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Development 122, 271-280 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 DEV3333

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Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos Eric S. Weinberg1,*, Miguel L. Allende1,†, Christina S. Kelly1, Aboulmagd Abdelhamid1, Tohru Murakami1, Peter Andermann1, O. Geoffrey Doerre1, David J. Grunwald2 and Bob Riggleman3 1Department of Biology, The University of Pennsylvania, Philadelphia, PA 19104, USA 2Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA 3Departments of Genetics and Cell Biology and Zoology, Washington State University,

Pullman, WA 99164, USA

*Author for correspondence †Present address: Department of Biology, Center for Cancer Research, E17-341, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge MA 02139, USA

SUMMARY We describe the isolation of the zebrafish MyoD gene and its expression in wild-type embryos and in two mutants with altered somite development, no tail (ntl) and spadetail (spt). In the wild-type embryo, MyoD expression first occurs in an early phase, extending from mid-gastrula to just prior to somite formation, in which cells directly adjacent to the axial mesoderm express the gene. In subsequent phases, during the anterior-to-posterior wave of somite formation and maturation, expression occurs within particular regions of each somite. In spt embryos, which lack normal paraxial mesoderm due to incorrect cell migration, early MyoD expression is not observed and transcripts are instead first detected in small groups of trunk cells that will develop into aberrant myotomal-like structures. In ntl embryos, which lack notochords and tails, the early phase of MyoD expression

is also absent. However, the later phase of expression within the developing somites appears to occur at the normal time in the ntl mutants, indicating that the presomitogenesis and somitogenesis phases of MyoD expression can be uncoupled. In addition, we demonstrate that the entire paraxial mesoderm of wild-type embryos has the potential to express MyoD when Sonic hedgehog is expressed ubiquitously in the embryo, and that this potential is lost in some of the cells of the paraxial mesoderm lineage in no tail and spadetail embryos. We also show that MyoD expression precedes myogenin expression and follows or is coincident with expression of snail1 in some regions that express this gene.

INTRODUCTION

degree (Venuti et al., 1995), but are blocked in later differentiation steps (Hasty et al., 1993; Nabeshima et al., 1993). Although the myogenic bHLH proteins are essential for the establishment of muscle cell precursors and their differentiation, much less is known about the processes of commitment of mesodermal precursors to the myogenic lineage. The zebrafish embryo provides a powerful system to study formation of skeletal muscle and the role of the notochord and neural tube in this process. Zebrafish embryos homozygous for the no tailb160 (ntl) mutation, a molecular lesion in the homolog of the mouse Brachyury (T) gene (Schulte-Merker et al., 1994), do not form a notochord or tail (Halpern et al., 1993). Although mutant embryos do form somites, these structures lack muscle pioneer cells and have a different shape than in wild-type embryos (Halpern et al., 1993). Embryos homozygous for another mutation, spadetail (spt), fail to form normal trunk somites as a consequence of aberrant cell movement of precursor cells (Kimmel et al., 1989; Ho and Kane, 1990). Cells that normally form paraxial mesoderm by convergence towards the dorsal side of the gastrula incorrectly move to the tail in spt embryos.

Trunk skeletal muscle in vertebrates is derived from the myotomal component of somites, segmental structures formed from the paraxial mesoderm in an anterior-to-posterior sequence. A group of structurally related proteins, the myogenic basic helix-loop-helix (bHLH) family of transcription factors, has a major regulatory role in myogenesis (reviewed in Emerson, 1993; Weintraub, 1993; Olson and Klein, 1994). Genes encoding four myogenic bHLH proteins have been found in mammals: MyoD, myogenin, myf-5 and MRF4 (also known as herculin or myf-6). These genes encode proteins with a basic domain that mediates sequence-specific DNA binding and a helix-loop-helix domain that regulates dimerization. Gene knock-out experiments in mice have shown that either MyoD or myf-5 is sufficient for muscle formation (Rudnicki et al., 1992; Braun et al., 1992), but the loss of both genes results in a failure to form myoblasts (Rudnicki et al., 1993). Mice lacking the myogenin gene form myoblasts that are capable of early stages in muscle differentiation to some

Key words: myogenesis, somite, MyoD, myogenin, snail, Sonic hedgehog, bHLH protein, zebrafish, Brachyury, notochord

