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 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 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 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 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 . 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 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 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. M. and to standard methods (Pharmacia). GST/Pax3(CT) was also further M. G., unpublished results). purified from SDS-polyacrylamide gels using UV shadowing and In this study, we show that Pax3 and Lbx1 are coexpressed in elutrap (Schleicher and Schuell) elution. H6/Pax3(CT) was purified all migrating hypaxial muscle precursors. We have examined the from BL21(DE3)plysS bacteria using a denaturing urea procedure function of Lbx1 in hypaxial muscle precursors by inactivating (Qiagen) and was column renatured prior to elution with an imidazole −/− gradient. Rats (anti-Pax3) were injected and boosted twice with SDS- the Lbx1 gene in mice. Homozygous Lbx1 mice lack most treated GST-Pax3, boosted once with soluble GST/Pax3(CT) and appendicular muscles; however, six forelimb flexors and two boosted twice with soluble H6/Pax3(CT). Rabbits (anti-Lbx) were hindlimb extensors are still present in mutant newborns. In injected and boosted three times with soluble GST-fusion protein. addition, diaphragm and tongue muscles still form, High titre sera were pooled and used for affinity purification. Soluble demonstrating Lbx1 is required only for limb muscle GST-Pax3(CT) and GST-LbxI120 were coupled to a 1:1 mixture of development. The extensive loss of limb muscles in Lbx1−/− mice Affigel 10 and 15 according to manufacturer’s instructions (Biorad). results from a cell migration defect. This defect is not due to the Antibody was affinity purified according to the method of Harlow and premature differentiation of limb muscle precursors or impaired Lane (1988), concentrated by ultrafiltration, adjusted to 10% glycerol cell motility. Rather, the observed changes in cell migration and 1 mg/ml BSA then pre-absorbed with GST-whole cell extract demonstrate that Lbx1 is required for muscle precursors to beads, which were generated by coupling whole bacterial extracts of GST overproducing bacteria (Pharmacia) to Affigel 10 and 15. migrate laterally into the limbs. As a result, misplaced muscle precursors are found at the posterior-ventral and dorsal margins Antibody staining of the forelimbs and hindlimbs, respectively, thereby generating Embryos were rinsed for 1-5 hours in PBS (4°C), fixed for 1 hour in the residual hypaxial muscles that are present in Lbx1−/− mice. 4% paraformaldehyde (4¡C), and then rinsed overnight in PBS before Lbx1 controls limb muscle precursor migration 415 being equilibrated in 25-30% sucrose (4¡C) prior to embedding in At forelimb levels in E9.5-E10 embryos, Lbx1 staining was OCT (Tissue-Tek). Cryostat sections (12-20 µm) were dried and observed in the ventral lip of the dermomyotome, in cells that treated as follows: PBS at room temperature (RT), 3× 5 minutes; are delaminating (Fig. 1A). Large numbers of Pax3+/Lbx1+ methanol (−20¡C), 5 minutes; air dry; 3SB (PBS, 0.3% BM blocker cells were scattered throughout the proximal region of the limb, (Boehringer Mannheim) heat inactivated sera: 5% fetal calf, 5% goat, confirming that Lbx1 is expressed in presumptive migrating 1% chick) with 0.2% Triton X-100, 1 hour; primary antibody (rabbit limb muscle precursors (Fig. 1B). All Lbx1+ muscle precursors anti-EGFP and rabbit anti-MyoD (Santa Cruz Biotechnology), mouse coexpressed Pax3 and all migratory Pax3+ cells expressed anti-Pax7 and mouse anti- (Developmental Studies + + Hybridoma Bank) in 3SB/0.1-0.2% Triton X-100, overnight (4¡C); Lbx1. Pax3 /Lbx1 cells were seen in both the ventral and PBST (PBS, 0.1% Tween 20), 3× 10 minutes (RT); secondary dorsal muscle masses of the limb (Fig. 1B). Complete co- antibodies (Cy2-donkey anti-rat and Cy3-goat anti-rabbit (Jackson expression of Lbx1 and Pax3 was also seen in hypaxial muscle Laboratories), biotinylated goat anti-mouse (Vector Laboratories) in precursors that migrate ventrally into the diaphragm and 3SB/0.1-0.2% triton, 2-3 hours (RT); PBST, 3× 10 minutes (RT); tongue (Fig. 7, data not shown). Thus, co-expression of Pax3 streptavidin-Cy5 (Jackson Laboratories) in PBST, 1-2 hours (RT); and Lbx1 was observed in all migrating muscle precursors. PBST, 3× 10 minutes (RT). Sections were dehydrated in an Myogenin expression was examined in sections through ethanol/xylene series before mounting with DPX (BDH). Images were the forelimb that had been stained with antibodies to Pax3 recorded as three single tracks (red, blue, green) using a Zeiss LSM and Lbx1 to determine at the cellular level whether limb 510 laser scanning microscope. All figures were assembled using + Adobe Photoshop. myoblasts express Lbx1 and Pax3. Although Myogenin Histological and anatomical analysis of newborn mice Fixed newborns were dissected and muscle connections to bones were traced to identify muscles according to Gilbert (1968) and Gray’s Anatomy (Williams et al., 1989). For histology, newborn pups were killed and cut open along the abdominal midline and fixed for at least 2 days in 4% paraformaldehyde prior to dehydration in an ethanol series (8-12 hours each step). Embryos were transfered to Histoclear (8-12 hours) and then paraffin (2 hours and 12 hours) prior to embedding. 6 µm sections collected at regular intervals were stained with hematoxylin and eosin prior to mounting.

