Large Scale Genetics in a Small Vertebrate, The Zebrafish

8/13/2019 Large Scale Genetics in a Small Vertebrate, The Zebrafish

1/7

In t. J .U c\ . B io I. ~ o: 2 21 -2 27 (1 99 6) 22 1

Large scale genetics in a sm all vertebrate, the zebrafishPASC AL H AFFTER * and CHR ISTIANE N USSLE IN -VO LHAR D

M ax -P la nc k-/n stitu t fu r E ntw ic klu ng sb io lo gie , T ub in gen , G erm an y

ABSTRACT The systematic isolation and characterization of mutants inDrosophila has enor-mously facilitated the analysis of molecular mechanisms underlying developmental pathways inthe embryo. A similar approach is presently being used to study embryonic development of thezebrafish, which is becoming a mainstream model organism for vertebrate development. W ith itsgenetic versatility and sophisticated embryology, zebrafish offers the possibility to rapidlyincrease our knowledge of vertebrate development and add to what we have learned from othervertebrate m odel organism s.

K E Y W O R D S: zebra ji sh , mutagenes is , 1 .. le r/ ebmledro flojnne fl /, nn lJ ryunic dn lt 'l upmen t, s a/ural ion SCH'f 'l l

Introduction

The mystery of our own origin and mode of development hasfor over 100 years stim ulate d interest in vertebrate develo pm ent.F undam ental processes of early de velopm ent appear conservedam ong the vertebrates. By studying a particular vertebrate m od-el orga nism w e can therefo re draw conclusions a bout verteb ratedevelopment in general and ultimately come up with modelsabout our own development. Genes encoding key functions indevelopment show a high degree of conservation with each oth-er. Once we have identified a developmentally important gene

from one vertebrate we can easily clone the homolog from othervertebrates and study its function in the vertebrate which is them ost practical for the selected approach. This review is about am utational approach towards the study of vertebrate develop-m en t u sin g th e z eb ra fis h, D an io r erio , as a system .

Genes that may be important for vertebrate developmenthave, so far, m ainly been identified using two approaches. The1irst approach was based on the finding that many genes wilhimportant functions in the development of Drosophilamelanogaster have conserved counterparts in vertebrates.Frequently whole fam ilies of vertebrate genes were isolated bym olecular screening for hom olo gy to specificDrosophila genes.The obvious lim itation of this approach is that vertebrate genesw ithout hom olog y to p reviously cloned genes w ill g o u ndetected .The second approach is to isolate genes displaying interestingspatial or tem poral expression patterns in the em bryo. The func.tion of these genes is studied by generating loss.of.functionm u1ations using the pow erlul ES -cell based knockout technolo-gy in the mouse (Mansour e f a l.. 1988). Som e 01 these knock-

outs display interesting specific defects in em bryonic develop-ment, whereas others have disappointingly little or no effect.Genes whose function in development is not modulated at thelevel of expression may be missed by approaches that arebased on diffe rentia l ge ne expression pattern s.

The spectacular progress in understanding the embryonicdevelopm ent of Drosophila is m ainly due to a genetic approach(N Osslein-Volha rd and W ieschau s, 198 0). S ys1 em alic m u1ation -al screens were carried out in which genes w ere identified basedon the phenotype caused by a mutation in these genes. Thisapproach proved to be very successful and allowed the identifi-cation of most if not all genes required for early development(JOrgens e f a l., 1984; NOssle in-Volhard e f a l., 1 984; Wiesc hau se t a l. , 1984). The key feature for such saturation screens was thehighly developed genetics of Drosophila. T his in clu de s a largecollection of visible marker mutations in the adult which had

be en accum ulated by geneticists over the years. Th e a vailabilityof balancer chromosomes enormously facilitated saturationscreens covering particular chrom osom es and the subsequentmaintenance of mutant stocks. The detailed genetic maps andthe cytological maps of giant polytene chromosomes haveallowed rapid cloning of Ihe mutated genes. In spite of 1hesophisticated genetic m ethods available inDrosophila, it is stillnot possible to create loss-of-function m utations by targetedgene disruption. Nevertheless, for many developm ental biolo-gists Drosophila is still the m odel organism of choice.

