Here, we have demonstrated that cells in the zebrafish dermomyotome are responsive to BMP signaling throughout their early development, and that BMP can regulate the timing of differentiation of these cells. In the presence of ectopic, extra BMP, myogenic precursors are inhibited from differentiating into MRF-expressing myoblasts. However, although BMP is sufficient to inhibit myogenic differentiation, it does not appear to be necessary. The inhibition of BMP signaling does not affect myogenesis, suggesting that there is redundancy in the inhibition of myogenic differentiation.
BMP Regulates the Development of Myogenic Precursors at Multiple Stages of Their Development
The progression of expression of BMP pathway members during somite development in zebrafish suggests that BMP may regulate the development of dermomyotomal cells throughout their development, including their spatial positioning in the anterior row of the epithelial somite and after they reach the lateral surface. We have shown that throughout muscle development, the domain of bmp2b expression is adjacent to the developing dermomyotome (Fig. 1). During early development of the dermomyotome, bmp2b is expressed in the presumptive Rohon-Beard neurons, adjacent to the developing paraxial mesoderm (Fig. 1A, A′, D). At the end of segmentation, bmp2b and bmp4 are expressed in distinct domains adjacent to the dorsal and ventral aspects of the somite (Fig. 1G, H). Near the dorsal somite, the dorsal ectoderm expresses bmp2b and bmp4, and near the ventral somite, the lateral plate mesoderm also expresses bmp2b and bmp4.
The expression of BMP signaling components also supports the idea that BMP signaling regulates the development of myogenic precursors throughout their development. During somitogenesis, somitic cells express BMP signal transduction genes, including bmpr1b (Alk6; Fig. 1B, B′, E) and smad5 (Fig. 1C, C′, F). Interestingly, smad5 is expressed most abundantly by cells in the anterior portion of the somite, which have been shown to give rise to the Pax7-expressing myogenic precursors on the lateral surface of the somite during later stages of development. Smad1 is expressed in a wider domain, with expression throughout the somite during early somite development (Dick et al., 1999; Muller et al., 1999). Activated Smad is also distributed throughout the somite at the earliest detectable stages where dermomyotome precursors are present (Fig. 2A), and the pattern of Smad activation correlates with the combined expression of smad1 and smad5 mRNAs. At 24 hr, smad5-expressing cells are on the surface of the myotome, corresponding to the location of Pax7-expressing dermomyotomal cells, suggesting that cells much later in maturation can still respond to BMP signaling. Activated Smad is found in only a subset of the Pax7-expressing cells on the surface of the somite, similar to that found for chick (Faure et al., 2002), suggesting that BMP signaling is regulating the development of at least some of the dermomyotomal cells at any given moment, specifically at the dorsal and ventral extremes of the somite. Therefore, pSmad labeling shows a “snapshot” of which cells are responding at that moment to BMP. As this population is continuously replenished as they differentiate (i.e., not static), a significant proportion of dermomyotome cells will be influenced by BMP signaling throughout development. Our work corroborates and extends previous findings on the expression of BMPs and Smads in the developing zebrafish somite (Dick et al., 1999).
The effect of overexpression of BMP on the dermomyotome supports our interpretation that the zebrafish dermomyotome is responsive to BMP signaling throughout its development. BMP overexpression at the 3S stage led to an expansion of the pax3-expressing anterior domain (Fig. 4A, B), and a reduction in the myoD-expressing posterior domain (Fig. 4C, D). As the anterior domain gives rise to dermomyotome, these effects suggest that BMP promotes the development or maintenance of dermomyotome precursors at the expense of myotome, while they are still at the anterior border of newly formed somites. While both Pax7 and MEF2 cell numbers in hs-bmp2b 3S heat-shocked embryos returned to control levels by 24 hr (Fig. 5G), the continued reduction of multinucleated fibers and increase in somite angle indicate a lasting effect of ectopic bmp2b overexpression. BMP overexpression at late segmentation stages led to a similar expansion of the dermomyotome (Fig. 3). The expansion of dermomyotome in the anterior somites of these embryos, while not as extensive as in posterior somites, indicates that dermomyotomal cells remain responsive to BMP quite late in their development.
