Biol. Cel l , Vol 76, 1992 219

I N F L U E N C E OF T H E O N C O P R O T E I N v - e r b A ON QUAIL MYOBLAST PROLIFERATION AND DIFFERENTIATION.

CASSAR-MALEK l,~abelle, MARCHAL Soph ie , WRUTN1AK C h a n t a l , SAMARUT d a e q u e s * , CABELLO G 6r a r d . Un i t6 de Dl f f6 renc la t lon Ce l lu la i r e et C r o l s s a n c e , 1NRA. Place Via la , 3 4 0 6 0 MONTPELLIER C e d e x 1. * Lab. Biologic Mol6cu la i r e et Ce l l u l a l r e , CNRS UMR49 , INRA ENS Lyon, 4 6 a l l6e tl ' l talle, 6 9 3 6 4 Lyon C~dex 07

N u m e r o u s d a t a ev idence t h a t the e x p r e s s i o n of the v- e rbA o n e o g e n e I n h l b l t s the d i f f e r en t i a t i on of e r y t b r o l d cell p r e c u r s o r s (1) a n d t he e x p r e s s i o n of spec i f ic e r y t h r o c y t l c g e n e s (2). It Is genera l ly a s s u m e d t h a t the v -e rbA p r o t e i n Is ab le to a n t a g o n i z e t he effects of t he T 3 / n u c l e a r r e c e p t o r c o m p l e x e s on gene t r a n s c r i p t i o n .

As we h a v e s h o w n t h a t T3 i n f l u e n c e s quai l m y o b l a s t p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n (3), we h a v e s t u d i e d the In f luence of v -e rbA In o u r c u l t u r e model . We have Infec ted m y o b l a s t s wi th a r e c o m b i n a n t r e t r o v i r u s ca r ry ing the v-erbA o n c o g e n e . In t he a b s e n c e of T3 (and p r o b a b l y of r e t l n o i c acid), v - e rbA s ign i f i can t ly I n c r e a s e s the p ro l i fe ra t ion r a t e of m y o b l a s t s . M o r e o v e r . It e n h a n c e s a c c u m u l a t i o n of a c e t y l c h o l i n e r e c e p t o r s a n d of m y o s i n h e a v y c h a i n , m e a s u r e d as d i f f e ren t i a t ion m a r k e r s .

T h e s e r e s u l t s b r i n g t he f i r s t e v i d e n c e t h a t v - e r b A c o u l d a f f e c t ce l l p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n I n d e p e n d e n t l y of T3 a n d r e t l no lc acid . They r a i s e a lso the fol lowing q u e s t i o n s : Is v - e r b A ab le to s t i m u l a t e m y o b l a s t d i f f e r e n t i a t i o n ? Al te rna t ive ly , does It f a v o u r the e m e r g e n c e of m y o b l a s t s wi th h i g h d i f f e r en t i a t i on po ten t i a l i t i e s , f rom a h e t e r o g e n c o u s m y o b l a s t p o p u l a t i o n ?

(1) GANDRILLON O., JURDIC P., PAIN B.. DESBOIS C., MAD,JAR J.,l., MOSCOVICI M.G.. MOSCOVICI C. et SAMARUT d. (1989}. Cell 58, 115-121. {21 PAIN B., MELET F., JURDIC P. et SAMARUT J. (1990}. Tile New Biologist 2. 284-294. [31 MARCHAL S.. CASSAR-MALEK I., WRUTNIAI{ C.. CABELLO G. Molecular control of tile cell cycle ENS Lyon, 6-8 dulllel 1992

DIFFERENTIATION OF THE RABBIT EMBRYO CORNEAL EPITHELIUM: EXPRESSION OF THE ACIDIC KERATIN K12. CHALOIN-DUFAU Catherin~ 1, SUN Tung-Tien 2 and DHOUAILLY Danielle 1 . 1- Laboratoire de Biologie de la Diffdrenciation Epithdliale. Universitd Joseph Fourier. CERMO. BP 53X. 38041 GRENOBLE. 2- Epithelial Biology Unit. Department of Dermatology. New York Universi~/ School of Medicine. NEW YORK.

The K3/K12 keratins pair serves as biochemical marker for an advanced slage of "corneal-type" epithelial differenliation (Sun et al., 1984). Using an AK12 monoclonal antibody (Chaloin-Dufau et aL, 1990) highly specific for the acidic keratin K12, we have isolated a cDNA of this keralin from a ~.gll 1 rabbit corneal epithelium library. Sequential expression of the K12 keratin and of its mRNA was studied during embryonic deve lopment using bolh immunofluorescent slaining and in situ hybridization. The appearance of the protein and lhe activation of the corresponding gene are strictly synchronized: they appear first in Ihe suprabasal layer of central corneal epithelium at 17 days of gestation, and reach the basal epithelial layer six days later (23 days of gestation). Therefore, the regulation of the acidic keratin K12 gene appears to be primarily at the transcriptional level.