Until recently, a system atic m utational approach to study ver-tebrate developm ent was not considered feasible. Am phibiansand chicken, although the favorite experimental organisms ofembryologists for many years, cannot be used for geneticapproaches due to their size and generation time. The mousewith its long-standing tradition of genetic research and its pow-erlul ES-cell based gene knockoul technology has providedusw ith m any insights into vertebrate developm ent. However, due tothe intrauterine developm ent of its em bryos and the sm all num -ber of progeny obtained from one genetic cross, it is im practical

.-lbbrl'viatiolls u s e d in thism eth anesulfo nate; R ..\P D,

p ol> nH :ra ~e c ha in re aclio n:

J a p e r : E XU , e th yln itr o:o >o ur ea ; L \lS , e th yl-random amplified polymorphic DX.\; PCR,S SR , ~im ple ~equ encl' repeal~.

* Ad dre ss fo r r ep rin ts : M ax -P la nc k-In stitu t fu r E ntw ic klu ng sb io lo gie, S pe ma nn stra l3 e 3 5/1 11 ,7 20 76 T ub in ge n. G er man y. FAX : 4 9.7 07 1.6 01 38 4.

021~-6282/96/S03.00()

L 'H C P rePrint~iJ in S r ~ iT


2/7

222 P. Haffter alldC. Niiss/eill- Vo/hard

/

caudal

I/

Fig. 1. 5 day old zebrafish em bryo. Tissues and organs that are visible u nder a sim ple d is se ct in g m ic ro sc op e a re i nd ic at ed .

to p er fo rm la rg escale sa turat ion screens fo r m u ta tio ns a ffe c t in ge a r l y development.

The zebrafish is becom ing a n in c re a s in g ly p o p u la rmodelorganism for studying vertebrate developm ent (Kim mel, 1989;M ullins an d N Osslein-Volhard, 1993; D rievere t a l. , 1994). It hasm an y p ro p e r t ie s th a t m ak e it a n id e a lo rg an is m fo r s ys te m at icmutational approaches. It has a short generation tim e of 2-4months and can be kept at a high density with relatively littlem ain te na nc e. O ne p a ir p ro d u c e s o n a v e ra g e200 progeny atweekly in te rv als w h ic h g re a t ly fa c il i ta te sgenetic analysis.Embryonic development is r ap idand s yn ch ro no us . E xt er na l f er -t i lization provides easy acces s t o t he e m br yo s t hr ou gh ou t d ev el -o pm en t. In a dd itio n z eb ra fis h e mb ry os a re la rg e a nd tra ns pa r-ent. This allows all tissues and organs to be obselVed duringd ev elo pm en t u nd er a s im ple d is se ctin g m ic ro sc op e w ith ou t p rio rmanipulations of the embryo (Fig. 1). This includes the majorsubdivi si ons o f the b ra in the neu ra l t ube fl oo rp la te notocho rdsomites heart jaw gills eyes otic vesicles fins blood liver gutand pigmenta t ion .

A number of genetic methods have been developed by G.Streisinger and his colleagues (Kimm el, 1989). These includegenetic tricks such as the production of haploid em bryos andgynog enetic diploid em bryos w ho se geno me is fully d erivedfrom the m aterna l genom e (S tre isin ger e t a l., 1981). B oth tech-n iq ue s w ere u se d b y la bo ra to rie s b as ed in E ug en e O re go n tois ola te a s ig nific an t n um be r o f in te re stin g m uta tio ns fo llo win g y -ra y m u ta ge ne sis d em o ns tra ti ng t he m u ta bi li ty o f th e z eb ra fi shg en om e (K im m el e t a l. , 1989 ; Fel senfeld e t a l. , 1990; Westerf ie lde t a l., 1 99 0; H atta e t a l., 1 99 1; H alp er n e t a l., 1993). A methoda ll owing succes sful reg en eration of fro zen sperm was alsodeveloped (in Westerfield, 1993). This will in the near futurebecome a very important feature o f z eb ra fi sh g en eti cs s in ce alarge n um ber of g enetic stocks can b e kept w ith out con tinuou sturnover.

In addition to the ge netic ve rsa tility zeb ra fish also o fferse xc elle nt e xp erim en ta l te ch niq ue s w hic h w ere m ain ly d ev el-oped by C. Kimmel and his collaborators in Eugene (Oregon)(Kimmel and Warga, 1986, 1988; Kimmelef al., 1990). Thisincludes a detailed fate map of the early zebrafish embryo andsophisticated e m br yo lo gy s uc h a s l in ea ge tr ac in g a nd tr an sp la n-tation.