Embryos injected with hs-bmp2b and heat shocked at various stages display marked changes from control that cannot be accounted for by heat shock alone (Fig. 5G). With respect to somite 17, hs-bmp2b-injected embryos heat shocked at the 17S stage show more dramatic changes than those heat shocked at the 3S stage, which we interpret as a partial recovery of 3S heat-shocked embryos to near control levels during the post-heat-shock interval before fixation at 24 hr. Embryos heat shocked at the 17S stage apparently do not have sufficient time to recover before fixation at 24 hr. Taken together, the maturity assays, lack of an effect on proliferation, shifted myoD expression patterns, and increase in dermomyotome domain suggest that bmp2b is likely causing a delay in differentiation of dermomyotome cells (Pax7+) into muscle fibers (as determined by MEF2 labeling). This hypothesis is supported by the 25% increase in Pax7+ cells after 17S heat shock combined with a concomitant 25% decrease in MEF2+ cells (Fig. 5G). While we cannot exclude the possibility that overexpression of BMP may cause an increase in the number of cells being directed down the dermomyotome route, our data strongly suggest that a developmental delay in muscle differentiation is the most likely action of BMP overexpression. This hypothesis also closely mirrors the pattern found in amniotes (Reshef et al., 1998; Amthor et al., 2006).
Other morphological indicators of somite maturity (multinucleated fibers and somite angle) were also greatly affected by ectopic bmp2b overexpression. The number of triplet fibers was the only measure where hs-bmp2b-injected embryos heat shocked at either the 3S or 17S stages were not significantly different from each other, indicating the recovery of these embryos is not complete, and that somite differentiation is affected long after bmp2b exposure, even though Pax7+ and MEF2+ numbers had returned to control levels. While there was a significant increase in somite angle in both 3S and 17S hs-bmp2b-injected embryos, little is known regarding the underlying developmental mechanism ultimately responsible for regulating somite angle, and the significance of a change in somite angle (shape) requires further investigation.
Regulation of Muscle Development by BMP Signaling
There are similarities and differences between our results and those from previous studies in amniotes, in which the role of BMP signaling in somite patterning has been extensively investigated. In both zebrafish and amniotes, BMP2 and BMP4 are expressed in tissues dorsal (dorsomedial) and ventral (ventrolateral) to the dermomyotome, during the time when the dermomyotome is undergoing both proliferation and differentiation into muscle fibers (Sela-Donenfeld and Kalcheim, 2002), and in both amniotes and zebrafish, the effect of BMP signaling is to delay or inhibit the differentiation of dermomyotome cells into muscle fibers. Moreover, in both amniotes and zebrafish, BMP inhibits premature differentiation of myogenic precursors both in the epithelial somite and later in the fully formed epithelial dermomyotome. In chick, ectodermal BMP expression during segmentation prevents premature expression of MRFs in the somite (Linker et al., 2003). After formation of the dermomyotome, BMP from the lateral plate mesoderm inhibits the differentiation of myogenic precursors in the hypaxial dermomyotome (Pourquié et al., 1996; Reshef et al., 1998; Amthor et al., 1999). In zebrafish, we have shown that ectopic BMP signaling during segmentation increases pax3 expression while dermomyotomal precursors are still in the anterior domain of the somite. After the dermomyotomal cells reach the lateral surface of the somite, ectopic BMP expression can also delay the differentiation of myogenic precursors into MRF expressing myoblasts.
One distinct difference between the function of BMP in myogenesis in zebrafish and amniotes is the spatial requirement of BMP. In amniotes, BMP signaling represses the differentiation of cells mainly in the ventrolateral hypaxial dermomyotome, which provides cells to the limb musculature and body wall. This is presumably to delay the differentiation of cells that migrate into the limb, ensuring that there are enough precursors to support muscle growth there. In the epaxial somite, BMP signaling to the dermomyotome is blocked by the secreted inhibitor Noggin, which allows for earlier differentiation of the epaxial dermomyotome compared to the hypaxial dermomyotome. However, this inhibition can be overcome by overexpression of BMP, which lateralizes the epaxial somite and causes a delay in the differentiation of myogenic precursors, indicating that both the epaxial and hypaxial somite are responsive to BMP signaling (Pourquié et al., 1996; Reshef et al., 1998). In zebrafish, the spatial contribution of the dermomyotome to the epaxial and hypaxial musculature is still unclear, and cells in both the dorsal and ventral dermomyotome appear to be actively responding to BMP signaling. In axolotl, an anamniote tetrapod, BMP inhibition results in decreased Pax7 throughout the lateral surface. In addition, overexpression of BMP leads to upregulation of Pax7, similar to zebrafish (Epperlein et al., 2007). Therefore, the function of BMP signaling in inhibition of myogenic differentiation may be basal, and may have been modified in the amniote lineage to regulate development of mainly the lateral dermomyotome.