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In an attempt to learn more about the factors that regulate myogenesis, we have initiated studies on the myogenic bHLH gene family in the zebrafish. We report here the isolation and sequence of a cDNA encoding MyoD, and show that the gene is expressed in several characteristic phases, including an initial phase unique to teleost embryos. To position the MyoD gene in the regulatory pathway leading to the differentiation of muscle, we also compared its expression pattern with myogenin and snail1 and studied the effects on expression of MyoD of mutations in two genes, spt and ntl, which have effects on myotome formation in the zebrafish. Our additional findings, that the entire paraxial mesoderm of wild-type embryos has the potential to express MyoD when Sonic hedgehog is expressed ubiquitously in the embryo and that many of the cells of the paraxial mesoderm lineage in ntl and spt embryos have lost this potential, indicate that although proximity of paraxial mesoderm to a source of Shh is sufficient to activate MyoD, key parts of this induction pathway are defective in the mutant embryos. MATERIALS AND METHODS Zebrafish methods Wild-type and brass zebrafish (Danio rerio) obtained from EkkWill WaterLife Resources (Gibbonston, FL) were raised under standard conditions at 28.5°C (Westerfield, 1993). The brass mutant line is particularly well suited for in situ hybridization because of delayed pigmentation. We obtained spadetailb106 (spt) and no tailb160 (ntl) heterozygous fish from the University of Oregon. The terms spt and ntl are used to refer to embryos homozygous for the mutation. Among the progeny of crosses between heterozygotes, heterozygous and homozygous wild-type siblings (approximately three-fourths of the embryos) were indistinguishable from the embryos obtained from the brass mutant line, and all were considered as ‘wild-type’ embryos in this study with respect to somite morphogenesis and gene expression. Stages are given as hours of development after fertilization. Cloning of the MyoD cDNA Degenerate oligonucleotide primers were designed for conserved regions of the MyoD bHLH region. A sense strand primer (5′ GCTCGAGCAA(A/G)GTIAA(T/C)GA(G/A)GCITT(T/C)GA 3′) encoding the amino acid sequence SKVNEAFE and an antisense strand primer (5′ TGGAAGCTTTCIAT(A/G)TAICG(T/G/A)ATIGC(G/A)TT 3′) encoding the amino acid sequence NAIRYIES were synthesized. Polymerase chain reaction (PCR) amplifications were performed using zebrafish genomic DNA as a template with an annealing temperature of 40°C for 2 minutes and 30 cycles. Under these conditions, a DNA fragment of approximately 120 bp was amplified and then cloned into a Bluescript (Stratagene) vector. Sequencing revealed that the inserts of several of these clones encoded amino acids identical to the helixloop-helix region of murine MyoD. The insert of one of these clones was used to screen a 20-28 hour zebrafish embryonic lambda-ZAP cDNA library (constructed by B. Riggleman and K. Helde; inserts are cloned into the EcoRI and XhoI sites). Of 400,000 plaques screened, two hybridizing clones were purified. Plasmids containing the cDNA sequences were prepared by in vivo excision from the lambda ZAP clones as recommended by the manufacturer. The two clones contained 1.65 kb inserts that had identical sequences at the 5′ and 3′ ends. Both strands of one of these cDNAs (pZMD3) were sequenced by the chain termination method (the nucleotide sequence appears in the EMBL Nucleotide Sequence Database under accession number Z36945). In situ hybridization to whole embryos and RNA injection Digoxigenin-labeled probes were synthesized by in vitro transcription using T3 and T7 polymerases and digoxigenin-11-UTP (Boehringer-

Mannheim) in the reaction. Embryos were fixed in 4% paraformaldehyde in PBS for 12-16 hours at 4°C. We used a modified version (M. Westerfield, personal communication) of the in situ hybridization protocol of Schulte-Merker et al. (1992). snail1 transcripts were detected using a probe prepared from the cDNA clone of Thisse et al. (1993). Whole embryos were observed and photographed using a Wild MC3 stereo dissecting microscope. For observation at higher magnification, dissected embryos were viewed under DIC optics using a Reichert Polyvar-2 compound microscope. RNA was synthesized in vitro from a zebrafish shh sequence cloned in pSP64T (Krauss et al., 1993), generously supplied by P. Ingham. Injections of RNA (approximately 1 nl of a 100 µg/ml solution) were carried out using an immobilizing device designed by one of us (E. S. W.) (described in Westerfield, 1993).

RESULTS Structural analysis of zebrafish MyoD Sequence analysis demonstrated that the zebrafish MyoD cDNA encodes a conceptual protein of 275 amino acids in length (Fig. 1). An ATG 187 nt from the 5′ end of the cDNA provides the most-likely initiation codon. This ATG, followed immediately by a G and preceded by the sequence ACC, lies within a plausible translational initiation sequence (Kozak, 1987). The zebrafish putative MyoD protein is 75.6% identical to the Xenopus MyoD homolog Xlmf1 (Hopwood et al., 1989; Scales et al., 1990; Harvey, 1990) and 72.4% identical to mouse MyoD1 (Davis et al., 1987). Within the bHLH region, the degree of identity between the three predicted MyoD proteins is greater than 94% with three amino acid differences between the zebrafish and each of the other two sequences. Interestingly, several large insertions occur in the mouse gene that are not found in either the Xenopus gene or the zebrafish gene. Because of the lineage relationship between teleosts, amphibians and mammals, the most likely molecular explanation for these differences is that a set of insertions occurred in the mammalian lineage after divergence from the amphibian lineage.

MyoD expression in wild-type embryos To examine the timing of MyoD expression and the spatial distribution of MyoD transcripts during zebrafish development, we performed in situ hybridizations on whole embryos using digoxigenin-labeled probes. The expression of the gene occurs in several phases. In the first phase, which lasts from midgastrula to just before somite formation, the gene is activated in rows of cells (termed adaxial cells) adjacent to the notochord or notochord precursor mesoderm. MyoD transcript is first detected in small triangular patches on each side of the embryonic shield at about 7-7.5 hours (Fig. 2A). Transverse sections through the shield and surrounding tissue (data not shown) indicate that MyoD expression at this stage is located in the hypoblast (cells that have involuted). At this time, the axial mesoderm cells that express goosecoid have migrated toward the animal pole and are far removed from the MyoDexpressing cells (data not shown). By 8.5 hours, the patches of cells containing MyoD transcript elongate and form a pair of narrow longitudinal rows, which lie on either side of the prospective notochord (Fig. 2B). At 10.5 hours, prior to the formation of somites, the rows of MyoD-expressing cells are two cells wide along the mediolateral axis (Figs 2C,3A), and increase in thickness along the dorsoventral axis from two cells (anteriorly) to six cells (posteriorly). The second phase of MyoD expression occurs between 10.5