An increasingly important reason for studying zebrafish as a

model organism is purely economical. The breeding and mainte-

nance of zebrafish is re latively cheap when compared to other

vertebrate organism s. Increasing economical considerations arelikely to contribute towards zebrafish becoming a mainstream

vertebrate model organism in the future .

G en etic sc re en s

G en etic s cre en s in d ip lo id o rg an is ms s uc h a s z eb ra fis h p os eth e p ro ble m that rece ssiv e m utations m ust be bred to h om ozy-gosity to uncover their phenotype. The ability to m ake haploidem bryos in zebrafis h m akes it p ossible to circ um vent this p ro b-lem (Stre is inger e t a l., 1981). Haploid em bryos are obtained bfertilizing eggs v t r o u s i n g U V-in ac tiv ate d s pe rm . 5 0 o f th ehaploid em bryos derived from a female heterozygous for a spcific m utation w ill reveal the phenotype of this m utation (Fig.2) .T he m ajo r a dva ntag es o f t his m eth od a re th at it c an b e d on e w i t ha smal l f ish fac il it yand mutan t car rie rs a re iden ti fi ed d ir ec tlya mo ng th e F 1 fe ma le fis h. H ow ev er, th e m ajo r d is ad va nta geth is m eth od is th at h ap lo id e mb ry os d is pla y a ra th er h ig h b acgro un d o f a bn orm al d ev elo pm en t a nd d ie a rou nd fiv e d ays aftefertiliza tio n. T his m ak es it im possib le to screen fo r m uta tio nscau sin g su btle d efects or m uta tio ns affectin g o rg an s o r tissu esat a la ter sta ge in d ev elo pm en t. M uta tio ns id en tified in h ap lo idscreens have to be recovered from the F 1 carrier before furtheran aly sis of th e m uta nt ca n b e p erfo rm ed .

W ith th e d ev elo pm en t o f efficien t system s to raise an d m aintain large num bers of independent lines of zebrafish it hasbecome feas ib le to perform large sca le muta tionalscreens uti-liz in g s ta nd ard c ro ss in g s ch em es to p ro du ce homozygousd ip lo id e mb ry os (F ig . 3 , M ullin se t a l., 1994; Solnica-Krezel etal., 1994). In such a schem e, the sperm atogonial cells of Gfo un de r m ale s a re m uta ge niz ed a nd th e m ale s a re th en o ut-cro sse d to w ild typ e fe ma le s, re su ltin g in a la rg e n um be ro f Ffish th at a re a ll h etero zyg ou s fo r a d ifferen t an d u nk no wn set on ew ly in du ce d m u ta tio ns . F r o m single pair matings between F1fis h, F 2 fa milie s a re ra ise d. H alf o f th e fish in s uch a n F 2 fa ma re h ete ro zy go us f or a p ar tic ula r m u ta tio n. Sibling crossesamong F 2 f is h willmatch two carr ie rsof a specif ic mutat ioninaq uarter of th e m atin gs an d th e m uta nt p hen oty pe w ill b e d is


3/7

Muta~n*~Treatmentof

~X~Sp


4/7

2 2 4 P H a ff le r a n dC . Niiulein- Vo wrd

TABLE 1

FREQUENCY OF MUTATIONS ISOLATED PER SCREENEDH APLO ID G EN OM E

Mutagen N um ber of ha ploidg en om es s cre en ed

Number of mutations Mutations perisolated haploid genome

EN UEM SX-rays

90 1.21 0.029 0.09

7444

10 3

G O m ales were mutagenized using either ENU, EMS or X-rays and out-crossed to wild-type. Following the standard crossing schem e shown inFigure 3. F3 progeny were screened for m utations with phenotypes thatare visible al em bryonic stages. The frequency of m utations per haploidg en om e s cr ee ne d are m uch higher for ENU than fo r E M S o r X -ra y s(Mullins e t a l., 1 99 4).

during DN A replication and thusdoes not produce m osaic prog-

eny. Mosaicism am ong the F1 fish would result in fewer fish with-in an F2 family carrying a specific mutation which wouldcausean enormous drop in the efficiency of screening. M utagenizedmales are therefore outcrossed three weeks or longer after themutagenic treatment, by which time no mosaicism am ong the F1progeny is observed. A potential problem of mutagenizing pre-meiotic germ cells are spermatogonial clones, which wouldresult in an identical m utation being isolated from two separateF2 families that derive from the same mutagenized founderm ale. In zebrafish this is very unlikely to happen, since fertiliza-tion occurs w ith a vast excess of sperm (S treisinger e t a l. , 1981)and the number of spermatogonial stem cells is estimated to bebetween 500 and 1000 (Mullins ela/., 1994). A s a preca ution,large numbers of F1 fish are nevertheless obtained from a rea-sonably high num ber of m utagenized m ales.

