In vertebrates, cells that eventually form the skeletal muscles of the trunk and limbs, differentiate from the dorsal part of the somite, the dermomyotome (Christ and Ordahl, 1995; Tajbakhsh and Buckingham, 2000; Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2005). Cells of the dermomyotome proliferate and form a pool of undifferentiated muscle progenitor cells. Cells located within the dorsomedial lip of the dermomyotome are the first to differentiate into skeletal muscle and migrate underneath the dermomyotome to form the medial region of the myotome (Denetclaw et al., 2001; Ordahl et al., 2001; Kahane et al., 2002; Venters and Ordahl, 2002; Gros et al., 2004); subsequently, cells from all four borders of the dermomyotome give rise to myotomal cells (Gros et al., 2004). Each step of the muscle differentiation program can be distinguished by the expression of specific transcription factors. The dermomyotome expresses the paired box transcription factors, Pax-3 and Pax-7 (Goulding et al., 1991; Jostes et al., 1991). As cells enter the myotome and differentiate into skeletal muscle, the expression of these factors diminishes, while expression of the myogenic bHLH transcription factors Myf-5 and MyoD are up-regulated (Sassoon et al., 1989; Hirsinger et al., 2001; Kiefer and Hauschka, 2001). Pax-3 has been documented to be upstream of MyoD expression in the trunk (Tajbakhsh et al., 1997), and forced expression of Pax-3 in somitic cells is able to induce MyoD expression and skeletal muscle differentiation in tissue culture (Maroto et al., 1997). MyoD and Myf-5 in turn are required for the differentiation of skeletal muscle throughout the body (Rudnicki et al., 1993), and forced expression of these myogenic basic helix–loop–helix (bHLH) transcription factors can induce skeletal muscle differentiation in a variety of cell types (Weintraub et al., 1989).
Many extrinsic signals have been documented to control the patterning, differentiation, expansion, and survival of skeletal muscle cells in the somite. Whereas intrinsic Wnt signals within the somite have been found to be necessary for the induction of MyoD (Linker et al., 2003), Sonic hedgehog (Shh) secreted by the notochord and ventral neural tube acts in concert with Wnt-1 and Wnt-3a secreted by the dorsal neural tube to promote myotome formation (Munsterberg et al., 1995; Stern et al., 1995; Fan et al., 1997; Ikeya and Takada, 1998; Borycki et al., 1999; Gustafsson et al., 2002). In addition, Shh promotes the survival and expansion of skeletal muscle precursors (Duprez et al., 1998; Teillet et al., 1998; Marcelle et al., 1999; Kruger et al., 2001) and is necessary for the maintained expression of myogenic bHLH genes in the myotome (Chiang et al., 1996; Coutelle et al., 2001; Teboul et al., 2003). Also, signals from the surface ectoderm, which can be mimicked by Wnt-4 or Wnt-6, can induce dermomyotome formation (Fan et al., 1997) and expression of MyoD (Tajbakhsh et al., 1998). Bone morphogenetic protein (BMP), which is secreted by the lateral plate mesoderm can act as an inhibitor of myogenesis and can be counteracted by the BMP antagonist, Noggin, which is expressed in the dorsomedial lip of the dermomyotome (Hirsinger et al., 1997; Marcelle et al., 1997; Reshef et al., 1998; Tonegawa and Takahashi, 1998). Interestingly, Shh and Wnt signals induce Noggin expression in the dorsomedial lip of the dermomyotome, which in turn stimulates dermomyotomal cells to enter the myotomal compartment (Hirsinger et al., 1997; Marcelle et al., 1997; Reshef et al., 1998).
In addition to Wnt, Shh and BMP signals, the Notch pathway has also been shown to influence myogenesis. Notch signaling is triggered by the interaction of the Notch receptor with one of its ligands, Delta or Serrate/Jagged (Mumm and Kopan, 2000). After ligand binding, the Notch receptor undergoes a proteolytic cleavage that releases the Notch intracellular domain (NICD) to the cytoplasm. The NICD translocates to the nucleus, associates with members of the CSL (CBF1/RBP-Jκ, Suppressor of hairless (Su(H)), LAG-1) family, and modulates the transcriptional activity of these proteins. In the case of CBF1/RBP-Jκ, association with the NICD turns this transcriptional repressor into an activator (Kao et al., 1998). The NICD-CSL complex activates the expression of downstream targets including members of the Hairy/Enhancer-of-Split family of bHLH transcription factors. In vitro studies in mouse cell lines have shown that forced expression of either Notch ligands or activated Notch receptor inhibits muscle differentiation (Kopan et al., 1994; Shawber et al., 1996; Luo et al., 1997; Kuroda et al., 1999; Nofziger et al., 1999; Wilson-Rawls et al., 1999). This inhibition is mediated by at least two pathways: one pathway operates upstream of MyoD and involves transcriptional activation of CSL and the Hairy-related transcription factors HES-1 and/or Hey-1 (Kuroda et al., 1999; Nofziger et al., 1999; Sun et al., 2001). The second pathway appears to operate independently of CSL (Nofziger et al., 1999). Consistent with this, the NICD itself can directly bind to and inhibit the transcriptional activity of MEF-2C, a transcription factor, which acts downstream of myogenic bHLH proteins to promote skeletal muscle differentiation (Wilson-Rawls et al., 1999).
