Expression of zebraﬁsh nk2.2is inﬂuenced by sonic hedgehog ... · shh/vhh-1 and the transcription factor axial are involved in the spatial regulation of expression of nk2.2shh/vhh-1

1755Development 121, 1755-1768 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate

hedgehog-1 and demarcates a zone of neuronal differentiation in the

embryonic forebrain

Katrin Anukampa Barth and Stephen W. Wilson

Developmental Biology Research Centre, Randall Institute, King’s College London, 26-29 Drury Lane, London WC2B 5RL, UK

We have isolated zebrafish nk2.2, a member of the Nk-2family of homeobox genes. nk2.2 is expressed in a continu-ous narrow band of cells along a boundary zone demar-cating the location at which two of the earliest nuclei in thebrain differentiate. This band of cells is located within afew cell diameters of cells expressing the signallingmolecule sonic hedgehog/vertebrate hedgehog-1 (shh/vhh-1). Injection of shh/vhh-1 RNA results in ectopic expressionof nk2.2 and concomitant abnormalities in the forebrain

and eyes. Moreover, cyclops mutant embryos, whichinitially lack neurectodermal expression of shh/vhh-1, showa concomitant lack of nk2.2 expression. Together, theseresults suggest a requirement of shh/vhh-1 protein for thespatial regulation of nk2.2 expression.

Key words: boundary, Nk2, hedgehog, axial, zebrafish, forebrain,neuronal differentiation

SUMMARY

INTRODUCTION

The last few years have seen significant advances in our under-standing of the mechanisms underlying anteroposterior anddorsoventral patterning of the hindbrain and spinal cord(Krumlauf et al., 1993; Smith, 1994). However, the morpho-genesis of more rostral brain regions is less well understood,and there is still debate over such basic issues as defining theneural axes in the forebrain. It has been demonstrated that,similar to the hindbrain, distinct neuromeres are present in thedeveloping forebrain (Puelles et al., 1987). However, contro-versy remains as to the number and exact positions of theforebrain neuromeres and whether they correspond to truesegmental subdivisions (Figdor and Stern, 1993; Puelles andRubenstein, 1993; Macdonald et al., 1994).

Because of its relative simplicity, the embryonic zebrafishCNS is well suited for studies of early forebrain development.By 24 hours of development (h), a simple scaffold of axontracts has been established by a small number of neurons atinvariant locations within the brain (Chitnis and Kuwada,1990; Wilson et al., 1990). We have recently shown thatneurons that pioneer this scaffold differentiate at boundaries ofgene expression domains (Macdonald et al., 1994). Forexample, the nuclei of the tract of the postoptic commissure(nTPOC) and of the medial longitudinal fasciculus (nMLF)both develop and extend axons along the ventral boundary ofpax6 and rtk1 expression.

In this study, we report the isolation of a member of the Nk-2 family of homeobox genes, termed nk2.2, which is expressedalong the boundary zone at which the nTPOC and nMLF dif-ferentiate. Like several other homeobox-containing gene

families, the Nk-2 family is characterised by an additionalconserved motif, the Nk-2 domain (Price et al., 1992). Thismotif, the prototype of which is found in the Drosophila NK-2 gene (Kim and Nirenberg, 1989), consists of at least 17amino acids located carboxyterminal to the homeobox. Sincethe isolation of the first vertebrate family member Nkx-2.1/TTF-1 (Guazzi et al., 1990; Lazzaro et al., 1991; Price etal., 1992), five other family members have been isolated inmice. Nkx-2.1 to Nkx-2.4 are all closely related (Price et al.,1992; Price, 1993), while Nkx-2.5 and Nkx-2.6 represent moredivergent members of the family (Lints et al., 1993). Inaddition to being expressed in the thyroid and lung, Nkx-2.1/TTF-1 is also transcribed in restricted regions of theforebrain, as is Nkx-2.2 (Lazzaro et al., 1991; Price et al.,1992). Nkx-2.5 is thought to be involved in heart development(Lints et al., 1993), while no detailed expression data have beenreported for Nkx-2.3, Nkx-2.4 and Nkx-2.6. Zebrafish nk2.2 ismost closely related to Nkx-2.2 and the Xenopus gene XeNk2(Saha et al., 1993).

In this study, we suggest that the signalling moleculeshh/vhh-1 and the transcription factor axial are involved in thespatial regulation of expression of nk2.2. shh/vhh-1 is thezebrafish homologue of the Drosophila hedgehog gene (Krausset al., 1993; Roelink et al., 1994), and axial (Strähle et al.,1993) is the homologue of mouse HNF-3β, a member of thewinged-helix family of transcription factors (Pani et al., 1992;Lai et al., 1993). shh/vhh-1 and axial/HNF-3β are bothinvolved in regulating the patterning of midline structures inmesoderm and in the ventral CNS (Ang and Rossant, 1994;Smith, 1994; Strähle and Blader, 1994; Weinstein et al., 1994).The notion that axial/HNF-3β is a key regulator of floorplate

1756 K. A. Barth and S. W. Wilson

development is supported by the finding that ectopicexpression of HNF-3β results in the ectopic appearance offloorplate markers (Sasaki and Hogan, 1994; see also Ruiz iAltaba and Jessel, 1992). It seems likely that shh/vhh-1 isresponsible for the induction of axial/HNF-3β in the pre-sumptive floorplate since ectopic expression of shh/vhh-1results in ectopic axial/HNF-3β expression (Echelard et al.,1993; Krauss et al., 1993; Roelink et al., 1994), while COScells secreting shh/vhh-1 induce floorplate differentiation inadjacent neuroectoderm (Roelink et al., 1994).

In zebrafish, analysis of embryos carrying the cyclopsmutation has also provided results consistent with the possi-bility that shh/vhh-1 and axial/HNF3β regulate floorplatedevelopment. The cyclops mutation affects specification of theventral midline of the CNS such that homozygous mutantembryos lack a floorplate and exhibit fusion of the eyes (Hattaet al., 1991). The cyclops gene may be involved in the sig-nalling pathway between mesoderm and neuroectoderm thatspecifies ventral CNS cell types (Hatta et al., 1991, 1994). Inagreement with this interpretation, neither shh/vhh-1 nor axialare initially expressed in the neurectoderm, while mesodermalexpression of these genes is present (Krauss et al., 1993;Strähle et al., 1993).

The timing and spatially restricted expression of nk2.2suggests that this gene may play a role in the regulation of azone of neuronal differentiation within the embryonic zebrafishforebrain. Furthermore, we present evidence suggesting thatshh/vhh-1 may be involved in the spatial regulation of nk2.2expression. We show that nk2.2 expression is initially absentfrom the neuroectoderm of cyclops mutant embryos which con-comitantly lack shh/vhh-1 expression and that overexpressionof shh/vhh-1 results in ectopic expression of nk2.2.

MATERIALS AND METHODS

Fish stocksBreeding fish were maintained at 28.5°C and embryos were collectedby natural spawning and staged up to 24h (30 somites) according toWesterfield (1993); beyond this time, embryonic stage is given ashours post fertilisation. Cyclops (cycb16) mutant carrier fish wereobtained from C. Kimmel and C. Nüsslein-Volhard.

