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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

Muscle precursors express lbx1b and lbx2. A-L: Muscle precursors transiently express lbx2. A-G, L: Expression of lbx2 at 70%-epiboly (A), 90%-epiboly (B), bud (C), segmentation (D), 24 hpf (E, L), 48 hfp (F, G). Whole-mounts. H-K: lbx2 (blue) and ntl (red) expression at 70%-epiboly (H), 90%-epiboly (I), bud (J), and segmentation (K). Flat-mounts. L: Transverse section, 24 hpf embryo, F59 (green) and lbx2 mRNA (blue). (A, H) lbx2 mRNA appears at 70%-epiboly adjacent to ntl expressing cells in blastoderm margin. lbx2 expression in paraxial mesoderm by end of gastrulation (B-C, I,). lbx2 expression later restricted to adaxial cells (J-K, brackets). (L) Subset of fast muscle cells expresses lbx2 in epaxial (black arrow) and hypaxial (black arrowhead) domains. F59 and lbx2 labeling shows differentiated slow muscle cells lose lbx2 expression (L, white arrowhead). (F) lbx2 expression in trunk disappears by 48 hpf (F, bracket). lbx2 expression in fin primordia (F-G, black arrowheads), hindbrain (F-G, white arrowhead), and hyoid (F, white arrow). M: Diagram of zebrafish muscle. Adaxial cells (K, bracket) migrate superficially and differentiate into slow muscle fibers (green). Then, fast (magenta) and medial fast fibers (red) differentiate. N-R: 5-somite (N), 18-somite (O), 24 hpf (P-Q) and 48 hpf (R). (Q) Higher magnification of P. lbx1b mRNA appears at 5-somite stage in neural tube (N), along rostral-caudal axis (O, arrow), then lateral to somites (P-Q), and later in fin (R). (A-D, N-O) Dorsal views, rostral towards the top; (E-F, P) lateral views, rostral toward the left, dorsal toward the top; (H-K) rostral toward the top; (L) dorsal toward the top; (G, R) dorsal views, rostral toward the left. Scale bars: (A-D, N-O, Q) 200 μm, (E-G, P, R) 100 μm, (H-L) 50 μm.
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Figure 1: Muscle precursors express lbx1b and lbx2. A-L: Muscle precursors transiently express lbx2. A-G, L: Expression of lbx2 at 70%-epiboly (A), 90%-epiboly (B), bud (C), segmentation (D), 24 hpf (E, L), 48 hfp (F, G). Whole-mounts. H-K: lbx2 (blue) and ntl (red) expression at 70%-epiboly (H), 90%-epiboly (I), bud (J), and segmentation (K). Flat-mounts. L: Transverse section, 24 hpf embryo, F59 (green) and lbx2 mRNA (blue). (A, H) lbx2 mRNA appears at 70%-epiboly adjacent to ntl expressing cells in blastoderm margin. lbx2 expression in paraxial mesoderm by end of gastrulation (B-C, I,). lbx2 expression later restricted to adaxial cells (J-K, brackets). (L) Subset of fast muscle cells expresses lbx2 in epaxial (black arrow) and hypaxial (black arrowhead) domains. F59 and lbx2 labeling shows differentiated slow muscle cells lose lbx2 expression (L, white arrowhead). (F) lbx2 expression in trunk disappears by 48 hpf (F, bracket). lbx2 expression in fin primordia (F-G, black arrowheads), hindbrain (F-G, white arrowhead), and hyoid (F, white arrow). M: Diagram of zebrafish muscle. Adaxial cells (K, bracket) migrate superficially and differentiate into slow muscle fibers (green). Then, fast (magenta) and medial fast fibers (red) differentiate. N-R: 5-somite (N), 18-somite (O), 24 hpf (P-Q) and 48 hpf (R). (Q) Higher magnification of P. lbx1b mRNA appears at 5-somite stage in neural tube (N), along rostral-caudal axis (O, arrow), then lateral to somites (P-Q), and later in fin (R). (A-D, N-O) Dorsal views, rostral towards the top; (E-F, P) lateral views, rostral toward the left, dorsal toward the top; (H-K) rostral toward the top; (L) dorsal toward the top; (G, R) dorsal views, rostral toward the left. Scale bars: (A-D, N-O, Q) 200 μm, (E-G, P, R) 100 μm, (H-L) 50 μm.

Mentions: To study the function of lbx genes in vertebrate skeletal muscle development, we examined expression of lbx1b and lbx2 in zebrafish. Previous analysis of lbx1a [24] showed that it is not expressed in the somites, so we excluded it from this study. lbx2 expression becomes detectable by the 70%–80% epiboly stage (Fig. 1A). Double labeling with no tail (ntl), which marks the blastoderm margin, reveals that lbx2 first appears adjacent to the margin (Fig. 1H) in the region that later contributes to head muscle and pronephros [25]. By the end of bud stage, lbx2 expression appears in paraxial mesoderm and adaxial cells (Fig. 1C, J, brackets), precursors of slow muscle cells and muscle pioneers [18]. lbx2 expression is not detected in the somites (Fig. 1D, K, rostral region), even though a subset of adaxial cells, the slow muscle precursors, migrates through the somites during segmentation stages [18,20,21].


