RESEARCH ARTICLE 403

Development 136, 403-414 (2009) doi:10.1242/dev.028019 Differential requirements for myogenic regulatory factors distinguish medial and lateral somitic, cranial and fin muscle fibre populations Yaniv Hinits*, Daniel P. S. Osborn* and Simon M. Hughes†

Myogenic regulatory factors of the Myod family (MRFs) are transcription factors essential for mammalian skeletal . However, the roles of each in myogenesis remain unclear, owing partly to genetic linkage at the Myf5/Mrf4 and to rapid morphogenetic movements in the amniote . In mice, Myf5 is essential for the earliest epaxial myogenesis, whereas Myod is required for timely differentiation of hypaxially derived muscle. A second major subdivision of the somite is between primaxial muscle of the somite proper and abaxial somite-derived migratory muscle precursors. Here, we use a combination of mutant and morphant analysis to ablate the function of each of the four conserved MRF in zebrafish, an organism that has retained a more ancestral bodyplan. We show that a fundamental distinction in somite myogenesis is into medial versus lateral compartments, which correspond to neither epaxial/hypaxial nor primaxial/abaxial subdivisions. In the medial compartment, Myf5 and/or Myod drive adaxial slow fibre and medial fast fibre differentiation. Myod-driven activity alone is sufficient for lateral fast somitic and pectoral fin fibre formation from the lateral compartment, as well as for cranial myogenesis. Myogenin activity is a significant contributor to fast fibre differentiation. Mrf4 does not contribute to early myogenesis in zebrafish. We suggest that the differential use of duplicated MRF paralogues in this novel two-component myogenic system facilitated the diversification of vertebrates.

KEY WORDS: Muscle, Zebrafish, Myosin, Slow, Fibre, Fast, Head, Fin, mrf4 (), , , Myogenin, Hedgehog, , , , hsp90,

INTRODUCTION Gensch et al., 2008; Haldar et al., 2008; Kassar-Duchossoy et al., Motility is a key determinant of evolutionary success. During 2004; Tajbakhsh et al., 1997). Owing to the extensive growth of vertebrate evolution, somitic, appendicular and head muscle has secondary muscle fibres, it has been difficult to determine whether undergone significant adaptation as fish evolved and moved onto specific differentiated muscle cells are lacking in single MRF land. One major division shared by all vertebrates is the mutant adults. An exception is the lack of tail epaxial muscle in dorsoventral segregation of the somitic myotome into epaxial and Myf5-null alleles (Kassar-Duchossoy et al., 2004), which hypaxial halves with distinct innervation. A second division of emphasises one overriding theme to emerge from murine MRF somitic myogenesis that emphasises the attachments of muscles to knockout studies of somite myogenesis: that Myf5 function is skeletal/connective tissue elements of somitic (primaxial) or lateral initially required for myogenin (Myog) and Myod expression and plate (abaxial) origin has also been recognised (Burke and Nowicki, myogenesis in the epaxial somite domain (Kablar et al., 1997; 2003). Human diseases and murine genetic manipulations that Kassar-Duchossoy et al., 2004; Tajbakhsh et al., 1997). affect specific muscle groups suggest that such differences in Perturbation of the Myf5/Mrf4 locus also prevents early hypaxial muscle form are underlain by distinct populations of myogenic cells Myod expression, possibly owing to loss of Mrf4 function (Kassar- (Sambasivan and Tajbakhsh, 2007). How muscle diversified at a Duchossoy et al., 2004; Tajbakhsh et al., 1997). Myod function is molecular evolutionary level is unclear, but changes in the required for early hypaxial myogenesis, which is probably utilisation and function of myogenic regulatory factors of the myod dependent on Pax3 (Kablar et al., 1997; Tajbakhsh et al., 1997). family (MRFs) are possible contributors. Analysis of murine MRF Consistent with this, Myod is the only MRF thought to be able to function has revealed that Myf5, Myod and Mrf4 (Myf6) are not, drive myogenesis in the absence of any other MRF, but is not individually, essential for viability (Kassar-Duchossoy et al., 2004; efficient (Kassar-Duchossoy et al., 2004; Rawls et al., 1995; Valdez Kaul et al., 2000; Rudnicki et al., 1992; Zhang et al., 1995). Yet et al., 2000). The consensus view remains that the combined mice lacking function of all three of these genes fail to make activity of several MRFs promotes myogenesis (Weintraub, 1993). skeletal muscle (Kassar-Duchossoy et al., 2004; Rudnicki et al., Null mutation in Myog, however, is perinatal lethal owing to a 1993). Lack of one MRF usually delays myogenesis until another failure of terminal differentiation of a large proportion of myoblasts MRF is expressed, although there is debate about the cell autonomy in some muscles. Mutants have significant myogenesis at early of compensation (Braun and Arnold, 1996; Braun et al., 1994; stages of development and there is efficient terminal differentiation of Myog-null myoblasts in cell culture (Hasty et al., 1993; Nabeshima et al., 1993; Venuti et al., 1995). Early reports presumed Randall Division for Cell and Molecular Biophysics and MRC Centre for that Mrf4, being structurally related to Myog and abundantly Developmental Neurobiology, New Hunt’s House, Guy’s Campus, King’s College London, SE1 1UL, UK. expressed in differentiated muscle fibres, permitted this differentiation. However, pairwise ablation of Myog and either Mrf4, *These authors contributed equally to this work Myod or Myf5/Mrf4 in mice did not worsen the Myog-null †Author for correspondence ([email protected]) phenotype (Rawls et al., 1995; Rawls et al., 1998). The role and

Accepted 17 November 2008 relationship of Myog to the other MRFs remains ambiguous. DEVELOPMENT 404 RESEARCH ARTICLE Development 136 (3)

