zebrafish hox genes: expression in the hindbrain - development

393Development 125, 393-406 (1998)Printed in Great Britain © The Company of Biologists Limited 1998DEV1185

Zebrafish hox genes: expression in the hindbrain region of wild-type and

mutants of the segmentation gene, valentino

Victoria E. Prince 1,*, Cecilia B. Moens 2, Charles B. Kimmel 2 and Robert K. Ho 1

1Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA2Institute of Neuroscience, University of Oregon, Eugene, OR 97401, USA∗ Author for correspondence (e-mail: [email protected])

Accepted 5 November 1997: published on WWW 13 January 1998

The developing hindbrain is organized into a series ofsegments termed rhombomeres which represent lineagerestricted compartments correlating with domains of geneexpression and neuronal differentiation. In this study, weinvestigate the processes of hindbrain segmentation andthe acquisition of segmental identity by analyzing theexpression of zebrafish hox genes in the hindbrains ofnormal fish and fish with a loss-of-function mutation inthe segmentation gene valentino (val, the homologue ofmouse kreisler; Moens, C. B., Cordes, S. P. Giorgianni, M.W., Barsh, G. S. and Kimmel, C. B. (1998). Development125, 381-391). We find that zebrafish hox genes generallyhave similar expression profiles to their murine and aviancounterparts, although there are several differences intiming and spatial extent of expression which mayunderlie some of the functional changes that haveoccurred along the separate evolutionary lineages of

teleosts and tetrapods. Our analysis of hox geneexpression in val− embryos confirms that the val geneproduct is important for subdivision of the presumptiverhombomere 5 and 6 territory into definitive rhom-bomeres, suggests that the val gene product plays acritical role in regulating hox gene transcription, andindicates that some neural crest cells are inappropriatelyspecified in val− embryos. Our analysis of gene expressionat several developmental stages has allowed us to infer dif-ferences between primary and secondary defects in thevalmutant: we find that extended domains of expression forsome hox genes are secondary, late phenomena potentiallyresulting from inappropriate cell mixing or lack of normalinter-rhombomeric interactions in the caudal hindbrain.

Key words: Zebrafish, Hox genes, Hindbrain, Rhombomere, kreisler,valentino,Segmentation

SUMMARY

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INTRODUCTION

Studies on a range of different vertebrates have revealed the rostrocaudal axis of the developing hindbrain is transiensubdivided into a series of reiterated segments termed rhbomeres (reviewed by Guthrie, 1996; Lumsden and Krumla1996) which play a pivotal role in organizing the structure afunction of the vertebrate head. Cellular studies in the chhave revealed that rhombomeres are lineage restricted cpartments (Fraser et al., 1990; Birgbauer and Fraser, 19which correspond to the segmental organization of reticuneurons, branchiomotor nerves and sensory ganglia (Lums1990). Rhombomeric organization also correlates with tformation and migration of the cranial neural crest (Lumsdet al., 1991; Schilling and Kimmel, 1994; Trainor et al., 1994Comparative studies have suggested that these general prties of hindbrain organization are conserved within the vtebrates (Gilland and Baker, 1993).

There appears to be a fundamental two-segment periodialong the rostrocaudal axis of the developing hindbrain whinfluences both the formation and the patterning of the rhobomeres. Studies in chick have shown that rhombomboundary formation requires alternating rhombomeric stat

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when odd and even-numbered rhombomere cells are juxposed, a third state is achieved leading to boundary formati(Guthrie and Lumsden, 1991; Guthrie et al., 1993). This twosegment periodicity is reflected by neuronal organizatio(Lumsden and Keynes, 1989), and by the expression patternsdevelopmental control genes. These genes include receptosuch as eph-like receptor tyrosine kinases, their ligands, atranscription factors such as the zinc-finger gene Krox-20 andthe Hox genes (reviewed by Lumsden and Krumlauf, 1996). Imouse and chick, the anterior limits of Hox gene expressiongenerally define two segment regions out of register with thodefined by the branchiomotor neuron pools (Wilkinson et a1989b; Hunt et al., 1991a,b). Seven rhombomeres have bedescribed for the zebrafish, each sharing a common internstructure (Hanneman et al., 1988; Trevarrow et al., 1990). Tearliest neurons to differentiate, the primary reticulospinaneurons that are born near the end of gastrulation, form a laddlike array that corresponds to locations at the centres of the invidual rhombomeres (Mendelson, 1986a,b; Metcalfe et a1986; Hanneman et al., 1988). The cranial motoneurons derfrom pairs of rhombomeres, for example, the trigeminal (Vtnerve) derives from r2 and r3, the facial (VIIth nerve) from r6and r7, and the abducens (VIth nerve) from r5 and r6 (Cha

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drasekhar et al., 1997; Trevarrow et al., 1990; Gilland aBaker, 1993). A similar pairwise derivation of motoneurons hbeen described for the mouse, although, interestingly, specific rhombomeres in which individual motoneurons diffeentiate vary between species (Gilland and Baker, 1993).

The Hox genes are implicated in conferring segmentidentity, both by homology to the Drosophilahomeotic genesand from functional approaches in vertebrates. For exammice with targeted disruptions of the Hoxb-1 gene showchanges in r4 identity (Goddard et al., 1996; Studer et 1996). Disruptions of the Hoxa-2 gene, which is normallyexpressed in r4-derived neural crest, lead to abnormal patting of the crest-derived second branchial arch cartilaelements (Gendron-Maguire et al., 1993; Rijli et al., 199When Hoxa-1 is overexpressed, either in transgenic mi(Zhang et al., 1994) or by ectopic expression in zebrafi(Alexandre et al., 1996), the phenotype is reminiscent of clashomeosis: r2 takes on aspects of r4 identity. However, the pnotypes of Hoxa-1disruptions hint at the possibility that somvertebrate Hox genes may not only play a role in conferrinsegmental identity but also in segmentation per se. The Hoxa-1 gene has an early anterior expression limit at the boundbetween rhombomeres 3 and 4 (r3/r4 boundary) but expresregresses rapidly out of the hindbrain. Two separate mutatifor this gene have been generated (Carpenter et al., 1993; Met al., 1993; reviewed by Wright, 1993) both lead to severe druption of the hindbrain between r4 and r7, with r5 beireduced or deleted. This potential dual role of the Hox genes,in setting up segmentation, as well as in conferring segmeidentity, may explain the apparent paucity of candidate smentation genes. Only two other candidate segmentation gehave been described, the zinc-finger transcription factor Krox-20, which is expressed in presumptive r3 and r5 (Wilkinsonal., 1989a), and the kreisler(kr) gene which encodes a bZIPtranscription factor (Cordes and Barsh, 1994). Targeted ruptions of Krox-20lead to loss of r3 and r5 (SchneideMaunoury et al., 1993; Swiatek and Gridley, 1993), and Krox-20 directly regulates transcription of Hoxa-2 and Hoxb-2(Sham et al., 1993; Nonchev et al., 1996). In kr mutant mice,the neural tube appears unsegmented caudal of the rboundary (Frohman et al., 1993; McKay et al., 1994).

Normal segmentation of the hindbrain is similarly disruptein zebrafish mutant for valentino (val) (Moens et al., 1996);val− embryos also lack visible hindbrain segmentation cauto the r3/r4 boundary. It has recently been demonstrated val is the zebrafish homologue of mouse kr (Moens et al.,1998). The val gene product is expressed throughout r5 andand mosaic analysis has suggested that val is required cell-autonomously for normal subdivision of r5 and r6 from a hypthetical common precursor region (Moens et al., 1996). In absence of functional val gene product, this common precursoregion is maintained as rX, a region of one rhombomerlength that lies between, yet fails to form boundaries with,and r7. Analysis of neurons and neuronal cell types within mutant hindbrain has shown that rX has a unique idenwhilst incorporating some aspects of the identities of bothand r6. For example, the primary reticulospinal neurons Miand MiD3, characteristic of r5 and r6 respectively, are bopresent in rX in the normal anteroposterior order, although spacing between these neurons is reduced. However, the ldifferentiating r5- and r6-specific cells of the abducens (V

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In this study we have investigated the processes of hindbrsegmentation and the acquisition of segmental identity analyzing the expression of zebrafish hox genes in the devel-oping hindbrain. To date, detailed expression analysis of oone such gene, hoxa1, has been reported (Alexandre et1996). We have carried out expression analyses for a furthehoxgenes; our results allow a comparative approach by corlation with available data from the mouse, human, chick, aXenopussystems and we find that the zebrafish hoxgenes sharemany expression properties with their tetrapod homologualthough some details of temporal and spatial pattern do difIn addition, we have investigated hox gene expression inmutants of the hindbrain segmentation gene val, and haverelated our results to those obtained with mutants of its murhomologue kr, allowing a comparison of the patterning mechanisms at work in the zebrafish and mouse hindbrains. We hused our results to address the apparent conundrum that saspects of segmental identity are maintained in the mutants the reticulospinal neurons are normally patterned) despite lof overt segmentation in the caudal hindbrain. Our detailanalysis of gene expression at a variety of developmenstages in mutant and wild-type embryos has helped to shed light on the detailed temporal changes in cell identity occurriduring rhombomere formation and patterning. Our resusuggest that the valgene product functions both in the segmentation process, by subdividing two rhombomeres frotheir common precursor, and in aspects of the acquisitionsegmental identity, by regulating hoxgene expression.

