
piuripcteat cells to particular
have observed that P19 EC
iavolved ia
committing
developmental pathways.
I
cells differeatiate entirely into
neuroas, giial astrocyte cells, aad fibroblast-like cells la
the preseace of non-toxic concentrations
10~ 7 M.
cf RA greater than
The tact that oligodendrocytes were act observed In
RA treated P19
preseace of
cultures may be a sin-pie
serum ia the medium.
consegueace of the
Oligodendrocytes,
while
sharing a coiion precursor with some astrocytes, differentiate only in cultures with
et al,
1983).
low concentrations cf serum (Faff
Neural cell types
treated P19 cultures.
induces P1* cells to
developmental pathway.
were aot observed ia un-
The simplest coaclusioa is
that RA
differeatiate along aa neuroectodermal
Thxs system snould,
useful tor studyiag comiiltmeat
therefore,
be
to neuroectodermal dlfferea-
tiation.
Several piece*
ol iafcrmatioa
would be
useful in
con-
structing a mcael of the events which occur whea EA is added
to a culture of P19 cells.
The first lavolves a^certainiag
- 106 -
107
the embryoaic
cell type which
is equiveleat to
P19 cells.
This would eaable us tc establish the mlalmum rumber of cell
type chaages uadergoae by the
P19 cells as they differenti-
ate into
that P19
aeuroas.
The fact
into extra-embryonic-like cells in
tc yolk sac la vivo
vitro and can contribute
(Rossant and McBurney,
that they are equivalent to ICM cells.
ceils late
cells differeatiate
1982)
suggests
Injectiag siagle ICM
geaetically distinct blastocysts has
showa that
the ICfc is partitioned into primitive endoderm aad primitive
ectoderm by 4 1/2 days and taat the latter cell type caa aot
give rise
to the former
after this txiue
(Gardaer,
However, soaa in vi;tjrc studies (Pederson et al, 1977:
dek,
1979)
capacity
1981).
Dzia-
suggest that primitive ectoderm does retala the
to dif fereatiate
short period.
lato
Therefore it Is
pris>itive
eadoderm for
a
possible that P19 cells may
be equivaleat tc an early primitive ectodermal cell In spite
of their oDility tc give rise
to small amounts of extra-em-
bryoaic actcderrt-llke ceils.
Tae commitment of P19 cells to
ditferentiate intc
which time
If cell
neural cells requires 48
the cells would be
dxvisioa is necessary
able tc divide
hours,
during
three times.
for commitment to
occur and
Pi* cells dc indetd represent ICM celis, then there would be
very uttie txme for a P19
tc an ectoderaal-llke
ceil to develop from er ICM-llke
and finally intc a
leurcepitheiial differentiation.
cell committed to
This time frame may argue
that Pi* cells are, in fact, ectodermal equivalents.
108
The correiatloa observed betweea
analogues to
induce the
differentiation ot
their reported ability to biad
gests that a
of RA
aad
as its first
bioohemical actioa
step,
binding
to the
A defect in cPAEP activity could explain the reces-
sivity
of the
RAC65 mutation,
RAC65 cells tc RA,
kiaetxcs ia
cells dc
the noa-respoasiveness
of
aad the appareat requirement for two hit
obtaiaiag the RAC65 mutaats.
act respond tc DMSO (Edwards,
Since it is
affect
F19 cells
to the cfiABP indirectly sug-
possible mechaaism for the
might lavolve,
CRABP.
the ability cf retlaoid
However,
RAC65
M£c.
thesis 1983).
difficult to understaad how DMSO
could biad or
CRABP,
this
suggests that
eveat(s) after blading tc cPABP.
ed for cRAfat activity.
The
01A1D3 (Edwards MSc.thesis,
the RAC65
block is
at
RAC65 cell should be test-
DMSO non-responsive cell line,
1983)
which does differentiate
into neuroas ia the preseace of RA,
should be useful la ia-
vestigating whether RA and DMSO act independently in the P19
cells.
Another possible mechaaism of actioa
aot Investigate
in this tnesis,
cell surface glycoproteins.
of RA,
would involve
which I did
changes la
Ia this type of model, RA would
eatar F19 c<allii and be converted to a retlayl phcsphate.
has beta suggested that a metabolite
It
of RA can form retiayl
phcsphate which can then act as a carrier for mannosyl -residues,
et al,
transporting them across the plasma ^embrace (De luca
1979).
In this manner,
the ceil surface glycopro-
109
teias of
tha P19
cell could
be modified.
would presumably cause surrouadiag
These changes
cells to act differently
ana trigger the chaages leading to aeural differentiatioa.
After the initial 48 hours of RA treatment, the P19 cells
must be aggregated
in order to differentiate
RA- is ao loager required.
fibrobiast-iike cells.
arraagemsat ot
ganization cf
Noa-aggregated cells develop iato
tresumabiy,
the three dimensional
the differentiating cells is
as it is in vivo.
into aeurcas.
importaat just
I have ao laformation on the spatial or-
the. differeatiatiug neural cell
types within
the aggregate, but it is tempting to compare it to the neural tube witn
neurons aad glial cells geaerated
ia aa lraer
layer and ^grating ourwards after they begin to differentiate.
This system offers exciting possibilities fcr studylag
aeural precursor cell types aad
for examing the cytodiffer-
eatiatici cf aeurcas and glial cells.
still not
knowr if aeurcas and
precursor.
In particular, it is
glial cells share
a cofctron
With this system, it should be possible tc idea-
tify this ceil typ^, if it does exist, by exaainjng the progeny of suitably labelled ceils.
Sdhards aad
show that EA,
duce P19
juscle.
trations.
."cBurney (19c<3)
have preseated
data which
at coaceatrations of 10~ a S and lower caa in-
cells to differentiate
into cardiac
end skeletal
Thuo, EA ha^ diffcreat effects at different concenBaden et ai (1982) aad Tickle et al (1982)
have
shown that BA can affect pattern formation ia developing and
no
regenerating limbs,
a developmental
iavolve gradleats of morphogeas.
different ccacentrations of
process which seems to
Oae can speculate that the
RA effective la the
P19 system
might represaat a gradient effect in v.iy.o.
In ccaclusioa,
the effect of RA
oa P19 cells appears to
be aa effect oa the determination events which commit pluripotent
ceils
to ectodermal
pathways
of
differentiation.
This system can be used to study the events involved in this
determiaatioa process aad also caa be exploited for studylag
aeural developmeat.
Appeadlx A
RESPONSE OF P19 CELLS TO DMISO
Tnis a^peaaix contains the preiiuiaary
report of the ef-
fect cf DMst oa the ditfwrcatiation cf P19 cells.
-111-
R c p n n l e d from N J I U K
\ >l 2w
Ni
^*- 7'/
Mu( rrullun Jounuih
Control of muscle and neuronal
differentiation in a cultured
embryonal carcinoma cell line
\l~
9 September
14o2
II
A
M. W . M c B u r n e j , E . M . V. Jones-Villeneuve,
M. K. S. E d w a r d s & P . J. A n d e r s o n
Departments of Medicine Biolog> and Biochemistry
Unnersm of Ottawa Ottawa Canada K1H 8M*i
Pluripotent murine embrvonal carcinoma cells can differentiate
in culture into man\ tissue types similar to those normally found
in earl> embryos 1 and may be useful in invest.gating some
developmental e>ents 12 . Central to our understanding of
embnonic de*elopment are explanations of cellular determination, that is, the commitment of early embryonic cells to form
divergent cell types. Of relevance is recent work with the F9
bne of embryonal carcinoma cells which suggests that certain
extra-embryonic cell tjpes are specifically formed following
treatment of undifferentiated cells with drugs3 4 and the manipulation of culture conditions*. We report here that the P19 line
of embryonic carcinoma cells6 may provide an analogous s> stem
in which drugs can be used to manipulate the formation of
tissues which normalU comprise the fetus. In the presence of
dimethyl sulphovide (DMSO) aggregates of P19 cells differentiate rapidly to form large amounts of cardiac and skeletal muscle
but no neurones or glia. We have pre>iousl> shown that in the
presence of high concentrations of retinoic acid (>5 x 10~7 M),
aggregates of these same cells develop into neuronal and glial
tissues but not muscle 7 . Thus, drugs can be used to generate
two quite different spectra of embryonic tissue tjpes from the
same population of embryonal carcinoma cells.
