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E L S E V I E R Mechanisms of Development 59 (1996) 73-87

Transcripts of Grg4 , a murine groucho-related ene, are detected in adjacent

t i ssues to o ther murine neurogen ic gen e ho m ologu es

during embryonic dev e lopm ent

K a r e n E . K o o p , L e a k M . M a c D o n a l d , C o r r i n n e G . L o b e *

De par tme n t o f B ioc he mis t ry , Ins t itu te . for Mo le c ular B io logy and Canc e r Re se arc h Group, M c Ma s te r Univ er si ty , 1280 M ain S t . W . ,

Hamilton, ON, L8S 4K1, Canad a

Received 13 March 1996; accepted 28 June 1996

A b s t r a c t

T he groucho-related genes (Grg) of the m ouse comprise at least four family mem bers. In Drosop hila, groucho i s one o f the neuro-

genic genes that participates in the Notch signalling pathway. The Groucho protein interacts with Hairy-related transcription factors to

regulate segmentation, neurogenesis and sex determination. Thus, by analogy to the Drosophila proteins, murine Grg proteins may

interact with mammalian Hairy and E(spl) homologues (Hes proteins) and take part in a signalling pathway downstream of murine

Notch. We have isolated m urine Grg4 cDNAs and examined Grg4 expression du ring embryogenesis. Transcripts of Grg4 were detected

in proliferating epithelial tissues undergoing mesenchymal induction, overlapping with Grg3, Notch1 an d Hes l expression. Grg4 wasalso expressed in the central nervous system and somites, but in cells adjacent to Grg3-, Notch1 -, and Hesl-expre ssing cells. This dis-

tinct pattern of expression suggests a role for Grg4 in later stages of cell differentiation than for the other mouse neurogenic gene

homologues.

Keywords: Differentiation; Nervous system; Expression; Notch; Groucho

1. I n t r o d u c t i o n

T h e r e g u l a t io n o f c e l l d e t e r m i n a t i o n a n d d i f f e r e n ti a t io n

i s c ri ti c a l t o t h e c o r r e c t d e v e l o p m e n t o f t h e e m b r y o . T r a n -

sc r ip t ion fac tors t ha t p romote ce l l de t e rmina t ion and d i f -

f e r e n t i a t i o n h a v e b e e n i d e n t i f i e d t h r o u g h m u t a t i o n a l

s c r e e n s o f Drosoph i la (N t i ss l e in-Volha rd and Wieschaus ,

1980; N i Jss l e in-Volha rd e t a l . , 1987) . These inc lude nu-

m e r o u s E b o x - b i n d i n g b a s i c h e l i x - l o o p - h e l i x ( b H L H )

fac tors . For example , i n Drosophi la , t h e p r o n e u r a l

Achae te - scu te p ro te ins d i rec t ce l l s a long a neura l l i neage

(Cabre ra e t a l . , 1987) and the musc le de t e rmina t ion fac -

to r , Naut i lus , d i rec t s ce l l s a long a myogenic l i neage (Ba te

et al . , 1993 ).

T h e b H L H p r o t e i n s a s so c i a t e d w i t h d e t e r m i n a t i o n

a l o n g p a r t i c u l a r l i n e a g e s a r e i n m a n y c a se s a n t a g o n i z e d

b y a n o t h e r f a m i l y o f b H L H p r o t e i n s , su c h a s t h e H a i r y -

l ike pro te ins (O hsak o e t a l. , 1994; Van Do ren e t a l. , 1994;

* Corresponding author. Tel. : +1 905 5259140, ext. 27335; fax: +1

905 5212955; e-mail: [email protected]

Brown e t a l . , 1995) . In Drosophi la , the H a i r y - l i k e p r o -

t e i n s i n c l u d e H a i r y , D e a d p a n a n d m e m b e r s o f t h e E n h a n -

ce r -of - sp l i t f ami ly . H a i ry- l ike pro te ins inh ib i t t he t ran-

sc r i p ti o n a l a c t i v a ti o n o f E b o x - b i n d i n g b H L H p r o t e i n s b y

b i n d i n g N - b o x p r o m o t e r e l e m e n t s a n d d i r e c t l y r e p r e s s i n g

t h e i r e x p r e s s i o n ( V a n D o r e n e t a l . , 1 9 9 4 ) . T h e y m i g h t

a l so in h i b i t t h e E b o x - b i n d i n g p r o t e i n s b y c o m p e t i t i o n f o r

d imer i za t ion pa r tne rs (Sasa i e t a l . , 1992) . The Ha i ry- l ike

t ransc r ip t ion fac tors have a c f i a rac t e r i s t i c Trp-Arg-P ro-

T r p ( W R P W ) se q u e n c e a t t h e C - t e r m i n u s , w h i c h i s i n -

v o l v e d i n b i n d i n g t o a n o t h e r p r o t e in , G r o u c h o , w h i c h a c ts

as a t r ansc r ip t iona l co- repres sor (Pa rous h e t a l. , 1994) .

G r o u c h o a n d H a i r y - r e l a t e d t r a n sc r i p t i o n f a c t o r s f o r m

p a r t o f t h e N o t c h s i g n a l li n g p a t h w a y . M e m b e r s o f t h e

N o t c h p a t h w a y a r e r e f e r r e d t o a s n e u r o g e n i c g e n e s , b e -

c a u se o f t h e i r ro l e i n d e s i g n a t in g e p i d e r m a l v e r su s n e u r a l

c e l l f a t e s ( C a m p o s - O r t e g a , 1 9 9 0 ) . H o w e v e r , t h e N o t c h

p a t h w a y a l so r e g u l at e s m y o g e n e s i s , g u t d e v e l o p m e n t , a n d

se v e r a l o t h e r c e l l d e t e r m i n a t i o n p r o c e s se s ( B a t e e t a l . ,

1 9 9 3 ; F o r t i n i a n d A r t a v a n i s - T sa k o n a s , 1 9 9 3 ; T e p a s s a n d

H a r t e n s t e i n , 1 9 9 5 ) . N o t c h i s a t r a n sm e m b r a n e p r o t e i n

0925-4773/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved

P I I S0925-4773 (96)005 82-5

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74 K.E. Koop et al. / Mechanisms o f Development 59 (1996) 73-87

w i t h e x t r a c e l l u l a r E G F r e p e a t s t h a t a c t a s a r e c e p t o r f o r

in t e rce l lu l a r s igna l l ing . In the case of neuro genes i s , Notc h

ac t iva t ion l eads to nuc lea r loca l i za t ion of a t ransc r ip t ion

f a c t o r , S u p p r e s s o r o f h a i r l e s s ( S u ( H ) ; F o r t i n i a n d A r -

t avani s -Tsakonas , 1994) . Su(H) then ac t iva te s t ransc r ip-

t i o n o f E(spl) genes (Er i ckson e t a l . , 1992; Meyers e t a l . ,

1 9 9 3 ) . T h e p r o t e i n p r o d u c t s o f t h e E(spl) genes in tu rn

inh ib i t t he A Sc - C p r o n e u r a l g e n e s , t h e r e b y l e a d i n g t h ece l l a long an ep ide rma l ra the r than a neura l l i neage

(Ar tavani s -Tsakonas and S impson , 1991; Pa roush e t a l . ,

1994) .

M a m m a l i a n h o m o l o g u e s o f t h e Drosophila n e u r o g e n i c

g e n e s h a v e b e e n i d e n t i f i e d . M a s h l a n d M a s h 2 , m a m m a l -

i a n h o m o l o g u e s o f A c h a e t e - s c u t e , p la y a r o l e i n c e l l

d i f f e r e n t ia t i o n in t h e n e r v o u s s y s t e m a n d t h e e c t o p l a c e n -

t a l cone , re spec t ive ly (Lo e t a l . , 1991; Gui l l emot e t a l . ,

1993 , 1994) . The musc le regula tory fac tors , f i r s t found

i n v e r t e b r a t e s ( M y f 5 , M y o D , M y o g e n i n , a n d M y f 6 )

and then iden t i f i ed in Drosophila (Naut i lus ) , a re bHLH

p r o t e in s i n v o l v e d i n m u s c l e c e l l d e t e r m i n a t i o n a n d d i f f e r -en t i a t ion (Braun e t a l . , 1989; Sas soon e t a l . , 1989; Wright

et a l . , 1989; Bate e t a l . , 1993). H es g e n e s , h o m o l o g u e s

o f hairy an d E(spl), h a v e b e e n i s o l a t e d f r o m r a t a n d

mouse (Akazawa e t a l . , 1992; Sasa i e t a l . , 1992) . The

s ing le groucho g e n e o f Drosophila i s represen ted by a

m u l t i g e n e f a m i l y i n h u m a n s (TLE genes ) (S t i fan i e t a l . ,

1 9 9 2 ) a n d in m o u s e (Grg gen es ) (Mal lo e t a l. , 1993; Miy-

asaka e t a l. , 1993; Leo n e t a l. , 1996) . A fam i ly of mam -

m a l i a n N o t c h h o m o l o g u e s h a s a l s o b e e n c h a r a c t e r i z e d

(Coffman e t a l . , 1990; E l l i s en e t a l . , 1991; Weinmas te r e t

a l . , 1991 , 1992; F ranco de l Amo e t a l . , 1992; Reaume e t

a l ., 1992; La rde l l i e t a l ., 1994) . Here w e re fe r to them a m m a l i a n h o m o l o g u e s o f t h e n e u r o g e n i c g e n e s a s t h e

m N G s .