Isolation of the nk2.2 cDNA cloneTo isolate zebrafish NK-2 homologues, primers to the region flankingthe homeobox and Nk-2 domain of the Xenopus XeNk-2 gene (Sahaet al., 1993) were used to amplify a 370 bp cDNA fragment fromXenopus cDNA (stage 17). The PCR fragment obtained was clonedand used to screen 1.2×106 recombinant clones of a zebrafish neurulastage cDNA library at low stringency (50% formamide, 6× SSC at37°C). A single clone containing 1.5 kb of cDNA was obtained,subcloned and sequenced using internal primers and the SequenaseVersion 2.1 sequencing kit (USB). Sequence data were analysed usingthe Genetics Computer Group Sequence Analysis Software Package,Version 7.0 (Devereux et al., 1984).

In situ hybridisation and immunohistochemistryAntisense digoxigenin-labelled RNA probes were synthesized usingthe digoxigenin (DIG) RNA labelling kit (Boehringer Mannheim).For nk.2.2, probes either comprising the entire 1.5 kb cDNA clone,or comprising a 630 bp 3′ region starting immediately downstream ofthe homeobox and including the Nk-2 domain gave best results. A710 bp probe derived from the 5′ region upstream of the homeobox

resulted in a spatially identical signal, but gave higher backgroundstaining. For axial, shh/vhh-1 and hlx-1, full-length cDNA probeswere synthesized. To decrease background staining, probes were frac-tionated over a G-50 (Sigma) drip column to remove unincorporatedDIG-UTP. Whole-mount in situ hybridisations were carried out asdescribed (Xu et al., 1994). After staining with NBT/X-phosphate(Boehringer Mannheim), embryos were refixed overnight in 4%paraformaldehyde/PBS, washed in PBS and cleared in 70% glycerol.Embryos were dissected from the underlying yolk and mounted in70% glycerol for photography. Immunohistochemistry was carriedout according to standard procedures (Wilson et al., 1990).

RNA injectionsshh/vhh-1 RNA for injections was derived from the pSP64T-shhplasmid kindly provided by J.-P. Concordet and P. Ingham; see Krausset al., 1993. RNA for injections was transcribed in vitro and severalpicoliters were injected at a concentration of 0.1 mg/ml into blastomeresof 1- to 4-cell stage embryos using a pressure-pulsed Picospritzer II(General Valve Corp.). To assess the extent of chimerism, lacZ RNAwas co-injected at a lower concentration of 20 µg/ml. For control injec-tions, RNA encoding β-galactosidase was injected at the same concen-tration (0.1 mg/ml). Analysis of β-galactosidase activity was performedon embryos that had been fixed for 10 minutes in 4% paraformalde-hyde, 0.5% glutaraldehyde at stages between 12 and 24 hours. Afterseveral washes in PBS, 0.1% Tween 20 (Sigma), embryos were rinsedin buffer A (1 mM MgCl2, 15 mM K3Fe(CN)6, 12 mM K4Fe(CN)6)and incubated at 37°C in buffer A containing X-gal (Stratagene) to afinal concentration of 800 µg/ml. After staining, embryos were washedseveral times, refixed and processed for in situ hybridisation.

RESULTS

nk2.2 is homologous to mouse Nkx-2.2 andXenopus XeNk-2Through screening a zebrafish neurula stage cDNA library witha fragment of XeNk-2, a single clone containing 1.5 kb ofcDNA was isolated and termed nk2.2 based on its homologyto murine Nkx-2.2 (Price et al., 1992) and Xenopus XeNk-2(Saha et al., 1993). The sequence of the 1470 bp nk2.2 cloneshows a single open reading frame with a coding potential of269 amino acids (Fig. 1A).

To define the extent of homology between nk2.2 and otherNk-2 gene family members, we compared the nk2.2 translationproduct to other sequences. Within the homeobox and Nk-2domain, nk2.2 is 100% identical to mouse Nkx-2.2 andXenopus XeNk-2 proteins, and 93% identical to the samedomains of the Drosophila NK-2 protein (Kim and Nirenberg,1989; Fig. 1B,C). Comparison of nk2.2 to the published 472bp genomic fragment of Nkx-2.2 shows that, allowing for apossible frameshift in the published Nkx-2.2 sequence, nk2.2and Nkx-2.2 are 93% identical over the entire published Nkx-2.2 sequence.

The region of high identity (94%) between nk2.2 andXenopus XeNK-2 comprises amino acid sequences bothaminoterminal (20 aa) and carboxyterminal (30 aa) to thedomain containing the homeobox and Nk-2 domain. To eitherside of this core region, the sequence diverges and the putativeproteins differ in size, with nk2.2 being 64 amino acids longerthan the published XeNk-2 protein. However, 58 amino acidsupstream from the reported translational start site of XeNk-2,there is another in frame ATG, which is identical to theproposed translational start site for nk2.2.

1757Zebrafish nk2.2 gene

A
nk2.2 is expressed from late gastrula in thepresumptive forebrain adjacent to cells expressingshh/vhh-1 and axialnk2.2 expression is first detected around 95% epiboly (9.5h) asa small patch of cells at the animal pole in the presumptivebrain (Fig. 2A). Between bud/1 somite (10h) and 3 somites(11h), the domain of expression is a narrow column of cells atthe midline of the condensing neural keel (Fig. 2B,C). At laterstages, cavitation of the neural keel bisects this column of cellsto generate bilaterally symmetrical stripes of expression. Pre-liminary data indicated that expression of axial (Strähle et al.,1993), shh/vhh-1 (Krauss et al., 1993) and the homeobox-con-taining gene, hlx-1 (Fjose et al., 1994) may be localized toregions neighbouring nk2.2 expression and so the evolvingpattern of nk2.2 expression was examined with respect to theexpression domains of these genes.
At 5 somites (11.7h), the nk2.2 expression domain can bedivided into two components. The rostral domain of expressionextends from the anterior end of the neural keel to the mid-dien-cephalon (Fig. 2E) and lies adjacent and dorsal to cells thatexpress both shh/vhh-1 and hlx-1 (Fig. 2D,F), but anterior to the

Fig. 1. Sequence, gene structure and deduced amino acid sequence ofthe zebrafish nk2.2 gene. (A) The nucleotide sequence of nk2.2 isshown along with the conceptual translation of the open readingframe. The putative translational initiation site (ATG) is indicated,the homeodomain is boxed, and the Nk-2 motif underlined (thickline). The 3′ polyadenylation consensus sequence is marked by a thinline. (B) Gene structure and partial restriction map of nk2.2. Thickbars at the 5′ and 3′ ends, representing 280 bp and 368 bprespectively, indicate untranslated regions, while the open boxrepresents the protein coding region. The homeobox (light stripedbox) and Nk-2 domain (dark striped box) are highlighted. (C) Amino acid sequence comparison of the nk2.2 homeobox andNk-2 domain to other members of the Nk-2 family in mouse,Xenopus and Drosophila as well as to a more distantly related mousegene, Dlx-1. Percentage of amino acid identity is given for the core17 amino acids of the Nk-2 domain and to the slightly larger regionof 21 amino acid that is identical between nk2.2, Nkx-2.2 andXeNK-2. Adapted from Price et al. (1992) and references therein andSaha et al. (1993).