RESULTS

Lbx1 and Pax3 are coexpressed in all migrating limb muscle precursors Previous RNA in situ analyses of Lbx1 expression during embryogenesis show Lbx1 is expressed in the ventral dermomyotome at cervical and limb levels in presumptive migratory muscle precursors and later in the tongue, diaphragm and limbs (Jagla et al., 1995; Mennerich et al., 1998; Dietrich et al., 1998; 1999). However, from these studies it was not clear whether Lbx1 and Pax3 marked different or identical populations of migrating limb muscle precursors, or if Lbx1 was expressed in all migrating muscle precursors. It was also not known if Lbx1 and Pax3 were coexpressed with muscle regulatory factors (MRFs) in differentiating muscle precursors, which would suggest a potential role Fig. 1. Expression of the Lbx1, Pax3 and Myogenin proteins in limb muscle in the differentiation or patterning of hypaxial precursors. (A,B) Sections through the forelimbs of E9.5 (A) and E10 (B) wild- muscles. Antibodies against mouse Pax3 and Lbx1 type embryos stained with antibodies to detect Lbx1 (red) and Pax3 (green). Lbx1- were therefore generated and used to analyze the expressing cells are seen delaminating from the dermomyotome (arrowhead) and expression of both proteins in developing embryos. migrating muscle precursors coexpress Pax3 and Lbx1 (arrow). The forming dorsal The Pax3 antibody detected Pax3 expression in and ventral muscle masses can be seen in B. (C) Section through the forelimb of an the dorsal neural tube, dorsal root ganglia, E10.5 embryo showing Pax3 (green), Lbx1 (red) and Myogenin (blue). Myogenin (arrowhead) is only expressed in the epaxial dermomyotome (dm) but not in dermomyotome and in scattered cells in the limb, Pax3+/Lbx1+ migrating limb muscle precursors (arrow). (D) Expression of Pax3 diaphragm and hypoglossal cord. Specific Lbx1 (green) and Myogenin (blue) in wild-type E11 limbs. While both genes are antibody staining was present in the marginal zone expressed in overlapping domains in the limb, very few Pax3+ cells co-express of the neural tube, as well as in migrating limb Myogenin. dm, dermomyotome; dmm, dorsal muscle mass; fl, forelimb; vmm, muscle precursors. ventral muscle mass. 416 M. K. Gross and others cells were clearly present in the developing epaxial 2B1 ES cell lines exhibited identical mutant phenotypes. dermomyotome (Fig. 1C,D) at E10.5, few Myogenin+ cells Heterozygous Lbx1−/+ offspring were fertile and exhibited no were present in the limb, consistent with previous studies gross developmental abnormalities. In contrast, homozygous showing that Myogenin expression in the limb begins at Lbx1−/− mutant mice died shortly after birth with striking defects E10.5 (Ott et al., 1991; Yee and Rigby, 1993). In E11 in the organization of the appendicular musculature. embryos, Myogenin+ cells were found interspersed with The cause of the perinatal lethality observed in Lbx1−/− mice Lbx1+/Pax3+ muscle precursors in both the dorsal and ventral is not known, although the respiratory problems observed in one muscle masses (Fig. 1D); however, very few, if any, cells were newborn mutant mice may be a major contributing factor. seen co-expressing Pax3 and Myogenin (Fig. 1D), or Lbx1 Homozygous Lbx1−/− embryos did not express the Lbx1 protein, and Myogenin (data not shown). Similar results were arguing that the Lbx1 mutation is a null allele. Nevertheless, it is obtained when Pax3, Lbx1 and MyoD expression was possible that the first 62 amino acids of the Lbx1 protein are compared (data not shown). From E11.5 onwards, Pax3 and translated in frame with EGFP and could therefore exhibit some Lbx1 were expressed predominantly in undifferentiated cells biological activity. that were located at the leading edge of migrating pools of limb muscle precursors. These results show that during limb muscle development muscle precursors that are initially Pax3+/Lbx1+/MRF− become Pax3−/Lbx1−/MRF+. Together, these data demonstrate that Pax3 and Lbx1 are not expressed in limb myoblasts and indicate that their downregulation may be required for hypaxial muscle precursors to differentiate. Generation of Lbx1 mutant mice To examine the function of Lbx1 in hypaxial muscle precursors, we generated a targeted mutation in the Lbx1 gene by homologous recombination. An EGFP reporter cassette was fused in frame into the Lbx1 coding region at aa 62 to trace migrating muscle precursors in Lbx1−/− mice. However, EGFP expression in both Lbx1−/+ and Lbx1−/− embryos was weak, requiring the use of an anti-EGFP antibody to detect cells that would normally express Lbx1. A frame shift mutation was also introduced into the homeobox of Lbx1 to ensure that no functional Lbx1 protein is produced (Fig. 2A). W9.5 ES cells were electroporated with the Lbx1 Fig. 2. Generation of Lbx1 knock-in mice. (A) Structure of the mouse Lbx1 locus, targeting vector and targeting vector. Two cell lines, mutated Lbx1 allele. The coding region of EGFP was cloned in frame into the first exon of Lbx1. The 6C1(A) and 2B1(B), were homeobox is indicated by a hatched box. Arrows mark transcription start sites. The 5′ and 3′ probes isolated that exhibited that were used for Southern analysis are shown as bars above the endogenous Lbx1 locus. Arrowheads homologous recombination at indicate PCR primers. The mutated Lbx1 locus is shown below the targeting vector. (B) Southern blots of EcoRI digested genomic DNA from ES cells, and wild-type and Lbx1 mutant mice derived from the Lbx1 locus (Fig. 2B). Both either the 6C1 cell line (A) or the 2B1 cell line (B). Genomic DNA digested with EcoRI gives a wild- cell lines when injected into type band of 10 kb with both probes. The 5′ probe detects an 8.5 kb band in the mutated Lbx1 allele. C57Bl6 blastocysts gave The 3′ probe detects a 5.3 kb band in the targeted Lbx1 allele. B, BglII; N, NotI; R, EcoRI; X, XhoI; germline transmission, and GFP, Green Fluorescent Protein; Neo, Neomycin resistance gene; PGK, PGK promoter sequences; TK, offspring from the 6C1 and HSV thymidine kinase gene; An, SV40 or PGK polyadenylation sites. Lbx1 controls limb muscle precursor migration 417