T h e f u n c t i o n a l i n t e r a c t i o n s b e t w e e n N o t c h a n d t h e

b H L H t r a n s c r i p t io n f a c t o r s a l s o a p p e a r t o b e c o n s e r v e d i n

v e r t e b r a t e s . T h e i n t r a c e l l u l a r d o m a i n o f m u r i n e N o t c h

i n h ib i ts n e u r a l a n d m y o g e n i c d i f f e re n t i a ti o n o f m o u s e

P19 EC ce l l s and repres ses t ransc r ip t iona l ac t iva t ion by

M y f 5 , M y o D a n d a n A c h a e te - s c u te h o m o l o g u e ( K o p a n e t

a l . , 1994; Nye e t a l . , 1994) . A l ink be tween Notch , Su(H)

a n d E ( s p l ) m a m m a l i a n h o m o l o g u e s w a s a l s o r e c e n t l y

d e m o n s t ra t ed : K B F 2 / R B P - J x , a h u m a n h o m o l o g u e o f

S u ( H ) , w a s s h o w n t o b i n d t h e i n t r ac e l lu l a r d o m a i n o f ac o n s t i t u t i v e ly - a c t i v e f o r m o f N o t c h a n d a c t i v a t e tr a n -

s c r i p t i o n o f a H e s l p r o m o t e r f r a g m e n t ( K a n i a e t a l . ,

1 9 9 0 ) . R e c o m b i n a n t H e s p r o t e i n s a r e a b l e t o b l o c k t r a n -

s c r i p t i o n a l a c t i v i t y o f a n E b o x - c o n t a i n i n g p r o m o t e r i n

v i tr o a n d m y o g e n i c c o n v e r s i o n o f 1 0 T 1 /2 c e ll s b y M y o D

(Akazawa et a l . , 1992; Sasai e t a l . , 1992; Ishibashi e t a l . ,

1993) . Thus , bo th the s t ruc ture and in t e rac t ions of the

n e u r o g e n i c g e n e f a m i l y m a y b e c o n s e r v e d f r o m Droso-

phi la t o v e r t e b r a t e s . H o w e v e r , t h e r e s u l t s f r o m g e n e t a r -

ge t ing of Notch, Mash a n d H es genes ind ica te a ro l e for

t h e m N G s i n r e g u l a t in g c e l l d i f f er e n t i a ti o n ( G u i l l e m o t e t

a l. , 1993 , 1994; Swia tek e t a l ., 1994; Conlo n e t al . , 1995;

I sh ibash i e t al . , 1995) , ra the r than in ce l l de t e rmina t ion a s

fo r Drosophila.

W e h a v e c l o n e d a f a m i l y o f m u r i n e g e n e s t h a t a r e

h o m o l o g u e s o f Drosophila groucho, h e r e r e f e r r e d t o a s

Grg, fo r groucho-related genes (Mal lo e t a l . , 1993) . Two

o f t h e s e g e n e s h a v e b e e n r e p o r t e d p r e v i o u s l y a n d d e s i g -

na ted Grg (Mal lo e t a l . , 1993) and A E S (Miyasak i e t a l ,

1 9 9 3 ). A f a m i l y o f h u m a n g r o u c h o - r e l a t e d g e n e s w a s a l s op r e v i o u s l y r e p o r t e d a n d d e s i g n a t e d TLE 1- 4 ( t ransduc in-

l ike e l ement ; S t i fan i e t a l . , 1992) . There fore , we have

a d o p t e d t h e d e s i g n a ti o n o f Grg f o r t h e m u r i n e g e n e s , a n d

n u m b e r e d t h e m c o n s i s t e n t l y w i t h t h e n u m b e r i n g o f t h e

h u m a n T L E g en e s .

W e p r e v i o u s l y s t u d i e d t h e e x p r e s s i o n p a t t e r n o f Grg3

(Groucho-related gene 3) d u r i n g m u r i n e e m b r y o g e n e s i s

a n d n o t e d t h a t i ts e x p r e s s i o n o v e r l a p p e d e x t e n s i v e l y w i t h

Notch1, s u g g e s t in g t h a t t h es e t w o g e n e s m a y h a v e a c o n -

se rved func t iona l in t e rac t ion (Leon e t a l . , 1996) . The ex-

pres s ion domains inc luded undi f fe ren t i a t ed ce l l s i n the

n e r v o u s s y s t e m a n d e p i th e l ia l s t r u c tu r e s u n d e r g o i n g m e s -e n c h y m a l in d u c t i o n . T h e r e f o r e , a s n o t e d f o r N o t c h p r e v i -

ous ly (We inma s te r e t a l. , 199 1) , Grg3 expres s ion i s a s so-

c i a t ed wi th ce l l s which a re commi t t ed to a pa r t i cu la r l i ne -

age bu t a re undi f fe ren t i a t ed and mi to t i ca l ly ac t ive .

H e r e w e r e p o r t t h e e x p r e s s i o n o f a n o t h e r groucho-

re l a t ed gene , Grg4. T h i s g e n e i s a l s o e x p r e s s e d d u r i n g

embryogenes i s in a pa t t e rn tha t sugges t s a ro l e in ce l l

d i f fe ren t i a t ion . However , i t s expres s ion domains a re d i s -

t inc t f rom Notch, Hes a n d o t h e r Grg g e n e s t h a t w e h a v e

e x a m i n e d , p a r t i c u l a r l y i n t h e d e v e l o p i n g c e n t r a l n e r v o u s

s y s t em ( C N S ) . I t s e x p r e s s io n i n t h e n e r v o u s s y s t e m s u g -

ges t s i t may in t e rac t w i th a t ransc r ip t ion fac tor ou t s ide oft h e k n o w n H e s f a m i l y t o e f f e c t n e u r o n a l d i f f e r e n t ia t i o n .

2 . R e s u l t s

2.1. Isolation o f a Grg4 cDNA

I n o r d e r t o i s o l a t e m o u s e h o m o l o g u e s o f Drosophila

groucho, w e u s e d P C R t o a m p l i f y c o n s e r v e d s e q u e n c e s i n

t h e W D 4 0 d o m a i n . T h e p r i m e r s w e r e c h o s e n b a s e d o n

sequence s imi l a r i ty be tween Drosophila a n d h u m a n s e -

quences (S t i fan i e t a l . , 1992) . Sequences represen t ing

three d i f fe ren t Grg t r ansc r ip t s were iden t i f i ed f rom theP C R p r o d u c t s ( L e o n e t a l . , 1 9 9 6 ) . T h e s e w e r e t h e n

u s e d t o s cr e e n a m o u s e c D N A l i b ra r y . S e v e r a l o f th e

c D N A s c o r r e s p o n d e d t o t h e m o u s e h o m o l o g u e o f TLE4,

w h i c h i s o n e o f t h e h u m a n groucho h o m o l o g u e s ( S t i f a n i

e t a l. , 1 9 9 2 ) , t h e r e f o r e w e d e s i g n a t e d t h e m o u s e g e n e

Grg4.

Grg4 e n c o d e s a p r o t e i n t h a t i s h i g h l y c o n s e r v e d w i t h

th e Drosophila G r o u c h o p r o t e i n a n d w i t h th e o t h e r

m a m m a l i a n G r o u c h o h o m o l o g u e s ( F i g . 1 ) . T h e f i v e c h a r-

a c t er i st ic d o m a i n s o f G r o u c h o a r e t h e Q , G / P , C c N , S / P

a n d W D 4 0 d o m a i n s ( S t i f a n i e t a l. , 1 9 9 2 ) . T h e W D 4 0 d o -

main i s l i ke ly used for in t e rac t ions wi th o the r p ro te ins

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K.E . K oop et al. M echanisms o[ Development 59 1996) 73-87 75

( N e e r e t a l ., 1 9 9 4 ) a n d i s v e r y h i g h l y c o n s e r v e d a m o n g a l l

o f t h e G r o u c h o h o m o l o g u e s ( > 9 6 % ) . T h i s s u g g e s t s th a t

e a c h G r g m e m b e r i n t e r a c t s w i t h t h e s a m e p r o t e i n s

t h r o u g h t h a t d o m a i n .

O n t h e o th e r h a n d , t h e S / P d o m a i n , w h i c h i s u s e d f o r

i n t e r a c t i o n s w i t h t h e H a i r y - r e l a t e d t r a n s c r i p t i o n f a c t o r s

( P a r o u s h e t a l ., 1 9 9 4 ) , i s n o t w e l l c o n s e r v e d b e t w e e n

G r g 4 a n d o t h e r G r g m e m b e r s . T h i s s u g g e s t s t h a t e a c hG r o u c h o h o m o l o g u e b i n d s to a u n i q u e s u b s e t o f H a i r y -

r e l a te d tr a n s c r i p ti o n f a c t o r s . H o w e v e r , t h e S / P d o m a i n o f

G r g 4 d o e s s h o w h i gh h o m o l o g y t o t h e S / P d o m a i n o f i ts

r a t a n d h u m a n c o u n t e r p a r t s , T L E 4 a n d E s p 2 , r e s p e c t i v e l y

( F i g . 1 ) ( S t i f a n i e t a l ., 1 9 9 2 ; S c h m i d t a n d S l a d e k , 1 9 9 3 ) ,

f u rt h er s u g g e s t in g t h at t h e a n a l o g o u s G r o u c h o h o m o l o g u e

f r o m e a c h m a m m a l i a n s p e c i e s b in d s t o a c o n s e r v e d t ra n -

s c r i p t i o n f a c t o r .

T h e Q d o m a i n i s g l u ta m i n e - r i c h a n d i s h i g h l y c o n -

s e r v e d b e t w e e n m o s t m e m b e r s o f t h e G r o u c h o f a m i l y .

W e h a v e f o u n d t h a t t h i s d o m a i n i s u s e d f o r d i m e r i z a t i o n

b e t w e e n G r g p r o t e i n s ( P i n t o a n d L o b e , u n p u b l i s h e d ) .