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and 12 hours, the period in which the first 6-7 somites rapidly Fig. 3C,E). In all fully formed somites, some cells in the ventral form. During this interval, MyoD transcripts are simultaneously region do not express the gene and some dorsal cells including detected in a series of 5-7 faint bands of cells that project those directly abutting the neural tube are also devoid of tranlaterally from the longitudinal rows (data not shown). We never script. It will be of interest to learn whether the expression zone observed a case in which fewer than 5 of these bands are of MyoD coincides with the zebrafish myotome once that unit is formed. The initiation of MyoD expression in the laterally probetter defined by lineage studies. jecting bands begins shortly after the time of first somite furrow The dynamic modeling of the somite characteristic of the formation (10.5 hours), and intensifies during the formation of third phase of MyoD expression, therefore, involves stereothe first six somites. At the end of this period, prominent bands typed changes within a single somite occurring over a several can be seen at the posterior of each formed somite. hour period. These changes in the MyoD pattern, which can be Between 12 and 30 hours, two somites are formed per hour observed at any one time by comparing somites along the in a regular fashion (Hanneman and Westerfield, 1989; Westanterior-posterior axis, are diagrammed schematically for a 9erfield, 1993). In a third phase of MyoD expression, starting at somite (13 h) embryo (Fig. 5): (a) an initial rapid extension of 12.5 hours (7- to 8-somite stage), additional bands of cells MyoD expression to the posterior lateral regions of the develexpress the gene (Figs 2D,3C). Typically at this time there are oping somite, (b) an intensification of expression throughout 7-8 pairs of prominent bands and two pairs of less intense the posterior half of the somite except in the outermost (lateral, bands located caudally (Fig. 3C). The prominent bands are dorsal and ventral) cell layer, (c) a broadening of expression, each located at the posterior of the already formed somites now covering most of the anterior-posterior axis of the somite adjacent to the furrow (Fig. 3C, arrows), and fainter pairs of at its medial extent, but still restricted to the posterior portion bands (Fig. 3C, open arrows) are at the posterior of a somite of the somite at more lateral positions and (d) a decrease in just undergoing furrow formation or in paraxial mesoderm yet transcript levels throughout the somite, about 3-4 hour after to form segmented structures. somite formation (not shown in Fig. 5 since this is obvious only The shapes of the somites, and the locations of MyoDin later embryonic stages). expressing cells within these somites, differ along the anteriorBy 24 hours, the level of MyoD transcript has dropped posterior axis and thus with respect to time since formation of markedly throughout the trunk and the strongest region of each somite. The most anterior four somites have their highest expression is found within the tail (Fig. 2F). In the trunk concentration of MyoD transcript in the somite centers (Fig. 3C, region, the gene is expressed at highest level in the middle arrowheads), the three more posterior somites have greatest con(with respect to the anteroposterior axis) of each chevroncentration in the row of cells one cell removed from the posterior shaped somite and more intensely in ventral regions with of the somite, and the most posterior well-formed somite has respect to its dorsoventral axis. highest expression in the posterior row of cells. The anterior MyoD transcripts are also expressed in several other somites are block shaped and contain a wedgeshaped zone of MyoD expression which fills the posterior-medial half of the block, whereas the posterior somites are more rounded and have a zone of expression of the gene which is more like a stripe. By the time 12-13 somites have formed (in the 14.5 hour embryo), there are as many strongly labeled lateral bands of cells as there are somites (Figs 2E,3E). In addition, there are two fainter pairs of stained bands that correspond to the positions of the posterior of the next two pairs of somites to form. The formation of the bands of MyoD-expressing cells thus continues to take place in an anterior-to-posterior wave, anticipating the formation of somites by 30-60 minutes. The somite shapes and the location of MyoDexpressing cells show a posterior-to-anterior agedependent change similar to that of the 12.5 hour embryo. The oldest somites, however, now have much less transcript. Comparison of somites 1-4 in the 12.5 and 14.5 hour embryos (Fig. 3C,E) illustrates the transient nature of expression of the gene in any one somite. Transverse sections of 12.5 and 14.5 hour embryos were examined to determine the extent of MyoD expression along the somite dorsoventral axis (data not Fig. 1. Sequence comparison between the predicted amino acid sequences of shown). It appears that the changes in MyoD zebrafish, Xenopus and mouse MyoD proteins. Sequences of the complete proteins are optimally aligned. Vertical bars indicate identical residues; the basic and HLH expression within the somite during these stages domains are indicated by brackets. are coincident with changes in somite shape (e.g.,