MATERIALS AND METHODS

ZebrafishZebrafish (Danio rerio) embryos were obtained from naturaspawnings and staged as described by Kimmel et al. (199valb337/valb337 embryos were produced by crossing valb337/val+ fishtogether, yielding wild-type and mutant embryos in a 3:1 ratio. Coparisons of the expression of hox genes in wild-type, heterozygousval+/− mutants and homozygous val−/− mutants showed that the het-erozygotes were indistinguishable from wild types.

Hox gene cloningRACE-PCR was carried out as previously described (Frohman, 199cDNA was reverse transcribed, using the Gibco-BRL Superscript according to manufacturer’s instructions, from 24-hour zebrafiembryo RNA prepared as described by Chomczymski and Sac(1987). PCR reaction conditions were : 1 cycle at 94°C 2 minutes;cycles at 94°C 1 minute, 45-50°C 2 minutes, 72°C 2.5 minutescycle at 72°C 10 minutes. PCR products were cloned into the PrompGEM-T cloning vector, or the EcoRV site of pBluescript SK(−)(Stratagene) according to manufacturer’s instructions.

Primers were designed based on published sequences of homecDNAs (Njølstad et al., 1988a; Rundstadler and Kocher, 1991; Miset al., 1996).

Primer sequences (names previously given to these genesindicated in parentheses):

hoxb1 (Misof et al., Z-3):5′ GCAAGTATCTGACGCGAGCAC and 5′ CACGGCGTGTG-

GAGAATTGCTGhoxa2 (Misof et al., Z-75):

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T R R R R I E I A H A L C L T E R Q I K I W F Q N R R M K W K K E N K

S R A R R V E I A A T L E L N E T Q V K I W F Q N R R M K Q K K R E RT R A R R V E I A A T L E L N E T Q V K I W F Q N R R M K Q K K R E KC R P R R V E I A A L L D L T E R Q V K V W F Q N R R M K H K R Q T QC R P R R V E I A A L L D L T E R Q V K V W F Q N R R M K H K R Q T QC R P R R V E I A A L L D L T E R Q V K V W F Q N R R M K H K R Q T EC R P R R V E I A A L L D L T E R Q V K V W F Q N R R M K H K R Q T TC R P R R V E M A N L L N L S E R Q I K I W F Q N R R M K Y K K D Q KC R P R R V E M A N L L N L S E R Q I K I W F Q N R R M K Y K K D Q KC R P R R V E M A N L L N L T E R Q I K I W F Q N R R M K Y K K D Q KC R P R R V E M A N L L N L T E R Q I K I W F Q N R R M K Y K K D Q KT R R R R I E I A H T L C L S E R Q V K I W F Q N R R M K W K K D H KT R R R R V E I A H T M C L S E R Q V K I W F Q N R R M K W K K D H KT R R R R V E I A H A L C L S E R Q I K I W F Q N R R M K W K K D H KT R R R R V E I A H T L C L S E R Q I K I W F Q N R R M K W K K D H K

omeobox sequences for 7 zebrafish hoxgenes with their likely murinech case the first few amino acids of the zebrafish sequences are taken; the PCR primers used to clone these cDNAs were based on theseences are compared to the Drosophila Antennapediasequence. Blacknserved with Antp. All sequences have been submitted to the EMBLos Y13944-13950.

5′ AACAAG/ATAAT/CCTG/TTGC/TCGGCG/C and 5′ GGC-CAAGGCGTGTGGAAATC

hoxb2 (Misof et al., Z-151):5′ AACAAG/ATAAT/CCTG/TTGC/TCGGCG/C and 5′

GGCGCAGGCGCGTTGAAATThoxb3 (Misof et al., Z-56):5′ TTCAACCGA/CTACCTGTGC/TCG and 5′ GGCCGAG-

GCGTGTGGAAATGhoxd3 (Misof et al., Z-92):5′ TTCAACCGA/CTACCTGTGC/TCG and 5′ GCCCCAGAA-

GAGTGGAGATGhoxx4 (Rundstadler and Kocher, zf26):5′ AGAGGTCTCGCACCGCCTAC and 5′

CCAGCAGGCTCTTGAGCTTGhoxb4 (Misof et al.,Z-17; Njølstad et al., zf13)5′ TTACAACCGCTATCTGACCCG and 5′ CAGAA-

GAAGGGTGGAAATCGCClones were screened by sequencing of double stranded temp

(Sequenase, US Biochemicals Inc.) from forward and reverse primthe most 5′ 400 bp (at minimum), of coding sequence and 3′ untrans-lated region, were then sequenced in both directions using inteoligonucleotide primers. Sequence analyses and comparisons performed using the Wisconsin genetics GCG software package. logenetic tree analysis was performed with the Megalign modulethe Laser gene programme (DNASTAR, Inc.). All the sequendescribed are available in the EMBL database under accesnumbers Y13944-13950. The sizes (in base pairs) of the cDNobtained for each gene are indicated as follows.

hoxb1, 1100; hoxa2, 1000; hoxb2, 900; hoxb3, 600; hoxd3, 800;hoxx4, 900; hoxb4, 1600.

In situ hybridizationIn situ hybridizations were performed essentially as previoudescribed (Thisse et al., 1993) with the following modificationEntire 3′RACE-PCR derived cDNAs were used as templates to sthesize antisense riboprobes with T7, T3 or SP6 RNA polymer(Promega) as appropriate; probes were not hydrolyzed. Proteinatreatment (10 µg/ml in PBT) time was reduced to approximately 3seconds per somite; the proteinase K reaction was stopped by retion in 4% paraformaldehyde in PBS.

Two colour in situ hybridizations were performed essentially described by Hauptmann and Gerster (1994); Jowett and Le(1994). Briefly, the antisense riboprobe to one gene was labeled digoxigenin-UTP (Boehringer Mannheim) and the second prolabeled with fluorescein-UTP (BoehringerMannheim), the probes were thenhybridized to the embryo simultaneously.The digoxigenin-labeled probe was visual-ized with anti-digoxigenin alkaline phos-phatase (Boehringer Mannheim) reactedwith NBT and BCIP to produce a bluecolour; glycine treatment (0.1 M glycine-HCl pH 2.2, 0.1% Tween-20, 10 minutes atroom temperature) was then used to removeanti-digoxigenin alkaline phosphataseconjugate before application of anti-FITCalkaline phosphatase conjugate (BoehringerMannheim) and detection with Fast Red(Sigma). In situ hybridizations were stoppedby washing in PBS, 20 mM EDTA. Doublestained embryos were mounted in 80%glycerol in PBS, single stained embryoswere dehydrated through a methanol series,cleared with benzyl benzoate and mountedin Permount. The zebrafish krox-20riboprobe was as previously described(Oxtoby and Jowett, 1993), the valentino

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RESULTS

Cloning of zebrafish hox genes expressed in thedeveloping hindbrainWe have obtained cDNAs of 7 zebrafish hox genes, fromparalogue groups 1 through 4, which are expressed withindeveloping hindbrain. The 3′ RACE-PCR technique (Frohman1993) was used to amplify cDNAs based on publishsequences of homeobox regions (Materials and MethoNested primers were targeted to the 5′ half of the homeoboxdomain and RT-PCR was used to amplify the region betwethe specific primers and the 3′end of the message. We havused this approach to obtain relatively long cDNAs (600-16bp; see Materials and Methods) facilitating further charactezation of the genes and the synthesis of specific riboprobeshave identified individual genes based upon sequence comison, and in the majority of cases by locating the genesspecific hox clusters (see below). Furthermore, similaritiebetween the expression patterns of our zebrafish genes anequivalent murine and chick genes are consistent with assignments. The sequences of the homeoboxes of thisolated genes are shown in Fig. 1.

