Neogenin regulates skeletal myofiber size and focal adhesion kinase and extracellular signal-regulated kinase activities in vivo and in vitro

Abstract

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.

Figure 1.

The Neo1^Gt allele is variably hypomorphic. (A) Map of the gene-trap insertion site in the Neo1 locus. The arrow indicates the insertion site of the secretory gene-trap vector in intron 7. The positions of the primers used for genotyping are represented by red and black arrowheads (F, forward primer for wild-type allele; I, reverse primer for wild-type allele and forward primer for mutant allele; R, reverse primer for mutant allele). Lines above primers represent PCR products shown in B. The dotted line in the mutant allele represents a deleted sequence, and the white region in the wild-type and mutant alleles represents an inverted sequence. Within the gene-trap vector, the vertical white rectangle indicates a transmembrane (TM) domain, the blue segment is β-geo, the green segment an internal ribosome entry site (IRES), and the purple segment is an alkaline phosphatase reporter gene (PLAP) (B) Genotype analysis of Neo1^+/+, Neo1^+/Gt, and Neo1^Gt/Gt embryos. A three-primer PCR produces a 704-base pair fragment from the wild-type allele and a 917-base pair fragment from the mutant allele. (C) Western blot analysis of neogenin protein production in embryos and MEFs. Heads from E13.5 embryos of the indicated genotype were removed, and extracts were blotted with antibodies to the neogenin intracellular domain. Extracts from MEFs of the indicated genotype were also analyzed. The ∼190-kDa band (arrow) represents full-length neogenin, and the ∼60-kDa band (arrowhead) may correspond to a processed form of the neogenin intracellular region. The ∼120-kDa band (asterisk) is a nonspecific band recognized in embryonic head, but not MEF, extracts. Extracts were also probed with a pan-Cadherin antibody as a loading control.

Figure 2.

Normal formation of myotomes in Neo1^Gt/Gt embryos. (A) Analysis of Neo1 expression by β-gal activity (blue color) in Neo^+/Gt embryos of the indicated stage. Neo1 is expressed ubiquitously but with particularly high levels in dorsal somites (arrows) and the dorsal aspect of the neural tube (arrowheads). (B and C) Whole-mount in situ hybridization of E10.5 (B) and E11.5 (C) embryos of the indicated genotype with a myogenin probe. The red brackets span the length of the myotome, with no significant difference seen between Neo1^+/+ and Neo1^Gt/Gt embryos.

Figure 3.

Reduced expression of muscle-specific proteins and diminished concentration of phospho-FAK and phospho-ERK in premuscle masses of E15.5 Neo1^Gt/Gt embryos. (A–E) Transverse sections through developing intercostal muscles (A and E), premuscle mass of the pectoralis (B and C) and premuscle mass of the trapezius (D) stained with antibodies against the indicated proteins. Within each panel, the lower micrographs are higher magnification views of the ones directly above and are delineated by the boxed areas in A and D. (F) Western blot analysis of production of muscle-specific and signaling proteins in extracts of dissected E15.5 hind limbs. Bars, 0.5 mm (A, top), 50 mm (A, bottom), and 50 mm (B–E).

Figure 4.

Neo1^Gt/Gt mice form small myofibers. (A) Cross-sections though the hind limbs of E18.5 Neo1^+/+ and Neo1^Gt/Gt mice stained with hematoxylin and eosin. Note that overall muscle patterning is not different between wild-type and mutant animals. R, radius; U, ulna. (B) Cross-sections though the hind limbs of E18.5 Neo1^+/+ and Neo1^Gt/Gt mice stained with hematoxylin and eosin. (C) Quantification of myofiber cross-sectional area (CSA) from B. Asterisk indicates different from control, p < 0.0001 by Student's t test. (D) Cross-sections though the hind limbs of P21 Neo1^+/+ and Neo1^Gt/Gt mice stained with hematoxylin and eosin. (E) Quantification of myofiber CSA from D. Asterisk indicates different from control, p < 0.0001 by Student's t test. Bars, 0.5 mm (A) and 50 mm (B and D).

Figure 5.

Defective differentiation of Neo1^Gt/Gt myoblasts in vitro. (A) Photomicrographs of Neo1^+/+ and Neo1^Gt/Gt myoblasts cultured in differentiation medium, and fixed and stained with an antibody to MHC. (B) Quantification of myotube formation by cell lines shown in A. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results and with multiple, independent isolates of the cells. Asterisks indicate different from control, p < 0.01 by Student's t test. (C) BrdU incorporation into proliferating Neo1^+/+ and Neo1^Gt/Gt myoblasts. Cultures were exposed to BrdU for 2 h, fixed, and stained with an antibody to BrdU. (D) Quantification of BrdU incorporation shown in C. (E) Photomicrographs of Neo1^+/+ and Neo1^Gt/Gt myoblasts cultured in growth medium (GM) or differentiation medium (DM) for 48 h and analyzed for apoptotic cells by TUNEL assay (green). Cultures were also stained with 4,6-diamidino-2-phenylindole to reveal nuclei (blue). (F) Quantification of myotube formation by cell lines shown in E. Values represent means of triplicate determinations ± 1 SD. Asterisk indicates different from Neo1^+/+ control, p < 0.01 by Student's t test. (G) Western blot analysis of production of muscle-specific and signaling proteins in extracts of differentiating Neo1^+/+ and Neo1^Gt/Gt myoblasts.

Figure 6.

Defective response to netrin signaling in Neo1^Gt/Gt and Cdo^−/− myoblasts. (A) Western blot analysis of phospho-FAK (p-FAK) and total FAK levels in Neo1^+/+ and Neo1^Gt/Gt myoblasts treated with netrin-2 for the indicated times. Lysates were also probed with an antibody to neogenin. (B) Western blot analysis of p-FAK and total FAK levels in Cdo^+/+ and Cdo^−/− myoblasts treated with netrin-2 for the indicated times. Lysates were also probed with an antibody to Cdo. (C) Western blot analysis of p-FAK and total FAK levels in Neo1^+/+ and Neo1^Gt/Gt myoblasts treated with RGMc for the indicated times. (D) Western blot analysis of p-FAK and total FAK levels in Cdo^+/+ and Cdo^−/− myoblasts treated with RGMc for the indicated times. (E) Western blot analysis of phospho-ERK2 (p-ERK2) and total ERK2 levels in Neo1^+/+ and Neo1^Gt/Gt myoblasts treated with RGMc for the indicated times. (F) Western blot analysis of p-ERK2 and total ERK2 levels in Cdo^+/+ and Cdo^−/− myoblasts treated with RGMc for the indicated times. (G) Western blot analysis of p-ERK2 and total ERK2 levels in Neo1^+/+ and Neo1^Gt/Gt myoblasts treated with RGMc for the indicated times. (H) Western blot analysis of p-ERK2 and total ERK2 levels in Cdo^+/+ and Cdo^−/− myoblasts treated with RGMc for the indicated times.