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Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-beta gene families.

Du SJ, Devoto SH, Westerfield M, Moon RT - J. Cell Biol. (1997)

Bottom Line: We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube.We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite.We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of Washington, School of Medicine, Seattle 98195, USA.

ABSTRACT
We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.

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Induction of slow muscle cells by zebrafish Sonic hedgehog (Shh) and Tiggy-winkle hedgehog (Twhh). (A, B, and C) Sections  (dorsal to the top) showing fluorescence localization of slow muscle cells labeled with F59, an anti–myosin heavy chain antibody, in embryos injected with frame shifted sonic hedgehog (Shhfs) (A), Shh (B), or Twhh (C). (D, E, and F) Sections (dorsal to the top) showing  fluorescence localization of muscle pioneer cells labeled with 4D9, an anti-engrailed antibody, in embryos injected with Shhfs (D), Shh  (E), or Twhh (F). (G, H, and I) Whole-mount Nomarski images showing muscle pioneer cells labeled with the 4D9 antibody in embryos  injected with Shhfs (G), Shh (H), or Twhh (I). Embryos in G, H, and I are oriented in side views, with anterior to the left and dorsal to  the top. Bars, 50 μm.
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Figure 2: Induction of slow muscle cells by zebrafish Sonic hedgehog (Shh) and Tiggy-winkle hedgehog (Twhh). (A, B, and C) Sections (dorsal to the top) showing fluorescence localization of slow muscle cells labeled with F59, an anti–myosin heavy chain antibody, in embryos injected with frame shifted sonic hedgehog (Shhfs) (A), Shh (B), or Twhh (C). (D, E, and F) Sections (dorsal to the top) showing fluorescence localization of muscle pioneer cells labeled with 4D9, an anti-engrailed antibody, in embryos injected with Shhfs (D), Shh (E), or Twhh (F). (G, H, and I) Whole-mount Nomarski images showing muscle pioneer cells labeled with the 4D9 antibody in embryos injected with Shhfs (G), Shh (H), or Twhh (I). Embryos in G, H, and I are oriented in side views, with anterior to the left and dorsal to the top. Bars, 50 μm.

Mentions: Previous studies have shown that ectopic expression of Hedgehog induces MyoD and supernumerary muscle pioneer cells, suggesting that it may play an important role in muscle development in zebrafish (Currie and Ingham, 1996; Hammerschmidt et al., 1996a; Weinberg et al., 1996). To examine directly whether hedgehog genes influence the development of muscle fiber type identity, we expressed zebrafish sonic hedgehog or tiggy-winkle hedgehog ectopically by injection of RNA into cleavage stage embryos. We then examined the developing embryos for induction of slow muscle cells using several monoclonal antibodies that recognize the entire population of slow muscle cells, including the muscle pioneers. F59 recognizes myosin heavy chain in fish (Miller et al., 1989); in zebrafish it specifically labels slow muscle fibers during the first day of development, and then later it also weakly labels fast muscle fibers (Devoto et al., 1996b). We also used zn5 (Trevarrow et al., 1990) and S58 (Crow and Stockdale, 1986) antibodies that also label slow but never label fast muscle fibers in zebrafish (Devoto et al., 1996b). We found that both Sonic hedgehog and Tiggy-winkle hedgehog induced the development of many extra slow muscle cells. Specifically, as in uninjected embryos, only one layer of slow muscle cells was present in the superficial layer of the somite in control embryos injected with frame-shifted sonic hedgehog RNA (Fig. 2 A), whereas in embryos injected with sonic hedgehog (Fig. 2 B) or tiggy-winkle hedgehog (Fig. 2 C) RNA, almost all cells in the somite differentiated into slow muscle. These ectopic slow muscle cells were also labeled by the S58 and zn5 antibodies, indicating that these cells had fully differentiated as slow muscle fibers (data not shown). Presumably, these extra slow muscle cells are formed at the expense of fast muscle because they occupy the locations where fast muscle cells normally form, and because nearly all the muscle cells in the somite exhibited these slow muscle properties. Both Sonic hedgehog (Fig. 2, E and H) and Tiggy-winkle hedgehog (Fig. 2, F and I) also induced extra muscle pioneer cells, as determined by labeling with the anti–engrailed monoclonal antibody, 4D9. In control embryos injected with frame-shifted sonic hedgehog, two to six muscle pioneer cells were normally present in each somite (Fig. 2, D and G) as in uninjected embryos, whereas Sonic hedgehog induced an average of 20 muscle pioneer cells per somite (88%, n = 87; Fig. 2, E and H), and Tiggy-winkle hedgehog induced an average of 10 muscle pioneer cells per somite (75%, n = 105; Fig. 2, F and I).


Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-beta gene families.