We have assigned zebrafish hoxb1, hoxb2, hoxb3and hoxb4to the hoxbcluster based upon their presence on a single Yclone (A. Fritz, unpublished; YAC library, L. Zon., personacommunication) which also contains the previously identifigenes hoxb5and hoxb6(previously designated ZF21and ZF22respectively; Njølstad et al., 1988b,c, 1990). Presence of genes on the YAC was determined by PCR with primers frwithin the 3′ untranslated sequence of each gene (data shown). Consistent with these results the zebrafish hoxb2andhoxb4 genes are absent in a mutant with a large deletencompassing the hoxb cluster (Andreas Fritz pers. commFritz et al., 1996). The homeobox sequence of zebrafish hoxb1has only a single amino acid difference to chick Hoxb-1, thezebrafish hoxb2 and hoxb3 homeobox sequences both sho100% identity to their mouse and human homologues. T

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zebrafish hoxb4 gene is 100% identical at the DNA level to thpreviously cloned zf-13gene (Njølstad et al., 1988a), this genwas previously incorrectly identified as hoxd4 based onsequence alignments (Misof et al., 1996). The homeobox ofzebrafish hoxb4 gene has just a single amino acid chancompared to its mouse and human homologues.

The homeobox of the gene we assign as zebrafish hoxd3 has100% amino acid identity with mouse and human Hoxd-3, andis linked to hoxd4 on hybrid chromosomes (Marc Ekkepersonal communication; Ekker et al., 1996). The homeoof the gene we identify as zebrafish hoxa2shows 100% aminoacid identity with mouse Hoxa-2 but also with Hoxb-2. Weassign this gene to the acluster because its identity with mousHoxa-2 is 67% over the entire 210 amino acids sequenccompared to only 41% with human Hoxb-2 (no Hoxc-2 orHoxd-2 genes have been reported from other species).addition, this gene shows diagnostic features of theHoxa-2expression pattern (see below) and we already have a candhoxb2gene. Finally, the last gene we analyzed, which we tezebrafish hoxx4,is 100% identical to the previously clonedzf-26 gene (Rundstadler and Kocher, 1991); this gene was pr

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ously identified as hoxb4based on sequence alignments (Miset al., 1996; Rundstadler and Kocher, 1991), however already have a clear example of a hoxb4 gene. The homeoboxof hoxx4shows 95% amino acid identity with both mouse ahuman Hoxa-4and phylogenetic tree analysis suggests hoxx4to be most closely related to the Hoxa-4 genes of other specieshowever we are unable to unambiguously assign this genthe a cluster.

Expression of zebrafish hox genes during normalhindbrain developmentWe analyzed the expression patterns of the 7 zebrafish hoxgenes by whole-mount in situ hybridization in normal zebrafiembryos at stages between 6 hours (50% epiboly) and 30 hof development (Fig. 2). In many cases double in situ hybrizations are shown using krox-20 (the zebrafish homologue omurine Krox-20; Oxtoby and Jowett, 1993) as a secomolecular marker to indicate the locations of rhombomereand 5. The expression patterns of the zebrafish hoxgenes showmany properties in common with their murine homologueconsistent with our gene assignments.


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Paralogue group 1 genesThe zebrafish hoxb1gene shares the characteristic expressdomain in rhombomere 4 of the developing hindbrain exhibiby the Hoxb-1genes of mouse, chick, human, Xenopusandanother teleost fish, the carp (Frohman et al., 1990; SundinEichele, 1990; Vieille-Grosjean et al., 1997; Godsave et 1994, Stevens et al., 1996; Fig. 2A). The onset of zebrahoxb1 expression is during gastrulation, between 80% a100% of epiboly with an anterior limit at approximately thlevel that will give rise to r4; this is similar to the situation the carp (Stevens et al., 1996), but unlike the situatdescribed for mouse and chick in which initial expressionconfined to the posterior of the embryo with the expressdomain then spreading forward until the anterior expresslimit is reached (Frohman et al., 1990; Sundin and Eiche1990). Up to the 1 somite (1s) stage, zebrafish hoxb1expression is continuous from pre-r4 posterior throughout CNS (Fig. 2Ai). At the 3s stage, expression is down-regulain the region immediately caudal to r4, this is clearly indicatby the reduced level of hoxb1staining in r5 (Fig. 2Aii); r5 iseasily identified by krox-20expression (red signal). The regression of hoxb1expression (continued down-regulation movinposterior from r4), a phenomenon which has also been repofor other species, continues during the next few hours of deopment (Fig. 2Aiii, iv); concomitant with this procesexpression levels in r4 become significantly up-regulated.the 10s stage, a low level of hoxb1expression is visible fromthe level of r7 and posterior (Fig. 2Aiii), but by the 15s sta

Fig. 2. Expression of hoxgenes in the developing hindbrain; whole-mount in situ hybridization with 7 different hoxgene riboprobes(purple signal). In most specimens krox-20was used as a secondmarker to allow orientation with respect to r3 and r5 (red signal).Embryos were dissected off the yolk and flat-mounted between coslips for photography. In all panels except E(i) only the hindbrainregion is shown, anterior is to the left. Scale bar, 50 µm, where noscale bar shown scale is equivalent to panel above. (A) hoxb1 isexpressed in presumptive r4 and posterior from the onset ofexpression at approximately 90% epiboly, through the 1 somite (1stage (i); by the 3s stage (ii) expression in r4 is up-regulated, notelies between the r3 and r5 krox-20 expressing domains (red); hoxb1expression in r5 and r6 is concomitantly down-regulated. At the 1stage (iii) the r4 expression domain has sharp borders, abutting thkrox-20 expression domains in r3 and r5; more posterior expressiois at significantly lower levels in r7 and posterior. At the 20s stage(iv) the same pattern persists, but expression posterior of r4 is nolonger apparent in the CNS. (B) hoxa2is expressed in presumptiver2 and r3 from the onset of expression at the 2s stage (i), the samexpression domain persists through the 5s stage (ii). At the 10s s(iii) high level expression persists in r2 and r3 (note sharp anteriorlimit of expression at r1/2 boundary); low level expression has sprposteriorly through r4 and r5 (note overlap with krox-20 expressiondomains). At the 20s stage (iv) neural expression persists in r2through r5, there are differences in expression levels between theindividual rhombomeres such that the highest level is in r2, a slighreduced level in r3, further reduced in r5 and lowest of all in r4.Neural crest migrating into the 2nd and 3rd branchial arches (arroalso expresses hoxa2. (C) hoxb2has an anterior expression limit onerhombomere length more posterior than that of its paralogue, hoxa2,at the r2/3 boundary. The onset of expression is at approximately1s stage (i) in presumptive r3 and r5, this was determined in othespecimens by colocalization with krox-20; however, to avoidobscuring the weak r5 expression domain the sample shown is asingle in situ with only the hoxb2probe. By the 3-4s stage (ii) the

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only the r4 expression domain is clearly visible in the CN(data not shown; Fig. 2Aiv). Transverse sections through r4,the 5s and 10s stages, reveal transient expression of hoxb1inneural crest cells migrating out of r4 (data not shown).

The hoxb1expression pattern shares some features with thof the paralagous gene hoxa1(Alexandre et al., 1996). Theonset of expression of hoxa1 is somewhat earlier, at 50%epiboly or germ ring stage. However, by tailbud stage the twgenes share rather similar expression domains with distianterior borders approximating to the location of presumptir4. As in other species hoxa1 expression rapidly regressescaudally, without leaving behind an r4-specific expressiodomain as seen forhoxb1.The differences in the expressionpatterns between the two genes are consistent with their assment to thehox a andb clusters respectively.