Here, we turn to zebrafish in the hope of finding shared and Morpholino knockdown divergent roles for individual MRFs in myogenesis. Each MOs (Gene-Tools) were injected into 1- to 2-cell stage embryos unless zebrafish somite generates at least four populations of muscle otherwise stated. MO sequences are: myog MO-1, 5Ј-GCTGGTTTA- fibres within the first 15 hours of somitogenesis, a timing and GAGTCCACCCGCTGTG-3Ј; myog MO-2, 5Ј-GGGTTGGTCTTCGAA- AAGCTCCATGT-3Ј; myod, 5Ј-ATATCCGACAACTCCATCTTTTTTG- diversity that are akin to amniote somite myogenesis (Kahane et Ј Ј Ј al., 2001; Kassar-Duchossoy et al., 2004). Two Hedgehog (Hh)- 3 ; and myf5, 5 -GATCTGGGATGTGGAGAATACGTCC-3 . dependent medial mononucleate slow muscle fibre types derive from the myf5- and myod-expressing adaxial cells lying adjacent RESULTS to the notochord (Blagden et al., 1997; Coutelle et al., 2001; Myf5 cooperates with Myod to drive slow Weinberg et al., 1996; Wolff et al., 2003). Approximately three myogenesis muscle pioneer fibres remain at the dorsoventral midline, whereas Knockdown of both Myf5 and Myod in zebrafish ablates adaxial ~20 superficial slow fibres migrate to the lateral myotome surface myog, mrf4, slow myosin heavy chain 1 (smyhc1) and desmin (Devoto et al., 1996). More lateral paraxial cells within the somite expression and slow muscle differentiation (Hammond et al., also express the MRFs myf5 and myod (Coutelle et al., 2001; 2007; Hinits et al., 2007; Maves et al., 2007) (Fig. 1). As myog is Weinberg et al., 1996) and give rise to two kinds of multinucleate expressed in adaxial cells, we tested its requirement for slow fast fibre: the Fgf8-dependent lateral fast fibres and the Fgf8- myogenesis in morphants. Two non-overlapping myog MOs independent medial fast fibres, a subset of which becomes the fast blocked Myog accumulation as detected by western -expressing cells as a result of later Hh signalling analysis and ablated nuclear Myog immunoreactivity (see Fig. S1 (Groves et al., 2005; Wolff et al., 2003). Both slow and fast in the supplementary material). At the 15-somite stage (15 ss), lineage cells also express myog and mrf4 (Hinits et al., 2007; myog morphants showed no change in any of the adaxial Weinberg et al., 1996). Using predicted null mutations in myf5 and markers examined (Fig. 1). A myod MO also reduced Myod mrf4 and morpholino antisense oligonucleotides (MOs) to accumulation (see Fig. S1 in the supplementary material) knockdown myod and myog function, we show that distinct (Hammond et al., 2007). Injection of sufficient doses of myod MO lineages use different combinations of MRFs to drive reduced myog expression and mildly delayed, but did not prevent, myogenesis. myf5 mutants die during larval growth, whereas mrf4 smyhc1 expression and slow muscle differentiation (Fig. 1). mutants are viable. Medial slow and fast fibres require either Expression of the thin filament genes α-actin and α-tropomyosin Myf5 or Myod to undergo myogenesis. Lateral myogenesis is (tpma) was not affected [Fig. 1; as for desmin (Maves et al., driven by Myod alone. No Mrf4-initiated myogenesis was 2007)]. Myf5 knockdown did not affect any of the markers observed. Our data show that Myf5 and Myod are likely to have examined (Fig. 1). Although mrf4 is Myf5/Myod-dependent, it is been the ancestral triggers of vertebrate myogenesis. However, not expressed until late in slow muscle differentiation and, neither Myf5/Myod- nor Myod/Myog-requiring cell types are therefore, does not trigger slow myogenesis (Hinits et al., 2007) restricted to epaxial or hypaxial somite domains. We speculate (see below). Pairwise loss-of-function of myog with either myf5 that the common ancestor of teleosts and amniotes had several or myod did not prevent slow fibre formation (see below). We kinds of somite skeletal muscle cell that utilised Myf5 and Myod conclude that either Myf5 or Myod alone can drive slow differently. myogenesis, but that Myod activity might be rate limiting in the wild-type condition.

MATERIALS AND METHODS Myf5 is dispensable for embryonic and larval Zebrafish lines and maintenance development Wild-type and transgenic lines Tg(acta1:GFP)zf13 (Higashijima et al., Injection of both myf5 and myod MOs blocked expression of late 1997) and Tg(mylz2:GFP) (Moore et al., 2007) were maintained on King’s College wild-type background, and staging and husbandry were myogenesis markers and diminished, but did not entirely eliminate, as described (Westerfield, 1995). myf5hu2022 and mrf4hu2041 were on a Tl expression of mRNAs encoding the earlier-expressed thin filament background and were genotyped by sequencing of PCR products (Fig. 1) (Hinits et al., 2007). As we have no antibody to amplified from fin clip or embryo genomic DNA using primers 5Ј- Myf5, we were concerned that the myf5 MO might not ablate GCAACTTGCGCTTCGTCTCC-3Ј and 5Ј-CATCGGCA GGCTGTAG - production of all Myf5 protein. The zebrafish strain myf5hu2022, in TAGTTCTC-3Ј for myf5, and 5Ј-TGAATCTGAAGCCCCGCAAC-3Ј which cysteine 59 of the myf5 gene has changed to a stop codon, was and 5Ј-ATTGCTCTGCTCC TGCTCATCC 3Ј-for mrf4. obtained from the Sanger Centre TILLING screen (Stemple, 2004) hu2022 In situ mRNA hybridisation, immunohistochemistry and western (see Fig. S2 in the supplementary material). myf5 is a predicted analysis null, truncating Myf5 upstream of the basic helix-loop-helix (bHLH) +/hu2022 In situ mRNA hybridisation and immunohistochemistry were performed as domain (Fig. 2A). Heterozygous myf5 incrosses yielded described (Hinits and Hughes, 2007). The fluorescein- or digoxigenin- clutches of normal size and the survival of embryos and larvae was tagged probes used were mef2ca and mef2d (Ticho et al., 1996), mrf4 (Hinits good throughout early development. No gross morphological et al., 2007), myf5 (Groves et al., 2005), myod and myog (Weinberg et al., defects were observed and larvae moved and fed normally (Fig. 2B). 1996), eng1a and eng2a (Ekker et al., 1992), mylz2, tpma and myhz1 (Xu et The similarity of myf5hu2022/hu2022 (hereafter called myf5hu2022) and al., 2000), pax3 (Hammond et al., 2007), meox1 and lbx2 (Neyt et al., 2000), myf5 morphants suggests that the latter represent strong loss-of- smyhc1 (Bryson-Richardson et al., 2005), actin (actc1, IMAGE 7284336), function (see below). hsp90a (IMAGE 7259827), prdm1 (Baxendale et al., 2004), (Morin- myf5hu2022 homozygotes survive to adulthood less well than their Kensicki and Eisen, 1997) and ptc1 (Concordet et al., 1996). Antibodies were against Myog (Devoto et al., 2006), Myod (Hammond et al., 2007), siblings; no adults have been found (Fig. 2C). Despite the lack of (Hinits and Hughes, 2007), MyHC (A4.1025), fast MyHC (EB165) early muscle defects, in situ mRNA hybridisation of myf5 at 15 ss (Blagden et al., 1997), slow MyHC [F59 or S58 (Devoto et al., 1996)] and revealed a reduction in myf5 mRNA accumulation, presumably GFP (Torrey Pines). Western analysis was performed as described (Blagden reflecting nonsense-mediated decay, which was not observed in