Fig. 2. Comparison of the developmental time course of nk2.2 expression in the rostral brain with that of shh/vhh-1 and axial. Whole-mountembryos hybridised with antisense RNA to nk2.2, shh/vhh-1, axial or hlx-1. Lateral views (except A,B) are shown with rostral to the left. In D-Y, the skin, yolk and eyes have been removed. (A,B) Frontal views (with dorsal up) showing nk2.2 expression (arrowheads) at 95% epiboly(9.5h) (A) and bud/1s (10h) stage (B). Dots outline the yolk plug in A. (C) Lateral view of nk2.2 expression at 3 somites (11h) and 5 somites(11.7h). (D) hlx-1 expression in the forebrain of a 5 somites (11.7h) embryo. (E-Y) Comparison of rostral brain expression domains of nk2.2(E,H,K,N,Q,T,W), shh/vhh-1 (F,I,L,O,R,U,X) and axial (G,J,M,P,S,V,Y) from 5 somites (11.5h) to 44-48h. The arrowheads in E-G indicate asmall groove in the mid-diencephalon at which the cephalic flexure will later form. Arrowhead in Q indicates the gap between rostral andcaudal nk2.2 expression domains. In V, the embryo was also labelled with an antisense RNA probe to wnt1 which is expressed in cells beneaththe epiphysis (Macdonald et al., 1994). Abbreviations: cb, cerebellum; cf, cephalic flexure; e, epiphysis; fp, floorplate; hy, hypothalamus; mb,midbrain; mdb, mid-diencephalic boundary; or, optic recess; p; anlage of the anterior pituitary; rd and cd, rostral and caudal domains of nk2.2expression; t, telencephalon; te, tegmentum; III, third ventricle. Scale bar=100 µm


domain of axial expression (Fig. 2G). The weaker caudaldomain of forebrain expression of nk2.2 (Fig. 2E,H) is directlydorsal to cells expressing both shh/vhh-1 and axial in the caudaldiencephalon and midbrain. The junction between the rostraland caudal domains overlies a small transverse groove in theventral neuroepithelium (Fig. 2E-G) at which the cephalicflexure will later form (see Fig. 2P), and corresponds to theanterior boundary of both axial expression and the presumptivefloorplate. We have previously described this position along therostrocaudal axis as the mid-diencephalic boundary (MDB,Macdonald et al., 1994), which may, at later stages, correspondto the zona limitans interthalamica described in other species(Puelles and Rubenstein, 1993; Rubenstein et al., 1994).

By 15 somites (16.5h), a dorsally directed deflection in theband of nk2.2-expressing cells at the MDB becomes apparent(Fig. 2H). By this stage, shh/vhh-1 is no longer detectable inthe ventralmost cells of the rostral forebrain (Fig. 2I), and therostralmost domains of shh/vhh-1 and nk2.2 expressionpartially overlap. However, within the caudal forebrain, nk2.2continues to be restricted to cells dorsal to the domains of bothshh/vhh-1 and axial (compare Fig. 2H to Fig. 2I and J). Fromthis stage onwards, the pattern of hlx-1 expression becomescomplex and highly dynamic (Fjose et al., 1994) and shows noobvious correlation with nk2.2 expression (not shown).

Between 22 (20h) and 28 somites (23h), the dorsal deflec-tion of nk2.2 expression at the MDB becomes more pro-nounced (Fig. 2K-N). Concurrent with this change, the

domains of shh/vhh-1 and axial expression extend furtherdorsally at the MDB (axial expression expands dorsally severalhours before shh/vhh-1), with the dorsal tip of expressioncoming to underlie the anterior epiphysis (Fig. 2L,M,O,P).During these stages, cavitation of the neural keel begins togenerate the ventricular system of the CNS and it becomesapparent that the anterior domain of nk2.2 and shh/vhh-1expression is located directly ventral to the optic recess/thirdventricle (Fig. 2K,L). The rostral domain of nk2.2 expressionthus overlaps the ventralmost cells within the diencephalicexpression domains of both pax6 and rtk1 (see Macdonald etal., 1994).

By 26-27h, a small gap between the rostral and caudaldomains of nk2.2 expression is visible (Fig. 2Q and see Fig.5G). This gap overlies the narrow dorsally directed finger-likeprojection of shh/vhh-1- and axial-expressing cells at the MDB(Fig. 2R,S, and see Fig. 5H). Throughout later developmentalstages, the expression domains of the three genes maintainsimilar spatial relationships as the forebrain undergoes furthermorphogenesis (Fig. 2T-Y). Finally, nk2.2 transcripts aredetected in the anlage of the anterior pituitary (Fig. 2Q) thoughexpression is transient and decreases during further develop-ment (Fig. 2T).

Low levels of nk2.2 transcripts are present in thehindbrain and in cells ventral to the notochordAlthough the most prominent site of nk2.2 expression lies within


Fig. 3. nk2.2 expression in the hindbrain and in a group of cells ventral to the notochord. Lateral views (A,B,E) with rostral to the left andtransverse sections (C,D) of 20-22 somites (19-20h) embryos from which the eyes and yolk have been removed. The alkaline phosphatasecolour reaction was developed 5-6 times longer than usual to reveal weak expression. The approximate levels of the sections shown in C and Dare indicated in A. (A) Low magnification view of the entire embryo. The arrow indicates very faint staining in the caudal spinal cord, and thearrowhead points to the group of cells shown in D and E. (B) nk2.2 expression in the brain. The arrow indicates the discontinuity in the band ofnk2.2-expressing cells. (C) Transverse section through the caudal hindbrain revealing expression in cells adjacent to the floorplate. (D)Transverse section near the hindbrain/spinal cord junction showing nk2.2 expression in cells beneath the notochord and hypochord (arrow).(E) Lateral view of the same group of cells as (D). Abbreviations: fb, forebrain; fp, floorplate; h, hypochord; hb, hindbrain; hy, hypothalamus;mb, midbrain; n, notochord; s, somite; sc, spinal cord; t, telencephalon. Scale bar: A,B=100 µm, C-E=20 µm.

the forebrain, lower levels of mRNA were also detected in morecaudal parts of the CNS, as well as in a cluster of cells ventralto the notochord (Fig. 3). Within the CNS, the column of nk2.2-expressing cells extends from the ventral optic stalk to the caudalspinal cord with highest expression rostrally, lower transcriptlevels in the hindbrain and only barely detectable expression inthe spinal cord (Fig. 3A). There is one small gap in this column

Fig. 4. Gene expression boundaries of nk2.2, shh/vhh-1 and axialdemarcate sites of neuronal differentiation and axogenesis in theforebrain and midbrain. Embryos are hybridised with nk2.2(A,B,E,F,L,M), shh/vhh-1 (C,G-I), axial (D,J,K,N) antisense RNAand HNK-1 antibody (brown labelling of neurons and axons).(A-D) Lateral views of sagittal hemisections with rostral to the leftand eyes removed. (A) nk2.2 expression with respect to the nTPOCand nMLF. The dark blue alkaline phosphatase reaction productmasks the nTPOC in A and C. (B) High magnification of nk2.2expression with respect to the nTPOC. The arrowheads indicate thecourse of the axons in the TPOC, and the white arrow indicatesHNK1 labelling within the nk2.2 expression domain. (C,D)Correlation of shh/vhh-1 (C) and axial (D) expression domains withthe locations of the nTPOC and nMLF. The arrows in D indicate afew axial-expressing cells dorsal and ventral to axons in the TPOC

of expression at the boundary between midbrain and hindbrain(Fig. 3B). Within the hindbrain, nk2.2 is expressed in severalcells to either side of the floorplate (Fig. 3C).