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Fig. 2. Localization of MyoD, myogenin, and snail1 transcripts in whole embryos. Wild-type embryos were hybridized with MyoD (A-F), myogenin (G-J), snail1 (K-N), or MyoD + snail1 (O-R) probes. spt (S-W) or ntl (X-B′) embryos were hybridized with MyoD probe. Vertical panel rows show embryos at a particular embryonic age: 7-7.5 hours (A), 8-8.5 hours (B,K,O,S,X), 10-10.5 hours (C,G,L,P,T,Y), 12-12.5 hours (D,H,M,Q,U,Z), 14-14.5 hours (E,I,N,R,V,A′), and 24 hours (F,J,W,B′). All embryos are viewed dorsally (either directly or obliquely) with their anterior end to the top except for the 24 hour embryos (F,J,W,B′) which are viewed laterally, anterior to the left. In C-E, open arrows indicate portions of the expression pattern which are absent at the position of corresponding open arrows in Y, Z and A′. In C, the arrowhead indicates cells in the tailbud that express MyoD. In K-N, arrowheads indicate regions of paraxial mesoderm and tailbud that express snail1 but not MyoD. In W, arrows indicate myotome-like bodies expressing MyoD and the arrowhead shows the position of the spade tail in which expression is absent. Scale bar in A indicates 200 µm; all panels at same magnification.

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Fig. 3. Dorsal views of the trunk regions showing MyoD expression during somite formation and development. 10.5 hour (A,B), 12.5 hour (C.D) and 14.5 hour (E.F,G) wild-type (A,C,E) ntl (B,D,F), or spt (G) embryos were dissected, yolk removed from under the trunk, and the tissue viewed and photographed under DIC optics. All embryos are shown with anterior to the left. An open arrow in A points to the region of expression connecting the two parallel rows of expressing cells. In C-F, open arrows indicate prospective somites either unsegmented or with only partially formed posterior furrows, solid arrows point to the most recently fully formed furrows, and arrowheads indicate older somites with changed shape and MyoD expression pattern. In G, arrows show expressing cells remote from the notochord, open arrow shows thick spade tail area devoid of expression of the gene. All panels are composites of views in which the plane of the notochord is kept in focus. Scale bar in A equals 100 µm, all panels at same magnification.

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locations outside of the trunk. Starting at approximately 30 hours, MyoD staining is detected in the fin bud primordia, and somewhat later, in at least three pairs of extraocular muscles and one pair of jaw muscle progenitors (Fig. 4), all sites of MyoD expression in the mouse (Goldhamer et al., 1992). MyoD expression persists at these sites during later stages of development, at least until 60 hours. We did not detect MyoD transcripts in the heart at any stage of development. Comparison of MyoD, myogenin and snail1 expression in wild-type embryos To position the MyoD gene in the myogenic regulatory pathway, we compared its expression pattern with myogenin, and with snail1, a gene shown to be expressed in zebrafish paraxial mesoderm and in developing somites (Hammerschmidt and Nüsslein-Volhard, 1993; Thisse et al., 1993). We have cloned and sequenced a zebrafish cDNA encoding a 256 amino acid myogenin-like protein (B. Riggleman, T. Murakami, D. Grunwald, and E. Weinberg, unpublished results) and performed in situ hybridizations using probes prepared from this template. Without exception, the expression of zebrafish myogenin follows that of MyoD and is observed only in cells expressing MyoD. Unlike MyoD, however, myogenin transcripts were first detected only at 10.5 hours, and only in the adaxial cells at anteroposterior positions at which the first 5-6 somites will form (Fig. 2G). At 12-12.5 hours, myogenin transcripts appeared in lateral bands of cells extending away from the adaxial cells (Fig. 2H), but in contrast to MyoD, expression is restricted to already formed somites. Moreover, the wedge-shaped groups of cells expressing myogenin, a subset of the cells expressing MyoD, extend laterally only 2-3 cells from the adaxial cells instead of the 5-6 cell-wide bands which express MyoD at this stage. During the subsequent formation of somites from unsegmented paraxial mesoderm, myogenin is expressed in the posterior of each somite during the second hour after somite furrow formation, thus lagging behind MyoD expression in this region by approximately two hours. These differences between MyoD and myogenin expression are diagrammed for a 9 somite (13 h) embryo in Fig. 5. Somites of older embryos also show these differences and, although the myogenin expression zone now constitutes a larger portion of each somite (Fig. 2I), the myogeninexpressing cells are still only a subset of those expressing MyoD. The expression pattern of the two genes is also distinct in that (1) at all stages, the more posterior adaxial cells do not begin to express myogenin until approximately 2 hours prior to formation of somites in that region, and (2) at 14 hours, and later, myogenin transcripts are still present at high levels in the more anterior older somites (Fig. 2I) whereas MyoD transcript levels decrease in this region (Figs 2E,3E). The zebrafish snail1 gene is expressed in the paraxial mesoderm cell lineage during gastrulation and in developing somites (Fig. 2K-N; Hammerschmidt and Nüsslein-Volhard, 1993; Thisse et al., 1993). Although the pattern of snail1 expression resembles that of MyoD to some extent, there are important differences. snail1 is expressed in some regions that do not express MyoD, namely in paraxial mesoderm located lateral to the adaxial cells in both presomitogenic and somitogenic embryos (Fig. 2K-N, arrowheads). MyoD, although always expressed in cells containing at least some snail1 transcript, is expressed at high levels in some cells in which snail1 expression is relatively low. For example, the rows of adaxial

cells expressing snail1 at high level in 10-14.5 hour embryos are one cell wide rather than the typically observed 2-cell-wide rows of MyoD-expressing cells, and the bands of cells expressing high levels of snail1 at the posterior of each somite of the 12.5 and 14.5 hour embryos are thinner than the corresponding MyoD-expressing cells (compare Fig. 2D,E with M,N).