et al., 1997). myf5 morphants (Isken and Maquat, 2007) (Fig. 2D). We conclude DEVELOPMENT Function of zebrafish MRFs RESEARCH ARTICLE 405

Fig. 1. Myf5 or Myod are required for slow fibre formation. Dorsal flatmounts of zebrafish embryos injected with the indicated MOs and uninjected controls (con), analysed at the 13- to 15-somite stage by whole- mount immunohistochemistry for MyHC (red) or in situ mRNA hybridisation for the indicated mRNAs (blue). Anterior to top. (Left panels) myf5+myod MOs ablated all slow myosin heavy chain (smyhc1 and slow MyHC, arrowheads), but did not completely prevent actin or tpma expression (arrowheads). myod MO slightly delayed smyhc mRNA and MyHC accumulation (arrows), whereas other single MOs had little effect. (Right panels) Single MRF MOs had no effect on adaxial expression of the other MRFs. However, the myod MO reduced myog signal in fast muscle precursors (white arrows). The box region is shown magnified beneath. Note the increase in signal for each MRF caused by its cognate MO (asterisks). s, .

that a wild-type zygotic myf5 locus is not essential for early MRFs are not required for adaxial prdm1 myogenesis, but might confer a selective advantage during growth expression and maturation of fish into adulthood. Which step in slow myogenesis is affected by loss of Myf5 and Myod Murine Myf5 is essential for Myog expression and the first epaxial function? Expression of most regulatory genes implicated in early myogenesis (Braun et al., 1994; Kassar-Duchossoy et al., 2004; myogenesis, such as myog and mef2d (presomitic adaxial markers) Kaul et al., 2000). Analysis of zebrafish myf5hu2022 mutants revealed and mrf4 and mef2ca (adaxial slow fibre markers), is ablated in double a 1- to 2-hour delay in early adaxial myog expression and a ~30- morphants or myf5 mutant;myod morphants (Fig. 1, Fig. 3A; data not minute delay in expression in fast muscle precursors (Fig. 2E). Thus, shown) (Hinits and Hughes, 2007; Hinits et al., 2007; Weinberg et al., Myf5 contributes to the rate of myogenesis. 1996). By contrast, expression of prdm1 and ptc1 mRNAs, which We used myf5hu2022 to examine the role of Myf5 in muscle encode proteins required for proper Hh-dependent slow fibre formation. All embryos from myf5hu2022 heterozygote crosses differentiation (Baxendale et al., 2004; Concordet et al., 1996), was expressed myod, myog, mrf4, mef2ca, pax3, prdm1, ptc1, smyhc1, unaffected by loss of Myf5 and Myod function, showing that the slow α-actin and tpma genes normally at 15 ss (see Fig. S2C in the muscle precursor cells were still present (Fig. 3A). Thus, Myf5 and supplementary material; data not shown). Where analysed, injection Myod drive a specific module of gene expression within slow muscle of myf5 MO gave the same result (Fig. 1; data not shown). When precursors. myod MO was injected into a myf5+/hu2022 incross, 25% of the resulting embryos lacked expression of slow terminal differentiation Myod, but not Myf5, drives cranial and pectoral markers, confirming the previous conclusion based on myf5 MO fin myogenesis (Fig. 3A; 59/243, for all quantitative data see Table S1 in the It has been reported, based on MO knockdown of myf5, that Myf5 supplementary material). Thus, Myf5 and Myod cooperate to drive is required for myogenesis of specific head muscles and brain

adaxial slow myogenesis in zebrafish. morphogenesis (Chen and Tsai, 2002; Lin et al., 2006). We analysed DEVELOPMENT 406 RESEARCH ARTICLE Development 136 (3)

cranial, fin and hypaxial myogenesis in myf5hu2022 embryos and larvae and did not detect any defects, consistent with the viability of some 3-week-old fry (Fig. 3B-D). Moreover, using the myf5 ATG MO, with a sequence identical to that reported previously (Lin et al., 2006), we only obtained head defects at high, probably toxic, MO concentrations (data not shown). As maternally expressed myf5 was not detected (Chen et al., 2001; Coutelle et al., 2001), myf5 is dispensable for non-somitic myogenesis in zebrafish embryos. We next asked whether Myod or Myog is required for cranial myogenesis. Most myod morphants completely lacked head muscle, myhz1 and myog expression and myosin accumulation prior to 72 hours post-fertilisation (hpf), without accompanying gross morphological defects of the brain and head (Fig. 3B-D). Some myod morphants had small amounts of residual muscle, possibly arising from incomplete knockdown (see below). Injecting myod MO into myf5hu2022 mutants gave a distribution of phenotypes indistinguishable from that upon injection into siblings (Fig. 3C,D). myog is highly expressed in head muscle anlage at 48 hpf, and myog morphants showed defects in head myogenesis similar to myod morphants, with the exception that the sternohyoideus was less affected (Fig. 3D). mrf4 is not expressed in head muscle until well after 48 hpf (Hinits et al., 2007). We conclude that Myod is the primary MRF driver of myog expression and early myogenesis in cranial muscles. Unlike most cranial muscle, fin muscle is somite derived and composed exclusively of fast fibres that express the lbx2 gene (previously known as or lbx1h) at early stages (Neyt et al., 2000; Patterson et al., 2008; Wotton et al., 2008). Myod knockdown prevented myog mRNA and myosin accumulation but not lbx2 expression in fin buds, whereas myf5hu2022 mutants appeared normal (Fig. 3E). Therefore, Myod is the major MRF required for fast pectoral fin myogenesis.