Paralogue group 2 genesThe zebrafish hoxa2gene has an anterior expression limit inthe CNS similar to those of the Hoxa-2genes of chick, humanand mouse, namely at the r1/r2 boundary (Prince and Lumsd1994; Vieille-Grosjean et al., 1997; Frasch et al., 1995). Tonset of detectable expression is at approximately the 2s st(Fig. 2Bi), and commences as a single hindbrain ‘stripe’ in thregion destined to give rise to r2 and r3. Similar to hoxb1, thisearly expression pattern differs from that observed in the chicwhere expression commences in the most posterior part of embryo and is gradually activated from posterior to anteriuntil the final r1/r2 boundary limit is reached (Prince an

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level of r5 expression has increased and there is low level expressionin r4 between the two high level domains, in addition there is lowlevel expression posterior to r6. By the 10s stage (iii) expression isconfined to rhombomeres 3, 4 and 5, with a step-wise expressiongradient from anterior to posterior (highest levels in r3). At the 20sstage (iv) this step-wise gradient persists and expression can also beseen in neural crest cells migrating out into the second branchial arch(arrow). (D) hoxb3has an onset of expression at the 1-2s stage,within the CNS posterior to r5, by the 3-4s stage (i) expressionoverlaps the r5 krox-20domain, the anterior-most expressiondomain, in presumptive r5 and r6, is at elevated levels compared tomore posterior expression. The expression limit and elevatedexpression levels in r5 and r6 are maintained through the 5s (ii) and10s (iii) stages. At the 10s stage (iii) neural crest migrating into the3rd branchial arch from the posterior part of r6, is also expressinghoxb3. By the 30 hour stage (iv) a low level expression domain in r4has become apparent (arrow). (E) hoxd3also has an expression onsetat the 1-2s stage (i) in the posterior most part of the embryo, by the3-4s stage (ii) expression has spread rapidly anteriorly to abut the r5expression domain of krox-20. This r5/6 expression limit ismaintained through the 10s stage (iii) and 20s stage (see Fig. 5I). By30 hours (iv) a more anterior expression domain in a smalllateral/ventral group of r5 cells is visible (arrow). (F) hoxx4 has anexpression onset at about the 1s stage (i) with an anterior limit lyingapproximately within r7, expression is at highest levels toward theanterior limit. This expression pattern is maintained through the 5s(ii) and 10s (iii) stages, with the anterior expression limit lyingwithin r7. Even by 30 hours no sharp anterior boundary has beenreached (iv). (G) hoxb4 has a similar expression onset to itsparalogue, hoxx4, at the 1s stage (i) approximately within r7. Thisexpression domain is maintained at the 5s stage (ii), by the 10s stage(iii) the limit of hoxb4 expression is slightly more anterior than thatof hoxx4, reaching the r6/7 boundary by the 15s stage (data notshown). The anterior expression limit is maintained at the r6/7boundary through 30 hours of development (iv).

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Lumsden, 1994). By the 10s stage, zebrafish hoxa2expressionhas expanded posteriorly into r4 and r5, although this posteexpression domain is at significantly reduced levels compato the r2+r3 domain (Fig. 2Biii). Note that by this stage texpression limits have become sharp, possibly reflecting time of morphological rhombomere boundary formation. Comencing at approximately the 12s stage (data not shown), clearly visible by the 20s stage (Fig. 2Biv), expression canseen in neural crest cells migrating out into the second and tbranchial arches (i.e. the hyoid and first gill arches). Expressis maintained in r2 through r5 at the 20s stage, but theredifferences in the expression levels in individual rhombome(Fig. 2Biv); in particular the expression level in r4 is reducrelative to the other rhombomeres.

Zebrafish hoxb2 also shows a similar anterior expressiolimit to that of its murine and human homologues, at the r2boundary (Wilkinson et al., 1989b; Hunt et al., 1991a; VieillGrosjean et al., 1997; Fig. 2C). The onset of zebrafish hoxb2expression is at approximately the 1s stage, and similahoxa2, expression commences at the anterior expression limin this case in pre-r3 (determined by double labeling with krox-20; data not shown). There is also expression in pre-r5 (Fig. 2ii); this colocalization of hoxb2and krox-20 is suggestive ofhoxb2 activation by the krox-20 gene product, as has beedescribed for the mouse (Sham et al., 1993). By the 10s stexpression is primarily localized to r3, r4 and r5 with higheexpression levels towards the anterior (Fig. 2Ciii); in somspecimens a region of low level expression was also noted fr7 and posterior. This pattern continues through the 20s s(Fig. 2Civ), with spinal cord expression levels graduadecreasing, and is maintained to at least 30h of developmFrom about the 12s stage, expression is seen in neural crestmigrating from r4 into the second arch. These cells are clevisible at the 20s stage (Fig. 2Civ). There is also transient lowlevel expression, between approximately the 12s and 18s stain neural crest cells migrating into the third branchial arch.

Paralogue group 3 genesThe zebrafish hoxb3gene shares an expression limit at the r4boundary with the group 3 paralogue genes of mouse, humand chick (Wilkinson et al., 1989b; Hunt et al., 1991a; VieillGrosjean et al., 1997; Itasaki et al., 1996; Fig. 2D). The onsehoxb3 expression is at the 1-2s stage, commencing posteriothe r5 krox-20 domain in both the CNS and the developinsomites (data not shown). By the 3-4s stage, the expresdomain expands forward to reach an anterior limit at the r4boundary (Fig. 2Di). At the 5s stage the most anterior expresdomain in r5+r6 is at significantly higher levels than moposterior (Fig. 2Dii). This expression pattern is maintained at 10s stage (Fig. 2Diii); neural crest cells just beginning to migrout of the posterior limit of r6, towards the 3rd branchial arcalso express hoxb3. From the 20s stage, a low level expressis observed more anteriorly, throughout r4 (see Fig. 5Expression of the paralogue group 3 genes in mouse and chas not been reported in r4 (Wilkinson et al., 1989b; Hunt et1991a; Itasaki et al., 1996). At the 30 hour stage, expreswithin r4 is still visible although it is now confined to the ventr(basal) part of the rhombomere (Fig. 2Div).

The expression of the zebrafish hoxd3gene also begins atthe 1-2s stage, in this case expression commences in the posterior part of the embryo (Fig. 2Ei), more in line with th

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expression patterns reported in tetrapod species. expression domain expands rapidly toward the anterior to rethe r5/r6 boundary by the 3-4s stage (Fig. 2Eii). This anteexpression limit, which is one rhombomere more posterior ththe limit reported for the mouse or human (Wilkinson et a1989b; Hunt et al., 1991a; Vieille-Grosjean et al., 1997), is vprominent and is maintained up to at least the 20s stage (2Eiii, 5I). However, by the 30 hour stage there is also limitexpression within r5; this expression is confined to a smregion in the basal plate of the rhombomere (Fig. 2Eiv). Llevels of hoxd3 expression were seen in neural crest cmigrating towards the 3rd branchial arch in some overstaispecimens at the 20s stage (data not shown).

Paralogue group 4 genes The murine Hoxa-4, Hoxb-4andHoxd-4genes are reported tohave anterior expression limits at the r6/r7 boundary, althouthere are differences in the expression levels of these gewithin their anterior domains (Wilkinson et al., 1989b; Hunt al., 1991a). The two zebrafish group 4 paralogues whichhave analyzed share this anterior expression limit. Tzebrafish hoxx4gene has an expression onset at the 1s stathe anterior limit of expression appears to be approximatwithin r7 by comparison to krox-20 (Fig. 2Fi). This genecontinues to have an anterior expression limit lying within but not reaching the r6/r7 boundary, up to at least the 30 hstage (Fig. 2Fii-iv). The zebrafish hoxb4gene has a similartime of onset and expression pattern at early stages (Fig. ii). By the 10s stage the anterior limit of hoxb4expression isa little ahead of that of hoxx4(compare Fig. 2Fiii and Giii). Atapproximately the 15s stage, hoxb4reaches an anterior limit atthe r6/r7 boundary (data not shown; Fig. 3G). This limit maintained until at least 30 hours of development (Fig. 2G

Analysis of valentino mutant embryos using the hoxgenesThe spatial limits of hox gene expression are very promineand robust; between the 5 and 20 somite stages of developthey provide useful molecular markers for all the rhombomboundaries from the r1/r2 boundary through to the r6boundary. We have made use of these markers to furanalyze the val mutant, in which presumptive r5 and r6 do nsubdivide but instead produce a region the length of a sinrhombomere, rX. The rX region has some features of bothand r6, including characteristic reticulospinal neurons; nevtheless, the identity of rX is unlike that of r5 and r6 basedmosaic analyses and marker gene expression (Moens e1996). We have used hox gene expression to investigate borhombomere identity and the nature of the interfaces betwrX and the adjacent rhombomeres.