The only site of nk2.2 expression outside the neuroepithe-lium is a patch of cells ventral to the hypochord at thehindbrain/spinal cord boundary (Fig. 3A). Expression is firstdetected in this location around 15 somites (16.5h), peaks at

and the positions of the sections shown in H and I are indicated in C.(E-N) Transverse sections. (E-G) nk2.2 (E,F) and shh/vhh-1 (G)expression at the level of the nTPOC. The arrowheads in F indicateimmunoreactive processes within the nk2.2 expression domain.(H-J) shh/vhh-1 (H,I), axial (J,K,N) and nk2.2 (L,M) expression atthe level of the nMLF (H and J are through the rostral part of thenucleus and I,K and L are through the caudal part of the nucleus).The arrowheads in M and N indicate immunoreactive processesconnecting to the ventricle. The section shown in M is from aslightly older embryo than in L. Abbreviations: cf, cephalic flexure;hy, hypothalamus; mb, midbrain; mdb, mid-diencephalic boundary;nMLF and MLF, the nucleus of the medial longitudinal fasciculusand its associated tract; nTPOC and TPOC, the nucleus of the tract ofthe postoptic commissure and its associated tract; or, optic recess; t,telencephalon. Scale bars for A,C,D,E, G-L and B,F,M,N=100 µm.



Table 1. Alterations in gene expression following injectionof RNA encoding shh/vhh-1

% Embryos affected Severelyabnormal

n* Total MDB MDB+eyes Mb % WT embryos**

nk2.2 114 63 37 26 37 7shh/vhh-1 59 34 34 66 11Axial 71 64 64 20 36 2hlx-1 12 67 67 33

Total n= 256 145 101 30 14 107 20

In total, 256 shh/vhh-1-injected embryos were analysed for changes in thepattern of gene expression.

‘total n=’ represents the number of embryos in each category.n* are the numbers of embryos examined for each gene.** are the numbers of severely abnormal embryos that were not included in

the analysis (see text). Scored embryos were either wild type (WT), or fellinto one of three classes of altered expression patterns: ‘MDB’ corresponds tochanges in gene expression domains at the mid-diencephalic boundary;‘MDB+eyes’ indicates ectopic gene expression in the eyes in addition toaltered expression at the MDB, and ‘Mb’ denotes widespread ectopic geneexpression in the midbrain (and sometimes the hindbrain) as well as at theMDB. In a few cases, embryos that showed ectopic expression of nk2.2 in theeyes and at the MDB also exhibited a small patch of ectopic expression in themidbrain (see text). Figures are given as percentages.

about 20-22 somites (19-20h) (Fig 3D,E) and diminishes by28-30 somites (23-24h). Because expression is transient, wehave not determined the fate of this group of cells.

nk2.2 expression demarcates a zone of neuronaldifferentiation in the rostral brainBoundaries between gene expression domains demarcate thesites at which the first neurons in the forebrain differentiate andextend axons (Macdonald et al., 1994). The dorsoventralposition of nk2.2 expression suggested that it may overlie theboundary at which neurons in the nTPOC and the nMLF dif-ferentiate. To test this possibility, we examined the formationof the nMLF and nTPOC with respect to sites of nk2.2,shh/vhh-1 and axial expression.

Mature neurons of the TPOC differentiate within the nk2.2expression domain (Fig. 4A,B,E,F). Indeed, many HNK1-immunoreactive radial processes connected to the ventricle liewithin the nk2.2 expression domain; these processes probablybelong to young neurons that still retain ventricular connec-tions (Fig. 4F). There is considerable overlap between theshh/vhh-1 and nk2.2 expression domains in the rostralforebrain though shh/vhh-1 expression extends furtherventrally and nk2.2 expression extends more laterally into theoptic stalk (compare Fig. 4E to G). While at least some of theneurons of the nTPOC appear to differentiate just within theshh/vhh-1 expression domain (compare Fig. 4F to G), matureneurons and axons are positioned at the edge of this domain(Fig. 4G). The axons of the TPOC initially trace a course alongthe ventral edge of cells expressing nk2.2 (Fig. 4B) and as theyapproach the mid-diencephalon, they extend into a domain ofcells expressing axial (Fig. 4D). A small region not express-ing axial (Fig. 4D) was usually observed at the point of entryof the leading TPOC axons into the domain of axial expression.

The nMLF differentiates along the ventral edge of the caudaldomain of nk2.2 expression (Fig. 4A,L,M). While manyimmunoreactive radial processes, probably belonging to youngneurons, lie within the nk2.2 expression domain, most and

perhaps all of the mature nMLF neurons lie just lateral to thedomain and do not express nk2.2 (Fig. 4M). Conversely, manyof the mature neurons lie just within the axial expressiondomain and do express this gene (Fig. 4J,K,N) whereas manyof the radial processes lie just dorsal to the axial expressiondomain (compare Fig. 4M to N). The shh/vhh-1 expressiondomain in the midbrain does not extend quite as far dorsal asthe axial expression domain with the result that there areusually a few non-expressing cells between the shh/vhh-1expression domain and the neurons of the nMLF (Fig. 4C,H,I).

Overexpression of shh/vhh-1 RNA results inelevated and ectopic nk2.2 expressionThe observation that all sites of nk2.2 expression are withinseveral cell diameters of cells expressing shh/vhh-1 raises thepossibility that shh/vhh-1 may be involved in the regulation ofnk2.2 expression. In order to determine if shh/vhh-1 can inducenk2.2, we analysed embryos that ectopically expressedshh/vhh-1 after injection of synthetic shh/vhh-1 RNA.

In total, 256 shh-injected embryos were examined for alter-ations in expression of nk2.2, shh/vhh-1, axial and hlx-1 (seeTable 1). More than half of the injected embryos had specificalterations in CNS expression domains (see below), a few hadminor deficiencies in the body axis (such as kinked noto-chords), while the remainder did not show any obvious defects.A further 20 injected embryos showed severely perturbeddevelopment and were not included in our detailed analysis.Similarly disturbed development was occasionally seen incontrol injections or in the wild-type background.

nk2.2 and axial expression was noticeably altered in abouttwo thirds, and endogenous shh/vhh-1 expression changed inone third of injected embryos (Table 1). Because widespreadectopic expression of shh/vhh-1 was not detected, we assumethat the injected shh/vhh-1 RNA was already degraded by thestage at which embryos were fixed. Almost invariably, alter-ations in the expression of all genes examined were apparentat the MDB. For nk2.2, the expression domain at the MDB wasbroader, extended further dorsal and mRNA levels wereelevated as compared to controls (compare Fig. 5A to 2Q). Insome cases, the gap between the rostral and caudal domains ofnk2.2 expression was enlarged (compare Fig. 5D to 2Q and5G). Ectopic nk2.2 transcripts were also observed in the eyes(see below), and occasionally in the midbrain (Fig. 5D). Noectopic nk2.2 expression was observed posterior to themidbrain or outside the CNS.