Myf5 drives hsp90a expression in fast muscle precursors We have shown that Myod is important for the formation of certain fast fibres in cranial, fin and somitic muscle. Yet myf5 is the first MRF expressed in early presomitic mesoderm and tailbud and persists in fast muscle precursors of the posterior somite (Chen et al., 2001; Coutelle et al., 2001; Stellabotte et al., 2007). Loss of myf5 function decreased expression of hsp90a in the anterior presomitic mesoderm, but other markers of this tissue, such as meox1 and myod, were unaffected (Fig. 3F; see Fig. S2C in the supplementary material). Anteriorly, in the nascent somites, hsp90a mRNA expression was upregulated, dependent on Myod activity (Fig. 3F). No defect in late muscle gene expression was found in myf5 mutants. Hsp90a is required for proper sarcomere assembly (Du et al., 2008; Hawkins et al., 2008). Thus, early Myf5 function may prime the somite for fast myogenesis, but is not essential for it. Fig. 2. myf5hu2022 mutants appear normal as embryos but fail to mature into adults. (A) Myf5 truncation predicted from the Myf5 and Myod are essential for fast myogenesis myf5hu2022 allele. The location of the introduced stop codon is shown; myf5 and myod double morphants lacked myog mRNA at 10 ss and B, basic domain; HLH, helix-loop-helix domain. (B) Genotyped zebrafish muscle at 15 ss (Hammond et al., 2007; Maves et al., 2007) (Fig. 1, embryos from a myf5+/hu2022 carrier incross show no phenotype at 5 Fig. 4A). By 24 hpf, however, small amounts of fast muscle formed days post-fertilisation (5d) as compared with the wild type (+/+). in the medial somite (Fig. 4B,C). Knockdown of Myod in myf5hu2022 (C) Survival at various ages of fish of each genoptype obtained from homozygotes further reduced fast muscle (Fig. 4B,D). The addition +/hu2022 myf5 incrosses. The numbers of embryos are shown. (D) In situ of myf5 MO did not worsen the phenotype of myod MO; myf5hu2022 mRNA hybridisation for myf5 revealed mildly reduced signal in putative mutants. Thus, the myf5 mutant revealed that myf5 morphants are myf5+/hu2022 (13/28) and lower levels in putative myf5hu2022/hu2022 (9/28) embryos. No such effect was observed in myf5 morphants (0/27). hypomorphic. (E) Embryos from an myf5+/hu2022 incross lack adaxial myog mRNA early It is likely that myod morphants also retain low levels of Myod (15/49), and later have a mild delay in myog mRNA accumulation in function, even though we detected little Myod protein (see Fig. S1 in fast muscle precursors correlated with reduced myf5 mRNA (20/74). the supplementary material). In the myf5hu2022 homozygote μ μ

Scale bars: 100 m in D; 150 m in E. background, myod MO either completely ablated or greatly reduced DEVELOPMENT Function of zebrafish MRFs RESEARCH ARTICLE 407

Fig. 3. Myf5 requirement differs between somitic, head and fin muscle development. In situ mRNA hybridisation and/or MyHC immunohistochemistry of control and morphant wild-type or myf5+/hu2022 incross embryos. Dorsal flatmounts with anterior to top (A,F), or whole-mounts with anterior to left in ventral (B, upper row, C,D), lateral (B, lower row) or dorsal (E) view. (A) myf5hu2022 mutants injected with myod MO show a loss of the adaxial slow muscle differentiation markers smyhc1, actin, tpma and mef2ca and MyHC immunostaining (black arrows), but retain other adaxial markers such as prdm1 and ptc1 (white arrows). (B-D) Head muscles are present in 80 hpf myf5hu2022 mutants but are missing from embryos injected with myod (B,C) or myog MO (C). Arrowheads indicate lack of ventral (green), dorsal (yellow), extra-ocular (red) and sternohyoideus (sh, white) muscles. All embryos show normal heart (h, blue arrow). Note the presence of sternohyoideus in myog morphants. All embryos from a myf5+/hu2022 incross have normal expression of myhz1 at 72 hpf (C) and myog at 48 hpf (D) in the head. myod morphants lack myog mRNA (19/21), although sternohyoideus was still detected in some embryos (6/21). (E) In pectoral fin, myog mRNA in muscle masses (pink arrowheads) and MyHC are ablated or reduced by loss of Myod, but not by loss of Myf5. Ibx2 mRNA is unaffected by myod MO. (F) Expression of hsp90a is delayed in fast muscle precursors (arrow) of the myf5hu2022 mutant and recovers in somites (arrowhead), but is drastically reduced throughout the axis in myf5hu2022 mutants injected with myod MO, both in slow and fast precursors. meox1 expression is unchanged in fast precursors.

fast muscle in individual somites. However, embryos entirely lacking quantity of medial fast cells that can express myog and differentiate. fast muscle were never observed; a few residual fast muscle cells were Clearly, the medially located residual muscle required less present in the medial region of many somites, but lateral to Myf5/Myod activity than did other fast muscle. undifferentiated cells that presumably represent adaxial cells or sclerotome (Fig. 4D). Residual fast muscle cells expressed myog by Mrf4 is not required for viability 20 ss, and went on to differentiate and accumulate myhz1, tpma and In the mouse, Mrf4 supports myogenesis of a subset of early muscle mylz2 mRNA at 24 hpf, but did not elongate or fuse (Fig. 4A,D). cells (Kassar-Duchossoy et al., 2004). Could Mrf4 activity account Lower doses of myod MO left more fast muscle in each somite. for the residual muscle in Myf5 plus Myod knockdown fish? hu2022 hu2041

Therefore, the level of Myod in myf5 mutants determines the TILLING yielded the mrf4 allele, a predicted null mutation in DEVELOPMENT 408 RESEARCH ARTICLE Development 136 (3)