We have compared the expression domain of the val genewith those of the hoxgenes in normal embryos, and analyzethe expression patterns of the hox genes in val− embryos atthree developmental stages: the 4-6 somite (5s) stage, thesomite (10s) stage and the 18-22 somite (20s) stage. At theearly stages it is not possible to identify mutants morpholocally and therefore double in situ hybridizations weperformed using krox-20 as a second marker; the r5 domainkrox-20 is radically reduced in val− embryos facilitating iden-tification of mutant embryos.


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Comparison of hox and val gene expressionpatternsComparison with the expression pattern of krox-20has shownthat val expression is localized to r5 and r6 between its onseexpression, at the end of gastrulation, and the 20s stage; this stage val expression decreases starting at the anterior an24 hours, the r5 domain has disappeared (Moens et submitted). At the 5s, 10s and 20s stages the r5+r6 expresdomain of val precisely corresponds to the high level expressdomain of hoxb3(compare Fig. 3A and C with B and D). valisalso expressed in the Mauthner neurons (Moens et al., 1998;3B) and in a subset of the r6-derived neural crest (Fig. 3B). neural crest expression domain is very restricted in comparto that of hoxb3 (compare Figs 3B and D). The posterior limof val expression at the r6/7 boundary corresponds to the maanterior limit of hoxb4expression (Fig. 3F).

Expression of hoxb1 and hoxb4 in val mutantsshows a gradual expansion of expression domainsacross region rXThe hoxb1gene has a characteristic expression domain in r4the normal embryo. At the 5 somite stage, the r4 expressdomain is clearly delimited but the borders of expression areyet totally sharp (Fig. 4A), presumably reflecting the incompleformation of morphological boundaries at this early stage. Thare no distinguishable differences in hoxb1expression betweenwild-type and val− embryos at the 5s stage (compare Fig. 4A aB). By the 10s stage the r4 expression domain has develosharp borders in normal embryos (see Fig. 2Aiii). In 10s val−

embryos the anterior expression limit of hoxb1 at the r3/r4boundary has become sharp, but the posterior border oexpression is less clearly defined and patchy expression caseen in the most rostral part of rX (Fig. 4C). By the 20s stthere is extensive expression of hoxb1within rX, although thisexpression is only present in scattered cells (Fig. 4D). No hoxb1-expressing cells are present within the small, localized domof krox-20 expression that remains within rostral/dorsal rX.

The hoxb4 gene represents a convenient marker for the rboundary, although this limit is not sharp until the 20s staResults of in situ hybridizations using hoxb1and hoxb4 probessimultaneously are shown in Fig. 4E-J. At the 5s stage, psumptive r5+r6 in both normal and mutant fish does not exphoxb1 (Fig. 4A,B), but hoxb1is expressed at a significant levewithin r7 and posteriorly and thus it is not possible to differetiate between the posterior hoxb1 domain and the overlyinghoxb4domain. Nevertheless, at the 5s stage, no significant ferences were noted in the size of the presumptive r5+r6 nexpressing domain between wild-type and mutant embryos (4A and B). By the 10s stage, hoxb1 expression in r7 andposterior is drastically reduced (see Fig. 2Aiii). In 10s wild-tyembryos there is a clearly demarcated non-expressing regbetween the hoxb1and hoxb4expression domains, corresponding to r5+r6 (Fig. 4E). In 10s val− embryos the size of this regionis reduced in comparison to normal embryos (compare Fig.and F), probably due to lack of separation of the presumpr5+6 domain into definitive r5 and r6 territories (Moens et a1996). By the 20s stage there is no clearly demarcated nexpressing region between the expression domains of hoxb1 andhoxb4in the val− embryos (compare Fig. 4G, I with H, J; dorsand lateral views). The expression within rX is at lower levthan in r4 or r7, again probably reflecting a ‘pepper and salt’ t

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distribution of expressing and non-expressing cells. There ismall region at the border of r4 and rX where hox expression isat a very low level; this region is at the correct location correlate with the small rX krox-20expression domain (compareFig. 4D and H). The krox-20expression domain in val− embryosis rather variable in precise size and level, but is confined to dorsal and anterior parts of rX; consistent with this observatithe low level hoxexpression domain is also confined to thiregion in a lateral view of a val− embryo (Fig. 4J). In situ hybrid-ization experiments using the hoxb4probe alone, confirmed thatexpression of this gene spreads gradually into the rX region (dnot shown), similar to our observations with hoxb1.

Expression of group 3 paralogues in val embryos:the val gene product may be required for high leveltranscription of hoxb3In normal embryos hoxb3has an anterior expression limit at ther4/r5 boundary. There is expression throughout the cauhindbrain and spinal cord but expression is at an elevated lewithin r5 and r6 (Fig. 2Diii), the two rhombomeres in which thval gene product is normally expressed (Fig. 3). In val− embryosthis elevated expression is lost at both the 5s stage (datashown) and the 10s stage (compare Fig. 5A and B), although level expression persists in rX. In addition, hoxb3does not showa clear anterior limit of expression in val− embryos, insteadexpression fades out at approximately the position of the r4interface (Fig. 5B). Analysis of multiple embryos, and correltion with the position of the otic vesicle, suggests that hoxb3expression may extend into the posterior part of r4 in val−

embryos, although low expression levels make this difficult assess. At the 20s stage the high level r5+r6 expression domis maintained in wild-type embryos, in addition to appearancea weak expression domain in r4 (Fig. 5C,E). The r4 expressdomain also appears in val− embryos (Fig. 5D), but a level ofexpression equivalent to that in normal r5+r6 is not attained rX (Fig. 5D,F). In approximately half the embryos examinethere is a slight elevation of the hoxb3expression level within rX(Fig. 5D), in the remaining half no difference was noted betwethe expression levels in rX and the more posterior neural tu(Fig. 5F). This lack of high level hoxb3 expression in those rhombomeres in which val is expressed suggests a role for the val gproduct in up-regulating hoxb3 transcription. Neural crestexpression of hoxb3is maintained in val− embryos, althoughinspection of multiple specimens suggests that there are fehoxb3-expressing cells in the mutant (compare Fig. 5C and D

The hoxd3gene is expressed with a clear anterior limit athe r5/r6 boundary in normal embryos (Fig. 5G – 10s stage– 20s stage). In val− embryos there continues to be hoxd3expression in rX (Fig. 5H,J). The anterior limit of hoxd3expression is somewhat diffuse in val− embryos at the 10sstage, similar to the situation for hoxb3, however, hoxd3expression does not extend throughout the rXkrox-20domainand thus cannot extend into r4 (compare Figs 5B and H). the 20s stage hoxd3expression is limited to approximately theposterior half of rX (Fig. 5J), suggesting some cryptic AP paterning information remains along the length of rX.

Expression of paralogue group 2 genes suggestsmismigration or mis-specification of cranial neuralcrest posterior to the otic vesicleThe paralogue group 2 genes are expressed in cranial ne

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Fig. 3. Comparison of expression of val (A,B)withhoxb3(C,D)and hoxb4(E,F) at the 10s stage (A,C,E)and 20s stage (B,D,F); double in situs with krox-20(red)to indicate r3 and r5; the precise extent of r5 is indicatedwith a bracket in A-D. (A) At the 10s stage val isexpressed in presumptive r5+r6 (rhombomeres arenumbered). (C) There is high level expression of hoxb3in the corresponding r5+r6 region, note expression inemergent neural crest at the r6 level (arrow). (E) hoxb4has not reached its anterior expression limit at this stage.(B) At the 20s stage val expression continues to belocalized to r5 and r6, there is also expression in theMauthner neurons in r4 (arrowheads) and low levelexpression in a small number of emergent neural crestcells (arrow). (D) The high level expression domain ofhoxb3 continues to co-localize with the val expressiondomain in r5 and r6, note expressing neural crest cells(arrow). (F) hoxb4has now reached its anterior limit atthe r6/7 boundary, corresponding to the posterior limit ofval expression.

