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 which 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. 2 A,B). At the trunk level, where the myotome is less mature, Numb is present in only a few cells (Fig. 2 E,F). In immature epithelial somites in the caudal regions of the embryo, we did not detect Numb protein (Fig. 2 H,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).

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Figure 2

Numb protein is asymmetrically localized in the myotome

Confocal microscope images displaying Numb (red) and DNA (green) localization at differing axial levels of a stage 17 chicken embryo. (A and B) Numb protein is specifically localized on the ventral side of myotomal cells (arrowheads) of an anterior somite. (C) Section in situ hybridization staining for MyoD RNA (blue) at the same axial level displayed in (A and B) shows MyoD expression throughout the myotome (arrowheads). (E and F) At the trunk level, Numb protein is distributed in crescents in a few myotomal cells (arrowheads). (G) Section in situ hybridization staining for MyoD RNA (blue) at the same axial level displayed in (E and F). (H and I) In immature epithelial somites, Numb protein is not observed. Medial region of the somite adjacent to neural tube is on right side of each image. DM, dermomyotome; Myo, myotome; NT, neural tube; s, epithelial somite.

In anterior somites at stage 22, Numb protein continues to show an asymmetric distribution (Fig. 3 A,B). Crescents of Numb protein could be seen in cells of the dorso-medial lip of the dermomyotome, specifically on the basal side (i.e., on the outer surface of the dorso-medial 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. 3 D,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. 3 C,D). Within the myotome, Numb protein co-localizes with MHC except for a narrow band along the ventral edge of the myotome where Numb, but not MHC, is found (Fig. 3D).

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Figure 3

Numb protein is asymmetrically localized to the basal surface of cells in the dorso-medial lip of the dermomyotome and is upregulated throughout the cytoplasm in mature cells of the myotome

(A and B) Confocal microscope images displaying Numb (red) and DNA (green) localization in anterior somites from a stage 22 chicken embryo. Crescents of Numb protein (red) are located on the basal surface of the dorso-medial lip of the dermomyotome (DL, arrowheads). High levels of Numb protein are present throughout the cytoplasm in the myotome. DM, dermomyotome; Myo, myotome. (B) Same image as in A displaying only Numb protein. (C) Section in situ hybridization staining for MyoD RNA (blue) at the same axial level displayed in (A and B) shows MyoD expression throughout the myotome. Dorso-medial lip is delineated by open arrowheads. (D) Confocal microscope images displaying Numb (red) and myosin heavy chain (MHC; green) localization in anterior somites from a stage 22 chicken embryo. Ventral edge of the myotome that lacks MHC immunoreactivity retains Numb staining (solid arrowheads). DL delineated by open arrowheads. (E) Confocal microscope images displaying Numb (red) and Pax7 (green) localization in anterior somites from a stage 22 chicken embryo. In the DL of the dermomyotome, crescents of Numb protein (red, open arrowheads) are found in cells that express Pax7 (green). Cells of the myotome (Myo) that do not express Pax7 show uniform cytoplasmic Numb protein accumulation. (F) Numb accumulates in cells that lie adjacent to a basal lamina. Numb (red) and Laminin (green) expression in an anterior somite from a stage 25 chick embryo. Dapi staining (blue) identifies nuclei of cells. (G, H, I) Confocal image of the dorso-medial lip of the dermomyotome in anterior somites of a stage 22 chick embryo showing numb (red) accumulation on the basal side of mitotic cells. Dashed line outlines border of somite. Asterisks (*) align with the myotome. Nuclei stained with Sytox green. Open arrowheads indicate Numb crescents (red) on the basal surface of mitotic cells in which chromosomes are condensed. (J, K) Confocal image of the BrdU incorporation (green) and Numb (red) accumulation in the dorso-medial lip of the dermomyotome in anterior somites of a stage 22 chick embryo. Proliferating cells in the dermomyotome and in the dorso-medial lip of the dermomyotome incorporate BrdU (green) and display cortical crescents of Numb protein (red). High levels of Numb accumulation in post-mitotic myotomal cells (BrdU-negative cells, indicated with *) is observed throughout their cytoplasm. Closed arrowhead indicates last BrdU+ cell in dorso-medial lip of the DM that borders the myotome and has asymmetric Numb protein. Note that Numb staining in J and K is poor due to treatment required to reveal BrdU antigenicity. Medial region of the somite adjacent to neural tube is on right side of each image