Fig. 4. Myf5 or Myod is required for medial fast myogenesis. In situ hybridisation for myog, myhz1, tpma, mylz2 or mef2d or immunohistochemistry for the indicated myosins in wild-type or myf5+/hu2022 incross zebrafish injected with the indicated MOs. Lateral views, anterior to left unless otherwise stated. (A) myog expression was delayed in embryos injected with myf5+myod MOs. Dorsolateral views, anterior to top. (B) MyHC was reduced in both epaxial and hypaxial domains in myod MO embryos, grossly reduced in double myf5+myod morphants and further diminished in myf5hu2022 mutants injected with myod MO. Note the complete absence of residual cells in some individual somites (dotted outline). (C) Residual differentiated muscle in myf5+myod morphants expressed fast but not slow MyHC at 24 hpf. Lateral view of confocal stack of somites 9-10. (D) Fast muscle markers were indistinguishable in 24 hpf myf5hu2022 homozygous mutants and their siblings (left column), reduced by myod MO in siblings (middle) and almost eliminated by myod MO in myf5hu2022 mutants (right). Inset illustrates that myf5 MO does not worsen the myod MO plus myf5hu2022 phenotype. mylz2 mRNA accumulation (red, arrowheads) and myog downregulation (blue) are delayed at ~20 ss in fast muscle. (Bottom panels) Transverse sections of double knockdowns show residual fast muscle (arrowheads) immediately lateral to adaxial and/or twist2-expressing sclerotomal cells (arrows). (E) (Left, top) mef2d mRNA initiates normally in pbcs in the myod morphant, but is restricted to the medial region of rostral somites (yellow brackets). In myf5+myod MOs, mef2d mRNA is absent. Dorsal flatmounts, anterior to top. (Left, bottom) Lack of both myf5 and myod ablates mef2d expression. Lateral views, anterior to top. (Right) By 22 ss, transverse sections (at the level of the bar on lateral flatmounts) show reduction of mef2d mRNA (purple) in myod or myog, but not in myf5, single morphants. Mef2 position is shown relative to slow fibres (brown). Note the expression of mef2d lateral to migrating slow fibres in the control and in myf5 and myog, but not myod, morphants (arrows).

which a stop codon in the basic domain eliminates the helix-loop- mutants could not be made). No additional loss of muscle was helix region essential for dimerisation and myogenic function (Fig. observed above that caused by the MOs in a wild-type background 5A; see Fig. S3 in the supplementary material). mrf4hu2041 mutant (Fig. 5D). Thus, Mrf4 does not drive the residual fast, or apparently fish were viable and fertile (Fig. 5B). Myosin accumulation in slow, any other, myogenesis prior to 24 hpf. fast, cranial and fin muscles occurred on time. Among a series of myogenic or muscle-specific genes examined at various Both epaxial and hypaxial somitic compartments developmental stages, none was altered in mrf4hu2041 homozygotes. have two modes of fast myogenesis Although we have no proof that the mrf4hu2041 allele is null, it In mouse somites, Mrf4 drives some lateral/hypaxial myogenesis, appears that mrf4 is not an essential gene in zebrafish. but Myod is also involved in this region (Kassar-Duchossoy et al., In myf5;myod morphants, which lack slow fibres, mrf4 mRNA 2004). In fish, Myod is the major myogenic gene in the lateral was not detected in the residual myog-expressing cells (Fig. 5C). To somite. Knockdown of Myod alone depletes lateral muscle in both eliminate the possibility that low-level Mrf4 drives residual medial epaxial and hypaxial somites (Fig. 4B,D) (Hammond et al., 2007). fast myogenesis, we injected myf5 and myod MOs into Expression of Myod was first observed in the lateral somite in hu2041

mrf4 mutants (owing to linkage of myf5 and mrf4, double posterior border cells (pbcs), but these cells only accumulated myog DEVELOPMENT Function of zebrafish MRFs RESEARCH ARTICLE 409

