Notch signaling has been implicated in the promotion of SM at the expense of striated muscle development from bipotent progenitors resident in the lateral DM [14]. To further assess whether endogenous Notch is necessary for this choice, its activity was inhibited in the lateral DM by focal electroporation (EP) of a dominant-negative (dn) form of MAML1, a co-factor within the Notch activation complex, or Dll1, which represses Notch signaling cell-autonomously [see Additional file 1: Figure S1A-C] [29]-[31]. Forty hours post-EP, control GFP-labeled cells were observed in the desmin-positive myotome and in the ventral sclerotome between the lateral DM and the cardinal vein likely en route to this blood vessel [see Additional file 1: Figure S1D and [14]]. In addition, a fraction of labeled cells had integrated into the blood vessel wall as SM cells as determined by expression of both desmin and smooth muscle actin (SMA) [see Additional file 1: Figure S1D-D”]. In striking contrast, inhibiting Notch activity promoted an increase in the proportion of myotomal colonization. Consistently, a marked decrease of the proportion of migratory cells was apparent in the sclerotome and blood vessels [see Additional file 1: Figure S1E-G]. These findings are in agreement with previously reported effects of Numb and further confirm a function of Notch in the segregation of muscle sublineages [14],[32].
Since SM development requires that progenitors emigrate from their epithelium of origin, migrate through the sclerotome and reach the target blood vessels, we asked whether different phases of this process depend upon the duration of Notch signaling. To this end, we first expressed a constitutively active form of Notch2 (aN2) for increasing periods of time in the lateral DM. As soon as four hours after EP, Notch signaling compromised the epithelial morphology of treated cells, evident by the loss of their typical elongated, pseudostratified appearance, and induced cell delamination when compared to control embryos (Figure 1A,D; 79.6 ± 2.5% in control DM compared to 56.7 ± 7.9 % in Notch-treated embryos; 6 ± 2.9% in sclerotome of control embryos compared to 28.2 ± 6.2% in Notch-treated embryos, N = 4 for each treatment, P <0.05). This was further enhanced by eight hours post-EP (Figure 1B,E; 75.6 ± 3.3% in control DM compared to 40.9 ± 5.1% in Notch-treated embryos; 13.5 ± 5.2% in sclerotome of control embryos compared to 44.8 ± 6.6% in Notch-treated embryos, N = 4 and 7, respectively, P ≤0.01). By 20 hours virtually all labeled cells had delaminated from the DM when compared to controls in which cells were still epithelial (Figure 1C,F; 63.7 ± 3.2% in control DM compared to 20.3 ± 5.7% in Notch-treated embryos; 16.4 ± 1.5 % in sclerotome of control embryos compared to 70.5 ± 5.7% in Notch-treated embryos, N = 6 and 5, respectively, P <0.01).
We next asked whether varying lengths of exposure affect final cell identities. To this end, an inducible version of aNotch2 (aN2) was prepared by subcloning into a tetracycline-sensitive plasmid followed by transfection into the lateral DM. Its expression was restricted by doxycycline treatment to the first 8 hours, 16 hours or 40 hours of a 40-hour total incubation period. Control-GFP-treated embryos exhibited a typical distribution of labeled cells (Figure 1G). Although eight hours exposure of aN2 induced excess delamination from the DM (Figure 1E), it did not prevent myotome colonization (Figure 1H). However, these cells were not dispersed throughout the myotome as in controls, but rather aggregated at a ventro-medial bulge abutting the sclerotomal border (Figure 1H). When exposed to aN2 for 16 hours, most labeled cells were in the sclerotome where they ectopically upregulated SMA/desmin, a feature never observed under control conditions, and some had already incorporated into the wall of the cardinal vein (Figure 1I). To discriminate whether this effect is due to the duration of Notch activity or, alternatively, to a positional effect induced within the sclerotome on cells initially stimulated to emigrate by Notch, Notch was conditionally activated twenty hours post-EP for eight hours only followed by fixation. Under control conditions, the few cells that exited the lateral DM by 28 hours did not express SMA/desmin [see Additional file 2: Figure S2A,A’ and [14]]. Late exposure to aN2 stimulated cell delamination from the DM yet was not sufficient to induce the muscle markers in cells located within the ventral sclerotome [see Additional file 2: Figure 2B,B’]. Thus, the ectopic muscle marker expression observed (Figure 1I) is likely a consequence of extended Notch activity rather than of an environmental effect stemming from the sclerotome through which the cells migrate.
