461

Fishing for genes controlling development Michael Granato* and Christiane NOsslein-Volhard
In recent years, the zebrafish has become a popular system for studying vertebrate development. Large scale mutant searches have led to the identification of >400 genes with unique functions during embryonic and larval development. A number of these genes play important roles in well studied processes, such as dorsoventral patterning of the early embryo, notochord formation and signaling, somitogenesis and neural specification. Other newly identified genes offer opportunities to investigate less well understood processes, including locomotion behavior, organogenesis, neural crest development and axonal pathfinding.

Addresses Max Planck InstitOt for Entwicklungsbiologie, Spemannstrasse 35/11t, 72076 T~bingen, Germany *Present address: University of Pennsylvania, Department of Cell and Developmental Biology, Stellar-Chance, Philadelphia, Pennsylvania 19104-6058, USA; e-mail: granatom@mail.med.upenn.edu Current Opinion in Genetics & Development 1996, 6:461-468 :~ Current Biology Ltd ISSN 0959-437X

important processes during cinbryogenesis [5-8]. T h e mcthodologx for large-scale mutant screens in zebrafish was later d e v e l o p e d and screens ~ e r e c o m p l e t e d recently in laboratories in Boston and Tfibingcn [9-14]. Together, more than 1500 mutations resulting in visible defects during c m b r w m i c and early larval d e v e l o p m e n t ~ c r c isolatcd, defining over 400 gencs required in a widc range of dcxclopmental processes [15-50]. In this revie~v, we discuss how these mutants can either contribute to our understanding of some well studied fundamental processes of vertebrate embryogcncsis or define starting points for a genetic and molcct,lar analysis of processes that are less well elucidated.

Results of the screens

Point mutations were induccd in D. n'rio ~ i t h the chemical mutagcn cthyhfitrosourca and m u t a g e n i z e d chromosomes were brcd to homozygosity in a three gcneration inbrceding scheme [11-14]. I~ixe embryos and larvae were screened at successive d e x e l o p m e n t a l stages for dcfects in a variety of morphological structures including the notochord, somitic boundaries, brain subdivisions, spinal cord, cardiovascular system, lixer, gut, muscles, eyes, ()tic vesicles, embryonic fin folds and derivatives of the neural crest cells (pigment cells, jaw and gill arches). In our laboratory in "l'fibingen, >4000 mutants were found [131. "l\~o thirds of these mutants show rather general abnormalities, such as rctardation and degeneration, and were discarded. To date, 900 of the 1200 mutants initially kept in the T t i b i n g e n screen have been assigned to 371 genes, resulting in an axerage allele frequency of 2.4 mutants per gene [13]. T h e n u m b e r of genes identified for each phenotypic class is givcn in T a b l e 1. In the Boston screen, somewhat lox~er numbers were attained with the same overall results.

Introduction
Profound insights into diverse d e v e l o p m c n t a l p a t h ~ a y s of the invertebrate model systems Drosophila me/ano~aster and L~/enol~ahditZr eleeans have been achieved by the mutational identification of genes and their s u b s e q u e n t molecular analysis. In many instances, gcnes with similar p h e n o t y p e s function in the same d e v c l o p m e n t a l pathxvay. l,arge scale scrccns have therefore been successfld in dissecting and understanding various processes, such as pattern formation in the Drosophila embryo, signal transduction and cell fate spccification in Drosophi/a and 6". el<gans (rcviexved in [1--l]). Progress in understanding the genetic control of vertebratc d c v e l o p m e n t has relied mainly on the analysis of genes isolated hv their homology to Drosop/ziM genes identified in mutant screens. This approach, howe~cr, focuses on conserved features betxveen invertebrates and vertebrates, neglecting those dcveh)pmental events t, n d e r - r e p r e s e n t e d in Drosophila mutant collections, such as organogcnesis or locomotion behavior, in addition to vertebrate-specific p h e n o m e n a , such as neural crest dexelopmcnt. To identit\" the genes controlling such processes in vertebrates, genetic approaches analogous to those use cJ in Diwsophi/a are necessary. In the zebrafish, Danio rerio--introduced by Strcisinger and co-workers as a vertebrate genetic s y s t e m - - g e n e t i c screens following T-ray mt,tagenesis h a v e been used to identify" a n u m b e r of embryonic mutants, including O,C/ops, spadetail and no rail, which have defects in

Dorsoventral patterning: who's who?