PI9 is a euploid (40 XY) embryonal carcinoma cell line
derived from a teratocarcinoma induced in C3H/He strain
mice 6 For the? experiments described below, we used
P19S1801A1, a ouabam-resistant and 6-thioguanine-resistant
subclone of P19 isolated without mutagenesis Suspensions of
dispersed cells were plated onto bacterial-grade plastic surfaces
to which cells do not adhere 8 Cells adhere to each other to
form small aggregates These aggregates were cultured in suspension for 4-5 days in the presence or absence of DMSO
They were then plated into tissue culture-grade plastic dishes
In the absence of drug, the plated aggregates contained
undifferentiated embrvonal carcinoma cells along with small
numbers of extra embryonic endodermal cells7 (Fig l a ) The
presence of DMSO in the culture medium produced effects
which became clear 1-2 days after plating, that is, 6-7 days
after initiation of the experiment In cultures exposed to 0 25%
(v/v) DMSO, most plated aggregates contained embryonal
carcinoma cells, rhythmically contracting muscle and fibroblasthke cells At concentrations of 0 5, 0 75 and 1 0% DMSO,
none of the plated aggregates contained embryonal carcinoma
cells (identified by morphology), virtually all contained areas
of rhythmically contracting muscle, and all contained cells with
fibroblast-like morphology (Fig lc) By 10-12 davs the amount
of contracting muscle had increased (Fig Id, e) Also at this
time many of the DMSO-treated aggregates developed areas
of bipolar myoblasts which fused into myotubes (Fig If) These
myotubes were usually non-contractile but often developed
spontaneous twitching activity by 14 days
Electron microscopy of the cells in DMSO-treated cultures
indicated that the rhythmically contracting cardiac muscle cells
contained glycogen granules, large numbers of mitochondria,
and numerous areas of thick and thin filaments which were not
organized into mature myofibrils (Fig la) The multinucleate
skeletal muscle cells were similar in appearance (Fig lb) Thus
both muscle types seemed to be immature Many of the nonmuscle cells had abundant rough endoplasmic reticulum and
n
\ ,. —ir/' SZ'r ,.
^
Fig 1 Phase contrast photomicrographs of live teratocarcinoma cells The
conditions for cell culture 7 ' and aggregation" have been described pre
viousl> Cell aggregates were formed from a stock culture of P19S1K01 Al
cells and parallel cultures were carried in a normal medium o medium
plus 10°o fetal bovine serum 1 ) b m medium containing ^ x l o M
retinoic acid and c f in medium containing 0 ^uo (\ \ I DMSO Acgrtkate^
were cultured in suspension in bacterial grade Petri dishes [or ^ da\s before
being plated on to tissue culture grade plastic surfaces Photographs were
taken 2 days (a-c) or 9 davs [d f) later a Untreated aggregates contain
embryonal carcinoma and a few extra embrvonic endoderm cells larrowl
b Aggregates of cells which had been cultured in the PKS n e of ** It M
retinoic acid contain neurones and asirocvu LJIJI t<JK
V u LJIC
cultured continuouslv in the presence of (1 S DMSO lonta n smill area
of rhvthmicallv contracting cardiac muscle (arrows m c wh LI- he >mt
more extensive with time id) At higher magnification are ( aicis of
rhythmically contracting mononucleate cardiac muscle and / multinucleate
skeletal muscle Scale bars 200 p.m
some were surrounded bv extracellular matrix which included
collagen fibres (Fig 2c)
The DMSO treated aggregates of P10S1801A1 cells
developed muscle but neither neurones nor gha Treatment of
the same cells with retinoic acid resulted in the development
of neurones (Fig 16), glial cells, fibroblast-like cells but no
muscle Cultures exposed to both retinoic acid (5 x 10 7 M i and
DMSO (0 5 or 1 0%) developed as if exposed onh to retinou
acid, that is, neurones and glia but no muscle were formed
Differentiated cultures contained more actin than did
untreated cultures (Table 1) Much of the actin in DMSO
treated cultures was a-actin, the type present onl\ in skeletal
and cardiac muscle cells9 Muscle-specific mvosin was also
detected in both cardiac and skeletal muscle by immunofluorescence using monoclonal antibodies directed against muscle
myosin About 15% of all cells were muscle myosin positive
in these cultures by 8 days but none were detected in untreated
or in retinoic acid-treated cultures
7
Presence of a actin in D M S O treated cult ures
Table 1
Treatment
Total actin*
a actin*
% Muscle
actin
Untreated day 7
Retinoic acid, day 7
D M S O day 7
D M S O day 11
1 91±007
231±003
2 62 ± 0 IS
3 0 5 ± 0 14
0 16 ± 0 02
030±002
052±001
0 6 8 ± 0 02
84
130
198
22 3
Aggregated cultures were prepared as described in Fig 1 legend Two days
after plating (day 7) or 6 davs after plating (day 11) the cultures were collected
for analysis The DMSO treated day 7 culture contained rhythmically contracting
but not multinucleate muscle Bv da\ ] ] both muscle types were present Proit-in
and peptide isolation was carried out as previously described 24 except for the
use of trypsin instead of chymotr>psm for peptide generation Trie actin contents
were calculated by measuring the amounts of male lal which co purified during
electrophoresis at pH 6 5 2 1 and 3 *> with tryptic peptides generated from muscle
actin Total actin values were calculated from the amounts of radioactive material
co-migrating with the two chemically modified peptides CmCys-Asp lie Asp He
Arg and CmCys Phe All known actins contain peptides which should co punf\
with these two a-Actin values were calculated from the amount of material
co-purifying with an 18 residue CmCys containing peptide generated from the
N terminal region of a actin Actins from other tissues differ from o actin jn
this region 9 so should not co-punfy with this peptide Total actin is higher in
differentiated than in undifferentiated cultures and there is more muscle specific
o actin in DMSO treated cultures The 8-13% of muscle actin present in
untreated and in retinoic acid treated cultures may represent a background of
radioactive label derived from non Q actin peptides which co purify with the
legitimate peptide
* mg actin per 100 mg total protein
Fig 2 Electron micrographs of some of the tissues formed in DMSO
treated cultures a A section through cardiac muscle shows bundles of
thick and thin filaments in longitudinal section (arrows) glycogen granules
(arrowhead) and numerous mitochondria b A section through a multinu
cleale mvotube shows the thick and thin filaments in cross section (arrow)
and glvcogen (arrowhead) Manv of the non muscle cells in these cultures
secreted collagen le i which was often seen forming part of an intercellular
matrix Scale bars 0 5 i*m
When lOjiM adrenaline was added to DMSO-treated cul
tures, the cardiac muscle responded b\ a 2-2 5-fold increase
in contraction frequency and some previously quiescent areas
of the culture were stimulated into rhythmic activity Therefore
/?- adrenergic receptors were present Such receptors are
apparently acquired by cardiac muscle after the acquisition of
spontaneous contractility"'