Lbx1 mutant mice lack most appendicular muscles the extremities of the limbs (Fig. 3). Hindlimbs were severely When heterozygous Lbx1 mice were bred, a number of reduced in girth, while the forelimbs were hyperflexed and newborn offspring with gross morphological defects were thinner than those of wild-type newborns (Fig. 3A,B). found dead in each litter. One live mutant pup was born and Anatomical dissection of mutant and wild-type P0 mice died within two hours. All abnormal mice were homozygous revealed a number of differences in the organization of the for the mutated Lbx1 allele. Lbx1−/− mice exhibited a skeletal musculature (Table 1). All hindlimb muscles were pronounced limb phenotype characterized by a severe missing, with the exception of the gluteus medius and one other reduction in muscle mass in the shoulders, pelvic girdle and unidentified hypaxial muscle. Two hindlimb suspension

Table 1. Dissection analysis of muscles of Lbx1 mutant newborns Forelimb muscle Status* Hindlimb muscle Status Muscle Status Lateral/Extensor Ilium-Leg Neck and Head supraspinatusa − gluteus medius (ilium)f + sternomastoid + infraspinatus − iliopsoas (iliacus)g − cleidomastoid + teres major − sartorius − sternohyoid + spinodeltoid − rectus femoris − geniohyoidm + triceps (3 heads) − thyrohyoid + ext. carpi ulnaris − Ischium-Leg mylohyoidm + ext. digit. communis − biceps femoris − digastricm + ext. digit. lateralis − semimembranosus − masseter + ext. carpi obliqus − semitendinosus − clavotrapezius + ext. carpi radialis longus − gracilis − scalenes + ext. carpi radialis brevis − splenius + Pubis-Leg intrinsic tonguen + Medial/Flexors adductor longish − subscapularisb + r adductor femorish − Abdominal biceps brachii + pectineush − rectus abdominusd + brachialis − unidentifiedh + external obliqued + coracobrachialis + internal obliqued + flexor carpi ulnarisc +Leg intercostal + flexor digit. profundusc + r femur-distali − diaphragm (peripheral)o + + pronator teres + tibia/fibula-distali − diaphragm (central)o + r

Suspensiond Suspensiond Axis Muscles pectoralis major + gluteus maximus − psoas minor + pectoralis minor + gluteus medius (vert.)f − along vertebral columnp + clavobrachialis − quadratus lumborumj + + spinotrapezius + iliopsoas (psoas)g − v latissimus dorsie − v caudofemoralisk − v serratus ventralis + pubococcygeusl + rhomboidius + iliococcygeusl − rhomboidius capitis +

*+, Present; −, not detected; ++, enlarged; r, reduced in size but connected appropriately; v, vestigial muscle incorrectly or not attached detected at the approximate location of the normal muscle. aSome striated strands observed in fascia on anterior scapular edge. bApproximately 20% of normal size. cProximal heads appeared normal but tapering distally was sudden and distal tendons were weak. dMuscles that connect the vertebral column or ribs to scapula, pelvis, or more distal appendicular bones; abdominal muscles connecting to the pelvis are listed seperately. eA small ribbon of striated muscle is connected by tendinous strap to the anterior proximal humerus; the approximate location of the muscle is similar to but the humerus attachment and layering with repect to a fat pad behind the limb differs from the normal lattissimus dorsi. fGluteus medius normally connects ilium and vertebrae to greater trochanter of femur; the size of this muscle and its connections to the ilium and greater trochanter appear normal but connections to the vertebrae were not detected. gIliopsoas normally connects lumbar vertebrae (psoas) and ilium (iliacus) with lesser trochanter of femur; a loosely connected muscle bundle between the normal lumbar insertion point of the iliopsoas and some tendons converging to the femur was observed. hAdductor muscle was clearly absent; a tiny muscle connecting the pubis to the lesser trochanter of femur was observed but could not be identified from the two texts cited; the attachment to femur did not fit descriptions of the three muscles connecting pubis to femur; these three muscles could not be cleanly dissected in normal newborns to allow a fourth muscle to be confirmed. iNo muscles connecting femur or tibia/fibula to more distal bones were observed. jApproximately 50-100% increase on bulk. kA small and variable band of striated muscle appears in facia connecting the vertebral column to lower leg. lHuman nomenclature according to Grays Anatomy. mMylohyoid, geniohyoid and digastric could not be dissected cleanly but seemed to be headed to their normal connections; the overall appearance of the musculature below the jaw was altered; mutant musculature was less integrated and did not result in a smooth packet of muscle from the mandible to the hyoid cartilage; jaw bones appeared slightly abnormal in shape. nTongues excised at the hyoid cartilage appeared slightly broader and and shorter than normal; sections at E13.5 show no apparent disruption of the pattern of MyoD-positive cells from base to tip of tongue. oCentral muscle bundle connecting central tendon to vertebral column was well formed but approximately 50% smaller; peripheral muscle fibers connecting central tendon to sternum and ribs were enlarged and appeared less well integrated with the tendonous membrane. pSuperficial appearance was normal; detailed dissection of muscles connecting the vertebrae to each other was not done. 418 M. K. Gross and others

