fishing for genes controlling development

8/13/2019 Fishing for genes controlling development

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F i s h in g fo r g e n e s c o n t r o ll in g d e v e l o p m e n tM ichael Gra nato and Chr ist iane NOssle in-VolhardI n r e c e n t y e a r s , t h e z e b r a f i s h h a s b e c o m e a p o p u l a r s y s t e mf o r s t u d y i n g v e r t e b r a t e d e v e l o p m e n t . L a r g e s c a l e m u t a n ts e a r c h e s h a v e l e d t o th e i d e n t if ic a t io n o f > 4 0 0 g e n e s w i thu n i q u e f u n c t i o n s d u r i n g e m b r y o n i c a n d l a rv a l d e v e l o p m e n t . An u m b e r o f t h e s e g e n e s p la y i m p o r t a n t r o l e s in w e l l s t u d i e dp r o c e s s e s , s u c h a s d o r s o v e n t r a l p a t t e r n i n g o f t h e e a r l ye m b r y o , n o t o c h o r d f o r m a t i o n a n d s i g n a l i n g , s o m i t o g e n e s i sa n d n e u r a l s p e c i f i c a t i o n . O t h e r n e w l y i d e n t i f i e d g e n e s o f f e ro p p o r t u n i ti e s t o i n v e s t i g a te l e s s w e l l u n d e r s t o o d p r o c e s s e s ,i n c l u d i n g l o c o m o t i o n b e h a v i o r , o r g a n o g e n e s i s , n e u r a l c r e s td e v e l o p m e n t a n d a x o n a l p a t h f i n d i n g .

A d d r e s s e sMax Planck Inst itOt for Entwicklungs bio log ie, Spem annstrasse 35/11t ,72076 T~bingen G e r m a n y*Present a ddress: Un iversi ty o f Pen nsylvan ia, Depa rtment o f C el l andDeve lopmental Bio logy, Stel lar-Chance, Ph i ladelph ia, Pennsy lvan ia19 104 - 605 8 , US A; e - ma i l: g ran a t o m@ m ai l. med . u p en n .ed uC u r r e n t O p i n io n i n G e n e t ic s D e v e l o p m e n t 1 9 9 6 , 6 : 4 6 1 - 4 6 8: ~ Cu r ren t B io l o g y L t d I S S N 09 59 - 43 7X

I n t r o d u c t i o nP r o f o u n d i n s ig h t s in t o d iv e r s e d e v e l o p m c n t a l p a t h ~ a y so f t h e i n v e r t e b r a t e m o d e l s y s t e m s Drosophila me/ano~astera n d L~/enol~ahditZr eleeans h a v e b e e n a c h i e v e d b y t h em u t a t i o n a l i d e n t i f i c a t i o n o f g e n e s a n d t h e i r s u b s e q u e n tm o l e c u l a r a n a ly s i s . In m a n y i n s t a n c e s , g c n e s w i t h s i m i l a rp h e n o t y p e s f u n c t i o n i n t h e s a m e d e v c l o p m e n t a l p a t h x v a y .l , a r g e s c a l e s c r c c n s h a v e t h e r e f o r e b e e n s u c c e s s f l d ind i s s e c t i n g a n d u n d e r s t a n d i n g v a r i o u s p r o c e s s e s , su c ha s p a t t e r n f o r m a t i o n i n t h e Drosophila e m b r y o , s i g n a lt r a n s d u c t i o n a n d c e l l f a t e s p c c i f i c a t i o n i n Drosophi/a a n d6 . el 4 0 0 0 m u t a n t s w e r e f o u n d[ 13 1. l \ ~o t h i r d s o f t h e s e m u t a n t s s h o w r a t h e r g e n e r a la b n o r m a l i t i e s , s u c h a s r c t a r d a t i o n a n d d e g e n e r a t i o n , a n dw e r e d i s c a r d e d . T o d a t e , 9 00 o f t h e 1 2 00 m u t a n t s i n i t i a ll yk e p t i n t h e T t i b i n g e n s c r e e n h a v e b e e n a s s i g n e d t o 3 71g e n e s , r e s u l t i n g i n an a x e r a g e a l l e l e f r e q u e n c y o f 2 .4m u t a n t s p e r g e n e [ 1 3 ]. T h e n u m b e r o f g e n e s i d e n t i f i e d fo re a c h p h e n o t y p i c c l a s s is g i v c n i n T a b l e 1. In t h e B o s t o ns c r e e n , s o m e w h a t l o x ~e r n u m b e r s w e r e a t t a i n e d w i t h t h es a m e o v e r a l l r e s u l t s .D o r s o v e n t ra l p a t te r n in g : w h o s w h o ?I n a m p h i b i a n s , t h e f o r m a t i o n a n d p a t t e r n i n g o f t hd o r s o v c n t r a l a x i s i n x o l x c s a n u m b e r o f w e l l c h a r a c t e r i z e di n d u c t i o n p r o c e s s e s . I n i t i a ll y , d o r s a l a n d v e n t r a l v e g e t a ls i g n a l s i n d u c e o v e r l y i n g b l a s t o m e r c s t o f o r m d o r s a l a n dx e n t ra l m e s o d e r m r e s p e ct i v e ly . T h e i n d u c e d d o r sa l m e s o -d e r l n , c o r r e s p o n d i n g t o t h e f u t u r e S p e m a n n o r g a n i ze r ,p a t t e r n s a d j a c e n t m e s o d e r m a n d i n d u c e s n e u r o e c t o d e r m .A d d i t i o n a l l y , a v e n t r a l s i g n a l a n t a g o n i z e s t h i s d o r s a l s i g n a la n d s p c c i f i e s v e n t r a l c el l fa t e s. A l t h o u g h n u m e r o u s g e n e sf r o m Xenopus h a v e b e e n i m p l i c a t e d i n t h e s e s i g n a l i n gp r o c e s s e s b y t h e i r e x p r e s s i o n p a t t e r n s a n d t h e i r a c t i v i t i e sw h e n e c t o p i c a l l y e x p r e s s e d i n e m b r y o s , i n m a n y c a s e st h e i r r o le s in t h e e m b r y o h a x e , to d a t e , n o t b e e n t e s t e di n a l o s s - o f - f u n c t i o n s i t u a t i o n .I n t h e z e b r a f i s h s c r e e n , e i g h t g e n e s w e r e i d e n t i f i e dw i t h p h e n o t y p e s t h a t s u g g e s t t h e y p l a y c e n t r a l r o le s


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4 6 2 P a t t e rn f o r m a t io n a n d d e v e l o p m e n t a l m e c h a n i s m s

T a b l e 1G e n e s i d e n t i f i e d i n t h e T [ i b i n g e n s c r e e n : p h e n o t y p i c g r o u p sP h e n o t y p i c S u b g r o u p G e n e s A l l e le s / R e f e r e n c e sg r o u p ( N ) g e n e sEar ly in Ep ib o ly 4 1 .0 [38 ]e m b r y o g e n e s i s E a r ly a r r e s t a n d n e c r o s i s 1 t 1 . 6 [ 3 5 ]B o d y a x e s D o r s a l i s e d 6 3 . 1 [ 3 3 ]

V e n t r a l i s e d 2 2 0 [ 3 4 ]O t h e r s 1 0 1 , 7 [ 3 7 ]P r e c o r d a l p l a t e a n d h a t c h i n g 4 1 . 0 [ 3 7 ]

M e s o d e r m N o t o c h o r d f o r m a t io n 4 2 . 3 [ 1 6 ]N o t o c h o r d d i f f e r e n t i a ti o n 6 7 . 5 [ 1 6 ]U n d u l a t e d n o t o c h o r d 5 2 . 6 [ 1 6 ]D e g e n e r a t i n g n o t o c h o r d a n d 3 1 . 3 [ 1 6 ]

l a t e n o t o c h o r d d e f e c t sS o m i t e f o r m a t i o n 4 4 . 8 [ 1 7 ]S o m i t e p a t t e r m n g 4 1 . 8 [ 1 7 ]

C N S F o r e b r a i n 3 1 . 3 [ 2 3 ]M i d b r a i n - h i n d b r a i n 2 3 . 5 [ 2 4 ]H i n d b r a i n 1 3 2 . 0 [ 2 5 ]B r a i n d e g e n e r a t i o n * 0 0 [ 2 6 ]S p i n a l c o r d 1 2 1 . 8 [ 2 7 ]

