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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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Dorsalin-1 blocks the development of muscle pioneer cells.  (A) β-gal expression in embryos injected with twhh-β-gal was monitored  by enzyme activity and Nomarski microscopy at early pharyngula stage  (∼24 h). At this stage, the expression  of β-galactosidase was notochord  specific in >90% (n = 120) of the injected embryos that expressed the  construct. We first detected β-galactosidase expression in the early segmentation stage (∼12 h), specifically  in notochord cells of injected embryos (84%, n = 57, data not shown).  (B) Immunolocalization with anti– c-myc antibody in embryos injected  with the DNA construct twhh-dsl-1myc. The twhh promoter drives expression of dsl-1myc in notochord cells  (arrowheads). (C and D) Double labeling with anti–c-myc antibody and anti-engrailed antibody, 4D9, in embryos injected with either the DNA construct twhh-bGFP (C)  or twhh-dsl-1myc (D). The bracket in D marks the region affected by Dorsalin-1. The dsl-1myc expressing notochord cells in D are indicated by the arrowheads. Muscle pioneer cells in C and D are indicated by arrows. Cells in only some regions of the embryos expressed  the transgenes (B and D, arrowheads), consistent with the mosaic expression of other injected DNAs (Westerfield et al., 1992). In 93%  (n = 54) of the embryos lacking muscle pioneer cells in some of their somites, nearby notochord cells expressed Dorsalin-1. Embryos  are oriented in side views, with anterior to the left and dorsal to the top. Bar, 50 μm.
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Figure 4: Dorsalin-1 blocks the development of muscle pioneer cells. (A) β-gal expression in embryos injected with twhh-β-gal was monitored by enzyme activity and Nomarski microscopy at early pharyngula stage (∼24 h). At this stage, the expression of β-galactosidase was notochord specific in >90% (n = 120) of the injected embryos that expressed the construct. We first detected β-galactosidase expression in the early segmentation stage (∼12 h), specifically in notochord cells of injected embryos (84%, n = 57, data not shown). (B) Immunolocalization with anti– c-myc antibody in embryos injected with the DNA construct twhh-dsl-1myc. The twhh promoter drives expression of dsl-1myc in notochord cells (arrowheads). (C and D) Double labeling with anti–c-myc antibody and anti-engrailed antibody, 4D9, in embryos injected with either the DNA construct twhh-bGFP (C) or twhh-dsl-1myc (D). The bracket in D marks the region affected by Dorsalin-1. The dsl-1myc expressing notochord cells in D are indicated by the arrowheads. Muscle pioneer cells in C and D are indicated by arrows. Cells in only some regions of the embryos expressed the transgenes (B and D, arrowheads), consistent with the mosaic expression of other injected DNAs (Westerfield et al., 1992). In 93% (n = 54) of the embryos lacking muscle pioneer cells in some of their somites, nearby notochord cells expressed Dorsalin-1. Embryos are oriented in side views, with anterior to the left and dorsal to the top. Bar, 50 μm.

Mentions: In initial experiments, we found that injection of Dorsalin-1 mRNA had a severe ventralizing effect during gastrulation, similar to that caused by injection of BMP4 mRNA (Hammerschmidt, et al., 1996b). Thus, to assess potential later effects on somite patterning, we expressed Dorsalin-1 in the notochord after gastrulation. Additionally, expression of Dorsalin-1 in the notochord localized the protein to the region of the somites, where we anticipated the lowest activity of the putative inhibitor of Hedgehog signaling. To express a potential inhibitor specifically in this region of the somites, we put dorsalin-1 under the control of a promoter from the tiggy-winkle hedgehog gene. The floor plate normally expresses Tiggy-winkle hedgehog. Paradoxically, we found that 5.2 kb of the 5′-flanking sequence from the tiggy-winkle hedgehog gene leads to expression of heterologous proteins, including β-galactosidase, specifically in the notochord (Fig. 4 A; a further characterization of this promoter is in progress). Thus, we used this promoter fragment to express Dorsalin-1 in the notochord.


