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Regulation of slow and fast muscle myofibrillogenesis by Wnt/beta-catenin and myostatin signaling.

Tee JM, van Rooijen C, Boonen R, Zivkovic D - PLoS ONE (2009)

Bottom Line: Deviation from proper muscle development or homeostasis results in various myopathic conditions.Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/beta-catenin signaling is essential for muscle fiber growth and maintenance.Epistatic analyses suggest a possible genetic interaction between Wnt/beta-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Center, Utrecht, The Netherlands.

ABSTRACT
Deviation from proper muscle development or homeostasis results in various myopathic conditions. Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/beta-catenin signaling is essential for muscle fiber growth and maintenance. In zebrafish embryos, gain-of-Wnt/beta-catenin function results in unscheduled muscle progenitor proliferation, leading to slow and fast muscle hypertrophy accompanied by fast muscle degeneration. The effects of Wnt/beta-catenin signaling on fast muscle hypertrophy were rescued by misexpression of Myostatin or p21(CIP/WAF), establishing an in vivo regulation of myofibrillogenesis by Wnt/beta-catenin signaling and Myostatin. Epistatic analyses suggest a possible genetic interaction between Wnt/beta-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.

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Related in: MedlinePlus

mstn is upregulated in LOF Wnt/β-catenin.(A) 2 ng Lef1 MO was injected at 1-cell stage into wild-type or axin1/apc1 embryos, and the number of somites was counted at 54 hpf. Two independent clutches of axin1/apc1 heterozygous incross were analyzed (Total n = 64), in which 10 were genotyped as axin1/apc1 homozygous. (B) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 36 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is upregulated in Lef1 morphants, corresponding to the in situ hybridization with mstn probe in bottom panels. Scale bar, 100 µm. (C) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is downregulated in axin1/apc1 embryos and upregulated in Lef1 morphants (D) Western blot on lysates collected from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graph shows upregulation of Mstn in Lef1 morphants. However, there is no significant difference in levels of Mstn protein in axin1/apc1 embryos versus wildtypes. (E) Misexpression of mstn mRNA downregulates the Wnt target gene lef1 in axin1/apc1 mutants shown with WISH for lef1 riboprobe. Scale bar, 100 µm.
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pone-0005880-g007: mstn is upregulated in LOF Wnt/β-catenin.(A) 2 ng Lef1 MO was injected at 1-cell stage into wild-type or axin1/apc1 embryos, and the number of somites was counted at 54 hpf. Two independent clutches of axin1/apc1 heterozygous incross were analyzed (Total n = 64), in which 10 were genotyped as axin1/apc1 homozygous. (B) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 36 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is upregulated in Lef1 morphants, corresponding to the in situ hybridization with mstn probe in bottom panels. Scale bar, 100 µm. (C) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is downregulated in axin1/apc1 embryos and upregulated in Lef1 morphants (D) Western blot on lysates collected from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graph shows upregulation of Mstn in Lef1 morphants. However, there is no significant difference in levels of Mstn protein in axin1/apc1 embryos versus wildtypes. (E) Misexpression of mstn mRNA downregulates the Wnt target gene lef1 in axin1/apc1 mutants shown with WISH for lef1 riboprobe. Scale bar, 100 µm.

Mentions: As expression profiling in Mstn loss-of-function (LOF) identified modulation of Wnt- pathway components [33], we examined for possible genetic interaction between the two pathways, by using gain and loss of Wnt/β-catenin signaling. To establish a suitable genetic means for analysis of mstn mRNA upon loss of Wnt/β-catenin function, we first tested whether morpholino (MO)-mediated knock-down of Lef1 [22], which is upregulated in axin1/apc1 mutants (Fig. 1B) would rescue their aberrant somitogenesis. Knockdown of Lef1 with 2 ng MO in wild-types resulted in loss of a number of somites (59%, n = 54) at 54 hpf (Fig. 7A), suggesting that Lef1 is required for normal somitogenesis. Notably, in 50% of Lef1-MO-injected axin1/apc1 mutants, the normal number of somites was restored, establishing a mechanistic link between Lef1 hyperactivity and somite loss (Fig. 7A).