Since Numb protein accumulates in a crescent pattern in cells located at the dorso-medial 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 dorso-medial lip of the dermomyotome (Fig. 3E). Only cells in the dorso-medial 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 dorso-medial lip of the dermomyotome (Pax-7⁺) and the myotome (Pax-7⁻) (Fig. 3E). Interestingly, Numb protein distribution changed at this boundary from cortical crescents in the Pax-7-positive dorso-medial lip dermomyotomal cells to more uniform cytoplasmic expression in the Pax-7-negative myotomal cells (Fig. 3E). Numb expression in cells located in the dorso-medial lip of the dermomotome seemed to be greatest on the basal surface of these cells (see for example Figs. 3A and 3B). 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 dorso-medial lip of the dermomyotome, while 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 dorso-medial lip of the dermomyotome

Since 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 to that found in the neural tube (about 1/10 as many). Nevertheless cells with mitotic chromosomes were located in both the cleft of the dorso-medial 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 dorso-medial 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 (3 examples are shown in Fig. 3G-I). The remaining 5 mitotic cells did not have any detectable Numb. Similar findings were recently published by Venters and Ordahl who observed that Numb specifically accumulated on the basal side of cells in the dorso-medial lip of the dermomyotome undergoing mitosis in the basal-apical orientation (which are enriched in the dorso-medial 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, BrdU was used to label cells undergoing proliferation during a longer time span (i.e., 30 minutes prior to tissue fixation). Consistent with our analysis of condensed chromosomes, only cells of the dermomyotome and dorso-medial 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 post-mitotic as indicated by the lack of BrdU staining. Our observations suggest that, upon asymmetric cell division, basally located daughter cells in the dorso-medial lip of the dermomyotome specifically accumulate Numb protein. When cells from the dorso-medial lip join the myotome they become post-mitotic 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 if muscle differentiation leads to an induction in Numb mRNA expression, we employed 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 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 hours (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.

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Figure 4

Myogenic signals induce expression of Serrate-2 RNA but not that of Numb RNA

Somites I-III were explanted from a stage 10 chick embryo and infected with an avian retrovirus encoding either Green Fluorescence Protein (GFP), Wnt-3a, or MyoD as indicated and cultured in either the absence or presence of Shh (as indicated). After 5 days culture, the explants were harvested and gene expression was assayed by RT-PCR analysis.

To determine if 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).

MyoD may induce the accumulation of Numb protein by a post-transcriptional 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 post-transcriptional 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 and LNX in the myotome. Alternatively, because the Numb anti-sera we employed 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 dorso-medial lip of the dermomyotome, while cells within the maturing myotome accumulate Numb both on their basal surface and uniformly throughout their cytoplasm. It was recently reported that transplantation of lateral dermomyotomal cells into the region of the dorso-medial 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 dorso-medial 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 dorso-medial 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). Interestingly, 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. Over-expression 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 following over-expression 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 dorso-medial lip of the dermomyotome and the myotome. It will be interesting to determine whether the asymmetric distribution of Numb in dividing cells within the dorso-medial lip of the dermomyotome marks progeny cells that are specifically destined to give rise to the myotome (see Fig. 6B).

Cloning of RCAS-MyoD virus

The single myc-tagged mouse MyoD insert was isolated from pCS2-6mt-MyoD using Nco1 and Xho1 (blunted) and sub-cloned 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 one hour with virus prior to 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. 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: TRITC-donkey anti-rabbit antibody, FITC-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 hours 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 hours 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 6 one-hour 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 minutes, and incubated in diluted primary antibody overnight at 4°C. They were then washed with PBT, incubated with diluted secondary antibody for 2 hours 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 6 ten-minute 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.

We are very grateful to Y. Wakamatsu for the generous gift of antibodies and cDNA, to S. Evans and M. Spechler for technical assistance, and to W. Zhong for pointing out that the Numb antisera we employed 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 (GM054879) to support work on somitogenesis awarded to A.B.L.

Grant sponsor: National Institutes of Health; Grant number: GM054879.