ABSTRACT

Skeletal muscle derives from dorsal mesoderm formed during vertebrate gastrulation. Fibroblast growth factor (Fgf) signalling cooperates with Tbx transcription factors to promote dorsal mesoderm formation, but their role in myogenesis has been unclear. Using zebrafish, we show that dorsally derived Fgf signals act through Tbx16 and Tbxta to induce slow and fast trunk muscle precursors at distinct dorsoventral positions. Tbx16 binds to and directly activates the myf5 and myod genes, which are required for commitment to myogenesis. Tbx16 activity depends on Fgf signalling from the organiser. In contrast, Tbxta is not required for myf5 expression, but binds a specific site upstream of myod that is not bound by Tbx16 and drives (dependent on Fgf signals) myod expression in adaxial slow precursors, thereby initiating trunk myogenesis. After gastrulation, when similar muscle cell populations in the post-anal tail are generated from tailbud, declining Fgf signalling is less effective at initiating adaxial myogenesis, which is instead initiated by Hedgehog signalling from the notochord. Our findings suggest a hypothesis for ancestral vertebrate trunk myogenic patterning and how it was co-opted during tail evolution to generate similar muscle by new mechanisms.

This article has an associated ‘The people behind the papers’ interview.

INTRODUCTION

Sarcomeric muscle arose early in animal evolution and is today regulated by similar gene families in Drosophila and vertebrates e.g. mef2 genes (Taylor and Hughes, 2017). Skeletal myogenesis is initiated during gastrulation. Yet conserved bilaterian pathways leading specifically to skeletal (as opposed to cardiac or visceral) muscle have been hard to discern, perhaps because new regulatory steps have evolved in each phylum since their divergence.

A key step in vertebrate evolution was the chordate transition through which animals acquired notochord, post-anal tail, gill slits and dorsal neural tube, facilitating swimming (Brunet et al., 2015; Gee, 2018; Gerhart, 2001; Satoh et al., 2012). Throughout vertebrates, the notochord patterns the neural tube and paraxial mesodermal tissue by secreting Hedgehog (Hh) signals that promote motoneuron and early muscle formation (Beattie et al., 1997; Blagden et al., 1997; Du et al., 1997; Münsterberg et al., 1995; Roelink et al., 1994). Nevertheless, in the absence of either notochord or Hh signalling, muscle is formed in vertebrate somites (Blagden et al., 1997; Dietrich et al., 1999; Du et al., 1997; Grimaldi et al., 2004; Zhang et al., 2001). How might deuterostome muscle have formed prior to evolution of the notochord?

A change in function of the Tbxt gene, a T-box (Tbx) family paralogue, occurred during chordate evolution such that Tbxt now directly controls formation of posterior mesoderm, notochord and post-anal tail in vertebrates (Chiba et al., 2009; Showell et al., 2004). Hitherto, Tbxt may have distinguished ectoderm from endoderm and regulated formation of the most posterior mesendoderm (Arenas-Mena, 2013; Kispert et al., 1994; Woollard and Hodgkin, 2000; Yasuoka et al., 2016). In zebrafish, Tbxt genes also promote slow myogenesis (Coutelle et al., 2001; Halpern et al., 1993; Martin and Kimelman, 2008; Weinberg et al., 1996). Other Tbx genes, such as Tbx1, Tbx4, Tbx5, Tbx16 and Tbx6, also influence sarcomeric muscle development in both fish and amniotes (Chapman and Papaioannou, 1998; Griffin et al., 1998; Hasson et al., 2010; Kimmel et al., 1989; Manning and Kimelman, 2015; Nandkishore et al., 2018; Weinberg et al., 1996; Windner et al., 2015). For example, the Tbx6 family is implicated in early stages of paraxial mesoderm commitment, somite patterning and myogenesis (Bouldin et al., 2015; Chapman and Papaioannou, 1998; Kimmel et al., 1989; Manning and Kimelman, 2015; Nikaido et al., 2002; White et al., 2003; Windner et al., 2012). It is unclear, however, whether the Tbx genes promote myogenesis directly, and/or are required for earlier events in mesoderm development that are necessary for myogenesis.

In vertebrates, a key essential step in skeletal myogenesis is activation of myogenic regulatory factors (MRFs) encoded by the myf5 and myod genes (Hinits et al., 2009, 2011; Rudnicki et al., 1993). In mice, distinct myogenic cell populations initiate myf5 and myod expression in different ways, the genes being induced by distinct signals through different cis-regulatory elements in different skeletal muscle precursor cells. Once expressed, these MRF proteins have two functions: to remodel chromatin and directly enhance transcription of muscle genes (reviewed by Buckingham and Rigby, 2014). In zebrafish, as the anteroposterior axis forms and extends, de novo induction of myf5 and myod mRNAs in slow and fast muscle precursors occurs in tissue destined to generate each successive somite (Coutelle et al., 2001). Zebrafish myogenesis begins at about 75% epiboly stage when adaxial cells that flank the shield/organiser/nascent notochord (hereafter called pre-adaxial cells; see schematic in Fig. 1A), begin MRF expression (Hinits et al., 2009; Melby et al., 1996). Pre-adaxial cells express both myf5 and myod, converge to form two rows of adaxial cells flanking the notochord, become incorporated into somites and differentiate into slow muscle fibres (Coutelle et al., 2001; Devoto et al., 1996; Weinberg et al., 1996). Loss of either MRF alone is not sufficient to prevent slow myogenesis, but loss of both completely inhibits adaxial slow muscle formation (Hinits et al., 2009, 2011). Dorsal tissue immediately lateral to the pre-adaxial cells, the paraxial mesoderm (Fig. 1A), expresses myf5 and subsequently generates fast muscle once somites have formed, upregulating myod in the process. A key to understanding myogenesis in both paraxial and adaxial cells is thus the mechanism(s) by which myf5 and myod expression is regulated.

Intrinsic factors such as Tbx proteins likely interact with extrinsic positional cues within the embryo to pattern myogenesis. Fgf and Tbx function have long been known to interact to drive early mesendoderm patterning, but how directly they control early embryonic myogenesis remains unclear (Kimelman and Kirschner, 1987; Showell et al., 2004; Slack et al., 1987). Various Fgf family members are expressed close to myogenic zones during vertebrate gastrulation (Isaacs et al., 2007; Itoh and Konishi, 2007; Wilkinson et al., 1988). In zebrafish, Fgf signalling is required for mesendoderm formation, tailbud outgrowth and normal fast myogenesis (Draper et al., 2003; Griffin et al., 1995; Groves et al., 2005; Reifers et al., 1998; Yin et al., 2018). Fgf signalling is also involved in early expression of myf5 and myod in pre-adaxial cells, but the mechanism of initial induction of myf5 and myod is unknown (Ochi et al., 2008). Expression of several Fgfs has been detected in the chordoneural hinge (CNH; Fig. 1A) adjacent to pre-adaxial cells (Draper et al., 2003; Groves et al., 2005; Thisse and Thisse, 2005). Subsequently, Hh signalling from the ventral midline maintains MRF expression and progression of the pre-adaxial cells into terminal slow muscle differentiation (Coutelle et al., 2001; Hirsinger et al., 2004; Lewis et al., 1999). Here, we show how both Fgf and Hh extracellular signals cooperate with Tbx genes to control fast and slow myogenesis. In the trunk, Fgf signalling is required for the initiation of myogenesis and acts in cooperation with Tbx16/Tbxta function directly on myf5 and myod. In the tail, by contrast, direct MRF gene induction by Fgf is not required and the evolutionary novelty of midline-derived Hh signalling accounts for adaxial myogenesis.

RESULTS

Fgf signalling is essential for induction of adaxial myf5 and myod expression

Adaxial myogenesis is driven by successive Fgf and Hh signals. When Hh signalling was prevented with the Smoothened (Smo) antagonist cyclopamine (cyA) from 30% epiboly, myf5 and myod mRNAs in pre-adaxial cells were unaffected at 90% epiboly (Fig. 1A). In contrast, when Fgf signalling was inhibited with SU5402 from 30% epiboly both myf5 and myod mRNAs were lost (Fig. 1A) (Ochi et al., 2008). Persistence of the anterior mesodermal marker aplnrb (Zeng et al., 2007) showed that the lack of MRFs was not due to failure of gastrulation caused by SU5402 treatment (Fig. 1A, Fig. S1; see Table S1 for quantification and Table S5 for a summary checklist of results of this and subsequent experiments). In SU5402-treated embryos, aplnrb mRNA revealed the normal invagination of mesoderm and aplnrb-expressing cells flanking the organiser. However, both downregulation of aplnrb mRNA in paraxial trunk mesoderm that normally expresses myf5 alone, and pre-adaxial aplnrb upregulation were absent in SU5402-treated embryos (Fig. 1A). Thus, early Fgf signalling is required for the initiation of skeletal myogenesis in future trunk regions.

Fig. 1.

Inhibition of Fgf signalling blocks initial induction of myod and myf5 expression. ISH for myod and myf5 in control untreated, cyA-treated (100 µM) and SU5402-treated (60 μM) wild-type or mutant embryos, shown in dorsal view, anterior to top. (A) Adaxial (arrows) and paraxial myod and myf5 mRNAs are lost upon SU5402 treatment from 30% to 80 or 90% epiboly (dashes indicate approximate position of the germ ring) but are unaffected by cyA treatment. The anterior mesoderm marker aplnrb is normally downregulated in paraxial presomitic cells expressing myf5 (white dashes) and upregulated in adaxial cells (arrows). Both changes were absent after SU5402 treatment. Schematics illustrate the location of equivalent cell types at two successive stages. CNH, chordoneural hinge (hatched); mpcs, muscle precursor cells; PSM, presomitic mesoderm (brackets). (B) Smob641 mutants retain pre-adaxial myod mRNA at 6ss even after cyA treatment, but lack pre-adaxial myod mRNA at 15ss. ptc1 (ptch2) mRNA downregulation shows that both smo mutation and cyA treatment (shown after longer colour reaction) fully block Hh signalling throughout the axis. Scale bars: 50 µm.

Fig. 1.

Inhibition of Fgf signalling blocks initial induction of myod and myf5 expression. ISH for myod and myf5 in control untreated, cyA-treated (100 µM) and SU5402-treated (60 μM) wild-type or mutant embryos, shown in dorsal view, anterior to top. (A) Adaxial (arrows) and paraxial myod and myf5 mRNAs are lost upon SU5402 treatment from 30% to 80 or 90% epiboly (dashes indicate approximate position of the germ ring) but are unaffected by cyA treatment. The anterior mesoderm marker aplnrb is normally downregulated in paraxial presomitic cells expressing myf5 (white dashes) and upregulated in adaxial cells (arrows). Both changes were absent after SU5402 treatment. Schematics illustrate the location of equivalent cell types at two successive stages. CNH, chordoneural hinge (hatched); mpcs, muscle precursor cells; PSM, presomitic mesoderm (brackets). (B) Smob641 mutants retain pre-adaxial myod mRNA at 6ss even after cyA treatment, but lack pre-adaxial myod mRNA at 15ss. ptc1 (ptch2) mRNA downregulation shows that both smo mutation and cyA treatment (shown after longer colour reaction) fully block Hh signalling throughout the axis. Scale bars: 50 µm.

As trunk myogenesis proceeds, Hh signalling becomes essential for adaxial slow myogenesis. Blockade of Hh signalling with cyA or in smo mutants (which lack Smoothened, an essential component of the Hh signal transduction pathway), led to loss of myod mRNA from adaxial slow muscle at the 6 somite stage (ss), whereas paraxial fast muscle precursors were unaffected (Fig. 1B). Nevertheless, myod mRNA transiently accumulated in pre-adaxial cells of presomitic mesoderm (PSM) destined to make trunk somites, but was then lost in anterior PSM (Fig. 1B) (Barresi et al., 2000; Lewis et al., 1999; Osborn et al., 2011; van Eeden et al., 1996, 1998). Thus, as suggested previously (Coutelle et al., 2001; Ochi et al., 2008), in the wild-type situation trunk pre-adaxial myod expression is maintained and enhanced by Hh. In contrast, during tail myogenesis at 15ss and thereafter, no pre-adaxial myod expression was detected in smo mutants or cyA-treated embryos (Fig. 1B, Fig. S2). These data suggest that whereas Hh is necessary for induction of adaxial myogenesis in the tail, Fgf-like signals initiate myod expression in trunk pre-adaxial cells.

Additional evidence emphasises the greater reliance on Hh in tail myogenesis. In shha mutants at 24 hours post-fertilisation (hpf), slow muscle was lost from tail but remained in trunk somites, suggesting that slow muscle in tail is more sensitive to reduction in Hh activity (Fig. 2A,B). Moreover, injection of myod or myog mRNA into embryos lacking Hh signalling was able to rescue slow myogenesis in trunk but not in tail (Fig. S3). Similarly, absence of notochord-derived signals in noto (flh) mutants, in which the nascent notochord loses notochord character and converts to muscle (Coutelle et al., 2001; Halpern et al., 1995), ablated tail but not trunk slow muscle (Fig. 2C). Treatment of noto mutants with cyA shows that myod expression is initiated in trunk pre-adaxial cells adjacent to the transient pre-notochordal tissue, but fails to be maintained owing to the blockade of floorplate-derived Hh signals (Fig. 2D). Taken together, these data show that Hh can initiate and then maintain MRF gene expression, but that other signals initiate slow myogenesis in the trunk.

Fig. 2.