muscles (quadratus lumborum, pubococcygeus) were retained, suggesting that all other suspension muscles are hypaxially derived. In the forelimbs, all extensor muscles were absent; however, a number of flexor muscles were still present (Table 1). Both extensors and flexors connecting phalanges, metacarpals, and carpals were not detected (Fig. 4C,D). Wrist flexors were all present but reduced in size. Two of three elbow flexors, the biceps brachii and coracobrachialis, were also present. A loss of extensors was also observed in muscles that surround the scapula. Muscles on the lateral (extensor) side of the scapula (supraspinatus, infraspinatus, spinodeltoid, teres major) which are derived from the dorsal muscle mass were absent, whereas the subscapularis muscle on the medial (flexor) side was only reduced in size. All muscle suspending the forelimb from the body with the exception of the clavobrachialis and latissimus dorsi muscles were present and normal, suggesting that their formation does not require Lbx1. One difference that was seen in the organization of proximal forelimb flexors in Lbx1−/− mice was the loss of the brachialis muscle, which is one of the two principal flexors of the elbow Fig. 3. Comparison of newborn wild-type and homozygous mutant joint. Further differences were seen in the anatomy of the flexor Lbx1 mice. (A,B) Sideviews of newborn wild-type and homozygous muscles that connect the humerus, radius and ulna to the carpal Lbx1−/− mutant pups showing the external features of their forelimbs (A) and hindlimbs (B). The arrows mark the limbs and show the loss bones. These flexor muscles, while still present and correctly of limb muscles in homozygous Lbx1−/− mice. attached, were truncated along the proximodistal axis of the limb. Muscle tissue was observed only at the proximal ends of

Fig. 4. Limb muscle development in wild-type and Lbx1−/− mice. (A,B) Hematoxylin and eosin stained cross sections (6 µm) through the forelimbs of a wild-type (A) and a Lbx1 mutant (B) mouse at P0. Both sections are located approximately 800 µm from the ulna-carpal joint. (A) In wild-type embryos, flexor (f) and extensor (e) muscle groups are both present. (B) In Lbx1−/− mice, only tendons (*) are present. (C,D) Hematoxylin and eosin stained mid-sagittal sections through the forelimb paws of wild-type (C) and mutant (D) P0 mice. Striated muscle (m) is only present in the paws of wild-type mice. (E-L) Longitudinal sections through E13.5 wild-type (E,G,I,K) and Lbx1−/− (F,H,J,L) embryos stained with an antibody to MyoD (red). (E,F) Flexor muscles are present in the upper forelimbs of wild-type and Lbx1−/− embryos. (G,H) Sections from midway between the elbow and wrist showing flexor muscles at this level are present in wild-type embryos and largely absent from Lbx1−/− embryo (the arrowhead marks a single flexor muscle). (I) A section located more distal to those shown in G and H showing flexor muscles extend further toward the wrist in wild-type embryos. (J) Section through the head of the humerus (h) showing the limb is contiguous with the body at this level and that MyoD+ muscle cells are located adjacent to the ulna (u, see arrowhead). (K) Section through the tibia (t) and fibula (f) showing muscles in the lower hindlimb of a wild- type embryo. (L) Cross section through the hindlimb of a Lbx1−/− embryo at the same level as K. Note the complete absence of MyoD+ cells. e, extensor muscles; f, flexor muscles; fb, fibula; h, humerus; im, intercostal muscles; m, muscle; r, radius; s, skin; t, tibia; u, ulna. Lbx1 controls limb muscle precursor migration 419

Fig. 5. Migration of hypaxial muscle precursors in Lbx1 mutant mice. (A,B) Cross sections through the forelimb of Lbx1−/+ (A) and Lbx1−/− (B) embryos at E9.5 stained with antibodies to Pax3 (green), Myogenin (blue) and c-Met (red). Cells in the ventral dermomyotome coexpress Pax3 and c-Met in both wild-type and Lbx1−/− embryos. c-Met is also expressed in cells migrating from the ventral lip of the dermomyotome. Examples of Pax3+ cells that are migrating into the forelimb are shown (arrowheads in A). In Lbx1−/− embryos, Pax3+ cells are beginning to migrate ventrally instead of entering the limb (arrowhead in B). (C,D) Cross sections through the forelimb of Lbx1−/+ and Lbx1−/− embryos at E10.5 showing Pax3+ cells in the limb of Lbx1−/+ (C) but not Lbx1−/− (D) embryos. The arrows point to c-Met expression in the dorsal limb that is still present in Lbx1−/− embryos. (E,F) Adjacent sections to C and D showing Pax3+/EGFP (Lbx1)+ cells in the limbs of a Lbx1−/+ embryo (see arrows in E). In E10.5 Lbx1−/− embryos, Pax3+/EGFP(Lbx1)+ cells (yellow) do not migrate into the forelimb (see arrow in F). (G) Cross section through the hindlimb at E10.5 showing Pax3+/EGFP (Lbx1)+ cells migrating into the hindlimb of a Lbx1−/+ embryo. (H) Cross section through the hindlimb of an E10.5 Lbx1−/+ embryos showing Pax3+/EGFP (Lbx1)+ cells are still located dorsally close to the dermomyotome (see arrow). Abbreviations: dm, dermomyotome; fl, forelimb; hl, hindlimb. both bones, giving way to tendon midway between the elbow was compared at increasing distances from the junction of the and wrist. In wild-type P0 mice, skeletal muscle tissue extends humerus and ulna bones (Fig. 4E-I). In Lbx1−/− embryos, three-quarters of the way from the elbow to the wrist (Fig. extensor muscle anlagen were completely absent at this stage, 4A,B). In contrast to the severe loss of appendicular muscles in while the developing flexor muscles showed a substantial Lbx1−/− mice, the deep muscles of the back, intercostal muscles reduction in their size midway between the wrist and elbow and ventral body wall muscles were similar to age matched (Fig. 4G,H). In wild type embryos, MyoD expression extended wild-type and heterozygous littermates (data not shown). further toward the wrist (Fig. 4I). Sections through E13.5 Lbx1−/− embryos at the level of the elbow, show the anlagen Appendicular muscles are missing in mutant for those muscles that are retained in mutants, i.e. biceps embryos brachii, coracobrachialis and wrist-flexors, were located To determine whether skeletal muscles develop in the limbs of between the humerus/ulna and ribs (Fig. 4J). This shows that Lbx1 mutant mice and are subsequently lost or whether these proximal forelimb flexor muscles form when the elbow joint is limb muscles never form, the pattern of differentiating muscles no longer separated from the body wall and suggests a model in E13.5 mouse embryos was analyzed. At this time, myoblasts to explain the selective development of some flexor muscles in in the limb have segregated into distinct populations that mark Lbx1−/− mice. the developing muscle anlagen. A complete series of cross Muscle precursors do