X-rays and y-rays also produce high mutation rates inzeb ra fi sh (Chak raba rt i e t a l., 1983). In contrast to ENU, howev-er, m utations induced by X -rays or y-rays are m uch m ore difficultto recover, resulting in a lower frequency of recoverable muta-t ions (Mull in s e t a l. , 1994). This indicates that large deletions orchromosome breaks are induced. Deletions are often desired,because breakpoints serve as physical landm arks which facili-tates cloning of a m utated gene. However, in saturation screens,point mutations are favored over deletions, because only m uta-tions affecting single loci allow unam biguous association of spe-cific pheno types w ith single genes.

In a small-scale pilot screen, the efficiencies of ENU, EMSand X-rays at inducing recoverable m utations in zebrafish werecompared to each other (Table 1). ENU yielded about 1.2 muta-tions per haploid genom e, whereas only 0.02 and 0.09 m utationsper haploid genom e could be recovered after mutagenesis withEMS or X-rays respectively. This demonstrated that ENU is avery potent m utagen for recovering m utations in zebrafish.

The outcome of saturation screens

In Drosophila, saturation screens identified most if not allgenes with unique and indispensable function in embryonic

development (JOrgens et al., 1984 ; NOss le in -Vo lh ard et al..1984; Wieschaus e l a l. , 1984). They represent only a sm all fraction of all the essential genes in the fly. The sam e should be tru

for the large-scale screens presently carried out with zebrafishIn a sm all-scale pilot screen, 70% of the identified m utants wefound to display rather general defects (Mullins ela l.. 1 99 4).These genes are likely to encode proteins or genes that arerequired in m any cell types. The rem aining 30% of the mutanhad specific defects in em bryonic developm ent or organogene-sis. The num ber of mutations causing late lethality was foundbe sim ilar within the order of m agnitude to the number of embronic and early larval mutations (P.H. and F.v.E., unpublishedata). This suggests that the fraction of genes with specific anunique functions in early developm ent is about 15% of the toof all the essential gen es.

In a saturation screen often whole groups of genes involvein a specific developmental process are identified. This allow

one to study the functional relationship of these genes to eacother. Establishing epistatic relationships w ithin such a groupm utants allows ordering these genes in a developmental pathway. Double mutants will not only be useful in establishinepistatic relationships, but also reveal redundant functions caried by separate genes. Double mutant analysis in zebrafishenorm ously facilitated by the large num ber of progeny producefrom a single m ating.

Most genes encoding redundant functions will be missedthese screens. A prom inent example of redundant function ism yogenesis, where M yf-5 and M yoD can in part substitute for oano ther (Rudn icki e t a l. , 1993). Both genes need to be mutated toshow a complete loss of skeletal m uscle. M any genes with intee stin g e xp re ss io n p atte rn s h av e d is ap po in tin gly little o r n o v is ibeffect on development when m utated in the mouse. In many caes, this could be due to re dundancy and the fun ctional counterpa rs till re ma in s to b e id en tifie d. T he ta rg ete d g en e-k no ck ou t te ch noogy in the m ouse and the random m utational approach carried oin zebrafish are therefore com plem entary approaches tow ardid en tify in g d ev elo pm en ta lly im po rta nt g en es .

Some genes that form part of a developmental pathwayencode proteins that are required in many cell types, and willm issed in screens for specific loss-ot-function phenotypesHow ever, m utants identified in saturation screens serve as entpoints into developmental pathways, allowing the gaps tofilled using biochem ical approaches. The m utants will therebserve as valuable tools in elucidating the function of biochemcally characterized proteins in a sp ecific d evelop men tal processWhole mount antibody staining and in situ h yb rid iz atio n te chniques, which are well established in zebrafish (W estertield1 99 3), w ill g re atly fa cilita te s uc h e xp erim en ts .