E s p 2 , t h e ra t h o m o l o g o f G r g 4 , h a s a l a r g e g a p w i t h i n t h e

Q d o m a i n , i n d i c a t i n g t h a t i t m i g h t n o t d i m e r i z e w i t h t h e

o t h er G r g p r o te i ns . T h e G r g 4 c D N A s w e i s o la t e d e x t e n d

o n l y i n t o t h e G / P d o m a i n . T h e s e q u e n c e r e p o r t e d f o r h u -

m a n T L E 4 a l s o d o e s n o t e x t e n d t o t h e Q o r G / P d o m a i n s( S t i fa n i e t a l. , 1 9 9 2 ) s o i s n o t a v a i la b l e f o r c o m p a r i s o n .

2 .2 . E a r l y ex p r e s si o n o f G r g 4

G r g 4 e x p r e s s i o n w a s a n a l y z e d b y w h o l e m o u n t i n s i t u

h y b r id i za t io n o f m o u s e e m b r y o s w i th d i g o x y g e n i n -

l a b e ll e d a n t is e n s e R N A p r o b e s ( W i l k i n s o n a n d N i e t o ,

1 9 9 3 ) . S i g n a l s w e r e l o c a l i z e d m o r e p r e c i s e ly b y c r y o s e c -

t i o n in g t h e e m b r y o s . E m b r y o s o f 1 0 .5 d a y s p o s t c o i t u m

A

i CAAACAAACGACTCCATCAAGAGCTCTTCCGTATCCCCATCAGCCAGTTTCCGGGGTTCTGAGAAACATAGGAACTCTACAGACTATTCCTCAGAGAGCAAAAAGCAGAAAACAGAAGAAG•nThrAsnAsp•erI•eLysserser•erva•serPr•serA•aserPheArgG•yserG•uLysHisArgAsnserThrAspTyrser•erG•u•erLysLysG•nLysThrG•uG•u 40

121 AAAGAAATTGCAGCTCGCTATGACAGTGATGGTGAGAAGAGTGATGACAACTTGGTA•TTGATGTGTCCAATGAGGATCCATCTTCCCCCCGAGGAAGCCCAGCACATTCCCCAAGGGAG

LysG•uI•eA•aA•aArgTyrAspSerAspG•yG•uLysSerAspAspAsnLeuVa•Va•AspVa•SerAsnG•nAspPr•SerSerPr•ArgG•ySerPr•A•aHisSerPr•ArgG•u 80

241 AATGGCCTGGACAAGACACGACTCCTCAAGAAAGATGCCCCCATCAGCCCCGCTTCTGTTGCATCTTCCAGCAGTACTCCTTCCTCCAAATCCAAAGAGCTTAGCCTTAATGAAAAGTCT

AsnGlyLeuAspLysThrArgLeuLeuLysLysAspA•aPr•I•eSer•r•A•aSerVa•A•aSerSerSerSerThrPr•SerSerLysSerLysG•uLeuSerLeuAsnG•uLysSer 12 0

361 ACTACTCCTGTTTCAAA•TCCAATACCCCTACTCCACGAACTGATGCACCTACCCCTGGCAGTAATTCTACTCCTGGACTGAGGCCTGTACCTGGAAAGCCACCAGGTGTGGACCCCTTA

ThrThrPr•Va•SerLysSerAsnThrPr•ThrPr•ArgThrAspA•aPr•ThrPr•G•ySerAsnSerThrPr•G•yLeuArgPr•Va•Pr•G•yLysPr•Pr•G•yVa•AspPr•Leu 160

481 GCATCAAGCCTGAGGACCCCGATGGCTGTACCTTGTCCATATCCAACCCCTTTTGGGATCGTGCCCCATGCTGGGATGAACGGAGAGCTGACCAGCCCCGGAGCCGCCTATGCTGGGCTA

A•aSerSerLeuArgThrPr•MetA1aVa•Pr•CysPr•TyrPr•ThrPr•PheG•yI•e•a•Pr•HisA1aG•yMetAsnG•yG1uLeuThrSerPr•G1yA1aA•aTyrA•aG1yLeu 200

601 CACAACATCTCCCCCCAGATGAGTCGAGCTGCTGCTGCAGCTGCGGCAGCAGCAGCCTATGGAAGATCACCTGTGGTGGGATTTGATCCACACCATCACATGCGAGTGCCAGCAATACCT

HisAsnI•eSerPr•G1nMetSerArgA1aA•aA•aA•aA•aA•aA•aA•aA•aA•aTyrG•yArgSerPr•Va•Va•G•yPheAspPr•HisHisHisMetArgVa•Pr•A•a•1e•r• 240

721 CCAAATCTAACAGGCATTCCAGGAGGAAAACCAGCATACTCCTTTCACGTCAGGTCCGATGGCCAGATGCAGCCTGTCCCTTTCCCCCCTGATGCCCTCATTGGACCAGGGATCCCCCGA

Pr•AsnLeuThrG•yI•ePr•G•yG•yLysPr•A•aTyrSerPheHisVa•ArgSerAspG•yG•nMetG•nPr•Va1Pr•PhePr•Pr•AspA1aLeuI•eG1yPr•G•yI1ePr•Arg 28 0

841 CACGCTCGACAGATCAACACCCTCAACCATGGGGAGGTGGTGTGTGCAGTGACCATCAGCAACCCCACAAGGCACGTGTACACAGGTGGCAAGGGCTGTGTTAAGGTCTGGGACATCAGC

Hi•A•aArgG•nI•eAsnThrLeuAsnHisG•yG•uVa•Va•Cy•A•aVa•ThrI•eSerAsnPr•ThrArgHisVa•TyrThrG•yG•yLysG•yCysVa•LysVa•TrpAspI•eSer 320

961 CACCCTGGCAACAAGAGCCCAGTCTCTCAGCTGGACTGTCTGAACAGGGATAACTACATCCGTTCCTGCAGATTGCTCCCTGATGGTCGCACCTTAATTGTTGGAGGGGAAGCCAGCACA

HisPr•G•yAsnLysSerPr•Va•SerG1nLeuAspCysLeuAsnArgAspA•nTyrI•eArgSerCysArgLeuLeuPr•AspG•yArgThrLeuI•eVa•G•yG•yG•uA•aSerThr 360

1081 CTGTCCATCTGGGACCTGGCAGCTCCAACTCCACGCATCAAGGCAGAGCTGACATCCTCAGCCCCTGCCTGCTATGCTCTGGCCATCAGCCCCGACTCCAAGGTCTGCTTCT•ATGCTGC

LeuSerI•eTrpAspLeuA•aA•aPr•Thr•r•ArgI•eLysA•aG•uLeuThrSerSerA•aPr•A•aCysTyrA•aLeuA•aI•eSerPr•AspSerLysVa•CysPheSerCysCys 40 0

1201 AGCGACGGTAACATCGCAGTGTGGGATCTGCACAAGGAGACTCTGGTGAGGCAATTCCAGGGGCACACAGATGGAGCCAGCTGTATTGACATTTCTAATGATGGCACCAAGCTCTGGACA

SerAspGynAsnI•eA•aVa•TrpAspLeuHisLysG•uThrLeuVa•ArgG•nPheG•nG•yHisThrAspG•yA•aSerCysI•eA•pI•eSerAsnAspG•yThrLysLeuTrpThr 44 0

1321 GGTGGTTTGGACAACACTGTGAGGTCCTGGGACCTGCGTGAAGGGCGGCAGCTGCAGCAACATGACTTCACCTCTCAGATCTTTTCACTTGGCTATTGCCC~ACTGGAGAGTGGCTTGCA

G•yG•yLeuAspAsnThrVa•ArgSerTrpAspLeuArgG•uG•yArgG•nLeuG•nG•nHisA•pPheThrSerG•nI•ePheSerLeuG•yTyrCysPr•ThrG•yG•uTrpLeuA•a 480

1441 GTGGGGATGGAGAATAGCAATGTGGAAGTATTGCATGTCACCAAACCAGACAAATACCAGTTGCATCTTCATGAGAGCTGTGTGCTGTCACTCAAGTTTGCCCACTGTGGCAAATGGTTT

Va•G•yMetG•uAsnSerAsnVa•G•uVa•LeuHisVa•ThrLy•Pr•AspLysTyrG•nLeuHisLeuHisG•uSerCysVa•LeuSerLeuLysPheA•aHisCysG•yLysTrpPhe 520

1561 GTAAGCACTGGAAAGGACAACCTTCTGAATGCTTGGAGGACGCCTTATGGGGCCAGCATATTCCAGTCCAAAGAATCCTCATCGGTGCTTAGCTGTGACATCTCTGTGGATGACAAGTAC

Va1SerThrG•yLysAspAsnLeuLeuAsnA•aTrpArgThrPr•TyrG•yA•aSerI•ePheG•nSerLysG•uSerSerSerVa•LeuSerCysAspI1eSerVa•AspAspLysTyr 56 0

1681 ATTGTCACTGGCTCTGGGGACAAGAAAGCTACGGTTTATGAAGTTATTTATTAAGGACAAATCTTCGTGCAAACTGGACTCCTCCTCATAGCACTTTGCGCTGTTGTACTTTTCTGTTCA

IleValThrGlySerGlyAspLysLysAlaThrValTyrGluValIleTyr 577

1801 CCCCTCTCCCATTCTAAAACCAAGGATTTCCAATACTCATTGCAGTTGTGGAGTTTAATCCTTCCATAGCCCACTTCCTCCTTGCTATTGAATTGTGAATCTCATTAAGAACTGTGATAC

1921 AAATCTTCAG

Fig. 1. (A) Nucleotide and am ino acid sequences of Grg4. The nucleot ide sequence of the Grg4 DNA is given with its deduced amino acid sequence

shown below. T he region of W D40 dom ain-coding sequence , which was am plif ied by PC R and subsequently used as a probe, is und erlined. The nu m-

ber of the f irst nucleot ide and the last am ino acid o f each row are shown at the le ft and .r ight , respect ive ly. (B) Am ino a cid sequence comparison of

mouse G rg4 prote in with Drosoph i laGroucho, mouse Grg3a, rat Esp2, and huma n TL E4 and TLE3 prote ins . The sequences w ere al igned using the

DNST AR software package. Residues identical to the corresponding res idue in G rg4 are shown as a d ash (-) ; gaps and unkn own 5' sequence are le ft

blank. For complete prote in sequences, the num ber of the last amino acid o f each row is shown at the r ight . The v arious dom ains, as def ined by Stifani

et al . (1992) , are labeled at the le ft . The GenBa nk access ion num ber for the Grg4 DNA i s U61363.