Myod and Myog drive fast muscle differentiation Whereas myog is expressed at low levels in slow muscle precursors, it is abundant just before terminal differentiation in both medial and lateral fast precursors, both in wild-type and manipulated embryos (Figs 1 and 4). For example, myog mRNA accumulated prior to mylz2 mRNA in residual medial fast fibres of myod MO-injected myf5hu2022 mutant embryos (Fig. 4D). Upon injecting both myog and myod MOs into myf5hu2022 mutant embryos, all muscle was ablated (Fig. 6A). We conclude that Myog accumulation is essential for residual medial fast fibre differentiation. Could activation of myog expression in the medial somite of myf5 mutant;myod morphant embryos occur independent of residual Myod activity? We think not, for three reasons. First, although myog mRNA appears in medial fast fibres prior to their differentiation, Myog knockdown alone has no detectable effect on fast myogenesis (Fig. 1, Fig. 6D). Second, knockdown of Myf5 and Myod prevents all early myog expression, including that in the medial somite region that is destined to make the first fast muscle. Third, the occurrence of individual somites entirely lacking muscle in myf5 mutant;myod morphant embryos argues against a specific myog-dependent population in the medial region of each somite. We propose that residual Myod activity drives myog expression, but proof will await Fig. 5. Mrf4 has no role in trunk muscle differentiation. a null myod mutant. (A) Schematic illustrating the location of the mutation encoded by We next tested whether Myod or Myf5 alone can drive fast hu2022 mrf4hu2041. (B) Proportion of mrf4hu2041/hu2041 (red), mrf4hu2041/+ (green) myogenesis. Injection of myog MO into myf5 mutant embryos or mrf4+/+ (blue) in progeny from heterozygote crosses. The number of did not detectably reduce muscle (Fig. 6B). By contrast, when myog embryos of each genotype is shown. Homozygote mutants survive well and myod MOs were co-injected into α-actin:GFP transgenic and without obvious defects. (C) Whole-mount in situ mRNA embryos, essentially all fast muscle was ablated (Fig. 6C,D). Slow hybridisation of myf5+myod morphants or myod MO-injected muscle fibres were still present in normal numbers and accumulated hu2022 myf5 mutants reveal that residual medial fast fibres express myog GFP, slow Myosin heavy chain (MyHC) and Mef2. However, the (red) but not mrf4 (purple) at 22 ss, but accumulate mrf4 mRNA after somewhat U-shaped somites had shorter, disorganised slow fibres terminal differentiation at 24 hpf. Lateral view, anterior to left. (D)In and less Engrailed, a marker of muscle pioneer cells (Fig. 6C, Fig. situ mRNA hybridisation for myhz1 (purple) in embryos from a mrf4+/hu2041 incross reveals that injection of myf5+myod MOs fails to 7C). Both myhz1 transcript and fast MyHC were essentially absent ablate all muscle irrespective of mrf4 genotype. from myog;myod morphants (Fig. 6D). Therefore, Myod drives fast muscle differentiation in the absence of Myog. Moreover, Myf5 drives significant fast muscle differentiation in the absence of Myod through activation of myog expression. and mef2d mRNAs at 15 ss, just prior to becoming fast muscle (Fig. 1, Fig. 4E). Myod is required for pbc myog expression (Maves et al., Hedgehog signalling can drive medial fast 2007). However, to ablate mef2d mRNA, Myf5, which is transiently myogenesis expressed in pbcs, also had to be removed (Fig. 4E). Rostrally, A unique population of medial fast fibres, defined by weak however, myod morphants lacked lateral mef2d mRNA, suggesting Engrailed expression and previously designated MFFs, are that without Myod lateral cells cannot express myog, maintain mef2d dependent on midline Hh signalling at relatively late stages (Wolff or undergo terminal differentiation (Fig. 4E). et al., 2003). We therefore examined the Hh dependence of our Regulation of myog and mef2d in the medial somite is different. residual medial fast cells. The Hh-signal-blocking drug cyclopamine Both Myod and Myf5 are involved in this first fast myogenesis, completely ablated residual myhz1 expression in myf5hu2022 mutants because myog mRNA was decreased at 10 ss in both myf5 mutants injected with myod MO (Fig. 7A). Thus, Hh can promote medial fast and myod morphants (Fig. 1, Fig. 2E). Yet, neither Myod nor Myf5 cell differentiation, perhaps by enhancing residual Myod activity. is essential for medial fast myogenesis because expression of myog Hh is not required for most medial fast myogenesis. Treatment of and mef2d was detected medially at 15 ss in single morphants and myod morphants with cyclopamine produced little if any change in myf5hu2022 (Fig. 1, Fig. 4E). As the fast muscle differentiation myhz1 expression (Fig. 7A). Thus, Myf5 and Myog can trigger fast markers mylz2 and myhz1 were almost absent at late somitogenesis muscle differentiation without Hh. stages from embryos lacking Myf5 and Myod (Fig. 4), the simplest We could not detect Engrailed in residual fast fibres in interpretation of these observations is that, as in adaxial cells, Myf5 myf5hu2022 mutants injected with myod MO or in myf5;myod and Myod each contribute to medial fast myogenesis but, given double morphants (Fig. 7B; data not shown). Thus, the residual time, either alone is sufficient. Thus, the medial cells that make the cells are not MFFs, as originally defined. However, the continued first fast muscle in the somite employ a distinct mode of presence of unmigrated adaxial cells expressing ptc1 in myogenesis involving Myf5 that distinguishes them from lateral myf5;myod morphants might reduce Hh signal strength in the fast precursors. Importantly, as with the lateral Myod-dependent region of the residual fast fibres, consistent with the original mode, the medial Myf5/Myod-dependent mode generates fast proposal that MFF Engrailed expression is triggered by enhanced muscle in the medial region of both epaxial and hypaxial Hh signalling once slow fibres migrate (Wolff et al., 2003).

compartments (Fig. 4B,D). Interestingly, myod MO alone prevented eng2a expression and DEVELOPMENT 410 RESEARCH ARTICLE Development 136 (3)

Fig. 6. Loss of Myod and Myog ablates somitic fast muscle. In situ mRNA hybridisation (A,B,E) or immunofluorescent confocal stacks (C,D) of 24 hpf wild-type (con), Tg(acta1:GFP) or myf5+/hu2022 incrosses injected with the indicated MOs. Lateral views, anterior to left. (A) Magnified regions (boxed) showing that lack of Myf5, Myod and Myog results in loss of all muscle. Knockdown of Myod and Myog results in loss of most fast, but not slow, muscle. Note that mylz2 (red) is weakly expressed in immature slow muscle. (B) Loss of Myog and Myf5 has little effect, if any, on fast myhz1 or slow smyhc1 expression. (C) Injection of myod+myog MOs into Tg(acta1:GFP) fish results in loss of fast muscle, as shown by loss of Mef2 staining and GFP medial to the slow fibre layer accompanied by somitic apoptosis as revealed with DAPI (blue in the optical transverse sections). Elongated mononucleate slow fibres differentiate and express GFP, slow MyHC and Mef2. myod morphants confirm the lateral reduction of fast muscle mass. (D) myod+myog MOs ablate fast MyHC in somites, whereas single MOs have a lesser effect. (E) Titration of myod+myog MOs progressively diminishes residual myhz1-expressing cells. The lower panels are high-magnification images of mid-trunk somites (boxed).

reduced Engrailed protein in the somite, without obviously contrast to murine head muscle. The data reveal strong similarities, affecting eng1a mRNA (Fig. 7B). Moreover, myod;myog double but also significant differences, in the myogenic programmes morphants lacked eng2a and had little eng1a mRNA (Fig. 7B). regulating specific fibre populations across vertebrate phylogeny.