DMSO was not demonstrably cytotoxic to the P19S1801A1
cells at concentrations effective in differentiation experiments
Figure 3 shows that the efficiency of colony formation was
unaffected by DMSO at concentrations up to 1 0% Virtually
all colonies formed in DMSO contained only embryonal carcinoma cells In other experiments, monolayers of cells were
cultured for 20 days in 1% DMSO without change in growth
rate or morphology At the end of this 20-day period, the
DMSO-treated cells were aggregated in the presence or absence
of DMSO (0.5%) Those aggregates formed in the absence of
DMSO did not differentiate while those cultured in the drug
formed muscle and fibroblasts in the usual way Thus, it seems
that the DMSO had no effect on the P19S1801 A l cells cultured
as monolayers and that both the drug and cell aggregation are
necessary for muscle differentiation DMSO could be removed
after 2-3 days but cardiac muscle still developed at 6-7 days
as in the continuous presence of the drug
The effects of DMSO described above were observed not
onl\ on P19S1801A1 cells,'but also on the parental P19 cells
and on all of the subclones from this line which were tested
However, DMSO had no effect on the differentiation of the
embryonal carcinoma cell lines F 9 ' \ OC15S1 1 2 and C 8 6 S l i :
whereas some clones of P10 cells11 appear to form an excess
of neurones in the presence of DMSO (G D Paterno and
M W M , unpublished) Variation has also been obser\ed in
the response of different embr>onal carcinoma lines to retinoic
acid 17 and to aggregation m the absence of drugs 1
DMSO is an inducer of Friend cell differentiation14 as are
6-thioguanine ] \ butyrate 16 and ouabain 17 The effects reported
above for DMSO have also been observed with non-toxic
concentrations of 6-thioguanme and butyrate but not with
ouabain Another Friend cell inducer, hexamethvlene bisacetamide (HMBA), has previously been shown to influence
the differentiation of some other embryonal carcinoma cell
lines 1 * ,g but we have not tested this compound with P19 cells
Many papers have reported the formation of a limited range
of cell types following spontaneous or induced differentiation
of lines of teratocarcinomas and of embryonal carcinoma
cellO : \ but it is not clear whether this is the result of differential selection or of the occurrence of a limited number of
Concentration of DMSO {%*/»)
Fig. 3 The efficiency of colony formation of P19S1801A1 embryonal
carcinoma cells m the presence of DMSO indicates the absence of toxical
at concentrations of less than 1% (v/v) About 200 cells were introduced
into replicate 60 mm diameter disnes containing \anous concentrations of
DMSO dissolved in a medium supplemented with 10°/o fetal bovine serum
and 10 4 M $ mercaptoethanol Incubation was for 8 days at 37 °C AU
colonies consisted of cells with embryonal carcinoma morphology Those
colonies formed in 1 S% DMSO were smaller than control colonies indicat
mg that at these concentrations the rate of cell proliferation was decreased
3
determinative events. We think it is unlikely that differential
selection can account for our observations because: (1) the cells
did not differentiate into embryonic cell types in the absence
of drugs, (2) the cell types formed in DMSO-treated cultures
were substantially different from those formed by the same
cells in parallel cultures exposed to retinoic acid, (3) neither
drug appeared to be toxic, (4) all subclones responded to both
drugs, (5) the drugs were effective even when cultures were
exposed to them for 48 h at the beginning of an experiment,
and (6) DMSO did not inhibit the formation of neurones in
cultures exposed to both retinoic acid and DMSO. The simplest
interpretation of these data seems to be that each drug acts by
'inducing' uncommitted embryonal carcinoma cel^ to
differentiate along a limited number of developmental avenues.
If the drugs act by bringing about intracellular changes which
mimic the results of certain embryonic decisions, it may be
possible to use the drugs to identify parts of the cellular decisionmaking apparatus.
This work was supported by grants from the NCI of Canada
and the MRC of Canada. We thank Irwin Schweitzer and Kem
Rogers for their help. Antibody to glial fibrillar protein was
the gift of Dr V. Kalnins and antibody to muscle-specific myosin
was provided by Drs D. Morgenstern, D. A. Fischman and P.
Memfield.
Received 10 January, accepted IS June 1982
1. Graham, C. F in Concepts in Mammalian Embryogenests (ed Sherman, M I ) 313-394
(MIT Press, Cambridge, 1977)
Martin. G R Science 209, 768-775 (1980)
Strickland, S & Mahdavi, V Cell 15, 393-403 (1978)
Strickland. S . Smith, K K 4 Marotti. K R Cell 12, 347-355 (1980)
Hogan, B L M , Taylor, A & Adamson. E Nalurt 291,23S-237 (1981)
McBurney. M W & Rogers. B J Devi Biol 89,503-508(1982)
Jones-Villeneuve E M V , McBurney. M W Rogen, K A & Kalnins, V 1 J Cell
Biol (in the press )
8 Martin, G R & Evans. M J Proc nam Acad Sci USA 72, 1441-1445 (1975)
9 Vandekerckhovc, J & Weber, K Eur J Biochem. 113, 595-603 (1981)
10 Lipshultz, S , Shanfeld, J AChacko.S Proc nam Acad Sci USA 78,288-292(1981)
11 Bernstine, E G , Hooper, M L , Grandchamp, S &. Ephnissi, B Prvc nam Acad Sci
USA 70,3899-3903(1973)
12 McBurney, M W / all Physiol 89, 441-456 (1976)
13 McBurney. M W & Stnitt, B J Cell 21, 357-364 (1980)
14 Fncnd, C . Schcr. W . Holland, J G i S a l o . T Proc nam Acad Sci USA 68,378-383
(1971)
15 Guselia.J F & Housman, D Cell 8, 263-269 (1976)
16 Leder, A & Leder, P 0 ( 1 5 , 3 1 9 - 3 2 2 ( 1 9 7 5 )
17 Bernstein, A., Hunt. D M , Cnchley, V &Mak,T W Cell 9, 375-381 (1976)
18 Jakob, H , Dubois, P , Eisen, H A Jacob, F Cr hebd Seanc Acad Sci, Pans 286D,
109-111(1978)
19 Speere, W C , Birdwell, C R & Dnon. F J Am J Path 97, 563-584 (1979)
20 Gearhart.J G * Mine, B Cell 6,61-66 (1975)
21 Vandenberg, S R , Herman, M M , Ludwin, S K & Bignami. A Am. J Palh 79,
147-168(1975)
22 PfeiHereral / Cell Biol 88,57-66(1981)
23 Darmon, M , Bottcnstcin, J & Sato, G Devi Biol 85,463-473(1981)
24 Anderson, P J Biochem. J 179, 425-430 (1979)
2
3
4
5
6
7
REFERENCES
Abnay, E.R., P.P. Bartlett, and M.C. Raff. 1981. Astrocytes,
ependymai cells, and oligodendrocytes develop cn schedule
in dissociated cell cultures of ea'bryonlc rat brain. Dev.
Biol. oJ: 301-310.
Adasison, E..D. 1976. Isozyme transitions of creatine
pfaosphokinasie, aldolase, and phosj-hoglycerate mutase in
differentiating mouse cells. J. Embryol. exp. Morphol.
35: 355-367.
Adamson, E.D., M.J. Evans, and G.G. Hagrane. 1977.
tiiocb.eiu-.cal markers of tne progre&s of differentiation In
cloned teratocarcinoma cell lines. Eur. J. Biochem.
Jl»
607-615.
Adamson, E.D., and C F . Graham. 1980. Loss of tu&erogenicity
and gain of differentiated function by embryonal
carcinoma cellt. Results and Problems in Cell
Differentiation. 11: ^90-297.
Artzt, K., P. Dubois, C. Bennett, H. Ccndamlne, C. Babinet,
and f. Jacob. 1973. Surfaca antigen ccimon tc mouse
^ cleavage eibryos and primitive teratocarcinoma cell ir
culture. Proc. Natl, flcaa. Sci. U.S.A. 20: 2988-2992.
