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Lbx2 regulates formation of myofibrils.

Ochi H, Westerfield M - BMC Dev. Biol. (2009)

Bottom Line: Moreover, knockdown of Lbx2 results in malformation of muscle fibers and reduced fusion of fast precursors, although no obvious effects on induction or specification are observed.Expression of myofilament genes, including actin and myosin, requires the engrailed repressor domain of Lbx2.Our results elucidate a new function of Lbx2 as a regulator of myofibril formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA. harukiochi@bs.naist.jp

ABSTRACT

Background: Skeletal muscle differentiation requires assembly of contractile proteins into organized myofibrils. The Drosophila ladybird homeobox gene (lad) functions in founder cells of the segmental border muscle to promote myoblast fusion and muscle shaping. Tetrapods have two homologous genes (Lbx). Lbx1 functions in migration and/or proliferation of hypaxial myoblasts, whereas the function of Lbx2 is poorly understood.

Results: To elucidate the role of Lbx in vertebrate myogenesis, we examined Lbx function in zebrafish. Zebrafish lbx2 transcripts appear in newly formed paraxial mesoderm and become restricted to adaxial cells, precursors of slow muscle. Slow muscles lose lbx2 expression as they differentiate, while a subset of differentiating fast muscle cells transiently expresses lbx2. Fin and hyoid muscle express lbx2 later. In contrast, lbx1b expression first appears lateral to the somites at late segmentation stages and is later restricted to fin muscle. Morpholino knockdown of Lbx1b and Lbx2 suppresses hypaxial muscle development. Moreover, knockdown of Lbx2 results in malformation of muscle fibers and reduced fusion of fast precursors, although no obvious effects on induction or specification are observed. Expression of myofilament genes, including actin and myosin, requires the engrailed repressor domain of Lbx2.

Conclusion: Our results elucidate a new function of Lbx2 as a regulator of myofibril formation.

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Related in: MedlinePlus

Lbx2 and Lbx1b function in hypaxial muscle development. A: Splice donor MOs against lbx2 or lbx1b inhibit correct splicing of lbx2 or lbx1b, respectively. RT-PCR was performed using bud stage (lbx2) or segmentation stage (lbx1b) embryos. Spliced bands and unspliced bands in the lbx-splice donor-MO lane indicate aberrantly spliced message increases and correctly spliced message decreases. Arrows indicate primers. En: Engrailed domain, HD: homeodomain. Arrows indicate specific primers for amplification of correctly spliced or unspliced lbx genes. B-G: Expression of myod in control (ctrl) embryos (B, C), lbx2-MO injected embryos (D, E). lbx1b-MO injected embryos (F, G). The white arrowhead indicates the sternohyoideus primordium (B) and the white arrows indicate fin muscle precursors. myod expression in fin bud is suppressed by lbx2-MO or lbx1b-MO (B: 100%, n = 36; D: 12%, n = 56; F: 16%, n = 24). H-M: Expression of fgf8 in ectodermal cells of the fin bud. Control embryos (H, I, 100%, n = 12), lbx2-MO injected embryos (J-K, 100%, n = 12), lbx1b-MO injected embryos (L-M, 100%, n = 15). (B, D, F, H, J, L) Dorsal views, rostral towards the left, (C, E, G. I, K, M) lateral views, rostral toward the left, dorsal toward the top. Scale bar: (B-M) 100 μm.
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Figure 2: Lbx2 and Lbx1b function in hypaxial muscle development. A: Splice donor MOs against lbx2 or lbx1b inhibit correct splicing of lbx2 or lbx1b, respectively. RT-PCR was performed using bud stage (lbx2) or segmentation stage (lbx1b) embryos. Spliced bands and unspliced bands in the lbx-splice donor-MO lane indicate aberrantly spliced message increases and correctly spliced message decreases. Arrows indicate primers. En: Engrailed domain, HD: homeodomain. Arrows indicate specific primers for amplification of correctly spliced or unspliced lbx genes. B-G: Expression of myod in control (ctrl) embryos (B, C), lbx2-MO injected embryos (D, E). lbx1b-MO injected embryos (F, G). The white arrowhead indicates the sternohyoideus primordium (B) and the white arrows indicate fin muscle precursors. myod expression in fin bud is suppressed by lbx2-MO or lbx1b-MO (B: 100%, n = 36; D: 12%, n = 56; F: 16%, n = 24). H-M: Expression of fgf8 in ectodermal cells of the fin bud. Control embryos (H, I, 100%, n = 12), lbx2-MO injected embryos (J-K, 100%, n = 12), lbx1b-MO injected embryos (L-M, 100%, n = 15). (B, D, F, H, J, L) Dorsal views, rostral towards the left, (C, E, G. I, K, M) lateral views, rostral toward the left, dorsal toward the top. Scale bar: (B-M) 100 μm.

Mentions: To examine Lbx function in myogenesis, we knocked down Lbx2 or Lbx1b activity using specific splice blocking morpholino oligonucleotides (MOs). To confirm the specificity of the MOs, we performed reverse transcriptase PCR on mRNA from MO injected embryos. We find that injection of lbx2 or lbx1b splice donor MO results in production of aberrantly spliced lbx2 or lbx1b transcripts, respectively (Fig. 2A), indicating that splicing of lbx2 and lbx1b is blocked by the specific lbx splice donor MOs.