DISCUSSION Role of zebrafish MRFs By providing for a first comparison of a vertebrate species with the Predicted null mutations in myf5 and mrf4 demonstrate that these mouse, our data give insight into the evolution of MRF function. The genes are not essential for early myogenesis. Loss of Myf5 single MRF gene in Drosophila and C. elegans is not required for delayed myog and hsp90a expression in slow and fast muscle muscle differentiation (Balagopalan et al., 2001; Chen et al., 1994). precursors, but this soon recovered due to Myod action. In mice, In deuterostomes, MRFs have greater importance: the single Ciona Mrf4, not Myod, appears to permit recovery of epaxial muscle in MRF drives myogenesis (Meedel et al., 2007). The duplicated Myf5loxP/loxP mutants, after a longer delay (Kassar-Duchossoy et MRFs in mice and zebrafish are essential for skeletal myogenesis, al., 2004). In fish, unlike mice, ablation of both myf5 and myod but can partially compensate for one another. led to loss of essentially all muscle formation. Interpretation of There are two major modes of myogenesis in the early zebrafish murine Myf5 knockouts has been plagued by cis effects on the (Fig. 7D). In the ‘medial’ mode, Myf5 and Myod act together to drive linked Mrf4 gene (Kassar-Duchossoy et al., 2004). Because our slow myogenesis and the first fast myogenesis in the medial somite. myf5-null allele is a single base change and can be phenocopied In the ‘lateral’ mode, Myod is uniquely required for lateral muscle by MO knockdown, we can rule out cis effects on mrf4, which is precursor myogenesis, including that in the pectoral fin. We also show likewise linked to myf5 in zebrafish. Perhaps unsurprisingly, that myog expression is dependent on Myf5 and Myod and contributes mutants are more effective than morphants. In myf5hu2022 mutants to both medial and lateral modes of fast muscle differentiation. injected with myod MO, medial Hh signalling permits low levels

Zebrafish cranial muscles develop by the Myod-dependent mode, in of residual Myod to activate myog and thereby drive some DEVELOPMENT Function of zebrafish MRFs RESEARCH ARTICLE 411

Fig. 7. Residual fast fibres after Myf5 plus Myod knockdown are hedgehog dependent. In situ mRNA hybridisation (A,B) and immunofluorescent confocal stacks (C) of 23 ss, 24-26 hpf wild-type, Tg(mylz2:GFP) or myf5+/hu2022 incross embryos. Lateral views, anterior to left. (A) Cyclopamine (CyA) treatment of embryos from a myf5+/hu2022 incross injected with myod MO ablates residual myhz1 expression (arrows, magnified in insets). Fast muscle still forms in the absence of Myod and Hh signals. Note the altered somite shape, paralleling the absence of slow fibres. (B) Expression of eng1a and eng2a (arrowheads) is missing from myf5hu2022 mutants injected with myod MO and is reduced in myod+myog double morphants. Eng1a is not affected by myf5 mutation (revealed by weak myf5 mRNA expression) or myod MO alone, although eng2a is drastically reduced in the myod morphant. (C) Tg(mylz2:GFP) embryos injected with myod MO show reduction of both the weak Engrailed protein in MFF nuclei (yellow arrowheads) and the strong Engrailed expression of muscle pioneers (blue arrowheads). (D) Schematic illustrating how the cells generated in medial and lateral modes of myogenesis in the early zebrafish somite (left) each contribute to several later compartments (right). Note that dermomyotomal contributions are not defined because cell lineage is largely unknown.

myogenesis. Mrf4 does not account for residual muscle. We mutagenised individuals, mutation of a linked gene could account conclude that either Myf5 or Myod is required for myog for the phenotype. The mrf4 coding sequence is, however, wild- expression and muscle differentiation throughout the early type in myf5hu2022 mutants (data not shown), and mrf4 is expressed zebrafish embryo. normally. As with mice lacking Mrf4, fish mrf4 mutants are viable and fertile, in contrast to a recent MO analysis (Wang et al., 2008). Our MRFs and slow myogenesis mutation is predicted to disrupt both mrf4 transcripts upstream of the MRFs are an essential part of the slow muscle programme. HLH domain required for myogenesis (Hinits et al., 2007; Wang et Knockdown of Myf5 and Myod ablates early slow myogenesis al., 2008). Murine Mrf4 initiates myogenesis in certain hypaxial (Hammond et al., 2007; Maves et al., 2007) (Fig. 1). The myf5 cells in embryonic somites (Kassar-Duchossoy et al., 2004). Fish mutation confirms this result. Loss-of-function of either myf5 or mrf4 expression is restricted to differentiated muscle fibres (Hinits myod alone delays but ultimately does not prevent slow (and medial et al., 2007). Even though ectopic Mrf4 expression is potently fast) myogenesis. Myog is not essential, supporting the original view myogenic (see Fig. S3B in the supplementary material), we have no that the combined activity level of various MRFs drives muscle evidence for an early myogenic role of Mrf4 in zebrafish. formation (Weintraub, 1993). Zebrafish lacking myf5 function fail to thrive during larval Several genes important for slow myogenesis require little or no phases. Although it has been reported that Myf5 is required for MRF. Like myf5 and myod themselves, prdm1 and ptc1 are Hh- the formation of most head muscles (Lin et al., 2006), we observe dependent genes involved in adaxial slow myogenesis (Baxendale no such defects in myf5 mutants. Head, fin and trunk muscles et al., 2004; Concordet et al., 1996; Coutelle et al., 2001; Koudijs et appear to form normally in myf5 mutants, as in our myf5 al., 2008; Weinberg et al., 1996). In MRF knockdown embryos, morphants. We suspect, therefore, that the defects in brain and adaxial prdm1 expression is normal at 15 ss, apparently recovering head musculature previously reported after Myf5 knockdown from an earlier defect (Liew et al., 2008). As Prdm1 is not required (Chen and Tsai, 2002; Lin et al., 2006) reflect MO toxicity. To for MRF expression (Baxendale et al., 2004) (our unpublished date, the cause of death in myf5hu2022 mutants is unknown. observations), Hh signalling may bifurcate, promoting MRF hu2022

Because the myf5 allele was found by TILLING in expression to trigger muscle formation and prdm1 to drive slow DEVELOPMENT 412 RESEARCH ARTICLE Development 136 (3) character. The reduction of Engrailed in myod morphants (Fig. 7) with mammals, teleosts retain a bodyplan more similar to that of suggests that MRF activity might also contribute to aspects of primitive teleostomi (bony fish and tetrapods), so we suggest that differentiated slow fibre character. lateral Myod-driven myogenesis was an ancestral trait of teleostomi.