Paralleling the changes observed for nk2.2 expression, axialand shh/vhh-1 expression domains also extended furtherdorsal, and mRNA levels were higher and detected in a widerstripe of cells at the MDB of injected embryos (Fig. 5B,C,E,F).In severe cases, the width of the band of tissue expressingshh/vhh-1 and axial at the MDB expanded from the 1-2 cellsnormally observed (Fig. 5H) to 10 or more cell diameters (Fig.5E,F,I). Embryos examined for changes in axial expression atearlier stages indicated that cells in the mid-diencephalonexpressed the gene earlier in injected embryos than in controls(Fig. 5J). About one third of injected embryos examined foraxial expression also exhibited ectopic expression in themidbrain and/or hindbrain as has been previously observed(Krauss et al., 1993). hlx-1 was also overexpressed at the MDBin 8 out of 12 embryos examined (not shown). Embryos thatexhibited changes in gene expression domains also showed


Fig. 5. Injection of shh/vhh-1 RNA results in elevated and ectopic expression of nk2.2, axial and shh/vhh-1. Whole-mount embryos with rostralto the left. (A-F) Lateral views of 24h shh/vhh-1-injected embryos showing nk2.2 (A,D), axial (B,E) and shh/vhh-1 (C,F) expression. Eyes havebeen removed. (D-F) Examples of embryos affected more severely than those in A-C. (G,H) Dorsal views showing the gap in nk2.2 expressionat the MDB of uninjected wild-type embryos (G) and the complementary expression of axial at the same location (H). The eyes are removed inH. (I) Dorsal view of expanded axial expression domain at the MDB in an shh/vhh-1-injected embryo. (J) Lateral view of axial expression in ancontrol (left) and shh/vhh-1-injected 12 somite embryos. (K) Detection of β-galactosidase activity (blue) in embryos injected with β-galactosidase-encoding RNA. Some embryos were also examined for nk2.2 expression (eg. dark blue label in the forebrain of embryo at bottomleft). (L) Higher magnification of the tail region of embryo seen bottom right in K. Blue cells are positive for β-galactosidase. Abbreviations:bl, blood; cf, cephalic flexure; h, hypochord; hy, hypothalamus; mb, midbrain; mdb, mid-diencephalic boundary; n, notochord; or, optic recess;sc, spinal cord; sk, skin; t, telencephalon; y, yolk; III, third ventricle. Scale bar=100 µm.


morphological defects in the anterior brain. In particular, thecavity of the third ventricle appeared reduced (compare Fig. 5Iand H) and the development of the eyes was abnormal (seebelow and Krauss et al., 1993).

Changes in gene expression were usually not apparent at thesites at which the nTPOC and nMLF differentiate. Forinstance, nk2.2 expression never expanded ventrally into thehypothalamus or floorplate. However, in a few cases, nk2.2expression was disrupted in the midbrain and in these embryoswe also observed disruption of the nMLF from being a tight

Fig. 6. Ectopic expression of nk2.2 in the optic primordia of shh/vhh-1-in(A-D) Whole-mount 22-24h shh/vhh-1-injected embryos hybridised withexpression throughout the optic primordia. (A) Focussed at the level of tharrowhead indicates the normal position of the optic stalk and the arrow ibrain. (C) Ventral view of an shh/vhh-1-injected embryo with ectopic nk2view of an shh/vhh-1-injected embryo with nk2.2 expression throughout texpression in normal 22-26 somites (20-22h) embryos. (G-H) Eye morphThe lens is reduced in H and absent in I. Ventrorostral eye development acephalic flexure; ch, choroid fissure; hy, hypothalamus; l, lens; mb, midbprimodia; or, optic recess; os, optic stalk; pe, pigment epithelium; pnr, prsurface ectoderm; t, telencephalon. Scale bar: A-F=100 µm, G-I=50 µm.

column of neurons to being a much more widely scatteredgroup of cells (not shown).

To ascertain that changes in gene expression were not dueto the effects of injecting RNA per se, we examined embryosinjected with RNA encoding β-galactosidase for changes innk2.2 expression. Of 99 injected control embryos, 94 weremorphologically normal with unchanged expression patterns,while 5 embryos showed non-specific defects.

We examined the distribution of injected RNA in 42embryos that had been injected with both RNA encoding

jected embryos correlates with impaired eye development. antisense RNA to nk2.2. (A,B) Lateral views showing ectopic nk2.2e eye and (B) focussed through the eye and onto the brain. The whitendicates the dorsocaudal limit of fusion of the optic primordia to the.2 expression in the anterior part of the optic primordia. (D) Frontalhe optic primordia. (E,F) Dorsal (E) and frontal (F) views of nk2.2ology in living normal (G) and shh/vhh-1-injected (H,I) 30h embryos.nd pigment formation is affected in both embryos. Abbreviations: cf,

rain; mdb, mid-diencephalic boundary; nr, neural retina; op, opticesumptive neural retina; ppe, presumptive pigment epithelial layer; se,


shh/vhh-1 and β-galactosidase by assaying the distribution ofenzyme activity. In all cases, β-galactosidase-positive cellswere widely distributed throughout the embryo (Fig. 5K) anddetected in all tissue layers (Fig. 5L).

Ectopic expression of nk2.2 in the eyes of shh/vhh-1-injected embryos correlates with abnormal eyedevelopmentIn normal embryos, nk2.2 is expressed in the proximal, ventralpart of the optic stalk, but not within the eyes (Fig. 6E,F). Incontrast, 41% of shh/vhh-1-injected embryos in which nk2.2expression was altered, exhibited ectopic nk2.2 expression inthe eyes. The extent of this expression was variable, sometimesbeing detected throughout the optic primodia (Fig. 6A,D), inother cases restricted to more medial and ventral parts of thedeveloping eyes (Fig. 6C). Although nk2.2 expression spreadlaterally into the eyes of injected embryos, it was neverdetected in the hypothalamus or within dorsal regions of thetelencephalon (Fig. 6A,C,D).

In embryos that exhibited ectopic nk2.2 expression in theoptic primordia, normal eye development was impaired, andeyes remained fused to the brain (Fig. 6A,B,D). The area offusion extended dorsocaudally from the normal position of theoptic stalk to near the MDB (Fig. 6A,B). In addition, the opticprimordia frequently failed to invaginate to form an optic cupand showed abnormal development of the presumptive neural

Fig. 7. Expression of nk2.2, shh/vhh-1 and axial in homozygous mutant ctransverse sections (B,C) of embryos hybridised with nk2.2, axial or shh/wild-type and cyclops mutant embryos. The transverse sections shown inthe dorsal surface of the embryo in B is an artefact. (D) 30 somites (24h)axial and shh/vhh-1. The arrowheads indicate a small cluster of axial andat the dorsal tip of the mid-diencephalic furrow of a 30 somite (24h) cycldiencephalic furrow of a 40-44h cyclops mutant embryo. Abbreviations: diencephalic boundary; mdf, mid-diencephalic furrow; ov, optic vesicle;

and pigment layers of the retina (compare Fig. 6D with F).Indeed, the abnormal optic primodia of injected embryos moreclosely resembled the undifferentiated optic vesicles of muchyounger normal embryos. Possibly as a consequence ofabnormal optic cup formation, the lens was frequently reducedin size or sometimes even absent from the eyes of injectedembryos (Fig. 6G-I).

cyclops mutant embryos that lack shh/vhh-1 andaxial expression in the neuroectoderm exhibit aconcomitant loss of nk2.2 expressionThat overexpression of shh/vhh-1 leads to ectopic induction ofnk2.2 suggests that shh/vhh-1 may be required for the normalinduction of nk2.2 expression. To investigate this possibility,we examined nk2.2 expression in embryos homozygous for thecyclops mutation. The cyclops mutation prevents specificationof the ventral midline in the CNS (Hatta et al., 1991) and, atearly stages, mutant embryos do not express shh/vhh-1 withinthe CNS (Krauss et al., 1993).