Successive Fgf and Hh signals drive trunk slow myogenesis. (A) Immunodetection showing slow fibre reduction is greater in tail than trunk in shha mutants. Insets show individual fibres magnified. (B) Quantification of experiment in A. Upper graph shows mean±s.d. (n=10 embryos of each genotype). Lower graph shows the fraction remaining in the mutant. (C) Trunk-specific residual slow muscle in noto mutant. (D) 5ss embryos from a noton1 heterozygote incross treated with cyA at 30% epiboly stage, showing loss of adaxial myod mRNA in anterior presomitic mesoderm (arrowheads), but retention in the most posterior pre-adaxial mesoderm (arrows) flanking the chordoneural hinge (white outline). (E) Exposure to cyA diminishes myf5 and myod mRNAs in adaxial cells in anterior PSM (arrowheads). Exposure to SU5402 (50 µM) at the tailbud stage ablates residual pre-adaxial myod and myf5 mRNAs in cyA-treated 8ss embryos (arrows). Expression of paraxial myod in fast muscle precursors (asterisks) is not affected by cyA but is decreased by SU5402. Scale bars: 50 µm.

Fig. 2.

Successive Fgf and Hh signals drive trunk slow myogenesis. (A) Immunodetection showing slow fibre reduction is greater in tail than trunk in shha mutants. Insets show individual fibres magnified. (B) Quantification of experiment in A. Upper graph shows mean±s.d. (n=10 embryos of each genotype). Lower graph shows the fraction remaining in the mutant. (C) Trunk-specific residual slow muscle in noto mutant. (D) 5ss embryos from a noton1 heterozygote incross treated with cyA at 30% epiboly stage, showing loss of adaxial myod mRNA in anterior presomitic mesoderm (arrowheads), but retention in the most posterior pre-adaxial mesoderm (arrows) flanking the chordoneural hinge (white outline). (E) Exposure to cyA diminishes myf5 and myod mRNAs in adaxial cells in anterior PSM (arrowheads). Exposure to SU5402 (50 µM) at the tailbud stage ablates residual pre-adaxial myod and myf5 mRNAs in cyA-treated 8ss embryos (arrows). Expression of paraxial myod in fast muscle precursors (asterisks) is not affected by cyA but is decreased by SU5402. Scale bars: 50 µm.

We next tested whether Hh-independent myod expression and myf5 upregulation in trunk pre-adaxial cells requires Fgf signalling. Treatment with cyA left residual pre-adaxial myod and myf5 mRNA flanking the base of the notochord at trunk levels, but ablated adaxial expression in slow muscle precursor cells (Fig. 2E). The residual expression was ablated when, in addition to cyA, SU5402 was used to block Fgf signalling from 30% epiboly (Fig. 2E). Application of SU5402 alone diminished myf5 and myod mRNA accumulation up to the tailbud stage, but caused little if any reduction of adaxial myf5 and myod mRNAs in the tailbud region at 6ss, after midline shha function had commenced (Fig. 1B) (Krauss et al., 1993). Nevertheless, SU5402 greatly diminished myod expression in somitic fast muscle precursors and reduced the extent of myf5 expression in paraxial PSM (Fig. 1B) (Groves et al., 2005; Reifers et al., 1998).

Fgf3, Fgf4, Fgf6a and Fgf8a collaborate to promote MRF expression

To identify candidate Fgf regulators of pre-adaxial myogenesis, the expression patterns of fgf3, fgf4, fgf6a and fgf8a were investigated in wild-type embryos (Fig. S4A). As reported previously, fgf4, fgf6a and fgf8a mRNAs were all detected in the posterior dorsal midline at 80% epiboly, followed by fgf3 in the chordoneural hinge (CNH) and posterior notochord (Fig. S4A) (Kudoh et al., 2001; Thisse and Thisse, 2005; Yamauchi et al., 2009). These Fgfs are candidate regulators of myf5 and myod.

Each Fgf was knocked down with previously validated antisense morpholino oligonucleotides (MOs) in wild-type embryos (Fig. S4B). At 80% epiboly, there was little or no decrease of myf5 or myod mRNA in individual Fgf morphants or fgf8a mutant embryos (Fig. S4B). Combinatorial knockdown of several Fgfs led to progressively more severe loss of myod mRNA and reduction of the raised pre-adaxial and paraxial levels of myf5 mRNA (Fig. 3A, Fig. S4C,D) and pre-adaxial aplnrb mRNA (Fig. S4E). Thus, specific Fgfs collaborate to drive the initial expression of myod and myf5 in pre-adaxial and paraxial cells.

Fig. 3.

Dorsally expressed Fgfs drive paraxial myogenesis. (A-D) ISH for myod and myf5 (A,C) or aplnrb (D) mRNAs at 80% epiboly or tbxta (red) and myod (blue/brown) at the tailbud stage (tb) (B). (A) Reduction of myod and myf5 mRNAs in dual and triple Fgf MO-injected wild-type embryos. Arrowheads indicate nascent adaxial cells. (B) In contrast to 80% epiboly (compare with Fig. S4C), at the tailbud stage, cyA treatment ablates anterior adaxial myod mRNA, but leaves pre-adaxial expression intact (arrowheads). Injection of fgf4+fgf8a MOs ablate residual myod mRNA. (C) fgf4 or fgf6a mRNA injection upregulates myod and myf5 mRNAs around the marginal zone (arrows). Note the widening of the unlabelled dorsal midline region (brackets). Insets show the same embryos viewed from the vegetal pole. (D) fgf4 mRNA injection downregulates aplnrb mRNA around the marginal zone (white dashes) but not anteriorly (brackets) in the dorsalised embryo. Scale bars: 100 µm.

Fig. 3.

Dorsally expressed Fgfs drive paraxial myogenesis. (A-D) ISH for myod and myf5 (A,C) or aplnrb (D) mRNAs at 80% epiboly or tbxta (red) and myod (blue/brown) at the tailbud stage (tb) (B). (A) Reduction of myod and myf5 mRNAs in dual and triple Fgf MO-injected wild-type embryos. Arrowheads indicate nascent adaxial cells. (B) In contrast to 80% epiboly (compare with Fig. S4C), at the tailbud stage, cyA treatment ablates anterior adaxial myod mRNA, but leaves pre-adaxial expression intact (arrowheads). Injection of fgf4+fgf8a MOs ablate residual myod mRNA. (C) fgf4 or fgf6a mRNA injection upregulates myod and myf5 mRNAs around the marginal zone (arrows). Note the widening of the unlabelled dorsal midline region (brackets). Insets show the same embryos viewed from the vegetal pole. (D) fgf4 mRNA injection downregulates aplnrb mRNA around the marginal zone (white dashes) but not anteriorly (brackets) in the dorsalised embryo. Scale bars: 100 µm.

By tailbud stage, however, fgf4+fgf8a MO treatment alone had little effect on myod mRNA accumulation, presumably due to the presence of Hh in the midline (Fig. 3B). Congruently, cyA-treatment reduced anterior adaxial myod mRNA, but pre-adaxial expression persisted after blockade of Hh signalling (Fig. 3B). Pre-adaxial myod mRNA was ablated in cyA-treated embryos injected with fgf4+fgf8a MO (Fig. 3B). Thus, expression of Fgf4 and Fgf8a in the shield, CNH and posterior notochord, provides a spatiotemporal cue for pre-adaxial myogenic initiation in the tailbud.

To test the ability of Fgfs to promote myogenesis further, we generated ectopic Fgf signals by injection of fgf4 or fgf6a mRNA into wild-type embryos, and analysed myf5 and myod mRNA at 80% epiboly (Fig. 3C). Both myod and myf5 mRNAs were upregulated in more ventral regions at levels comparable to those in adaxial cells of control embryos, despite the absence of Hh signalling in these regions. Overexpression of fgf4 mRNA upregulated myf5 mRNA in an initially uniform band around the embryo that extended towards the animal pole for a distance similar to that of myod mRNA in adaxial cells of controls (Fig. 3C). myod was less easily induced, but was upregulated in similar regions of the mesoderm. Fgf4-injected embryos became ovoid, with germ ring constriction that stretched and broadened the notochord (although the numbers of DAPI-stained notochord cells appeared normal). There was a positive correlation between the extent of myf5 and myod mRNA upregulation and the extent of deformation. aplnrb mRNA persisted in animal regions of the mesoderm, but was downregulated where myf5 mRNA was induced nearer to the margin, revealing that anterior/cranial mesoderm is present but resistant to Fgf-driven MRF induction and aplnrb suppression (Fig. 3D). Thus, Fgf4 dorsalised the embryo, converting the entire posterior paraxial and ventral mesoderm to a myogenic profile with some regions expressing only myf5 and others expressing also myod, particularly around the germ ring. Fgf6a overexpression also induced ectopic MRFs in cells around the germ ring, which then appeared to cluster (Fig. 3C). Taken together, these data show that posterior/dorsal Fgf signals initiate MRF expression in both pre-adaxial slow and paraxial fast muscle precursors in pre-somitic mesoderm.

MRFs are initially induced by Fgfs, Tbxta and Tbx16

Zebrafish Tbx genes, including tbxta and tbx16 [formerly called no tail (ntla) and spadetail (spt), respectively], potentially mediate Fgf signalling in gastrulating embryos (Amaya et al., 1993; Griffin et al., 1995; Smith et al., 1991; Sun et al., 1999). tbx16 is suppressed by a dominant negative Fgf receptor (FgfR) (Griffin et al., 1998). However, whether tbxta and tbx16 activities are altered by SU5402 treatment, which also blocks FgfR function, is unclear (Rentzsch et al., 2004; Rhinn et al., 2005). Wild-type embryos at 30% epiboly were therefore exposed to SU5402 and subsequently fixed at 80% epiboly or 6ss to investigate expression of tbxta and tbx16 (Fig. 4A). Application of 10 μM SU5402 diminished tbxta mRNA in notochord, and tbxta and tbx16 mRNAs in the germ ring (particularly in dorsal paraxial regions) at 80% and in the tailbud at 6ss (Fig. 4A), and 30 μM SU5402 abolished tbxta and tbx16 mRNAs throughout the trunk (Fig. 4A). Thus, Fgf-like signalling is required for normal Tbx gene expression in mesoderm.

Fig. 4.

Redundant Fgf/Tbx and Hh signals are required for MRF induction. ISH of drug and/or MO-treated embryos. (A) In 10 µM SU5402-treated wild-type embryos, tbxta and tbx16 transcripts are decreased (arrows) at 80% epiboly, but almost normal at 6ss. Both transcripts are absent in 30 µM SU5402-treated embryos at 6ss. (B) Adaxial myod expression (black arrowheads) is completely ablated in tbxta or tbx16 morphants at 80% epiboly, and myf5 expression is greatly decreased (black arrowheads). fgf8a mRNA is ablated in the posterior notochord of tbxta morphants (blue arrowheads), but upregulated in tbx16 morphants around the germ marginal zone at 80% and in the posterior notochord at 6ss (yellow arrowheads). Expression of fgf3 and fgf4 is absent in posterior notochord of tbxta morphants, but enhanced in that location in tbx16 morphants (green arrowheads). (C) At 6ss, pre-adaxial myod expression (black arrowheads) is lost in tbxta morphant tailbud, and diminished in tbx16 morphants. Injection of tbx16 MO, but not tbxta MO, reduces adaxial myf5 mRNA to the level observed in paraxial mesoderm (orange arrowheads), whereas tbxta MO but not tbx16 MO upregulates myf5 mRNA in the posterior tailbud (asterisks). Tbx16 MO abolishes pre-adaxial myf5 mRNA in cyA-treated embryos (white arrowheads). Adaxial myf5 and myod transcripts recover in tbxta morphants, but are ablated by cyA-treatment (red arrowheads). CyA-treatment of tbx16 morphants ablates adaxial myod expression throughout the axis (cyan arrowheads), leaving only residual paraxial myod and myf5 expression (pink arrowheads). Scale bars: 100 µm.

Fig. 4.

Redundant Fgf/Tbx and Hh signals are required for MRF induction. ISH of drug and/or MO-treated embryos. (A) In 10 µM SU5402-treated wild-type embryos, tbxta and tbx16 transcripts are decreased (arrows) at 80% epiboly, but almost normal at 6ss. Both transcripts are absent in 30 µM SU5402-treated embryos at 6ss. (B) Adaxial myod expression (black arrowheads) is completely ablated in tbxta or tbx16 morphants at 80% epiboly, and myf5 expression is greatly decreased (black arrowheads). fgf8a mRNA is ablated in the posterior notochord of tbxta morphants (blue arrowheads), but upregulated in tbx16 morphants around the germ marginal zone at 80% and in the posterior notochord at 6ss (yellow arrowheads). Expression of fgf3 and fgf4 is absent in posterior notochord of tbxta morphants, but enhanced in that location in tbx16 morphants (green arrowheads). (C) At 6ss, pre-adaxial myod expression (black arrowheads) is lost in tbxta morphant tailbud, and diminished in tbx16 morphants. Injection of tbx16 MO, but not tbxta MO, reduces adaxial myf5 mRNA to the level observed in paraxial mesoderm (orange arrowheads), whereas tbxta MO but not tbx16 MO upregulates myf5 mRNA in the posterior tailbud (asterisks). Tbx16 MO abolishes pre-adaxial myf5 mRNA in cyA-treated embryos (white arrowheads). Adaxial myf5 and myod transcripts recover in tbxta morphants, but are ablated by cyA-treatment (red arrowheads). CyA-treatment of tbx16 morphants ablates adaxial myod expression throughout the axis (cyan arrowheads), leaving only residual paraxial myod and myf5 expression (pink arrowheads). Scale bars: 100 µm.