In amphibians, the formation and patterning of the dorsovcntral axis inxolxcs a n u m b e r of well characterized induction processes. Initially, dorsal and ventral vegetal signals induce overlying blastomercs to form dorsal and xentral mesoderm respectively. T h e induced dorsal mesoderln, corresponding to the future S p e m a n n organizer, patterns adjacent m e s o d e r m and induces neuroectoderm. Additionally, a ventral signal antagonizes this dorsal signal and spccifies ventral cell fates. Although numerous genes from Xenopus have been implicated in these signaling processes by their expression patterns and their activities when ectopically expressed in embryos, in many cases their roles in the embryo haxe, to date, not been tested in a loss-of-function situation. In the zebrafish screen, eight genes were identified with p h e n o t y p e s that suggest they play central roles

462

Pattern formation and developmental mechanisms

Table 1

to anterior structures, are redticed [34]. In particular, the

Genes identified in the T[ibingen screen: phenotypic groups.

Phenotypic group Subgroup Genes (N) 4 1t 6 2 10 4 4 6 5 3 4 4 3 2 13 0 12 15 8 14 2 6 9 8 13 13 4 13 9 8 7 13 17 7 14 10 7 12 18 15 Alleles/ genes 1.0 1.6 3.1 20 1,7 1.0 2.3 7.5 2.6 1.3 4.8 1.8 1.3 3.5 2.0 0 1.8 2.0 1.1 1.3 15 1,0 1.3 3.6 1.5 4.7 2.3 4.4 36 3 1 3 1.9 3.6 11 29 1.5 1.6 1.8 2.8 1.7 References [38] [35] [33] [34] [37] [37] [16] [16] [16] [16] [17] [17] [23] [24] [25] [26] [27] [15] [29] [29] [29] [29] [23] [30] [30] [36] [36] 127] [21] [21 ] [21 ] [21 ] [28] [21] [21 ] [21] [31] [32] [20] [20]

Early in Epiboly embryogenesis Early arrest and necrosis Body axes Dorsalised Ventralised Others Precordal plate and hatching Notochord formation Notochord differentiation Undulated notochord Degenerating notochord and late notochord defects Somite formation Somite pattermng Forebrain Midbrain-hindbrain Hindbrain Brain degeneration* Spinal cord Blood Heart morphology Heart beat Circulation Liver, gut and kidney Eye Otoliths Ear morphology and lateral line organ Fins Skin Curly tails P~gment cell number and pattern Melanin pigmentation Melanophore differentiation Melanophore shape Xanthophores Iridophores Two cell types affected Three cell types affected Flatheads and posterior arches Hammerheads and anterior arches Reduced motility and muscle striation Reduced motility and normal striation Body shape Pigment pattern Eyes Fins Pigmentation Pathfinding Mapping

Mesoderm

CNS

ventrolateral mesoderm in ditlo mutants, marked by rc'rl expression, spans more than two thirds of the margin, compared to half in a xxild-typc embryo (Fig. 1B and B') and the dorsal prestimptive neuroectoderm, marked b v ,/ZYZ7 expression, is strongly reduced. Alterations arc already evident before the onset of g,astrulation, showing that diHo and melterie's provide the blastula with patterning instructions, similar to ttlose attributed to Spcmann's organizer in Xenopus. Interestingly, the size and t\ltc of the signaling tissue, the shield region (which is the zcbrafish equivalent of Spemann's organizer), is not unafl'cctcd in mutant embryos (Fig. 1B and B'); this suggests that a reduction of patterning signals emanating from the shield might account for the defects obscrxcd in d/no and menv'd~'s l-nutants.