not migrate into the limb in sections through forelimbs and hindlimbs of wild-type and − − Lbx1−/− embryos was stained with an antibody to MyoD. Lbx1 / embryos MyoD+ cells were absent from the hindlimbs of Lbx1−/− The expression of Lbx1 in migrating muscle precursors and the embryos, except for the anlage of the gluteus medius that was loss of appendicular muscles in E13.5 Lbx1−/− embryos led us located lateral to the ileum and one other unidentified muscle. to ask whether limb muscle precursors are impaired in their The number and size of MyoD+ muscle anlagen in the forelimb ability to migrate in Lbx1−/− mice. The distribution of Pax3+ 420 M. K. Gross and others muscle precursors was analyzed in a complete series of until after Lbx1 and Pax3 are downregulated (Fig. 1, data not sections through the limbs of E9.5 and E10.5 wild-type, shown). Therefore, Lbx1 may be required to delay Lbx1−/+ and Lbx1−/− embryos. At E9.5 a clear difference was in migrating hypaxial muscle precursors, and seen in the distribution of Pax3+ cells in wild-type versus Lbx1 premature muscle differentiation may cause the defects in mutant embryos. Whereas in wild-type embryos, Pax3+ cells Lbx1−/− mice. To test this hypothesis, E9.5 Lbx1−/− mutant had already invaded the limb, in Lbx1 mutant embryos these embryos were examined for evidence of precocious cells were clustered in the trunk, just beneath the ventrolateral myogenesis. Myogenin+ cells were observed beneath the lip of the dermomyotome (Fig. 5A,B). dorsal dermomyotome in both Lbx1−/+ and Lbx1−/− embryos The differences observed in the migration of limb muscle at E9.5, consistent with the early expression of Myogenin in precursors were even more pronounced at E10.5. In wild-type epaxial myoblasts described elsewhere (Fig. 5A,B). However, (not shown) and Lbx1−/+ embryos (Fig. 5C,E) Pax3+ cells had no Myogenin+ cells were seen amongst the Pax3+/EGFP+ already segregated into dorsal and ventral populations. In cells that had delaminated and failed to enter the limb in Lbx1 contrast, no Pax3+ cells were seen in the forelimbs of E10.5 mutants. At E10.5, two populations of Myogenin+ cells were Lbx1−/− mutant embryos. Instead, an enlarged stream of Pax3+ detected in the body wall adjacent to the limbs in both wild- cells was observed immediately adjacent to the ventral aspect type and mutant embryos (Fig. 5C,D, arrowhead). However, of the limb at posterior forelimb levels (Fig. 5F arrow). These there was no increase in the number of Myogenin+ cells in Pax3+ cells expressed EGFP, demonstrating that the cells Lbx1−/− embryos. Furthermore, the EGFP+ population of cells that normally express Lbx1 fail to migrate into the limbs of did not express Myogenin (Fig. 5 compare D and F). These Lbx1−/− embryos. When Pax3 expression was examined in results argue that the cell migration defect in Lbx1 mutants is sections through the hindlimbs of E10.5 Lbx1−/− embryos, a not caused by hypaxial muscle precursors differentiating pool of delaminated Pax3+/EGFP+ cells was seen just ventral prematurely. to the dermomyotome (Fig. 5H). In comparable sections through Lbx1−/+ hindlimbs, Pax3+/EGFP+ cells were seen in Altered cell migration leads to the formation of some the limb (Fig. 5G). In Lbx1−/− embryos, hindlimb muscle appendicular muscles precursors delaminate, but fail to migrate far from the ventral Our observation that Pax3+/Lbx1+ cells migrate ventrally at edge of the dermomyotome. forelimb levels (Fig. 5F), but not at hindlimb levels (Fig. In mice lacking c-Met, limb muscle precursors fail to 5H), led us to investigate whether this difference could migrate into the limb, resulting in the complete loss of limb account for the different patterns of muscle development that muscles (Bladt et al., 1995; Maina et al., 1996) and raising occur in the forelimbs and hindlimbs of Lbx1−/− mutants. the possibility that c-Met is no longer expressed in Lbx1−/− Expression of Pax3 and Myogenin was analyzed in a embryos. When c-Met expression was examined at E9.5, complete series of serial sections through E11.5 forelimbs strong c-Met staining was observed in the ventral and hindlimbs to identify populations of migrating muscle dermomyotome in both normal and Lbx1−/− embryos (Fig. precursors (Pax3+) and myoblasts (Myogenin). In wild-type 5A,B). c-Met receptor expression was also seen in E11.5 embryos, large numbers of Pax3+ cells were seen delaminating muscle precursors, demonstrating that the within the limb as expected. These cells were located both failure of limb muscle precursors to enter the limb in dorsally and ventrally where they had begun to separate into Lbx1−/− embryos is not due to the loss of c-Met. This finding muscle anlagen (Fig. 6A). When sections through the is consistent with the different morphologies of the ventral posterior forelimbs of Lbx1−/− mice were analyzed, a large dermomyotome which we observe in Lbx1 mutant embryos pool of Pax3+ cells was observed immediately adjacent to compared to that reported for c-Met mutant embryos. In the ventral aspect of the limb (Fig. 6B). These muscle Lbx1−/− embryos, muscle precursors delaminate from the precursors (Fig. 6B arrow) did not appear to be migrating dermomyotome, whereas in c-Met−/− embryos these cells into the limb, and instead clustered at the junction of body retain their epithelial morphology (Dietrich et al., 1999). wall and ventral limb. Myogenin+ cells were distributed Together, these results suggest that Lbx1 and c-Met regulate throughout this posterior-ventral pool of Pax3+ cells, different morphogenetic events in migrating muscle indicating limb muscle precursors had already begun to precursors. Interestingly, we also noted that antibodies to c- differentiate adjacent to the ventral limb. Furthermore, this Met did not detect large numbers of Pax3+ cells that co- pool of cells appear to be sufficiently close to the limb to express c-Met in the limb proper, suggesting that c-Met is receive normal muscle patterning signals, thereby giving rise downregulated once muscle precursors delaminate from the to the biceps brachii and