Lbx2 regulates formation of myofibrils.

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

Muscle precursors express lbx1b and lbx2. A-L: Muscle precursors transiently express lbx2. A-G, L: Expression of lbx2 at 70%-epiboly (A), 90%-epiboly (B), bud (C), segmentation (D), 24 hpf (E, L), 48 hfp (F, G). Whole-mounts. H-K: lbx2 (blue) and ntl (red) expression at 70%-epiboly (H), 90%-epiboly (I), bud (J), and segmentation (K). Flat-mounts. L: Transverse section, 24 hpf embryo, F59 (green) and lbx2 mRNA (blue). (A, H) lbx2 mRNA appears at 70%-epiboly adjacent to ntl expressing cells in blastoderm margin. lbx2 expression in paraxial mesoderm by end of gastrulation (B-C, I,). lbx2 expression later restricted to adaxial cells (J-K, brackets). (L) Subset of fast muscle cells expresses lbx2 in epaxial (black arrow) and hypaxial (black arrowhead) domains. F59 and lbx2 labeling shows differentiated slow muscle cells lose lbx2 expression (L, white arrowhead). (F) lbx2 expression in trunk disappears by 48 hpf (F, bracket). lbx2 expression in fin primordia (F-G, black arrowheads), hindbrain (F-G, white arrowhead), and hyoid (F, white arrow). M: Diagram of zebrafish muscle. Adaxial cells (K, bracket) migrate superficially and differentiate into slow muscle fibers (green). Then, fast (magenta) and medial fast fibers (red) differentiate. N-R: 5-somite (N), 18-somite (O), 24 hpf (P-Q) and 48 hpf (R). (Q) Higher magnification of P. lbx1b mRNA appears at 5-somite stage in neural tube (N), along rostral-caudal axis (O, arrow), then lateral to somites (P-Q), and later in fin (R). (A-D, N-O) Dorsal views, rostral towards the top; (E-F, P) lateral views, rostral toward the left, dorsal toward the top; (H-K) rostral toward the top; (L) dorsal toward the top; (G, R) dorsal views, rostral toward the left. Scale bars: (A-D, N-O, Q) 200 μm, (E-G, P, R) 100 μm, (H-L) 50 μm.
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Figure 1: Muscle precursors express lbx1b and lbx2. A-L: Muscle precursors transiently express lbx2. A-G, L: Expression of lbx2 at 70%-epiboly (A), 90%-epiboly (B), bud (C), segmentation (D), 24 hpf (E, L), 48 hfp (F, G). Whole-mounts. H-K: lbx2 (blue) and ntl (red) expression at 70%-epiboly (H), 90%-epiboly (I), bud (J), and segmentation (K). Flat-mounts. L: Transverse section, 24 hpf embryo, F59 (green) and lbx2 mRNA (blue). (A, H) lbx2 mRNA appears at 70%-epiboly adjacent to ntl expressing cells in blastoderm margin. lbx2 expression in paraxial mesoderm by end of gastrulation (B-C, I,). lbx2 expression later restricted to adaxial cells (J-K, brackets). (L) Subset of fast muscle cells expresses lbx2 in epaxial (black arrow) and hypaxial (black arrowhead) domains. F59 and lbx2 labeling shows differentiated slow muscle cells lose lbx2 expression (L, white arrowhead). (F) lbx2 expression in trunk disappears by 48 hpf (F, bracket). lbx2 expression in fin primordia (F-G, black arrowheads), hindbrain (F-G, white arrowhead), and hyoid (F, white arrow). M: Diagram of zebrafish muscle. Adaxial cells (K, bracket) migrate superficially and differentiate into slow muscle fibers (green). Then, fast (magenta) and medial fast fibers (red) differentiate. N-R: 5-somite (N), 18-somite (O), 24 hpf (P-Q) and 48 hpf (R). (Q) Higher magnification of P. lbx1b mRNA appears at 5-somite stage in neural tube (N), along rostral-caudal axis (O, arrow), then lateral to somites (P-Q), and later in fin (R). (A-D, N-O) Dorsal views, rostral towards the top; (E-F, P) lateral views, rostral toward the left, dorsal toward the top; (H-K) rostral toward the top; (L) dorsal toward the top; (G, R) dorsal views, rostral toward the left. Scale bars: (A-D, N-O, Q) 200 μm, (E-G, P, R) 100 μm, (H-L) 50 μm.
Mentions: To study the function of lbx genes in vertebrate skeletal muscle development, we examined expression of lbx1b and lbx2 in zebrafish. Previous analysis of lbx1a [24] showed that it is not expressed in the somites, so we excluded it from this study. lbx2 expression becomes detectable by the 70%–80% epiboly stage (Fig. 1A). Double labeling with no tail (ntl), which marks the blastoderm margin, reveals that lbx2 first appears adjacent to the margin (Fig. 1H) in the region that later contributes to head muscle and pronephros [25]. By the end of bud stage, lbx2 expression appears in paraxial mesoderm and adaxial cells (Fig. 1C, J, brackets), precursors of slow muscle cells and muscle pioneers [18]. lbx2 expression is not detected in the somites (Fig. 1D, K, rostral region), even though a subset of adaxial cells, the slow muscle precursors, migrates through the somites during segmentation stages [18,20,21].

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