Organs

Body shape Pigment cells

Jaw and gills Motility

Mutations in somitahun, s~e'ir/, p~<~'mil, snai/kous< /ost-a-#sz, and miJli ,#// result in a common dorsalizcd phcnlltypc which is complcmcntarx to the ventralizcd phcnotxpcs of gli#o alld men,'des. Structures derived normally From the dorsolatcral regions of the blastula (the paraxial n-icsodcrm) and ncttrocctoderma] structures arc expanded, x~hcrcas vcntrall\ dcrixcd structures (such its the tail, t~roncphros and blood) are either reduced or absent I33i. In the strongest dorsalizcd phcnotxpcs, both the midbrain and hhldbrain region of" the ncurocccodcrm (Fig. 1(: and (]') and rhc somitcs extend arotlild the entire circumf`crcncc of the c m b r \ o , ~vhcrcas hlood and the proncphros arc missing. 'l'hc mutant alleles of these six genes d/splaT similar and ovcrlappin~ phctlotvpcs, althott~h the extent to which each particular nltltallt is affected diftcrs. Scxcral mutations arc d(imhlallt in cictdition to I)cing rcccssi\c and illutations in t~vo genes, pL<<rlail and .w.u/lil<li:l///. result in mutcrnal-cf]'cct phcnotvpcs, su~e;csting thole the six ,~cncs function in a dosage-sensitive pathwax that starts durin~z oogencsis. T h e p a t t m a v promotes ventral cell faces, counteracting ti~c dorsal/zing illftilcncc tit'rtislo and///e'/7rdem and the combinatorial function ma\ establish p~lsiti~mal int'~lrlnation along the dorsovcntral axis o|" tile blastula 1331.

Adults

4 3 4

1.0 2.7 1

(a) (a) (a) (a) (a) [19} [18]

Retrnotectal Total

4 7 371

1.8 3.3 24

This class of mutants was not kept systematically [26]. (a) C N0ssleln-Volhard et al, unpublished data. N, number of identified genes

in both d o r s m c n t r a l patterning of the mcsodcrm aiid neural induction. \ h i t a t i o n s in d/no and imvr,r/r~ rcsttlt in a vcntralization of the blastula, meaning that xcntral fates, x~hich contribute to more posterior strttctures in the embryo, arc expanded; in contrast, dorsal fates, givin<< rise

'l'hc phenocypcs of the dorsalizcd zcbrafish nllltalltS circ renlinisc'cnl: of the rl<'capci/ial)D:<.,D ~roilp iif" ,<ones in I)rosopkihz that arc required to establish a dccapcntaplc~ic a c m i t \ ~radicnc in the dorsal hctlf" <if" the embryo 151-54]. lnacti\ation <if" tilt" ~crtcbrcttC htmlohlt~tic of dccapcntaplcgic, bone morphogcnctic protein 4 IBXIP41, catlscs a rccittcti(in of` xcntral-postcrit~r I l l C s ( i d c r l l / i n I/liL'C 153] x~hcrcas cctopic cxprcssi~m oF ILIIP4 in Xrlm/m.~ rcslths in a dorsalization of" xcntral mcsodcrm. 'l'his stl~csts dlat I7.111>4 is one candiclatc l]n- [hc zcbtaf]sh dorsalicd i/ltilTanl.s: other Ceil/dictate genes inch/de those chat encode tl~c B]klP receptor, and ,~sk-,:,, \\nt-M and c \ c l , x~hcrcas molecules stich its N f ) ( i ( i l N , ( : I t ( ) R I ) I N , F ( ) I . I , I S T A ' I ' I N hat c sinlilar acti\ itics t{i diH~J and/UClT,rD:c when expressed cctopicall) in Xrtiopiis embr\tis 15f>-59i.

Fishingfor genes controllingdevelopmentGranatoand NOsslein-Volhard 463

l,inkagc analysis b e t w c c n these genes and thc mutations will reveal if anx of these cloned gcncs correspond to the gencs dcscribcd hcrc.