O r g a n s B l o o d 1 5 2 . 0 [ 1 5 ]H e a r t m o r p h o l o g y 8 1 . 1 [ 2 9 ]H e a r t b e a t 1 4 1 . 3 [ 2 9 ]C i r c u l a t i o n 2 1 5 [ 2 9 ]L i v er , g u t a n d k i d n e y 6 1 , 0 [ 2 9 ]E y e 9 1 . 3 [ 2 3 ]O t o l i t h s 8 3 . 6 [ 3 ]E a r m o r p h o l o g y a n d l a te r a l 1 3 1 . 5 [ 3 0 ]

l i n e o r g a nF i n s 1 3 4 . 7 [ 3 6 ]S k i n 4 2 . 3 [ 3 6 ]

B o d y s h a p e C u r l y t a i ls 1 3 4 . 4 1 2 7 ]P i g m e n t P ~ g m e n t c e l l n u m b e r a n d 9 3 6 [ 2 1 ]c e l l s p a t t e r n

M e l a n i n p i g m e n t a t i o n 8 3 1 [ 2 1 ]M e l a n o p h o r e d i f f e r e n t i a t io n 7 3 [ 2 1 ]M e l a n o p h o r e s h a p e 1 3 1 . 9 [ 2 1 ]X a n t h o p h o r e s 1 7 3 . 6 [ 2 8 ]I r i d o p h o r e s 7 1 1 [ 2 1 ]T w o c e l l t y p e s a f f e c t e d 1 4 2 9 [ 2 1 ]T h r e e c e l l t y p e s a f f e c t e d 1 0 1 . 5 [ 2 1 ]

J a w a n d F l a t h e a d s a n d p o s t e r i o r a r c h e s 7 1 . 6 [ 3 1 ]g i l l s H a m m e r h e a d s a n d a n t e r i o r a r c h e s 1 2 1 . 8 [ 3 2 ]M o t i l i ty R e d u c e d m o t i l i ty a n d m u s c l e 1 8 2 . 8 [ 2 0 ]

s t r ia t ionR e d u c e d m o t i li ty a n d n o r m a l 1 5 1 . 7 [ 2 0 ]s t r ia t ion

A d u l t s B o d y s h a p e 4 1 . 0 ( a )P i g m e n t p a t t e r n 3 2 . 7 ( a )Eyes (a )Fins 4 1 (a)P i g m e n t a t i o n ( a )

R e t rn o - P a t h f i n d i n g 4 1 . 8 [ 1 9 }t e c t a l M a p p i n g 7 3 . 3 [ 1 8 ]T o t al 3 7 1 2 4

T h i s c l a s s o f m u t a n t s w a s n o t k e p t s y s t e m a t i c a l l y [ 2 6 ] . ( a ) C N 0 s s l e l n - V o l h a r d et alu n p u b l i s h e d d a t a . N , n u m b e r o f i d e n t i f ie d g e n e s

i n b o th d o r s m c n t r a l p a t t e r n i n g o f t h e mcsodcrm iidn e u r a l i n d u c t i o n . \ h i t a t i o n s i n d / n o a n d i m v r , r / r ~ r c s t t l ti n a v c n t r a l i z a t i o n o f t h e b l a s t u la , m e a n i n g t h a t x c n t r a lf a t e s , x~ h ic h c o n t r i b u t e t o m o r e p o s t e r i o r s t r t t c t u r e s i n t h ee m b r y o , a r c e x p a n d e d ; i n c o n t r a s t , d o r s a l f a t e s , gi vin


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Fishing or gen es controllingdevelopment Granatoand NOsslein Volhard 46 3

l , i n k a g c a n a l y si s b e t w c c n t h e s e g e n e s a n d t h c m u t a t i o n sw i ll r e v e a l i f a n x o f th e s e c l o n e d g c n c s c o r r e s p o n d t o t h eg e n c s d c s c r i b c d h c r c .

A x i s f o r m a t i o n a n d s i g n a l in gD u r i n g g a s t r u l a t i o n , d e r i v a t i v e s o f t h c t h r e e g e r m l a v c rsb e c o m e a r r a n g e d i n a p a ra l l e l n r d e r a l o n g t h e a n t e r o -p o s t e r i o r a x is , o u t l i n i n g t h c b a s i c b o d y p l a n o f th ev c r t c b r a t c e m b r y o . T h e p r e c h o r d a l p l a t e in t h e h e a d a n dt h c n o t o c h o r d i n th c t r u n k ( a x i a l m c s o d e r m ) o c c u p x ac e n t r a l p o s i t i o n a n d t h u s c o n t a c t t i s s u e s o f a l l g e r m l a y er s :t h e n e u r a l p l a t e ( n e u r o e c t o d e m l ) , t h e s o m i t e s ( p a r a x ia lm c s o d e r m ) a n d t h e g u t ( e n d ( ) d e r m ) . T h e n o t o c h o r d i sr e q u i r e d f i) r t h e s p e c i f i c a t i o n a n d m a i n t e n a n c e o f c e l lf a t e s in t h e s o m i t e s , a n d t h e i n d u c t i o n o f t h e f l o o r p l a t ea n d m o t o r n e u r o n s i n t h e x e n t r a l s p in a l co r d ( r e v i e w e d i n[ 6 0, 61 ] ). T h e m u t a n t p h e n o t y p e s o f a n u m b c r o f z c b r a f i shg e n e s r c f e c t t h i s c e n t r a l r o le o f t h e n n t o c h o r d i n t h ep a t t e r n i n g o f a d j a c e n t t i ss u e s a n d s u g g e s t s t h a t t h e g e n e sm a y r c p r c s c n t c n m p o n c n t s o f a c o m p l e x n c t ~ o r k b y w h i c ht h c a x i al m c s o d c m l c x c r t s d i f f e re n t p a t t e r n i n g i n f lu c n c c so n t h e s u r r o u n d i n g t i s s u e s .W h e r e a s m u t a t i o n s i n t h e f l o a t i n g k e a d , m o m o , n o t a i /nd d o t g e n e s r e q u i r e d f o r n o t o c h o r d f o r m a t i o n a l s o

a f f e c t p a t t e r n i n g o f th e s o m i t e s , m u t a t i o n s m 1 3 o t h e rg e n e s d i s p l a y d e j e c t s i n t h e s o m i t e s a n d / o r t h c n e u r a lp l a t e b u t n o t i n t h c n o t o c h o r d , f h c s c g e n e s c o m p r i s es m a l l g r o u p s w i t h c o m m o n p h e n o t y p i c f ' ca t u rc s , a l t h o u g hi n d i x i d u a l p h e n o t y p e s m a y i n c l u d e a d d i t i o n a l d e f e c t s .C e l l t ra n s p l a n t a ti o n e x p e r i m e n t s , d e s i g n e d t o d e t e r m i n et h e t i s s u e s i n w h i c h i n d i v i d u a l g e n e s a r e r e q u i r e d , a l l o wo n e t o a s s i g n s p e c i f i c ro l e s tB r t h e s e g e n e s . I n s e v e r a li n s t a n c c s , p h e n o t y p e s a r e r e s t r i c t e d t o s u b r c g i n n s o ft h e a n t e r o p o s t e r i o r ax i s, s u g g e s t i n g d i f f e r e n c e s i n t h ep a t t e r n i n g o f h e a d , t r u n k , a n d t a i l [ 16 , 2 7] .T h e n o t o c h o r d i s l a c k i n g in e m b r y o s m u t a n t f i) r m o m oo r f l o a t i n g k e a d . A l t h o u g h a x i al m c s o d c r m a l c e l ls e x p r c s sn o t o c h o r d m a r k e r s i n i t i a ll y , t h e y s o o n s w i t c h t h e i r f a t e t ob e c o m e p a r a x ia l m e s o d e r m [ 1 6, 49 , 62 ,6 3 ]; t h i s s u g g e s t s ar o le f o r b o t h g e n e s i n t h c s p e c i f i c a t i o n o f a x i al m c s o d e r m a lc e l l s . I n n o t a i / a n d d o c m u t a n t s , n o t o c h o r d p r e c u r s o r sa r e p r e s e n t b u t f a il to d i f f e r e n t i a t e [ 16 ]. A l t h o u g h t h cp a t t e r n i n g o f t h e v e n t r a l n c u r a l t u b e i s d i s r u p t e d i nt h e a b s e n c e o f t h e n o t o c h o r d i n m o m o a n d f l o a t i n g k e a dm u t a n t s , t h c u n d i f f e r e n t i a t e d n o t o c h o r d i n n o t a i / a n d d o cm u t a n t s i s s u f f i c i e n t fo r fl o o r p l a t e in d u c t i o n . I n m u t a n t so f al l f o u r g e n e s , h o w e x c r , a b s c n c e o f a d i f f c r c n t i a t e dn o t o c h o r d a f f e c ts i n d u c t i o n o f a s p e c i a l i s e d c l a ss o f m u s c l ec c l l s a n d p a t t e r n i n g o f t h e s o m i t c s i n t o a d n rs a l an d ax e n t r a l p a r t [ 1 6 , 6 3 , 6 4 ] ._ M u t a t i o n s i n s i x l o c i - - c t / a m a e / e o n , y o u - t o o , so n i c y o u ,y ou , u -hoot , a n d ~ / ) o l w - - c a u s c s i m i la r d e je c t s i n t h es o m i t e s b u t n o t o c h o r d d e x e l o p m e n t i s u n a f f e c t e d [ 17 ].C e l l t r a n s p l a n t a t i o n c x p c r i m e n t s i n d i c a t e t h a t s o m i t c