In vertebrate embryos, Notch receptors and ligands are widely expressed in the somite. Notch-1 RNA is expressed in the pre-somitic mesoderm and later is concentrated in the medial portions of the dermomyotome and myotome in both chick (Hirsinger et al., 2001) and mouse (Williams et al., 1995) embryos, whereas mouse Notch-2 and -3 are expressed in the dermomyotome of more mature somites (Williams et al., 1995). Chick Delta-1 is found in the caudal portion of each somite in both the dermomyotome and myotome, and chick Serrate-2 is expressed in the differentiating myotome (Hirsinger et al., 2001). Although mouse mutants bearing mutations in Notch-1 or Delta-1 develop abnormalities in somite formation, they do not possess obvious defects in skeletal muscle differentiation (Conlon et al., 1995; de la Pompa et al., 1997; Hrabe de Angelis et al., 1997). These results suggest that various Notch ligands and receptors may display functional redundancy during skeletal muscle formation. However, mice mutant for CBF1/RBP-Jκ lack somitic expression of the myogenic factor, myogenin (Oka et al., 1995), supporting a role for the Notch pathway in modulating muscle cell differentiation in vivo. Furthermore, forced expression of Delta-1 in chick embryos inhibits expression of MyoD and blocks skeletal muscle differentiation in both the myotome and developing wing bud (Delfini et al., 2000; Hirsinger et al., 2001).
Because Notch and its ligands are expressed in the somite while myotome formation occurs, it seems plausible that Notch signaling may be actively repressed in this somitic domain. One mechanism for the inhibition of Notch signaling is through expression of the Notch inhibitor Numb. Numb blocks Notch signaling by binding to the C-terminus of the NICD and has been reported to both prevent its nuclear translocation (Frise et al., 1996; Guo et al., 1996; Wakamatsu et al., 1999) and induce its endocytosis (Santolini et al., 2000; Berdnik et al., 2002). In dividing myoblasts and neuroblasts of the fly, Numb protein is asymmetrically localized in a cortical crescent on one side of a dividing cell and is required for specification of distinct muscle (Ruiz Gomez and Bate, 1997; Carmena et al., 1998) and neural (Frise et al., 1996; Spana and Doe, 1996) cell fates. Recently, a role for Numb in vertebrate myogenesis was shown in postnatally derived muscle satellite cells (Conboy and Rando, 2002). Numb is localized asymmetrically in the daughters of these dividing muscle progenitor cells and its expression correlates with induction of myogenic regulatory factors, cell cycle withdrawal, and loss of Pax-3 expression (Conboy and Rando, 2002). Moreover, Numb overexpression promotes the differentiation of skeletal muscle progenitors (Conboy and Rando, 2002). Despite this new information regarding the role of Numb in postnatal vertebrate myogenesis, its role during muscle development in the embryo has not been ascertained.
Here, we examine the dynamics of Numb RNA and protein expression during somitic myogenesis in chick embryos. Numb is asymmetrically localized to the basal surface of dividing cells in the dorsomedial lip of the dermomyotome, which contains precursors for both the dermomyotome and myotome, In addition, Numb is expressed throughout the cytoplasm of differentiated muscle cells in the myotome. We find that signals and transcription factors that promote skeletal muscle formation induce the accumulation of Numb protein (by possibly a post-transcriptional mechanism) and simultaneously up-regulate the expression of the Notch ligand Serrate-2. Together, these findings indicate that dividing cells within the dorsomedial lip of the dermomyotome display asymmetric accumulation of Numb protein and that induction of the myotome by skeletal muscle inducing signals results in the high uniform expression of Numb protein throughout the cytoplasm and the simultaneous expression of Serrate-2 in this tissue. The coordinated expression of both a Notch antagonist (i.e., Numb) and ligand (Serrate-2) within the myotome ensures that Notch signaling is selectively inhibited within this somitic domain and may be promoted in the surrounding dermomyotome and sclerotome.