MyoD expression in spadetail embryos In embryos homozygous for sptb104, cells that normally converge toward the dorsal midline to form the paraxial mesoderm migrate incorrectly into the tail region (Kimmel et al., 1989; Ho and Kane, 1990). These spt embryos lack normal trunk somites, but do form tail somites. We were interested in determining whether the ability to activate myogenic genes is lost in the inappropriately migrating cells of the paraxial mesoderm lineage and whether non-paraxial mesodermal cells that come to be situated alongside or near the notochord in the mutant embryos can express MyoD. We routinely observed a lack of staining with MyoD probe during gastrulation in about one-fourth of the progeny of crosses of spt heterozygotes (Fig. 2S). Because all progeny of wild-type crosses stain at these stages, these unstained embryos are presumably spt homozygotes. At 10 hours, definitive spt embryos (identified by an accumulation of cells near the yolk plug closure) show no evidence of MyoD expression (Fig. 2T) in contrast to their wild-type siblings. These results indicate that, during gastrulation, the cells that lie adjacent to the developing notochord (presumably mostly if not entirely nonparaxial mesoderm cells) in spt embryos do not activate the MyoD gene. Moreover, misdirected spt cells, now located in the posterior of the embryo, also do not accumulate any MyoD transcript at this time. We observed that small clumps of trunk cells express the MyoD gene in 12.5 and 14.5 hour spt embryos (Fig. 2U,V). Dorsal views (Fig. 3G) and transverse sections (data not shown) of these embryos show that MyoD is expressed in groups of cells located alongside, and in some cases at a distance from, the notochord. The groups of stained cells are not always bilaterally arranged, but irrespective of whether or not they are adjacent to the notochord (which is usually bent and sometimes bifurcated), they are situated alongside the pial surface of the developing spinal cord. It is not clear what the origin of these MyoD-expressing cells is. In many cases, the cells resemble the adaxial cells of the wild-type embryo, but in some cases the cells appear to be mesenchymal. The shape of the expression zones is in sharp contrast to the ordered lateral bands of cells that express the gene in wild-type siblings at the same developmental age. In the 24 hour embryo, MyoD expression is detected in clumps of cells located at variable positions throughout the trunk (Fig. 2W, arrows) in myotomallike structures corresponding to those described by Kimmel et al. (1989). It is probable that these groups of cells are derived from MyoD-expressing adaxial cells or mesenchymal cells located alongside the spinal cord in the 14.5 hour spt embryo. The most prominent site of MyoD expression in the 24 hour spt embryo is in the proximal portion of the tail (Fig. 2W). Here, staining is as intense as in similarly situated cells in wildtype embryos (Fig. 2F), but the expression pattern is different. Although there is some periodicity in expression along the anteroposterior axis of the spt tail region, a much broader and less ordered array of cells express the gene along the dorsoven-

Expression of MyoD in the zebrafish embryo tral axis in comparison to expression in the wild-type tail. Interestingly, there is no expression of the gene in the flattened spade tail itself (Fig. 2W, arrowhead), which contains many of the mis-migrated cells (Kimmel et al., 1989), even though the notochord extends into this region. We also compared MyoD and snail1 expression in spt embryos (data not shown). As previously reported (Hammerschmidt and Nüsslein-Volhard, 1993; Thisse et al., 1993), snail1 expression is mostly absent from spt paraxial mesoderm in gastrula embryos. We found snail1 to be expressed in the region alongside the notochord of 12.5 and 14.5 hour embryos (similar to results of Thisse et al., 1993), but at a lower level and in a wider group of cells than for MyoD (Fig. 2U,V).

MyoD expression in the no tail mutant We decided to investigate the expression of MyoD in embryos homozygous for the ntlb160 allele since the mutation has been shown to cause a number of somite defects including the failure to form muscle pioneer cells (Halpern et al., 1993). There is a striking difference in the early expression of MyoD in wild-type and ntl embryos. Approximately one-fourth of the progeny of crosses of ntl heterozygotes fail to show staining with MyoD probe in the gastrula stages (Fig. 2X). When we did parallel hybridizations with progeny of wild-type strains, every embryo at the 8 hour stage gave the typical pattern discussed above (e.g., Fig. 2B). Since the Ntl phenotype only becomes apparent at late gastrula, these results are only inferential. At 10 hours we could show, however, that all morphologically distinguishable ntl homozygotes, which lack notochords, either lack MyoD transcripts or have a smaller and weaker region of expression. By 10.5 hours, approximately one-fourth of the progeny showed faint expression in shortened rows of adaxial cells (Figs 2Y,3B). The ntl embryos, therefore, first express MyoD at detectable levels between 10 and 10.5 hours of development. The rows of expressing cells at this stage are 1-2 cells in thickness, as in the wild-type embryos, but the rows are only about one-third as long and the level of transcript is lower (Figs 2Y,3B). The posterior extent of the rows of expressing cells was somewhat variable, but often was at an anteroposterior position near where the fifth or sixth somite would subsequently form. The rows of expressing cells are separated by two to three nonexpressing cells which are probably precursor cells that have failed to form a recognizable notochord (Halpern et al., 1993). In contrast to the early phase of MyoD expression, which differs dramatically in wild-type and ntl embryos, the subsequent phases of expression of the gene are quite similar. Just as in the wild-type embryos, bands of cells express the gene in the posterior of each new somite of ntl embryos. The patterns of these bands in ntl 12.5 hour (Figs 2Z,3D) and 14.5 hour (Figs 2A′,3F) embryos resemble those of wild-type embryos at the same ages. A major difference in the mutant embryos, however, is the lack of expressing adaxial cells in the posterior of the embryo (Fig. 2Z,A′, open arrows). Another difference is that the transcripts disappear from the older anterior somites more rapidly in the ntl embryos than in wild-type embryos (compare Fig. 3E and F). The dynamic nature of MyoD expression within the wildtype somites is also seen in the ntl embryos. The laterally projecting bands form about 30-60 minutes in advance of overt somite formation (Fig. 3F, open arrows), and the bands of expressing cells at the posterior of the newly formed somite adjacent to the furrow (Fig. 3F, arrows) are at the same