coracobrachialis flexor muscles, dermomyotome (Fig. 5A). but not the brachialis. Since Pax3+ muscle precursors were only present adjacent to the posterior forelimb at E11.5, we Muscle precursors do not differentiate prematurely − − conclude that all hypaxial muscle precursors in the anterior in Lbx1 / mutants half of the forelimb, including the precursors that would Myogenesis in the mouse begins at E9.5 in the dorsal normally give rise to extensors, instead migrate ventrally and dermomyotome (Ott et al., 1991); however, it is delayed in enter the septum transversum. Consistent with this the ventral lip of the dermomyotome, especially at limb and hypothesis, there is an increase in the number of Pax3+ cells cervical levels where Lbx1 is expressed. Expression of MyoD in the diaphragm at E11.5 (Fig. 7). and Myogenin in the limb does not begin until after muscle We did not observe a similarly located pool of Pax3+ cells precursor cells have finished migrating, suggesting at hindlimb levels in E11.5 Lbx1−/− embryos (Fig. 6C,D), myogenesis is not compatible with long-range cell migration. confirming our observations that muscle precursors are unable Furthermore, our results show that Myogenin is not expressed to migrate at hindlimb levels (Fig. 5G,H). Nevertheless, an Lbx1 controls limb muscle precursor migration 421

Fig. 6. Expression of Pax3 and Myogenin in E11.5 wild-type and Lbx1−/− embryos. (A,B) Pax3 (green) and Myogenin (blue) expressing cells in the forelimbs of wild-type (A) and Lbx1−/− (B) embryos. Sections are at the level of the posterior forelimb. An abnormal pool of Pax3+ cells adjacent to the ventral forelimb is marked by an arrow in B. A second more medial population of Pax3+ cells is also visible (arrowhead). The arrowheads in A mark migrating Pax3+ cells within the limb. (C,D) Sections through the hindlimb of a wild-type (C) and a Lbx1−/− (D) embryo showing Pax3+ (green) and Myogenin+ (blue) cells. Note the extensive migration of Pax3+ cells into the hindlimbs of the wild-type embryo (arrowhead in C), but not the Lbx1−/− embryo. Pax3+ and Myogenin+ cells are still present dorsally in Lbx1−/− embryos (D). (E,F) Expression of Pax3 (green) and Myogenin (blue) in the trunk at hindlimb levels in Lbx1−/− embryos (F) and wild-type embryos (E). Increased numbers of Myogenin+ cells at located at the ventral edge of the myotome in Lbx1−/− embryos (arrow). dm, dermomyotome; fl, forelimb; hl, hindlimb. increase in the number of Myogenin+ cells at the ventral edge of the epaxial dermomyotome was noted in Lbx1−/− embryos (Fig. 6E,F arrows) suggesting that cells that normally migrate laterally into the limb instead congregate just ventral to the epaxial dermomyotome. These cells may contribute to the gluteus medius muscle that is still present in the hindlimb and connects the ilium to the femur (see Table 1). This muscle is located lateral to the pelvic bone, indicating that it is an extensor and is derived from dorsal muscle precursors. In addition, the quadratus lumborum, a hindlimb suspension muscle, is enlarged in Lbx1−/− mice, suggesting hypaxial muscle precursors that do not migrate contribute to this muscle. Tongue and diaphragm muscles form in Lbx1−/− mice migration of these two populations of hypaxial muscle Lbx1 and Pax3 are not only expressed in limb muscle precursors is altered in Lbx1−/− mice, E10.5 and E11.5 embryos precursors that migrate laterally into the limb buds, but also in were examined for the presence of migrating Pax3+ cells in the muscle precursors that migrate ventrally into the thorax to diaphragm and second branchial arch. In E11.5 wild-type and generate the diaphragm muscles and into the branchial arches Lbx1−/− embryos, Pax3+ muscle precursors were seen in the to form the intrinsic tongue muscles (Fig. 7; Dietrich et al., diaphragm. However, the diaphragms of Lbx1−/− embryos at 1999; Mackenzie et al., 1998). To determine whether the ventral E11.5 consistently contained more Pax3+ cells (Fig. 7A,B),

Fig. 7. Tongue and diaphragm muscle development in Lbx1 mutant mice. (A,B) Cross sections through the developing diaphragm at E11.5 showing Pax3+ (green) muscle precursors that have migrated into the septum transversum (see arrows). Most of the Pax3+ cells co- express Pax7 (blue). The Lbx1−/− diaphragm (B) contains more muscle precursors that the wild-type diaphragm (A). (C,D) Sections at the level of the first and second branchial arch showing Pax3+ (green) cells migrating into the tongue anlage. Pax3+ cells that are weakly Lbx1+ (yellow) can be seen in C, showing Pax3+ tongue muscle precursors coexpress Lbx1. (E,F) Hematoxylin and eosin stained longitudinal sections through the thorax of wild-type (E) and Lbx1−/− (F) newborn mice, showing that muscle is present in the diaphragm (d) of Lbx1−/− mice. (G,H) Hematoxylin and eosin stained longitudinal sections through the face and tongue showing the tongue musculature is normal in Lbx1−/− mice. d, diaphragm; da, dorsal aorta; mh, mylohyoid muscle; st, septum transversum; t, tongue. 422 M. K. Gross and others suggesting that the muscle precursors that normally enter the less severe than those seen in Sp embryos. However, unlike anterior forelimb migrate inappropriately into the septum Lbx1 mutants, the tongue and diaphragm muscles as well as all transversum. The migration of tongue muscle precursors was appendicular muscles are missing (Bladt et al., 1995; Maina et also examined at E10.5 for evidence of changes in their al., 1996). migratory behaviour. A delay was seen in the migration of Although Pax3 is required for the migration of limb muscle Pax3+ cells into the second branchial arch in E10.5 embryos precursors (Daston et al., 1996), there is evidence that Pax3 (Fig. 7C,D). However, by E11.5 large numbers of Pax3+ cells also plays an early role in the differentiation of hypaxial muscle were present within the tongue primordium of Lbx1−/− mice precursors (Maroto et al., 1997, Tajbakhsh et al., 1997). In Sp (data not shown). embryos the loss of hypaxial muscles is accompanied by a Anatomical and histological investigation of newborn