~touting kern/, no rail,

Axis f o r m a t i o n and signaling

During gastrulation, derivatives of thc three germ lavcrs become arranged in a parallel nrder along the anteroposterior axis, outlining thc basic body plan of the vcrtcbratc embryo. T h e prechordal plate in the head and thc notochord in thc trunk (axial mcsoderm) occupx a central position and thus contact tissues of all germ layers: the neural plate (neuroectodeml), the somites (paraxial mcsoderm) and the gut (end()derm). T h e notochord is required fi)r the specification and maintenance of cell fates in the somites, and the induction of the floorplate and motorneurons in the xentral spinal cord (reviewed in [60,61]). T h e mutant p h e n o t y p e s of a n u m b c r of zcbrafish genes r c f e c t this central role of the nntochord in the patterning of adjacent tissues and suggests that the genes may rcprcscnt cnmponcnts of a complex nct~ ork by which thc axial m c s o d c m l cxcrts different patterning influcnccs on the surrounding tissues. Whereas mutations in the floating kead, momo, no tai/ and dot genes required for notochord formation also affect patterning of the somites, mutations m 13 other genes display dejects in the somites and/or thc neural plate but not in thc notochord, f h c s c genes comprise small groups with common p h e n o t y p i c f'caturcs, although indixidual p h e n o t y p e s may include additional defects. Cell transplantation experiments, designed to d e t e r m i n e the tissues in which individual genes are required, allow one to assign specific roles tBr these genes. In several instanccs, p h e n o t y p e s are restricted to subrcginns of the anteroposterior axis, suggesting differences in the patterning of head, trunk, and tail [16,27]. T h e notochord is lacking in embryos mutant fi)r momo or floating kead. Although axial mcsodcrmal cells exprcss notochord markers initially, they soon switch their fate to become paraxial mesoderm [16,49,62",63"]; this suggests a role for both genes in thc specification of axial mcsodermal cells. In no tai/ and doc mutants, notochord precursors are present but fail to differentiate [16]. Although thc patterning of the ventral ncural tube is disrupted in the absence of the notochord in momo and floating kead mutants, thc undifferentiated notochord in no tai/and doc mutants is sufficient for floorplate induction. In mutants of all four genes, howexcr, abscnce of a diffcrcntiated notochord affects induction of a specialised class of muscle cclls and patterning of the somitcs into a dnrsal and a xentral part [16,63,64]. _Mutations in six loci--ct/amae/eon, you-too, sonic you, you, u-hoot, and ~ / ) o l w - - c a u s c similar dejects in the somites but notochord d e x e l o p m e n t is unaffected [17]. Cell transplantation c x p c r i m e n t s indicate that somitc

pattcrning is d e p e n d e n t on the wild-type function of and doc in the notochnrd, whereas you-too and a-hoot are required autonomously in the snmitcs. Mutations in seven other genes affect flnorplate and motorneuron d e x c l o p m e n t in the ventral spinal cord, without obvious defects in the notochord and somites [27]. In particular, sckma/spal, as with oue-evedpinkead and the previously described O'c/ops mutants, lack a floorplatc [7,27,37] whereas maintenance but not formation of the floorplate is affected in mutants of detour; igaana, s~ma/hans and monorail, suggesting that these genes function to establish or to maintain proper organization of the zebrafish midline in thc brain and spinal cord [271. T h e s e genes may encodc signals emanating from the notochord or alternatively they may bc involved in rccciving and interpreting the signals in the ventral spinal cord and thc somites. A prime candidate for an inductixe signal produced by thc notochord is the secrcted Sonic hedgehog molecule (reviewed in [65]), and genetic linkage analysis bctwecn Sonic hedgehog and genes required for somitc pattcrning and f n o r p l a t c d e v e l o p m e n t is in progress.

Genetics of o r g a n o g e n e s i s and l o c o m o t i o n behavior: getting off t h e g r o u n d ?