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positions as in the wild-type embryo. In contrast to the presomitic phase of MyoD expression, the subsequent expression phases are therefore not grossly altered by the ntl mutation. In the 24 hour ntl embryo, we observe somites to be blocklike or columnar-shaped (Fig. 2B′), as previously reported (Halpern, 1993), instead of the wild-type chevron-shape. But similar to wild-type myotomes at this stage, those of the ntl embryo express MyoD at highest level in the middle (with respect to anteroposterior axis) of each segment and throughout the whole of its dorsoventral extent. In the most posterior 2 or 3 somites, a large zone created by fusion of the paired myotomes beneath the spinal cord (Halpern et al., 1993) expresses MyoD. We also compared MyoD and snail1 expression in the ntl embryo (data not shown). snail1 expression is reduced in the paraxial mesoderm of ntl embryos (Hammerschmidt and Nüsslein-Volhard, 1993; Thisse et al., 1993). In contrast to the complete absence of MyoD expression in adaxial cells of gastrulas (Fig. 2X) and in the adaxial cells located posterior to formed somites in 10, 12.5 and 14.5 hour ntl embryos (Fig. 2Y,Z,A′), we did observe a low but definite level of snail1 transcripts in these cells (data not shown). These results are another indication that, although there are similarities between the expression of MyoD and snail1, the two genes are not necessarily coordinately regulated. Response to ectopic Sonic hedgehog in wild-type, ntl and spt embryos Since ectopic expression of the shh gene in the chick and mouse results in a repatterning of the somites (Johnson et al., 1994; Fan and Tessier-Lavigne, 1994), we were interested in investigating the effects of shh overexpression and ectopic expression on MyoD gene regulation in the zebrafish embryo. Although a major effect of ectopic expression of shh in the chick dorsal segmental plate was an expansion of the sclerotome within the developing somites, ectopic dorsal expression of the gene also results in a lateral expansion and disorganization of the myotomal region of the somite as indicated by the increased number of cells expressing MyoD and desmin (Johnson et al., 1994). We had interest in whether a similar expansion of the MyoD expression zone would be obtained by ectopic Sonic hedgehog expression in the zebrafish, and also whether ntl and spt embryos responded in a way similar to wild-type embryos. We injected in vitro synthesized shh RNA (prepared using a zebrafish shh clone isolated by Krauss et al., 1993) into 1- to 4-cell-stage embryos and observed by in situ hybridization that the injected transcripts appeared to persist in all regions of the embryo during the first day of development (data not shown). Wild-type embryos injected with shh RNA had an expanded zone expressing MyoD in gastrula and somitogenesis stage embryos. This additional expression was restricted to the paraxial mesoderm and the contiguous region in the tailbud (Fig. 6E-H). At 10, 12 and 14 hours, the whole paraxial mesoderm of injected embryos expresses MyoD, including the anterior portions of the already formed somites and posterior yet unsegmented regions that do not normally express the gene at these particular embryonic stages (compare Fig. 6B-D with F-H). Although there is an even higher level of expression in lateral bands of cells within already formed somites (Fig. 6F,G), these bands are difficult to see because of the high level of transcript spread throughout the paraxial mesoderm. The

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lateral borders of induced MyoD expression are very sharp and appear to be the same as for expression of snail1 in similarly staged uninjected embryos (Fig. 2L-N). Although the differences in MyoD expression in wild-type, spt and ntl embryos are probably not due to differences in shh expression in the mutant embryos (shh expression does occur in spt notochord [our observations] and ntl notochord precursor cells, as well as in wild-type notochord [Krauss et al., 1993]), it was of interest to note whether the paraxial mesoderm cells of the mutant embryos were competent to respond to ectopic Sonic hedgehog. In the case of spt, the main question was whether ectopic Shh could induce MyoD in the mesenchymal cells that were located in positions normally occupied by the paraxial mesoderm cells and in the mis-migrated tail cells of paraxial mesoderm lineage. Although we did find that there was intensified MyoD expression in 12 and 14 hour embryos in groups of cells alongside the notochord and neural tube, at positions similar to MyoD-expressing cells in uninjected spt embryos, most of the mesenchymal cells lateral to the notochord as well as the mismigrated tailbud cells did not express MyoD (compare Fig. 6I,J). In the case of ntl, the main interest was to determine whether injection of shh RNA would overcome the deficiency in MyoD expression in adaxial cells. Although we did find that ectopic Shh resulted in expansion of the region of MyoD expression in ntl embryos, this was observed only in the anterior paraxial mesoderm of embryos undergoing somitogenesis (Fig. 6L) and not in the caudal unsegmented paraxial mesoderm of these embryos (open arrow). That such embryos were truly Ntl in phenotype was checked using DIC optics to confirm the absence of a notochord. Moreover, injection of shh RNA into ntl embryos did not result in MyoD expression in the gastrula stage. Despite the competence of such regions to respond to ectopic Sonic hedgehog in wild-type embryos, these regions of similarly staged ntl embryos appear not to be competent to express MyoD. DISCUSSION