nk2.2 expression was absent from the forebrain of all cyclopsmutant embryos examined between 10 somites (14h) and 24somites (21h) (n=23; Fig. 7A,B,C). shh/vhh-1 and axialexpression were also absent at comparable stages confirmingprevious results (Krauss et al., 1993; Strähle et al., 1993).However, in 30 somites (24h) and older mutant embryos, asmall patch of cells expressed nk2.2 (12/16 embryos examined),

yclops embryos. Lateral views (A,D,E,F) with rostral to the left, andvhh-1 anti-sense RNA. (A-C) nk2.2 expression in 18 somite (18h) B and C are at the level of the diencephalon. The small dark patch on cyclops mutant embryos hybridised with antisense RNA to nk2.2, shh/vhh-1-expressing cells in the forebrain. (E) shh/vhh-1 expressionops mutant embryo. (F) nk2.2 expression at the tip of the mid-cb, cerebellum; e, epiphysis; fe, fused eye; l, lens; mdb, mid-t, telencephalon; te, tectum. Scale bar=100 µm.


shh/vhh-1 (14/19) and axial (14/15) at the tip of the furrow thatforms in place of the MDB (see Macdonald et al., 1994; Patelet al., 1994) in cyclops mutant embryos (Fig. 7D-F).

Low levels of nk2.2 expression were also detected in morecaudal regions of the CNS of cyclops embryos from about 16somites (14h) (data not shown). Similar observations havebeen made for shh/vhh-1 (Krauss et al., 1993) and axial(Macdonald et al., 1994).

DISCUSSION

We have described the isolation and characterisation of thezebrafish nk2.2 gene. In common with all members of the Nk-2 family of homeobox genes, nk2.2 contains a conservedsequence characteristic for this family, the Nk-2 domain. Thehigh conservation of the Nk-2 domain suggests that it isimportant for the function of Nk-2 proteins. The nature of thisfunction is unknown although it has been suggested that theNk-2 domain could be involved in mediating protein-proteininteractions (Price et al., 1992).

nk2.2 is most closely related to mouse Nkx-2.2 (Price et al.,1992) and Xenopus XeNk-2 (Saha et al., 1993). The observedhomology at the amino acid level appears to be paralleled bythe conservation of the expression patterns among nk2.2, Nkx-2.2 and XeNk-2, although there are some differences betweenour interpretation of expression patterns and others. However,these differences probably reflect the fact that previous studieshave not analysed expression patterns in such great detail.Indeed, recent reanalysis of Nkx-2.2 expression in mousesuggests a very close similarity in expression between this geneand nk2.2 within the developing forebrain (Rubenstein et al.,1994; Rubenstein, personal communication).

While expression domains of nk2.2, Nkx-2.2 and XeNk-2appear to be similar in the CNS, nk2.2 exhibits one additionalsite of expression not reported for the mouse and frog homo-logues. This patch of nk2.2-expressing cells is located in aregion ventral to the hypochord near the hindbrain/spinal cordboundary. The transient and weak nature of expression at thissite may explain why it has not been described in other species.

nk2.2 expression delineates a zone of neuronaldifferentiation in the rostral brainMany of the early neurons in the rostral zebrafish CNS differ-entiate at boundaries between regulatory gene expressiondomains (Macdonald et al., 1994). For instance, the nTPOCand nMLF are both positioned at the ventral boundary ofexpression of the receptor tyrosine kinase, rtk1, and the pairedbox transcription factor, pax6. These observations raised thepossibility that cells at the interface between adjacentexpression domains may have an identity distinct from that ofeither of the neighbouring domains (Wilson et al., 1993). nk2.2is expressed in a band of cells at the interface where both thenTPOC and nMLF differentiate suggesting that this gene maybe involved in the establishment or maintenance of the identityof cells at a zone of neuronal differentiation.

From studies performed mainly in Drosophila, at least twoclasses of genes have been shown to be important in regulat-ing neurogenesis; proneural genes influence whether ectoder-mal cells become epidermis or neural tissue while neurogenicgenes influence which of the neural cells differentiate as

neurons (Jimenez and Modolell, 1993). Several members of thebasic helix-loop-helix family of transcription factors act asproneural genes (Jan and Jan, 1993), while signallingmolecules including Notch and Delta function in the neuro-genic pathway (Ghysen et al., 1993). Although nk2.2expression defines several regions where neurons differentiate,it is unlikely that it functions as a neurogenic gene. Forinstance, nk2.2 is expressed in a continuous longitudinal bandwithin the rostral brain at which early neuronal differentiationis only observed in two discrete sites. Therefore, many of thecells that express nk2.2 do not appear to be in the develop-mental pathway leading to early neuronal differentiation.However, it is possible that nk2.2 functions in combinationwith other genes to regulate the distribution of the earliestneurons in the brain (Barth and Wilson, 1994).

Our results indicate that cells at a boundary region aredistinct from adjacent cells in terms of gene expression, thoughit remains unknown if this distinction extends to differences inmorphology or cell surface properties, as has been documentedfor boundary cells between rhombomeres in the hindbrain(Heyman et al., 1994). Although there are no publisheddescriptions of cell surface proteins restricted to boundary cellsin the forebrain, several such proteins are expressed in spatiallyrestricted domains that respect these boundaries (Allendoerferet al., 1994; Redies and Gänzler, 1994.

shh/vhh-1 influences the expression of nk2.2All sites of nk2.2 expression in the CNS lie within several celldiameters of cells that express shh/vhh-1. The observations thatall changes in the pattern of shh/vhh-1 expression are accom-panied by complementary changes in nk2.2 expression, andthat overexpression of shh/vhh-1 ectopically induces nk2.2,suggest that secreted shh/vhh-1 may be required for nk2.2expression. Hence, the spatially restricted domain of nk2.2expression may arise due to the limited diffusion of shh/vhh-1 protein within the neuroectoderm. The Drosophila hedgehogprotein has recently been shown to be cleaved into two activeforms and it is likely that one has short-range, and one haslonger range activities (Lee et al., 1994). Similar cleavage ofzebrafish shh/vhh-1 occurs (Lee et al., 1994) though it remainsunknown over what range the two protein species may signaland so it is premature to speculate which of the two proteinsmay be involved in regulating nk2.2 expression.

Further support for a possible requirement of shh/vhh-1 forthe induction of nk2.2 expression is derived from the observedlack of nk2.2 transcripts in young cyclops mutant embryos,which lack shh/vhh-1 expression in the CNS. Although recentresults have shown that the mesoderm of cyclopic embryos isaffected (Thisse et al., 1994), the primary consequence of themutation appears to be the incorrect specification of ventralmidline cells in the CNS (Hatta et al., 1991, 1994). We suggestthat a secondary consequence of the failure to specify ventralmidline tissue is a failure to induce nk2.2 in more lateral cells.

In older homozygous cyclops mutant embryos, the partialrecovery of shh/vhh-1 and axial expression is accompanied bylate expression of nk2.2. The recovery of ventral midline geneexpression in the neuroectoderm of cyclops mutant embryos isnot understood but suggests that signalling between mesodermand ectoderm may not be completely blocked by the mutation.It also remains unknown if the recovery of gene expression isaccompanied by changes in the phenotype of midline cells.