Fig. 4. hoxb1(purple) and krox-20(red) expression in wild-type (A) andval− (B,C,D) embryos, rhombomeresare numbered. At the 5s stage, thereare no obvious differences in thehoxb1expression pattern betweennormal (A) and mutant (B) embryos.At the 10s stage the posterior limit ofhoxb1expression in r4 is sharp inwild-type embryos (see Fig. 2Aiii),but not in mutant embryos (C). By the20s stage there is diffuse hoxb1staining throughout rX (D). hoxb1andhoxb4 probes were combined to showexpression of both of these genes inwild-type (E,G,I) and val− (F,H,J)embryos. At the 10s stage (E) and the20s stage (G – dorsal view; I – lateralview) there is a clear zone of non-expression overlying r5 and r6 inwild-type embryos. In val− embryos at10s (F) the size of this domain isreduced and some expressing cells arevisible within the domain. By the 20sstage (H – dorsal view; J– lateralview) there is expression throughoutmost of rX, although expression isreduced in dorsal rX in the regionwhere krox-20expression is expectedto localize (indicated by bracket in J).Scale bar, 50 µm.


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Fig. 5. Expression of paralogue group3 genes in wild-type (A,C,E,G,I) andval− (B,D,F,H,J) embryos, r4 isindicated on each specimen. (A-F) Insitu hybridization with hoxb3. At the10s stage hoxb3 has a high levelexpression domain in r5+r6 (A, arrowindicates r4/5 boundary), this domain islacking in the val− embryos (B). By the20s stage a low level expressiondomain is apparent in r4 (C,D), highlevel expression continues in wild-typeembryos in r5+r6 (C) next to the oticvesicle (o), the comparable rXexpression in val− embryos continuesto be at a significantly lower level (D).Neural crest cells migrating posteriorto the otic vesicle and expressinghoxb3 are indicated with arrows. E andF show double in situ hybridizationswith krox-20at the 20s stage, toconfirm rhombomere locations (theposterior krox-20expression domainsare bracketed in each case). (G-J) Insitu hybridization with hoxd3and krox-20. In wild-type embryos at 10s (G)and 20s (I) hoxd3expression reachesthe r5/6 boundary to abut the r5 krox-20expression domain. In val− embryosat 10s (H) hoxd3 expression ismaintained in rX where, similar tohoxb3, it does not overlap with thesmall rX krox-20expression domain.At 20s (J) expression is confined to theposterior half of rX, arrowheadindicates the approximate location ofthe r4/rX interface. Note variable levels of krox20expression in rX, as discussed in text (compare J with F). Scale bar, 50 µm, where no barshown, scale is equivalent to adjacent or above panel.

crest migrating anterior to the otic vesicle into the secobranchial arch, and to a lesser extent posterior of the vesicle into the third branchial arch (Figs 2Biv, Civ). Secoarch neural crest derives from the level of r4 and r5, third aneural crest from r5, r6 and r7 levels (Schilling and Kimm1994). In normal embryos hoxb2 expression in neural cresmigrating into the 3rd arch is transient and by the 20s stagno longer visible (Fig. 6A). By contrast, in 20s stage val−

embryos there continues to be hoxb2 expression in post-

Fig. 6. Paralogue group 2 gene expression in wild-type (A,C) andval− (B,D) embryos at the 20s stage reveals a population of hoxexpressing cranial neural crest migrating posterior to the otic vesiIn wild-type embryos (A), hoxb2 is expressed in r3, r4 and r5 and incranial neural crest deriving from r4 and migrating anterior to theotic vesicle (o) into the 2nd branchial arch. In val− embryos (B) theotic vesicle (o) is reduced in size; in addition to the normalpopulation of cranial neural crest migrating anterior of the vesicle,there is a subpopulation possibly deriving from rX, which expressehoxb2but migrates posterior to the otic vesicle toward the 3rd arch(arrow). In wild-type embryos (C) hoxa2 is expressed in r2, r3, r4and r5, and in cranial neural crest migrating into the 2nd and 3rdbranchial arches. In val− embryos (D) expression levels are reducedin rX, however there are increased expression levels in neural cremigrating into the 3rd branchial arch (arrow). Scale bar, 50 µm.

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migrating neural crest (Fig. 6B). Similarly, hoxa2continues tobe expressed in wild-type 3rd arch neural crest at the 20s st(Fig. 6C), but expression is at higher levels in val− postoticneural crest cells (Fig. 6D). The otic vesicle of the val− embryosis reduced in anteroposterior length, but maintains its approimate normal location. The postotic migrating neural crest val− embryos appears to derive from approximately the Alevel of rX and may represent a misdirected neural crest poulation that would normally migrate anterior of the otic vesicle

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or alternatively a mis-specified crest population that migrating in the normal direction but has taken on an idenmore appropriate to r4-derived neural crest. Either interpretion is consistent with the observation that 3rd arch cartilaelements in val− embryos take on some characteristics normal 2nd arch elements (Moens et al., 1998).

We also analyzed the expression of the paralogue grougenes in the hindbrain at 5s, 10s and 20s, in order to furevaluate the changing identity of rX. Specifically, we wishedassess to what extent rX identity is correctly specified earlydevelopment, and to what extent rX takes on aspects oidentity, as revealed by the gradually attained mis-expressiothe r4 marker hoxb1within rX (described above). Expression ohoxa2in r4 and r5 is apparent at the 10s stage (Fig. 2Biii), the expression levels are approximately equivalent betwthese two rhombomeres and thus hoxa2does not usefully dif-ferentiate between r4 and r5 identity at this stage. In 10s sval− embryos, hoxa2expression is at a slightly reduced level rX in comparison to r4 or r5 expression levels in normal embry(data not shown), perhaps reflecting lack of krox-20expressionin the mutant; krox-20 may be involved in up-regulating hoxa2

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as has been shown for the mouse (Nonchev et al., 1996). hypothesis leads to the prediction that the small domain of krox-20 expression in rX should be coincident with expression hoxa2at levels found in wild-type r5; unfortunately due to threlatively low level of hoxa2expression in this region it has nobeen possible to show this conclusively. By the 20s stage, hoxa2expression reaches high levels in normal r5, yet remains at levels in rX (compare Fig. 6C and D), thus rX is again showia rather similar expression profile to normal r4 at this later sta

The relative expression levels of hoxb2in r4 and r5 changewith developmental stage. At the 5s stage the expression lein r5 is significantly higher than that in r4 (Fig. 2Cii), but at th10s and 20s stages this situation has reversed (Fig. 2Biii, 6A). In val− embryos at the 5s stage, high levelhoxb2expressionin rX is confined to the small domain of krox-20expression (datanot shown), again consistent with the idea that krox-20 mdirectly up-regulate hoxb2 expression as has been described fthe mouse (Sham et al., 1993). At the 10s stage, hoxb2expression in rX of val− embryos once again seems confined the domain of krox-20expression (data not shown), by this stagr4 expression has been up-regulated, yet the posterior part ocontinues to show low level expression of hoxb2, and thus dnot share r4 identity. By the 20s stage, hoxb2expression levelsin rX of val− embryos have increased to approximately r4 leveand are significantly higher than expression levels in normal(compare Fig. 6A and B). Thus, once again by the 20s stageseems to have acquired some aspects of the identity of r4.

DISCUSSION

We have analyzed the expression of 7 different hox geneswithin the developing hindbrain of wild-type and val−

zebrafish. We find many similarities between the expressdomains of the hox genes of zebrafish and other speciehowever, some distinct differences in timing, anterior limiand levels of expression do exist; these changes mayreflected in the observed interspecies modifications in nroanatomy. We find that many aspects of hoxgene expressionare relatively normal in val− embryos at early stages beforerhombomere boundary formation. This early phase expression may correspond to the time at which the retilospinal neurons acquire segmental identity, and hence observation is consistent with the retention of normal patteing of these early born neurons in val mutants. By contrast,later in development we observe inappropriate hox geneexpression within rX, the region in val− embryos that liesbetween, but fails to form boundaries with, r4 and r7. This lainappropriate expression may reflect lack of normal interhombomeric interactions or inappropriate cell mixing alonthe anteroposterior (AP) extent of the val− hindbrain.