Tbxta is required for normal myf5 and myod expression in posterior regions during tailbud outgrowth, partly due to loss of notochordal Hh signalling, which normally maintains a high level of tbx16 mRNA accumulation in adaxial cells in anterior PSM (Fig. S5) (Coutelle et al., 2001; Weinberg et al., 1996). To test whether Tbx genes are required for MRF expression at earlier stages, each Tbx gene was knocked down and MRF and Fgf expression analysed at 80% epiboly. Tbxta knockdown reduced expression of dorsal midline Fgfs, ablated myod mRNA and reduced myf5 mRNA accumulation in pre-adaxial cells to the level in paraxial regions (Fig. 4B). However, Tbxta knockdown had little effect on either fgf8a or myf5 mRNAs in more lateral paraxial mesoderm or the germ ring (Fig. 4B). This correlation raised the possibility (addressed below) that Tbxta may drive MRF expression through induction of Fgf expression. Tbx16 knockdown, on the other hand, ablated pre-adaxial myod mRNA and reduced both pre-adaxial and paraxial myf5 mRNA without reduction of Fgf expression (Fig. 4B). Indeed, both germ ring fgf8a mRNA at 80% epiboly and dorsal midline fgf3, fgf4 and fgf8a mRNAs in the tailbud at 6ss appeared to be increased (Fig. 4B), as previously described (Warga et al., 2013). As Tbx16 expression persists in tbxta mutants (Amack et al., 2007; Griffin et al., 1998), these data raise the possibility that Tbx16 is required to mediate the action of Fgf signals on myogenesis.

tbx16 null mutants show a failure of convergent migration of mesodermal cells into the paraxial region (Ho and Kane, 1990; Molven et al., 1990), which, by reducing mesodermal cells flanking the CNH, may contribute to the reduction in MRF mRNAs observed at 80% epiboly. Nevertheless, tbx16 mutants generate enough paraxial mesoderm that reduced numbers of both paraxial fast muscle and adaxially derived slow muscle fibres arise after Hh signalling commences (Honjo and Eisen, 2005; Weinberg et al., 1996). To investigate whether Tbx16 is required for initial induction of myf5 and/or myod expression in pre-adaxial cells, we titrated MO to reduce tbx16 function to a level that did not prevent accumulation of significant numbers of trunk mesodermal cells and examined myf5 and myod expression at 6ss (Fig. 4C). In tbx16 morphants, myod mRNA was readily detected in adaxial cells adjacent to notochordal Hh expression (Fig. 4C, cyan arrowheads). Treatment of these tbx16 morphants with cyA to block Hh signalling, however, ablated adaxial myod expression, leaving only weak myod in paraxial somitic fast muscle precursors (Fig. 4C, cyan and pink arrowheads). In contrast, treatment of control embryos with cyA left pre-adaxial myod mRNA intact (Fig. 4C, black arrowheads). These findings show that Fgf-driven pre-adaxial myod expression flanking the CNH requires Tbx16 function.

Adaxial myf5 expression also requires Tbx16. Tbx16 knockdown reduced myf5 mRNA accumulation in the posterior tailbud, and also diminished the upregulation of myf5 mRNA in pre-adaxial and adaxial cells (Fig. 4C, orange arrowheads). Addition of cyA to tbx16 morphants had little further effect on myf5 expression (Fig. 4C, white arrowheads). In contrast, cyA treatment alone reduced adaxial myf5 mRNA in the anterior PSM, but did not affect the myf5 upregulation in pre-adaxial cells or tailbud myf5 expression (Fig. 4C, white arrowheads). Additional knockdown of Tbx16 in cyA-treated embryos prevented pre-adaxial myf5 upregulation (Fig. 4C, white arrowheads). Thus, Tbx16 is required for Fgf to upregulate both myf5 and myod in pre-adaxial cells.

Both pre-adaxial and anterior PSM adaxial myod expression were also absent in tbxta morphants, but recovered in somites, again due to midline-derived Hh signalling (Fig. 4C) (Coutelle et al., 2001). In marked contrast, Tbxta knockdown upregulated myf5 mRNA in the tailbud (Fig. 4C, asterisks), presumably reflecting loss of tailbud stem cells that lack myf5 mRNA. Taken together, the data strongly suggest that tbx16 is required for midline-derived Fgf signals to induce myod and upregulate myf5 in pre-adaxial cells in the tailbud. In contrast, the loss of MRF expression in tbxta mutants could be simply explained by loss of midline-derived Fgf signals, and/or might require some other Tbxta-dependent process.

myf5 and myod induction by Fgf signalling requires Tbx16

To test rigorously whether Tbx16 is required for Fgf to induce MRFs, fgf4 mRNA was injected into embryos from a tbx16 heterozygote incross. Whereas Fgf4 upregulated myf5 and myod mRNAs all around the germ ring in siblings, in sequence-genotyped tbx16 mutant embryos no upregulation was detected (Fig. 5A). It is clear that mesoderm was present in tbx16 mutants because the mRNAs encoding Aplnrb, Tbxta, Tbx16 and Tbx16-like (formerly known as Tbx6 and then Tbx6-like) are present in tbx16 mutants (Fig. S6; Griffin et al., 1998; Morrow et al., 2017). The effect of Fgf4 does not appear to act by radically altering tbxta or tbx16 gene expression (Fig. 5B). Thus, Tbx16 is required for Fgf-driven expression of MRFs in pre-somitic mesoderm.

Fig. 5.

Tbx16 is necessary and sufficient for MRF induction. ISH of manipulated embryos at 80% epiboly stage. Dorsal views. Insets show ventral views. (A) myf5 and myod mRNAs flank the dorsal midline in siblings (sib), but are absent or greatly diminished in tbx16−/− mutants. fgf4 mRNA injection widened notochord and induced ectopic myod and myf5 mRNA around the germ marginal zone of siblings, but did not rescue expression in tbx16−/− embryos. (B) tbxta mRNA reveals widened notochord (bars) in wild-type embryos injected with fgf4 mRNA. Both tbxta and tbx16 mRNAs show clumping in the germ ring after overexpression of fgf4. (C,D) ISH at 80% epiboly stage for myf5 and myod mRNAs in wild-type control or tbx16 mRNA-injected embryos treated with SU5402 at 10 µM (C) or 60 µM (D). myf5 and myod mRNAs are ectopically induced in posterior mesoderm by Tbx16 expression, but decreased by administration of 10 µM SU5402 in wild type, and rescued in SU5402-treated embryos by overexpression of tbx16. High dose SU5402 prevents MRF expression, even after tbx16 mRNA injection. Scale bars: 100 µm.

Fig. 5.

Tbx16 is necessary and sufficient for MRF induction. ISH of manipulated embryos at 80% epiboly stage. Dorsal views. Insets show ventral views. (A) myf5 and myod mRNAs flank the dorsal midline in siblings (sib), but are absent or greatly diminished in tbx16−/− mutants. fgf4 mRNA injection widened notochord and induced ectopic myod and myf5 mRNA around the germ marginal zone of siblings, but did not rescue expression in tbx16−/− embryos. (B) tbxta mRNA reveals widened notochord (bars) in wild-type embryos injected with fgf4 mRNA. Both tbxta and tbx16 mRNAs show clumping in the germ ring after overexpression of fgf4. (C,D) ISH at 80% epiboly stage for myf5 and myod mRNAs in wild-type control or tbx16 mRNA-injected embryos treated with SU5402 at 10 µM (C) or 60 µM (D). myf5 and myod mRNAs are ectopically induced in posterior mesoderm by Tbx16 expression, but decreased by administration of 10 µM SU5402 in wild type, and rescued in SU5402-treated embryos by overexpression of tbx16. High dose SU5402 prevents MRF expression, even after tbx16 mRNA injection. Scale bars: 100 µm.

Tbx16 requires Fgf-like signalling to rescue myf5 and myod expression

The results so far show that tbx16 function is necessary for Fgf to induce myf5 and myod (Fig. 5A). To determine whether increased Tbx16 activity is sufficient to induce MRFs, Tbx16 was overexpressed. Injection of tbx16 mRNA caused ectopic expression of both myf5 and myod in the germ marginal zone (Fig. 5C). Notably, Tbx16 overexpression induced myf5 mRNA in a much broader region than was observed for myod mRNA.

We next tested whether Tbx16 could induce expression of myf5 or myod in the absence of Fgf signalling. Exposure to a high dose of SU5402, which downregulates endogenous tbx16 and tbxta mRNA (Fig. 4A), prevented MRF induction by injection of tbx16 mRNA (Fig. 5D). Nevertheless, when tbx16 mRNA was injected into low dose (10 µM) SU5402-treated embryos, which normally have reduced MRF expression, the level of myf5 and myod mRNAs was rescued (Fig. 5C). However, tbx16 mRNA was less effective at ectopic MRF induction in the presence of SU5402 (Fig. 5C). These results demonstrate that Fgf signalling cooperates with Tbx16 activity in inducing expression of myf5 and myod in pre-adaxial cells at gastrulation stages. Moreover, Tbx16 requires Fgf-like signals to induce MRF gene expression.

myf5 and myod are direct transcriptional targets of Tbx16

In order to determine further the regulatory relationship between Tbx16 and myf5 and myod, we interrogated our previously published chromatin immunoprecipitation and sequencing (ChIP-seq) experiments for endogenous Tbx16 at 75-85% epiboly stage (see Materials and Methods) (Nelson et al., 2017). Our analyses revealed a highly significant peak at −80 kb upstream (myf5 distal element, 5DE) and two peaks proximal to myf5 (proximal elements, 5PE1,5PE3) (Fig. 6A,B; Table S3). Cross-referencing to published histone modification ChIP-seq data (Bogdanovic et al., 2012) revealed that 5DE and 5PE1 overlapped significant H3K27ac and H3K4me1 peaks (Table S3), suggesting that these are functionally active enhancers. Tbx16 ChIP-qPCR confirmed the validity of the 5DE and 5PE1 ChIP-seq peaks (Fig. 6C). These putative enhancers are likely to regulate myf5, the promoter of which has a H3K4me3 mark, rather than the adjacent myf6 gene, which is not expressed at 80% epiboly and does not have a H3K4me3 mark. Each peak showed significant conservation to regions adjacent to myf5 in other fish species (Fig. 6A,B). Thus, ChIP-seq peaks corresponding to histone marks indicative of enhancer activity suggest evolutionarily conserved mechanisms of myf5 regulation in fish.

Fig. 6.

myf5 is a direct transcriptional target of Tbx16. (A,B) ChIP-seq on wild-type embryos at 75-85% epiboly reveals endogenous Tbx16 and Tbxta binding events within 120 kb flanking the myf5 transcriptional start site (TSS). H3K4me3 indicates TSSs. H3K4me1 indicates putative enhancers. H3K27ac indicates active enhancers. Known transcripts with exons (black) and introns (arrowheads) are indicated. Purple and cyan boxes indicate validated and other mentioned Tbx16 binding sites, respectively. (C) ChIP-qPCR validation of Tbx16 peaks on myf5 distal element (5DE) and proximal element 2 (5PE1). Error bars indicate s.e.m. for biological triplicate experiments. (D) Schematic of Tbx16 direct-target assay. (E) Wild-type embryos injected with tbx16-GR mRNA treated with CHD±DEX. CHD alone permits wild-type myf5 expression at 75-80% epiboly, whereas CHD+DEX induced mosaic ectopic myf5 expression with strong (white arrowheads, comparable to wild-type pre-adaxial level) and weak (black arrowheads, comparable to wild-type paraxial level) staining. Numbers indicate the fraction of embryos with the expression pattern(s) shown. Inset shows an unusual induction of myf5 in anterior regions that was not observed with myod. (F) Injection of tbx16 mRNA (200 pg) into embryos from a myf5hu2022/+ heterozygote incross led to ectopic upregulation of myod mRNA in the dorsal germ ring (arrows) irrespective of genotype. Numbers indicate fraction of embryos showing ectopic mRNA/total analysed. Scale bars: 100 µm.

Fig. 6.

myf5 is a direct transcriptional target of Tbx16. (A,B) ChIP-seq on wild-type embryos at 75-85% epiboly reveals endogenous Tbx16 and Tbxta binding events within 120 kb flanking the myf5 transcriptional start site (TSS). H3K4me3 indicates TSSs. H3K4me1 indicates putative enhancers. H3K27ac indicates active enhancers. Known transcripts with exons (black) and introns (arrowheads) are indicated. Purple and cyan boxes indicate validated and other mentioned Tbx16 binding sites, respectively. (C) ChIP-qPCR validation of Tbx16 peaks on myf5 distal element (5DE) and proximal element 2 (5PE1). Error bars indicate s.e.m. for biological triplicate experiments. (D) Schematic of Tbx16 direct-target assay. (E) Wild-type embryos injected with tbx16-GR mRNA treated with CHD±DEX. CHD alone permits wild-type myf5 expression at 75-80% epiboly, whereas CHD+DEX induced mosaic ectopic myf5 expression with strong (white arrowheads, comparable to wild-type pre-adaxial level) and weak (black arrowheads, comparable to wild-type paraxial level) staining. Numbers indicate the fraction of embryos with the expression pattern(s) shown. Inset shows an unusual induction of myf5 in anterior regions that was not observed with myod. (F) Injection of tbx16 mRNA (200 pg) into embryos from a myf5hu2022/+ heterozygote incross led to ectopic upregulation of myod mRNA in the dorsal germ ring (arrows) irrespective of genotype. Numbers indicate fraction of embryos showing ectopic mRNA/total analysed. Scale bars: 100 µm.