Axeirod, fa.R., and D. Bennett. 19b2. A siwpl^fied method for
obtaining embryonic stem c^ll lines from blastocysts.
Abstract fros korkshop on teratocarcinoaa steir cells,
10th Cold Spring Harbour Conference on Cell
Proliferation.
Bennett, D. 1975. The 1 lecus of the mouse. Cell.
451-454.
6:
Berastine, E.t.., w.L. Hooper, S. GrancLamp, ana B. Ephrussi.
1973. Aikaiice phosphatase activity in mouse teratoma.
Proc. Natl. Acad. Sci. U.S.A. 2£: 3899-3903.
Blgnami, A., L.F. Eng, L. Oahl, and C.l. Uyeda. 1972.
Localization cf the glial fibrillar acidic protein in
astrocytes by ladiuncfluoresence. brain Res. 43:
429-435.
Bizzinl, B. 1979. Tetanus toxin. Microbio. Rev.
224-240.
- 115 -
4^:
116
Bollag, W., and A. Matter. 1981. Fron vitamin A to retinoids
in experimental and clinical oncology: achievements,
failures, and outlooks Ann. N.Y. Acad. Sci. 3jj>2: 9-23.
Boulder Committer. 1970. Embryonic veterbrat© central
nervous system: revised terminology. Anat. Rec. 166*
257-262.
Brinster, R.L. 1974. The effect of cells transferred into
the mouse blastocyst on subsequent development. J. Exp.
Med. 140: 1049-1056.
Chader, G,J., B. higgert, P. Russell, and ft. Tanaka. 1981.
Retinoid binding proteins of retina and retinoblastoma
cells in culture. Ann. N.Y. Acad. Sci. 359: 115-133.
Chopra, D.P., and L.J. wilkcff. 1977. Reversal by vitamin A
analogues (retinoids) of hyperplasia induced by N-methylS'-nitroN-nitrosoguanidine in mouse prostate organ
culture. J. Natl. Cancer Inst. 5b: 923-930.
Chytil, F., and D.E. Ong. 1979. Cellular retinol- and
retinoic acia-bindlng proteins in vitamin a action.
Proc. 38: 2510-2514.
Fed.
Connolly, J.A., V.I. Kalnins, D.H. Cleveland, and K.».
Klrschner. 1978. Intracellular localization of the high
molecular veigbt microtubule accessory protein by
indirect immunofluorescence. J. Cell Bicl. 76: 781-786.
Cowan,fc.tt.1978. Aspects of neural development. Int. Rev.
Physiol. 17: 149-191.
Crcce, C M . , A. Linnenbach, K. Heufaner, J.£. tarres- D.H.
Marguilies, E. Apella, and J. G. Seidman. 1981. Control
of expression of histocoirpatibility antigens (H-2) and
beta-2-microglcbulin in F9 teratoccrclnoma stem cells.
Proc. Natl. Acad. Sci. U.S.A. 78: 5754-5758.
Damjancv, I., D. Solter, «. Belicza, and N. Skreb. 1971a.
Teratomas obtained through extrauterine growth of seven
day mouse embryo-'. J. Natl. Cancer Inst. 46: 471-480.
Daiiijancv, I., D. Loiter, and N. Skreb. 1971b.
Teratocarcidogenes:s as related tc the age of embryos
grafted under the kidney capsule, hilhelm Roux' Archiv.
162: 28b-290.
Daraon, M., J. Pottenstein, and G. Sato. 1981. neural
differentiation following culture cf embryonal carcinoma
cells In a serum-free defined medium. Dev. Biol. 85:
463-473.
117
De Laca, L.«. 1977. The direct involvment of vitairln A in
giycosyl transfer reactions ofraafi.malidnmembranes.
Vltam. Horn. 3.5: 1-57.
De Luca, L.M., f.v. Bhat, N. Sasak, and S. Adarao. 1979.
BiosynthteSis cf phosphoryl derivatives of vitairln A In
biological membranes. Fed. Proc. 3&~ 2535-3539.
De Luca, L.M., and S.S. Shapiro, eds. 1981. Modulation cf
Cellular Interactions oy Vitamin A and Derivatives
(Retinoids). Ann. N. Y. Acad. Sci. 359: 1-4/8.
Detrey, M.J., J.D. Gearbart, ana B. Mintz. 1977a. Cell
surface catigens cf totipotent mouse teratocarcinoma
cells grcwn in vi,tr.o: their relation to embryo, adult,
and tumour antigens. Dev. Biol. 55: 359-374.
Dewey, M.J., D.S. Martin, G.R. Martin, and B. flintz. 1977b.
Mosaic mic^ with teratocarcinoma-derived mutant cells
deficient in hypoxanthine pbosphoribosyitransferase.
Proc. Hdtl. Acad. Sci. U.S.A. 2i: b564-5568.
Dewey, M.J., R. Filler, and B. Mlntz. 1978. Protein patterns
or devtlcpmertally totipotent mouse teratocarcinoma ceils
and normal eabryo cells. Dev. Biol. 65.: 171-182.
Dewey, M.J., and 6. Mintz. 1980. Teratocarcinoma cells as
agents f.r producing mutant mice. Results and Problems
in Cell Differentiation. 11: 275-282.
Dimpfel, e., F.T.C Huang, and E. Habermanii. 1977.
Gangiiosides in nervous tissue cultures ana binding of
12s
I-labelied tatanud toxin-d neuronal marxer. J.
.leurocfaei. 29.: 329-334.
Diwan, £•£., and L.C. Stevens. 1976. Development cf
teratomas frcm tht ectoderm of mouse egg cylinders. J.
Natl. Cancer lr,dt. 57: 937-939.
Ducibella, 1., D.F Albertini, E. Anderoon. and J.E. Blggers.
1975. Tr,e preivplantatj.cnffiimaalianembryo:
characterxzaticr, cf intercellular junctions and their
appearance during development. Dev. Biol. 45: 231-250.
Duclbelli, T., anc E . anderscn. 1y75. Ceil shape and
aeinbiane changts in the eight cell aiou&>e embryo:
prerequisites for th= ircrphogeaa&is of the blastocyst.
Dev. Biol. 42: 45-5o.
D u m a , I., J.f. McoidS, B. Jakob, E.L. Bendetti, and F.
Jacoo. 1979. Junctional modulation in souse eirbryonal
carcinoma ceils by tab fragments of rabbit anti-embryoaal
carcinoma call serur. Froc. Natl. Acad. Sci. t.S.A. 26:
33o7-33^1.
118
Dziadek, M. 1979. cell differentiation in Isolated inner
cell masses of mouse blastocysts in vitro: onset of
specific gene expression. J. Embryol. exp. Mcrph. 53:
367-379.
Edwards, N.K.S. 1983. Induced dif f erentlatior. of embryonal
carcinoma cells. MSc. thesis, University of Ottawa,
Ottawa, Ont. pp 1-106.
Edwards, M.K.S., and M.W. McBurney. 1983. The concentration
of retinoic acid determines the differentiated cell types
formed by a teratocarcinoma cell line. Dev. Bicl. 98:
187-191.
Eisenbarth, G.S., F.S. iaish, and M. Nirenberg. 1979.
Monoclonal antibody to a plasma membrane cell antigen cf
neurons. Proc. Nat. Acad.Sci. U.S.A. J^z
4913-4917.
Ellman, G.L., K.D. counez, V. Andres, and K.M. Featherstone.
1961. A new and rapid colorimetric determination of
acetylcholinesterase activity. Biochem. Pharmacol. 7:
88-95.
Eng, L.F., J.J. Vanderhaeghen, A. Bignami, and B. Gerstl.