Although we suggest that shh/vhh-1 influences nk2.2expression, other molecules may also be involved in the spatialregulation of expression of this gene since overexpression ofshh/vhh-1 results in spatially restricted ectopic expression ofnk2.2; thus only a subset of neural cells exposed to shh/vhh-1respond by inducing nk2.2. However, we cannot rule out thepossibility that spatially restricted ectopic nk2.2 expressionmay be explained by position-specific differences inexogenous shh/vhh-1 RNA or protein stability or processing.

The spatial relationship between cells that express nk2.2 andcells expressing shh/vhh-1 differs slightly between the rostraland caudal domains of nk2.2 expression in the forebrain. In thecaudal domain, cells expressing nk2.2 and shh/vhh-1 arediscrete populations whereas rostrally the expression domainsof these genes overlap. If shh/vhh-1 is involved in the inductionof nk2.2 transcription, then it is of interest to consider whynk2.2 expression is not induced within all shh/vhh-1-express-ing cells. One possibility is that other gene(s) may repressnk2.2 expression within many of the cells that express shh/vhh-1 and thus it could be the absence of such factors rostrally thatallows overlap of nk2.2 and shh/vhh-1 expression.

Do nk2.2 and shh/vhh-1 expression domains definethe rostro-caudal axis of the brain?Temporal analysis of shh/vhh-1 and nk2.2 expression indicatesthat both genes are transcribed to the anterior tip of the con-densing neural keel. As the forebrain differentiates, it becomesapparent that the anteriormost point of expression lies imme-diately ventral to the optic recess. These observations supportthe hypothesis that the anterior end of the brain is the opticstalk region and that the telencephalon is a dorsal structure(Ross et al., 1992; Puelles and Rubenstein, 1993; Hatta et al.,1994; Rubenstein et al., 1994), not a discrete neuromere rostralto the diencephalon.

Although nk2.2 is expressed further dorsally in rostralcompared to caudal regions of the brain, we believe that theposition at which this gene is expressed may be equivalentthroughout the CNS in terms of the molecular mechanisms thatregulate dorsoventral patterning. Indeed, in the early neuralkeel, there is little difference in the dorsoventral position ofshh/vhh-1 or nk2.2 expression between the rostral brain andmore caudal regions. It is only at later stages that the distinc-tion between more dorsally positioned expression in the dien-cephalon and ventral expression in more caudal regionsbecomes apparent.

Two prominent features of forebrain morphogenesis con-tribute to the spatial changes in gene expression in the dien-cephalon. The first is the expansion of the hypothalamus andthe associated development of the cephalic flexure; the secondis the development of the MDB. Fate mapping studies of theneural plate in Xenopus suggest that the hypothalamus derivesfrom midline cells (Eagleson and Harris, 1990), and so weassume that the hypothalamic cells derive from the early poolof shh/vhh-1-expressing midline cells. The later expansion ofthe hypothalamic region is not well understood in any speciesand it is unknown if it occurs by recruitment of more dorsalforebrain cells or by exaggerated proliferation of the mostventral cells.

The shift in gene expression associated with the develop-ment of the MDB takes place after the neural keel hascondensed and continues throughout early forebrain morpho-

genesis. Over time, there is a gradual dorsal extension in axialexpression, followed by a comparable change in shh/vhh-1expression and complemented by a dorsal deflection of nk2.2expression either side of the MDB. At least two possibilitiescould account for the changes in gene expression at the MDB.Either cells could migrate from ventral regions to more dorsalpositions at the MDB or, alternatively nk2.2, axial andshh/vhh-1 expression may be gradually induced in progres-sively more dorsal cells within the mid-diencephalon. Indeed,the precocious expression of axial in dorsal cells at the MDBof shh/vhh-1-injected embryos suggests that dorsal cells maybe responsive to inductive signals at stages before axial isnormally expressed. This would suggest that in normalembryos, the temporal availability of inductive signals maycontribute to the regulation of gene expression at the MDB.

We thank Phil Ingham, Jean-Paul Concordet, Stefan Krauss, UweSträhle, Anders Fjose, Denis Duboule and Claudio Stern for probesor antibodies, David Grunwald and R. Riggleman for the cDNAlibrary and Nigel Holder, Rachel Macdonald and Roger Patient forcomments on the manuscript. This study was initiated with funds fromthe Medical Research Council and supported by the Wellcome Trust.S. W. was a Science and Engineering Research Council AdvancedResearch Fellow and is a Wellcome Senior Research Fellow.

The databank accession number for nk2.2 cDNA sequence isX85977.

REFERENCES

Allendoerfer, K. L., Tole, S. and Patterson, P. H. (1994). Biochemicalcharacterization of FORSE-1, a positionally-restricted proteoglycan in thedeveloping CNS. Soc. Neurosci. Abstr. 20, 691.

Ang, S.-L. and Rossant, J. (1994). HNF-3β is Essential for node andnotochord formation in mouse development. Cell 78, 561-574.

Barth, K. A. and Wilson, S. W. (1994). Specification of neuronal identity inthe embryonic CNS. Semin. Dev. Biol. 5, 349-358.

Chitnis, A. B. and Kuwada, J. Y. (1990). Axonogenesis in the brain ofzebrafish embryos. J. Neurosci. 10, 1892-1905.

Devereux, J., Haeberli, P. and Smithies, O. (1984). A Comprehensive set ofsequence analysis programs for the VAX. Nucl. Acids Res. 12, 387-395.

Eagleson, G. W. and Harris, W. A. (1990). Mapping of the presumptive brainregions in the neural plate of Xenopus laevis. J. Neurobiol. 21, 427-440.

Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J.,McMahon, J. A. and McMahon, A. P. (1993). Sonic Hedgehog, a memberof a family of putative signalling molecules is implicated in the regulation ofCNS polarity. Cell 75, 1417-1430.

Figdor, M. and Stern, C. D. (1993). Segmental organization of embryonicdiencephalon. Nature 363, 630-633.

Fjose, A., Izpisua-Belmonte, J. C., Fromental-Ramain, C. and Duboule, D.(1994). Expression of the zebrafish gene hlx-1 in the prechordal plate andduring CNS development. Development 120, 71-81.

Ghysen, A., Dambly-Chaudiere, C., Jan, L. Y. and Jan, Y. N. (1993). Cellinteractions and gene interactions in peripheral neurogenesis. Genes Dev. 7,723-733.

Guazzi, S., Price, M., De Felice, M., Damante, G., Mattei, M.-G. and DiLauro, R. (1990). Thyroid nuclear factor 1 (TTF-1) contains ahomeodomain and displays a novel DNA binding specificity. EMBO J. 9,3631-3639.

Hatta, K., Kimmel, C. B., Ho, R. K. and Walker, C. (1991). The cyclopsmutation blocks specification of the floorplate of the zebrafish centralnervous system. Nature 350, 339-341.

Hatta, K., Püschel, A. W. and Kimmel, C. B. (1994). Midline signalling in theprimordium of the zebrafish anterior central nervous system. Proc. Natl.Acad. Sci. USA 91, 2061-2065.

Heyman, I., Kent, A. and Lumsden, A. (1994). Cellular morphology andextracellular space at rhombomere boundaries in the chick embryohindbrain. Devel. Dyn. 198, 241-253.