Differences between teleost and tetrapod hox geneexpression may reflect differing neuroanatomy ordevelopmental mechanismsThe expression patterns of mouse and zebrafish Hox genes aresummarized in Fig. 7. Despite broad similarities in expressipatterns between these species, we have noted several disancies. For example, the onsets of zebrafish hoxgene expressionare generally relatively late, at the beginning of somitogenecompared to gastrulation stages in other species. In orde

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make useful cross-species comparisons regarding the timinHox gene function, the most useful parameter to consider istime at which the anterior limit of expression is reached; diffences between the posterior expression domains may not any functional role as in general Hoxgenes seem to function aor close to their anterior limits of expression (reviewed McGinnis and Krumlauf, 1992). For the majority of the zebrafigenes we analyzed, expression appeared to commence witfinal anterior limit already set. This is quite different to thgeneral situation reported in tetrapod vertebrates whexpression commences in the posterior of the embryo gradually spreads forward until the final anterior limit is reach(Deschamps and Wijgerde, 1993). We would therefore sugthat the apparent late onset of zebrafish hoxgene expression doesnot correlate with late function; for example, Hoxb-1expressionreaches its anterior limit in the murine neurectoderm at the oof neurulation (Frohman et al., 1990), an approximately equalent stage to that at which zebrafish hoxb1is first expressed withits final anterior limit already set. It is possible that the rapdevelopment of the zebrafish embryo precludes detection oequally rapid anterior spread of hoxexpression; for example, forhoxd3, a gene which does show an anterior spread of expresthe anterior expression limit shifts from the posterior most pof the embryo to the hindbrain within one hour (Fig. 2D). Altenatively, the general lack of preliminary posterior expressionzebrafish hox genes may reflect differences in the mechanisused to activate Hox expression in zebrafish and mice.

We find several differences in relative expression levelsindividual rhombomeres between fish and mouse (Fig. 7Murine Hoxa-2and Hoxb-2have high expression levels in r3and r5, correlating with Krox-20 expression domains; indeeKrox-20has been shown to directly activate Hoxa-2andHoxb-2 expression (Sham et al., 1993; Nonchev et al., 1996). Inzebrafish, hoxb2 expression is first localized to r3 and r5shortly after the onset of krox-20 expression (Oxtoby andJowett, 1993), suggesting similar mechanisms are at wHowever, by the 10 somite (10s) stage, r5 expression of hoxb2is at relatively low levels, and hoxa2expression does not attainhigh levels in r5 until the 20s stage. For both of these genexpression in the CNS posterior to r5 is at very low levels, in other species these genes are expressed caudally alonlength of the hindbrain and spinal cord.

The major disparity we note between zebrafish and moHoxgene expression domains is for the paralogue group 3 g(Fig. 7A). Up to at least the 20s stage, zebrafish hoxd3 has aclear anterior expression limit at the r5/r6 boundary, at lastages of development there is a small hoxd3expression domainin r5, but this is confined to a small population of ventro-latecells (Fig. 2Eiv). Conversely, mouse Hoxd-3 has an anteriorlimit one rhombomere more anteriorly, at the r4/r5 boundaalthough the r5 expression is at a lower level than more posteexpression (Fig. 7A). The hoxb3 gene of the zebrafish does the same anterior limit of expression as its murine homologat the r4/r5 boundary (Fig. 7A), at least until late stages wa low-level r4 domain of expression appears. However, in mouse there is an elevated expression domain of Hoxb-3confined to r5 (Sham et al., 1993), but we find elevated hoxb3expression in the zebrafish in both r5 and r6. Observed difences in neuronal architecture between murine and zebrahindbrains may reflect the differences we observe in Hox geneexpression. For example, in the zebrafish, the motor nucle

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the abducens (cranial nerve VI) lie in r5 and r6 (Gilland aBaker, 1993; Moens et al., 1996), whereas in mouse abducens nucleus is confined to r5; it is interesting that in einstance the abducens nucleus colocalizes with the high leexpression domain of hoxb3. In addition, the cell bodies of thefacial nuclei (cranial nerve VII) lie within r6 and r7 of thezebrafish (Chandrasekhar et al., 1997), but within r4 and r5the mouse. Such differences in rhombomere identity presuably need to be patterned at the level of gene expression,thus differences in Hoxgene expression between mouse and fiat this rhombomeric level may not be unexpected. Indeed, existence of such interspecies differences in Hox geneexpression was accurately predicted from comparative studof neuroanatomy (Gilland and Baker, 1993).

Changes in hox gene expression during val− embryodevelopment may suggest cell mixing acrossrhombomere interfacesExpression of hoxb1 and hoxb4 changes dramatically duringdevelopment of val− embryos. At early stages, shortly after thnormal onset of val expression (10 hours; Moens et al., 1998the r4 hoxb1expression domain is indistinguishable in wild-typand val− embryos, as are the r7 hoxb4and hoxx4expressiondomains. This suggests no requirement for the valgene productin setting up r4 and r7 identity. However, between the 5s and stages, expression of both hoxb1and hoxb4becomes progres-sively more apparent in rX, although remaining at lower levethan in the normal r4 and r7 territories, which could reflect a dtribution of expressing and non-expressing cells (summarizedFig. 7). This observation can be explained in one of two waeither cells within the rX territory have an incorrect positionspecification that allows them to activate inappropriate markeor hox gene expressing cells are crossing the r4/rX and r7interfaces between the 5s and 20s stages.

The timing of val expression, and its location in r5 and r(Moens et al., 1998; Fig. 3), might suggest a role for the val geneproduct in down-regulatinghoxb1expression within these tworhombomeres. However, our observations do not support idea as we see clear absence of hoxb1expression in presump-tive r5+r6 in 5s stage val− embryos (Fig. 4B). Thus, earlyexpression of hoxgenes seems to be specified normally in thabsence of val gene product; the possibility remains, howevethat the val gene product is involved in ensuring that hoxb1doesnot become re-activated in the r5+r6 region later in developmeHence, if inappropriate hoxgene expression in rX is a manifestation of incorrect specification, this must be a relatively laeffect, perhaps resulting from lack of normal inter-rhombomeinteractions in the absence of definitive r5 and r6. The altertive idea, that cells may migrate into rX from adjacent rhombomeres, is supported both by the apparent gradual spreaectopic gene expression through rX suggesting cell movemand by the results of experiments which suggest that the r4and r7/rX interfaces are not fully formed boundaries. In val−

embryos Moens et al. (1996) report an absence of morpholocal boundaries caudal to the r3/r4 boundary, and loss expression of the boundary marker mariposa.Guthrie and col-leagues (Guthrie and Lumsden, 1991; Guthrie et al., 1993) fothat when two ‘like’ rhombomeres are juxtaposed, cells cmigrate across the interface; boundary formation requires juxposition of alternating odd- and even-numbered rhombomstates. The results of Moens et al. (1996) are consistent with

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idea and further suggest that both of these two states absolutely required for boundary formation to occur, i.e.odd nto even leads to a boundary, but neither odd (r7) nor even next to unspecified territory (rX) can cause boundary formatiopresumably due to lack of recognition between the adhessystems of presumptive and definitive rhombomeres.

It is interesting to note that hoxb1expression does not spreadinto the region of rX where krox-20 expression is maintain(Fig. 4D,H,J), suggesting that this region maintains r5-liproperties incompatible with hoxb1expression. This observa-tion suggests that if cell mixing is occurring across the r4/interface then cells must down-regulate hoxb1 as they passthrough the krox20expression region, or, perhaps more likelthat they must migrate ventral of this dorsally restrictedomain. Currently, there are arguments to support or refeither of the two hypotheses we propose to explain the gradchanges in molecular identity of rX (i.e. incorrect positionspecification of rX versus cell mixing across the r4/rX anr7/rX interfaces); resolution of this question will ultimatelrequire cell labelling and time-lapse analyses.

Changes in hox gene expression in val− embryosmay indicate regulation of hox gene expression bythe val gene productThe major differences in hox gene expression between normaand val− embryos are summarized in Fig. 7. Loss of high levhoxb3gene expression in rX suggests that the val gene productmay be involved in up-regulating transcription of this gene. Trelative timings of gene expression are consistent with this idin normal embryos high level hoxb3expression is present in r5and r6 from the 3-4s stage, shortly after the onset of valexpression (Fig. 3; Moens et al., 1998). A similar interpretatiwas proposed by McKay et al. (1994) based upon analysisHoxa-3 expression in the mouse kr mutant. Furthermore, thekrgene product (the murine homologue of val) has recently beenshown to interact with enhancer elements for Hoxb-3which arerequired for transcriptional activity (Manzanares et al., 199Lack of high level hoxb3expression correlates with the lack oan abducens nucleus in val− fish. As described above, the highlevel hoxb3expression domain colocalizes with the abducenucleus in both zebrafish and mouse; these observations suggest a possible role for the hoxb3gene product in abducensspecification. The recently demonstrated role of murine Hoxb-1in specification of the facial nerve provides a precedent for thypothesis (Goddard et al., 1996; Studer et al., 1996).