()ur understanding of the genctic control of processes occurring late in vertebrate cmbryogenesis - - such as inner organ formation, patterning of the hcad and the brain, gcncration of p i g m c n t cell patterns, sensory organ d e x e l o p m e n t , formation and growth of paired a p p e n d a g e s and establishmcnt of neural circuits undcrlying simple locomotion b e h a v i o r - - a r c lcss well adxanced. Such events take place within the first six days of zebrafish d e v e l o p m e n t and can directly bc observed in lixe embryos or larvae. Approximately half of the genes identified in the f 0 b i n g e n screen affect thcsc late proccsses ( T a b l e 1) [15,2(t,21,23-26,29-32,42-48]. In this section, we discuss the potential of genes with functions in intcrnal organ formation and locomotion behaxior as examples. ()rganogenesis involves many d e v e l o p m e n t a l mechanisms, including induction, cell type differentiation and morphngcnesis. Mutant p h e n o t y p e s arc useful because they facilitate the dissection of complex events into individual steps. ()f particular interest is a collection of mutant p h e n o t y p c s covering a broad range of events during cardiovascular d e v e l o p m e n t and function. T h e s e define at least 9 genes required for hematopoesis [15,43] and 25 genes ~ i t h functions in the various c o m p o n e n t s of the cardimascular system, including fusion of the two bilateral heart primordia, chamber formation, looping, matrix d e v e l o p m e n t and sizing during heart morphogenesis, contractility and rhythm of heart function, and vessel formation [29,42] (Fi R. I N and N'- O and O'). M a n \ of these genes will be valuable in understanding cardiovascular malfunctions in humans, as the mutant p h e n o t y p e s rescinble human congenital cardiovascular diseases [39].

464

Pattern formation and developmental mechanisms

Figure 1

Fishing for genes controlling development Granato and N0sslein-Volhard

465

Figure 1 Zebrafish mutants. (A) Schematic of epiboly. Structure of the embryo at high blastula stage (left) and 70% epiboly. (e, epiblast; h, hypoblast; evl, enveloping layer; ysl, yolk syncitial layer.) (B) Wild-type and (B') dino mutant at 70% epiboly, goosecoid (gsc) and eve mRNAs localization, animal pole view, dorsal right. (C) Wild-type and (C') swirl mutant at 10 somite stage, stained with Pax 2 (mb, midbrain; p, pronephros; op, optic vesicle; ot, otic vesicle). (D) Wild-type, (D') floating head and (D") momo mutants at the somite stage, stained with anti ntl (brown) and MyoD (blue). (1=) Skeletal staining of 6 month old wild-type and (l=') fused somites mutant fish (ha, hemo-arch; ct, centron; na, neural arch). (F) Lateral view of a 24 hour old wild type and (F') sleepy mutant. (G) Antibody staining visualizing the muscle pioneer cells in wild type and (G') you mutant embryos. (N) Zotx-2 staining in wild type and (H') masterblind mutants. (I) Zn5 antibody staining of the optic nerves in wild type and (1') detour mutants. Wild-type (1, K) and yugiri mutant embryos (I'K') using light microscopy in (J, J') or acridine orange (AO) staining for cell death in (K, K'). (L) Wild-type and white tail (L') mutant embryo stained with 3A10 antibody (Ro12, identified neuron in hindbrain; m, Mauthner cell). Expression of Otx-1 in the otic vesicle of wild-type (M; arrow) and absence of expression in van gogh (M') mutants. Live images (N, N') and sagittal sections (O, O') of wild-type hearts (N, O) and santa (N', O') mutants at two days of development (a, atrium; v, ventricle; o, outflow tract). Antibody staining of (P) wild-type and (P') unplugged mutant embryos (MIP, middle primary motoneuron; CIP, caudal primary motoneuron). Cartilage stains of day 5 (O.) wild-type and (O') schmerle mutant larvae. (R) Wild-type and (R') never mind mutant larvae at day 5 of development, injected with Dil (green) and DiO (red) to visualize their retinotectal projections.