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7 6 K.E. Koop et al. /Mechanisms of Development 59 (1996) 73--87

BG r o u c h o

G r g 3 a

E s p 2

T L E 3

G r o u c h o

G r g 3 a

E s p 2

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 3

G r o u c h oG r g 3 a

G r g 4

E s p 2

T L E 4

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 4

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 4

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 4

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 4

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 4

T L E 3

G r o u c h o

G r g 3 a

G r g 4

E s p 2

T L E 4

T L E 3

M Y p S P V R H P A A G G P P P Q G P I K F TI A D T L E R I K E E F N F L Q A H Y H S I KL E C E K L S N E K T E M Q R H Y V M Y Y 6 7

M C P C G L Q A P H Q P G Q P G F K F T V A E S C D R I K D E F Q F L Q AQ Y H S L K V E Y D K L A N E K T E M Q R H YV M Y Y 6 4

M Y R R R A H P A P H Q P A Q P F K F T I S E S C D R I K E E F Q F L Q AQ Y H S L K L E C E K L A S E K T E M Q R H YV M Y Y 6 4

M Y P Q G R H P A P H Q P G Q P G F K F T V A E S C D R I K D E F Q F L Q AQ Y H S L K V E Y D K L A N E K T E M Q R H YV M Y Y 6 5

E M S Y G L N V E M H K Q T E I A K R L N TL I N Q L L P F L Q A D H Q Q Q V L Q A V E RA K Q V T M Q E L N L I I G Q Q I H A Q 1 3 2

E M S Y G L N I E M H K Q T E I A K R L N TI L A Q I M P F L S Q E H Q Q Q V A Q A V E RA K Q V T M T E L N A I I G Q Q Q L Q A Q 1 3 0

E M S Y G L N I E M H K Q A E I V K R L N AI C A Q V I P C L S Q E Q Q Q L Q A Q 1 0 5

E M S Y G L N I E M H K Q T E I A K R L N TI L A Q I M P F L S Q E H Q Q Q V A Q A V E RA K Q V T M T E L N A I I G Q Q Q L Q A Q 1 3 1

Q V P G G P P Q P M G A L N P F G A L G A T M G L P H G P Q G L L N K P P E HH R P D I K P T G L E G P A A A E E R L 1 9 1

H L S H A T H G P P V Q L P P H P S G L Q PP G I P P V T G S S S G L L A L G A L G S Q A H L A V K D E K N H H E L D H R E R E - S T 1 9 7

Q T N D S I K

H L L T W T W S A C A S D T T P L G F S Q P F H P S G S S A G L L A L S S A L G G Q S H LP I K D E K K H H D N D H Q R D R . . . . 1 7 1

H L S H A T H G P P V Q L P P H P S G L Q PP G I P P V T G S S S G L L A L G A L G S Q A H L T V K D E K N H H E L D H R E R E - S A 1 9 8

R N . . . . A D - E K Y R T - S P L - I E N D - - R R - D - K L Q E D E . . . . . Q D . . . . . A - - M E S H - - - P N G E - V S 2 5 6

N N . . . . . E - L K A . . . . . , - . - - - . - , - - , - . - - - . , , , . . . . . . D . . . . . . . . . . . . . . A T - - V S . . . . . 2 6 7

S S S V S P S A S F R G S E K H R N S T DY S S E S K K Q K T E E K E I A A R Y D S D GE K S D D N L V V D V S N E D P S S P R G S P A H S P

. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . 2 4 2

N N . . . . . E - L - A . . . . . , - . - - - . - . - - , - . - - - , , , , . . . . . . D . . . . . . . . . . . . . . A T - - V . . . . . . 2 6 8

M - V R D R E S - N G E - L E K P S S S G I- Q E R - P - R S G S S - - R . . . . L - T K D M - - P G - - G A - A R . . . . N 3 1 9

P . . . . . . A - G . . .. . . T . . .. . .. . .. . .. . . T - D- G H- D -- S -- G L . . .. . .. . 3 2 2

R E N G L D K T R L L K K D A P I S P A S V A S S S S T P S SK S K E L S L N E K S T T P V S K S N T P T PR

. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 2 9 6

P . . . . . . A - S . . .. . . T . . .. . .. . .. . .. . . T - D- G H- D -- S -- G L . . .. . .. . 3 2 3

A A - - A - - V - P K Q M M - Q G - - - A G - - G A - Y Q R - A D 3 5 2

N . . . . . . T S T . . . . . S M . . . . . . M - - I G I M - - A . . . . I T L T S S - - A - - A M M S - H E - - - S . . . . S - 3 8 7

T D A P T P G S N S T P G L R P V P G KP p G V D p L A S S L R T P M A V P C P Y P T P F G I V P H A G M N G E L T S P G A

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 3 5 8

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

N . . . . . . T S T . . . . . S M . . . . . . M - - I G I M - - A . . . . I S I T S S - A A - - A M M S - H E - - - S . . . . . . 3 8 8

P Y Q R P P S D P . . . . P - P M P Y - - - A - V - T N G - - H P S A L T . . . . . . . . . 3 9 8

. . . . . . . P S . . . . . . . . . . . . . . . . . M - S F G A V . . . . . P P - - A T G L - S S - A S . . . . . . . . . . . 4 5 0

A Y A G L H N I S P Q M S R A A A A A AA A A A Y G R S P V V G F D P H H H M R V P A I P P N L T G I P G GK P A Y S F H

. . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . 4 1 9

. . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . P . . . . . . . . . . . . . . . . . . M - S F G A V . . . . . P P - - A T G L - S S - A S . . . . . . . . . . . 4 5 1

M N G E - S L . . . . . . . . . . V - V . . . . . . . . . . . . S . . . . . . . . . . . . . . K Y . . . . . . . . . . . . . . . Q . . . . N . . . . . . . . . . . 4 7 9

- S A . . . . . . . . . . H - - - A . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . .. . . . . . I . . . . Q - - S - - - I S . . . . . . . . 5 3 1

v R S D G Q M Q P v P F • • D A L I G PG I P R H A R Q I N T L N H G E v v C A V T I SN P T R H v Y T G G K G C v K V W D I S H P GN K S P V S Q L D c L N R D

- S A . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . T D . . . . . . . . . . . . . . . . 5 0 0

- S A . . . . . . . . . . . . P . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . .

- S A . . . . . . . . . . H - - - A . . . . . . . . . . . . . . S - - G . . . . . . . . . . S . . . . . . . . . . . . I . . . . Q - - S - - - I . . . . . . . . . 5 3 2

. . . . . V K . . . . . . . . . . . . . . . N . . . . . . . S . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . N - I . . . . 5 6 0

. . . . . . K . . . . . G . . . . . . . . . . . T . . . . . S . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . A . . . . . . N Q . . . . . 6 1 2

N Y I R S C R L L P D G R T L I V G G EA S T L S I W D L A A P T P R I K A E L T S SA P A C Y A L A I S P D S K V C F S C C S D G NI A V W D L H K E T L V R Q

. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . N Q . . . . . 5 8 1

. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . N Q . . . . .

- - M - - - K - H . . . . . . . . . . . G - - - T . . . . . S . . . . . . . . . . . . . . . . . . . . . . .. A . . . . . . . . . . . . . . . . . . N Q . . . . . 6 1 3

. . . . . . . . . . . . . . P - - S R . . . . . . . . . . . . . 7 . . . . . . . . . . . . S . . . . . . . . . . . . D . . . . . . . . . H . . . . . A S . . . . . 6 4 1

. . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . S . . . . . . . H . . . . . . 6 9 3

F Q G H T D G A S C I D I S N D G T K L W T G G L D N T V R S W D L R E GR Q L Q Q H D F T S Q I F S L G Y C P T G E W L A V G M E N S N V E V L H V T K P D K Y

. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 6 6 2

. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .

. . . . . . . . . . . . . . H . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . S . . . . . . . H . . . . . . 6 9 4

i

. . . . . . . . . . . . R - - A . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . T . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . 7 1 9

. . . . . . . . . . . . . . . y . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . 7 7 1

Q L H L H E S C V L S L K F A H C G K WF V S T G K D N L L N A W R T P Y G A S I F Q S K E S S S V L S C D I S V D D K Y I V T G S G D K K A T V Y E V I Y

. . . . . . . . . . . . . . . . . . . . . . R P . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . A . . . . . . . . . . . . . . . . . . . . . 7 4 0

. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .

. . . . . . . . . . . . . . . Y . . . . . . . . . . . . . . . . . . . .. . . . . S . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . 7 7 2

Q d o m a i n

G P d o m a i n

C o N d o m a i n

S P d o m a i n

W D 4 0 d o m a i n

F i g . l b .

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K.E. Koop et al. / Mechanism s of Development 59 (1996) 73- 87 77

Fig. 2. Express ion of mouse neu rogenic gene homo logues in 7.5 dpc embryos. (A) Grg4 t ranscr ipts are present in the lateral mesod erm (arrow) and the

amnion. (B) Grg3 i s also expressed in the lateral mesoderm, whereas (C) Hesl i s expressed in the neural plate and around the node (arrowhead) . (D)

Notchl , in contras t , i s expressed in pre-somit ic mesoderm. For al l panels , embryos are or iented with anter ior to the lef t and the posi t ion o f the node is

indicated with an arrowhead.