Regulation of slow and fast muscle myofibrillogenesis by Wnt/beta-catenin and myostatin signaling.

Tee JM, van Rooijen C, Boonen R, Zivkovic D - PLoS ONE (2009)

mstn is upregulated in LOF Wnt/β-catenin.(A) 2 ng Lef1 MO was injected at 1-cell stage into wild-type or axin1/apc1 embryos, and the number of somites was counted at 54 hpf. Two independent clutches of axin1/apc1 heterozygous incross were analyzed (Total n = 64), in which 10 were genotyped as axin1/apc1 homozygous. (B) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 36 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is upregulated in Lef1 morphants, corresponding to the in situ hybridization with mstn probe in bottom panels. Scale bar, 100 µm. (C) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is downregulated in axin1/apc1 embryos and upregulated in Lef1 morphants (D) Western blot on lysates collected from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graph shows upregulation of Mstn in Lef1 morphants. However, there is no significant difference in levels of Mstn protein in axin1/apc1 embryos versus wildtypes. (E) Misexpression of mstn mRNA downregulates the Wnt target gene lef1 in axin1/apc1 mutants shown with WISH for lef1 riboprobe. Scale bar, 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690692&req=5

pone-0005880-g007: mstn is upregulated in LOF Wnt/β-catenin.(A) 2 ng Lef1 MO was injected at 1-cell stage into wild-type or axin1/apc1 embryos, and the number of somites was counted at 54 hpf. Two independent clutches of axin1/apc1 heterozygous incross were analyzed (Total n = 64), in which 10 were genotyped as axin1/apc1 homozygous. (B) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 36 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is upregulated in Lef1 morphants, corresponding to the in situ hybridization with mstn probe in bottom panels. Scale bar, 100 µm. (C) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is downregulated in axin1/apc1 embryos and upregulated in Lef1 morphants (D) Western blot on lysates collected from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graph shows upregulation of Mstn in Lef1 morphants. However, there is no significant difference in levels of Mstn protein in axin1/apc1 embryos versus wildtypes. (E) Misexpression of mstn mRNA downregulates the Wnt target gene lef1 in axin1/apc1 mutants shown with WISH for lef1 riboprobe. Scale bar, 100 µm.
Mentions: As expression profiling in Mstn loss-of-function (LOF) identified modulation of Wnt- pathway components [33], we examined for possible genetic interaction between the two pathways, by using gain and loss of Wnt/β-catenin signaling. To establish a suitable genetic means for analysis of mstn mRNA upon loss of Wnt/β-catenin function, we first tested whether morpholino (MO)-mediated knock-down of Lef1 [22], which is upregulated in axin1/apc1 mutants (Fig. 1B) would rescue their aberrant somitogenesis. Knockdown of Lef1 with 2 ng MO in wild-types resulted in loss of a number of somites (59%, n = 54) at 54 hpf (Fig. 7A), suggesting that Lef1 is required for normal somitogenesis. Notably, in 50% of Lef1-MO-injected axin1/apc1 mutants, the normal number of somites was restored, establishing a mechanistic link between Lef1 hyperactivity and somite loss (Fig. 7A).

Bottom Line: Deviation from proper muscle development or homeostasis results in various myopathic conditions.Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/beta-catenin signaling is essential for muscle fiber growth and maintenance.Epistatic analyses suggest a possible genetic interaction between Wnt/beta-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Center, Utrecht, The Netherlands.

ABSTRACT
Deviation from proper muscle development or homeostasis results in various myopathic conditions. Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/beta-catenin signaling is essential for muscle fiber growth and maintenance. In zebrafish embryos, gain-of-Wnt/beta-catenin function results in unscheduled muscle progenitor proliferation, leading to slow and fast muscle hypertrophy accompanied by fast muscle degeneration. The effects of Wnt/beta-catenin signaling on fast muscle hypertrophy were rescued by misexpression of Myostatin or p21(CIP/WAF), establishing an in vivo regulation of myofibrillogenesis by Wnt/beta-catenin signaling and Myostatin. Epistatic analyses suggest a possible genetic interaction between Wnt/beta-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.

Show MeSH
Related in: MedlinePlus