We next tested whether Tbx16 is able to positively regulate myf5 directly by using a dexamethasone (DEX)-inducible system to activate Tbx16 in the absence of translation (Kolm and Sive, 1995; Martin and Kimelman, 2008) (Fig. 6D). Ectopic expression of myf5 around the germ ring, and of myod in a narrower domain flanking the base of the notochord, was observed upon Tbx16 activation (Fig. 6E). Interestingly, across the set of cycloheximide (CHD)+DEX-treated embryos, ectopic myf5 mRNA was induced to a higher level than elsewhere (as assessed by staining intensity) in a similar region to ectopic myod mRNA, suggesting that Tbx16 was able to induce two aspects of pre-adaxial character (myod expression and upregulation of myf5) directly in this region of the embryo. To confirm this result, we tested whether Myf5 is required for Tbx16 to induce myod expression. When tbx16 mRNA was injected into myf5 mutant or heterozygote embryos, ectopic myod mRNA was observed flanking the base of the notochord in about 50% of mutant or heterozygote embryos, but appeared to be more readily induced in wild-type siblings (Fig. 6F). Thus, Tbx16 is necessary for MRF expression and can induce both myf5 and myod independently, as long as Fgf signalling is active. Moreover, a feed-forward mechanism operates by which Tbx16 induction of myf5 augments, but is not essential for, induction of myod. In summary, Tbx16 directly induces MRF expression in gastrulating mesoderm and is particularly potent in the pre-adaxial region that normally retains high tbx16 expression.

Tbxta is essential for pre-adaxial but not paraxial myogenesis

Whereas the entire paraxial PSM expresses myf5, pre-adaxial cells upregulate myf5 and are the first cells to express myod. Tbxta and Tbx16 have similar DNA-binding recognition sequences (Garnett et al., 2009; Nelson et al., 2017). Congruently, we observed a prominent Tbxta ChIP-seq peak at the 5DE −80 kb site upstream of myf5, and minor peaks at the proximal sites (Fig. 6A,B; Table S3). Because of the role of Tbx16 and Tbxta in myod expression, we also examined the myod locus for Tbx protein binding. We found multiple sites occupied by Tbxta and Tbx16 either individually or in combination (Table S3). Notably, only one site (DDE3) displayed strongly significant H3K4me1 and H3K27ac peaks and this was only occupied by Tbxta and not by Tbx16 (Fig. S7, Table S3). However, an additional site (DDE1) showed significant occupancy by Tbx16 and Tbxta concurrent with H3K4me1 but not H3K27ac (Fig. S7, Table S3). These findings indicate that differential direct binding of Tbxta and Tbx16 may control both myf5 and myod expression at the inception of skeletal myogenesis.

Is Tbxta also required for MRF expression in response to Fgf? Overexpression of Fgf4 in tbxta mutants successfully induced myf5 mRNA and suppressed aplnrb mRNA widely in the posterior mesoderm except in a widened dorsal midline region, showing that the introduced Fgf4 was active (Fig. 7A,B). However, myod expression was not rescued in tbxta mutants in the dorsal pre-adaxial region of Fgf4-injected embryos, or elsewhere around the germ ring (Fig. 7A). Moreover, even an increased dose of 225 pg fgf4 mRNA/embryo failed to rescue myod mRNA in tbxta mutants. Importantly, tbxta heterozygotes showed significantly less extensive induction of myod mRNA in response to Fgf4 than did their wild-type siblings (P=0.0001 χ2-test; Fig. 7C; Table S4). Therefore, Tbxta is essential for myod induction in pre-adaxial cells independently of its role in promoting expression of midline Fgfs.

Fig. 7.

Tbxta is essential for Fgf4-driven induction of myod but not myf5. Embryos from a tbxta+/− incross injected with 150 pg fgf4 mRNA or control. (A) Tbtxa−/− mutants lack myod mRNA (arrows) but retain myf5 mRNA in presomitic mesoderm (white dashes). Fgf4 induced myf5 and myod mRNAs throughout the posterior mesoderm in siblings (sib; white dashes), but failed to induce myod mRNA in mutants. (B) Fgf4 suppressed aplnrb mRNA in posterior mesoderm above the germ ring (white dashes) in both tbxta−/− mutants and siblings. In A,B, individually genotyped embryos are shown in lateral view, dorsal to right. (C) Scoring of myod mRNA accumulation in response to Fgf4 in a tbxta+/− incross. Expanded: ventral expansion, generally all around germ ring as in A. Adaxial/faint: either wild-type pattern, or reduced intensity in a small proportion of mutants that was not significantly altered by Fgf4. Left: absolute number of embryos analysed from two experiments to emphasise the lack of induction in mutants (see Table S3). Right: alternative display to highlight the reduced response in heterozygotes compared with wild type (χ2 test). (D) Adaxial upregulation of tbx16 mRNA is lost in tbxta−/− mutants (arrowheads). Fgf4 upregulates tbx16 mRNA throughout ventral posterior mesoderm (arrows) and causes mesodermal cell aggregation (asterisks). tbxta−/− mutants accumulate less tbx16 mRNA than siblings and have less expression on the dorsal side (brackets). Scale bars: 100 µm.

Fig. 7.

Tbxta is essential for Fgf4-driven induction of myod but not myf5. Embryos from a tbxta+/− incross injected with 150 pg fgf4 mRNA or control. (A) Tbtxa−/− mutants lack myod mRNA (arrows) but retain myf5 mRNA in presomitic mesoderm (white dashes). Fgf4 induced myf5 and myod mRNAs throughout the posterior mesoderm in siblings (sib; white dashes), but failed to induce myod mRNA in mutants. (B) Fgf4 suppressed aplnrb mRNA in posterior mesoderm above the germ ring (white dashes) in both tbxta−/− mutants and siblings. In A,B, individually genotyped embryos are shown in lateral view, dorsal to right. (C) Scoring of myod mRNA accumulation in response to Fgf4 in a tbxta+/− incross. Expanded: ventral expansion, generally all around germ ring as in A. Adaxial/faint: either wild-type pattern, or reduced intensity in a small proportion of mutants that was not significantly altered by Fgf4. Left: absolute number of embryos analysed from two experiments to emphasise the lack of induction in mutants (see Table S3). Right: alternative display to highlight the reduced response in heterozygotes compared with wild type (χ2 test). (D) Adaxial upregulation of tbx16 mRNA is lost in tbxta−/− mutants (arrowheads). Fgf4 upregulates tbx16 mRNA throughout ventral posterior mesoderm (arrows) and causes mesodermal cell aggregation (asterisks). tbxta−/− mutants accumulate less tbx16 mRNA than siblings and have less expression on the dorsal side (brackets). Scale bars: 100 µm.

Although Fgf4 injection did not radically alter the location of tbx16 or tbxta mRNA (Fig. 5B), we noticed that the higher level of tbx16 mRNA in adaxial compared with paraxial cells was not obvious in Fgf4-injected wild-type embryos, with high levels present at all dorsoventral locations, presumably because pre-adaxial character was induced widely in posterior mesoderm (Fig. 5B). Nevertheless, as Fgf4 injection into tbxta mutants induced myf5 but not myod mRNA (Fig. 7A), it seems that Tbxta is essential for progression from myf5 to myod expression.

Two hypotheses could explain the lack of myod expression in Fgf4-injected tbxta mutants. First, despite the apparent lack of Tbxta protein in adaxial cells (Ochi et al., 2008; Schulte-Merker et al., 1994a), Tbxta might act directly on myod. Alternatively, Fgf-driven Tbxta activity might act indirectly in pre-adaxial cells to upregulate Tbx16 and thereby drive myod expression. We therefore examined the ability of Fgf4 to upregulate Tbx16 in tbxta mutants. Fgf4 enhanced tbx16 mRNA throughout the posterior mesoderm in siblings, with the exception of the widened notochordal tissue that contained nuclear Tbxta protein and failed to upregulate MRFs (Figs 7D and 8D). In tbxta mutants, Fgf4 also enhanced tbx16 mRNA in the ventral mesoderm, but a broader dorsal region did not express tbx16. Moreover, the level of tbx16 mRNA appeared to be lower than in siblings (Fig. 7D). Thus, Fgf4-injected tbxta mutants lack both Tbxta and Tbx16 upregulation in pre-adaxial cells. We conclude that, whereas Fgf-driven induction of lateral myogenic tissue requires Tbx16 but not Tbxta, induction of pre-adaxial character (marked by upregulated myf5 and myod mRNAs) requires both Tbx genes.

Fig. 8.

Tbx16 is essential for Fgf4-driven upregulation of tbx16 and suppression of tbxta. Embryos from a tbx16+/− incross injected with 150 pg fgf4 mRNA or control. (A) By 24 hpf, Fgf4-injected embryos have disorganised heads and, although lacking obvious trunk or tail, some contain twitching muscle. (B) ISH for col1a2 for dermomyotome/connective tissue and myhz1 for skeletal muscle revealed muscle in fgf4-injected sibs, but not in tbx16−/− mutants. Boxed areas are magnified to show the alternating pattern of aggregated muscle and connective tissue in Fgf4-injected siblings, but the reduced col1a2 and absent myhz1 mRNA in Fgf4-injected mutants. Note the aggregation of posterior mesoderm cells into strands around the yolk. (C) ISH for tbx16 mRNA in embryos from a tbx16+/− incross at around 90% epiboly. Nonsense-mediated decay of the mutant transcript is apparent (arrow). fgf4 RNA injection increases tbx16 mRNA in paraxial mesoderm, widens dorsal axial notochord domain (asterisks) and causes aggregation of paraxial cells in siblings, but suppresses residual tbx16 transcript in mutants. (D) Immunodetection of Tbxt protein and tbx16 mRNA in Fgf4-injected and control embryos from a tbx16+/− incross. The Fgf4-injected tbx16−/− mutant (bottom) reveals nuclear Tbxt protein in the entire posterior mesoderm. Residual tbx16 mRNA in the prechordal region (arrows) but absence in posterior mesoderm demonstrates the genotype. (E) Widespread upregulation of shha mRNA reveals the notochord-like character of posterior mesoderm in Fgf4-injected tbx16−/− mutant. Scale bars: 100 µm.

Fig. 8.

Tbx16 is essential for Fgf4-driven upregulation of tbx16 and suppression of tbxta. Embryos from a tbx16+/− incross injected with 150 pg fgf4 mRNA or control. (A) By 24 hpf, Fgf4-injected embryos have disorganised heads and, although lacking obvious trunk or tail, some contain twitching muscle. (B) ISH for col1a2 for dermomyotome/connective tissue and myhz1 for skeletal muscle revealed muscle in fgf4-injected sibs, but not in tbx16−/− mutants. Boxed areas are magnified to show the alternating pattern of aggregated muscle and connective tissue in Fgf4-injected siblings, but the reduced col1a2 and absent myhz1 mRNA in Fgf4-injected mutants. Note the aggregation of posterior mesoderm cells into strands around the yolk. (C) ISH for tbx16 mRNA in embryos from a tbx16+/− incross at around 90% epiboly. Nonsense-mediated decay of the mutant transcript is apparent (arrow). fgf4 RNA injection increases tbx16 mRNA in paraxial mesoderm, widens dorsal axial notochord domain (asterisks) and causes aggregation of paraxial cells in siblings, but suppresses residual tbx16 transcript in mutants. (D) Immunodetection of Tbxt protein and tbx16 mRNA in Fgf4-injected and control embryos from a tbx16+/− incross. The Fgf4-injected tbx16−/− mutant (bottom) reveals nuclear Tbxt protein in the entire posterior mesoderm. Residual tbx16 mRNA in the prechordal region (arrows) but absence in posterior mesoderm demonstrates the genotype. (E) Widespread upregulation of shha mRNA reveals the notochord-like character of posterior mesoderm in Fgf4-injected tbx16−/− mutant. Scale bars: 100 µm.

Fgf action on Tbx16 suppresses dorsoposterior axial fate

Finally, we examined the wider effect of Fgf signalling when skeletal muscle cannot form in the absence of tbx16 function. Excess Fgf action in embryos causes gross patterning defects (Kimelman and Kirschner, 1987; Slack et al., 1987). When Fgf4 was overexpressed in tbx16 heterozygote incross lays, some anterior mesodermal tissue formed and head tissues, such as eye and brain, were apparent, but trunk and tail mesoderm was grossly deficient (Fig. 8A). In siblings overexpressing Fgf4, some muscle was formed and truncated embryos were observed to twitch at 24 hpf. In contrast, no muscle was detected in tbx16 mutants overexpressing Fgf4 (Fig. 8B). Moreover, the residual expression of mutant tbx16 mRNA at 90% epiboly observed in uninjected embryos was lost upon Fgf4 overexpression (Fig. 8C). This suggests that the cells with tailbud character normally accumulating in tbx16 mutants were missing. Instead, widespread expression of Tbxta protein in nuclei far from the germ ring suggested that the entire posterior (but not anterior) mesoderm had converted to notochord, the most dorsal posterior mesoderm fate (Fig. 8D). Indeed, shha mRNA, a marker of notochord, was found to be broadly but weakly upregulated around the embryo in tbx16 mutant embryos injected with fgf4 mRNA, but not in their siblings (Fig. 8E). The data suggest that Fgf4 drives early involution of all posterior mesoderm precursors, leaving none to form a tailbud. In the presence of Tbx16, Fgf4 also dorsalises most involuted trunk mesoderm to muscle, whereas in the absence of Tbx16 Fgf4 converts most of the trunk mesoderm to notochord precursors.