MyoD expression in zebrafish compared with other vertebrates Zebrafish MyoD is unique among vertebrate myogenic genes in its pattern of early activation. Both zebrafish and Xenopus MyoD are expressed in a presomitogenic phase in contrast to the expression of MyoD in avian embryos, which takes place shortly after somites are formed (Pownall and Emerson, 1992), and in

Fig. 4. Localization of MyoD transcripts in 40 h embryos. Wild-type embryos, hybridized with MyoD probe as in Fig. 2, are shown in dorsal (A) and lateral (B) views. MyoD-expressing cells in the fin bud and in eye and jaw muscles are indicated respectively by arrows and arrowheads. Scale bar in A indicates 200 µm; both panels at same magnification.

the mouse, in which MyoD transcripts appear two days after somite formation (reviewed in Ontell et al., 1995). Activation of the Xenopus MyoD gene takes place throughout the entire paraxial mesoderm (Frank and Harland, 1991; Harvey, 1992; Hopwood et al., 1992) while, in the zebrafish, the gene transcripts are first found in only a small subset of paraxial mesodermal cells, the adaxial cells, and the expression zone spreads laterally in an anterior-to-posterior wave only just prior to somite formation. The second phase of zebrafish MyoD expression, the simultaneous appearance of transcripts in the posterior cells of the first 6-7 somites during their formation, also appears to be unique in comparison to expression of the gene in other vertebrate classes. The temporal distinction between this phase and the subsequent third phase, in which additional bands of cells express the gene in an anterior-to-posterior wave prior to furrow formation, suggests a fundamental difference between the most anterior 67 somites and the subsequently formed more posterior somites. Comparison of MyoD, myogenin and snail1 expression MyoD expression occurs during zebrafish embryogenesis far in advance of any overt myogenic differentiation, the first indication of which is the formation of actin myofibrils in the adaxial cell-derived muscle pioneer cells at 13-15.5 hours (Felsenfeld et al., 1991). Moreover, we demonstrate here that expression of the zebrafish myogenin gene is only detected in the embryo approximately 3 hours after the MyoD gene is activated in the embryo. MyoD expression, therefore, appears to mark specification of myogenic precursor cells rather than the onset of differentiation. Although our results are consistent with a role for MyoD as an upstream regulator of myogenin (reviewed in Weintraub, 1993; Olsen and Klein, 1994), MyoD expression cannot be sufficient for activation of the myogenin gene in the zebrafish embryo. During somite formation, myogenin expression follows that of MyoD by one to two hours, but only in a subset of the MyoD-expressing cells. Since little is known at present about differentiation of muscle within the myotome, we can only speculate that the localized myogenin expression may be related to the formation of a particular muscle type. The expression pattern of the snail1 gene in wild-type, spt and ntl embryos (Thisse et al., 1994; Hammerschmidt and Nüsslein-Volhard, 1994) resembles the expression of MyoD in

Fig. 5. Progression in expression of MyoD and myogenin during somite maturation. Schematic representation of MyoD (top somites) and myogenin (bottom somites) transcripts in a 9-somite (13 hour embryo), anterior to left. High transcript levels indicated in black, lower levels indicated by stippling. Prior to somite furrow formation, MyoD, but not myogenin, transcripts are detected in cells extending lateraly from the notochord (n) and adaxial cells. Stages a, b and c refer to the progression of MyoD expression during maturation of the somites, as described in the text. myogenin expression is limited to the adaxial cells until somites are between one and two hours old. At that time, expression is found in a subset of the MyoD-expressing cells.

