CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells

Abstract

Long-range migrating progenitor cells generate hypaxial muscle, for instance the muscle of the limbs, hypoglossal cord, and diaphragm. We show here that migrating muscle progenitors express the chemokine receptor CXCR4. The corresponding ligand, SDF1, is expressed in limb and branchial arch mesenchyme; i.e., along the routes and at the targets of the migratory cells. Ectopic application of SDF1 in the chick limb attracts muscle progenitor cells. In CXCR4 mutant mice, the number of muscle progenitors that colonize the anlage of the tongue and the dorsal limb was reduced. Changes in the distribution of the muscle progenitor cells were accompanied by increased apoptosis, indicating that CXCR4 signals provide not only attractive cues but also control survival. Gab1 encodes an adaptor protein that transduces signals elicited by tyrosine kinase receptors, for instance the c-Met receptor, and plays a role in the migration of muscle progenitor cells. We found that CXCR4 and Gab1 interact genetically. For instance, muscle progenitors do not reach the anlage of the tongue in CXCR4;Gab1 double mutants; this target is colonized in either of the single mutants. Our analysis reveals a role of SDF1/CXCR4 signaling in the development of migrating muscle progenitors and shows that a threshold number of progenitor cells is required to generate muscle of appropriate size.

Figure 1.

Generation of the Lbx1^GFP mutant allele. (A) Schematic representation of the targeting vector, the wild-type Lbx1 locus, and the mutated Lbx1 allele before and after removal of the neomycin (neo) cassette. The Lbx1 gene has two exons (yellow boxes); the first was interrupted by the insertion of a Gap43-GFP cassette (green box). In addition, a frame-shift mutation (indicated by a black line) was introduced into the BglII site of exon 2 that encodes the Lbx1 homeodomain. Neomycin (neo) and thymidine kinase (tk) cassettes present in the targeting vector were used for positive and negative selection. The positions of the probes, A and B, used for Southern analysis are shown by black and red bars, respectively. The predicted fragment sizes obtained after HindIII (H) and BamHI (B) digestion of genomic DNA are indicated. In addition, the following restriction enzyme sites are indicated: ClaI (C), Sse8387I (S), and NheI (Nh). (B) Southern blot analysis of HindIII-digested genomic DNA from wild-type and Lbx1^GFP/+ ES cells using probe A for hybridization (left) and BamHI-digested genomic DNA from wild-type, Lbx1^GFP/+, and Lbx1^GFP/GFP F1 animals using probe B for hybridization (right). (C) Immunohistological analysis of a forelimb section of an Lbx1^GFP/+ embryo at E10 stained with anti-Lbx1 (red) and anti-GFP (green) antibodies. Bar, 125 μm.

Figure 2.

Expression of CXCR4 and SDF1 in mouse and chick embryos. (A-F,H) In situ hybridization of chick embryos at HH25 (A,B) and mouse embryos at E10.25 (C-F) using probes specific for CXCR4 (A,C,E) and SDF1 (B,D,F). (G,H) Consecutive sections, displayed as mirror images, through the first branchial arch of wild-type mouse embryos at E10.25 were stained with antibodies against CXCR4 (red) and Lbx1 (green) (G), or hybridized with an SDF1-specific probe (H). CXCR4 and Lbx1 were coexpressed in muscle progenitors migrating toward the tongue anlage (arrowhead in G), whereas SDF1 transcripts were detected in the mesenchyme of the first branchial arch (arrows in H). Bars: A,B, 500 μm; C-H, 250 μm.

Figure 3.

Expression of CXCR4, Lbx1, and MyoD in limb muscle progenitors. (A-D) Sections of the forelimb of E10.25 (A,D), E10.75 (B), and E11.25 (C) mouse embryos stained with anti-CXCR4 (red) and anti-Lbx1 (green) antibodies. CXCR4⁺ and Lbx1⁺ cells are present in the muscle masses, and analysis at high magnification demonstrates that they are present in the same cells. (D) Note that not every Lbx1⁺ cell expressed CXCR4, but all CXCR4⁺ muscle progenitor cells were also positive for Lbx1. (A) In addition, CXCR4 was also present in limb endothelial cells (arrowhead). (E,F) Section of the forelimb of mouse embryo at E10.25 (E) and E11.25 (F) stained with anti-CXCR4 (red) and anti-MyoD (green) antibodies. CXCR4⁺/MyoD⁺ double-positive cells were very rare. (G) Schematic representation of gene expression in developing muscle progenitors. CXCR4 expression is induced after muscle progenitors have delaminated and have reached the limb, and is extinguished prior to their differentiation. Bars: A-C,E-F, 250 μm; D, 30 μm.

