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Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network.

Applebaum M, Ben-Yair R, Kalcheim C - BMC Biol. (2014)

Bottom Line: These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation.Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate.This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

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ABSTRACT

Background: Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors.

Results: Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other¿s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation.

Conclusions: Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

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Interactions of Notch with factors of the MRN.(A-C) aNotch2 expression in the central DM (brackets, left panels) promotes transcription of Id2 (A, N = 4), Id3 (B, N = 2) and FoxC2 (C, N = 3) compared to the contralateral sides. (D-G) Id2, Id3 and Pax7 affect Notch signaling. Dorsal views of whole-mount segments. (D) Basal activity of Hes1 reporter in the lateral DM (N = 21). Id2 (E) or Id3 (F) missexpression enhances Hes1 reporter activity (N = 13 and 8, respectively), whereas Pax7 inhibits signaling (G, N = 13). (D’-G’) Control RFP shows similar electroporation efficiency in all treatments. (H-K) Notch-induced EMT requires Snail1 activity. (H, H’) Cells co-electroporated with control-GFP and control scrambled siRNA are mostly found within the lateral DM with a few cells delaminating from the epithelial sheet 16 hours post-treatment (N = 7). (I, I’) aNotch2 induces labeled cells to undergo EMT and migrate towards blood vessels (N = 6). (J, J’) Snail1 knock-down maintains cells within the lateral DM, inhibiting delamination (N = 5). (K, K’) Snail1 knock-down hinders Notch-induced EMT, as transfected cells remain epithelial within the lateral DM (N = 4). Bar: (A-C) 100 μm, (H-K) 50 μm. CV, cardinal vein; DM, dermomyotome; EMT, epithelial-to-mesenchymal transition; EP, electroporation; ISH, in situ hybridization; RFP, red fluorescent protein.
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Figure 7: Interactions of Notch with factors of the MRN.(A-C) aNotch2 expression in the central DM (brackets, left panels) promotes transcription of Id2 (A, N = 4), Id3 (B, N = 2) and FoxC2 (C, N = 3) compared to the contralateral sides. (D-G) Id2, Id3 and Pax7 affect Notch signaling. Dorsal views of whole-mount segments. (D) Basal activity of Hes1 reporter in the lateral DM (N = 21). Id2 (E) or Id3 (F) missexpression enhances Hes1 reporter activity (N = 13 and 8, respectively), whereas Pax7 inhibits signaling (G, N = 13). (D’-G’) Control RFP shows similar electroporation efficiency in all treatments. (H-K) Notch-induced EMT requires Snail1 activity. (H, H’) Cells co-electroporated with control-GFP and control scrambled siRNA are mostly found within the lateral DM with a few cells delaminating from the epithelial sheet 16 hours post-treatment (N = 7). (I, I’) aNotch2 induces labeled cells to undergo EMT and migrate towards blood vessels (N = 6). (J, J’) Snail1 knock-down maintains cells within the lateral DM, inhibiting delamination (N = 5). (K, K’) Snail1 knock-down hinders Notch-induced EMT, as transfected cells remain epithelial within the lateral DM (N = 4). Bar: (A-C) 100 μm, (H-K) 50 μm. CV, cardinal vein; DM, dermomyotome; EMT, epithelial-to-mesenchymal transition; EP, electroporation; ISH, in situ hybridization; RFP, red fluorescent protein.

Mentions: 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).


Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network.

Applebaum M, Ben-Yair R, Kalcheim C - BMC Biol. (2014)

Interactions of Notch with factors of the MRN.(A-C) aNotch2 expression in the central DM (brackets, left panels) promotes transcription of Id2 (A, N = 4), Id3 (B, N = 2) and FoxC2 (C, N = 3) compared to the contralateral sides. (D-G) Id2, Id3 and Pax7 affect Notch signaling. Dorsal views of whole-mount segments. (D) Basal activity of Hes1 reporter in the lateral DM (N = 21). Id2 (E) or Id3 (F) missexpression enhances Hes1 reporter activity (N = 13 and 8, respectively), whereas Pax7 inhibits signaling (G, N = 13). (D’-G’) Control RFP shows similar electroporation efficiency in all treatments. (H-K) Notch-induced EMT requires Snail1 activity. (H, H’) Cells co-electroporated with control-GFP and control scrambled siRNA are mostly found within the lateral DM with a few cells delaminating from the epithelial sheet 16 hours post-treatment (N = 7). (I, I’) aNotch2 induces labeled cells to undergo EMT and migrate towards blood vessels (N = 6). (J, J’) Snail1 knock-down maintains cells within the lateral DM, inhibiting delamination (N = 5). (K, K’) Snail1 knock-down hinders Notch-induced EMT, as transfected cells remain epithelial within the lateral DM (N = 4). Bar: (A-C) 100 μm, (H-K) 50 μm. CV, cardinal vein; DM, dermomyotome; EMT, epithelial-to-mesenchymal transition; EP, electroporation; ISH, in situ hybridization; RFP, red fluorescent protein.
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Figure 7: Interactions of Notch with factors of the MRN.(A-C) aNotch2 expression in the central DM (brackets, left panels) promotes transcription of Id2 (A, N = 4), Id3 (B, N = 2) and FoxC2 (C, N = 3) compared to the contralateral sides. (D-G) Id2, Id3 and Pax7 affect Notch signaling. Dorsal views of whole-mount segments. (D) Basal activity of Hes1 reporter in the lateral DM (N = 21). Id2 (E) or Id3 (F) missexpression enhances Hes1 reporter activity (N = 13 and 8, respectively), whereas Pax7 inhibits signaling (G, N = 13). (D’-G’) Control RFP shows similar electroporation efficiency in all treatments. (H-K) Notch-induced EMT requires Snail1 activity. (H, H’) Cells co-electroporated with control-GFP and control scrambled siRNA are mostly found within the lateral DM with a few cells delaminating from the epithelial sheet 16 hours post-treatment (N = 7). (I, I’) aNotch2 induces labeled cells to undergo EMT and migrate towards blood vessels (N = 6). (J, J’) Snail1 knock-down maintains cells within the lateral DM, inhibiting delamination (N = 5). (K, K’) Snail1 knock-down hinders Notch-induced EMT, as transfected cells remain epithelial within the lateral DM (N = 4). Bar: (A-C) 100 μm, (H-K) 50 μm. CV, cardinal vein; DM, dermomyotome; EMT, epithelial-to-mesenchymal transition; EP, electroporation; ISH, in situ hybridization; RFP, red fluorescent protein.
Mentions: 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).

Bottom Line: These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation.Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate.This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors.

Results: Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other¿s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation.

Conclusions: Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

Show MeSH
Related in: MedlinePlus