1971. An aciaic protein Isolated from fibrous astrocytes.
Brain Res. 28: 351-354.
Evans, W.J. 1972. The isolation and properties of a clonal
tissue culture stain of aouse teratoma cells. J. Embryol.
exp. Morph. 28: 163-176.
Evans, «.J., and F.H. Kaufsean. 1981. Establishment in
culture tf pluripctentlal ceild from mouse embryos.
Nature 292: 154-156.
Evans, M.J., P.H. Lovell-Badge, P.L Stern, and M.G.
Stinnakre. 1979. Cell lineages of the mouse embryo and
embryonal carcinoma ceils; Fcrssman antigen distribution
and patterns cf prctein synthesis. INSERM Symposium 10:
115-129.
Featherstone, W . S . 1980. X Chromosome activity and embryonal
carcinoma cells. MSc. thesis. University of Ottawa,
Ottawa, Ont. pp 1-99.
Federotf, s.f acd L.C. Dc^ring. 1980. Colony culture ot
neural ceils as a s.ethod for the study cf cell lineages
in the developing central nervous system: the astrocyte
cell lineage. Curr. Topics Dav. Biol. 16: 283-304.
Fields, K.L. 1979. cell-type specific antigens of cells of
the central and peripheral nervous sy&tews. Curr. Topics
in Dev. B i d . 13: 237-257.
119
Finch, B.W., and £. Ephrussi. 1967. Retention cf nultiple
developmental potentialities by cells of a mouse
testicular teratocarcinoma during prolonged culture in
2itl2 and their extinction upon hybridization with cells
of permanent lines. Proc. Natl. Acad. Sci. U.S.A. 57:
615-621.
Fish, L.A., C.S. Baxter, and J.S. Bash. 1981. Murine
lymphocyte ccmitcgenesls by phorbol esters and its
inhibition by retinoic acid and Inhibitors of polyamine
biosynthesis. Toxicol. Appl. Pharmacol. 58: 39-47.
Fonnum, F. 1975. A rapid radiocnen&cal method for the
determination cf chcllne acetyltransferase. J. Biochem.
24: 407-409.
Franke, W.V., E. Schmld, K. Osborn, and K. Weber. 1978.
Different intermediate-sized filaments distinguished by
immunofluorescence microscopy. Proc. Natl. Acad. Sci.
U.S.A. .25: 5034-5038.
Franke, «.*., E. Schmid, K. Weber, and M. Osborn. 1979a.
HeLa cells contain intermediate-sized filaments of the
prekeratln type. Exp. Cell Res. 118: 95-109.
Franke, w.«., t. schmid, S. winter, P. Osborn, and K. Weber.
1979b. Widespread occurrance of the intermediate-sized
filaments of the vimentin type in cultured cells from
diverse vertebrates. Exp. Cell Res. 123: 25-46.
Fuchs, £., ana H. Green. 1*78. The expression of keratin
genes in epidermis and cultured epidermal cells. Cell 15:
887-897.
Fujlacto, H., T. Kurtmatsu, H. Urushihara, and K.O.
Yariagisa*a. 1982. Receptors to 'Cilochos biflorus'
agglutinin. A new surface marker common to
teratocarcinoma cells and pr^implantaticn mouse embryos.
Dirferentlation 22: 59-61.
Gachelln, G. 1976. Le teratocarcinome experimental de la
souris: une systeme ircdele pour 1'etude des relations
antrt ahz surface ctllulaires et differentiaticn
embryonnaire. Bull. Cancer 63: 95-110.
Gardner, R. L. 1961. In livo and in. vi.tr.o studies of cell
lineage ana cell determination in the early mouse embryo.
In: Ceilular Controls in Differentiation. C W . Lloyd and
D.A.. Re-as. tfl'.., Academic Press, London, pp 257-278.
Gautsch, J.*., and fl.C. Wilson. 1983. Delayed 'de novo*
methyiadon in teratccarcmoma suggests additional
ti£Sue-op=!Cific lechanisms for controlling gene
expressbiOL. Nature 301: 32-37.
120
Goodall, H., and M.H. Johnson. 1982. Use of
carboxyfluoresceln diacetate to study formation of
permeable channels between mouse blastomeres. Nature 295:
524-526.
Graoel, L.B., s.D. Rcsen, and G.R. Martin. 1979.
Teratocarcinoma stem cells have a cell surface
carbohdrate-binding component Implicated in cell-cell
adhesion. Cell 12: 477-484.
Grabal, L.B., M.S. Singer, G.R. Martin, and S.D. Rcsen.
1983. Teratocarcinoma stem ceil adhesion: the role cf
divalent cations and a cell surface lectin. J. Cell Biol.
96: 1532-1537.
Graham, C F . 1977. Teratocarcinoma cells and normal mouse
embryogenesis. In: Concepts in Mammalian Embryogenesis.
M.I. Sherman td. The M.I.T. Press, Cambridge, MA. pp
315-397.
Bartree, E.F. 1972. Determination of protein: a modification
of the Lcwry method which gives a linear photometric
response. Anal. Biochens. 48: 422-427.
Herbert, M . c , and C F . Graham. 1974. Cell determination and
biochemical differentiation of the early n-ouse embryo.
Curr. Top. Dev. Biol. 8: 151-178.
Heubner, K., A. Ilnnenbach, S. riiedner, G. Glenn, and C N .
Croce. 1981. Eeoxyribonuclease 1 sensitivity cf plasmid
gencmes in teratocarcinoma stem and differentiated cells.
Proc. Natl. Acad. Sci. U.S.A. 26: 5071-5075.
Hoffman, P.In., and K.J. Lasek. 1975. The slow component of
axocal transporc. Identification of major structural
polypeptlies ot the axon and their generality amoung
mammalian neurcns. J. Cell Biol. 66: 351-366.
Hogan, B.I.M., A. Taylor, and E. Adanscn. 1981. Cell
interactions modulate embryonal carcinoma cell
differentiation intc parietal or visceral endcderm.
Nature 291: 235-237.
Hsu, Y.C., J. Baskar, I.C. Stevens, and K.E. hash. 1974.
Development in vitro ct mous-j embryo^ from the two cell
stage to the early soailte stage. J. Embryol. exp. Morph.
31: 235-245.
lies, t.k,
1*77. fouse teratomas and embryoid bodies: their
induction and differentiation. J. fcmbryol. exp. Mcrphcl.
38: 63-75.
121
Illmensee, K., and B. Mlntz. 1976. Totlpotency and normal
differentiation of single teratocarcinoma cells cloned by
injection intc blastocysts. Proc. Natl. Acad. Sci.
U.S.A. 23: 549-553.
Jacob, F. 1*77. Bouse teratocarcinoma and embryonic
antigens. Immunol. Bev. 33.: 3-33.
Jacobson, M. 1978. Developmental Neurobiology, 2nd ed..
Plenum Press, New York, pp 27-55.
Jetten, A.M., and M.E.R. Jetten. 1979. Possible role of
retinoic acid binding protein in retinoic stimulation cf
embryonal carcinoma cell differentiation. Nature 228:
180-182.
Jetten, A.M., ti.t.R. Jetten, S.S. Shapiro, and J.P- Poon.
1979a. .Characterization of the action of retinoids on
aouse fibroblast cell lines. Exp. Ceil Res. 119:
289-299.
Jetten, A.M., M.E.R. Jetten, and M.I. Sherman. 1979b.
Stimulation of differentiation of several murine
embryonal carcinoma cell lines by retinoic acid. Exp.
Cell Res. 124: 381-391.
Jones-Villeneuve, E.M.V., M.w. McBurney, K.A. Rogers, and
V.I. Ralnms. 1982. Retinoic acid induces embryonal
carcinoma cells tc differentiate into neurons and glial
cells. J. Cell Biol. 24: 253-262.