DISCUSSION

The current work contains four main findings. First, that Tbx16 directly binds and activates myf5 regulatory elements to initiate skeletal myogenesis. Second, that Fgf signalling acts through Tbx16 to drive the initial myogenic events in the adaxial cell lineage, which subsequently require Hh signalling to complete myogenesis. Third, that Tbxta, the dorsal-most/posterior Tbx factor, binds directly to myod regulatory elements and also promotes dorsal midline expression of Fgfs, which subsequently cooperate to drive dorsal myogenesis through Tbx16. Fourth, that Fgf action through Tbx16 suppresses the dorsoposterior axial fate induced by Tbxta. Overall, Tbx transcription factors provide a crucial link between mesoderm induction and the initiation of myogenesis, which has profound implications for understanding the evolution of vertebrates.

Tbx genes and myogenesis

Building on previous evidence that Tbx16 upregulates myf5 expression (Garnett et al., 2009; Mueller et al., 2010), our findings show that myf5 and myod genes are direct targets of Tbx16. We also present evidence that myf5 and myod are direct targets of Tbxta. As MRF gene activity drives commitment to skeletal myogenesis in vertebrates, our findings place Tbx protein activity at the base of skeletal myogenesis in zebrafish (Fig. S8).

Before myogenesis, Tbx16 is required for migration of most trunk PSM cells away from the ‘maturation zone’ immediately after their involution (Griffin and Kimelman, 2002; Row et al., 2011). Our analysis of aplnrb-expressing mesodermal cells shows that most anterior (i.e. head) and posterior ventral (i.e. ventral trunk) mesoderm involution and migration occurs normally in both tbx16 and tbxta mutants. Indeed, some PSM eventually yielding muscle is formed in tbx16 mutant trunk (Amacher et al., 2002; Kimmel et al., 1989). PSM is more severely lacking in tbx16;tbxta or tbx16;tbx16l double mutants (Amacher et al., 2002; Griffin et al., 1998; Morrow et al., 2017; Nelson et al., 2017) or after Tbxtb knockdown in the tbxta mutant (Martin and Kimelman, 2008). Cooperation of Tbx proteins in PSM formation also occurs in Xenopus (Gentsch et al., 2013). It is likely, therefore, that all PSM formation and its accompanying myf5 expression requires Tbx proteins, which may help explain why tbx16 mutants have increased pronephric mesoderm (Warga et al., 2013). As Tbx16 is required for direct induction of myf5 and for pcdh8, msgn1, mespaa and tbx6 expression in PSM (Fior et al., 2012; Goering et al., 2003; Griffin and Kimelman, 2002; Lee et al., 2009; Morrow et al., 2017; Yamamoto et al., 1998), the data support previous proposals (Amacher and Kimmel, 1998; Griffin and Kimelman, 2002) of a role for Tbx16 in promotion of the earliest step in PSM formation, en route to myogenesis. These early actions of Tbx16 and Tbxta proteins have previously masked their direct myogenic actions in mutants.

Our data argue that once posterior (i.e. trunk) mesoderm forms, Tbx proteins are still required for MRF expression and normal myogenesis. Hh signalling from notochord acts to maintain adaxial MRF expression in wild type and, if Tbx-driven initiation fails, Hh can initiate myod and upregulate myf5 expression, thereby driving slow myogenesis (Blagden et al., 1997; Coutelle et al., 2001; Du et al., 1997). When Hh signalling is prevented, both Tbx16 and Tbxta are essential for initial pre-adaxial myod transcription. Conversely, Tbx16-induced ectopic myod expression is restricted to a narrower mesodermal region flanking the pre-adaxial cells, likely due to the restricted expression of smarcd3 (Baf60c) in this region (Ochi et al., 2008). Nevertheless, myod is induced by Tbx16 in the absence of Myf5, probably through direct binding to regulatory elements in the myod locus. It is possible that changes in chromatin structure in myf5 and myod loci accompanying posterior mesoderm formation facilitate Tbx16 access to its binding sites in these MRF genes.

Adaxial slow and paraxial fast myogenesis differ (Blagden et al., 1997; Devoto et al., 1996). Paraxial PSM expresses myf5 after involution, which requires Tbx16, but not Tbxta. However, fast myogenesis is delayed until somites form, perhaps through Tbx6 action (Windner et al., 2015). In contrast, pre-adaxial cells that generate slow muscle within PSM require both Tbxta and Tbx16 function for upregulation of myf5 and initiation of myod expression. Thus, distinct Tbx proteins are required for normal adaxial and paraxial myogenesis.

Residual muscle in tbx16 mutants is likely driven by Tbx16l (Morrow et al., 2017). However, as tbx16;tbx16l double mutants continue myod mRNA expression at a reduced level throughout the axis at 24 hpf (Morrow et al., 2017), we predict this is in adaxially derived slow muscle induced by Hh signalling.

To identify likely MRF enhancers at 80% epiboly, we have largely restricted our focus to robust Tbx16 and Tbxta ChIP-seq peaks that are co-incident with established histone marks indicative of active enhancers, H3K4me1 and H3K27ac. As many transcription factor-binding events may be non-functional, not all enhancers have H3K27ac (Pradeepa, 2017; Pradeepa et al., 2016) and a minority of embryonic cells are myogenic, additional Tbx16 and Tbxta ChIP-seq peaks beyond 5DE, 5PE1 and DDE3 may mark functionally important enhancers regulating myf5 and myod. Of particular note DDE1, −31 kb upstream of myod, with Tbx16 and Tbxta ChIP-seq peaks that colocalise with significant H3K4me1, may be functionally important.

Understanding of the elements driving specific aspects of zebrafish myf5 expression is limited. As with murine Myf5 (Buckingham and Rigby, 2014), our data suggest that elements ∼80 kb upstream of the transcription start site are required for myf5 expression. A BAC transgenic encompassing 5DE drives GFP expression in muscle, although analysis of shorter constructs has been confounded by cloning artefacts (Chen et al., 2001, 2007). We also observe Tbx-binding peaks far upstream of zebrafish myod. Upstream elements are known to initiate murine Myod (Myod1) expression in some embryonic regions, but whether these elements drive the earliest myotomal regulation of Myod is unknown (Chen and Goldhamer, 2004). Mouse Tbx6 and Tbxt family genes are also required for trunk/tail, but not cranial, myogenesis (Nandkishore et al., 2018). Moreover, other Tbx proteins bind similar DNA motifs and are required to pattern cranial, cardiac and limb muscle (Don et al., 2016; Knight et al., 2008; Lu et al., 2017; Papaioannou, 2014; Valasek et al., 2011). The extent to which Tbx genes act through similar binding elements to initiate MRF expression and myogenesis across vertebrates remains to be determined.

Fgf and myogenesis

Tbx16 is required for Fgf signalling to induce myf5 (Fig. 5A, Fig. S8). In its absence, Fgf drives all posterior mesoderm towards a notochord-like fate (Fig. 8D,E), probably via activation of Tbxta. Fgf signalling is required for expression of myf5 in tailbud paraxial PSM (Groves et al., 2005). Here, we show that Fgf is also required for the earliest myf5 expression in involuting trunk mesoderm and for the initiation of myf5 and myod expression in pre-adaxial cells destined to form the slow muscle of anterior somites. Our data provide further evidence that this MRF expression is subsequently maintained by Hh signalling, as the shield/tailbud-derived sources of Fgf recede from the adaxial cells (Coutelle et al., 2001; Osborn et al., 2011) (Fig. S8). We find that Fgf action is stronger in trunk (as opposed to tail) somites, consistent with (1) dwindling Fgf mRNA levels as tailbud outgrowth slows and (2) the unresolved issue of Hh-independent initiation of MRF expression in the anterior-most somites of murine smoothened mutants (Zhang et al., 2001). We suggest this MRF expression is triggered by Fgf in mouse, as in zebrafish.

Our data argue strongly that Fgf signalling not only promotes tbx16 expression, but also enhances the activity of the Tbx16 protein, constituting a feed-forward mechanism. The MRF-inducing activity of Tbx16 is suppressed by inhibition of Fgf signalling (Fig. 5C,D). Bearing in mind the existence of Tbx16l and Tbxta, this result is consistent with the finding that tbxta or tbx16 mutation sensitises embryos to Fgf inhibition (Griffin and Kimelman, 2003). As Tbx16 overexpression can expand PSM fates and reverse the effect of partial Fgf inhibition, a primary role of Fgf signalling is to cooperate with Tbx16 to drive expression of its target genes, including myf5. This understanding provides mechanistic insight into how the effects of Fgf on gastrulation movements and histogenesis are separated, as originally proposed (Amaya et al., 1993). Interestingly, Tbx16 overexpression rescues myf5 mRNA preferentially on the dorsal side of the embryo, suggesting that BMP and/or other signals continue to suppress PSM fates ventrally, and thus that Tbx16 does not act by simply suppressing the inhibitory effect of such signals.

Tbxta and Fgf appear to act in a positive-feedback loop to both maintain tailbud character and diversify PSM into pre-adaxial and paraxial. Tbxta is required for normal Fgf signalling from the midline to promote pre-adaxial myogenesis. Fgf overexpression drives myf5 in tbxta mutants, but cannot drive myod. ChIP shows sites upstream of myod preferentially bound by Tbxta. As Tbxta is not readily immunodetectable in these pre-adaxial cells (Hammerschmidt and Nüsslein-Volhard, 1993; Odenthal et al., 1996; Schulte-Merker et al., 1994a), Tbxta may either open the myod locus, act indirectly, or remain tightly bound despite low free concentration. In the absence of Tbx16, Fgf drives the entire germ ring towards notochord fate, preventing continued tailbud outgrowth. It seems, therefore, that Fgf/Tbx16 interaction is required to maintain tailbud stem cells and promote PSM formation. In a chordate ancestor, the evolution of interaction between Fgf/Tbx6/16-dependent muscle-forming tissue and Fgf/Tbxta-dependent dorsal organiser might be a key innovation leading to both tailbud stem cells and notochord.

Evolution of vertebrates

As efficient motility driven by sarcomeric muscle is found throughout triploblasts, it is likely that mesodermal striated muscle existed in the common ancestor of deuterostomes and protostomes. There is consensus that the appearance of neural crest, notochord and a post-anal tail were significant evolutionary steps for chordates (Gee, 2018). Already in cephalochordates at least two kinds of striated muscle had evolved in anterior somites (Devoto et al., 2006; Lacalli, 2002). Our evidence that initiation of both slow and fast myogenesis in the most anterior trunk is driven by Fgf/Tbx signalling indicates that a major function of this early mesodermal inducer was induction of trunk striated myogenesis, which may constitute an ancestral chordate character. Once Hh is expressed in maturing midline tissues, it triggers terminal differentiation of muscle precursors into functional muscle through a positive-feedback loop (Coutelle et al., 2001; Osborn et al., 2011) (Fig. S8). Parallel diversification of neural tube cells, also regulated by Hh (Placzek and Briscoe, 2018), may have generated matching motoneural and muscle fibre populations that enhanced organismal motility. With the evolution of a tbxta-dependent tailbud destined to make the post-anal tail and notochord, our data suggest that weakening Fgf signalling continued to induce myf5 expression and paraxial mesoderm character through tbx16, but was insufficient to induce adaxial myogenesis. The presence of Hh and Tbxta plus Tbx16, however, ensure that adaxial slow muscle is initiated in the zebrafish tail.

In the anterior somites of amniotes, as in zebrafish, Hh signalling maintains, rather than initiates, myf5 expression (Zhang et al., 2001). In more posterior somites of zebrafish, Xenopus and amniotes, Hh signalling drives myf5 initiation (Borycki et al., 1999; Grimaldi et al., 2004). Compared with zebrafish, however, murine Myf5 induction is further delayed until after somitogenesis, when Gli3 repressive signals in PSM have diminished (McDermott et al., 2005). In mouse, Tbxt (known as brachyury) is required for myogenesis and binds 20 kb downstream of Myod, but does not obviously control its expression (Lolas et al., 2014). No clear orthologue of tbx16 exists in mammals, although it clusters by sequence with Tbx6 genes. Mammalian Tbx6 suppresses neurogenesis in posterior paraxial mesoderm, suggesting that additional mechanisms have evolved that suppress early pre-somitic Myf5 expression (Chapman and Papaioannou, 1998). Indeed, possible low level Myf5 expression in PSM has long been a source of controversy (George-Weinstein et al., 1996; Gerhart et al., 2004). Thus, there has been diversification in how these Tbx genes regulate somitic myogenesis.

The ancestral situation seems clearer. In amphioxus, Tbx6/16 is expressed in tailbud and PSM (Belgacem et al., 2011). In Ciona, knockdown of Tbx6b/c/d leads to reduced MyoD expression, loss of muscle and paralysis (Imai et al., 2006). In Xenopus, both Tbx6 and VegT are implicated in early myogenesis (Callery et al., 2010; Fukuda et al., 2010; Tazumi et al., 2010), although some mechanisms may differ from those in zebrafish (Maguire et al., 2012). By adding our zebrafish findings, we show that in all major chordate groups Tbx-dependent gene regulation is central to skeletal myogenesis. The conserved involvement, yet divergent detail, of how Tbx16, Tbx6 and Tbxt genes regulate somitic myogenic diversity along the body axis are consistent with selective pressures on these duplicated Tbx gene families playing a key role in the diversification of myogenesis in the vertebrate trunk and tail, characters that gave these chordates their predatory advantage.