p a t t c r n i n g is d e p e n d e n t o n t h e w i l d - t y p e f u n c ti o n o f~tou t in kern/, no rail, a n d d o c i n t h e n o t o c h n r d , w h e r e a sy o u - t o o a n d a - h o o t a r e r e q u i r e d a u t o n o m o u s l y i n t h es n m i t c s . M u t a t i o n s i n s e v e n o t h e r g e n e s a f f e c t fl n o r p l a t ea n d m o t o r n e u r o n d e x c l o p m e n t i n t h e v e n t r a l s p i n a l c o r d ,w i t h o u t o b v i o u s d e f e c t s i n t h e n o t o c h o r d a n d s o m i t e s[ 2 7 ] . I n p a r t i c u l a r , s c k m a / s p a l , a s w i t h o u e - e v e d p i n k e a d a n dt h e p r e v i o u s ly d e s c r i b e d O c/ops m u t a n t s , l a c k a f l o o r p l a t c[ 7, 27 ,3 7 ] w h e r e a s m a i n t e n a n c e b u t n o t f o r m a t i o n o ft h e f l o o r p l a t e is a f f e c t e d i n m u t a n t s o f detour; igaana,s ~ m a / h a n s a n d m o n o r a i l , s u g g e s t i n g t h a t t h e s e g e n e sf u n c t i o n t o e s t a b l i s h o r t o m a i n t a i n p r o p e r o r g a n i z a t i o no f th e z e b r a f i s h m i d l i n e i n t h c b r a i n a n d s p i n a l c o r d[2 71 . T h e s e g e n e s m a y e n c o d c s i g n a l s e m a n a t i n g f r o mt h e n o t o c h o r d o r a l t e r n a t i v e l y t h e y m a y b c i n v o l v e d i nr c c c i v i n g a n d i n t e r p r e t i n g t h e s i g n a ls i n t h e v e n t r a l s p i n a lc o r d a n d t h c s o m i t e s . A p r i m e c a n d i d a t e f o r a n i n d u c t i x es i g n a l p r o d u c e d b y t h c n o t o c h o r d i s t h e s e c r c t e d S o n i ch e d g e h o g m o l e c u l e ( r e v i e w e d i n [ 6 5 ] ) , a n d g e n e t i c l i n k a g ea n a ly s is b c t w e c n S o n i c h e d g e h o g a n d g e n e s r e q u i r e df or so m i t c p a t t c r n i n g a n d f n o r p l a t c d e v e l o p m e n t i s i np r o g r e s s .G e n e t i c s o f o r g a n o g e n e s i s a n d l o c o m o t i o nb e h a v i o r : g e t t in g o f f t h e g r o u n d ?( ) u r u n d e r s t a n d i n g of t h e g e n c t i c c o n t r o l o f p r o c e s s e so c c u r r i n g l a t e i n v e r t e b r a t e c m b r y o g e n e s i s - - s u c h a si n n e r o r g an f o r m a t i o n , p a t t e r n i n g o f t h e h c a d a n d t h eb r a in , g c n c r a t i o n o f p i g m c n t c e l l p a t t e r n s , s e n s o r y o r g a nd e x e l o p m e n t , f o r m a t io n a n d g r o w t h o f p a i r e d a p p e n d a g e sa n d e s t a b l i s h m c n t o f n e u ra l c i r c u it s u n d c r l y i n g s i m -p l e l oc o m o t i o n b e h a v i o r - - a r c l cs s w e l l ad x a n c e d . S u c he v e n t s t a k e p l a c e w i t h i n t h e f i r s t s ix d a y s o f ze b r a f i s hd e v e l o p m e n t a n d c a n d i r e c tl y b c o b s e r v e d i n l ix e e m b r y o so r la r v ae . A p p r o x i m a t e l y h a l f o f t h e g e n e s i d e n t i f i e d i nt h e f 0 b i n g e n s c r e e n a f f e c t t h c s c l a t e p r o c c s s e s ( T a b l e 1 )[ 1 5 , 2 ( t , 2 1 , 2 3 - 2 6 , 2 9 - 3 2 , 4 2 - 4 8 ] . I n t h i s s e c t i o n , w e d i s c u s st h e p o t e n t i a l o f g e n e s w i t h f u n c t i o n s i n i n t c r n a l o r g a nf o r m a t i o n a n d l o c o m o t i o n b e h a x i o r a s e x a m p l e s .( ) r g a n o g e n e s is i n v o lv e s m a n y d e v e l o p m e n t a l m e c h a n i s m s ,i n c l u d i n g i n d u c t i o n , c e l l t y p e d i f f e r e n t i a t i o n a n d m o r -p h n g c n e s is . M u t a n t p h e n o t y p e s a r c u s e fu l b e c a u s e t h e yf a c i l it a t e t h e d i s s e c t i o n o f c o m p l e x e v e n t s i n t o i n d i v i d u a ls t e p s . ( ) f p a r t i c u l a r i n t e r e s t i s a c o l l e c t i o n o f m u t a n tp h e n o t y p c s c o v e r i n g a b r o a d r a n g e o f e v e n ts d u r i n gc a r d io v a s c u la r d e v e l o p m e n t a n d f u n c ti o n . T h e s e d e f i n ea t l ea s t 9 g e n e s r e q u i r e d f o r h e m a t o p o e s i s [ 15 ,4 3 ] a n d2 5 g e n e s ~ i t h f u n c t i o n s i n t h e v a r i o u s c o m p o n e n t s o ft h e c a r d i m a s c u l a r s y s t e m , i n c l u d i n g f u s i o n o f t h e t w ob i l a t e r a l h e a r t p r i m o r d i a , c h a m b e r f o r m a t i o n , l o o p i n g ,m a t r i x d e v e l o p m e n t a n d s i z in g d u r in g h e a r t m o r p h o -g e n e s i s , c o n t r a c t i l i ty a n d r h y t h m o f h e a r t f u n c t i o n , a n dv e s s e l f o r m a t i o n [ 2 9 ,4 2 ] ( F i R I N a n d N ' - O a n d O ' ) .M a n \ o f t h e s e g e n e s w i l l b e v a l u a b l e in u n d e r s t a n d i n gc a r d i o v a s c u l a r m a l f u n c t i o n s i n h u m a n s , a s t h e m u t a n tp h e n o t y p e s r e s c i n b l e h u m a n c o n g e n i t a l c a r d io v a s c u la rd i s e a s e s [ 3 9 ] .