Jorgensen, A.O., L. Subrahmaanyan, C Turnball, and ?.I.
Kalnins. 1976. Localization of neurofilament protein in
neuroblastoma calls by iismunfluorescent staining. Proc.
Natl. Acaa. Sci. U.S.A. 23: 3192-3196.
Kahan, B.W., and B. Ephrussi. 1970. Developmental
potentialities of clonal in vitro culture cf icuse
testicular teratomas. J. Natl. Cancer Inst. 44:
1015-1066.
K a l a m s , V.I., and J.A. Connolly. 1981. Applications of
imaunofluorescence In studies of cytoskeletal antigens.
Advances in Cellular Neurobiology 2: 393-460.
Kelly, 5.J. 1977. Studies of the developmental potential of
four and eight cell stage mouse blastcmeres. J. Exp.
Zocl. 200: 365-376.
Kf-mler,
R.,
C.
~— — — w
-' - »
— Guenet,
and
Guenet, and
and the T/t
U.S.A. 23:
Eabinet,
H. Coadamlne, C Gchelln, J.L.
_ _ — — ..-— —
F.
Jacob.
1*76. Embryonal carcinoma antigen
F. Jacob. 1*'
the mouse. Proc. Natl. Acad. Sci.
locus cf1 the
)84.
4080-4084.
r
w
v
122
Kemier, R., C. Babinet, H. Kisen, aad F. Jacob. 1977.
Surface antigen in early differentiation. Proc. Natl.
Acad. bcl. U.S.A. 24: 4449-4457.
Kleinsmith, L.J., and G.B. Pierce. 19t<+. Multipotentlality
of single embryonal carcinoma cells. Cancer Res. 24:
1544-1551.
Kuff, E.L., and J.h. Fewell. 1980. Induction cf neural-like
celle in cultures of F9 teratocarcinoma treated with
retinoic acid and dibutryl cyclic adenosine
monophosphate. DcV. Biol. 22: 103-115.
lane, E.S., B.L.M. Hcgan, M. Kurkinen, and J.I. Garrels.
1983. Cc-expressicn of vimentin and cytokeratins ia
parietal endoderm ct=ilb of the early mouse embryo. Nature
303: 701-704.
Lasnitzle*, I. 1976. Reversal of aiethylcholanthrene-induced
changes la mcute prostatas la viir£ by retinoic acid and
its analogues. Brit. J. Cancer 3.4: 239-248.
Lazarides, E. 1980. Intermediate filaments as mechanical
integrator^ of cellular space. Nature 283: 249-256.
Lazarides, t. 1982. Intermediate filaments: a chenically
heterogeneous, developaentally regulated class cf
prctelns. A m . Rev. Biccbem. 51: 219-250.
Lehman, J.M. Speers, W.C., Swartzendruber, D.E., and G.B.
Pierce. 1974. Neoplastic aifferentiaticn:
characteristics of cell lines derived from a murine
teratocarcincme. J. Cell Physiol. 84: 13-27.
Levine, A.J., a. Torisian, A.J. Sarokhan, and A.K. Teresky.
1974. Biochemical criteria for the in vitro
differentiation cf embryoid bodies produced by a
transplantable teratoma of asice. The production of
acetylcholine esterase and creatine phosf.rcklnese by
teratoma c d l c . J. Cell Physiol. 84: 311-318.
Levine, A.J. 198*. The nature of hoot-range restriction cf
SV40 and polyoma viruses in ambryonal carcinoma cells.
Curr. Topics ficrcbiol. Immunol, lfll: 1-30.
Le-vitt, P- i.L. Cooper, ana P Rakic. 1981. Coexistence of
neuronal aDd glial precursor cells in the cerebral
ventrlcuiai zcne of the fetdi monkey: at ultrastructural
ifiimuBoperoxidase analysis. J. Neurosci. 1: 27-39.
Liau, G., D.E. Ong, and F- Chytil. 1981. Interaction cf the
retinol/ cellular retlnol-bindlng complex with isolated
nuclei and nuclear component. J. Cell Biol. 91: 63-65.
123
Llea, R.K.fi., S.-H. Yen, G.D. Salomon, and ti.l. Sbelanskl.
1978. Intermediate filaments In nervous tissue. J. Cell
Biol. 2£: 637-645.
Llttlefleld, J.w. 1964. Selection of hybrids from matings of
fibroblasts in xitio ana their presumed recombinants.
Science 145: 709-710.
Lc, C.w. 1980. Gap junctions and development. In:
Development in Mammals. 4: M.H. Johnson, ed.
Elsevier/North-Holland Biomedical Press, pp 39-80.
Lo, C.W., and N.B. Gilula. 1980. PCC4/aza1 teratocarcinoma
stem cell differentiation in culture. 2. Morphological
characterization. Dev. Biol. 25: 93-111.
Loritz, F., A. Bernstein, and R.G. Miller. 1977. Early and
late volume changes during erythroid differentiation cf
cultured Frieno cells. J. Cell Physiol. 90: 423-438.
Lotan, R. 1980. The effects of vitamin A and its analogs
(retinoids) on normal and neoplastic cells. Biochem.
Biophys. Acta 60,5: 33-91.
Lotan, R., G. Newmann, and D. Lotan. 1980. Relationships
among retinoid structure, inhibition cf growth and
cellular retinoic acia binding protein in cultures of S91
meianoma cell&. Cancer Res. 40: 1097-1102.
Maden. M. 1982. Vitamin A and pattern formation in the
regenerating limb. Nature 295: 672-675.
Magnuscn, 1., A. Demsey, and C.W. Stackpole. 1977.
Characterization of intercellular junctions in the
preimplantation souse eisbryo by freeze-fracture and thin
section electron microscopy. Dav. Biol. 61: 252-261.
Martin, G.R. 1980. Teratocarcinomas and mammalian
embryogenesis. Selene* 209: 768-776.
Martin, G.R. 1981. Isolation of a pluripotent cell line from
early mouse embryos cultured in ueaium conditioned by
teratocarcinona stem ceils. Proc. Nati. Acad. Sci. 2£ :
7634-7b3t.
Martin, G.R., C.J.Epstein, B. Travis, G. Tucker, S. Yatsiv,
D.n. Martin, S. Cllft, and S. Cohe-n. 1978. X chromosome
mactlvaticn during differentiation of a feuale
teratocarcinoma .-item ceil. Nature 221: 329-333.
Martin, G.E., and M.J. Ev^ns. 1975a. Multiple
dilferentiaticn of clonal teratocarcinoma stem cells
following emtryola body formation in vitro. Ceil 6:
467-474.
124
Martin, G.R., and M.J. Evans. 1975b. Differentiation of
clonal lines of teratocarcinoma cells: formation of
embryola bodies In liitQ'
Proc. Natl. Acad. Sci. U.S.A.
22: 1441-1445.
Martin, G.R., Smith, S., and Epstein, C.J. 1978. Frotein
3ynthetxc patterns in teratocarcincma stem cells and
mouse embryos at early stages of development. Dev. Biol.
6.6: 8-16.
Martin, G.R., L.M. Wiley, and I. Damjanov. 1977. The
aevelopment of cystic emtryoid bodies in vi£ro from
clonal teratocarcinoma stea cells. Dev. Biol. 61:
230-244.
McBurney, M.W. 1976. Clonal lines of teratocarcinoma cells
in vitro: difterentiation and cytogenetic
characteristics. J. Cell. Physiol. 89: 441-456.
McBurney, M.W., an! E. D. Adamson. 1976. Studies on the
activity of the X chromosomes in female teratocarcinoma
celis In culture. Ceil £: 57-70.