Due to the transparency and accessibility o f z eb ra fis hem bryos, elegant cell lineag e tracing and tra nsplan tation expeiments can be carried out (Kim mel and W arga, 1988). Such tecniques allow one to study the migration of individual cellsgroups of cells in living mutant embryos and to study ceautonomous or non-autonomous requirements of mutatedgenes. The power of these techniques was dem onstrated when

F ig. 4. E xam ples of mutations with specific defects in the development of zebrafish embryos. AI embryos shown are 24 hours-old. (A IWildtype. (B I cycfops mutant with partial y fused eyes Hatta et a/..1993). (C ) Wildtype. (D ) cyclops mutant showing the absence of a floor pla Ha tta e t a l., 1993). IE ) Wildtype, (F ) no tail mutant which lacksa differen tiated n oto cho rd, h as no tai and ab norm ally shap ed som ite s Ha lp ern et1 99 3) . ( GI Wrldtype, (H ) spade tail m utant accum ulates trunk somitic m esoderm precursor cells in the ta il Ho et al., 1990).


5/7

Zebra fish genetics 225


6/7

226 P HajJrer alld C. Niiss/eill- Vo/hard

they w ere applied to a number of zebrafish m utants, three ofw hich are show n in Figure 4. cyclops m uta nt e mb ryo s have nofloorplate and are m issing part of the ventral forebrain resultingin p ar tia lly fused eyes (H atta et al., 1991). no tail mutantem bryos lack a differentiated notochord, have no tail and abnor-m ally shaped som ites (H alpern e t a l., 1993). spade tail mutantem bryos do not form somites in the trunk due to aberrant cellmigration during gast ru la tion leading to an accumula tiono f c ellsin the tail (Ho and Kane, 1990).These m utants have already giv-e n i nt er es ti ng in sig hts in to ce ll movements d u rin g g a st ru la tio nand the role of the floorplate and the notochord in dorsa-ventralpatterning. M any mutants with equally specific defects areexpected to be found in the ongoing s at ur atio n s cr ee ns .

How to clone the genes

A prerequisite for positional cloning of m utations w ill be ag en etic m ap o f th e z eb ra fis h g en om e. Two e ffo rts to wa rd s g en -erating such a m ap are under w ay. A R AP D (random am plifiedpolymorphic DNA) map using random decamer primers toamplify arbitrary DNA sequences by PCR was made by theg ro up o f J . P os tle th wa it (P os tle th wa it e t a l., 1994). This m apta ke s a dv an ta ge of th e po ss ib ility o f m akin g h ap lo id em bryo s,w hic h c irc um v en ts t he p ro ble m o f id en tify in g h ete ro zy go us in di-v id ua ls g en era lly p os ed b y th e R AP D m ap pin g te ch niq ue . In th ela bo ra to ry o f H . J ac ob , a n S SR (s im ple s eq ue nc e re pe ats ) m apusing PCR primers homologousto u niq ue s eq ue nc es fla nk in gC A re pe ats is b ein g g en era te d (Driever e t a l. , 1994), This map-ping technique can be done using diploid embryos since ita llo ws u na mb ig uo us d is tin ctio n o f h ete ro zy go us a nd h om oz y-gous individuals .

Both m aps will be invaluable in mapping mutations and

cloned genes to relative positions on the zebra f ish genom e.S om e m uta tio ns will turn out to be in previously identifiedg en es . A n e xa mp le fo r s uc h a fo rtu ito us m atc h is e xe mp lifie dbythe no tail gene, which was found to encode the zebrafishhom ologue of the m ouse TIBrachyury gene (Schu lt e-Merke r etal., 1994). Identifying closely linked molecular m arkers on agenetic m ap will provide a good starting point to undertake thepositional cloning of m utations. A num ber of laboratories arecurren tly cons truc ting genomic l ib ra ries appropria te for carryingo ut c hr om o so m al w a lk s c ov er in g s ev er al h un dr ed k ilo ba se sonth e 1 .6 xl0 9 b a se p air g en om e o f z eb ra fis h. T he p uff er fis h,Fugurubripes rubripes, h as a n orm al v erte bra te g en e re pe rto ire , b uta much lower amount of junk DNA such as repetitivesequences, pseudogenes and introns (Brennere t a l., 1 99 3) . If

gene order is conserved between the two fish species, thisw o uld d ra m atic ally s im p lif y p os itio na l c lo nin g, s in ce c hr om o so -m al walks could be done in the four tim es sm aller genom e ofthe pufferf ish.