shortening of the dermomyotomes (Bober et al., 1994; Daston Lbx1−/− mice confirmed the presence of a muscular diaphragm et al., 1996, Tajbakhsh et al., 1997) suggesting hypaxial muscle in Lbx1−/− mice (Fig. 7E,F). While small differences were precursors may either degenerate or are not specified correctly noted in the distribution of muscle tissue within the in the absence of Pax3. Additional evidence that Pax3 controls diaphragm, i.e. there was slightly more muscle laterally in the early specification of hypaxial muscle precursors comes mutant than in wild-type newborn mice (data not shown), the from the demonstration that all trunk muscles are lost in overall organization of the diaphragm in Lbx1−/− mutant mice Myf5/Pax3 mutant mice (Tajbakhsh et al., 1997). In c-Met was similar to newborn wild-type mice (Fig. 7 c.f. E,F). No mutant mice, the underlying cause of these muscle defects is significant difference was seen in the size or organization of the failure of cells in the ventrolateral dermomyotome to the tongue muscles in Lbx1−/− mice. The muscle fibres in the undergo an epithelial to mesenchymal transition (Dietrich et tongue were multinucleate and organized into the patterned al., 1999). Unlike c-Met, Lbx1 is not required for the bundles of muscle fibres that are characteristic for the intrinsic delamination of hypaxial muscle precursors. In Lbx1−/− muscles of the tongue (Fig. 7G,H). Taken together, these embryos, tongue and diaphragm muscle precursors migrate results demonstrate that while muscle precursors are unable to ventrally along their normal routes in the embryo, whereas migrate laterally into the limb, they still migrate along ventral appendicular muscle precursors delaminate, but fail to migrate routes into the septum transversum and branchial arches to laterally. form the muscles of the diaphragm and tongue, respectively. Further evidence that these genes control different steps in the development of hypaxial muscles comes from epistasis experiments. In Sp mutant embryos, the expression of c-Met DISCUSSION and Lbx1 is severely reduced in the ventral dermomyotome, demonstrating that both genes lie downstream of Pax3 We have examined the function of Lbx1 in the development of (Mennerich et al., 1998; Dietrich et al., 1999). However, it is hypaxial muscles by inactivating the Lbx1 gene in mice. Our not known if Pax3 directly regulates the expression of either results show that the ablation of Lbx1 causes the widespread, gene, and while potential binding sites for Pax3 have been but incomplete, loss of appendicular muscle and reveals a role identified in the c-Met promoter (Epstein et al, 1996), these for Lbx1 in directing the lateral migration of muscle precursors sites have not been shown to be functionally significant in vivo. into the limbs. Our findings and a model for how these muscle defects arise in Lbx1−/− mice are discussed below. Pax3, Lbx1 and c-Met regulate distinct aspects of hypaxial muscle development Inactivation of Lbx1 leads to the loss of most appendicular muscles, whereas diaphragm and tongue muscles are spared. While the loss of appendicular muscles that occurs in Lbx1 mutants is substantial, it is less severe than the muscle phenotypes seen in Pax3 and c-Met mutant mice. In Sp (Pax3−) mice, appendicular, tongue, diaphragm and ventral body wall muscles fail to form (Franz et al., 1993; Bober et al., 1994; Goulding et al., 1994; Tajbakhsh et al., 1997). Thus, Pax3 is necessary for the development of all hypaxial muscle precursors, Fig. 8. Model showing the migration routes of muscle precursors in wild-type and Lbx1−/− including those populations that do not embryos. The upper panels show the migration of Pax3+ muscle precursors at different axial undergo long range cell migration. The levels. The migratory routes of Pax3 cells in wild-type (middle panels) and Lbx1−/− (lower defects in c-Met mutant embryos are panels) embryos are shown. Abbreviations: L, lateral; V, ventral. Lbx1 controls limb muscle precursor migration 423 c-Met does not function upstream of Lbx1, since Lbx1 is still toward the ventral limb, but not enter it, whereas at hindlimb expressed in mice lacking c-Met or its ligand, Scatter Factor levels they are unable to migrate at all. (Dietrich et al., 1999). Furthermore, expression of c-Met and The absence of a ventral migratory pathway in the hindlimb Pax3 also does not depend on Lbx1 as seen in Fig. 5. This is is consistent with the accumulation of delaminated muscle consistent with the different muscle phenotypes seen in the precursors immediately below the dermomyotome in Lbx1−/− Lbx1−/− mice compared to c-Met−/− and Sp mice. Thus, Lbx1 embryos (Fig. 5H). EGFP+ cells are present in E10.5 Lbx1−/− and c-Met function independently downstream of Pax3 to embryos at hindlimb levels (Fig. 5H), demonstrating hindlimb control migration and delamination, respectively. hypaxial muscles are still specified, and in E11.5 Lbx1−/− embryos there are increased numbers of Myogenin+ cells near Lbx1 selectively regulates muscle precursor the ventral dermomyotome. In newborn Lbx1−/− mice, the migration along a lateral pathway into the limb gluteus medius muscle is present, demonstrating that this Lbx1 is expressed in all known migrating hypaxial muscle hypaxial muscle still develops at hindlimb levels. The dorsal precursors, and contrary to expectations, some populations of location of this muscle, argues that the precursors of the hypaxial muscle precursors do migrate in Lbx1−/− embryos. gluteus medius do not need to migrate extensively for it to Thus, Lbx1 does not specify the general property of long form, hence its presence in Lbx1−/− mice. Secondly, we observe range migration in hypaxial muscle precursors. However, a substantial increase in the size of the quadratus lumborum Lbx1 is required for the migration of muscle precursors into muscle, suggesting that non-migrating hypaxial muscle the limb field. Three models could account for the loss of precursors may also be recruited to contribute to this muscle. muscle precursors in the limbs of Lbx1−/− mice. First, Lbx1 