MATERIALS AND METHODS

Zebrafish lines and maintenance

Mutant lines fgf8ati282a (Reifers et al., 1998), noton1 (Talbot et al., 1995), smob641 (Barresi et al., 2000), tbxtab195 and tbx16b104 (Griffin et al., 1998) are likely nulls and were maintained on King's wild-type background. Staging and husbandry were as described previously (Westerfield, 2000). All experiments were performed under licences awarded under the UK Animal (Scientific Procedures) Act 1986 and subsequent modifications.

In situ mRNA hybridisation and immunohistochemistry

In situ mRNA hybridisation (ISH) for myf5 and myod was performed as described previously (Hinits et al., 2009). Additional probes were: fgf3 (Maroon et al., 2002), fgf4 (IMAGE: 6790533, https://www.ncbi.nlm.nih.gov/nuccore/BC128817), fgf6a (Thisse and Thisse, 2005), fgf8a (Reifers et al., 1998), tbxta (Schulte-Merker et al., 1994b) and tbx16 (Griffin et al., 1998; Ruvinsky et al., 1998). Anti-Ntla immunostaining was performed after ISH using rabbit anti-Ntla (Schulte-Merker et al., 1992; 1:2000) and goat anti-rabbit IgG-HRP-conjugated secondary antibody (PI-1000-1, Vector Laboratories).

Embryo manipulations

Embryos were injected with MOs (GeneTools LLC) as indicated in Table S2 to fgf3, fgf4, fgf6a, fgf8a, tbxta (Feldman and Stemple, 2001) or tbx16 (Bisgrove et al., 2005). Controls were vehicle or irrelevant mismatch MO. Cyclopamine (100 µM in embryo medium), SU5402 (at the indicted concentration in embryo medium) and vehicle control were added at 30% epiboly to embryos whose chorions had been punctured with a 30G hypodermic needle. A PCR product of fgf4 (IMAGE: 6790533) was cloned (primers in Table S2) into the SacI/SalI sites of pβUT3 to make mRNA for overexpression. One-cell-stage embryos were injected with 100-220 pg fgf4 mRNA (made with messageMachine), 50 pg fgf6a mRNA, 150 pg tbx16 mRNA (Griffin et al., 1998) or 150 pg tbx16-GR mRNA (Jahangiri et al., 2012). For hormone-inducible Tbx16 activation, mRNA corresponding to Tbx16 fused to the hormone-binding domain of glucocorticoid receptor (GR) was overexpressed in wild-type embryos. The resulting protein is held in the cytoplasm until DEX from shield stage stimulates GR nuclear translocation. In the presence of the translation inhibitor CHD (added prior to DEX at the germ ring stage), increased nuclear Tbx16 is expected to induce only direct targets of Tbx16 in a mosaic fashion. Embryos were treated with 10 μg/ml final concentration of CHD 2 h prior to collection at 75-80% epiboly. CHD caused ∼5% delay in epiboly, showing that it was active. After 30 min, 20 μM DEX was added for the remaining 1.5 h.

ChIP-seq and ChIP-qPCR

ChIP-seq data from GSE84619 and GSE32483 were analysed as reported previously (Nelson et al., 2017). Multiz Alignments & Conservation from UCSC Genome Browser (Haeussler et al., 2019) are shown beneath schematics. ChIP-seq peak height was measured in reads per million reads (RPM). ChIP-qPCR experiments were performed as previously reported (Jahangiri et al., 2012) using the primers in Table S2.

Acknowledgements

We are grateful to many members of the Hughes lab for advice and to Bruno Correia da Silva and his staff for care of the fish.

Footnotes

Author contributions

Conceptualization: S.M.H.; Methodology: D.P.S.O., K.L., S.J.C., A.C.N., Y.H., S.M.H.; Investigation: D.P.S.O., K.L., S.J.C., A.C.N., Y.H., S.M.H.; Resources: F.C.W.; Data curation: A.C.N.; Writing - original draft: S.M.H.; Writing - review & editing: D.P.S.O., A.C.N., F.C.W., Y.H.; Supervision: F.C.W., Y.H., S.M.H.; Funding acquisition: F.C.W., S.M.H.

Funding

S.M.H. is Medical Research Council Scientist with Programme Grant (G1001029 and MR/N021231/1) support. This work was also supported by grants from the British Heart Foundation (PG/14/12/30664 to Y.H. and S.M.H.; PG/13/19/30059 to F.C.W.). Deposited in PMC for immediate release.

Data availability

The ChIP-seq datasets can be found in GEO under accession numbers GSE84619 and GSE32483.

Peer review history

The peer review history is available online at https://dev.biologists.org/lookup/doi/10.1242/dev.184689.reviewer-comments.pdf