Numb mRNA Is Expressed in Somites
We initially examined Numb RNA expression during somite differentiation in the chick embryo and compared it with that of MyoD and its upstream activator, Pax3. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that Numb, Notch-1, and Pax3 transcripts were expressed throughout the presomitic mesoderm as well as in newly formed somites I-XII isolated from stage 14 chick embryos, while transcripts for MyoD were most highly expressed in the anterior somites (Fig. 1A). To further examine the localization of Numb transcripts within the somites, we performed in situ hybridization for Numb transcripts on transverse sections through either somite 10 from a stage 17 embryo or through a trunk level somite from a stage 25 embryo. While Numb RNA was uniformly expressed within the neural tube, it appears to be up-regulated within the developing myotome and expressed at lower levels in the sclerotome and dermomyotome (Fig. 1B,C).
Numb Protein Is Asymmetrically Localized During Somitic Myogenesis
To evaluate Numb protein distribution during somitic myogenesis, we performed immunofluorescent staining with antisera made against chick Numb and observed the localization of Numb protein using confocal microscopy (Wakamatsu et al., 1999). As this antisera was generated against a region of Numb that is also conserved in the mouse homolog of a related protein, Numblike (Zhong et al., 1997), it is possible that this antisera recognizes both Numb and Numblike in the chick. In anterior somites of a 3-day embryo (stage 17), Numb protein was present in the myotome (Fig. 2A,B). At the trunk level, where the myotome is less mature, Numb is present in only a few cells (Fig. 2E,F). In immature epithelial somites in the caudal regions of the embryo, we did not detect Numb protein (Fig. 2H,I). Therefore, despite the presence of Numb RNA at all somite levels, accumulation of Numb protein appears to be restricted to the myotome, where it initially accumulates along the ventral edge of myotomal cells adjacent to the sclerotome (Fig. 2A).
In anterior somites at stage 22, Numb protein continues to show an asymmetric distribution (Fig. 3A,B). Crescents of Numb protein could be seen in cells of the dorsomedial lip of the dermomyotome, specifically on the basal side (i.e., on the outer surface of the dorsomedial lip). In contrast, within the myotome, Numb protein was distributed throughout the cytoplasm, while very little Numb was seen in the more lateral dermomyotome (Fig. 3D,E). The region of high uniform cytoplasmic Numb protein expression corresponds to the MyoD and myosin heavy chain (MHC) -expressing regions of the maturing myotome (Fig. 3C,D). Within the myotome, Numb protein colocalizes with MHC except for a narrow band along the ventral edge of the myotome where Numb, but not MHC, is found (Fig. 3D).
Because Numb protein accumulates in a crescent pattern in cells located at the dorsomedial lip of the dermomyotome, where differentiated muscle cells first arise from the dermomyotome, we compared the expression of the dermomyotomal marker Pax-7 (Goulding et al., 1991) with that of Numb. Antibody staining for Pax-7 revealed nuclear localization in cells of the dermomyotome and in the dorsomedial lip of the dermomyotome (Fig. 3E). Only cells in the dorsomedial lip displayed crescents of Numb as well as nuclear Pax-7 (Fig. 3E, cells marked with arrowheads). We observed a sharp transition in Pax-7 expression at the boundary between the dorsomedial lip of the dermomyotome (Pax-7+) and the myotome (Pax-7−; Fig. 3E). Of interest, Numb protein distribution changed at this boundary from cortical crescents in the Pax-7–positive dorsomedial lip dermomyotomal cells to more uniform cytoplasmic expression in the Pax-7–negative myotomal cells (Fig. 3E). Numb expression in cells located in the dorsomedial lip of the dermomotome seemed to be greatest on the basal surface of these cells (see for example Fig. 3A,B). To ascertain if Numb expressing cells lie adjacent to a basal lamina, we evaluated the expression of Numb and Laminin in the anterior somites of stage 22 chick embryos. We observed that Numb expressing cells were indeed situated adjacent to a basal lamina in both the dorsal–medial lip of the dermomyotome and in the myotome proper (Fig. 3F).
In summary, whereas Numb mRNA expression is widespread in the somitic mesoderm, with a slight increase in the early myotome, Numb protein distribution is much more dynamic. Cortical crescents of Numb are present on the basal side of cells in the dorsomedial lip of the dermomyotome, whereas high amounts of Numb protein accumulate throughout the cytoplasm of differentiating myotomal cells.