The constellation of flexors retained in the forelimbs of serves to maintain limb precursors in an undifferentiated state Lbx1−/− mice is also very specific, and argues against a simple during migration, leading to the premature differentiation of model where Lbx1 specifies dorsal forelimb muscle precursors migratory cells in Lbx1 mutants. This is unlikely since MRF (extensors), but not ventral forelimb precursors (flexors). We expression was not observed in the proximal limb buds of observe that in Lbx1−/− embryos, Pax3+ muscle precursors still Lbx1−/− embryos between E9.5 and E10.5 (Fig. 5). Second, migrate ventrally at both forelimb levels. At anterior forelimb Lbx1 serves to activate cellular components required for cell levels, these cells enter the septum transversum and contribute to motility. However, the presence of ventrally migrating EGFP+ the diaphragm (Fig. 7). At more posterior levels they are unable cells in Lbx1−/− embryos demonstrates that Lbx1 is not to do so, and instead form a pool of cells immediately adjacent required for motility per se. Finally, Lbx1 could control the to the posterior forelimb (Fig. 6). Thus, the loss of all dorsal migration of hypaxial muscle precursors by allowing them to appendicular muscles at forelimb levels most likely reflects a respond to guidance cues along their migratory routes. The failure of these muscle precursors to migrate laterally into the observation that muscle precursors in Lbx1−/− embryos can limb. Not all forelimb flexor muscles are present in Lbx1 mutant still migrate ventrally, but not laterally, argues for this model, mice. While the biceps and coracobrachialis are retained and are and suggests that Lbx1 regulates responsiveness to a lateral morphologically normal, the brachialis is missing. In wild-type migration cue emanating from the limb bud. E10 embryos, few Pax3+ cells enter the ventral half of the limb + − − at anterior forelimb levels (Fig. 8). Conversely, many Pax3 cells Ontogeny of hypaxial muscles in Lbx1 / mice are present in ventral limb posteriorly (Fig. 8). However, by E10.5 Flexors and extensor muscles in the limbs are thought to arise the ventral muscle mass is evident both in the anterior and from the ventral and dorsal muscle masses, respectively. In posterior forelimb, indicating that cells in the ventral forelimb Lbx1−/− mice, six flexor muscles are retained in the forelimb relocate anteriorly. We propose that in Lbx1−/− embryos, muscle and two extensors are still present in the hindlimb. This raises precursors do not enter the posterior forelimb and are therefore the question as to whether the defects in appendicular muscle unable to migrate anteriorly, and as a result the brachialis muscle formation in Lbx1−/− mice are primarily due to changes in cell never forms. Consistent with this, there is no pool of Pax3+ cells migration, or whether Lbx1 has an additional role in specifying adjacent to the anterior forelimb in E11.5 Lbx1−/− embryos (data subsets of appendicular muscle precursors. Our detailed not shown). Nevertheless, muscle precursors that are present at examination of hypaxial muscle precursor migration, combined posterior forelimb levels can give rise to the biceps brachii and with an in depth analysis of the muscle defects in E13.5 and P0 coracobrachialis without migrating into the limb. Thus, the loss mutants, explains how certain appendicular muscles persist in of the lateral migration pathway is sufficient to account for the Lbx1 mutants without invoking an additional role for Lbx1 in abnormal patterning of elbow flexors. muscle specification. A model that outlines the migration routes Previous studies have shown that elements of the skeletal of muscle precursors in wild-type and Lbx1 mutant embryos is primordia are the likely source of the signals that pattern shown in Fig. 8. In this model, there is only a ventral migration skeletal muscles during development (see Kardon, 1998). This route at occipital levels. Two overlapping migratory cues are together with our observations, provides an explanation as to present at anterior forelimb levels in mammals, and these direct why some muscles form in Lbx1 mutant mice. At the time that cells to migrate laterally into the dorsal limb and ventrally into the forelimb muscle anlagen are forming, the elbow joint is the septum transversum (see Fig. 8), while at posterior forelimb connected to the body wall where migrating Pax3+ muscle levels, cells migrate along a ventrolateral route. At hindlimb precursors have congregated in Lbx1−/− embryos, thus allowing levels there is a lateral pathway, but no corresponding ventral them to be patterned by the adjacent appendicular skeleton (Figs route. With this model, we can explain the presence of all of 4 and 6). Although hindlimb muscle precursors do not appear the residual muscles in Lbx1−/− mice by assuming that Lbx1 is to migrate, they do detach from the dermomyotome and are required only for lateral migration, not ventral migration. Thus positioned close to the dorsal pelvis. Thus, in all cases it appears in Lbx1−/− embryos, forelimb muscle precursors can migrate that the muscles that form in Lbx1−/− mice do so because they 424 M. K. Gross and others are located near elements of the appendicular skeleton that play Pax3 modulates expression of the c-Met receptor during limb muscle an instructive role in patterning the developing muscle anlagen. development. Proc. Natl. Acad. Sci. USA 93, 4213-4218. The observation that abnormally migrating hypaxial muscle Fan, C.-M. and Tessier-Lavigne, M. (1994). Patterning of mammalian somites by surface ectoderm and notochord: evidence of sclerotome precursors still form anatomically correct muscles, indicates induction by a hedgehog homolog. Cell 79, 1175-1186. muscle precursors are naive and can be programmed to form Franz, T., Kothary, R., Surani, M. A., Halata, Z. and Grim, M. (1993). The different types of hypaxial muscles. Thus, it appears that the Splotch mutation interferes with muscle development in the limbs. 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