Zebrafish larva display distinct, stage-specific patterns of locomotion bchavi(m providing an interesting paradigm to s[tid\ the contr()l of" motor systems in vertebrates. Rcccnt progress in optical monitoring of ncural activity [00"] anti the xvcll studied ncuroarchitccturc of thc llerv()tls S\rStCl]l [~>7--73] allm~ us to ctetcrminc i, c'ic'o thc gross bchavioral function of identified ncnrons in zcbrafish. T h e identification of >48 g c n c s - - w h i c h function in distinct locomotion patterns, such as touch response, equilibrium control and rhythmic tail n/o~<cmcnts [ 2 0 J - - a d d s genetic approaches to the investigation of simple locomotion bchaxior in vertebrates. Some of the behavioral phcnotypcs can bc traccd back to defects in semitic muscle diffcrcntiation or degeneration (in which IH genes haxe been implicatcd), defects in physioh>gical components (e.g. nicotinic receptor [74])and defects in axonal elongation or pathfindin<~. For cxamplc, mutations in dia"a,~A'a and llnp/#,<d~7'd affect ax(mal chmgation and pathfinding of spinal nmt(~rnetir(ms, x v h c r c a s illti12;itiolls in Ill(Iv'/lo and ~true'rmind cause abnormal mapping of retinal axons on the optic tccmm (Fig. 1P and P'; R and R') [1H,201. Thus, although thcrc arc as vct Elr f'c\',cr behavioral mutants a\ailable than in C. e&<a/L~ (rex icx~cd in [751), the zcbrafish mntallts could bc nseftll in an approach tm~ard understanding the F,cnctic control of" neural ctex ch)pmcnt. Patterns of" h)con3otion that arc based on altcrnciting and r h y t h m i c m o v c m c n t s , s u c h :is s ~ v i m m i n g or c r a ~ l i n g , arc x~idcsprcad among xcrtcbrates but the idcntit\ of the neural c(mlponcnts and their roles in particular neural circuits is not complctel 3 tlnctcrstood. '1\~o ,~rotlps of. mutcints with unique bcha\ioral defects m a \ u;ivc chics about the comp(mcnts tinct their roles in neural circuits. Xlutations in 7 genes (the "accordion" gronp genes) cause simultclllC()ti', COlltractiollS ()f the mot(~r S\'StCIIlS Oil both sides ()f tim midlinc. In \~iltt-txpe cmbr\os, comnlissural illtcrncurons in tile spinal cord cnstire alternating muscle c()ntractions b \ a process termed 'reciprocal i n h i b i t i o n of the ;intclgonistic motor center', stigg{cstin~ that these s t \ o n .~cncs centre[ ()r mediate reciprocal inhibition.

t)crtnrbation of tile swimming rhythm, the rhvthn~ic undulation of the body, is sccn in mutants of ,~Tpaa, cad~,t, ,~7~ac<r/ out and t~e'itJl ta"ia', where thc tail of affcctcd larvae bcnds successi~clv to the same sidc [20]. In A}wop#s larvae, experimental scparathm of the two sides of the nor\otis sxstem reveals that both motor ccntcrs can gcncnltc swimming actixitv i n d e p e n d e n t l y but commissural intcrncurons, connecting both sides, arc essential for phasing their acti\itics. As a failure to coordinate antagonistic motor centers appears to c a u s e tile bchavioral locomotorv phcnotypcs in thcsc mutants, the three ,e,cncs m e n t i o n e d may play roles in the neural circuit generating or mediating rhx'thmicit\.