Numb Protein Is Localized to the Basal Side of Dividing Cells in the Dorsomedial Lip of the Dermomyotome
Because the dermomyotome constitutes a source of progenitors for embryonic skeletal muscle (Venters and Ordahl, 2002; Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2005), we wanted to correlate Numb protein expression with the mitotic activity of these cells. In cryosections of somites, the number of mitotic cells with condensed chromosomes was very rare compared with that found in the neural tube (approximately 1/10 as many). Nevertheless cells with mitotic chromosomes were located in both the cleft of the dorsomedial lip of the dermomyotome (Fig. 3G–I) as well as in the lateral dermomyotome (data not shown). Of 16 cells in the cleft of the dorsomedial lip that had condensed chromosomes, 11 displayed a localized crescent of Numb staining, which in all cases was localized to the basal side of the mitotic figures (three examples are shown in Fig. 3G–I). The remaining five mitotic cells did not have any detectable Numb. Similar findings were published recently by Venters and Ordahl who observed that Numb specifically accumulated on the basal side of cells in the dorsomedial lip of the dermomyotome undergoing mitosis in the basal–apical orientation (which are enriched in the dorsomedial lip of the dermomotome) and is absent from cells undergoing mitosis in a planar orientation (which are enriched in the dermomyotomal sheet; Venters and Ordahl, 2005).
Because of the rarity of mitotic figures, bromodeoxyuridine (BrdU) was used to label cells undergoing proliferation during a longer time span (i.e., 30 min before tissue fixation). Consistent with our analysis of condensed chromosomes, only cells of the dermomyotome and dorsomedial lip of the dermomyotome were BrdU labeled (Fig. 3J,K). Cells bearing crescents of Numb were BrdU labeled and, therefore, were about to undergo mitosis. Cells in the myotome, which displayed a homogeneous Numb distribution, were postmitotic as indicated by the lack of BrdU staining. Our observations suggest that, upon asymmetric cell division, basally located daughter cells in the dorsomedial lip of the dermomyotome specifically accumulate Numb protein. When cells from the dorsomedial lip join the myotome, they become postmitotic and express Numb protein more uniformly throughout their cytoplasm.
Exposure to Myogenic Stimuli Leads to Expression of the Notch Ligand Serrate-2 Without Significantly Altering Numb mRNA Levels
To determine whether muscle differentiation leads to an induction in Numb mRNA expression, we used RT-PCR analysis to assay Numb expression in somite cultures exposed to myogenic stimuli. In addition, we also assayed the expression of chick Serrate-2 (a homolog of mouse Jagged-2), as this Notch ligand has been reported to be expressed in the myotome (Hirsinger et al., 2001). Somites I–III were explanted from a stage 10 chick embryo and infected with an avian retrovirus encoding either green fluorescence protein (GFP) or Wnt-3a. Such explants were cultured in either the absence or presence of Sonic hedgehog (Shh) for 5 days. While others have observed that Wnt signals are able to induce the expression of the dermomyotomal marker Pax3 in murine presomitic mesoderm cultured for only 24 hr (Fan et al., 1997), when chicken somites are cultured for a more extended period of time (i.e., 5 days), both Wnt and Shh signals are apparently necessary to both maintain the expression of Pax3 and induce the expression of myogenic markers (Fig. 4, lanes 1–4). As Shh signals have been suggested to be required for the survival of the myotome in vivo (Teillet et al., 1998), the apparent requirement for Shh to maintain the expression of Pax3 in long-term somite cultures may similarly reflect a requirement for Shh to promote either the survival or proliferation of Pax3-expressing cells cultured in vitro. While the expression of Numb mRNA was not significantly affected by pro-myogenic signals, culture of somites with the combination of Wnt-3a and Shh induced both the skeletal muscle differentiation program and induced the robust expression of Serrate-2 (Fig. 4, lanes 1–4). Thus, somitic myogenesis correlates with the induction of the Notch ligand Serrate-2 but fails to significantly affect Numb mRNA levels.
To determine whether up-regulation of Serrate-2 is a corollary of somitic skeletal muscle differentiation, we examined if forced expression of MyoD in somites could similarly induce the expression of Serrate-2. Indeed, infection of somites with a retrovirus encoding MyoD led to both the induction of myosin heavy chain and Serrate-2 expression (Fig. 4, lane 6), indicating that MyoD can either directly or indirectly induce the expression of Serrate-2 in myotomal cells. In contrast, MyoD expression failed to significantly affect the expression level of Numb mRNA. In addition, we could detect low levels of the Notch ligands Delta-1 and Serrate-1 in these somite cultures, but their expression was not significantly affected by either muscle inducing signals or forced MyoD expression (data not shown). These results confirm previous in situ hybridization data showing that Serrate-2 is expressed in the differentiating myotome (Hirsinger et al., 2001).