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78 K.E. Koop et al. / Mechanisms of Development 59 (1996) 73-87

(dpc ) and o lde r were s ec t ioned f i r s t , fo l lowed by hybr id i -

za t ion wi th the RNA probe on s l ides (Wi lk inson and

Nieto, 1993).

The ea r l i e s t expres s ion of Grg4 was de tec t ed in 7 dpc

embryos , dur ing the ea r ly headfo ld s t age and pr ior to

somi te forma t ion (F ig . 2A) . A t th i s t ime the RNA probe

was de tec t ed in the migra t ing ex t raembryonic and l a t e ra l

mesoderm (F ig . 2A, a r row) , and in the amnion . Thi s ex-pres s ion was s imi l a r t o tha t o f anothe r groucho-related

gene , Grg3 (Fig. 2B) ( Leo n et a l. , 1996). In contras t ,

H es l at the same s tage is expressed in the neural pla te and

a round the node , and Notchl i s expressed in pre-somit ic

m e s o d e r m ( F ig . 2 C , D ) .

At 8.5 dpc, the Grg4 s ignal was s t rong in the neural

pla te in the primit ive s t reak region (Fig. 3A, ps) . The

neuroepi the l ium in the prospec t ive bra in a l so d i sp layed

high l eve l s o f expres s ion , bu t was re s t r i c ted to the pros -

encepha lon (F ig . 3A, concave a r rowhead) , a d i f fuse band

i n t h e m e s e n c e p h a l o n a n d m e t e n c e p h a l o n , a n d w e a k e r

s igna l s in two s t r ipes in the mye lencepha lon (F ig . 3A,a r rowheads ) . Grg4 was a l so expres sed in the presomi t i c

mesoderm and in newly formed somi tes , and expres s ion

cont inued in the cauda l ha l f each somi te (F ig . 3A) . Thi s

pa t t e rn ove r l aps expres s ion of Hesl i n the prosencepha-

Ion and the ros t ra l s t r ipe in the mesencepha lon (F ig . 3C,

a r rowheads ) , sugges t ing He s l may be a transc r ip t ion

fac tor w i th which Grg4 in t e rac t s in these t i s sues . Anothe r

ove r l apping dom ain of expres s ion was in the reg ion

where the f i r s t pha ryngia l pouch forms (F ig . 3A,C, p) ,

which may cor respond to neura l c re s t -de r ived mesen-chyme . In the CNS a t t he s ame s t age , Grg3 was expres sed

throughout the neura l p l a t e , bu t a t h ighes t l eve l s in the

midbra in (F ig . 3B) and Notch1 expres s ion was de tec t ab le

in the midbrain (Fig. 3D).

2.3. Grg4 expression during organogenesis (9.5-

12.5 dpc)

As the neura l t ube c losed (8 .5-9 .5 dpc ) , t he expres s ion

o f Grg4 became res t r ic ted, f i rs t dorsal ly (Fig. 4A, black

bracket) , and then to a dorso-la tera l regio n of the spinal

cord (F ig . 5C, a r rowhead) . Thi s expres s ion was d i f fe ren tf r o m o t h e r Grg and Hes genes repor t ed to da te , which a re

genera l ly expres sed more dorsa l ly in the neura l t ube (F ig .

,C

Fig. 3. Comparison of expression of mouse neurogenic gene homologues in 8.5 dpc embryos. (A) Grg4expression is detected in the rostral region ofthe forebrain (fb, concave arrowhead), a diffuse band in the mesencephalon (ms) and in the metencephalon (mt), and two faint bands in the m yelen-

cephalon (m y, arrowheads). The open neu ral plate in the primitive streak (ps) region also stains strongly for Grg4 ranscripts. Signal is presen t in thecaudal region of each som ite (arrows) and the prospective pharyngial pouch region (p). (B) Grg3at this tim e is expressed in the neural plate, lateral

and som itic mesoderm. (C) Hesl expression overlaps Grg4 in the forebrain (concave arrow) and band o f m esencephalon expression (arrowhead) aswell as the region of the prospective pharyngeal pouch (p). Expression of Hesl in the primitive streak region may correspond to tissue around thenode, by comparison to the expression around the no de at 7.5 dpc (see Fig. 2C). (D) Notch1 is expressed at low levels in the brain, in a restricted do-

main within the som ites, and at high levels in the preso mitic mesoderm.

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K.E. Koop e t al . / Mechanism s of Development 59 (1996) 73-8 7 79

Fig. 4. Express ion analysis in 9.5 dpc m ouse embryos. (A) Grg4 probe was detected in the telencephalon and olfactory placodes (co ncave arrowhead) ,

the dorsal region of the midbrain (mb) and hindbrain (hb) , the caudal par t of each somite (arrows) , the presomit ic mesoderm (white bracket) , the dorsal

neural tube (black bracket) , the open neural plate and the lateral mesoderm. (B,C) Grg3 an d He s l are also expressed in the telencephalon and olfactory

placodes , as wel l as a res t r icted domain of the som ites that does not over lap with Grg4 express ion. (D) Notch1 i s expressed pr imari ly in the presomit ic

mesod erm and at lower levels in a do rsal band of cel ls in the somites (arrows) .

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80 K.E. Koop et al. / Mechanism s ofDevelopment 59 (1996) 73-87

5D ,E ) (A ka z a w a e t a l. , 1995 ; Sa sa i e t a l ., 1992 ) . In t he

m i d b r a i n a n d h i n d b r a i n , G r g 4 w a s e x p r e s s e d d o r s a l l y

( F i g s . 4 A , m b , a n d 5 A , h b , a r r o w h e a d ) . A t 1 1 .5 d p c , a

f e w c e l l s i n t h e r e g i o n o f t h e f l o o r o f t h e m i d b r a i n a n d

h i n d b r a i n a l s o s t a i n e d f o r G r g 4 e x p r e s s i o n a n d t r a n s c r i p t s

c o u l d b e d e t e c t ed t h r o u g h o u t t h e v e n t r i c u l a r z o n e o f th e

CN S by 12 .5 dpc (F ig . 6 ) .

I n t h e f o r e b r a i n r e g i o n , t h e s t r o n g G r g 4 s igna l i n t he

n e u r o e p i t h e l i u m o f t he t e l e n c e p h a l o n w a s n e i g h b o r e d b y

e x p r e s s i o n i n t h e s u r f a c e e c t o d e r m o f t h e p r o s p e c t i v e

Fig. 5. Sections of 9.5 dpc embryos after whole mount in situ hybridization with a Grg4 antisense probe. (A) Grg4 signal is detected in the neuroepi-

thelium of the dorsal part of the hin dbra in (hb, arrowhead), the otic vesicle (ov), and in the most lateral portion o f the pharynx (ph, arrow ). (B) In the

tail region, Grg4 is expressed throughout the open neural plate (np), the presomitic mesoderm (arrowheads) and in the lateral mesoderm of the so-

matopleure and splanchnopleure (concave arrowheads and arrows, respectively). (C) Grg4 is expressed in a restricted dorso-lateral region of the neural

tube (arrowhead) and in the myotome (arrow), but no t in the dermatome. (D) By contrast, Grg3 is expressed in the dorsal-most part of the neural tube

and the ventricular zone next to the lum en, and in the dermamyotome of the somites (arrow). (E) Grg5 transcripts are localized to the dorsal-most part

of the neural tube and the dermatome compartment o f the somite (arrow).

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K.E. Koop et al. / Mechanisms ~)fDevelopment 59 (1996) 73-87 81

Fig. 6. Grg4expression in 12.5 dpc embryos. (A) Hybridization of a Grg4probe to a sagittal embryo section reveals expression throughout the ven-tricular zone o f the b rain and spinal cord and nasal epithelium. (B) Higher magnification of a parasagittal section show s expression in the ventricularzone of the hindbrain and spinal cord, and in the dorsal root ganglia. Anterior is at bottom , drg, dorsal root ganglia; m, mesencephalic vesicle; ne, nasal

epithelium; sc, spinal cord ; te, telencephalic vesicle; IV, fourth ventricle.

olfactory placodes (Fig. 4A, arrowhead) . Grg3, Grg5 and

Notchl are a lso expressed in the deve loping o l fac tory

placodes (Fig. 4B) (Franco del Amo et a l . , 1992; Reaume

et a l . , 1992; Leon et a l . , 1996) . The ear ly expression of

Grg4 there persisted a t la ter stages in the neural layer of

the olfactory epithelia (Fig. 6A, he) . Other expression in

the CNS was detected in the developing optic and otic

placodes, and subsequently in the lens and the neuroepi-

thelia of the developing eye and ear (Fig. 5A, ov, and data

not shown). In the per ipheral nervous system, Grg4 w as

expressed in the dorsal root ganglia and cranial ganglia(Fig. 6B, drg) . Again, this expression overlaps Grg3,

Grg5 and Notchl expression (Franco del Amo et a l . ,

1992; Reaume et al. , 1992; Leon et al. , 1996).

With in the pa raxial meso derm, express ion of Grg4 be-

gan in presomit ic meso derm (F ig . 4A, w hi te bracke t) and

in somites was re s t r ic ted to a por t ion of the myotomal

compartment (Fig. 5C, arrow). This is in contrast to Grg3,

which i s expressed throughout the myotome and de rma-

tome, and Grg5, which i s expressed in the de rmatome

(Fig. 5D,E, arrows) . O f the Hes genes reported, Hesl is

expressed in the somites (Fig. 4C, arrows) (Sasai e t a l . ,

1992) . However , we have not examined which region of

the somites i t is expressed in, therefore i t is not c lear

which of the Grg pro te ins Hes l may b e in te ract ing wi th

the re . Two of the genes tha t Hes and Grg pro te ins may

regulate , by analogy to the Drosophila proteins, are Myf5

an d MyoD, which encode bHLH musc le de te rmina t ion

factors. Myf5 and MyoD a re expressed in the myotome of

the somite (Braun et a l . , 1989; Sassoon et a l . , 1989) .