Figure 4.

Muscle progenitors are attracted by an ectopic source of SDF1. COS1 cells cotransfected with SDF1 and GFP expression plasmids were implanted into the right wing bud of chick embryos at HH19-20. The distribution of muscle progenitor cells was analyzed at HH25 in the untreated (A,D,G) and treated (B,E,H) contra-lateral limb by in situ hybridization using chCXCR4-specific (A,B), chPax3-specific (D,E), and chMyoD-specific (G,H) probes. (C,F,I) The positions of the GFP positive implants are shown and are also indicated by arrows. Note the aberrant position of the CXCR4⁺ and Pax3⁺ progenitor cells and the reduction of the MyoD signal in the limb implanted with SDF1-expressing cells. (J,K) COS1 cells transfected with GFP expression plasmid only (J) or COS1 cells cotransfected with SDF1 and GFP expression plasmids (K) were implanted into the limb bud, and the distribution of muscle progenitors was analyzed on sections after in situ hybridization using chCXCR4. The position of the implant is indicated. (L) Western blot analysis of supernatant from COS1 cells transfected with a plasmid encoding SDF1 (right lane); as a control, a plasmid encoding GFP was transfected (left lane). Bars, 500 μm.

Figure 5.

Migration of muscle progenitors along the hypoglossal cord. Sections of the first branchial arch of CXCR4^+/- (A), CXCR4^-/- (B), Gab1^-/- (C), and CXCR4^-/-Gab1^-/- (D) embryos at E10.75 were analyzed with anti-Lbx1 (red) and anti-MyoD (green) antibodies to identify muscle progenitor cells. In control embryos, muscle progenitors were observed along the hypoglossal cord (arrow) and colonized the mesenchyme of the first branchial arch, the target (arrowhead). Note the reduction in the numbers of muscle progenitors in the first branchial arch of CXCR4^-/- and Gab1^-/- embryos, and their absence in CXCR4^-/-Gab1^-/- embryos. Bar, 250 μm.

Figure 6.

Distribution of muscle progenitor cells in the limb. (A-D) Sections of forelimbs of CXCR4^+/- (A), CXCR4^-/- (B), Gab1^-/- (C), and CXCR4^-/-Gab1^-/- (D) embryos at E10.75 were analyzed with anti-Lbx1 (red) and anti-MyoD (green) antibodies. (E-H) Quantification of the numbers of Lbx1⁺ and TUNEL⁺ cells located in distinct domains of forelimbs of embryos with the genotypes CXCR4^+/- (blue bars) and CXCR4^-/- (green bars) (E,F); and Gab1^-/- (orange bars) and CXCR4^-/-Gab1^-/- (yellow bars) (G,H). For this, consecutive sections of E10.75 embryos were analyzed by immunohistochemistry, and the Lbx1⁺ or TUNEL⁺ cell numbers were counted on every third section in the different limb domains; (^★) p value < 0.007, n = 5; (^★★) p value < 0.005, n = 4 (see also Material and Methods for further details). (I) Schematic drawing of a developing limb and the four domains defined therein: dorsal proximal (I), dorsal distal (II), ventral proximal (III), and ventral distal (IV). Bar, 250 μm.

Figure 7.

Differentiated muscle groups of the limb and tongue. (A-D) Sections of the tongue of CXCR4^+/- (A), CXCR4^-/- (B), Gab1^-/- (C), and CXCR4^-/-Gab1^-/- (D) embryos at E13.5 stained with antibodies to myosin (green) and MyoD (red). (A) The intrinsic and extrinsic tongue muscles are indicated by an arrow and arrowhead, respectively. (E-H) Transverse sections through the proximal part of the lower forelimb of CXCR4^+/- (E), CXCR4^-/- (F), Gab1^-/- (G), and CXCR4^-/-Gab1^-/- (H) embryos at E13.5 stained with antibodies to myosin (green) and MyoD (red). (E) Indicated are extensor (ex) and flexor (fl) muscles; arrowhead and arrow point toward the radius and ulna, respectively. Bars, 250 μm.