Continuous expression for 40 hours completely prevented myotome colonization and most cells were already found in the cardinal vein co-expressing SM markers (Figure 1J).
Since a short eight hour exposure to aN2 induced excess delamination from the DM but did not prevent myotome colonization (Figure 1H), we sought to further examine the identity of these cells. Whole mount analysis confirmed that these cells translocated into the myotome but additionally showed that they failed to differentiate into unit-length myofibers when compared to their control GFP counterparts (Figure 2A-B). Furthermore, in contrast to control cells in which the progenitor marker Pax7 is downregulated upon exit from the DM (Figure 2C,C’,G), eight hours of Notch activity was sufficient to maintain low Pax7 expression for at least sixteen hours post-EP (Figure 2D,D’,G). Notch overexpression also maintained low Pax7 expression non-cell autonomously in untransfected cells that exited the DM. This might result from induction of Notch ligand(s) in cells adjacent to the transfected ones, which in turn stimulate Notch activity, that maintains Pax7, in cells neighboring them (Figure 2D,D’, arrowhead). In addition, short exposure to Notch stimulated proliferation of lateral DM cells, apparent by enhanced bromodeoxyuridine (BrdU) incorporation (Figure 2E,F,H). Taken together, short and limited exposure to Notch maintains the progenitor state and prevents differentiation. Therefore, Notch signaling harbors two sequential functions. First, with initial signaling it prevents striated myogenesis. Second, with extended signaling, it activates the SM program.
Id2, Id3, FoxC2 and Snail1 affect the choice between striated and SM issued from the lateral DM
Transient expression of Id2, Id3, FoxC2 and Snail1 in the lateral DM
To begin examining how Notch induces SM fate we surveyed for putative genes involved in segregation of muscle sublineages. First, in situ hybridization (ISH) was performed on the flank level of avian embryos to screen for candidate genes expressed in the lateral domain of the DM. Id2, Id3, FoxC2 and Snail1 mRNAs were already apparent at embryonic day (E)2.0 in the lateral aspect of the dorsal epithelial somite that is destined to become the DM [see Additional file 3: Figure S3A-D]. At E2.5 these factors were enriched in the lateral region of the DM, while being also transcribed to different extents in its medial aspect [see Additional file 3: Figure S3E-H]. At E3.5, although the lateral DM is still epithelial, it has completely repressed their expression [see Additional file 3: Figure S3, arrows in I-L]. Hence, these factors are transcribed early when the lateral DM mostly generates vascular SM fates and are downregulated when this region primarily generates myotomal muscle [14]. This prompted the hypothesis that these four factors might be involved in the segregation of vascular and myotomal muscle lineages issued from the lateral DM.
Transient missexpression of Id2, Id3, FoxC2 and Snail1 promotes SM at the expense of myotomal fates issued from the lateral DM
To begin investigating their function(s), we first adopted a gain of function approach. Tetracycline-inducible Id2, Foxc2 or Snail1 were missexpressed in the lateral somite at E2 for 20 hours, a time window corresponding to their endogenous transcription, and re-incubated for an additional day. In control-GFP-treated embryos, the majority of labeled progeny was located in the myotome. Cells were also observed in the ventral sclerotome and a small proportion already integrated into the blood vessel walls as desmin+/SMA + SM right outside the Qh1+ layer of endothelium (Figure 3A,F and Additional file 4: Figure S4A). In contrast, Id2, Foxc2 or Snail1 inhibited cell translocation into the myotome with an increased proportion of transfected cells in the sclerotome and at the SM layer of blood vessels as Desmin+/SMA + cells (Figure 3B,D,E,F and Additional file 4: Figure S4A).
When transiently missexpressed, Id3 had a weaker effect, yet constitutive expression for 44 hours yielded a similar outcome as that obtained with transient activation of Id2, Foxc2 or Snail1, whereby non-myotomal fates, particularly SM, were induced at the expense of the myotomal lineage (Figure 3C,F and Additional file 4: Figure S4A).
Since both Foxc2 and Snail1 also significantly reduced the proportion of progenitors that remained in the DM (Figure 3F), we examined an earlier time point to assess possible effects on cell delamination from the DM epithelium. Already by 16 hours, missexpression of Foxc2 induced EMT of labeled cells from the DM and promoted their migration through the sclerotome [see Additional file 5: Figure S5A,B,E]. An even stronger effect was observed with Snail1, which, in addition to loss of epitheliality, marked by ZO-1 staining, completely prevented myotome colonization [see Additional file 5: Figure S5C-E]. Thus, both Foxc2 and Snail1 trigger EMT of lateral DM progenitors as part of the generation of SM.
