Lbx1 Controls Limb Muscle Precursor Migration

Development 127, 413-424 (2000) 413 Printed in Great Britain © The Company of Biologists Limited 2000 DEV1499 Lbx1 is required for muscle precursor migration along a lateral pathway into the limb Michael K. Gross1,*, Laura Moran-Rivard2,*, Tomoko Velasquez1, Martin N. Nakatsu1, Krzysztof Jagla3 and Martyn Goulding2,‡ 1Molecular Neurobiology Laboratory, The Salk Institute, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA 2Biology Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA 3INSERM U. 384, 63001 Clermont-Ferrand, France *These two authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected]) Accepted 5 November; published on WWW 20 December 1999 SUMMARY In mammalian embryos, myogenic precursor cells emigrate that Lbx1 is not required for the specification of particular from the ventral lip of the dermomyotome and colonize the limb muscles, and the muscle defects that occur in Lbx1−/− limbs, tongue and diaphragm where they differentiate and mice can be solely attributed to changes in muscle precursor form skeletal muscle. Previous studies have shown that migration. c-Met is expressed in Lbx1 mutant mice and Pax3, together with the c-Met receptor tyrosine kinase and limb muscle precursors delaminate from the ventral its ligand Scatter Factor (SF) are necessary for the dermomyotome but fail to migrate laterally into the limb. migration of hypaxial muscle precursors in mice. Lbx1 and Muscle precursors still migrate ventrally and give rise to Pax3 are co-expressed in all migrating hypaxial muscle tongue, diaphragm and some limb muscles, demonstrating precursors, raising the possibility that Lbx1 regulates their Lbx1 is necessary for the lateral, but not ventral, migration migration. To examine the function of Lbx1 in muscle of hypaxial muscle precursors. These results suggest that development, we inactivated the Lbx1 gene by homologous Lbx1 regulates responsiveness to a lateral migration signal recombination. Mice lacking Lbx1 exhibit an extensive loss which emanates from the developing limb. of limb muscles, although some forelimb and hindlimb muscles are still present. The pattern of muscle loss suggests Key words: Lbx1, Pax3, Hypaxial muscles, Cell migration, Mouse INTRODUCTION 1997). The precursors for hypaxial muscles exhibit markedly different morphogenetic behaviours at different axial levels of The somitic mesoderm gives rise to multiple tissues in the the embryo. Cells in the ventral dermomyotome at limb and developing embryo including bone, connective tissue and cervical levels delaminate (Chevallier et al., 1977; Christ et al., muscle. Somites form as an epithelial ball of cells that later 1977; Christ and Ordahl, 1995; Mackenzie et al., 1998) and segregate into sclerotome and dermomyotome in response to undergo long range migration to form diaphragm, tongue patterning signals that arise from adjacent tissues (Dietrich et al., muscles and appendicular muscles. In contrast, hypaxial muscle 1993; Pourquie et al., 1993; Goulding et al., 1994; Fan and precursors at interlimb levels do not migrate. Instead, they retain Tessier-Lavigne, 1994; Munsterberg and Lassar, 1995). Cells in their epithelial morphology, forming a bud that extends ventrally the ventral half of the nascent somite undergo a transition from toward the midline to give rise to ventral body wall muscles epithelium to mesenchyme, forming the sclerotome, that (Parry, 1982; Christ et al., 1983). differentiates further to give rise to the axial skeleton. Cells in A number of genes that control the development and migration the dorsolateral half of the somite retain their epithelial of hypaxial muscles have been identified. Among these are the morphology, forming the dermomyotome, which contains paired domain transcription factor Pax3, which is expressed precursors for both the dermis and for skeletal muscles. throughout the dermomyotome, and c-Met, which is expressed Dermomyotome derived muscle precursors not only generate the in delaminating hypaxial muscle precursors (Bober et al., 1994; epaxial muscles that attach to the vertebral column, but also the Goulding et al., 1994; Williams and Ordahl, 1994). Analysis of hypaxial musculature of the limb, tongue, diaphragm and ventral Splotch (Sp, Pax3−) mutants shows Pax3 is required for the body wall (Christ and Ordahl, 1995). Whereas the epaxial normal development of all hypaxial muscles. Appendicular, muscles arise from cells in the medial dermomyotome, hypaxial tongue and diaphragm muscles are missing from homozygous Sp muscles are derived from precursors in the lateral half of the embryos, while the ventral body wall muscles are greatly reduced dermomyotome (Ordahl and Le Douarin, 1992; Denetclaw et al., in size (Franz et al., 1993; Tajbakhsh et al., 1997). The loss of 414 M. K. Gross and others appendicular muscles in Pax3 mutant embryos is primarily due MATERIALS AND METHODS to a failure of muscle precursors to migrate into the limb (Daston et al., 1996). However, Pax3 also regulates muscle cell Generation of knock-in mice differentiation (Maroto