Conclusions: limitations and perspectives of the screen

A main limitation of the ebrafish screen, ~hich applies to any cnetic screen in vertebrates, is the amotlnt of \',r()Fl-; and the capacit T of the animal facilit\ required to breed and anal\ze large ntlmbcrs of mutagenizcd lines. In the "l'{ibin<acn fish screen, -320(t lines were screened, resulting in an allele fre(lucnc ) of 2.4 n l n t a n t s per gone. T h i s is approximatcl'/ half the allele frcqticnc}' tllat ]las bccn a c h i m c d in the zygotic screens of Drosopkila [76-7S]. :\lthough the allele frc(luencv varies aml)n~ different phcnotypic classes in zcbrafish, on average it is certainly less than that rc(luired for saturation, indicating dmt a snl)stantial fTaction (if" zcbrafish g;cnes tlavc escaped iclcntification. :\nalvsis o|" :.JR c~.cn larger iltlmber of ebrafish Iincs, }m~cvcr, would have pushed the anll)tint of work rind the capacity of the ;Inilnal facilit\ r . v c r their ~ orkahlc limits. 'l'hc xcbrafish cmbrxo and larvae helve a large degree of complexit), conlparccl to the cuticle pattern in D/o,Ol')ilila lar~ac. In zcbratish, nlan\ structural |'CCltlires arc scored ,,ith case, %~hcrcas oti~ers arc less conspictlous and ma\ thcreforc rcquirc illorc carcflil analysis t'()r nltltanrs to bc consisrcnth detected. In addition, complex phcnotypcs arc not cllxva\s recognized as being of interest, b e c a tl s e

466

Pattern formation and developmental mechanisms

thcv may be parallclcd b \ rather gcncral detects, such as retardation or degeneration, obscrxcd in approximatcly two thirds of all mutants identified. T h e s e t\vo reasons can, in part, explain why the degree of saturation varics among different phcnotxpic classcs.

8.

Kimmel CB, Kane DA, Walker C, Warga RM, Rothman MB: A mutation that changes cell movement and cell fate in the zebrafish embryo. Nature 1989, 337:358-362. Mullins MC, NL~sslein-Volhard C: Mutational approaches studying embryonic pattern formation in the zebrafish. Curr Opin Genet Dev 1993, 3:648-654. Driever W, Stemple D, Schier A, Solnica-Krezel L: Zebrafish: genetic tools for studying vertebrate development. Trends Genet 1994, 5:152-159. Mullins MC, Hammerschmidt M, Haffter P, N0sslein-Volhard C: Large-scale mutagenesis in the zebrafish: in search of genes controlling development in a vertebrate. Curr Biol 1994, 4:189-202. Solnica-Krezel L, Schier AF, Driever W: Efficient recovery of ENU-induced mutations from the zebrafish germline. Genetics 1994, 136:1401-1420. Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, Odenthal J, Van Eeden FJM, Jiang Y-J, Heisenberg C-P et aL: The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 1996, in press. Driever W, Solnica-Krezel L, Schier AF, Neuhauss SCE Malicki J, Stemple DL, Stainier DYR, Zwartkruis F, Abdelilah S, Rangini Z et aL: A genetic screen for mutations affecting embryogenesis in zebrafish. Development 1996, in press. Ransom DG, Haffter P, Odenthal J, Brownlie A, Vogelsang E, Brand M, Van Eeden FJM, Furutani-Seiki M, Granato M, Hammerschmidt M e t al.: Characterization of zebrafish mutants with defects in embryonic hematopoiesis. Development 1996, in press. Odenthal J, Haffter P, Vogelsang E, Brand M, Van Eeden FJM, Furutani-Seiki M, Granato M, Hammerschmidt M, Heisenberg C-P, Jiang Y-J et aL: Mutations affecting the formation of the notchord in the zebrafish, Danio rerio. Development 1996, in press. Van Eeden FJM, Granato M, Schach U, Brand M, Furutani-Seiki M, Haffler P, Hammerschmidt M, Heisenberg C-P, Jiang Y-J, Kane DA et aL: Mutations affecting somite formation and patterning in the zebrafish Danio rerio. Development 1996, in press. Trowe T, Klostermann S, Baier H, Granato M, Crawford AD, Grunewald'B, Hoffmann H, Karlstrom RO, Meyer SU, Richter S et al.: Mutations disrupting the ordering and topographic mapping of axons in the retinotectal projection of the zebrafish, Danio rerio. Development 1996, in press. Karlstrom RO, Trowe "I", Klostermann S, Baier H, Brand M, Crawford AD, Grunewald B, Haffter P, Hoffman H, Meyer S U e t aL: Zebrafish mutations affecting retinotectal axon pathfinding. Development 1996, in press. Granato M, Van Eeden FJM, Schach U, Trowe T, Brand M, Furutani-Seiki M, Haffter P, Hammerschmidt M, Heisenberg C-R Jiang Y-J et al.: Genes controlling and mediating locomotion behaviour of the zebrafish embryo and larva. Development 1996, in press. Kelsh RN, Brand M, Jiang Y-J, Heisenberg C-P, Lin S, Haffter P, Odenthal J, Mullins MC, Van Eeden FJM, Furutani-Seiki M e t aL: Zebrafish pigmentation mutants and the processes of neural crest development. Development 1996, in press. Baier H, Klostermann S, Trowe T, Karlstrom RO, NLissleinVolhard C, Bonhoeffer F: Genetic dissection of the retinotectal projection. Development 1996, in press. Heisenberg C-P, Brand M, Jiang Y-J, Warga RM, Beuchle D, Van Eeden FJM, Furutani-Seiki M, Granato M, Haffter P, Hammerschmidt Met al.: Genes involved in forebrain development in the zebrafish, Danio rerio. Development 1996, in press. Brand M, Heisenberg CoP, Jiang Y-J, Beuchle D, Furutani-Seiki M, Granato M, Haffter R Hammerschmidt M, Kane DA, Kelsh RN : Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development 1996, in press. Jiang Y-J, Brand M, Heisenberg C-P, Beuchle D, Furutani-Seiki M, Kelsh RN, Warga RM, Granato M, Haffter P, Hammerschmidt M et al.: Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio. Development 1996, in press. Furutani-Seiki M, Jiang Y-J, Brand M, Heisenberg C-P, Houart C, Beuchl~ D, Van Eeden FJM, Granato M, Haffter P, Hammerschmidt