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4 6 4 P a t t er n f o r m a t i o n a nd d e v e l o p m e n t a l m e c h a n i s m s

F i g u r e


5/8

F i s h i n g f o r g e n e s c o n t r o l li n g d e v e l o p m e n t G r a n a t o a n d N 0 s s le in - Vo l ha r d 4 6 5

F i g u r e 1

Zebra f ish mutants . A) Sch em at ic o f ep ibo ly . St ruc ture o f the embryo a t h igh b las tu la s tage ( le f t) and 7 0% ep ibo ly . (e , ep ib las t ; h , hypoblas t ;ev l , enve lop ing layer ; ys l , yo lk sync it ia l l ayer .) (B) W i ld - typ e an d (B ' ) d i n o m u t a n t a t 7 0 % e p i b o l y , g o o s e c o i d (gsc ) and e v e mRNAs loca l i za t ion ,a n i m a l p o l e v i e w , d o r s a l r i g h t . ( C ) W i l d - t y p e a n d ( C ' ) s w i r l mutant a t 10 somi te s tage , s ta ined w i th Pax 2 (mb, midbra in ; p , pronephros; op ,opt ic ves ic le ; o t , o t ic ves ic le ) . (D) W i ld - type , (D ' ) f lo t i n g h e d a n d ( D ) m o m o mutants a t the somi te s tage , s ta ined w i th ant i n t l (b row n) andMyo D (b lue ) . (1= ) Ske le ta l s ta in ing o f 6 m onth o ld w i ld -type and ( l= ' ) f u s e d s o m i te s mutant f i sh (ha , hemo-arch; c t , cent ron; na , neura l a rch) .( F ) L a t e r a l v i e w o f a 2 4 h o u r o l d w i l d t y p e a n d ( F ' ) s l e e p y mutant . (G ) Ant ibody s ta in ing v isua l iz ing the musc le p ioneer ce l ls in w i ld type and( G ' ) y o u m u t a n t e m b r y o s . ( N ) Z o t x - 2 s t a in i n g i n w i l d ty p e a n d ( H ' ) m s t e r b li n d mutants . ( I ) Z n5 ant ibody s ta in ing o f the opt ic nerves in w i ldtype and (1 ' ) d e t o u r m u t a n t s . W i l d - t y p e ( 1 , K ) a n d y u g i r i mutant em bryos ( I 'K ' ) us ing l ight microscopy in (J , J ' ) o r acr id ine orang e (AO ) s ta in ingfor ce l l dea th in (K, K ' ) . (L ) Wi ld - type and w h i t e t i l (L ' ) m utant embryo s ta ined w i th 3A 10 ant ibody (Ro12 , ident i fi ed neuron in h indbra in ; m,Mau thne r ce l l) . Express ion o f O tx -1 in the o t ic ves ic le o f w i ld - type (M ; a rr ow ) and a bse nce o f express ion in v n g o g h ( M ' ) m u t a n t s. L i v e i m a g e s( N , N ' ) a n d s a g i tt a l s e c t io n s ( O , O ' ) o f w i ld - ty p e h e a r ts ( N , O ) a n d s n t (N ' , O ' ) m utants a t tw o days o f deve lopment (a , a t r ium; v , vent r ic le ;o , out f lo w t rac t ) . Ant ibody s ta in ing o f (P) w i ld - type and (P ' ) u n p l u g g e d mutant embryos (M IP, midd le pr imary motoneuron; CIP , cauda l pr imarymotoneuron) . Car t i l age s ta ins o f day 5 (O .) w i ld - type and (O ' ) s c h m e r l e m u t a n t l a r v a e . ( R ) W i l d - t y p e a n d ( R ' ) n e v e r m i n d mutant la rvae a t day 5of deve lopmen t , in jec ted w i th Di l (g reen) a nd D iO ( red) to v isua l i ze the ir re t inotec ta l pro jec t ions .

Zebrafish larva display distinct, stage-specific patterns ofl o c o m o t i o n b c h a v i ( m p r o v i d i n g a n i n t e r e s t i n g p a r a d ig m t os[tid\ the contr()l of motor systems in vertebrates. Rcccntprogress in optical mon itori ng of ncural activity [00 ] antithe xvcll stud ied ncu roa rch itc ctu rc of thc llerv()tls S\rStCl]l[~>7--73] allm~ us to c te tcrm inc i , c ' ic 'o thc gross bchavioralfunction of identified ncnro ns in zcbrafish. Th e identifica-tion of >48 gc nc s-- wh ic h funct ion in dis tinct locomotionpatterns, such as touch response, eq uili bri um control andrhythmic tail n/o~gical components (e.g. nicotinic recep-tor [74])and defects in axonal elongation or pathfindin


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4 6 6 P a t te r n f o r m a t i o n a n d d e v e l o p m e n t a l m e c h a n i s m s

thcv may be parallclcd b\ rather gcncral detects suchas retardation or degen erati on obscrxcd in approxima tclytwo thirds of all mutan ts identified . Thes e t\vo reasons canin part explain why the degree of saturation varics amon gdifferent phcnotxpic classcs.T h e ' l ' i i b i n g e n a n d B o s t o n s c r e e n s h a ~ c f l~ c us e d o n p h c -n o t y p c s t h a t a r c x i s i b l c u n d e r a s t e r e m i c r o s c o p e b u t m o r es p c c i a l i z c d s c r e e n s u s i n g p a r t i c u l a r a s s a y s a r c f 'c a s i b l c . I : o rc x a m p l e , i n c o n j u n c t i o n w i t h t h c m o r p h o l o g i c a l s c r e e ni n T t i b i n g e n , m u t a t i o n s a f f e c t i n g r e t i n o t e c t a l p r o j e c t i o nh a v e b c c n i s o l a t e d b y a n t e r o g r a d c l a b e l i n g o f a x o n sw i t h f l u o r c s c e n t d y e s [22] G e n e t i c s c r e e n s d c s i g n e d t oi d e n t i f \ c h a n g e s i n c x p r c s s i o n p a t t c r n s b y x ~ ho lc r e tr o ti v s i t u o r b y a n t i b o d x l a b e l i n g a rc i n p r o g r e s s an d s h o u l du n c m c r g c n e s w i t h m o r e s u b t l c p h e n o t y p c s [ 79 ] , O n cs h ( m l d k c c p i n m i n d , h o x ~ e v c r , t h a t a l a r g ; e n u m b c ro f m u t a g c n i z e d f is h f a m i l i es a rc n e e d e d t o a c h i e v c ar e a s o n a b l e d e g r e e o f s a tu r a t i o n f o r a n y g i v c n p h e n o t y p i ct r a i t .T h e z e b r a f is h g e n e s i d e n t i f i e d i n t h e s c r e e n s p c r f l~ r m e d i nT f i bi n ~ 4 cn a n d B o s t o n f l ) rm a f o u n d a t i o n f l) r s u b s e q u e n td e t a i l e d a n a l y s i s o f a w h o l e v a r i e t y o f d e v e l o p m e n t a lp r o c e s s e s , s o m e o f t h e m s p e c i f ic to v e r t e b r a t e s . T h ew e l l d e s c r i b e d c x p e r i m c n t a l a d v a n t a g e s o f t h e z e b r a f i s he m b r y o s t o g e t h e r w i t h t h e a v a i l a b i l i t y o f l i n k a g e m a p so f t h e ~ c n o m c [ 8 0 - 8 2 ] , t h e a b i l i t y to fl, n c r a t e t r a n s g c n i cl i n e s [ 8 3 , 8 4 - 8 8 ] , r e c e n t a d v a n c e s i n p o s i t i o n a l c l o n i n ~t e c h n i q u e s a n d s t r a t e g i e s , a n d o n g o i n g p r o g r e s s i n ~ , c n e r -a t i n g ~ i r u s - m e d i a t c d i n s e r t i o n a l m u t a g c n e s i s [ 84 ] s h o u l dr a p i d l y e x t e n d o u r k n o x~ l ed g c o f v e r t e b r a t e d e v e l o p m e n t .

c k n o w l e d g e m e n t s\ V e w o u l d l i k e t o t h a n k C P H c i s c n b c r e , , J ( ) d c n t h a l , I : V E c d c n , KRossna~cl, I) Gihnour, S Jcsuthasan, XI \lullins, T Nit isn, R Sommcr,J S c h o o l f o r m a n 3 h e l p f u l s u g g e s t i o n s o n t h e m a n u s c r i p t .