Du SJ, Devoto SH, Westerfield M, Moon RT - J. Cell Biol. (1997)

Induction of slow muscle cells by zebrafish Sonic hedgehog (Shh) and Tiggy-winkle hedgehog (Twhh). (A, B, and C) Sections  (dorsal to the top) showing fluorescence localization of slow muscle cells labeled with F59, an anti–myosin heavy chain antibody, in embryos injected with frame shifted sonic hedgehog (Shhfs) (A), Shh (B), or Twhh (C). (D, E, and F) Sections (dorsal to the top) showing  fluorescence localization of muscle pioneer cells labeled with 4D9, an anti-engrailed antibody, in embryos injected with Shhfs (D), Shh  (E), or Twhh (F). (G, H, and I) Whole-mount Nomarski images showing muscle pioneer cells labeled with the 4D9 antibody in embryos  injected with Shhfs (G), Shh (H), or Twhh (I). Embryos in G, H, and I are oriented in side views, with anterior to the left and dorsal to  the top. Bars, 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139815&req=5

Figure 2: Induction of slow muscle cells by zebrafish Sonic hedgehog (Shh) and Tiggy-winkle hedgehog (Twhh). (A, B, and C) Sections (dorsal to the top) showing fluorescence localization of slow muscle cells labeled with F59, an anti–myosin heavy chain antibody, in embryos injected with frame shifted sonic hedgehog (Shhfs) (A), Shh (B), or Twhh (C). (D, E, and F) Sections (dorsal to the top) showing fluorescence localization of muscle pioneer cells labeled with 4D9, an anti-engrailed antibody, in embryos injected with Shhfs (D), Shh (E), or Twhh (F). (G, H, and I) Whole-mount Nomarski images showing muscle pioneer cells labeled with the 4D9 antibody in embryos injected with Shhfs (G), Shh (H), or Twhh (I). Embryos in G, H, and I are oriented in side views, with anterior to the left and dorsal to the top. Bars, 50 μm.
Mentions: Previous studies have shown that ectopic expression of Hedgehog induces MyoD and supernumerary muscle pioneer cells, suggesting that it may play an important role in muscle development in zebrafish (Currie and Ingham, 1996; Hammerschmidt et al., 1996a; Weinberg et al., 1996). To examine directly whether hedgehog genes influence the development of muscle fiber type identity, we expressed zebrafish sonic hedgehog or tiggy-winkle hedgehog ectopically by injection of RNA into cleavage stage embryos. We then examined the developing embryos for induction of slow muscle cells using several monoclonal antibodies that recognize the entire population of slow muscle cells, including the muscle pioneers. F59 recognizes myosin heavy chain in fish (Miller et al., 1989); in zebrafish it specifically labels slow muscle fibers during the first day of development, and then later it also weakly labels fast muscle fibers (Devoto et al., 1996b). We also used zn5 (Trevarrow et al., 1990) and S58 (Crow and Stockdale, 1986) antibodies that also label slow but never label fast muscle fibers in zebrafish (Devoto et al., 1996b). We found that both Sonic hedgehog and Tiggy-winkle hedgehog induced the development of many extra slow muscle cells. Specifically, as in uninjected embryos, only one layer of slow muscle cells was present in the superficial layer of the somite in control embryos injected with frame-shifted sonic hedgehog RNA (Fig. 2 A), whereas in embryos injected with sonic hedgehog (Fig. 2 B) or tiggy-winkle hedgehog (Fig. 2 C) RNA, almost all cells in the somite differentiated into slow muscle. These ectopic slow muscle cells were also labeled by the S58 and zn5 antibodies, indicating that these cells had fully differentiated as slow muscle fibers (data not shown). Presumably, these extra slow muscle cells are formed at the expense of fast muscle because they occupy the locations where fast muscle cells normally form, and because nearly all the muscle cells in the somite exhibited these slow muscle properties. Both Sonic hedgehog (Fig. 2, E and H) and Tiggy-winkle hedgehog (Fig. 2, F and I) also induced extra muscle pioneer cells, as determined by labeling with the anti–engrailed monoclonal antibody, 4D9. In control embryos injected with frame-shifted sonic hedgehog, two to six muscle pioneer cells were normally present in each somite (Fig. 2, D and G) as in uninjected embryos, whereas Sonic hedgehog induced an average of 20 muscle pioneer cells per somite (88%, n = 87; Fig. 2, E and H), and Tiggy-winkle hedgehog induced an average of 10 muscle pioneer cells per somite (75%, n = 105; Fig. 2, F and I).

Bottom Line: We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube.We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite.We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of Washington, School of Medicine, Seattle 98195, USA.

ABSTRACT
We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.

Show MeSH
Related in: MedlinePlus