C lo nin g of m utation s is o f co urse m uch e as ier if th e m uta tedgene is tagged by an insertion. Insertional m utagenesis inze bra fis h is n ot y et p rac tica ble d ue to the lo w e fficien cy o f in te -g ra tio n in to th e g en om e (S tu art e t a l. , 1988; Culp e t a l., 1 99 1),R ec en tly, e nc ou ra gin g p ro gre ss h as b ee n m ad e b y u sin g re tro -v iral vectors to infec t zebraf ish ce lls (Burnse t a l. , 1 99 3; U n et a l. ,1 99 4) . H ig h r ate s o f in te gra tio n a llo w in g th e is ola tio n o f in se rt io na lle le s b y no nco mp lem en ta tio n w ou ld en orm ou sly ex pe ditee ffo rts to wa rd s th e c lo nin g o f m uta te d g en es .

T he future of zebrafish

Having a mutant and its affected gene at hand are majorsteps in analyzing a developmental process. Drosophila pro-vides an excellent example of how having bothstimulatesresearch to understand the m echanisms underlying specificdevelopm ental program s. W ith its great em bryology, zebrafishw ill become an even m ore attractive m odel organism to studyvertebrate development when genes and their correspondingm u ta nt s a re a va ila ble .

The experience gained from systematic m utational screensw ill greatly im prove the use of zebrafish as a genetic system . Ina dd itio n to yie ld in g m uta nts w ith in te restin g p hen otyp es in d eve lopm ent, the screens w ill also produce a large collection of viablem utatio ns w ith visib le phenotypes in the embryo or the adult.These w ill serve as valuable genetic m arkers. an d like the mark.er mutations in Drosophila w ill a llo w m ore so phistica te d g en eticexperim ents in zebrafish. M ore specialized screens involvingparticular assays directed at m ore specific aspects of develop-m en t are fea sib le . A n exce lle nt e xam ple of su ch a sp ecific a ssa yis the screen presently being carried out in the laboratory of FB on ho ef fe r in T C lb in ge n ( pe rs on alcommunication), in w hicha nte ro gr ad e la be lin g of re tin ote cta l a xo ns is pe rio rm ed to visu al-iz e m uta tio ns a ffe ctin g re tin ote cta l p ro je ctio ns in z eb ra fish .

M utations identified tw enty years ago in saturation screens inDrosophila stHl occupy an enorm ous number of scientists whoare interested in developmental mechanisms at molecular lev-els. The potential offered by zebra fishas a genetic system islikely to stim ulate its use as a m odel organism for vertebratedevelopment.

Acknowledgments

We th an k Cosima Fabian and E lisab eth Vogelsang for ph otographsan d Robert G eisler, M ichael G ranato, Jorg G rosshans and M ary M uffinsfor critical comments on the manuscript. We also thank FriedrichB on ho effe r fo r co mm un ica tin g re su lts p rio r to p ub lica tio n.

References

BRENNER, S ., E LG AR , G ., S AN DF OR D , R ., M AC RA E, A ., V EN KATE SH , B . aAPARACIO , S . (199 3) . Cha rac te ri za ti on o f the p u ff er /i sh Fugu) genom e a s acompactmodelvertebrategenome.Nature366: 265-268.

B UR NS , J.C ., F RIED MAN N, 1., D RIE VE R, w ., BU RR AS CAN O, M . and Y EJ .K . ( 19 93 ). Ve si cu la r s to m at iti s v ir us G g ly co pr ote in p se ud oty pe d r et ro vi ravectors - c on ce ntra tio n to v ery high tite r a nd e fficie nt g en e tra nsfe r in tom am m alia n a nd n on ma mm alia n c en s.P ro c. N at . A ca d. S ci. U SA 9 0: 8 03 3-8037.

C HA KR AB AR TI, S .. S TR EIS IN GE R. G ., S IN GE R. F. a nd WAL KE R, C . (1 98Frequen c y o f gamma- ray induce d s pec if ic l ocus and rece s sive l etha l mu ta ti onsin ma tu re germ c ell s o f th e z eb ra fi sh , B ra ch yD an io r er io . G e ne ti cs 1 03 : 1 09 .124.