Inhibition of BMP Signaling Does Not Affect the Development of the Zebrafish Dermomyotome
Inhibition of BMP signaling using a dominant-negative BMP receptor has no apparent effect on the development of myogenic precursors in zebrafish (Fig. 6E, F). The lack of an effect of inhibiting BMP signaling on dermomyotomal cells was surprising given the strong effect of BMP overexpression. Inhibition of BMP signaling during gastrulation, at a level high enough to perturb dorsoventral patterning and phenocopy a known bmp4 mutant (Stickney et al., 2007), did not significantly reduce Pax7 expression in the somites, although it did appear that embryos with overexpression of Chordin had increased Myogenin expression (Fig. 6A). This is consistent with our hypothesis that BMP inhibits the transition of myogenic precursors into MRF expressing myoblasts, but it leaves unexplained why there is no effect on Pax7 expression.
We also examined the expression of Pax7 and MRFs in embryos in which the inhibition of BMP signaling was restricted to the segmentation period, and found no significant change. One possibility is that dermomyotome cells are still receiving sufficient BMP signaling, even when the dominant-negative BMP receptor is expressed. This is supported by the lack of a significant decrease in the number of cells expressing both Pax7 and pSmad following heat-shock induction of the dominant-negative BMPR1a transgene (both between 20–25% of all Pax7+ cells). Although the overall level of pSmad is reduced by approximately 40%, this change is not reflected in the Pax7 population. Therefore, it is possible that the level of BMP signaling remaining after heat-shock induction of the dominant-negative receptor is sufficient for development and maintenance of the dermomyotome.
A second possibility is that there is redundancy in the regulation of differentiation of cells in the dermomyotome. Because of the importance of precisely regulating the timing of development of myogenic precursors, there may be multiple mechanisms ensuring that muscle differentiation occurs at the correct time and place. Fewer factors have been identified that negatively regulate muscle differentiation, compared to the number of factors identified that promote this process. In addition to BMP, Notch signaling negatively regulates the differentiation of muscle precursors in mouse and chick (Holowacz et al., 2006; Schuster-Gossler et al., 2007). Myostatin, a TGF-beta family member, is also required for the proper regulation of myogenic proliferation in chick, mouse, and zebrafish, and in the absence of myostatin, muscle develops to 2 or 3 times its normal size (McPherron et al., 1997; Xu et al., 2003; Amthor et al., 2006). If there were redundancy in the regulation of differentiation of muscle precursors, we would still expect to see an effect of overexpression of BMP, as BMP would be sufficient to repress the differentiation of muscle precursors. However, removing the BMP signal would not necessarily have an effect, as the secondary signal would be capable of appropriately regulating the differentiation of muscle precursors in the absence of BMP.
Molecular Regulation of the Zebrafish Dermomyotome
Despite the relatively small size, rapid development, and simple organization of the zebrafish somite, multiple signals regulate somite patterning, as in amniotes. Fgf8 signaling promotes the differentiation of myogenic precursors into myoblasts. The inhibition of Fgf8 signaling results in an up-regulation of pax3 and Pax7, and a down-regulation of MRFs (Groves et al., 2005; Hammond et al., 2007). The expression of Fgf8 in the somite is regulated by Retinoic Acid (RA), and exogenous RA also results in an up-regulation of myoD and a down-regulation of pax3 (Hamade et al., 2006; Hammond et al., 2007).
Hedgehog (Hh) also positively regulates the differentiation of myogenic precursors. Loss of Hh signaling leads to an increase in pax3 and Pax7 expression in the somites, and a decrease in differentiation (Feng et al., 2006; Hammond et al., 2007). Hh and BMP act in opposing ways in various developmental contexts, including in patterning of the somite (Marcelle et al., 1997). In zebrafish, BMP and Hh have been proposed to act in opposing ways in the development of slow muscle fibers, through the action of Scube2 (Du et al., 1997; Kawakami et al., 2005). The effect on muscle precursors following overexpression of BMP closely resembles the effect on muscle precursors following inhibition of Hh signaling. Based on the similarities between the effect of removing Hh and adding BMP, it appears that these signals may also be regulating the differentiation of myogenic precursors in opposite ways. However, it is not clear if BMP and Hh are acting in opposing ways at the same time.
Understanding how BMP, Hedgehog, Fgf8, and Retinoic Acid signaling integrate to regulate the dermomyotome will be critical to understanding how a balance between proliferation and differentiation is maintained during muscle development and growth.