Attenuation of Id2/3, FoxC2 and Snail1 activities stimulates myotome formation at the expense of SM
To examine the physiological functions of the genes, specific inhibitory RNAs were implemented. Inhibitory double-stranded RNAs (dsRNAs) directed against Id2 and Id3 were focally electroporated into the lateral DM. Analysis performed 40 hours post-EP revealed that inhibition of Id2/3 preserved a higher proportion of cells in the lateral DM and reduced their exit towards the sclerotome, when compared to the behavior of DMs that received a control dsRNA. Consequently, a lower proportion of labeled cells reached the SM layer of blood vessels (Figure 4A,B,D and Additional file 4: S4B). In addition, dsRNAs to Id2/3 stimulated premature myocyte differentiation that was already apparent 16 hours after transfection [see Additional file 6: Figure S6A,B]. Both short and long-term effects were completely rescued by co-transfecting the dsRNAs with exogenous Id2/3 [see Additional file 6: Figure S6C-E].
Repressing FoxC2 activity with a dsRNA or with a dominant negative FOXC2-Engrailed fusion protein resulted in an increased proportion of GFP-labeled, desmin + myotomal cells out of total labeled cells, at the expense of SM (Figure 4A,C,D and Additional files 4 and 7: S4B, S7A-E). Moreover, precocious myotome colonization and presence of partial as well as full-length myocytes was already observed 16 hours after EP when virtually no myofibers were yet observed in controls [see Additional file 7: Figure S7A,B,F,G]. Introducing exogenous Foxc2 completely abrogated the effects of Foxc2 knock-down by the dsRNA on myotome colonization [see Additional file 7: Figure S7H-I compared to Figure 4A,C]. Interestingly, both over-expression and knock-down of FoxC2 strongly reduced the proportion of cells remaining in DM (Figures 3D,F; 4C,D and Additional file 7: S7), but while the former promoted the SM fate, the latter induced the myotomal fate, consistent with previous mouse data [22].
Similarly, repressing Snail1 activity with siRNAs designed against Snail1 inhibited cell migration through the sclerotome and subsequent SM development (Figure 4E-G and Additional file 7: Figure S4C). As expected, loss of Snail1 maintained cells within the lateral epithelium (Figure 4E-G, Additional file 8: S8) suggesting that it mainly affects cellular EMT. Moreover, co-electroporation of siRNA to Snail1 along with the full-length gene rescued the excessive myogenic differentiation caused by attenuation of Snail1 activity [see Additional file 8: Figure S8C,F,G], further substantiating the specificity of this loss of function approach.
Together, both gain and loss of function data implicate Id2/3, FoxC2 and Snail1 as central factors regulating segregation of striated muscle versus SM progenitors from the lateral DM.
Id2/3, FoxC2 and Snail1 integrate into a Muscle Regulatory Network
Id2 and Id3 repress the myogenic function of Myf5 and activate FoxC2 transcription
Myf5, a bHLH transcription factor, is expressed in the lateral DM of avian embryos prior to MyoD[11]. Id2/3 are bHLH inhibitors whose repression promoted myogenesis (Figure 3). Since Id2/3 are co-expressed with Myf5, we examined whether the observed inhibition of myogenesis by the Id proteins can be explained by repression of Myf5 activity [33]. Over-expressing Myf5 induced precocious myocyte differentiation and this effect was completely abolished when Myf5 was over-expressed with either Id2 or Id3 (Figure 5A-D).
Furthermore, Id2 or Id3 induced FoxC2 mRNA, as exemplified in the central region of the DM that lacks endogenous transcription (Figure 5E-G). This function of Id proteins seemed to be independent of Id-Myf5 interactions, since over-expressing Myf5 in the lateral DM did not inhibit endogenous FoxC2 expression (Figure 5H,I). Thus, Id2 and Id3 have a two-pronged action, inhibiting the myogenic lineage by repressing Myf5 activity and up-regulating FoxC2 expression via a yet undefined mechanism.