Jan, Y. N. and Jan, L. Y. (1993). HLH proteins, fly neurogenesis, andvertebrate myogenesis. Cell 75, 827-830.

Jimenez, F. and Modolell, J. (1993). Neural fate specification in Drosophila.Curr. Op. Gen. Devel. 3, 626-632.

Kim, Y. and Nirenberg, M. (1989). Drosophila NK-homeobox genes. Proc.Natl. Acad. Sci. USA 86, 7716-7720.

Krauss, S., Concordet, J.-P. and Ingham, P. (1993). A functionallyconserved homolog of the Drosophila segment polarity gene hh is expressedin tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444.

Krumlauf, R., Marshall, H., Studer, M., Nonchev, S., Sham, M. H. andLumsden, A. (1993). Hox homeobox genes and regionalisation of thenervous system. J. Neurobiol. 24, 1328-1340.

Lai, E., Clark, K. L., Burley, S. K. and Darnell, J. E. Jr. (1993). Hepatocytenuclear factor 3/fork head or ‘winged helix’ proteins: a family oftranscription factors of diverse biologic function. Proc. Natl. Acad. Sci. USA90, 10421-10423.

Lazzaro, D., Price, M., De Felice, M. and Di Lauro, R. (1991). Thetranscription factor TTF-1 is expressed at the onset of thyroid and lungmorphogenesis and in restricted regions of the foetal brain. Development113, 1093-1104.

Lee, J. J., Ekker, S. C., von Kessler, D. P., Porter, J. A., Sun, B. I. andBeachy, P. I. (1994). Autoproteolysis in hedgehog protein biogenesis.Science 266, 1528-1537.

Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I. and Harvey, R. P. (1993).Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitorcells and their myogenic descendants. Development 119, 419-431.

Macdonald, R., Xu, Q., Barth, K. A., Mikkola, I., Holder, N., Fjose, A.,Krauss, S. and Wilson, S. W. (1994). Regulatory gene expressionboundaries demarcate sites of neuronal differentiation and reveal neuromericorganisation of the zebrafish forebrain. Neuron 13, 1039-1053.

Pani, L., Overdier, A., Porcella, A., Qian, X., Lai, E. and Costa, R. H.(1992). Hepatocyte nuclear factor 3b contains two transcriptional activationdomains, one of which is novel and conserved with the Drosophila fork headprotein. Mol. Cell. Biol. 12, 3723-3732.

Patel, C., Rodriquez, L.C. and Kuwada, J.Y. (1994). Axonal outgrowthwithin the abnormal scaffold of brain tracts in a zebrafish mutant. J.Neurobiol. 25, 345-360.

Price, M., Lazzaro, D., Pohl, T., Mattei, M.-G., Ruther, U., Olivo, J.-C.,Duboule, D. and Di Lauro, R. (1992). Regional expression of thehomeobox gene Nkx-2.2 in the developing mammalian forebrain. Neuron 8,241-255.

Puelles, L., Amat, J. A. and Martinez-de-la-Torre, M. (1987). Segment-related, mosaic neurogenetic pattern in the forebrain and mesencephalon ofearly chick embryos: i. topography of AChE-positive neuroblasts up to stageHH18. J. Comp. Neurol. 266, 247-268.

Puelles, L. and Rubenstein, J. L. R. (1993). Expression patterns of homeoboxand other putative regulatory genes in the embryonic mouse forebrainsuggest a neuromeric organization. Trends in NeuroScience 16, 472-479.

Redies, C. and Gänzler, S. (1994). Neuromeric expression of R-cadherin andthe formation of nuclei in the developing chicken forebrain. Soc. Neurosci.Abstr. 20, 252.

Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz iAltaba, A., Tanaba, Y., Placzek, M., Edlund, T., Jessel, T. M. and Dodd,J. (1994). Floor plate and motor neuron induction by vhh-1, a vertebratehomolog of hedgehog expressed by the notochord. Cell 76, 761-755.

Ross, L. S., Parrett, T. and Easter, S. S. Jr. (1992). Axonogenesis andmorphogenesis in the embryonic zebrafish brain. J.Neurosci. 12, 467-482.

Rubenstein, J. L. R., Martinez, S., Shimamura, K. and Puelles, L. (1994).The embryonic vertebrate forebrain: the prosomeric model. Science 266,578-580.

Ruiz i Altaba, A. and Jessel, T. M. (1992). Pintallavis, a gene expressed in theorganizer and midline cells of frog embryos: involvement in the developmentof the neural axis. Development 116, 81-93.

Ruiz i Altaba, A., Prezioso, V. R., Darnell, J. E. and Jessell, T. M. (1993).Sequential expression of HNF-3β and HNF-3α by embryonic organizingcenters: the dorsal lip/node, notochord and floorplate. Mech. Devel. 44, 91-108.

Saha, M. S., Michel, R. B., Goulding, K. M. and Grainger, R. M. (1993). AXenopus homeobox gene defines dorsal-ventral domains in the developingbrain. Development 118, 193-202.

Sasaki, H. and Hogan, B. L. M. (1993). Differential expression of multiplefork head related genes during gastrulation and axial pattern formation in themouse embryo. Development 118, 47-59.

Sasaki, H. and Hogan, B. L. M. (1994). HNF-3β as a regulator of floor platedevelopment. Cell 76, 103-116.

Smith, J. C. (1994). Hedgehog, the Floorplate, and the Zone of PolarizingActivity. Cell 76, 193-196.

Strähle, U. and Blader, P. (1994). Early neurogenesis in the zebrafish embryo.FASEB J. 8, 692-698.

Strähle, U., Blader, P., Henrique, D. and Ingham, P. (1993). Axial, azebrafish gene expressed along the developing body axis, shows alteredexpression in cyclops mutant embryos. Genes Dev. 7, 1436-1446.

Thisse, C., Thisse, B., Halpern, M. and Postlethwait, J. H. (1994). goosecoidexpression in neurectoderm and mesoderm is disrupted in zebrafish cyclopsgastrulas. Dev. Biol. 164, 420-429.

Weinstein, D. C., Ruiz i Altaba, A., Chen, W. S., Hoodless, P., Prezioso, V.R., Jessel, T. M. and Darnell, J. E. Jr. (1994). The winged-helixtranscription factor HNF-3β is required for notochord development in themouse embryo. Cell 78, 575-588.

Westerfield, M. (1993). The Zebrafish Book. University of Oregon Press. Wilson, S. W., Placzek, M. and Furley, A. (1993). Border disputes: do

boundaries play a role in growth cone guidance? Trends in NeuroScience 16,316-322.

Wilson, S. W., Ross, L., Parrett, T. and Easter, S. S., Jr. (1990). Thedevelopment of a simple scaffold of axon tracts in the brain of the embryoniczebrafish, Brachydanio rerio. Development 110, 121-145.

Xu, Q., Holder, N., Patient, R. and Wilson, S. W. (1994). Spatially regulatedexpression of three receptor tyrosine kinase genes during gastrulation in thezebrafish. Development 120, 287-289.

(Accepted 17 February 1995)

Documents

Expression of zebraﬁsh nk2.2is inﬂuenced by sonic hedgehog ... · shh/vhh-1 and the transcription factor axial are involved in the spatial regulation of expression of nk2.2shh/vhh-1