McBurney, K.W., and B.J. Strutt. 1979. Fusion of embryonal
carcinoma cells tc fibroblast cells, cytoplasts, and
karycplasts. Exp. Cell Res. 124: 171-180.
McBurney, M.W., and B.J. Strutt. 1980. Genetic activity of X
chromosomes in pluripotent female teratocarcincma cells
and their differentiated progeny. Cell 21: 357-364.
McBurney, M.n., E.M.V. Jones-Villeneuve, M.K.S. Edwards, and
P.J. Anaerson. 1982. Control of muscle and neuronal
differentiation in a cultured embryonal carcincma cell
line. Nature 299: 165-167.
McBurney, M.W., and B.J. Rogers. 1982. Isolation cf male
embryonal carcinoma celis and their chromosome
replication patterns. Dev. Bioi. 8*: 503-508.
Mintz, B., A. Cronroiller, and P.P. Custer. 1978. Somatic
ceil origin cf teratocarcinomas. Proc. Natl. Acad. Sci.
U.S.A. 75: 2t34-2b36.
Mintz, B., and K. Illmensee. 1975. Normal genetically mosaic
mice prooucea from malignant teratocarcinoma cells. Proc.
Matl. Acai. Sol. U.S.A. 22: 3585-3589.
Mirsky, R. 1982. The use of antibodies tc define and study
major call types in the central and peripheral nervous
aystems. In: NeuroitrmunoiDgy. J.Brocket, ed., Flanum
Preti, London, pp 141-1o1.
125
Mirsky, R., L.M.B. Wendon, P. Black, C Stclkin, and D.
Bray. 197o. Tetanus toxin: a cell surface marker for
neurcns in culture. Brain Res. 148: 251-259.
Moore, B.W., v.J. Perez, and M. Gearing. 1968. Assay and
regional distribution of a soluble protein cnaracteristlc
of the nervous system. J. Neurccheu. 15: 265-272.
Muraaatsu, 1., G.Gachelin, J.F. Nicolas, H. Condamine, H.
Jakob, and F. Jacob. 1978. Carbohydrate structure and
cell differentiation: unique properties of fucosylglycopeptides Isolated from embryonal carcinoma cells.
Proc. Natl. Acad. Sci. U.S.A. 25: 2315-2319.
Nicolas, J.F., t. Dubois, H. Jakob, J. Gaiilard, and F.
Jacob. 1*75. Teratocarinome de la souris:
differentiation en culture d'une lignee de cellules
primitives a pctentialites multiples. Ana. Micrcbiol.
(Paris) 126A: 3-22.
Nicolas, J.-F., P. Kemler, and F. Jacob. 1981. Effects of
antleiBtryonal carcinoma cell seruu. on aggregation and
metabolic cooperation between teratocarcinoma cells. Dev
Biol. 81: 127-132.
Niwa, 0., Y. Yakcta, H. Ishida, and I. Sugahara. 1983.
Independent rcecnanisits involved in the suppression of the
Maloney leukemia virus genome during diifereatlatiea of
murine teratocarcincma cells. Cell 3.2i 1105-1113.
Ogou, S., T. Okada, and M. Takeichl. 1982. Cleavage stage
mouse embryos share a common cell adhesion system with
teratocarclacma cells. Dev. Biol. 92: 521-528.
Papalcanncu, V.E., M.W. McBurney, R.L. Gardner, and M.J.
Evans. 1975. Fate of teratocarcinoma cells injected into
early mouse embryos. Nature 258: 70-73.
Pauiin, D., N. Forest, and J. Parreau. 1980. Cytoskeletal
proteiri0 as markers cf differentiation in mouse
teratocarclacma cells. J. Mol. Bicl. 144: 95-101.
Pauiin, D., H. Jakob, i. Jacob, K. Weber, and M. Osborn.
19o2. In viiro differentiation of mouse teratocarcinoma
cells monitored by intermediate filament expression.
Differentiation 22: 90-99.
Pauiin, D., J. Eerreau, H. Jakob, F. Jacob, and M. Yaniv.
1979. Tropomyosin synthesis accompanies formation of
actin filaments in en.bryondl carcinoma cells induced to
diflerentiate ty hexaaii ethylene biaacetamide. froc. Natl.
Acad. Sc: . U.S.A. 76: 1c>91-1895.
126
Pawson, B.A. 1981. A historical introduction tc the
chemistry of vitamin A and its analogs (retinoids). Ann.
N.Y. Acad. Sci. 359: 1-8.
Pederson, R.A., A.F. Spindle, aad L.M. Wiley. 1977.
Regeneration of endoderm by ectoderm Isolated from mouse
blastocysts. Nature 22P.: 435-437.
Pfelffer, S.E., H. Jakob, K. Mlkoshlta, f. Dubois, J.L.
Guenet, J.F. Nicolas, J. Gaillard, G. Chevance, and F.
Jacob. 1981. Differentiation of a teratocarcincma line:
preferential development of cholinergic neurons. J. Cell
Blcl. 88: 57-66.
Pierce, G.B., and T.F. Beals. 1964. The ultrastructure of
primordial ger0 cells cf the fetal testes and cf
embryonal carcinoma cells of mice. Cancer Ees. 24:
1553-1567.
Pierct, G.B., and F.J. Dixon. 1959. Testicular teratomas 2:
reratocarclncma an ascitic fluid. Cancer 12: 584-589.
Pierce, G.B., F.J. Djxon, and E.L. Verney. 1960.
Teratocarcincgenic and tissue-forming potentials of the
cell typet composing neoplastic bodies. Lab. Irvest. 9:
viy.o 583-602.
Raff, C , F.H. Miller, and M. Noble. 1983. A glial
progenitor cell that develops in vltr.c into an astrocyte
or an oligodendrocyte depending on culture medium.
Nature 3,03: 390-396.
Raju, T., A. Bignami, and D. Danl. 1981. In vivo and in
litre differentiation of neurons and astrccytes in the
rat eabryo. lsmunoflucrescence study with neurofilament
and glial filairent antlsera. Dev. Biol. 85: 344-357.
Raklc, ir. 1*72. Mode of cell migration tc the superficial
layers of fetal monkey cortes. J. Comp. Neurol- 145:
61-84.
Rakic, P. 1^74. Neuron* in Rhesus monkey visual ccrtex:
systematic relation between time cf origin and eventual
iispcsioion. bcieace 183.: 425-427.
Reisntr, Y., G. Gachelin, t.. Dubois, J.-F. Nicolas, N.
Sharon, ana F. Jacob. 1977. Interaction cf peanut
agglutinin, a lectin specific for non-reducing terminal
D-galactosyl residues with enbryonai carcincma cells.
Dev. Biol. 61: 20-27.
Rizzino, A. 19o3. Two fiultipotent eittryonal carcincma cell
lines irreversibly differentiate in defined media. Eev.
Biol. £5: 126-136.
127
Rosenthal, M.D., s. f. Wishnow, and G.H. Sate. 1970. In
IltlQ growth and differentiation cf clonal populations of
atultipotent mouse cells derived from a transplantable
testicular teratocarcinoma. J. Natl. Cancer Inst. 44:
1001-1014.
Rcssant, J., and ft.w. McBurney. 1982. The developmental
potential cf a euploid teratocarcinoma cell line after
blastocyst Injection. J. Embryol. exp. Morph. 2fi:
99-112.
Rcssant, J., and V.E. Papaloaanou. 1977. The biology of
embryogenesis. in: Concepts in Mammalian Embryogenesis.
M.I. Sherman ed. The M.I.T. Press, Cambridge, Cass, pp
1-36.
Scnachner, M. 1982a. Glial antigens and the expression of
neuroglial phenotypea. Treads ia Neurcsci. 5: 225-228.