FoxC2 inhibits the myogenic program by repressing Pax7
In the mouse, the balance of Foxc2 and Pax3/7 was shown to control vascular versus myotomal development, respectively [22]. We examined whether this balance operates similarly in the avian embryo. First, over-expression of Pax7 in the lateral DM promoted extensive myotome colonization and myogenic differentiation while inhibiting cell migration and SM production that was apparent under control conditions (Figure 6A,B). Since FoxC2 and Id2/3 displayed an opposite phenotype to that of Pax7 (Figures 3, 4, Additional files 6 and 7: S6, S7), we predicted they would negatively regulate Pax7. Indeed, FoxC2 and Id2 attenuated Pax7 expression in the DM, at the transcript and/or at the protein levels (Figure 6C-G and Additional file 9: Table S1). Conversely, Pax7 had no effect on FoxC2 or Id transcription [see Additional file 9: Table S1]. However, Pax7 repressed Snail1 mRNA and vice-versa, suggesting a negative feedback loop between the latter factors [see Additional file 9: Table S1].
Altogether, FoxC2, Id2/3 and Snail1 comprise a MRN that negatively influences the activity of distinct myogenic genes to favor SM at the expense of striated muscle development from the lateral DM.
Notch signaling interacts with components of the MRN
Since both Notch and the factors comprising the MRN were found to be necessary and sufficient for generating vascular fates, we examined possible interactions. In order to assess the possibility that Notch signaling affects expression of these factors, we turned to the central sheet of the DM, the compartment closest to the lateral DM, in which these pro-SM factors are not endogenously expressed [see Additional file 3: Figure S3]. Indeed, aN2 was sufficient to ectopically upregulate Id2, Id3 and FoxC2 mRNAs in this compartment of the DM 10 hours following transfection compared to the contralateral sides and to GFP-controls (Figure 7A-C, Additional file 10: S9).
Next, we examined whether there is feedback modulation of the factors on Notch signaling. To this end, we implemented a Notch reporter construct, comprising the mHes1 promoter driving GFP expression [34]. An avian homologue of Hes1, hairy2, is indeed expressed in the lateral DM [14], signifying active Notch signaling in this compartment. Id2 and Id3 ectopically enhanced reporter activity indicating that it augments Notch signaling (Figure 7D-F and see [34]; 2.7 ± 0.6 and 3.54 ± 0.2-fold for Id2 and Id3 over control values, N = 6 and 5, P <0.01 and P <0.05, respectively). Thus, a positive feedback is formed, whereby Notch signaling activates Id expression and Id in turn strengthens Notch signaling. Interestingly, no marked increase of Notch signaling was monitored in response to FoxC2 up-regulation [see Additional file 9: Table S1], yet Pax7 was found to silence reporter activity (Figure 7D,G, 0.16 ± 0.9-fold compared to control, N = 7, P <0.01). Thus, although FoxC2 does not enhance Notch signaling, by inhibiting Pax7 (Figure 6C-G) it presumably permits a basal level of Notch activation.
In addition, over-expression of Snail1 rapidly promoted cell delamination from the lateral DM (Figure 3, Additional file 5: S5), whereas loss of Snail1 activity for 40 hours had the opposite effect (Figure 4 and Additional file 8: Figure S8). Since Notch similarly promoted EMT (Figure 1), we sought to examine whether Notch triggers cell delamination independently of Snail1 activity. A 16-hour exposure to aN2 promoted delamination and migration of the labeled cells when compared to the control GFP-treated epithelium (Figure 7H,I and Figure 1). In contrast, silencing Snail1 repressed cell delamination (Figure 7J, 4 F-G) and aN2 missexpression was unable to rescue this phenotype (Figure 7K). However, aN2 did not up-regulate Snail1 transcription in the DM [see Additional file 9: Table S1]. Thus, in lateral DM progenitors, the endogenous activity of Snail1 is essential for Notch-induced EMT.
Positive regulation of the MRN by BMP signaling
BMP4, secreted from the LPM, signals the nearby lateral DM to promote vascular development and inhibit terminal muscle differentiation [11],[14],[15]. To test whether BMP regulates Id2, Id3, FoxC2 and Snail1, the secreted BMP antagonist noggin was electroporated into the LPM. Inhibition of BMP signaling completely abolished expression of Id2, Id3, FoxC2 and Snail1 in the adjacent lateral DM compared to the contralateral intact sides (Figure 8A-D), demonstrating that BMP signaling is necessary for maintaining transcription of these factors.
We next asked whether BMP modulates Notch signaling. Electroporation of a BMP4-encoding plasmid substantially activated the GFP signal from the Hes1 promoter (Figure 8E,F). Reciprocally, aN2 enhanced BMP activity, as monitored by expression of a specific BMP reporter, BRE::GFP [35] (Figure 8G,H). Hence, a positive regulatory relationship exists between these two signaling pathways in the hypaxial region of the DM. Collectively, the data presented provide evidence for the existence of a regulatory network underlying lineage segregation from the lateral DM (Figure 8I).