Expression of MyoD in the zebrafish embryo some but not all respects. Differences between the expression of the two genes, as outlined above, indicate that, although snail1 could potentially be a regulator of MyoD expression, the absence of a close quantitative correlation of expression of the two genes would suggest that other factors play a more definitive role in setting MyoD expression levels. The spt gene is required for early MyoD expression During gastrulation, cells that are fated to become myotome migrate from lateral positions toward the dorsal axis (Kimmel et al., 1990; Warga and Kimmel, 1990). In spt embryos, however, these cells do not converge normally and migrate into the tail region instead (Kimmel et al., 1989; Ho and Kane, 1990). Under these circumstances, the myotomal precursors do not express MyoD until much later. The range of effects produced by the spt mutation is not fully understood, and it is possible that spt affects myotomal specification as well as migration. However, the late expression of MyoD in at least some of the misdirected cells in the tails of spt embryos argues against this effect. Instead, the lack of MyoD expression in early spt embryos suggests that MyoD is regulated by the position of myogenic progenitors rather than by an autonomous clock that initiates MyoD expression at a specific time in these cells regardless of position. Although it has already been shown that the migration of cells of the paraxial mesoderm lineage is a specific autonomous function of the spt gene (Ho and Kane, 1990), similar transplantation experiments will be necessary to determine whether spt myotomal precursors can express MyoD when placed close to the axial mesoderm. The early phase of MyoD expression is absent in ntl embryos Our results showing an absence of the early phase of MyoD expression in ntl embryos are consistent with, but do not prove, a role of the axial mesoderm in the regulation of the MyoD gene. In fact, zebrafish floating head (flh) mutant embryos, which lack notochord, do express MyoD during gastrulation (Halpern et al., 1995). Thus, the early phase of MyoD is not dependent on development of a notochord per se. Rather, the ntl gene product must provide another function, independent of proper organization and development of the notochord, required for the early expression of MyoD. This function could conceivably be autonomous to the paraxial mesoderm cells since the ntl gene is expressed throughout the germ ring (SchulteMerker et al., 1992) including cells that give rise to the paraxial mesoderm (Kimmel et al., 1990). An alternative hypothesis is that the ntl gene product is required for signalling from the notochord precursor cells and that such signaling can occur in flh mutant as well as in wild-type embryos. The latter alternative would accommodate a role of the axial mesoderm in signaling MyoD activation, an attractive idea in light of our finding of a lack of MyoD expression in misdirected cells of the paraxial mesoderm lineage in spt embryos. These alternative hypotheses could be tested by transplantation experiments.

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Once somite formation begins, there are only minor differences between MyoD expression in wild-type and ntl embryos, although myotomes are aberrantly shaped. One important difference in the somites is that ntl myotomes lack muscle pioneer cells. Evidence for the importance of the notochord in zebrafish myotome formation comes from genetic mosaics produced by transplantation, which reveal that wild-type notochordal cells or their precursors could induce neighboring mutant cells to form muscle pioneers and normal-appearing somites (Halpern et al., 1993). It may be that the early expression of MyoD and the formation of muscle pioneer cells are linked. The muscle pioneer cells are normally first recognized alongside the notochord at 13 hours (Felsenfeld et al., 1991). It is likely that their precursors are similarly situated close to the notochord at earlier developmental times. Thus, muscle pioneer cells may arise from the early MyoD-expressing population of cells. Furthermore, ntl embryos fail to form muscle pioneers and fail to express MyoD in the adaxial cells prior to somite formation. A non-autonomous MyoD-inducing function of the ntl gene needs to be demonstrated, however, to support a functional connection between MyoD expression and muscle pioneer cell formation. The entire wild-type paraxial mesoderm is competent to express MyoD Ubiquitous distribution of injected shh RNA in wild-type embryos results in high levels of MyoD expression in a

Fig. 6. Effect of ectopic Sonic hedgehog expression on MyoD expression in wildtype and mutant embryos. Wild-type control embryos (A-D), wild-type embryos injected with shh RNA at the 1- to 4-cell stage (E-H), spt control embryos (I) and spt embryos injected with shh RNA (J), and ntl control embryos (K) and ntl embryos injected with shh RNA (L) were hybridized with MyoD probe. Embryonic ages of the embryos are 8 hours (A,E), 10 hours (B,F), 11 hours (K,L), 12 hours (C,G,I,J) and 14 hours (D,H). In F,L, open arrows indicate the posterior unsegmented region in which expression differs in injected wild-type and ntl embryos. Scale bar in A indicates 200 µm; all panels at same magnification.

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broadened region of gastrula paraxial mesoderm and throughout the paraxial mesoderm of somitogenic embryos. That the ectopic MyoD expression is limited to paraxial mesoderm and a contiguous band of cells in the tailbud indicates that a particular regulatory network which can respond to Sonic hedgehog is in place in only these embryonic regions. An expansion of the region of MyoD expression in response to ectopic shh expression has also been observed in chick myotomes (Johnson et al., 1994) and a role for shh is consistent with a notochord/ floor plate signal as one of the requirements for somite myogenesis (Münsterberg and Lassar, 1995). However, the inability of misdirected spt paraxial mesoderm lineage cells to respond to ectopic Shh indicates that competence to express MyoD most likely requires additional factors absent in these mis-migrated cells. Although the proximity of paraxial mesoderm to a source of Shh is sufficient to activate MyoD in wild-type embryos and in ntl paraxial mesoderm areas undergoing somite formation, the failure of ntl posterior paraxial mesoderm to respond to Shh indicates that either additional axial mesodermal signals (lacking in these regions in the ntl embryo) are required for MyoD expression, or that Ntl itself is necessary for Shh induction of MyoD expression in this region. We thank Marnie Halpern and Chuck Kimmel for providing mutant lines and fixed embryos, Monte Westerfield for sharing his unpublished in situ hybridization protocol, to the whole University of Oregon group for their help and advice during a visit in which we initiated work on the ntl embryos and to Phil Ingham for sending us the shh expression plasmid. This work was supported in part by a grant from the University of Pennsylvania Research Foundation to E. S. W., an NSF grant to D. J. G., an NIH training grant fellowship to M. L. A., a Damon Runyon Walter Winchell Foundation fellowship to B. R. and a Peace Fellowship grant from the Egyptian government to A. A.

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(Accepted 17 October 1995)