References

Amacher
,
S. L.
and
Kimmel
,
C. B
. (
1998
).
Promoting notochord fate and repressing muscle development in zebrafish axial mesoderm
.
Development
125
,
1397
-
1406
.
Amacher
,
S. L.
,
Draper
,
B. W.
,
Summers
,
B. R.
and
Kimmel
,
C. B
. (
2002
).
The zebrafish T-box genes no tail and spadetail are required for development of trunk and tail mesoderm and medial floor plate
.
Development
129
,
3311
-
3323
.
Amack
,
J. D.
,
Wang
,
X.
and
Yost
,
H. J
. (
2007
).
Two T-box genes play independent and cooperative roles to regulate morphogenesis of ciliated Kupffer's vesicle in zebrafish
.
Dev. Biol.
310
,
196
-
210
.
Amaya
,
E.
,
Stein
,
P. A.
,
Musci
,
T. J.
and
Kirschner
,
M. W
. (
1993
).
FGF signalling in the early specification of mesoderm in Xenopus
.
Development
118
,
477
-
487
.
Arenas-Mena
,
C
. (
2013
).
Brachyury, Tbx2/3 and sall expression during embryogenesis of the indirectly developing polychaete Hydroides elegans
.
Int. J. Dev. Biol.
57
,
73
-
83
.
Barresi
,
M. J.
,
Stickney
,
H. L.
and
Devoto
,
S. H
. (
2000
).
The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity
.
Development
127
,
2189
-
2199
.
Beattie
,
C. E.
,
Hatta
,
K.
,
Halpern
,
M. E.
,
Liu
,
H.
,
Eisen
,
J. S.
and
Kimmel
,
C. B
. (
1997
).
Temporal separation in the specification of primary and secondary motoneurons in zebrafish
.
Dev. Biol.
187
,
171
-
182
.
Belgacem
,
M. R.
,
Escande
,
M. L.
,
Escriva
,
H.
and
Bertrand
,
S
. (
2011
).
Amphioxus Tbx6/16 and Tbx20 embryonic expression patterns reveal ancestral functions in chordates
.
Gene Expr. Patterns
11
,
239
-
243
.
Bisgrove
,
B. W.
,
Snarr
,
B. S.
,
Emrazian
,
A.
and
Yost
,
H. J
. (
2005
).
Polaris and Polycystin-2 in dorsal forerunner cells and Kupffer's vesicle are required for specification of the zebrafish left-right axis
.
Dev. Biol.
287
,
274
-
288
.
Blagden
,
C. S.
,
Currie
,
P. D.
,
Ingham
,
P. W.
and
Hughes
,
S. M
. (
1997
).
Notochord induction of zebrafish slow muscle mediated by Sonic Hedgehog
.
Genes Dev.
11
,
2163
-
2175
.
Bogdanovic
,
O.
,
Fernandez-Minan
,
A.
,
Tena
,
J. J.
,
de la Calle-Mustienes
,
E.
,
Hidalgo
,
C.
,
van Kruysbergen
,
I.
,
van Heeringen
,
S. J.
,
Veenstra
,
G. J.
and
Gomez-Skarmeta
,
J. L
. (
2012
).
Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis
.
Genome Res.
22
,
2043
-
2053
.
Borycki
,
A. G.
,
Brunk
,
B.
,
Tajbakhsh
,
S.
,
Buckingham
,
M.
,
Chiang
,
C.
and
Emerson
,
C. P.
Jr
(
1999
).
Sonic hedgehog controls epaxial muscle determination through Myf5 activation
.
Development
126
,
4053
-
4063
.
Bouldin
,
C. M.
,
Manning
,
A. J.
,
Peng
,
Y.-H.
,
Farr
,
G. H
., III
,
Hung
,
K. L.
,
Dong
,
A.
and
Kimelman
,
D.
(
2015
).
Wnt signaling and tbx16 form a bistable switch to commit bipotential progenitors to mesoderm
.
Development
142
,
2499
-
2507
.
Brunet
,
T.
,
Lauri
,
A.
and
Arendt
,
D
. (
2015
).
Did the notochord evolve from an ancient axial muscle? The axochord hypothesis
.
BioEssays
37
,
836
-
850
.
Buckingham
,
M.
and
Rigby
,
P. W
. (
2014
).
Gene regulatory networks and transcriptional mechanisms that control myogenesis
.
Dev. Cell
28
,
225
-
238
.
Callery
,
E. M.
,
Thomsen
,
G. H.
and
Smith
,
J. C
. (
2010
).
A divergent Tbx6-related gene and Tbx6 are both required for neural crest and intermediate mesoderm development in Xenopus
.
Dev. Biol.
340
,
75
-
87
.
Chapman
,
D. L.
and
Papaioannou
,
V. E
. (
1998
).
Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6
.
Nature
391
,
695
-
697
.
Chen
,
J. C.
and
Goldhamer
,
D. J
. (
2004
).
The core enhancer is essential for proper timing of MyoD activation in limb buds and branchial arches
.
Dev. Biol.
265
,
502
-
512
.
Chen
,
Y.-H.
,
Lee
,
W.-C.
,
Liu
,
C.-F.
and
Tsai
,
H.-J
. (
2001
).
Molecular structure, dynamic expression, and promoter analysis of zebrafish (Danio rerio) myf-5 gene
.
Genesis
29
,
22
-
35
.
Chen
,
Y.-H.
,
Wang
,
Y.-H.
,
Chang
,
M.-Y.
,
Lin
,
C.-Y.
,
Weng
,
C.-W.
,
Westerfield
,
M.
and
Tsai
,
H.-J
. (
2007
).
Multiple upstream modules regulate zebrafish myf5 expression
.
BMC Dev. Biol.
7
,
1
.
Chiba
,
S.
,
Jiang
,
D.
,
Satoh
,
N.
and
Smith
,
W. C
. (
2009
).
Brachyury null mutant-induced defects in juvenile ascidian endodermal organs
.
Development
136
,
35
-
39
.
Coutelle
,
O.
,
Blagden
,
C. S.
,
Hampson
,
R.
,
Halai
,
C.
,
Rigby
,
P. W.
and
Hughes
,
S. M
. (
2001
).
Hedgehog signalling is required for maintenance of myf5 and myoD expression and timely terminal differentiation in zebrafish adaxial myogenesis
.
Dev. Biol.
236
,
136
-
150
.
Devoto
,
S. H.
,
Melancon
,
E.
,
Eisen
,
J. S.
and
Westerfield
,
M
. (
1996
).
Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation
.
Development
122
,
3371
-
3380
.
Devoto
,
S. H.
,
Stoiber
,
W.
,
Hammond
,
C. L.
,
Steinbacher
,
P.
,
Haslett
,
J. R.
,
Barresi
,
M. J.
,
Patterson
,
S. E.
,
Adiarte
,
E. G.
and
Hughes
,
S. M
. (
2006
).
Generality of vertebrate developmental patterns: evidence for a dermomyotome in fish
.
Evol. Dev.
8
,
101
-
110
.
Dietrich
,
S.
,
Schubert
,
F. R.
,
Gruss
,
P.
and
Lumsden
,
A
. (
1999
).
The role of the notochord for epaxial myotome formation in the mouse
.
Cell Mol. Biol.
45
,
601
-
616
.
Don
,
E. K.
,
de Jong-Curtain
,
T. A.
,
Doggett
,
K.
,
Hall
,
T. E.
,
Heng
,
B.
,
Badrock
,
A. P.
,
Winnick
,
C.
,
Nicholson
,
G. A.
,
Guillemin
,
G. J.
,
Currie
,
P. D.
, et al. 
. (
2016
).
Genetic basis of hindlimb loss in a naturally occurring vertebrate model
.
Biol. Open
5
,
359
-
366
.
Draper
,
B. W.
,
Stock
,
D. W.
and
Kimmel
,
C. B
. (
2003
).
Zebrafish fgf24 functions with fgf8 to promote posterior mesodermal development
.
Development
130
,
4639
-
4654
.
Du
,
S. J.
,
Devoto
,
S. H.
,
Westerfield
,
M.
and
Moon
,
R. T
. (
1997
).
Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-β gene families
.
J. Cell Biol.
139
,
145
-
156
.
Feldman
,
B.
and
Stemple
,
D. L
. (
2001
).
Morpholino phenocopies of sqt, oep, and ntl mutations
.
Genesis
30
,
175
-
177
.
Fior
,
R.
,
Maxwell
,
A. A.
,
Ma
,
T. P.
,
Vezzaro
,
A.
,
Moens
,
C. B.
,
Amacher
,
S. L.
,
Lewis
,
J.
and
Saude
,
L
. (
2012
).
The differentiation and movement of presomitic mesoderm progenitor cells are controlled by Mesogenin 1
.
Development
139
,
4656
-
4665
.
Fukuda
,
M.
,
Takahashi
,
S.
,
Haramoto
,
Y.
,
Onuma
,
Y.
,
Kim
,
Y. J.
,
Yeo
,
C. Y.
,
Ishiura
,
S.
and
Asashima
,
M
. (
2010
).
Zygotic VegT is required for Xenopus paraxial mesoderm formation and is regulated by Nodal signaling and Eomesodermin
.
Int. J. Dev. Biol.
54
,
81
-
92
.
Garnett
,
A. T.
,
Han
,
T. M.
,
Gilchrist
,
M. J.
,
Smith
,
J. C.
,
Eisen
,
M. B.
,
Wardle
,
F. C.
and
Amacher
,
S. L
. (
2009
).
Identification of direct T-box target genes in the developing zebrafish mesoderm
.
Development
136
,
749
-
760
.
Gee
,
H
. (
2018
).
Across the Bridge: Understanding the Origin of the Vertebrates
.
London
:
The University of Chicago Press
.
Gentsch
,
G. E.
,
Owens
,
N. D.
,
Martin
,
S. R.
,
Piccinelli
,
P.
,
Faial
,
T.
,
Trotter
,
M. W.
,
Gilchrist
,
M. J.
and
Smith
,
J. C
. (
2013
).
In vivo T-box transcription factor profiling reveals joint regulation of embryonic neuromesodermal bipotency
.
Cell Rep.
4
,
1185
-
1196
.
George-Weinstein
,
M.
,
Gerhart
,
J.
,
Reed
,
R.
,
Flynn
,
J.
,
Callihan
,
B.
,
Mattiacci
,
M.
,
Miehle
,
C.
,
Foti
,
G.
,
Lash
,
J. W.
and
Weintraub
,
H
. (
1996
).
Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro
.
Dev. Biol.
173
,
279
-
291
.
Gerhart
,
J
. (
2001
).
Evolution of the organizer and the chordate body plan
.
Int. J. Dev. Biol.
45
,
133
-
153
.
Gerhart
,
J.
,
Neely
,
C.
,
Stewart
,
B.
,
Perlman
,
J.
,
Beckmann
,
D.
,
Wallon
,
M.
,
Knudsen
,
K.
and
George-Weinstein
,
M
. (
2004
).
Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage
.
J. Cell Biol.
164
,
739
-
746
.
Goering
,
L. M.
,
Hoshijima
,
K.
,
Hug
,
B.
,
Bisgrove
,
B.
,
Kispert
,
A.
and
Grunwald
,
D. J
. (
2003
).
An interacting network of T-box genes directs gene expression and fate in the zebrafish mesoderm
.
Proc. Natl. Acad. Sci. USA
100
,
9410
-
9415
.
Griffin
,
K. J.
and
Kimelman
,
D
. (
2002
).
One-Eyed Pinhead and Spadetail are essential for heart and somite formation
.
Nat. Cell Biol.
4
,
821
-
825
.
Griffin
,
K. J.
and
Kimelman
,
D
. (
2003
).
Interplay between FGF, one-eyed pinhead, and T-box transcription factors during zebrafish posterior development
.
Dev. Biol.
264
,
456
-
466
.
Griffin
,
K.
,
Patient
,
R.
and
Holder
,
N
. (
1995
).
Analysis of FGF function in normal and no tail zebrafish embryos reveals separate mechanisms for formation of the trunk and tail
.
Development
121
,
2983
-
2994
.
Griffin
,
K. J.
,
Amacher
,
S. L.
,
Kimmel
,
C. B.
and
Kimelman
,
D
. (
1998
).
Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes
.
Development
125
,
3379
-
3388
.
Grimaldi
,
A.
,
Tettamanti
,
G.
,
Martin
,
B. L.
,
Gaffield
,
W.
,
Pownall
,
M. E.
and
Hughes
,
S. M
. (
2004
).
Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis
.
Development
131
,
3249
-
3262
.
Groves
,
J. A.
,
Hammond
,
C. L.
and
Hughes
,
S. M
. (
2005
).
Fgf8 drives myogenic progression of a novel lateral fast muscle fibre population in zebrafish
.
Development
132
,
4211
-
4222
.
Haeussler
,
M.
,
Zweig
,
A. S.
,
Tyner
,
C.
,
Speir
,
M. L.
,
Rosenbloom
,
K. R.
,
Raney
,
B. J.
,
Lee
,
C. M.
,
Lee
,
B. T.
,
Hinrichs
,
A. S.
,
Gonzalez
,
J. N.
, et al. 
. (
2019
).
The UCSC genome browser database: 2019 update
.
Nucleic Acids Res.
47
,
D853
-
D858
.
Halpern
,
M. E.
,
Ho
,
R. K.
,
Walker
,
C.
and
Kimmel
,
C. B
. (
1993
).
Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation
.
Cell
75
,
99
-
111
.
Halpern
,
M. E.
,
Thisse
,
C.
,
Ho
,
R. K.
,
Thisse
,
B.
,
Riggleman
,
B.
,
Trevarrow
,
B.
,
Weinberg
,
E. S.
,
Postlethwait
,
J. H.
and
Kimmel
,
C. B
. (
1995
).
Cell-autonomous shift from axial to paraxial mesodermal development in zebrafish floating head mutants
.
Development
121
,
4257
-
4264
.
Hammerschmidt
,
M.
and
Nüsslein-Volhard
,
C
. (
1993
).
The expression of a zebrafish gene homologous to Drosophila snail suggests a conserved function in invertebrate and vertebrate gastrulation
.
Development
119
,
1107
-
1118
.
Hasson
,
P.
,
DeLaurier
,
A.
,
Bennett
,
M.
,
Grigorieva
,
E.
,
Naiche
,
L. A.
,
Papaioannou
,
V. E.
,
Mohun
,
T. J.
and
Logan
,
M. P
. (
2010
).
Tbx4 and tbx5 acting in connective tissue are required for limb muscle and tendon patterning
.
Dev. Cell
18
,
148
-
156
.
Hinits
,
Y.
,
Osborn
,
D. P.
and
Hughes
,
S. M
. (
2009
).
Differential requirements for myogenic regulatory factors distinguish medial and lateral somitic, cranial and fin muscle fibre populations
.
Development
136
,
403
-
414
.
Hinits
,
Y.
,
Williams
,
V. C.
,
Sweetman
,
D.
,
Donn
,
T. M.
,
Ma
,
T. P.
,
Moens
,
C. B.
and
Hughes
,
S. M
. (
2011
).
Defective cranial skeletal development, larval lethality and haploinsufficiency in Myod mutant zebrafish
.
Dev. Biol.
358
,
102
-
112
.
Hirsinger
,
E.
,
Stellabotte
,
F.
,
Devoto
,
S. H.
and
Westerfield
,
M
. (
2004
).
Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors
.
Dev. Biol.
275
,
143
-
157
.
Ho
,
R. K.
and
Kane
,
D. A
. (
1990
).
Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors
.
Nature
348
,
728
-
730
.
Honjo
,
Y.
and
Eisen
,
J. S
. (
2005
).
Slow muscle regulates the pattern of trunk neural crest migration in zebrafish
.
Development
132
,
4461
-
4470
.
Imai
,
K. S.
,
Levine
,
M.
,
Satoh
,
N.
and
Satou
,
Y
. (
2006
).
Regulatory blueprint for a chordate embryo
.
Science
312
,
1183
-
1187
.
Isaacs
,
H. V.
,
Deconinck
,
A. E.
and
Pownall
,
M. E
. (
2007
).
FGF4 regulates blood and muscle specification in Xenopus laevis
.
Biol. Cell
99
,
165
-
173
.
Itoh
,
N.
and
Konishi
,
M
. (
2007
).
The zebrafish fgf family
.
Zebrafish
4
,
179
-
186
.
Jahangiri
,
L.
,
Nelson
,
A. C.
and
Wardle
,
F. C
. (
2012
).
A cis-regulatory module upstream of deltaC regulated by Ntla and Tbx16 drives expression in the tailbud, presomitic mesoderm and somites
.
Dev. Biol.
371
,
110
-
120
.
Kimelman
,
D.
and
Kirschner
,
M
. (
1987
).
Synergistic induction of mesoderm by FGF and TGF-β and the identification of an mRNA coding for FGF in the early Xenopus embryo
.
Cell
51
,
869
-
877
.
Kimmel
,
C. B.
,
Kane
,
D. A.
,
Walker
,
C.
,
Warga
,
R. M.
and
Rothman
,
M. B
. (
1989
).
A mutation that changes cell movement and cell fate in the zebrafish embryo
.
Nature
337
,
358
-
362
.
Kispert
,
A.
,
Herrmann
,
B. G.
,
Leptin
,
M.
and
Reuter
,
R
. (
1994
).
Homologs of the mouse Brachyury gene are involved in the specification of posterior terminal structures in Drosophila, Tribolium, and Locusta
.
Genes Dev.
8
,
2137
-
2150
.
Knight
,
R. D.
,
Mebus
,
K.
and
Roehl
,
H. H
. (
2008
).
Mandibular arch muscle identity is regulated by a conserved molecular process during vertebrate development
.
J. Exp. Zool. B Mol. Dev. Evol.
310B
,