Schaehner, M. 1982b. Cell type-specific surface artigens in
the mammalian nervous system. J. Neurochem. 39: 1-8.
Schindier, J., K.I. Katthaei, aad M.I. Sherman. 1981.
Isolation and characterization of mouse mutant embryonal
carcinoma cells which fail to differentiate in the
pretense of retinoic acid. troc. Natl. Acad. Sci. U.S.A.
2fi: 1077-1060.
Schlaepfer, W.W. 1977. Immunological and ultrastructural
studies or neurofilaments isolated frca rat peripheral
nerve. J. Call Biol. 2i: 226-24C.
Scott, K.F.f., F.L. Meykeus, and D.H. Russel. 1982.
Retinoids increase transglutaminase activity and inhibit
ornltcine aacarboxylase activity m Chinese hamster ovary
ceils anc ia melanoma cells 3tiiaulatad to differentiate.
Proc. Natl. Acad. Sci. U.S.A. 22: 4093-4097.
Snell, G.D., and I.e. Stevens. 1966. Early embryology. In:
Biology of the Laboratory Mouse. 2nd ed. E.I. Green ed.
McGraw-Hill, New York, pp 205-245.
Solter, D., N. Sdaitis, I. Damjanov, and H. Kaprowski. 1975.
Control of teratocarcinogeaeais In: Teratomas end
Differentiation M.I. Sherman and D. Solter, eds.,
Acaaeaic Press Inc., New York, pp 139-159.
Solter, D., and Damjanov, I. 1979. Teratocarcinomas rarely
develop from embryos transplanted into athyudc sice.
Nature 228: 554-555.
Solter, D., ana E.B. Knowies. 137c. Monoclonal antabody
defining i stacespecific mouse embryonic antigen
(SSEA-1). Proc. Natl. Acad. ocl. U.i.A. 25: 5565-55b9.
128
Somaer, I., and M. Schaehner. 1981. Monoclcnai antibodies
(01 to 04) to oligodendrocyte cell surfaces: ar
immunocytologlcal study in the central nervous system.
Dev. Biol. 83.: 311-327.
Spears, W . C , C R . Birdweli, and F.J. Dixon. 1979.
Chemically induced bidirectional differentiation of
ambryonai carcinoira cells in jltEfi* Am. J. Pathol.
563-584.
92:
Spora, M.B., G.H. Clamon, N.M. Dunlop, D.L. Newton, J.M.
Smith, and U. Saffiotti. 1975. Activity of vitamin A
analogues in ceil cultures of mouse epidermis and organ
cultures of hamster trachea. Nature 253: 47-50.
Stern, P.L., G.R. Martin, and M.J. Evans. 1975. Cell surface
antigens of clonal teratocarciaota cells at various
stages of ulffereatlation. Cell 6: 455-465.
Stevens, L.C 1960. Embryonic potency of embryoid bodies
derived from a transplantaole testicular teratoma of
mice. Dev. Biol. 2: 285-297.
Stevens, L.c. 1967. Origin cf testicular teratomas from
priffordial germ cells in mice. J. Natl. Cancer last.
549-552.
38:
Stevens, L.C 1968. The development of teratomas from
iatratesticular grafts of tubal mouse eggs. J. Fmbryol.
sxp. Mcrphcl. 20: 329-341.
Stevens, L.c. 1970a. Experimental production of testicular
teratomas in sice of strains 129, A/He, and tbeir F1
hyor^ds. J. Natl. Cancer Inst. 44: 923-929.
Stevens, L.C. 1970b. The development cf transplantable
teratocarcincasas from intratasticular grafts cf pre- and
postlmplantatj.on mouse embryos. Dev. Biol. 2.1' 364-382.
Stevens, 1 , C , and Little, C C . 1954. Spontaneous testicular
teratomas in an inbred strain of trice. Proc. Natl. Acad.
Sci. Wash. 40: 108C-1087.
-Stevens, i.e., and G.B. Pierce. 1y75. Teratomas: definitions
ana terminology. In: Teratomas and Differentistioa. M.I.
Snerman ana D. Solter, eds. pp 13-15.
Stevens, L . C , and D.S. Varnuia. 1974. The development cf
teratoma^ from parthogenetically activated ovarian mouse
eggs. Dev. Biol. 3,2: 369-380.
129
Stewart, C.L., H. Stuhlman, D. Jahner, and R. Jaenlsch.
1982. De. novo, methylation, expression, and infectlvity
of retroviral genomes introduced into embryonal carcinoma
celis. Proc. Natl. Acad. Sci. U.S.A. 29: 4098-4102.
Stanners, C.P., G. Elicieri, and H. Green. 1971. Two types
ot ribosoitiis in souse-hamster hybrid cells. Nature 230:
52-54.
Strickland, S., and V. Mahdavi. 1978. The induction of
differentiation in teratocarcinoma stem cells by retinoic
acid. Cell 15: 393-403.
Strickland, S., K.K. Smith, and K.R. Marotti. 1980. Hormonal
induction cf differentiation in teratocarcinoma stem
ceils: generation cf parietal eadcderm ty retinoic acid
and dlbutryl cAMP. Cell 21: 347-355.
Sun, T.-Ts, ana H. Green. 1978. Immuncfluorescent staining
of keratin fibers in cultured cells. Cell 14: 469-476.
Taksichi, M., T. Atsuail, c Yosheita, K. Uno, and T.S.
Okada. 1*c1. Selective adhesion cf embryonal carcinorca
cells ana differentiated cells by Ca + + dependent sites.
Dev. Biol. 82: 340-350.
Tarkowski, A.K., and J. Wroblewska. 1967. Development of
blaston.tr es cf mouse eggs isolated at the four and eight
cell stage. J. Embryol. exp. Morphol. 18: 155-180.
Tickle, C , B. Alberts, L. «olpert, and J. Lee. 1982. Local
application of retinoic acid tc the limb bud srimics the
action cf the polarizing region. Nature 296: 564-566.
Verma, A.p.., fa. M. Pice, B.G. Shapas, and t.K. Boutwell.
1978. Inhibition of 12-0-tetraaecanoyiphorbol-13-acetete
xnducea ornithine decarboxylase activity in mcuse
epideriria by vitaain A analogues (retinoids). Cancer
Ret. 38: 793-801.
Wetb, C G . 1*c0. Characterization of antlsera against mouse
teratocarcinoma OTT 6050: mclecular species recognized on
embryoid bodies, preinplantation embryos, and sperm. lev.
Bicl. 26: 203-214.
Siikotf, I.J., J.C Peckham, E.A. Dalwaage, R.W. Pcwry, and
D.r. Chopri. 1976. Evaluation of Vitamin i> analogues in
•uodulating epitk-ellal diffarantiaticn oi 13 day chick
embryo metatarsal skir explaitb. Cancer Fe«. 36,:
:»64-97*.
130
Wilson, S.H., B.K. Schreir, J.L. Farber, E.J. Ttompson. P.N.
Rosenberg, A.J.Blume, and M.W. NlreaDtrg. 1972. Markers
for gene expression in cultured cells froa the nervous
system. J. Eiol. Chem. 242: 3159-3169.
Yen, S.-H., D. Dahl, M. Schaehner, and M.I. Shelanskl. 1976.
Biochemistry cf the filaments of the brain. Proc. Natl.
Acad. Sci. U.S.A. 2i: 529-533.
Zomzely-Neurath, C.E., and W.A. Walker^ 1980. Nervous
system-specific proteins: 14-3-2 protein, neurcn-specific
enclase, and S-100 protein. In: Proteins of the Nervous
System, *ad ed. R.A. Bradshaw and D.M. Schneider eds.,
Raven Press, New York, pp 1-57.