Differential MRF activity in medial and lateral fast Modes of myogenesis provide insight into somite myogenesis evolution In wild-type fish, terminal differentiation of fast fibres is delayed Medial and lateral somite myogenesis are fundamentally distinct until well after somite border formation and follows myog (Fig. 7D). Our findings do not reveal differences in MRF function expression in fast precursors (Blagden et al., 1997; Devoto et al., that correspond either to the traditional epaxial/hypaxial division 1996; Weinberg et al., 1996; Xu et al., 2000). Maves et al. (Maves of the somite, or to the more recent primaxial/abaxial proposal et al., 2007) showed that Myod promotes timely myog and myhz1 (Burke and Nowicki, 2003). Zebrafish have retained an ancestral expression in fast muscle precursors in vivo, which we confirm. mode of somite myogenesis in which midline-derived Hh However, knockdown of Myod alone does not prevent fast muscle signalling induces slow muscle fibres that contribute to both differentiation (Hammond et al., 2007). To ablate fast muscle it is dorsal/epaxial and ventral/hypaxial regions, which differ in necessary to knockdown both Myod and Myf5, which prevents innervation (Devoto et al., 2006; Eisen, 1991; Lacalli, 2002). myog expression. Indeed, knockdown of Myod and Myog ablates Similarly, Hh-dependent MFFs (Wolff et al., 2003) and additional fast muscle differentiation, but leaves slow fibres intact. As myf5 ‘medial mode’ fast fibres are present in both epaxial and hypaxial mRNA accumulates only transiently as somites form, it seems that regions. These medial fibre populations are primaxial and form myog is a target of both Myf5 and Myod that amplifies MRF activity through either Myf5 or Myod activity. However, Myod-dependent during fast myogenesis, as first speculated for specific muscle ‘lateral mode’ fibres [which appear Fgf8-dependent (Hammond et lineages by Weintraub (Weintraub, 1993). al., 2007)] also form primaxially, again in both epaxial and Lateral somite myogenesis is distinct from medial based on (1) a hypaxial portions. We speculate that duplication of the ancestral specific requirement for Myod to drive myog expression and trigger myf5/myod gene in an invertebrate might have facilitated the differentiation (Hammond et al., 2007; Maves et al., 2007), (2) the evolution of novel lateral myogenesis under distinct myod differential requirement for Fgf signalling (Groves et al., 2005; regulation. Hamade et al., 2006) and (3) the finding that myf5 and myod Muscle in the pectoral fin, hypaxial yolk region and possibly sequentially activate mef2d expression in lateral cells just prior to sternohyoid, arises from migratory muscle precursors that originate their differentiation. Mef2d is not essential for fast myogenesis in the lateral somite (Haines et al., 2004; Neyt et al., 2000). The (Hinits and Hughes, 2007). We hypothesise that, together with other majority of such migratory precursors, reminiscent of the abaxial Myod targets, Mef2d works to promote lateral myogenesis in fish, division of amniote somites (Burke and Nowicki, 2003), are Myod- as suggested from studies in culture (Penn et al., 2004). dependent. Moreover, the intrinsic head muscles share Myod Expression of both myog and mef2d persists in medial somite cells dependence with these abaxial populations. As the abaxial division after Myod knockdown, but is lost if Myf5 is also removed. Medial arises from the lateral somite, Myod dependence appears to be the fast fibre precursors require the lowest levels of Myf5 and Myod. common theme in zebrafish lateral somitic muscle. The Hh dependence of residual myogenesis in myf5hu2022;myod Are homologues of adaxial cells and/or medial fast fibres present morphants suggests that Hh promotes Myod-driven myog in amniotes? These medial mode populations are unified by expression. Hh signalling is required in several aspects of fast fibre Myf5/Myod and Hh dependence. Unlike in fish, mouse Myod does differentiation (Henry and Amacher, 2004; Wolff et al., 2003). not contribute to early epaxial/medial myogenesis in the trunk or tail Perhaps some medial fast fibres depend on two phases of Hh action (Kassar-Duchossoy et al., 2004). However, other tetrapods, such as for their differentiation, analogous to the two phases of Hh action in birds and frogs, have retained Myod expression in Hh-dependent slow myogenesis (Wolff et al., 2003). medial cells (Borycki et al., 1998; Grimaldi et al., 2004; Hacker and Guthrie, 1998; Hopwood et al., 1991). In mice, nevertheless, epaxial Myod drives fin and head myogenesis myogenesis is dependent on Hh signalling from the ventral midline Myod has an important role in abaxial pectoral fin and head muscle (Borycki et al., 1999). Thus, changes in MRF utilisation were formation in zebrafish. Mice lacking Myod are viable and fertile selected during evolution. Perhaps mammals have lost adaxial cells, without reported head muscle defects, but do show a delay in limb leaving Myf5- and Hh-dependent homologues of medial fast fibres myogenesis, similar to, but less severe than, that in fish (Kablar et to generate the early myotome. al., 1998; Kablar et al., 1997; Rudnicki et al., 1992). In both species, both Myf5 and Myod are expressed in fin and head muscle We thank many colleagues for reagents, and particularly Robert Knight for head muscle identification and Kuoyu Li for Fig. 2E. The TILLED mutants were precursors. However, in fish myf5 becomes downregulated in head generously provided by E. Busch-Nentwich and D. Stemple through EU ZF- muscle anlage prior to the expression of muscle structural genes (Lin MODELS funding. S.M.H. is a member of MRC Scientific Staff with Programme et al., 2006). Myod is essential at these locations for myog Grant and EU MYORES support. Deposited in PMC for release after 6 months. expression, which itself is required for normal head myogenesis. Supplementary material Interestingly, Myog knockdown failed to phenocopy Myod Supplementary material for this article is available at knockdown in primaxial muscle, despite affecting mef2d expression, http://dev.biologists.org/cgi/content/full/136/3/403/DC1 perhaps indicating greater Myod activity in the somite. References Pectoral fin muscle derives from the rostral somites (Neyt et al., Balagopalan, L., Keller, C. A. and Abmayr, S. M. (2001). Loss-of-function 2000). As with lateral fast primaxial muscle, Myod is required for mutations reveal that the Drosophila nautilus gene is not essential for embryonic early fin muscle, which is entirely fast (Patterson et al., 2008). Thus, myogenesis or viability. Dev. Biol. 231, 374-382. fin muscle fibres may have an origin, molecular mechanism of Baxendale, S., Davison, C., Muxworthy, C., Wolff, C., Ingham, P. W. and Roy, S. (2004). The B-cell maturation factor Blimp-1 specifies vertebrate slow- myogenesis and fate similar to those of the lateral somitic fibres twitch muscle fiber identity in response to Hedgehog signaling. Nat. Genet. 36,

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