Numb Protein Levels Are Dramatically Enhanced in Somite Cultures Exposed to Myogenic Stimuli
As opposed to the striking up-regulation in Serrate-2 mRNA expression by either myogenic-inducing signals or forced MyoD expression, levels of Numb mRNA were not significantly modulated by either of these myogenic stimuli (Fig. 4, lanes 1–6). Therefore, we set out to determine whether signals that induce somitic myogenesis specifically regulate the cytoplasmic distribution of Numb protein. Antibody staining of somite cultures revealed that various myogenic stimuli do indeed enhance the levels of Numb protein accumulation (Fig. 5). Addition of either Shh (n = 6; Fig. 5A) or Wnt3a virus alone (n = 6; not shown) did not lead to any notable increase in Numb protein. In contrast, the combination of both Wnt-3a and Shh, which induces somitic myogenesis (Munsterberg et al., 1995; Fig. 4, lane 4), led to a marked increase in the amount of Numb staining in 19 of 20 cultures (Fig. 5B,C). These Numb-positive, multi-nucleate cells resembled myotubes and could be co-stained with an anti-MHC antibody (data not shown). Another way to induce somitic myogenesis is by co-culture of somites I-III with the overlying ectoderm and addition of Noggin-conditioned medium (Reshef et al., 1998). While somites cultured with either Noggin-conditioned medium alone (n = 6; not shown) or with only the surface ectoderm (n = 6; Fig. 5D) did not contain a substantial amount of either Numb or MHC, somites cultured with both ectoderm and Noggin-conditioned medium displayed a robust increase in expression of both Numb and MHC in the resultant myotubes (17 of 20 cultures; Fig. 5E,F). Of interest, although Numb and MHC were present in the same cells, exact overlap of the two proteins occurred in only a few domains (Fig. 5E), indicating that the two proteins occupy different cellular compartments.
Overexpression of the bHLH transcription factor MyoD can lead to skeletal muscle differentiation in several cell types (Davis et al., 1987; Weintraub et al., 1989). To see if the enrichment in uniform cytoplasmic Numb protein was downstream of MyoD, we infected chick somites with a MyoD-expressing retrovirus. While a control virus did not lead to any myogenesis or detectable Numb expression (n = 12; Fig. 5G), the MyoD-expressing virus induced the formation of large myotubes that expressed high levels of both Numb and MHC proteins in all treated cultures (n = 12; Fig. 5H,I). Hence, the high uniform cytoplasmic distribution of Numb protein induced by myogenic stimuli such as Wnt plus Shh, or ectoderm plus Noggin, is likely to occur downstream of MyoD expression.
In summary, Numb protein is expressed uniformly throughout the cytoplasm in somitic cells undergoing myogenesis. Myogenic stimuli that induce MyoD expression (such as Wnt plus Shh or ectoderm plus Noggin) also increase Numb protein levels, but fail to significantly alter the expression of Numb RNA. The enhancement of Numb protein levels by muscle-inducing cues likely occurs downstream of MyoD expression because forced expression of MyoD in somite cultures also leads to the accumulation of Numb protein throughout the cytoplasm.
Differential Numb Protein Expression and Localization in Dermomyotomal Versus Myotomal Cells
In this study, we describe the expression of Numb, an inhibitor of Notch signaling, during muscle formation in the chick embryo. We have found that Numb protein is asymmetrically localized to the basal surface of cells within the dorsomedial lip of the dermomyotome. The dorsomedial lip of the dermomyotome has been shown to contain progenitor cells that either remain within this structure or give rise to the myotome (Denetclaw et al., 2001; Ordahl et al., 2001; Kahane et al., 2002; Venters and Ordahl, 2002; Gros et al., 2004). Fate mapping (Cinnamon et al., 2001; Denetclaw et al., 2001; Gros et al., 2004) and gene expression analyses (Hirsinger et al., 2001; Kassar-Duchossoy et al., 2005; Relaix et al., 2005) have suggested that proliferating Pax-3/Pax-7-positive dermomyotomal cells give rise to Myf-5/MyoD–positive cells in the myotome. Pax-3–positive dermotomal progeny are similarly thought to give rise to Myf-5/MyoD–expressing myoblasts in the limb (Amthor et al., 1998; Delfini et al., 2000) and indeed dermomyotomal cells recently have been fate-mapped to give rise to satellite cells (Gros et al., 2005). While cells that remain within the dorsomedial lip of the dermomyotome maintain the expression of Pax-3/Pax-7 and continue to proliferate, those that migrate into the myotome lose expression of these Pax family members, activate expression of Myf-5 and MyoD, withdraw from the cell cycle, and express skeletal muscle structural proteins such as myosin heavy chain. Prior work has suggested that the BMP antagonist noggin may control the transition of a Pax-3–positive dermomyotomal cell into a MyoD-positive myotomal cell (Hirsinger et al., 1997; McMahon et al., 1998; Reshef et al., 1998; Amthor et al., 1999). Recent work has suggested that Notch signals may control this transition in satellite cells, which constitute a stem cell population for adult skeletal muscle (Conboy and Rando, 2002) and that Notch signaling is required for BMP-induced inhibition of myogenic differentiation (Dahlqvist et al., 2003). Indeed, forced expression of Delta-1 in the somite can also block the developmental maturation of embryonic muscle precursors (Hirsinger et al., 2001), raising the possibility that the dermomyotomal and myotomal compartments of the somite may be subject to differing levels of endogenous Notch signaling.