Thus , Grg4 express ion in the myot ome ma y p lay a ro le in

the regulation of Myf5 and MyoD express ion .

Another si te of Grg4 express ion dur ing th is pe r iod was

in the pharynx. The expression was restr ic ted to la teralreg ions of the pharynx and ne ighbor ing m esenc hym e

(Fig. 5A, arrow), which appeared to coincide with the

format ion of the pharyngea l pouches .

2.4. Late expression of Grg4 (14.5-1 6.5 dpc)

At 14 .5-16 .5 dpc , many of the s i te s of Grg4 expres-

sion (Figs. 7, 8 and 9) were similar to Notch1, Hesl and

Grg3 (Fig. 9) (Franco del Amo et a l . , 1992; Reaume et

al. , 1992; Sasai et al. , 1992; Leon et al. , 1996). This in-

cluded expression in the whisker foll ic les (Fig. 7A, wh),

neural layers of the nasosensory epithelia (Fig. 7D, ne) ,

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K.E. Koop et al. / Mechanisms (~['Development 59 (1996) 73-87 83

Fig. 8. Grg4 expression in the trunk of 14.5 dpc embryos. (A) Grg4 signal is located in the dorsal root ganglia, the lung epithelium, adrenal medulla,

kidney, and endoderm lining of the gut. (B) A highe r magnification showing Grg4 signal in the epithelial cells of the lung. (C) Close-u p of the adrenal

gland and kidney, showin g the very high signal in the adrenal medulla, ad, adrenal gland; drg, dorsal root ganglia; ki, kidney; lu, lung.

some t issues i t overlaps with expression of the other

mouse neurogenic gene homologues (mNGs) . This i s

most evident in developing epithel ia l t issues a t 12.5-

16.5 dpc. Common regions of expression were the sal i-

vary gland, nasosensory, lung and kidney epithel ia , the

enamel epithel ium of the tooth progenitors and the neural

layer of the re t ina. A s noted previously, these t issues areepithel ia l in nature and Undergo mesenchymal induction

to acquire their characterist ic morphologies (Wessel ls ,

1977; Weinmaster et al., 1991; Leon et al., 1996). An-

other si te of Grg4 expression undergoing similar induc-

t ion are the la teral ends of the pharynx in the branchial

region, which evaginate to form the pharyngial pouches

(Hogan et al., 1986). Expression in these tissues suggests

the mNGs play a role in cel l-cel l s ignall ing during

epithel ia l /mesenchymal interact ions, a role the neu rogenic

genes of Drosophilamay also have (Tepass and Harten-

stein, 1995).

Grg4 was also expressed in the developing nervoussystem, suggest ing that i t plays a role in neuronal differ-

entia t ion. Transcripts of Grg4 were first detected in the

prospective brain a t 8.5 dpc. T his exp ression appeared

very similar to that of Hesl in the rostral forebrain and in

a diffuse stripe in the ro of of the midbrain so i t is possible

that Grg4 interacts with Hesl there . The bands of Grg4expression in the midbrain and hindbrain suggest that i t

may function in early brain pat terning or mark the earliest

cel l populat ion to differentia te . During neural tube closure

and cell differentiation in the spinal cord, Grg4 expres-

sion is initially expressed throughout the neural plate,

then becom es dorsal ly restric ted after neural tube closure ,

fol lowed by restric t ion to a dorso-la teral region at 9.5-

10.5 dpc. Finally, at 12.5 dpc, expression is limited to the

ventricular zone in the spinal cord. This changing ex-

pression pat tern may reflect the dynamics of cel l differ-

entia t ion in the spinal cord, with motor neurons first dif-

ferentia ting ventral ly a t 9.5 dpc, then the comm issural

neurons m ore dorsal ly a t 10.5 dpc, and final ly the sensor yneurons in the dorsal-mo st domains a t 11.5-12.5 dpc.

Thus, Grg4 expression appears to precede cel l differen-

tiation in the spinal cord.

Most intriguing is the Grg4 expression in specific lay-

ers of the developing cerebral cortex. The layers of the

cerebral cortex develop by the migrat ion of neurons, after

their last mitot ic division, from the ventricular zone out-

wards to the molecular layer (marginal zone), beginning

at approxim ately 14.5 dpc. A fter migrat ion, cel ls are

gradually displaced inwards as other cells arrive later at

the molecular layer. This gives rise to the ' inside-out '

pat tern of the final six layers o f the cort ical pla te (Rakic ,1972, 1988). The neurons in each of these layers differ-

entia te to a characteristic varie ty of n eurons (Dehni e t a l. ,

1995; Ishibashi et al., 1995 ).

Grg4 expression by the earl iest neurons to migrate to

the cort ical pla te , fol lowed by expression in cel ls that

have been displaced inwards, could represent e i ther of

two possible functions for Grg4 protein there . It may act

to specify differentia t ion to one part icular neuronal type,

or i t may play a more general role in regulat ing the matu-

ration of a blast cell to a differentiated cell. The expres-

sion of Grg4 in other types of prol iferat ing cel l popula-

t ions, including the spinal cord (see above), during devel-

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84 K.E. Koop et al. / Mechanisms o f Development 59 (1996) 73- 87

Fig. 9. Expression of mouse neurogenic gen e homologues in the head region of 16.5 dpc mouse embryos. (A) Grg4 expression is strongest in the in-

fragranular region of the cortical plate in the cerebral cortex. Expression is also detected in the midbrain, cranial ganglia, external granular (outer ger-

minal) layer of the cercbellum, w hisker follicles, nasal epithelium, to oth bud and salivary glands. (B) Higher magnification of Grg4 expression show-

ing restriction of the signal to the infragranular laye r of the cortical plate. (C,E) Grg3 and Hesl are expressed in overlapping tissues to Grg4, except for

the expression in the cerebral cortex. (D) Grg3 expression in the cerebral cortex is in the ventricular zone and the supragranular layer of the cerebral

cortex, while (F) Hesl expression is only in the ventricular zone. cb, cerebellum; cc, cerebral cortex; CPi, infragranular layer of the cortical plate; CPs,

supragranular laye r of the cortical plate; ro b, midbrain; ne, nasal epithelium; sg, salivary gland; tb, toothbud; v z, ventricular zone; wh, w hisker folli-

cles; V, trigeminal ganglia.

o p m e n t i n d i c a t e s a g e n e r a l f u n c t i o n t o r e g u l a t e c e l l d i f -

f e r e n t i a t i o n . T h e o t h e r m N G s a r e e x p r e s s e d i n t h e p r o l i f -

e r a t i n g c e l l s o f t h e v e n t r i c u l a r z o n e a n d t h e n e w l y a r r i v e d

n e u r o n s o f t h e c e r e b r a l c o r t e x . T h e r e f o r e , e a c h g e n e

s e e m s t o p l a y a r o l e i n a p a r t i c u l a r s t a g e o f d i f f e r e n t i a t i o n

r a t h e r t h a n i n s p e c i f y i n g n e u r a l l i n e a g e s . T h i s i s f u r t h e r

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K.E. Koop et al. / Mechanisms of Development 59 (1996) 73-87 85

su p p o r t e d b y t h e o b se r v a t i o n t h a t Grg4, as we l l a s t he

o t h e r m N G s , a r e e x p r e s se d i n t h e o u t e r g r a n u l a r l a y e r o f

the ce rebe l lum a t 16 .5 dpc ( see F ig . 9 ) . A t a round b i r th to

3 w e e k s p o s t p a r t u m , n e u r o b l a s ts m i g r a t e i n f r o m t h e

g r a n u l a r l a y e r t o f o r m t h e c o r t i c a l l a y e r s o f t h e c e r e b e l -

l u m , a n a l o g o u s t o t h e m i g r a t i o n o f c e l l s f r o m t h e v e n -

t r i cu la r zon e of t he ce rebra l cor t e x ( I sh ibash i e t a l ., 1995)

A de ta i l ed ana lys i s o f Grg4 e x p r e s s i o n d u r i n g d e v e l o p -m e n t o f t h e c e r e b r a l c o r t e x a n d o t h e r c o r t i c a l t i s su e s o f

t h e b r ai n , su c h a s t h e c e r e b e l lu m a n d h i p p o c a m p u s , w h i c h

d e v e l o p p o s t n a t a l ly , sh o u l d r e v e a l w h a t so r t o f r o l e Grg4

p l a y s i n C N S d e v e l o p m e n t .

A n o t h e r t i s su e w h e r e Grg4 i s expressed in ad jacen t

r a t h e r t h a n o v e r l a p p i n g t i ssu e s t o t h e o t h e r m N G s i s in t h e

somi tes . Grg4 t r ansc r ip t s a re re s t r i c t ed to the myotome

c o m p a r t m e n t o f t h e so m i t e s , u n l ik e a n y o f t h e o t h e r c h a r-

a c t e r i z e d m N G s . Grg3 i s e x p r e s se d i n t h e d e r m a m y o -

t o m e , Grg5 i s e x p r e s se d i n t h e d e r m a t o m e , a n d Notchl

and Hesl a r e e x p r e s se d i n a r e s t r i c t e d d o r sa l d o m a i n o f

t h e so m i ti c m e so d e r m .T h e e x p r e s s i o n o f Grg4 i n d i f f e r e n t i a t i n g n e u r o n s o f

t h e c o r t e x a n d m y o g e n i c c e l ls o f t h e so m i t e is su r p r i si n g

i n l i g h t o f t h e r o l e o f n e u r o g e n i c g e n e s i n o p p o s i n g t h e

a c t iv i t y o f b H L H c e l l d e t e r m i n a t i o n f a c to r s , su c h a s M y f 5

and MyoD (Kopan e t a l . , 1994; Nye e t a l . , 1994) . Thi s

su g g e s t s G r g 4 h a s a d i s t i n c t f u n c t i o n f r o m t h e o t h e r

G r o u c h o - r e l a t e d p ro t e in s . G r g 4 m a y b e i n t e r a c ti n g w i t h a

pa r t i cu la r Hes pro te in to p romote , r a the r t han inh ib i t , ce l l

d e t e r m i n a t i o n a l o n g t h e p r o - m y o g e n i c / n e u r a l p a t h w a y .