Lbx2 regulates formation of myofibrils.

Ochi H, Westerfield M - BMC Dev. Biol. (2009)

Lbx2 and Lbx1b function in hypaxial muscle development. A: Splice donor MOs against lbx2 or lbx1b inhibit correct splicing of lbx2 or lbx1b, respectively. RT-PCR was performed using bud stage (lbx2) or segmentation stage (lbx1b) embryos. Spliced bands and unspliced bands in the lbx-splice donor-MO lane indicate aberrantly spliced message increases and correctly spliced message decreases. Arrows indicate primers. En: Engrailed domain, HD: homeodomain. Arrows indicate specific primers for amplification of correctly spliced or unspliced lbx genes. B-G: Expression of myod in control (ctrl) embryos (B, C), lbx2-MO injected embryos (D, E). lbx1b-MO injected embryos (F, G). The white arrowhead indicates the sternohyoideus primordium (B) and the white arrows indicate fin muscle precursors. myod expression in fin bud is suppressed by lbx2-MO or lbx1b-MO (B: 100%, n = 36; D: 12%, n = 56; F: 16%, n = 24). H-M: Expression of fgf8 in ectodermal cells of the fin bud. Control embryos (H, I, 100%, n = 12), lbx2-MO injected embryos (J-K, 100%, n = 12), lbx1b-MO injected embryos (L-M, 100%, n = 15). (B, D, F, H, J, L) Dorsal views, rostral towards the left, (C, E, G. I, K, M) lateral views, rostral toward the left, dorsal toward the top. Scale bar: (B-M) 100 μm.
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Related In: Results  -  Collection

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Figure 2: Lbx2 and Lbx1b function in hypaxial muscle development. A: Splice donor MOs against lbx2 or lbx1b inhibit correct splicing of lbx2 or lbx1b, respectively. RT-PCR was performed using bud stage (lbx2) or segmentation stage (lbx1b) embryos. Spliced bands and unspliced bands in the lbx-splice donor-MO lane indicate aberrantly spliced message increases and correctly spliced message decreases. Arrows indicate primers. En: Engrailed domain, HD: homeodomain. Arrows indicate specific primers for amplification of correctly spliced or unspliced lbx genes. B-G: Expression of myod in control (ctrl) embryos (B, C), lbx2-MO injected embryos (D, E). lbx1b-MO injected embryos (F, G). The white arrowhead indicates the sternohyoideus primordium (B) and the white arrows indicate fin muscle precursors. myod expression in fin bud is suppressed by lbx2-MO or lbx1b-MO (B: 100%, n = 36; D: 12%, n = 56; F: 16%, n = 24). H-M: Expression of fgf8 in ectodermal cells of the fin bud. Control embryos (H, I, 100%, n = 12), lbx2-MO injected embryos (J-K, 100%, n = 12), lbx1b-MO injected embryos (L-M, 100%, n = 15). (B, D, F, H, J, L) Dorsal views, rostral towards the left, (C, E, G. I, K, M) lateral views, rostral toward the left, dorsal toward the top. Scale bar: (B-M) 100 μm.
Mentions: To examine Lbx function in myogenesis, we knocked down Lbx2 or Lbx1b activity using specific splice blocking morpholino oligonucleotides (MOs). To confirm the specificity of the MOs, we performed reverse transcriptase PCR on mRNA from MO injected embryos. We find that injection of lbx2 or lbx1b splice donor MO results in production of aberrantly spliced lbx2 or lbx1b transcripts, respectively (Fig. 2A), indicating that splicing of lbx2 and lbx1b is blocked by the specific lbx splice donor MOs.

Bottom Line: Moreover, knockdown of Lbx2 results in malformation of muscle fibers and reduced fusion of fast precursors, although no obvious effects on induction or specification are observed.Expression of myofilament genes, including actin and myosin, requires the engrailed repressor domain of Lbx2.Our results elucidate a new function of Lbx2 as a regulator of myofibril formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA. harukiochi@bs.naist.jp

ABSTRACT

Background: Skeletal muscle differentiation requires assembly of contractile proteins into organized myofibrils. The Drosophila ladybird homeobox gene (lad) functions in founder cells of the segmental border muscle to promote myoblast fusion and muscle shaping. Tetrapods have two homologous genes (Lbx). Lbx1 functions in migration and/or proliferation of hypaxial myoblasts, whereas the function of Lbx2 is poorly understood.

Results: To elucidate the role of Lbx in vertebrate myogenesis, we examined Lbx function in zebrafish. Zebrafish lbx2 transcripts appear in newly formed paraxial mesoderm and become restricted to adaxial cells, precursors of slow muscle. Slow muscles lose lbx2 expression as they differentiate, while a subset of differentiating fast muscle cells transiently expresses lbx2. Fin and hyoid muscle express lbx2 later. In contrast, lbx1b expression first appears lateral to the somites at late segmentation stages and is later restricted to fin muscle. Morpholino knockdown of Lbx1b and Lbx2 suppresses hypaxial muscle development. Moreover, knockdown of Lbx2 results in malformation of muscle fibers and reduced fusion of fast precursors, although no obvious effects on induction or specification are observed. Expression of myofilament genes, including actin and myosin, requires the engrailed repressor domain of Lbx2.

Conclusion: Our results elucidate a new function of Lbx2 as a regulator of myofibril formation.

Show MeSH
Related in: MedlinePlus