C UL P, P., N O SS LE IN -V OL HA RD , C . a nd H O PK IN S, N . (1 99 1). H ig h-fre qu eng er m-lin e tra ns mis sio n o f p la sm id D NA s eq ue nc es in je cte d in to fe rtiliz edzebral ish eggs. P ro c. N atl . A ca d. S ci . U S A88: 7953-7957.

O RIE VE R, W., S TE MP LE , D .. S CH IE R, A . a nd S OL NJ CA -K RE ZE L, L (1 99Zebra fi sh : gene ti c t oo ls for s tudying ve rt eb ra te d e ve lo p men t.T ren ds G en et. 5 :152-159.

ENDO, A . a n d IN G AL LS , T.H . 196B).C h ro m os om e s o lth a z eb ra f is h,a m od el lo rcyt ogene ti c, em bryo log ic , and ecolog ic s tudy.J . Hered i ty 59: 382-384.

FElSENFELD. A.l., W ALKER, C.. W ESTERFIELD, M., KIM MEL,C. andSTREISI NGER, G. (19 90) . Mu ta ti on s a ff ec ti ng sk el et al muscl e myof ib ri l s tr utur e in the zebraf is h . D eve lo pm en t 1 08 :443-59.


7/7

H AL PE RN , M E, H O, R .K ., WAL KE R, G . an d K IM ME L, C .B _(1993). I nd u ct ion o fm uscle pioneers and f100r plate is distinguished by the zebralish no-tail m uta-

tion. Cell 75 : 99 -111.

HATTA, K., KIM MEL, C.B., HO, R.K. and W ALKER, C. (1991). The cyclops m uta-

tion blocks specification of the floor plate of the zebrafish central nervous sys-

tem. Nature 350: 339-341.

H O, A .K . and KA NE, D .A. (1990). Cell-autonom ous action of zebrafish spt.1 m uta-tio n in s pe ci fic m es od erm al p re cu rs ors . Nature 348: 728-30.

JENKINS, J.B. (1967). The induction of mosaic and complete dumpy m utants in

Drosoph il a me lanogast er with ethylmethanesulfonate. M uta t. R es . 4 : 9 0- 92 .

JURGENS, G., W IESCHAUS, E. and NUSSLEIN.VO LHARD, C. (1984). Mutations

affecting the pattem of the larval cuticle inDrosoph il a me lanogast er II. Zygotic

l oc i o n the thi rd c h romosome. R ou x A rc h. D e v. B io i.193: 283-295.K IM ME L, G .B . (1 98 9). G en etic s a nd e arly d ev elo pm en t o f z eb ra lish .Tre nd s G e ne t.

5 : 2 83 -8 .

KIM MEL, C.B. and W ARGA, R.M.(1986), T is su e s pe cif icc ell lin ea ge s o rig in ate in

t he g as tru la o f t he z eb ra fis h, Science 231: 356-368.

KIM MEL, C.B. and W ARGA, A.M . (1988). Cell lineage and developmental poten-

tia l o f c ells in th e z eb ra fis h e mb ry o. Tre nd s G e ne t. 4 : 68 -74 .

KIMMEL, C.B., KANE, DA, W ALKER,CW ARGA, R.M. and ROTHMAN, M.B.

(1989). A m utation that changes cell m ovem ent and cell fa te in th e z e b ra fls h

embryo. Nature 337: 358-362 .

KIM MEL, C.B., W ARGA, A.M. and SCHILLING , T.F. (1990). Origin and organiza-t io n o f t he z eb ra fis h f ate m a p.Development 708: 581-94

LlN, S., GAIANO, N_, CULP, P., BURNS, J.C., FRIEDMANN, 1., YEE, J-K. and

H OP KIN S. N . (1 99 4). In te gra tio n a rn : g erm -lin e tra ns mis sio n o f a p se ud otyp ed

r et ro vi ra l v ec to r i n z eb ra fi sh . Science 2 65 : 6 66 -6 69 .

M ANSOUR, S.L.. THO MAS, K.A. and CAPECCHI, M.R. (1988). Disruption of the

proto-oncogene int.2 in m ouse em bryo-derived stem cells: a general strategy

fo r targ etin g m uta tio ns to n on -se le cta ble g en es . Nature 3 36 : 3 48 -3 52 .