The cells of the dorsomedial medial lip of the dermomyotome are fated to give rise to progeny cells in both the dermomyotome and myotome (Ordahl et al., 2001; Gros et al., 2004). We speculate that such cells may undergo asymmetric cell division to give rise to either another proliferating dermomyotomal progenitor cell or a nascent myotomal cell, which will ultimately withdraw from the cell cycle. Interestingly, we have found that dividing cells that lie in the cleft of the dorsomedial lip of the dermomyotome accumulate a crescent of Numb protein on their basal side. Thus, it seems most likely that the Numb expressing cells that arise in the cleft give rise to nascent myotomal cells. More mature myotomal cells that lie ventral to these progenitors contain a higher level of Numb protein, which is uniformly localized throughout their cytoplasm (see Fig. 6A). We propose that asymmetric cell division, as revealed by Numb protein distribution, is an intrinsic property of muscle progenitor cells. Consistent with our results, Numb was also found to be asymmetrically localized in Pax-7–expressing muscle progenitor cells in adult regenerating skeletal muscle (Conboy and Rando, 2002).
These striking differences in Numb protein expression and localization in proliferating dermomyotomal versus postmitotic myotomal cells suggests that modulation of Notch signaling by Numb may act to modulate whether dermomyotomal precursors continue to proliferate or differentiate into postmitotic myotomal cells, as has been suggested to be the case in satellite cells (Conboy and Rando, 2002). The asymmetric inheritance of a cortical crescent of Numb located on the basal side of progenitor cells in the dorsomedial lip of the dermomyotome may designate which progeny cells differentiate into myotome. In contrast, the high cytoplasmic level of Numb in differentiated skeletal muscle cells which is first observed in the myotome of stage 22 embryos may play a role in the maintenance of the differentiated phenotype but likely does not play a role in its induction. It has been shown recently that mitotically active Pax3/Pax7-expressing cells that do not express myogenic bHLH factors migrate into the myotome upon de-epithelialization of the dermomyotome (Kassar-Duchossoy et al., 2005; Relaix et al., 2005). Although we have not assayed the expression of Numb in such cells, it seems plausible that these myotomal cells remain mitotically active (and fail to differentiate), because they fail to express Numb.
MyoD May Induce the Accumulation of Numb Protein by a Posttranscriptional Mechanism
We have found that signals that promote somitic myogenesis, such as Wnt plus Shh, ectoderm plus Noggin, or forced expression of MyoD all induce uniform cytoplasmic accumulation of Numb protein in the resultant myotubes. Interestingly, each of these myogenic inducing regimens fails to significantly affect the levels of Numb RNA, suggesting that they act by a posttranscriptional mechanism to induce Numb protein levels. Several molecules have been reported to regulate Numb protein translation as well as stability. Musashi-1 binds Numb mRNA and prevents translation (Imai et al., 2001), while two other proteins, Siah-1 and LNX have been reported to target Numb for ubiquitin-dependent degradation (Susini et al., 2001; Nie et al., 2002). Thus, it is possible that MyoD may induce accumulation of Numb protein (in the absence of significantly affecting Numb RNA levels) by modulating the expression or activity of either Musashi-1, Siah-1, or LNX in the myotome. Alternatively, because the Numb antisera we used also recognizes Numblike, it is possible that MyoD may induce the expression of Numblike in myotubes, which would account for the increased Numb/Numblike immunostaining within these cells.
We observed an asymmetric distribution of Numb protein on the basal surface of cells located within the dorsomedial lip of the dermomyotome, while cells within the maturing myotome accumulate Numb both on their basal surface and uniformly throughout their cytoplasm. It was reported recently that transplantation of lateral dermomyotomal cells into the region of the dorsomedial lip of the dermomyotome, resulted in the induction of Numb protein on the basal surface of these cells (Venters and Ordahl, 2005). Taken together, our results and those of Ordahl's group suggest that signals present in the dorsomedial lip of the dermomyotome are sufficient to promote the asymmetric accumulation of Numb in dermomyotomal cells (Venters and Ordahl, 2005) and that subsequent expression of myogenic bHLH factors induces a uniform cytoplasmic accumulation of Numb protein in progeny cells that enter the myotome proper.