F o r e a c h G r g f a m i l y m e m b e r , t h e S / P d o m a i n , w h i c h i n -

t e rac t s w i th the t ransc r ip t ion fac tor , i s h igh ly va r i ab le

b e t w e e n G r g f a m i l y m e m b e r s . T h e r e f o r e , e a c h i s l ik e l y toin t e rac t w i th a pa r t i cu la r Ha i ry- l ike (Hes) t r ansc r ip t ion

f a c t o r . I n t h e c a se o f G r g 4 a n d a c o r r e sp o n d i n g H e s p r o -

t e in , t h i s may hav e d i f fe ren t re su l t s on ce l l d i f fe ren t i a t ion

t h a n, f o r e x a m p l e , a G r g / H e s c o m b i n a t i o n i n t h e v e n t r i c u -

l ar z o n e o f t h e c o r t e x a n d d e r m a m y o t o m e .

A f u r t h e r i n d i c a t i o n t h a t G r g 4 m a y h a v e a d i s t i n c t

f u n c t i o n f r o m t h e o t h e r G r g p r o t e i n s i s t h a t i t m a y l a c k

p a r t o f th e Q d o m a i n . T h e s e q u e n c e o f th e r a t h o m o l o g u e

o f G r g 4 ( E sp 2 ) r e v e a l s t h a t a 2 5 a m i n o a c i d g a p i s p r e se n t

i n t h e Q d o m a i n ( S c h m i d t a n d S l a d e k , 1 9 9 3 ) . W e h a v e

f o u n d t h a t t h e G r g p r o t e i n s c a n d i m e r i z e t h r o u g h a r e g i o n

o f t h e Q d o m a i n t h a t i n c lu d e s t h e 2 5 a m i n o a c i d s m i s s i n gi n ra t E sp 2 ( P i n t o a n d L o b e , su b m i t t ed ) . O n e o f t h e G r g

f a m i l y m e m b e r s , Grg5, e n c o d e s a p r o t e in w i th o n l y t h e Q

a n d G / P d o m a i n s . T h e G r g 5 p r o t e in d i m e r i z e s e f f i c ie n t l y

w i t h th e o t h e r G r g p r o t e in s . H o w e v e r i t l a c k s t h e S / P a n d

W D 4 0 d o m a i n s t o i n t e r a c t w i t h t h e b H L H t r a n sc r i p t i o n

f a c t o rs a n d h e t e r o l o g o u s p r o t e in s , r e sp e c t i v e l y . T h e r e f o r e ,

t h e f u n c t i o n o f G r g 5 m a y b e t o r e g u l a t e t h e a c t i v i t y o f

o t h e r G r g p r o t e i n s . I f G r g 4 , l i k e E sp 2 , i s m i s s in g so m e o f

t h e s e q u e n c e r e q u i r e d f o r d i m e r i z a t i o n , i t m a y b e f r e e o f

th i s i n t e rac t ion wi th Grg5 an d the regula t ion tha t t he o the r

fu l l - l eng th Grg pro te ins a re sub jec t t o .

T h e m u l t ip l e m e m b e r s o f th e Notch, Hes an d Grg

f a m i l ie s c o u l d i n t e r a c t i n v a r i o u s c o m b i n a t i o n s t o a c h i e v e

di f fe ren t e f fec t s i n regula t ing ce l l d i f fe ren t i a t ion dur ing

d e v e l o p m e n t. T h e Grg4 e x p r e s s i o n p a t t e r n a n d s e q u e n c e

su g g e s t i t h a s a r o l e d i s t i n c t f r o m t h e o t h e r G r g p r o t e i n s .

T h e c o m p a r i so n o f e x p r e s s i o n b e t w e e n t h e n e u r o g e n i c

g e n e h o m o l o g u e s i n d i c a t e s p o s s i b l e i n t e r a c t i o n s w h e r e

d i f f e r e n t m N G s a r e c o - e x p r e s se d . F u t u r e w o r k t o d e t e r -

m i n e w h e t h e r t h e p r o t e i n p r o d u c t s d o i n t e r a c t w i l l b ed o n e u s i n g i n v i t r o a n d i n v i v o f u n c t i o n a l s t u d ie s .

4. Experim ental procedures

4.1. PCR of the WD40 domain

Grg c D N A s e n c o d i n g 1 5 3 a m i n o a c i d s f r o m th e

W D 4 0 d o m a i n w e r e a m p l if i e d f r o m a n 1 1 .5 d p c m o u s e

n e u r a l t u b e c D N A l i b r a r y ( g i f t o f P . G r o s ) u s i n g n e s t e d

P C R . F r o m t h e c D N A l i b r a r y , 1 . 2 5 x 1 0 6 p f u w e r e f i r s t

a m p l i f i e d t o y i e l d a 4 9 9 b p f r a g m e n t u s i n g t h e f o l l o w i n g

p ri m er s: 5 '- G C A T C A A G G C T G A G C T G A C G - 3 ' an d 5 '-G C T C C A T A A G G C G T C C T C C A - 3 ' ( O p e r o n T e c h n o l o -

g i e s) . F i v e p e r c e n t o f t h is p r o d u c t w a s t h e n a m p l i f i e d

u s i n g t h e p r i m e r s 5 ' - T C C T C G G C T C C A G C C T G T T A - 3 '

a nd 5 ' - G G C A T F G A G A A G G T T G T C T T - 3 ' , p r o d u ci n g a

4 5 9 b p f r a g m e n t . T h e r e a c t i o n c o n d i t i o n s f o r P C R w e r e :

5 0 m M K C 1 , 1 0 m M T r i s -H C l ( p H 9. 0 a t 2 5 °C ) , 0 .1 %

T r i t o n X - 1 0 0 , 1 .5 m M M g C 1 2, 2 0 0 n M d N T P s , a n d 3 n M

o f e a c h p ri m e r . T h e r e a c t i o n w a s h e a t e d a t 9 4 ° C f o r 5 m i n

p r i o r to t h e a d d i ti o n o f 5 U o f T a q p o l y m e r a s e ( P r o m e g a ) .

T h i r t y P C R c y c l e s c o m p r i s e d o f 1 m i n a t 9 4 ° C , 2 m i n a t

5 0 ° C , a n d 3 m i n a t 7 2 ° C w e r e p e r f o r m e d i n a P e r k i n s -

E l m e r / C e t u s D N A t h e r m a l c y c l e r . A n a d d i t io n a l 7 m i ne x t e n s i o n p e r i o d a t 7 2 ° C w a s i n c l u d e d a t t h e e n d o f c y -

cl ing.

4.2. Isolation of Grg4 cDNAs

D N A i n s e r t c o n t a i n i n g t h e G r g 4 W D 4 0 d o m a i n , o b -

t a i n e d b y P C R a m p l i f i c a t i o n , w a s u se d a s a p r o b e t o

sc r e e n a m o u se 1 1 .5 d p c n e u r a l t u b e l i b r a r y ( g i f t o f D r . P .

Gros) . Ge l -pur i f i ed inse r t was l abe led to a spec i f i c ac t iv -

i t y o f 1 - 5 × 1 0 8 c p m / / z g u s i n g a r a n d o m p r i m e r k i t

( B R L ) . P h a g e w e r e p l a t e d o n t o 2 2 × 2 2 c m P e t r i d ish e s

a n d d u p l i c a te c o p i e s w e r e l i ft e d o n t o H y b o n d - N n i t ro c e l -l u l o se f i lt e rs . T h e f i lt e rs w e r e d e n a t u r e d ( 0 . 5 M N a O H ,

1 .5 M NaC1), neu t ra l i zed (0 .5 M Tr i s -HC 1, 1 .5 M NaC I)

a n d r i n se d i n 2 × S S C ( 1 × S S C i s 1 5 0 m M N a C 1,

1 5 m M so d i u m c i tr a t e, p H 7 ) . F il t e rs w e r e t h e n b a k e d a t

8 0 ° C f o r 9 0 m i n . P r e - h y b r i d i z a t i o n w a s c a r r i e d o u t f o r

1 2 - 1 6 h a t 6 5° C in 4 × S E T - l % S D S (1 × S E T is

1 5 0 m M N aC 1, 3 0 m M T r i s- H C 1 p H 8 , 1 m M E D T A ) .

D e n a t u r e d p r o b e w a s t h e n a d d e d t o a f i n a l c o n c e n t r a t i o n

o f - 1 × 1 0 5 c p r n / m l a n d t h e f i l te r s w e r e h y b r i d i z e d a t

6 5 ° C o v e r n i g h t . T h e y w e r e t h e n w a sh e d i n 0 . 2 × S S C /

1 % S D S a t 6 5 ° C t h r e e t im e s f o r 3 0 m i n a n d e x p o se d t o

X - r a y f i l m . P l a q u e s f r o m r e g i o n s o f p o s i t i v e s ig n a l s w e r e

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86 K.E. Koop et al. / Mechanisms of Development 59 (1996) 73-87

r e - s c r e e n e d u s i n g t h e s a m e c o n d i t i o n s . I s o l a t e d p o s i t i v e

p h a g e c l o n e s w e r e t h e n u s e d f o r e x c i s i o n f r o m t h e l a m b d a

Z A P v e c t o r t o p r o d u c e c D N A i n s e r t s i n t h e p B l u e s c r i p t

v e c t o r ( S t r a t a g e n e ) . I n s e r t s w e r e s e q u e n c e d u s i n g t h e

S a n g e r d i d e o x y n u c l e o t id e m e t h o d w i th T 7 D N A p o -

l y m e r a s e ( P h a r m a c i a B i o t e c h ) .