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)

Dorsalin-1 blocks the development of muscle pioneer cells.  (A) β-gal expression in embryos injected with twhh-β-gal was monitored  by enzyme activity and Nomarski microscopy at early pharyngula stage  (∼24 h). At this stage, the expression  of β-galactosidase was notochord  specific in >90% (n = 120) of the injected embryos that expressed the  construct. We first detected β-galactosidase expression in the early segmentation stage (∼12 h), specifically  in notochord cells of injected embryos (84%, n = 57, data not shown).  (B) Immunolocalization with anti– c-myc antibody in embryos injected  with the DNA construct twhh-dsl-1myc. The twhh promoter drives expression of dsl-1myc in notochord cells  (arrowheads). (C and D) Double labeling with anti–c-myc antibody and anti-engrailed antibody, 4D9, in embryos injected with either the DNA construct twhh-bGFP (C)  or twhh-dsl-1myc (D). The bracket in D marks the region affected by Dorsalin-1. The dsl-1myc expressing notochord cells in D are indicated by the arrowheads. Muscle pioneer cells in C and D are indicated by arrows. Cells in only some regions of the embryos expressed  the transgenes (B and D, arrowheads), consistent with the mosaic expression of other injected DNAs (Westerfield et al., 1992). In 93%  (n = 54) of the embryos lacking muscle pioneer cells in some of their somites, nearby notochord cells expressed Dorsalin-1. Embryos  are oriented in side views, with anterior to the left and dorsal to the top. Bar, 50 μm.
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Figure 4: Dorsalin-1 blocks the development of muscle pioneer cells. (A) β-gal expression in embryos injected with twhh-β-gal was monitored by enzyme activity and Nomarski microscopy at early pharyngula stage (∼24 h). At this stage, the expression of β-galactosidase was notochord specific in >90% (n = 120) of the injected embryos that expressed the construct. We first detected β-galactosidase expression in the early segmentation stage (∼12 h), specifically in notochord cells of injected embryos (84%, n = 57, data not shown). (B) Immunolocalization with anti– c-myc antibody in embryos injected with the DNA construct twhh-dsl-1myc. The twhh promoter drives expression of dsl-1myc in notochord cells (arrowheads). (C and D) Double labeling with anti–c-myc antibody and anti-engrailed antibody, 4D9, in embryos injected with either the DNA construct twhh-bGFP (C) or twhh-dsl-1myc (D). The bracket in D marks the region affected by Dorsalin-1. The dsl-1myc expressing notochord cells in D are indicated by the arrowheads. Muscle pioneer cells in C and D are indicated by arrows. Cells in only some regions of the embryos expressed the transgenes (B and D, arrowheads), consistent with the mosaic expression of other injected DNAs (Westerfield et al., 1992). In 93% (n = 54) of the embryos lacking muscle pioneer cells in some of their somites, nearby notochord cells expressed Dorsalin-1. Embryos are oriented in side views, with anterior to the left and dorsal to the top. Bar, 50 μm.
Mentions: In initial experiments, we found that injection of Dorsalin-1 mRNA had a severe ventralizing effect during gastrulation, similar to that caused by injection of BMP4 mRNA (Hammerschmidt, et al., 1996b). Thus, to assess potential later effects on somite patterning, we expressed Dorsalin-1 in the notochord after gastrulation. Additionally, expression of Dorsalin-1 in the notochord localized the protein to the region of the somites, where we anticipated the lowest activity of the putative inhibitor of Hedgehog signaling. To express a potential inhibitor specifically in this region of the somites, we put dorsalin-1 under the control of a promoter from the tiggy-winkle hedgehog gene. The floor plate normally expresses Tiggy-winkle hedgehog. Paradoxically, we found that 5.2 kb of the 5′-flanking sequence from the tiggy-winkle hedgehog gene leads to expression of heterologous proteins, including β-galactosidase, specifically in the notochord (Fig. 4 A; a further characterization of this promoter is in progress). Thus, we used this promoter fragment to express Dorsalin-1 in the notochord.

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