R ef e r en ces an d r eco m m en d ed r ead i n gP a p e r s o f p a r t i c u l a r i n t e r e s t , p u b l i s h e d w i t h i n t h e a n n u a l p e r i o d o f r e v i e w ,h a v e b e e n h i g h l i g h t e d a s :

o f s p e c i a l i n t e r e s t, ,. o f o u t s t a n d i n g i n t e r e s t1

2.3 .

4

5 .6 .

S t Johns ton D , NL iss l e in -V o l hard C : T h e o r i g i n o f p a t t e r n a n dp o l a r i t y in the D r o s o p h i l a e m b r y o . C e i l 19 9 2 , 6 8 : 2 0 1 - 2 1 9 .C h a l f i e M : T o u c h r e c e p t o r d e v e l o p m e n t a n d f u n c t i o n i nCaenorhabdi t i s e legans . J Neurobio l 1 9 9 3 , 2 4 : 1 4 3 3 - 1 4 4 1 .P a r k h u rs t S M , M e n e e l y P M : S e x d e t e r m i n a t i o n a n d d o s a g ec o m p e n s a t i o n : l e s s o n s f r o m f l i e s a n d w o r m s . Science 1 9 9 4 ,2 6 4 : 9 2 4 - 9 3 2 .K a y n e P S , S t e r n b e r g P W : R a s p a t h w a y s i n C e l e g a n s Cur tOpin Genet Dev 1 9 9 5 , 5 : 3 8 - 4 3 .K i m m e l C B : G e n e t i c s a n d e a r l y d e v e l o p m e n t o f z e b r a f i s h .ends Genet 1 9 8 9 , 5 : 2 8 3 - 2 8 8 .H a l p e r n M E , H R K , W a l k e r C , K i m m e l C B : I n d u c t i o n o f m u s c l ep i o n e e r s a n d f l o o r p l a t e I s d i s t i n g u i s h e d b y t h e z e b r a f i s hno - ta f t m u t a t i o n . Cei l 1 9 9 3 , 7 5 : 9 9 - 1 1 1 .H a t t a K , K i m m e l C B , H R K , W a l k e r C : T h e c y c l o p s m u t a t i o nb l o c k s s p e c i f i c a ti o n o f t h e f l o o r p l a t e o f t h e z e b r a f i s h c e n t r a ln e r v o u s s y s t e m . Nature 1 9 9 1 , 3 5 0 : 3 3 9 - 3 4 1 .

8 . K i m m e l C B , K a n e D A , W a l k e r C , W a r g a R M , R o t h m a n M B : Am u t a t i o n t h a t c h a n g e s c e l l m o v e m e n t a n d c e l l f a t e i n t h ez e b r a f i s h e m b r y o . Nature 1 9 8 9 , 3 3 7 : 3 5 8 - 3 6 2 .9 . Mu l l i ns MC , NL~ss l e in -V o l hard C : M u t a t i o n a l a p p r o a c h e s s t u d y i n ge m b r y o n i c p a t t e r n f o r m a t i o n i n t h e z e b r a f i s h . Curr Opin GenetDev 1 9 9 3 , 3 : 6 4 8 - 6 5 4 .1 0 . D r i e v e r W , S t e m p l e D , S c h i e r A , S o l n i c a - K r e z e l L : Z e b r a f i s h :

g e n e t i c t o o l s f o r s t u d y i n g v e r t e b r a t e d e v e l o p m e n t . TrendsGenet 1 9 9 4 , 5 : 1 5 2 - 1 5 9 .1 1 . M u l l i n s M C , H a m m e r s c h m i d t M , H a f f t e r P , N 0 s s l e i n - V o l h a r d C :L a r g e - s c a l e m u t a g e n e s i s i n th e z e b r a f i s h : i n s e a r c h o f g e n e sc o n t r o l l i n g d e v e l o p m e n t i n a v e r t e b r a t e . Cur r B i o l 1 9 9 4 ,4 : 1 8 9 - 2 0 2 .12. S o l n i c a - K r e z e l L , S c h i e r A F , D r i e v e r W : E f f i c ie n t r e c o v e r y o fE N U - i n d u c e d m u t a t i o n s f r o m t h e z e b r a f i s h g e r m l i n e . Genetics1 9 9 4, 1 3 6 : 1 4 0 1 - 1 4 2 0 .1 3 . H a f f t e r P , G r a n a t o M , B r a n d M , M u l l i n s M C , H a m m e r s c h m i d t M ,K a n e D A , O d e n t h a l J , V a n E e d e n F J M , J i a n g Y - J, H e i s e n b e r g C - Pet aL: T h e i d e n t i f i c a t i o n o f g e n e s w i t h u n i q u e a n d e s s e n t i a lf u n c t i o n s i n t h e d e v e l o p m e n t o f t h e z e b r a f i s h D a n i o rer io .Development 1 9 9 6 , i n p r e s s .1 4 . D r i e v e r W , S o l n ic a - K r e z e l L , S c h i e r A F , N e u h a u s s S C E M a l i c k i J ,S t e m p l e D L , S t a i n i e r D Y R , Z w a r t k r u i s F , A b d e l i la h S , R a n g i n i Zet aL: A g e n e t i c s c r e e n f o r m u t a t i o n s a f f e c t i n g e m b r y o g e n e s i s

in zebraf ish. Development 1 9 9 6 , i n p r e s s .1 5 . R a n s o m D G , H a f f t e r P , O d e n t h a l J , B r o w n l i e A , V o g e l s a n gE , B r a n d M , V a n E e d e n F J M , F u r u t a n i - S e i k i M , G r a n a t o M ,H a m m e r s c h m id t M e t al.: C h a r a c t e r i z a t io n o f z e b r a f i s h m u t a n t sw i t h d e f e c t s i n e m b r y o n i c h e m a t o p o i e s i s . Development 1 9 9 6 , i np r e s s .1 6 . O d e n t h a l J , H a f f t e r P , V o g e l s a n g E , B r a n d M , V a n E e d e n F JM ,F u r u t a ni -S e i k i M , G r a n a t o M , H a m m e r s c h m i d t M , H e i s e n b e r gC-P , J i ang Y -J et aL: M u t a t i o n s a f f e c t i n g t h e f o r m a t i o n o f t h en o t c h o r d i n t h e z e b r a f i s h Da nio rer io . Development 1 9 9 6 , i np r e s s .1 7 . V a n E e d e n F J M , G r a n a t o M , S c h a c h U , B r a n d M , F u r u t a n i - S e i k i M ,H a f f l e r P , H a m m e r s c h m i d t M , H e i s e n b e r g C - P , J i a n g Y - J , K a n e D Aet aL: M u t a t i o n s a f f e c t i n g s o m i t e f o r m a t i o n a n d p a t t e r n i n g i nt h e z e b r a f i s h Da nio rer io . Development 1 9 9 6 , i n p r e s s .1 8 . T r o w e T, K l o s t e r m a n n S , B a i e r H , G r a n a t o M , C r a w f o r d A D ,G r u n e w a l d ' B , H o f f m a n n H , K a r l s tr o m R O , M e y e r S U , R i c h te r

S e t a l : M u t a t i o n s d i s r u p t i n g t h e o r d e r i n g a n d t o p o g r a p h i cm a p p i n g o f a x o n s i n t h e r e t i n o t e c t a l p r o je c t i o n o f th ez e b r a f i s h D a n i o rerio. Development 1 9 9 6 , i n p r e s s .1 9 . K a r l s t ro m R O , T r o w e I , K l o s t e r m a n n S , B a i e r H , B r a n d M ,C r a w f o r d A D , G r u n e w a l d B , H a f f te r P, H o f f m a n H , M e y e r S U e t aL:Z e b r a f i s h m u t a t i o n s a f f e c t i n g r e t i n o t e c t a l a x o n p a t hf in d in g .Development 1 9 9 6 , i n p r e s s .2 0 . G r a n a t o M , V a n E e d e n F J M , S c h a c h U , T r o w e T , B r a n d M ,F u r u t a n i - S e ik i M , H a f f t e r P, H a m m e r s c h m i d t M , H e i s e n b e r g C - RJiang Y-J et al.: G e n e s c o n t r o l l i n g a n d m e d i a t i n g l o c o m o t i o nb e h a v i o u r o f t h e z e b r a f i s h e m b r y o a n d l a r v a . Development1 9 9 6 , i n p r e s s .2 1 . K e l s h R N , B r a n d M , J i a n g Y - J , H e i s e n b e r g C - P , L i n S , H a f f t e r P ,O d e n t h a l J , M u l l i n s M C , V a n E e d e n F J M , F u r u t a n i - S e i k i M e t aL:Z e b r a f i s h p i g m e n t a t i o n m u t a n t s a n d t h e p r o c e s s e s o f n e u r a lc r e s t d e v e l o p m e n t . Development 1 9 9 6 , i n p r e s s .22 . B a i e r H , K l os te rman n S , Trow e T, K ar l s t r om RO, NL i ss le i n -