4.3. Wh ole-mo unt in s i tu hybridizat ion of 7 .5-10 .5 dpcembryos

F V B / N m i c e w e r e m a t ed a n d t h e d ay o f t h e p lu g w a s

t a k e n a s 0 . 5 d p c . E m b r y o s o f 7 . 5 - 1 0 . 5 d p c w e r e d i s s e c te d

i n to P B S a n d f i x e d o v e r n i g h t a t 4 ° C i n 4 % p a r a f o r m a l d e -

h y d e i n P B S . T h e f o l l o w i n g d a y t h e y w e r e p a s s ed t h r o u g h

a g r a d e d m e t h a n o l s e r i e s a n d t h e n s t o r e d i n m e t h a n o l a t

- 2 0 ° C .

T h e p r e h y b r i d i z a t i o n a n d h y b r i d i z a t i o n c o n d i t i o n s f o r

w h o l e m o u n t i n s i t u s w e r e e x a c t l y a s d e s c r i b e d ( W i l k i n -

s o n a n d N i e t o , 1 9 9 3 ) . W a s h e s t h e f o l l o w i n g d a y w e r e

t w i c e f o r 3 0 m i n a t 7 0 ° C i n s o l u t i o n 1 ( 5 0 % f o r m a m i d e ,5 × SSC, pH 4~5, 1% SDS ) , 10 min a t 70°C in a 1 :1 mix

o f s o l u t io n 1 a n d s o l u t io n 2 ( 0 .5 M N a C I , 1 0 m M T r i s -

H C 1 , p H 7 . 5 , 0 . 1 % T w e e n - 2 0 ) , t h r e e 5 m i n w a s h e s i n

so lu t ion 2 , two 30 min incuba t ions a t 37°C in so lu t ion 2 ,

a 5 min wash in so lu t ion 2 , 5 min in so lu t ion 3 (50% for -

m a m i d e , 2 × S S C , p H 4 . 5 ) , t w o 3 0 m i n w a s h e s i n s o lu -

t io n 3 a t 6 5 ° C , t h r e e 5 m i n w a s h e s i n T B S T ( 1 3 7 m M

N aC 1, 2 5 m M T r i s - H C l , p H 7 .6 , 3 m M K C I , 0 . 1%

T w e e n - 2 0 ) a n d a 9 0 m i n b l o c k i n 1 0 % s e r u m / T B S T .

R N a s e A w a s t y p i c a l l y n o t a d d e d t o t h e s o l u t io n 2 w a s h e s

b e c a u s e w e f o u n d t h i s p r o d u c e d a h i g h e r s i g n a l w i t h o u t

i n t r o d u c i n g a b a c k g r o u n d p r o b l e m . E m b r y o s w e r e t h e ni n c u b a t e d o v e r n i g h t a t 4 ° C w i t h a l k a l i n e p h o s p h a t a s e -

c o u p l e d a n t i - d i g o x y g e n i n a n t i b o d y ( B o e h r i n g e r - M a n n -

h e i m ) . T h e a n t i b o d y w a s p r e - b l o c k e d f o r 9 0 m i n a t 4 ° C

w i th 1 3 d p c e m b r y o p o w d e r a n d 1 % s e ru m i n T B S T . A f -

t e r o v e r n i g h t i n c u b a t i o n w i t h a n t i b o d y , e m b r y o s w e r e

w a s h e d t h r e e t i m e s f o r 5 m i n a n d f o u r t im e s f o r 1 h i n

T B S T , a n d t h e n w a s h e d o v e r n i g h t in T B S T . T h e f o l lo w -

i n g d a y e m b r y o s w e r e w a s h e d t h r e e t i m e s f o r 1 0 m i n i n

N T M T ( 1 0 0 m M N aC 1, 1 0 0 m M T r is -H C 1 , p H 9 .5 ,

5 0 m M M g C I 2 , 0 . 1 % T w e e n - 2 0 , 2 m M l e v a m i s o l e ) a n d

t h e n s ta i n e d f o r 2 - 1 2 h i n 3 3 7 . 5 / z g / m l N B T / 1 7 5 / z g / m l

B C I P i n N T M T . A f t e r s t a i n i n g , e m b r y o s w e r e w a s h e d i nP B T a n d s t o r e d in 8 0 % g l y c e r o l / P B T at 4 ° C . T h e y w e r e

p h o t o g r a p h e d u s i n g i l lu m i n a t e d l i g h t o n a L e i c a W i l d

M 3 C d i s s e c t i n g m i c r o s c o p e .

A f t e r p h o t o g r a p h i n g e m b r y o s , t h e y w e r e c r y o p r o t e c t e d

i n 2 0 % s u c r o s e / P B S , t h e n f r o z e n a n d s e c t i o n e d a t 1 0 / z m

o n a L e i t z c r y o s t a t at - 2 0 ° C . S l id e s w e r e d i r e c t ly

m o u n t e d i n 7 % g e l a t i n / 5 0 % g l y c e r i n a n d p h o t o g r a p h e d

o n a Z e i s s A x i o p h o t m i c r o s c o p e w i t h D I C o p t i c s .

4.4. In s i tu hybridizat ion o f emb ryo sect ions

E m b r y o s o f 1 0 . 5 - 1 6 . 5 d p c w e r e d i s s e c te d i n t o P B S

a n d f i x e d o v e r n i g h t i n 4 % P F A / P B S a t 4 °C . T h e n e x t d a y

they were r insed th rough a graded e thanol s e r i e s , xy lene ,

a n d t h e n w e r e p a r a f f i n e m b e d d e d . E m b r y o b l o c k s w e r e

s e c t i o n e d a t 2 0 / ~ m . T h e s l i d e s w e r e p r e - h y b r i d i z e d , h y -

b r i d i z e d , i n c u b a t e d w i t h a n t i b o d y a n d w a s h e d a s d e -

s c r ib e d ( W i l k in s o n a n d N i e t o , 1 9 9 3 ) , e x c e p t t h a t R N a s e A

was no t added to the so lu t ion 2 washes . The a lka l ine

p h o s p h a t a s e s t a in i n g r e a c t i o n w a s a l l o w e d t o p r o c e e d f o r6 h to ove rn igh t . Fo l low ing a r inse in PB S and wa te r ,

s l i d e s w e r e a i r - d r i e d , m o u n t e d i n P e r m o u n t a n d p h o t o -

g r a p h e d o n a Z e i s s A x i o p h o t w i t h D I C o p t i cs .

4.5. Digoxygenin-labeled RNA probes

T h e D N A t e m p l a t e u s e d f o r g e n e r a t io n o f t h e G r g 4

p r o b e w a s t he 4 5 9 b p s e q u e n c e f r o m t h e W D 4 0 d o m a i n

o b t a i n e d b y P C R . G r g 3 a n d G r g 5 p r o b e s w e r e f r a g m e n t s

c o v e r i n g t h e Q d o m a i n ( L e o n e t a l. , 1 9 9 6 ) . E a c h o f t h e s e

G rg p r o b e s d o e s n o t b i n d t o t r a n s c r ip t s o f o t h e r m e m b e r s

o f t h e G rg f a m i l y u n d e r t h e h y b r i d i z a t i o n c o n d i t i o n sused . The H e s l (Sasai e t a l . , 1992) and No t c h l ( R e a u m e

e t a l . , 1992) probes were k ind ly provided by Drs . H .

K a g e y a m a a n d J . R o s s a n t, r e s p e c t i v e l y . D N A s w e r e l i n e -

a r i z e d a n d R N A s y n t h e s i s w a s d i r e c t e d u s i n g T 7 , T 3 o r

S P 6 R N A p o l y m e r a s e , i n c l u d i n g d i g o x y g e n i n - l a b e l e d

n u c l e o t i d e m i x a s r e c o m m e n d e d b y t h e s u p p l i e r s

( B o e h r i n g e r - M a n n h e i m ) . P r o d u c t s w e r e a n a l y z e d o n a

1 % a g a r o s e g el a n d a p p r o x i m a t e l y 1 / ~ g /m l o f th e d i g o x y -

g e n i n - l a b e l e d a n t i s e n s e R N A w a s u s e d f o r h y b r i d i z a t i o n

o f e m b r y o s a n d s l id e s .

Acknowledgements

W e t h a n k D r s . R o g e r J a c o b s a n d A n d r a s N a g y f o r t h e

u s e o f e q u i p m e n t a n d f o r h e l p fu l c o m m e n t s o n t h e m a n u -

sc r ip t , Jod i S tead for s ec t ion ing the whole mount em-

bryos , Cosmo Leon for a s s i s t ance wi th the in s i tu hybr id i -

z a t io n s , D r . P . G r o s f o r t h e e m b r y o n i c n e u r a l t u b e c D N A

l ibra ry , Dr . J . Rossan t fo r the No t c h c D N A a n d D r . R .

K a g e y a m a f o r t h e H e s l an d H e s 3 c D N A s . W e a l s o t h a n k

t h e a n o n y m o u s r e v i e w e r s o f th e m a n u s c r i p t f o r t h e ir v e r y

c o n s t r u c t iv e a n d t h o u g h t f u l f e e d b a c k . T h i s w o r k w a s

s u p p o r t e d b y a n o p e r a t i n g g r a n t f r o m t h e M e d i c a l R e -

s e a r ch C o u n c i l o f C a n a d a . C L i s a s ch o l a r o f t h e M e d i c a lR e s e a r c h C o u n c i l o f C a n a d a .

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