Numb Protein May Act to Block Notch Signals in Both the Myotome and Cells of the Dorsomedial Lip of the Dermomyotome
Numb is a cytoplasmic protein that binds to the intracellular domain of the Notch receptor and antagonizes Notch signaling (Frise et al., 1996; Guo et al., 1996; Wakamatsu et al., 1999). Numb also binds α-adaptin, a protein involved in receptor-mediated endocytosis (Santolini et al., 2000). Of interest, when mutant forms of α-adaptin are present in Drosophila embryos, they cause phenotypes similar to those seen in numb mutant embryos (Berdnik et al., 2002). During myogenesis in Drosophila, asymmetric segregation of Numb protein influences the fate of muscle progenitor cells in the larval mesoderm (Carmena et al., 1998). Loss of Numb results in duplicated or deleted muscle founder cells, reminiscent of Notch gain-of-function phenotypes. Overexpression of Numb in satellite cells derived from regenerating adult mouse myofibers causes cell cycle exit and skews cells toward muscle differentiation (Conboy and Rando, 2002). This is a converse phenotype to that observed after overexpression of an activated Notch receptor in either myogenic cell lines (Kopan et al., 1994) or in satellite cells (Conboy and Rando, 2002). Thus, Numb apparently blocks the receipt of Notch signals in a variety of cellular contexts, suggesting that Notch signals are blocked or at least attenuated in Numb expressing cells in the dorsomedial lip of the dermomyotome and the myotome. It will be interesting to determine whether the asymmetric distribution of Numb in dividing cells within the dorsomedial lip of the dermomyotome marks progeny cells that are specifically destined to give rise to the myotome (see Fig. 6B).
MyoD Coordinates the Expression of a Notch Ligand and a Notch Antagonist Within the Myotome
We have found that signals that promote myogenesis, such as Wnt plus Shh, or forced expression of MyoD also induce expression of the Notch ligand, Serrate-2. In Xenopus embryos, Rupp and colleagues have similarly shown that MyoD induces the expression of Delta-1 (Wittenberger et al., 1999). Secreted forms of Jagged-1 and -2, the mouse homologues of Serrate-1 and -2, have been shown to inhibit myogenesis in a muscle cell line (Shawber et al., 1996; Luo et al., 1997). Whereas it initially seems paradoxical that myotomal cells express a molecule capable of inhibiting skeletal muscle differentiation, we propose that accumulation of Numb protein in these same cells blocks the effects of Notch signaling specifically in myotomal cells. Thus, it seems most likely that Serrate-2 is secreted by the myotome and activates the Notch signaling pathway in either adjacent dermomyotomal or sclerotomal cells, both of which lack Numb protein. What then is the utility of Notch ligands secreted by differentiated skeletal muscle cells? Analogous to other developmental systems, it seems plausible that Notch signals provided by the differentiated myotome may act to maintain the proliferation of dermomyotomal precursor cells. In this way, a balance between the number of skeletal muscle progenitors in the dermomyotome and differentiating myotomal cells can be maintained. Consistent with this hypothesis, regenerating skeletal muscle is known to up-regulate the expression of Delta-1 and, thereby, promote the proliferation of Pax-3–positive satellite cell precursors (Conboy and Rando, 2002). Studies are in progress to determine whether Notch signals are similarly necessary to promote the proliferation of dermomyotomal cells.
In Situ Hybridization
Whole-mount in situ hybridization for Numb (Wakamatsu et al., 1999) and MyoD (Lin et al., 1989) was carried out as previously described (Stern, 1998). Some stained embryos were embedded in OCT and sectioned (15- to 25-μm thick).
Cloning of RCAS-MyoD Virus
The single myc-tagged mouse MyoD insert was isolated from pCS2-6mt-MyoD using Nco1 and Xho1 (blunted) and subcloned into the Nco1/Sma1 site of Slax13 (Riddle et al., 1993). The Slax-13 insert was transferred as a Cla1 fragment into RCASBP-A (Hughes et al., 1987).
Embryo and Explant Culture
Embryos were staged according to (Hamburger and Hamilton, 1951). Somitic tissue was dissected and cultured for 4 to 5 days as described (Munsterberg et al., 1995). RCAS viruses were harvested from chick embryo fibroblasts, concentrated and titered according to (Morgan and Fekete, 1996). Somites were incubated on ice for 1 hr with virus before plating (108 to 109 viral particles per ml).