V o l h a r d C , B o n h o e f f e r F : G e n e t i c d i s s e c t i o n o f t h e r e t i n o t e c t a lprojection. Development 1 9 9 6 , i n p r e s s .2 3 . H e i s e n b e r g C - P , B r a n d M , J i a n g Y - J , W a r g a R M , B e u c h l eD , V a n E e d e n F J M , F u r u t a n i - S e ik i M , G r a n a t o M , H a f f t e rP , H a m m e r s c h m i d t Met a l . : G e n e s i n v o l ve d i n f o r e b r a i nd e v e l o p m e n t i n t h e z e b r a f i s h D a n i o rerio. Development 1 9 9 6 ,i n p ress .2 4 . B r a n d M , H e i s e n b e r g C o P , J i a n g Y - J , B e u c h l e D , F u r u t a n i - S e ik i M ,G r a n a t o M , H a f f t e r R H a m m e r s c h m i d t M , K a n e D A , K e l s h R N :M u t a t i o n s i n z e b r a f i s h g e n e s a f f e c t i n g t h e f o r m a t i o n o f t h eb o u n d a r y b e t w e e n m i d b r a i n a n d h i n d b r a i n . Development 1 9 9 6 ,i n p ress .2 5 . J ia n g Y - J , B r a n d M , H e i s e n b e r g C - P , B e u c h l e D , F u r u t a n i - S e i k i M ,K e l s h R N , W a r g a R M , G r a n a t o M , H a f f t e r P , H a m m e r s c h m i d t M

e t a l : M u t a t i o n s a f f e c t in g n e u r o g e n e s i s a n d b r a in m o r p h o l o g yi n th e z e b r a f i s h D a n i o rerio. Development 1 9 9 6 , i n p r e s s .2 6 . F u r u t a n i - S e i k i M , J i a n g Y - J , B r a n d M , H e i s e n b e r g C -P , H o u a r t C ,B e u c h l ~ D , V a n E e d e n F J M , G r a n a t o M , H a f f t e r P , H a m m e r s c h m i d t


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M, Granato M, Haffter R Ham merschmidt M, Heisenberg C-P,Jiang Y-J et aL: The z eb ra f i s h ea r l y a r res t mu tan t s . Development1996, in press.36. Van Eeden FJM, Gran ato M, Schach U, Brand M, Furutani-Seik i M,Haffte r P, Hammerschmidt M, Heisenberg C-P, J iang Y-J. Kane DAet al.: G ene t i c ana l y s i s o f f in f o rm a t i on i n the z e b ra f i s h , D a n i orerie. Development 1996, in press.37. Hamm erschmidt M, Pelegri E Mul l ins MC, Kane DA, Brand M, VanEeden FJM, Furutani-Seik i M, G rana to M, Ha ffter P, HeisenbergC-P : Mu ta t i ons a f f ec t i ng morpho gene s i s du r i ng gas t ru l a t i onand t a i l f o rma t i on i n the z eb ra f i s h , Dan io rer io . Development1996, in press.38. Kane DA, Hamm erschmidt M, Mul l ins MC, Maischein H-M,Brand M , Van Eeden FJM, Furutani-Seik i M, Gran ato M, HaffterP, Heisenberg C -P et al.: The z eb ra f i s h ep i bo l y mu tan t s .Development 1996, in press.39. Weinstein BM, Stemple DL, Driever W, Fishman MC : gr id lock al o c a l i z ed he r i t ab l e v as c u l a r pa t t e rn i ng de fec t i n t he z eb ra f i s h .N a t M e d 1995 , 1 : 1143 -1147 .40. Broc kerh off SE, Hurley JB, Janssen-Bienhold U. NeuhaussS, Dr iever W, Dowl ing J : A behav iora l sc reen for i so la t ingz eb ra f i s h mu tan t s w i t h v i s ua l s y s tem de fec t s . Proc Nat l AcadSc i USA 1 9 95 , 9 2 : 1 0 5 4 5 - 1 0 5 4 9 .41. Mal icki J, Neuhauss SCF, Sch ier AF, Solnica-Krezel L, StempleDL, Stainier DY R, Abdel ilah S, Zwartkruis F, Rangini Z. DrieverW : M u ta t i ons a f f ec t ing dev e l opm en t o f the z eb ra f i s h re t ina .Development 1996, in press.42. Stainier DY R, Fouquet B, Che n JN, Warren KS, Weinstein BM,Mei ler S. Mohideen MPK, Neuhauss SCF, Solnica-Krezel L, SchierAF et aL: Muta t i ons a f f ec t i ng t he f o rma t i on and f unc t i on o f t hec a rd i ov as c u l a r s y s tem i n t he z eb ra f i s h embry o . Development1996, in press.43. Weinstein BM, Sch ier AE Abdel ilah S, Mal icki J, Solnica-Krezel L,Stemple DL, Stainier D YR , Zwartkruis F, Driever W, Fishman MC :

Hema topoe t i c mu ta t i ons i n t he z eb ra f i s h . Development 1996, inpress.44. Abdel i lah S, Moutcast leoShah E, Harvey M, Solnica-Krezel L,Schier AF, Stemple DL, Mal icki J, Neuhauss SC F, ZwartkruisE Stainier DYR et aL: Muta t i ons a f f ec t i ng neu ra l s u rv i v a l i nz eb ra f i s h Dan ie rer io . Development 1996, in press.45. Schier AF, Neuhauss SCF, Harvey M, Mal icki J, Solnica-Krezel L,Stainier DY R, Zwartkruis F, Abdel i lah S, Stemple DL, Rangini Zet al.: M u t a t i o n s a f fe c t in g t h e d e v e l o p m e n t o f t h e e m b r y o n i cz eb ra f i s h b ra in . Development 1996, in press.46. Mal icki J, Sch ier AE Solnica-Krezel L, Stemple DL, Neuhauss SCF,Stainier DY R, Abdel ilah S, Ra ngini Z, Zwartkruis F, Driever W:Mu ta t i ons a f f ec t i ng dev e l opmen t o f t he z eb ra f i s h ea r .Development 1996, in press.47. Pack M, Solnica-Krezel L, Mal icki J, Neuhauss SCF, Schie r AEStemple DL, Dr iever W, Fishman MC: Mu tat ion s a f fec t ingd e v e l o p m e n t o f z e b r a f is h d i g e s t i v e o r g a n s . Development 1996,in press.48. Neuhauss SCF, Solnica-Krezel L, Schier AF, Zwartkruis F, StempleDL, Mal icki J, Abdel i lah S, Stainier DY R, Driever W: M uta t ion sa f f ec t i ng c ran i o fac i a l dev e l opm en t i n z eb ra f i s h . Development1996, in press.49. Stemple DL, Solnica-Krezel L, Zwartkruis E Neuhauss SCF, Sch ier