The zebrafish hoxd3gene is normally expressed in r6 but noin r5. We observe expression of hoxd3in rX, perhaps implyingthat some specific characteristic of zebrafish r5 normaprevents hoxd3expression. At the 20s stage, hoxd3expressionappears to be confined to the posterior half of rX suggestthat some elements of normal AP patterning are retained althe length of rX; only the posterior half of rX exhibits the r6like property of maintaining hoxd3expression. This result isconsistent with other observations that suggest some identity is retained by rX. For example, in mosaic experimenif wild-type cells are placed in rX they are only able to exprekrox-20if they lie in the anterior portion, close to r4. Similarlythe reticulospinal neurons retain their normal identity and Aorder in val mutants.

Our expression analyses of hoxa2and hoxb2do not suggestregulation of the paralogue group 2 genes by the val gene

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product. In normal 20s stage embryos, hoxa2 expression isupregulated in r5. In val− embryos there is no equivalentelevated expression in rX, perhaps reflecting lack of krox-20expression; Krox-20 has been shown to directly upregulaHoxa-2 transcription in mouse r5 (Nonchev et al., 1996Expression of hoxb2in both r3 and r5 of normal embryosshortly follows the onset of krox-20 expression, and rXexpression in val− embryos is limited to the krox-20-expressingregion, again consistent with a role for Krox-20 in activatintranscription of this gene as shown in the mouse (Sham et 1993). However, hoxb2has a relatively low level of expressionin r5 of normal 20s stage zebrafish. In val− embryos the rXexpression of hoxb2is at a higher level than normal r5expression, more similar to the expression level in r4. It seelikely that the gradual attainment of r4-like aspects oexpression for the paralogue group 2 genes in rX, may reflthe same processes as do the changes in hoxb1 expressiondiscussed above; namely aberrant cell mixing or incorrect pitional specification.

Expression of paralogue group 2 genes suggestsaberrant migration or specification of cranial neuralcrest in val− embryosIn wild-type embryos, the neural crest cells which migraposterior to the otic vesicle toward the third branchial arcexpress hoxb3 and, at low levels, hoxd3and val. In val−embryosthis population of cells expresses hoxa2and hoxb2; molecularmarkers characteristic of neural crest cells that migrate anteto the otic vesicle at equivalent stages in normal zebrafiembryos. A similar caudally migrating crest cell population warevealed by CRABP I staining in the mouse kr mutant (McKayet al., 1994). We suggest that these neural crest cells tranhox information appropriate to the second arch into the thiarch, thus leading to a cartilage transformation. In val− fish andkr− mice, the 3rd arch cartilage shows shape changes sugtive of an anterior transformation towards 2nd arch charactetics (Moens et al., 1998; Frohman et al., 1993). The aberrhox expression status of neural crest migrating into the thiarch may reflect inappropriate cell migration, resulting fromshortening of the hindbrain in val− and kr− embryos, thusallowing primarily r4-derived 2nd arch neural crest to inappropriately mix with more caudally derived 3rd arch crest. Altenatively, aberrant hox expression in neural crest might resulfrom inappropriate specification of the neural crest primordiumAccording to its hoxexpression status, rX has some aspects r4 identity and thus rX-derived neural crest cells migrating inthe 3rd arch may be incorrectly specified with 2nd arch identiHowever, it should be noted that these r4-like aspects of rX hoxexpression are all seen well after the time at which neural crcells migrate away from the neural keel.

Analysis of hox gene expression in val− embryos ata variety of stages suggests primary and secondarydefects in hindbrain patterningOur detailed expression analyses at several developmental sthave allowed us to determine differences between primary asecondary defects in valmutant zebrafish. For example, wepropose that loss of high levelhoxb3expression in val− mutantswhich occurs from the earliest stages, likely reflecting a diretranscriptional regulation of hoxb3by the val gene product, rep-resents a primary defect. However, we propose that change


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expression of other hoxgenes are due to secondary defects. Fexample, our analyses of several different developmental stahas revealed that hoxb1 expression posterior to r4 in val−

embryos is indistinguishable from that of wild-type embryos early stages. However, at later stages there is an ectopic posextension of hoxb1 expression into rX of val− embryos.Similarly, hoxb2 expression in rX of val− embryos becomesincreasingly similar to the expression in r4 with developmentime. These results suggest that rX only gradually attains aspof r4-like identity during its development. Our interpretation thchanges in hoxb1expression are a secondary defect extends observations in kr− mice where a posterior extension ofHoxb-1expression (Frohman et al., 1993; McKay et al., 1994) led to idea that the territory posterior to normal r4, analogous to has aspects of r4 identity.

Our results indicate that some aspects of hoxgene expressionin the val−hindbrain are specified appropriately, but later in devopment there are secondary changes in expression, perhapsresult of inappropriate cell mixing or lack of normal inter-rhombomeric interactions. Frohman et al. (1993) noted that expresof Hoxb-1 overlapped with that of Hoxb-4 and Hoxb-3 in kr−

mice; they interpreted this result to mean that the region posteto r4 has a mixed identity, with properties of more than one rhobomere. Our results with val− zebrafish suggest that any mixeidentity of rX is a secondary event; at early times no regiexpresses markers appropriate to more than one rhombomlevel. As many aspects of hox gene expression seem relativelnormal in val− embryos at early stages, this might suggest thhox-dependent AP patterning events that occur early wouldcorrespondingly insensitive to val genotype. This idea issupported by the disposition of the early born reticulospinneurons in val− embryos: the wild-type complement of neuronis present and they retain the normal AP order. Similarly, as hoxexpression does not begin to differ markedly between wild-tyand val− embryos until after the 5s stage, this may also suggthat the proposed function of the valgene product in subdividinga presumptive r5+r6 territory occurs after this time. In mosexperiments in which wild-type cells are placed in val− embryos,the wild-type cells lying in r4 and r7 have not been seen to enrX; however, our suggestion that rX gradually takes on a mr4-like identity, predicts that if such manipulated embryos weallowed to develop further, then rX might gradually becomcolonized by wild-type cells from adjacent r4 and r7.

There are many similarities between the val and kr pheno-types, although a few differences are apparent. For examelevated levels of apoptotic cell death have been observethe r4 region of kr embryos (McKay et al., 1994); no such cedeath has been found in val− embryos analyzed at the 20s stag(Moens et al., 1996). Another difference lies in the detailsthe expression profiles of the val and krgene products; afterrhombomere boundary formation val continues to be expressedthroughout r5 and r6 (Moens et al., 1998), whereas at equlent stageskr expression is confined to r5 plus the anterior paof r6 (Cordes and Barsh, 1994). These differences betwmouse and zebrafish do not seem extensive enough to suga fundamentally different role for the val and kr gene products.We propose that in both systems the val/kr gene product isnecessary for allowing subdivision of presumptive r5 and into definitive rhombomeres. Additionally, by regulating hoxgene expression, the val gene product presumably has aimportant role in imparting final segmental identity; thus th

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late differences in expression profiles of val and krmay haveprofound effects on mature neuroanatomy.

We are extremely grateful to Andreas Fritz, Len Zon and MaEkker for assisting us and sharing information regarding mappingthe hoxgenes. We would also like to thank Anand Chandrasekhar sharing unpublished observations and Laure Bally-Cuif for commeon the manuscript. V. E. P. and C. B. M. are Human Frontiers ScieProgram long-term fellows. This work was supported by a BaO’Connor Starter Scholar Research Award from the March of Dimto R. K. H. who is a Rita Allen Foundation Scholar, by a donatiofrom the Rathmann Family Foundation to the Molecular BiologDepartment at Princeton University, and by NIH grants ROHD34499 to R. K. H and NS17963 to C. B. K.

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zebrafish hox genes: expression in the hindbrain - development

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