355
-
369
.
Kolm
,
P. J.
and
Sive
,
H. L
. (
1995
).
Efficient hormone-inducible protein function in Xenopus laevis
.
Dev. Biol.
171
,
267
-
272
.
Krauss
,
S.
,
Concordet
,
J.-P.
and
Ingham
,
P. W
. (
1993
).
A functionally conserved homologue of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos
.
Cell
75
,
1431
-
1444
.
Kudoh
,
T.
,
Tsang
,
M.
,
Hukriede
,
N. A.
,
Chen
,
X.
,
Dedekian
,
M.
,
Clarke
,
C. J.
,
Kiang
,
A.
,
Schultz
,
S.
,
Epstein
,
J. A.
,
Toyama
,
R.
, et al. 
. (
2001
).
A gene expression screen in zebrafish embryogenesis
.
Genome Res.
11
,
1979
-
1987
.
Lacalli
,
T. C
. (
2002
).
The dorsal compartment locomotory control system in amphioxus larvae
.
J. Morphol.
252
,
227
-
237
.
Lee
,
H.-C.
,
Tseng
,
W.-A.
,
Lo
,
F.-Y.
,
Liu
,
T.-M.
and
Tsai
,
H.-J
. (
2009
).
FoxD5 mediates anterior-posterior polarity through upstream modulator Fgf signaling during zebrafish somitogenesis
.
Dev. Biol.
336
,
232
-
245
.
Lewis
,
K. E.
,
Currie
,
P. D.
,
Roy
,
S.
,
Schauerte
,
H.
,
Haffter
,
P.
and
Ingham
,
P. W
. (
1999
).
Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling
.
Dev. Biol.
216
,
469
-
480
.
Lolas
,
M.
,
Valenzuela
,
P. D. T.
,
Tjian
,
R.
and
Liu
,
Z
. (
2014
).
Charting Brachyury-mediated developmental pathways during early mouse embryogenesis
.
Proc. Natl. Acad. Sci. USA
111
,
4478
-
4483
.
Lu
,
F.
,
Langenbacher
,
A.
and
Chen
,
J.-N
. (
2017
).
Tbx20 drives cardiac progenitor formation and cardiomyocyte proliferation in zebrafish
.
Dev. Biol.
421
,
139
-
148
.
Maguire
,
R. J.
,
Isaacs
,
H. V.
and
Pownall
,
M. E
. (
2012
).
Early transcriptional targets of MyoD link myogenesis and somitogenesis
.
Dev. Biol.
371
,
256
-
268
.
Manning
,
A. J.
and
Kimelman
,
D
. (
2015
).
Tbx16 and Msgn1 are required to establish directional cell migration of zebrafish mesodermal progenitors
.
Dev. Biol.
406
,
172
-
185
.
Maroon
,
H.
,
Walshe
,
J.
,
Mahmood
,
R.
,
Kiefer
,
P.
,
Dickson
,
C.
and
Mason
,
I
. (
2002
).
Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle
.
Development
129
,
2099
-
2108
.
Martin
,
B. L.
and
Kimelman
,
D
. (
2008
).
Regulation of canonical Wnt signaling by Brachyury is essential for posterior mesoderm formation
.
Dev. Cell
15
,
121
-
133
.
McDermott
,
A.
,
Gustafsson
,
M.
,
Elsam
,
T.
,
Hui
,
C. C.
,
Emerson
,
C. P.
, Jr
and
Borycki
,
A. G
. (
2005
).
Gli2 and Gli3 have redundant and context-dependent function in skeletal muscle formation
.
Development
132
,
345
-
357
.
Melby
,
A. E.
,
Warga
,
R. M.
and
Kimmel
,
C. B
. (
1996
).
Specification of cell fates at the dorsal margin of the zebrafish gastrula
.
Development
122
,
2225
-
2237
.
Molven
,
A.
,
Wright
,
C. V.
,
Bremiller
,
R.
,
De Robertis
,
E. M.
and
Kimmel
,
C. B
. (
1990
).
Expression of a homeobox gene product in normal and mutant zebrafish embryos: evolution of the tetrapod body plan
.
Development
109
,
279
-
288
.
Morrow
,
Z. T.
,
Maxwell
,
A. M.
,
Hoshijima
,
K.
,
Talbot
,
J. C.
,
Grunwald
,
D. J.
and
Amacher
,
S. L
. (
2017
).
tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis
.
Dev. Dyn.
246
,
759
-
769
.
Mueller
,
R. L.
,
Huang
,
C.
and
Ho
,
R. K
. (
2010
).
Spatio-temporal regulation of Wnt and retinoic acid signaling by tbx16/spadetail during zebrafish mesoderm differentiation
.
BMC Genomics
11
,
492
.
Münsterberg
,
A. E.
,
Kitajewski
,
J.
,
Bumcrot
,
D. A.
,
McMahon
,
A. P.
and
Lassar
,
A. B
. (
1995
).
Combinatorial signaling by sonic hedgehog and Wnt family members induces myogenic bHLH gene expression in the somite
.
Genes Dev.
9
,
2911
-
2922
.
Nandkishore
,
N.
,
Vyas
,
B.
,
Javali
,
A.
,
Ghosh
,
S.
and
Sambasivan
,
R
. (
2018
).
Divergent early mesoderm specification underlies distinct head and trunk muscle programmes in vertebrates
.
Development
145
,
dev160945
.
Nelson
,
A. C.
,
Cutty
,
S. J.
,
Gasiunas
,
S. N.
,
Deplae
,
I.
,
Stemple
,
D. L.
and
Wardle
,
F. C
. (
2017
).
In Vivo regulation of the zebrafish endoderm progenitor niche by T-box transcription factors
.
Cell Rep.
19
,
2782
-
2795
.
Nikaido
,
M.
,
Kawakami
,
A.
,
Sawada
,
A.
,
Furutani-Seiki
,
M.
,
Takeda
,
H.
and
Araki
,
K
. (
2002
).
Tbx24, encoding a T-box protein, is mutated in the zebrafish somite-segmentation mutant fused somites
.
Nat. Genet.
31
,
195
-
199
.
Ochi
,
H.
,
Hans
,
S.
and
Westerfield
,
M
. (
2008
).
Smarcd3 regulates the timing of zebrafish myogenesis onset
.
J. Biol. Chem.
283
,
3529
-
3536
.
Odenthal
,
J.
,
Haffter
,
P.
,
Vogelsang
,
E.
,
Brand
,
M.
,
Van Eeden
,
F. J. M.
,
Furutani-Seiki
,
M.
,
Granato
,
M.
,
Hammerschmidt
,
M.
,
Heisenberg
,
C. P.
,
Jiang
,
Y. J.
, et al. 
. (
1996
).
Mutations affecting the formation of the notochord in the zebrafish, Danio rerio
.
Development
123
,
103
-
115
.
Osborn
,
D. P.
,
Li
,
K.
,
Hinits
,
Y.
and
Hughes
,
S. M
. (
2011
).
Cdkn1c drives muscle differentiation through a positive feedback loop with Myod
.
Dev. Biol.
350
,
464
-
475
.
Papaioannou
,
V. E
. (
2014
).
The T-box gene family: emerging roles in development, stem cells and cancer
.
Development
141
,
3819
-
3833
.
Placzek
,
M.
and
Briscoe
,
J
. (
2018
).
Sonic hedgehog in vertebrate neural tube development
.
Int. J. Dev. Biol.
62
,
225
-
234
.
Pradeepa
,
M. M
. (
2017
).
Causal role of histone acetylations in enhancer function
.
Transcription
8
,
40
-
47
.
Pradeepa
,
M. M.
,
Grimes
,
G. R.
,
Kumar
,
Y.
,
Olley
,
G.
,
Taylor
,
G. C.
,
Schneider
,
R.
and
Bickmore
,
W. A
. (
2016
).
Histone H3 globular domain acetylation identifies a new class of enhancers
.
Nat. Genet.
48
,
681
-
686
.
Reifers
,
F.
,
Bohli
,
H.
,
Walsh
,
E. C.
,
Crossley
,
P. H.
,
Stainier
,
D. Y.
and
Brand
,
M
. (
1998
).
Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis
.
Development
125
,
2381
-
2395
.
Rentzsch
,
F.
,
Bakkers
,
J.
,
Kramer
,
C.
and
Hammerschmidt
,
M
. (
2004
).
Fgf signaling induces posterior neuroectoderm independently of Bmp signaling inhibition
.
Dev. Dyn.
231
,
750
-
757
.
Rhinn
,
M.
,
Lun
,
K.
,
Luz
,
M.
,
Werner
,
M.
and
Brand
,
M
. (
2005
).
Positioning of the midbrain-hindbrain boundary organizer through global posteriorization of the neuroectoderm mediated by Wnt8 signaling
.
Development
132
,
1261
-
1272
.
Roelink
,
H.
,
Augsburger
,
A.
,
Heemskerk
,
J.
,
Korzh
,
V.
,
Norlin
,
S.
,
Ruiz i Altaba
,
A.
,
Tanabe
,
Y.
,
Placzek
,
M.
,
Edlund
,
T.
,
Jessell
, ,
T. M.
(
1994
).
Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord
.
Cell
76
,
761
-
775
.
Row
,
R. H.
,
Maitre
,
J.-L.
,
Martin
,
B. L.
,
Stockinger
,
P.
,
Heisenberg
,
C.-P.
and
Kimelman
,
D
. (
2011
).
Completion of the epithelial to mesenchymal transition in zebrafish mesoderm requires Spadetail
.
Dev. Biol.
354
,
102
-
110
.
Rudnicki
,
M. A.
,
Schnegelsberg
,
P. N.
,
Stead
,
R. H.
,
Braun
,
T.
,
Arnold
,
H. H.
and
Jaenisch
,
R
. (
1993
).
MyoD or Myf-5 is required for the formation of skeletal muscle
.
Cell
75
,
1351
-
1359
.
Ruvinsky
,
I.
,
Silver
,
L. M.
and
Ho
,
R. K
. (
1998
).
Characterization of the zebrafish tbx16 gene and evolution of the vertebrate T-box family
.
Dev. Genes Evol.
208
,
94
-
99
.
Satoh
,
N.
,
Tagawa
,
K.
and
Takahashi
,
H
. (
2012
).
How was the notochord born?
Evol. Dev.
14
,
56
-
75
.
Schulte-Merker
,
S.
,
Ho
,
R.K.
,
Herrmann
,
B.G.
and
Nüsslein-Volhard
,
C.
(
1992
).
The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo
.
Development
116
,
1021
-
1032
.
Schulte-Merker
,
S.
,
Hammerschmidt
,
M.
,
Beuchle
,
D.
,
Cho
,
K. W.
,
De Robertis
,
E. M.
and
Nüsslein-Volhard
,
C
. (
1994a
).
Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos
.
Development
120
,
843
-
852
.
Schulte-Merker
,
S.
,
van Eeden
,
F. J. M.
,
Halpern
,
M. E.
,
Kimmel
,
C. B.
and
Nüsslein-Volhard
,
C
. (
1994b
).
no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene
.
Development
120
,
1009
-
1015
.
Showell
,
C.
,
Binder
,
O.
and
Conlon
,
F. L
. (
2004
).
T-box genes in early embryogenesis
.
Dev. Dyn.
229
,
201
-
218
.
Slack
,
J. M.
,
Darlington
,
B. G.
,
Heath
,
J. K.
and
Godsave
,
S. F
. (
1987
).
Mesoderm induction in early Xenopus embryos by heparin-binding growth factors
.
Nature
326
,
197
-
200
.
Smith
,
J. C.
,
Price
,
B. M.
,
Green
,
J. B.
,
Weigel
,
D.
and
Herrmann
,
B. G
. (
1991
).
Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction
.
Cell
67
,
79
-
87
.
Sun
,
X.
,
Meyers
,
E. N.
,
Lewandoski
,
M.
and
Martin
,
G. R
. (
1999
).
Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo
.
Genes Dev.
13
,
1834
-
1846
.
Talbot
,
W. B.
,
Trevarrow
,
B.
,
Halpern
,
M. E.
,
Melby
,
A. E.
,
Farr
,
G.
,
Postlethwait
,
T. J.
,
Kimmel
,
C. B.
and
Kimelman
,
D
. (
1995
).
A homeobox gene essential for zebrafish notochord development
.
Nature
378
,
150
-
157
.
Taylor
,
M. V.
and
Hughes
,
S. M
. (
2017
).
Mef2 and the skeletal muscle differentiation program
.
Semin. Cell Dev. Biol.
72
,
33
-
44
.
Tazumi
,
S.
,
Yabe
,
S.
and
Uchiyama
,
H
. (
2010
).
Paraxial T-box genes, Tbx6 and Tbx1, are required for cranial chondrogenesis and myogenesis
.
Dev. Biol.
346
,
170
-
180
.
Thisse
,
B.
and
Thisse
,
C
. (
2005
).
Functions and regulations of fibroblast growth factor signaling during embryonic development
.
Dev. Biol.
287
,
390
-
402
.
Valasek
,
P.
,
Theis
,
S.
,
DeLaurier
,
A.
,
Hinits
,
Y.
,
Luke
,
G. N.
,
Otto
,
A. M.
,
Minchin
,
J.
,
He
,
L.
,
Christ
,
B.
,
Brooks
,
G.
, et al. 
. (
2011
).
Cellular and molecular investigations into the development of the pectoral girdle
.
Dev. Biol.
357
,
108
-
116
.
van Eeden
,
F. J.
,
Granato
,
M.
,
Schach
,
U.
,
Brand
,
M.
,
Furutani-Seiki
,
M.
,
Haffter
,
P.
,
Hammerschmidt
,
M.
,
Heisenberg
,
C. P.
,
Jiang
,
Y. J.
,
Kane
,
D. A.
, et al. 
. (
1996
).
Mutations affecting somite formation and patterning in the zebrafish, Danio rerio
.
Development
123
,
153
-
164
.
van Eeden
,
F. J.
,
Holley
,
S. A.
,
Haffter
,
P.
and
Nüsslein-Volhard
,
C
. (
1998
).
Zebrafish segmentation and pair-rule patterning
.
Dev. Genet.
23
,
65
-
76
.
Warga
,
R. M.
,
Mueller
,
R. L.
,
Ho
,
R. K.
and
Kane
,
D. A
. (
2013
).
Zebrafish Tbx16 regulates intermediate mesoderm cell fate by attenuating Fgf activity
.
Dev. Biol.
383
,
75
-
89
.
Weinberg
,
E. S.
,
Allende
,
M. L.
,
Kelly
,
C. S.
,
Abdelhamid
,
A.
,
Murakami
,
T.
,
Andermann
,
P.
,
Doerre
,
O. G.
,
Grunwald
,
D. J.
and
Riggleman
,
B
. (
1996
).
Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos
.
Development
122
,
271
-
280
.
Westerfield
,
M
. (
2000
).
The Zebrafish Book - A Guide for the Laboratory use of Zebrafish (Danio rerio)
:
University of Oregon Press
.
White
,
P. H.
,
Farkas
,
D. R.
,
McFadden
,
E. E.
and
Chapman
,
D. L
. (
2003
).
Defective somite patterning in mouse embryos with reduced levels of Tbx6
.
Development
130
,
1681
-
1690
.
Wilkinson
,
D. G.
,
Peters
,
G.
,
Dickson
,
C.
and
McMahon
,
A. P
. (
1988
).
Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse
.
EMBO J.
7
,
691
-
695
.
Windner
,
S. E.
,
Bird
,
N. C.
,
Patterson
,
S. E.
,
Doris
,
R. A.
and
Devoto
,
S. H
. (
2012
).
Fss/Tbx6 is required for central dermomyotome cell fate in zebrafish
.
Biol. Open
1
,
806
-
814
.
Windner
,
S. E.
,
Doris
,
R. A.
,
Ferguson
,
C. M.
,
Nelson
,
A. C.
,
Valentin
,
G.
,
Tan
,
H.
,
Oates
,
A. C.
,
Wardle
,
F. C.
and
Devoto
,
S. H
. (
2015
).
Tbx6, Mesp-b and Ripply1 regulate the onset of skeletal myogenesis in zebrafish
.
Development
142
,
1159
-
1168
.
Woollard
,
A.
and
Hodgkin
,
J
. (
2000
).
The caenorhabditis elegans fate-determining gene mab-9 encodes a T-box protein required to pattern the posterior hindgut
.
Genes Dev.
14
,
596
-
603
.
Yamamoto
,
A.
,
Amacher
,
S. L.
,
Kim
,
S. H.
,
Geissert
,
D.
,
Kimmel
,
C. B.
and
De Robertis
,
E. M
. (
1998
).
Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm
.
Development
125
,
3389
-
3397
.
Yamauchi
,
H.
,
Miyakawa
,
N.
,
Miyake
,
A.
and
Itoh
,
N
. (
2009
).
Fgf4 is required for left-right patterning of visceral organs in zebrafish
.
Dev. Biol.
332
,
177
-
185
.
Yasuoka
,
Y.
,
Shinzato
,
C.
and
Satoh
,
N
. (
2016
).
The mesoderm-forming gene brachyury regulates ectoderm-endoderm demarcation in the coral acropora digitifera
.
Curr. Biol.
26
,
2885
-
2892
.
Yin
,
J.
,
Lee
,
R.
,
Ono
,
Y.
,
Ingham
,
P. W.
and
Saunders
,
T. E
. (
2018
).
Spatiotemporal coordination of FGF and Shh signaling underlies the specification of myoblasts in the zebrafish embryo
.
Dev. Cell
46
,
735
-
750.e734
.
Zeng
,
X. X.
,
Wilm
,
T. P.
,
Sepich
,
D. S.
and
Solnica-Krezel
,
L
. (
2007
).
Apelin and its receptor control heart field formation during zebrafish gastrulation
.
Dev. Cell
12
,
391
-
402
.
Zhang
,
X. M.
,
Ramalho-Santos
,
M.
and
McMahon
,
A. P
. (
2001
).
Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node
.
Cell
105
,
781
-
792
.

Competing interests

The authors declare no competing or financial interests.

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