AF, Malicki J, Stainier D YR , Abde lilah S, Ran gini Z, Mou ntcastle-S hah E , D r iev e r W : Mu ta t i ons a f f ec t i ng de v e l opm en t o f t heno toc ho rd i n z eb ra f i s h . Dev e l opmen t 1996, in press.50. Solnica-Krezel L, Stemple DL, Mountcast le-Shah E, Rangini Z,Neuhauss S CF, Mal icki J, Schier AE Stainier DY R, ZwartkruisE Abdel i l ah S, Dr iever W: Mutat ions a f fec t ing ce l l fa tes andc e l l u l a r rea r rangem en ts du r i ng gas t ru l a t i on i n z eb ra f i s h .Development 1996, in press.51. Arora K, NLissle in-Volhard C: A l tered mi to t i c dom ains revealf a t e m a p c h a n g e s i n Dros oph i l a e m b r y o s m u t a n t f o rz y go t i c do rs ov en t ra l pa t t e rn i ng genes . Development 1992,1 1 4 : 1 0 0 3 - 1 0 2 4 .52. Ferguson E, Anderson K: Dec apen tap l eg i c a c t s a s a m o r p h o g e nto o rgan i z e do rs a l - v en t ra l pa t t e rn i n t he D r o s o p h i l a embry o .Ce i l 1992 , 71 :451 -461 .53. Franco is V, Solloway M , O Neill JW, Em ery J, Bier E:Do rs a l - v en t ra l pa t t e rn i ng o f t he D r o s o p h i l a e m b r y o d e p e n d s

on a pu ta t i v e nega t i v e g row th f ac to r enc oded by t he s ho r tgas t ru l a t i on gene . G enes Dev 1 9 9 4 , 8 : 2 6 0 2 - 2 6 1 6 .54. Wharton KA, Ray RP, Gelbart WM: An ac t i v i ty gra d ien t o fdec apen tap l eg i c i s nec es s a ry f o r t he s pec i f i c a t i on o f do rs a lpa t t e rn e l emen ts i n t he D r o s o p h i l a embry o . Development 1993.1 1 7 : 8 0 7 - 8 2 2 .55. Winnier G, Blessing M, Labosky PA, Hog an BLM: Bonem o r p h o g e n e t i c p r o t e in - 4 i s r e q u i re d f o r m e s o d e r m f o r m a t i o nand pa t t e rn i ng i n t he mous e . Genes Dev 1 9 9 5 , 9 : 2 1 0 5 - 2 1 1 6 .This authors of this pape r have used homologous recombination in embry-onic stem cel ls to inactivate the mouse BM P4 g ene; the resul t ing phe notypeof mutant em bryos is analyzed using m olecular markers. The resul ts sug gestthat BM P4 is a key component in di f ferent p roce sses in early mouse devel-opment, beginning wi th gastrulat ion and mesoderm formation.56. Sasai Y, Lu B, Steinbeisser H, De Robert is E: Re gu lat ion ofneu ra l i nduc t i on by t he C h d and B m p - 4 an tagon i s t i c pa t t e rn i ngs igna ls in Xenopus . Nature 1995 . 376 :333 -336 .5? Smi th WC, Knecht AK, Wu M, Har iand RM: Sec reted nog ginp ro te i n m i m i c s t he S pem ann o rga n i z e r in do rs a l i z i ng X e n o p u sm e s e d e r m . Nature 1993 , 361 :542 -549 .58. Lamb TM, Knecht AK, Smith WC , Stachel SE. Economides AN,Stah l N, Yancopolous GD, Har land RM: Neura l i nduc t ion by th es ec re ted po l y pep t i de nogg i n . Science 1993 , 262 :713 -718 .59. Hemm ati-Brivanlou A, Kel ly OG, Melton DA: Fol l is tat in, anan tagon i s t o f ac ti vi n, i s ex p res s ed i n t he S pem ann o r gan i z e rand d isp lays d i rec t neura l i z ing ac t i v i ty . Cel l 1994 , 77 :283 -295 .60. Bumcrot DA, McM ahon AP: Sem i te d i f fe re nt ia t ion : Son ic s igna lss om i t es . Cur r B i o / 1995 . 5 : 612 -614 .61. P laczek M, Fur ley A: Neu ra l deve lopm ent : pat te rn ing casca desin the neura l tube. Cur r B i o11996 6 : 5 2 6 - 5 2 9 .62. Talbot WS, Trevarrow B, Halpern ME, Melby AE, Fa rr G, Postlethwait JH, Jowett T, Kimmel CB, Kimelman D: Theo r g a n i z e r - sp e c i f ic h o m e o b o x g e n e f l oa t i ng head i s es s en t i a lf o r no toc ho rd dev e l opmen t i n t he z eb ra f i s h . Nature 1995,3 7 8 : 1 5 0 - 1 5 7 .


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4 6 8 Pa t te r n fo r m a t i o n a n d d e v e l o p m e n ta l m e c h a n i s m s

T h i s r e p o r t p r e s e n t s th e p h e n o t y p i c a n a l y s i s o f t h e z e b r a f is h f loat ing headm u t a n t a n d s h o w s t h a t f loat ing head i s th e z e b r a f i sh h o m o l o g u e o f t h e h o m e -o b o x g e n e Xnot. f loat ing head m u t a n t s l a c k a d i f f e r e n t i a t e d n o t o c h o r d a n dm u s c l e t is s u e o c c u p i e s t h e m i d l in e o f th e s e a n i m a l s i n s t e a d - s u g g e s t i n gt h a t f loat ing head r e g u l a t e s n o t o c h o r d p r e c u r s o r c e l l f a t e s .6 3 . H a l p e r n M E , T h i s s e C , H o R K , R i g g l e m a n B , T r e v a r r o w B , W e i n b e r g E S , P o s t l e t h w a i t J H , K i m m e l C B : C e l l a u to n o m o u s

respecification o f a x ia l m e s o d e r m i n z e b r a f i s h f l oa t i ng headm u t a n t s . Development 1 9 9 5 , 1 2 1 : 4 2 5 7 - 4 2 6 4 .I n t h i s w o r k , c e l l t r a n s p l a n t a t i o n t e c h n i q u e s a r e u s e d t o d e m o n s t r a t e t h a t t h ef loat ing head g e n e a c t s c e l l a u t o n o m o u s l y i n t h e n o t o c h o r d . M o l e c u l a r m a r k -e r s c h a r a c t e r i s t i c o f a x ia l m e s o d e r m a r e e x p r e s s e d i n ti a ll y i n m u t a n t e m b r y o s ,b u t e x p r e s s i o n d o e s n o t p e r s i s t, s u g g e s t i n g t h a t f loat ing head i s r e q u i r e d f o rm a i n t a i n in g d e v e l o p m e n t o f t h e n o t o c h o r d - f o r m i n g a x ia l m e s o d e r m .6 4 . H a l p e r n M E , H o R K , W a l k e r C , K i m m e l C B : I n d u c t i o n o f musclepioneers and floor plate i s d is t i n g u i s h e d b y t h e z e b r a f i s h n otai l m u t a t i o n . Cei l 1 9 9 3 , 7 5 : 9 9 - 1 1 1 .6 5 . C o n c o r d e t J P, I n g h a m P : P a t t e r n i n g goes Sonic. Nature 1 9 9 5 ,3 7 5 : 2 7 9 - 2 8 0 .6 6 . F e t c h o J R , O ' M a l l e y D M : V i s u a l i z a t i o n o f active neural circuits i n t h e s p i n a l c o r d o f i n t a c t z e b r a f i s h . J Neurophysio] 1 9 9 5 ,7 3 : 3 9 9 - 4 0 6 .A c l e v e r m e t h o d i s u s e d t o m o n i t o r n e u r a l a c t i v it y o n a m i l li s e c o n d t i m e s c a l ei n l i ve zebra f i sh l a r vae. Ca l c i um green c l ex t ran was i n j ec ted i n t o t he ax i a lm u s c u l a t u r e o r i n t o t h e s p i n a l c o r d t o l a b e l p o p u l a t i o n s o f n e u r o n s . T h ed e x t r a n - c o n j u g a t e d c a l c i u m i n d i c a t o r p e r m i t s t h e v i s u a l i z a t i o n o f t h e n e u r a la c t i v it y o f i n d i v id u a l n e u r o n s .6 7 . K u w a d a J Y, C h i t n is A , L i n d a m e r L A : S t e r e o t y p e d p a t h f i n d i n gb y i d e n t if ie d g r o w t h c o n e s i n t h e e m b r y o n i c s p i n a l c o rd . S ocNeurosc i Abs t r 1 9 8 7 , 1 3 : 1 2 2 0 .6 8 . C h i t n is A B , K u w a d a . IY : A x o n o g e n e s i s in t h e b r a in o f z e b r a f i s he m b r y o s . J Neurosc i 1 9 9 0 , 1 0 : 1 8 9 2 - 1 9 0 5 .6 9 . W i l s o n S W , R o s s LS , P a r r e tt T , E a s t e r S J : T h e d e v e l o p m e n t o fa s i m p l e s c a f f o l d o f axon tracts i n t h e b r a i n o f t h e embryonicz e b r a f i s h , B rac hy dan i o re r io . D ev e l opmen t 1 9 9 0 , 1 0 8 : 1 2 1 - 1 4 5 .7 0 . R o s s L S , P a r r e t t T , E a s t e r S S : Axonogenesis andmorphogenes is i n th e e m b r y o n i c z e b r a f i s h b r a i n . J Neurosc i1 9 9 2 , 1 2 : 4 6 7 - 4 8 2 .7 1 . K u w a d a J Y, B e r n h a r d t R R , N g u y e n N : D e v e l o p m e n t o f spinalneurons and tracts i n t h e z e b r a f i s h e m b r y o . J Comp Neu ro l1 9 9 0 , 3 0 2 : 6 1 7 - 6 2 8 .7 2 . K u w a d a J Y, B e r n h a r d t R R , C h i t n is A B : P a t h f i n d i n g b y i d e n t i f i e dg r o w t h c o n e s i n t h e s p i n a l c o r d o f z e b r a f i s h e m b r y o s .J Neurosc i 1 9 9 0, 1 0 : 1 2 9 9 - 1 3 0 8 .7 3 . B e r n h a r d t R R , C h i t n i s A B , L i n d a m e r L , K u w a d a J Y : I d e n t i f i c a t i o no f spinal neurons i n t h e embryonic and larval z e b r a f i s h .J Comp Neu ro l 1 9 9 0 , 3 0 2 : 6 0 3 - 6 1 6 .7 4 . W e s t e r f i e l d M , L iu D W , K i m m e l C B , W a l k e r C : P a t h f i n d i n g andsynapse formation i n a z e b r a f i s h m u t a n t l a c k i n g f u n c t i o n a la c e t y l c h o l i n e r e c e p t o r s . Neuron 1 9 9 0 , 4 : 8 6 7 - 8 7 4 .7 5 . T h o m a s . IH : T h e m i n d o f a w o r m . Science 1 9 9 4 ,2 6 4 : 1 6 9 8 - 1 6 9 9 .7 6 . J ~ J r g e n sG , W i e s c h a u s E , N L ~ s s l e i n - V o l h a r d C : M u t a t i o n s a f f e c t i n gt h e p a t t e r n o f t h e l a r v a l c u t ic l e i n Dros oph i l a me l anogas te r :