CHALOIN-DUFAU C., SUN T-T., DHOUAILLY D. (1990). Cell Different. Develop. 32, 97-108. SUN T-T., EICHNER R., SHERMER A., COOPER D., NELSON WG., WEISS RA. (1984). In "Cancer cell', vol 1, The Iransformed phenolype,Levine A, Topp W, Vande Woude G, Watson JD, eds, Cold Spring Harbor, 169-176.

DOWN R E G U L A T I O N OF CDC2 EXPRESSION IN T I l E D E V E L O P I N G QUAIL N E U R O R E T I N A

g lLLET Germain, ESPANEL Xavier, STETTLER Olivier*, TAVITIAN Bertrand*, BRUN Gilbert

ENS Lyon, 46 allde d 'hal ie 69364 Lyon cddex 07

*SHFJ,CEA, 4 place du G al Leclerc 91401 Orsay cdde.r In the d e v e l o p i n g quai l n e u r o r e t i n a , n u m e r o u s cel l d ivis ions are observed before embryonic day E7 while no mitosis can be detected after day E8 sugges t ing that a s t r ingent negat ive control of cell d iv is ions occurs at this stage, ht an a t tempt to unders tand the molecular events i nvo lved in this g rowth arrest , cdc2 express ion was analysed in the deve lop ing quail neurore t ina . We have observed, both at the m R N A and at the protein levels, a high express ion of cdc2 before day E8 followed by a marked decrease after day E9. This suggests a negat ive regulat ion of cdc2 express ion at the t ranscr ipt ional level. The amount of the Rb protein is also down regulated

fol lowing the same kinetics as p34cdc2 . The synchronous

decrease of p34 cdc2 and Rb could be a major event leading to postmitot ic neurona l precursors . In situ hybr id iza t ion

e x p e r i m e n t s us ing a synthet ic 35S o l igonucleo t ide as a probe, have c o n f i r m e d this nega t ive cont ro l of cdc2 express ion . An in tense and hom ogeneous labe~lling was observed in all cell layers before day E7 and no labell ing could be detected after day E8. However , at day E7 the inner nuc lear layer was sti l l label led sugges t ing that pos tmi to t i c a m a c r i n e neurona l p recurso r s sti l l exp res s cdc2. Therefore, in some cell populations of the developing

neurore t ina , p34 ode2 could be involved in the early steps of d i f ferent ia t ion occuring immediately after the arrest of cell division.

HORMONAL CONTROL OF CELL-TO-CELL COlVlMUNICATION AND OF CONNEXIN GENE

EXPRESSION IN THE THYROID. MUNARI-SILEM Yvonne(l), GUERRIER Annie(l), FROMAGET

Catherine(2), KRUTOVSKIKH Vladimig3), EL AOUMARI Abdelhakim(2), GROS Daniel(2),

ROUSSET Bernard(I) (ll INSERM U. 197, Facultd de Mddecine A. Carrel, Lyon; (2) L.A. CNRS 179, Facultd des Sciences de Luminy, Marseille; (3) International Agency for Research Cancer, Lyon.

Gap junctions (Gj) are constituted by the aggregation of many hydrophilic channels which mediate direct cell-to-cell communication. Gj are involved in metabolic coupling and in the control of cell growth and differentiation. In some tissues, the synthesis of Gj constituting protein: connexin (Cx) and the "functioning" (i.e. opening or closure) of the Gj channels seem to be under homlonal dependance. In thyroid, we attempted to determine 1) whether cultured thyroid ceils are able to reconstitute functional gap junctions, 2) whether channel gating is regulated by TSH, 3) whether Cx gene expression is regulated by TSH. The direct cell-to-cell cormrtunication was studied by microinjection of a fluore:;cent probe (Lucifer Yellow) in pig thyroid cells cultured for 1 to 8 days in the presence or the absence of TSH.The expression of Cx mRNA was analyzed by northern blot using eDNA probes for the two best studied Cx: Cx32 and Cx43. The content and the distribution of the two Cx were analyzed by immunofluorescence using polyclonal antipepdde antibodies for Cx32 and Cx43. The level of cell-to-cell communication remained high tot up to I0 days in cells cultured with TSH, while it rapidly decreased in cells cultured without TSH. Acute TSH treatment rapidly increased the communication between cells cultured in the absence of TSH; this effect was dose- and time-dependent. High amounts of the 1.6 kb Cx32 tmRNA were detected in freshly isolated thyrocytes (ITC); the level of this transcript decreased in cells cultured in the absence of TSH and remained high in the presence of TSH. ITC contained only trace amounts, of the 3 kb Cx43 transcript. Interestingly, the amount of this transcript increased in cells cultured in the presence of TSH. The amount of Cx43 protein detected by western blot or immuno-fluorescence was higher in cells cultured in the presence of TSH than in the absence of TSH.

These results demonstrated that: 1) Cultured thyroid epithelial cells rapidly reconstitute functional Gj which appeared positively modulated by TSH. 2) .Thyroid cells which express very preferentially Cx32 "in situ", still express Cx32 and progressively express Cx43 in culture. 3) The expression of these two Cx was positively.modulated by TSH. In conclusion, TSH regulates both Gj gating and the expression of their constitutive proteins.