Immunohistochemistry and Confocal Microscopy
Rabbit anti-Numb antibody (Wakamatsu et al., 1999) was used at a dilution of 1:400. Hybridoma supernatants (1:10) containing monoclonal anti-myosin heavy chain (MF20), 12-101, and anti-Pax-7 antibodies, and purified mouse anti-BrdU antibody (1:200) were purchased from the Developmental Studies Hybridoma Bank, University of Iowa. Rabbit anti-laminin (1:50 dilution, #ab11575) and goat anti-numb (1:1000 dilution, #ab4147) antibodies were obtained from Abcam. Sytox Green (Molecular Probes) was sometimes used as a nuclear counterstain (50 nM) and added to the secondary antibody solution. Secondary antibodies (1:250) were as follows: tetrarhodamine isothiocyanate–donkey anti-rabbit antibody, fluorescein isothiocyanate–donkey anti-mouse and Cy3-goat anti-mouse from Jackson Immunoresearch; Alexa 568–goat anti-rabbit and Alexa 488–goat anti-mouse antibodies from Molecular Probes.
Embryos were fixed for 2 hr at room temperature in 4% paraformaldehyde in PBS, washed in PBT (PBS with 0.1% Tween-20), incubated in blocking buffer (PBT, 5% goat serum, 1% horse serum, 0.1% sodium azide) for 2 hr at room temp, then incubated in diluted primary antibody in blocking buffer at 4°C overnight with gentle rotation. The following day, they were rinsed in PBT and subjected to six 1-hr washes with PBT. Secondary antibody incubations and subsequent washes were carried out similarly. Embryos were sliced transversely at a thickness of 1 mm using a #10 scalpel blade and placed on a slide in wells created with perforated strips of Sylgard. Mounting medium (Biomeda Gelmount) was added and a coverslip was placed on top.
After fixation and PBT washes, some embryos were frozen in OCT and sectioned at a thickness of 20–25 μm. BrdU staining was carried out as previously described (Hirsinger et al., 2001). Sections were washed with PBT, incubated in blocking buffer for 30 min, and incubated in diluted primary antibody overnight at 4°C. They were then washed with PBT, incubated with diluted secondary antibody for 2 hr at room temp, washed with PBT and mounted. For staining of explant cultures, explants embedded in collagen gels were fixed with 4% paraformaldehyde for 15 min at room temp, washed 6 times for 10 min with PBT, and blocked with blocking buffer. Primary and secondary antibody incubations were carried out at 4°C overnight, with six 10-min PBT washes after each incubation. Confocal microscopy was performed using a Nikon TE300 microscope with Perkin–Elmer spinning disk confocal attachments and Metamorph Image Acquisition software.
RT-PCR Analysis of Molecular Markers
RNA was extracted from explants using the Qiagen RNeasy Mini Kit and Qiagen DNAse reagents according to the manufacturer's instructions except that for each sample, 100 ng of carrier RNA (Qiagen) was added to the lysis buffer. RT reactions and polymerase chain reaction (PCR) analysis were carried out as previously described (Munsterberg et al., 1995; Maroto et al., 1997). PCR primers that were not previously published are as follows: Numb (Wakamatsu et al., 1999) 5′-ccg ggc gtt ctc ata cat ctg-3′, 5′-tgg ggg ttg ctc att tcc ttc-3′ (408 bp); Notch-1 (Wakamatsu et al., 1999) 5′-cgc ctc ccc tta cta cca ctg-3′, 5′-ccg tcc cac tca cac tca aa-3′ (453 bp product); Serrate-1 (Myat et al., 1996) 5′-ccc cga taa ata cca gtg ttc-3′, 5′-ggt ttg ccc tca cat tca ttc-3′ (312 bp); Serrate-2 (Hayashi et al., 1996) 5′-gcg acg aaa atg gaa aca aag-3′, 5′-cag ggg tta gat aca caa gca-3′ (400 bp); Delta-1 (Henrique et al., 1997) 5′-gcc gac ccc gcc ttc agc aac-3′, 5′-aca gag cag cct tcc ccg tag-3′ (272 bp). We used 23 cycles to assay glyceraldehyde -3-phosphate dehydrogenase (GAPDH) and 28–30 cycles for other markers. Amplifications were tested to ensure that they were roughly linear and that no PCR product was obtained without the addition of RT.
We thank Y. Wakamatsu for the generous gift of antibodies and cDNA, S. Evans and M. Spechler for technical assistance, and W. Zhong for pointing out that the Numb antisera we used may also recognize Numblike. Confocal microscopy was carried out at the Nikon Imaging Center at Harvard Medical School and was much facilitated through the help of its director, Dr. J. Waters Shuler. Most of all, we are indebted to the following members of the Lassar laboratory for helpful discussions: G. Di Rocco, J.B. Lazaro, H. Kempf, D.-W. Kim, R. Sohn, and E. Tzahor. T.H. was supported by a joint fellowship from the Foundation for Gene and Cell Therapy and the Canadian Institute for Health Research. L.Z. was supported by an NIH postdoctoral fellowship. This work was funded by a grant from the NIH to support work on somitogenesis awarded to A.B.L.