9.

10.

T h e 'l'iibingen and Boston screens ha~c fl~cused on phcnotypcs that arc xisiblc under a steremicroscope but more spccializcd screens using particular assays arc f'casiblc. I:or cxample, in conjunction with thc morphological screen in Ttibingen, mutations affecting retinotectal projection have bccn isolated b y anterogradc labeling of axons with fluorcscent dyes [22]. Genetic screens dcsigned to i d e n t i f \ changes in cxprcssion pattcrns by x~holc retrot iv situ or by antibodx labeling arc in progress and should u n c m c r gcnes with more subtlc p h e n o t y p c s [79"], Onc sh(mld k c c p in mind, hox~evcr, that a larg;e n u m b c r of mutagcnized fish families arc n e e d e d to achievc a reasonable degree of saturation for any givcn phenotypic trait.
The

1 1.

12.

13.

14.

15.

zebrafish genes identified in the screens pcrfl~rmed in Tfibin~4cn and Boston fl)rm a foundation fl)r s u b s e q u e n t detailed analysis of a whole variety of d e v e l o p m e n t a l processes, some of them specific to vertebrates. T h e well described cxperimcntal advantages o f the zebrafish
embryos together with the availability of linkage maps

16.

of the ~cnomc [80-82], the ability to fl,cncrate transgcnic lines [83",84-88], recent advances in positional clonin~ techniques and strategies, and ongoing progress in ~,cnerating ~irus-mediatcd insertional mutagcnesis [84] should r a p i d l y extend our knox~ledgc of vertebrate development.

17.

1 8.

Acknowledgements
\Ve would like to thank CP Hciscnbcre,, J ()dcnthal, I:V Ecdcn, K

19.

Rossna~cl, I) Gihnour, S Jcsuthasan, XI \lullins, T Nitisn, R Sommcr,

J School for man 3 helpful suggestions on the manuscript. 20.

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