et al., 1997; Tajbakhsh et al., 1997) and The Lbx1 targeting vector was assembled using the pKSloxPNT vector the dysgenesis of all hypaxial muscles in Sp embryos, including (provided by A. Joyner) that contains HSV TK and lox-P flanked those that do not migrate, suggests Pax3 may also be required for Neomycin gene cassettes in a Bluescript KS backbone. Genomic the differentiation or survival of hypaxial muscle precursors. The sequences encompassing the mouse Lbx1 gene were isolated from a c-Met receptor tyrosine kinase is also expressed in migratory 129SV genomic phage library. The coding region of EGFP was cloned in frame into a NotI site at aa 62 of the mouse Lbx1 protein. A 4.1 kb muscle precursors, and mice lacking c-Met exhibit a muscle XhoI fragment containing EGFP-pA (upstream arm) and a 3.6 kb NotI phenotype that is similar to the Sp muscle phenotype, except that fragment (downstream arm) were cloned seperately into pKSloxPNT the ventral body wall muscles are still present (Bladt et al., 1995). to generate the Lbx1 targeting vector. A frameshift in the Lbx1 c-Met is expressed in the ventral lip of the dermomyotome as homeobox was created by filling in the BglII site in exon 2 of the muscle precursors are delaminating (Daston et al., 1996; Bladt et downstream arm prior to its insertion into the targeting vector. al., 1995). In c-Met mutant mice, muscle precursors fail to W9.5 embryonic stem cells were maintained on primary fibroblast delaminate, and the dermomyotomes remain elongated at limb feeder layers supplemented with LIF. 2×107 ES cells were levels (Dietrich et al., 1999). A similar phenotype is also seen in electroporated with 25 µg of the Lbx1 targeting vector after mice lacking Scatter Factor (SF), the ligand for c-Met (Bladt et linearization with SalI. Fifty clones were screened by Southern al., 1995; Dietrich et al., 1999). Thus, c-Met activation is analysis using an upstream external probe (Fig. 1). Two clones with a recombined Lbx1 allele were identified by Southern analysis and by necessary for cells in the ventrolateral dermomyotome to undergo PCR analysis using primers for Neo and the Lbx1-EGFP boundary. an epithelial to mesenchymal transition prior to migration. Both clones were injected into C57Bl6 blastocysts to generate Pax3 and c-Met are also expressed in non-migratory chimeras. Germline founders and F1 generations were generated on a populations of dermomyotomal cells. Pax3 is expressed C57Bl6 background. Mice and embryos were genotyped by PCR using throughout the dermomyotome (Goulding et al., 1994; Daston et tail or visceral yolk sac DNA. Primers MKG396 (CAGCTGCA- al., 1996), while c-Met is expressed in the ventral dermomyotome GAAGCCAGGACTG; 12 ng/µl), MKG321 (CCGGACACGCTGA- at interlimb levels and in cells located at the dorsal tips of the ACTTGTGG; 12 ng/µl), and MKG333 (ATGACTTCCAAGGAGG- dermomyotome (Yang et al., 1996). Consequently, factors other ACGGCA; 24 ng/µl) were used in a 25 µl reaction containing Taq than Pax3 and c-Met must specify and control the migratory buffer (1.6 mM MgCl2, 0.2 mM dNTPs, 10% DMSO) and 1.25 Units behaviour of hypaxial muscle precursors. The Lbx1 gene is a Taq polymerase (Perkin-Elmer). Amplification of mutant and wild- type Lbx1 alleles generated diagnostic bands of 315 and 445 bp, candidate for regulating the migratory behaviour of these cells. respectively. Lbx1 encodes a homeodomain transcription factor that is expressed specifically in hypaxial muscle precursors that are Generation of antibodies to Pax3 and Lbx1 destined to migrate from the ventrolateral dermomyotome at Rat anti-Pax3 and rabbit anti-Lbx1 antibodies were generated against limb, cervical and occipital levels (Jagla et al., 1995). bacterial fusion proteins containing 122 aa of the Pax3 C terminus and Subsequently, cells expressing Lbx1 leave the ventrolateral 120 aa of the mouse Lbx1 protein that includes the homeodomain, dermomyotomes and migrate into the limb buds and diaphragm respectively. A 365 bp PvuII fragment from the mouse Pax3 C terminus at lower cervical and limb levels, and toward the pharynx at was inserted into SmaI/XhoI fill in vector pGEX4T-2 to generate occipital levels (Mennerich et al., 1998; Dietrich et al., 1999). GST/Pax3(CT). A fragment encoding His6-Pax3(CT) was inserted into the NheI/B cut vector pET11d. A BglII-EarI fragment from exon2 Thus, expression of Lbx1 is restricted to hypaxial muscle of the downstream mouse genomic clone was inserted into the BglII- precursors that undergo long range cell migration. In addition, EarI sites of the human LBX1 cDNA to create BS-Lbx1(C-mm). An Lbx1 is not expressed in the dermomyotome in Sp embryos, Ecl136II/Xho fragment of BS-Lbx1(C-mm) was inserted into the demonstrating Lbx1 lies downstream of Pax3 and may therefore SmaI/XhoI cut vector pGex4T2 to generate GST/Lbx(I120). Soluble contribute to the loss of hypaxial muscles that occurs in Sp GST fusion proteins were purified from BL21(DE3) bacteria according embryos (Mennerich et al., 1998; Dietrich et al., 1999; L.

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