M ULLINS, M .C. and NOSSlEIN-VOLHARD, C. (1993). M utational approachess tu dy in g e mb ryo nic p atte m fo rm atio n in th e ze bra fish . C ur ro O pin . G en et. Dev.

3 : 6 4 8- 6 54 .

MULLINS, M.C., HAMMERSCHMIDT, M., HAFFTER, P. and NUSSLEIN-

Zebra fish genetics 227

VOLHA RD , C . (1994) . l arge .s ca le m uta g e n e s is in th e z e b ra fis h : in s e a rc hg en es c on tro llin g d ev elo pm e nti n a ve rt eb ra te . CurroBioI.4 : 189-202 .

N U SS LE IN -V O LH A RD , C . a nd W IE S CH A US , E . ( 19 80 ). M u ta tio ns a ffe cti ng s egm e nt n um b er a nd p ol ar it y inDrosophila . Nature2 87 : 7 95 .8 01 .

N OS SL EIN .V OlH AR D, C ., W IE SC HA US , E . a nd K lU DIN G,H . 1 98 4). M u ta tio nsa ffe c tin g th e p a tte rn o f th e la rv a lcu ti cl e i n Drosophila melanogaster 1.Zygotic

loc i on the secondchromosome.RouxArch.Dev.BioI.193:267-282.P OS TL ET HWA IT, J .H ., JO HN SO N. S L, M ID SO N, C .N ., TAL BO T. W. S., G AT ES , M .

B AL LIN GE R. E W., A FR IC A, D . A N DR EW S , R ., C AR L, 1 ., E IS EN , J .S ., H OR NE ,S., K IM MEL , C.B., H UTC HIN SO N, M .. JO HNSO N, M . and RO DRIG UEZ , A(1994).A geneticlin ka ge m a p fo r th e z eb ra fis h.Science264: 699-703.

RUDNICKI, M .A ., S GH NE GE lS BE RG , P.N .J., ST EA D, R .H ., B RA UN , 1.,A RN OL D, H -H . a nd J AE NIS CH ,R . 1 99 3).M yo D o r M yf- 5 is re qu ire d fo r th eformat io n o f ske le ta l musc le .Cell 75: 1351.1359

S CH ULTE -M ER KE R, S ., VAN E ED EN , F.J .M ., H AL PE RN , M .E ., K IM ME L,C.B.and NOSSLEINNOLHARD,C.(1994). n o tall nrl)is the zebrafishh o m o l o g u eo f th e m ou s eT Brachyury)gene. D ev elo pm en t 1 20 : 1 00 9. 10 15 .

S OlN IC A-K RE ZE l, l ., S CH IE R, A .F. a nd D R lE VE R,W . 1 99 4). E ffic ie nt re co ve ryofENU -inducedmutation sfrom the zebrafishg e r m l i n e . Genetics 136: 1401.1420.

S TR EIS IN GE R, G ., WAL KE R, C ., D OW ER .N _, K NA UBE R, D. and SIN GER, F.

(1981). Product ion o f c lo n e s o f h o m oz y g o u s d ip lo id z e b ra fis h BrachyDan;orerio).Nature291: 2 9 3 - 2 9 6 .

STUART,GW M c M U RR AY, ,V. a nd W E ST E RF IE L D,M. ( 1988) . Rep li ca ti on ,i nt eg ra ti on and s ta b le ge rm - li ne t ransm i ss ion o f for eigns eq ue nc es in je cte d in tear ly zebraf ish embryos . D eve lopmen t 10 :403 -412.

W E ST ER FIE LD , M . (1 99 3).T h e Z e br af is h B o o k U niv ers ity o f O re go n P re ss ,Eugene.

W EST ERFIE lD , M ., lIU . O W., K IM ME L. C.B . and WAL KE R,C. 1 9 9 0 ) .P a t h f i n d i n g and synapseformat ion in a zebraf ish mutant lacking functionalacetylcholine receptors. Neuron 4 : 8 67 .7 4 .

WIESCHAUS,E., NOSSLEIN-VOLHARD,C . a nd J OR G EN S ,G. (1984) . Mutationsa ff ec ti ng the pa tt ern o f t he l arva l cu ti cl e i nDrosophila melanogaster III. Zygoticl oc i on the X-ch romos omea n d f ou rt hchromosome.RouxArch.Dev.Bioi. 193.296-307.

Documents

Large Scale Genetics in a Small Vertebrate, The Zebrafish