I I . Z y g o t i c l o c i o n t h e t h i r d c h r o m o s o m e . Rouxs Arch Dev B io l1 9 8 4 , 1 9 3 : 2 8 3 - 2 9 5 .7 7 . W i e s c h a u s E , N L i s s l e in - V o l h a r d C , J ~ r g e n s G : M u t a t i o n s a f f e c t i n gthe pattern of the larval cuticle in Dros oph i l a me l anogas te r : II1.Z y g o t i c l o c i o n t h e X chromosome and four th chromosome.Rouxs Arch Dev B io l 1 9 8 4 , 1 9 3 : 2 9 6 - 3 0 7 .7 8 . N L i ss le i n- V o lh a r d C , W i e s c h a u s E , K l u d i n g H : M u t a t i o n s a f f e c t i n gthe pattern o f t h e l a r v a l c u t ic l e i n Dros oph i l a me l anogas te r : I.Z y g o t i c l o c i o n t h e second chromosome. Rouxs Arch Dev B io l1 9 8 4 , 1 9 3 : 2 6 7 - 2 8 2 .7 9 . H e n i o n P D , R a i b l e D W , B e a t t i e C E , S t o e s s e r K L , W e s t o n JA , E i sen JS : Screen for mutations affecting develop men t o fz e b r a f i s h n e u r a l c r e s t . Dev Genetics 1 9 9 6 , 1 1 : 1 1 - 1 7 .T h e a u t h o r s p r e s e n t p r e l i m i n a r y r e s u l t s f r o m a g e n e t i c s c r e e n i n w h i c hp a r t h e n o g e n e t i c d ip l o i d e m b r y o s a r e e x a m i n e d b o t h f o r v i si b le p h e n o t y p e sa n d f o r c e l l u l a r d e f e c t s i n n e u r a l c r e s t d e r i v e d t i s s u e s t h a t a r e r e c o g n i z e di m m u n o h i s t o c h e m i c a l l y .8 0 . K n a p i k E W , G o o d m a n A, A t k in s o n O S , R o b e r t s C T , S h i o z a w a M ,S i m C U , W e k s l e r - Z a n g e n S , T r o ll ie t M R , F u t re l l C , I n n e s B A et a/.:A reference cross f o r z e b r a f i s h Dan i o re r i o ) a n c h o r e d w i t hs i m p le s e q u e n c e l e n g th p o l y m o r p h i s m s . Development 1 9 9 i np r e s s .8 1 . P o s t l e th w a i t J H , J o h n s o n S L , M i d s o n C N , T a l b o t W S , G a t e sM, B a l l i nger E W, A f r i ca D , A ndrews R , Car l T , E i sen . IS eta/. :A g e n e t i c l i n k a g e m a p f o r t h e z e b r a f i s h . Science 1 9 9 4 ,2 6 4 : 6 9 9 - 7 0 3 .8 2 . J o h n s o n S L , M i c l s o n C N , B a l li n g e r E W , P o s t l e t h w a i t . IH :I d e n t i f i c a t i o n o f R A P D p r i m e r s t h a t reveal extensivep o l y m o r p h i s m s between laboratory strains o f z e b r a f i s h .Genomics 1 9 9 4 , 1 9 : 1 5 2 - 1 5 6 .8 3 . A m s t e r d a m A , L i n S , H o p k i n s N : The e q u o r e a v i c t o r i a green fluorescent protein can be used as a reporter in live z e b r a f i s he m b r y o s . Dev B /o /1996 , 1 7 1 : 1 2 3 - 1 2 9 .T h e a u t h o r s d e m o n s t r a t e t h a t g r e e n f l u o r e s c e n t p r o t e i n , w h e n i n j e c t e d a s ap l a s m i d o r a s a t r a n s g e n e a f t e r p a s s a g e , c a n b e v i s u a l i z e d i n l i v e e m b r y o sa p p r o x i m a t e l y o n e h o u r a f t e r z y g o t i c t r a n s c r i p t i o n b e g i n s . I n t ra n s g e n i c l in e s ,f l u o r e s c e n c e c a n b e o b s e r v e d i n m a n y e m b r y o n i c t is s u e s f o r s e v e r a l d a y saf t e r i n j ec t i on .8 4 . L i n S , G a i a n o N , C u l p P , B u r n s J C , F r i e d m a n n T , Y e e J + K , H o p k i n sN : Integration and germ l ine transmission o f a pseudotypedr e t r o v i r a l v e c t o r i n z e b r a f i s h . Science 1 9 9 4 , 2 6 5 : 6 6 6 - 6 6 9 .8 5 . L in S , Y a n g S , H o p k i n s N : / a c Z e x p r e s s i o n in g e r m l i n etransgenic zebrafish can b e d e t e c t e d i n l iv i n g e m b r y o s . DevB i o11994 , 1 6 1 : 7 7 - 8 3 .8 6 . H o p k i n s N : H i g h t i t e r s o f r e t r o v i r u s ( v e s i c u l a r s t o m a t i t i sv i r u s ) p s e u d o t y p e s , a t l a s t . P roc Na t /A c ad S c i US A 1 9 9 3 ,9 0 : 8 7 5 9 - 8 7 6 0 .8 7 . L in S , L o n g W , C h e n .I, H o p k i n s N : P r o d u c t i o n o f g e r m - l i n ec h i m e r a s i n z e b r a f is h b y c e l l tr a n s p l a n t s f r o m g e n e t i c a l lypigmented t o a l b i no e m b r y o s . Proc Nat l Acad Sc i USA 1 9 9 2 ,8 9 : 4 5 1 9 - 4 5 2 3 .8 8 . C u l p R N e s s l e in - V o l h a rd C , H o p k i n s N : H i g h - f r e q u e n c y g e r m - l i n etransmission o f p l a s m i d D N A s e q u e n c e s i n j e c t e d i n to f e r ti li z e dz e b r a f i s h e g g s . Proc Nat l Acad Sc i USA 1 9 9 1 , 8 8 : 7 9 5 3 - 7 9 5 7 .

fishing for genes controlling development

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