The Splotch mutation interferes with muscle ... - Springer Link

Anat Embryol(1993) 187:153 160

Anatomyand

Embryology

9 Springer-Verlag 1993

The Splotch mutation interferes with muscle development in the limbs T. Franz 1, R. Kothary 2, M.A.H. Surani 3, Z. Halata 4, and M. Grim s l AnatomischesInstitut, Abteilungfiir Neuroanatomie, Universiffits-KrankenhausEppendorf, Martinistrasse 52, W-2000 Hamburg 20, Germany 2 Institut du Cancer de Montreal, 1560, rue Sherbrooke est, Montreal, P.Q., Canada H2L 4MI 3 Department of Molecular Embryology,AFRC Institute of Animal Physiologyand Genetics Research, Cambridge Research Station, Babraham, Cambridge, UK 4 AnatomischesInstitut, Abteilungffir funktionelleAnatomic, Universiffits-KrankenhausEppendorf, Martinistrasse 52, W-2000 Hamburg 20, Germany 5 Institute of Anatomy,Charles University,U nemocnice3, CS-128 00 Prague 2, CSFR Accepted October 13, 1992

Summary. Homozygosity for the Splotch mutation causes neural tube and neural crest defects in mice. It has been demonstrated that Splotch mutant mice carry mutations in the homeodomain of the Pax-3 gene. Pax-3 is expressed in the neural tube, some neural crest derivatives, the mesenchyme of the limb bud and the somites. We have examined the development of the somite-derived skeletal muscles in homozygotes carrying the Splotch (Sp ~n) mutation. Our results suggest that the Splotch mutation affects the development of skeletal muscles in a region-specific way: 1. The expression of the CMZ transgene in homozygotes reveals a disorganisation of the dermomyotome in whole stained embryos. 2. The axial musculature is reduced in size along a rostro-caudal gradient. 3. The muscle anlagen in the limbs develop much more slowly. Muscles of the head and the ventral body wall are normally developed in the mutant on day 13.5 of gestation. Recently, it has been shown that the myogenic precursors of the limbs are derived from the lateral half of the somite. The specific disturbance of muscle development in the limbs of Splotch mutants thus suggests a role for Pax-3 in the organisation of the somite, the production of trophic factors in the limb mesenchyme or an alteration of myogenic and mesenchymal cells. Key words: Splotch mutation muscle - Mouse

Pax-3 gene - Skeletal

Introduction Homozygosity of the Splotch mutation (Sp) on chromosome I of the mouse causes defects in the closure of Correspondence to : T.

Franz

the neural tube (Auerbach 1954) and defects in neural crest-derived tissues such as pigment cells (Auerbach 1954), spinal ganglia (Auerbach 1954; Moase and Trasler 1989), Schwann cells (Franz 1990; Grim et al. 1992), sympathetic trunk (Auerbach 1954) and the septum of the truncus arteriosus (Franz 1989). Homozygous mutants die on day 14 of gestation presumably because of the malformation of the cardiac outflow tract (Auerbach 1954; Franz 1989). Heterozygotes are fully viable and fertile and only show a white belly spot. Several radiation-induced alleles, e.g. Sp TM and Sp zn, have been found that reproduce the phenotype observed in the spontaneously arisen Splotch mutant (Sp/Sp). Most recently, it has been demonstrated that the Sp zH Splotch mutation shows a small deletion in the homeodomain of the Pax-3 gene (Epstein et al. 1991). The Pax3 gene is a routine homologue of the drosophila paired gene family. Its pattern of expression in the mouse during embryogenesis makes it likely that Pax-3 is involved in pattern formation and organogenesis of mammals. The Pax-3 gene is expressed in the developing central nervous system including the diencephalon, midbrain, hindbrain, and spinal cord as well as neural crest derivatives such as spinal ganglia and mesenchymal cells in the pharyngeal arches (Goulding et al. 1991). Phenotypic effects are, however, not observed in the cranial anlagen of Splotch homozygous mutants, although expression of Pax-3 is detected there in wildtype embryos. Pax-3 expression has also been observed in the somites from their formation until their dissolution, and in the limb bud mesenchyme (Goulding et al. 1991). It has previously been noted that mutations at the Splotch locus can cause alterations of the cell cycle and morphometric parameters in the somitic mesoderm (Yang and Trasler 1991). In this report, we address the question whether the Pax-3 mutation in Sp TM homozygous embryos might

154 cause a defect in somite-derived m y o g e n i c precursor cells.

Materials and methods

Mice. Sp TM is a radiation-induced allele of the Splotch locus that reproduces the Splotch mutant phenotype described for the original Splotch mutant (Auerbach 1954). Sp TM heterozygous breeding pairs in a (C3H x 101)F1 background were obtained from MRC Radiobiology. The HCMV-IEP-lacZ transgene (human cytomegalovirus immediate early gene promoter - lacZ, for short called CMZ) (Kothary et al. 1991) was introduced into Sp TM mutants by crossing mice of the transgenic CMZ33 line with Splotch heterozygotes. Transgenic Splotch heterozygotes were identified by the presence of a white belly spot and the presence of the transgene in the genomic DNA isolated from tail biopsies. The transgene was identified by Southern hybridization of genomic DNA with a lacZ-specific probe using standard procedures (Sambrook et al. 1989).

Whole-embryo staining for lacZ activity. Embryos were removed from the uterus and dissected from the yolk sac and amnion. Embryos on day 11.5 of gestation were fixed in 3% paraformaldehyde for 10 rain and then permeabilized and whole-stained for lacZ activity as described (Kothary et al. 1991). Embryos were then embedded in methacrylate (Technovit 7100, Kulzer, FRG) according to the manufacturer's recommendations. Sections of 2 to 3 gm thickness were cut on a Reichert rotation microtome and counterstained with nuclear fast red. Preparation of histological specimens. Pregnant females were killed by cervical dislocation and the embryos removed from the uterus. The embryos were freed from the visceral yolk sac and amnion and fixed by immersion in either Carnoy's solution for haematoxylin-eosin stains or in 4% paraformaldehyde for immunocytochemistry. Embryos were then dehydrated in graded alcohols and embedded in paraffin (Paraplast). Sections of 4 gm thickness were cut on a Reichert and Jung (FRG) rotation microtome. The sections were stained with haematoxylin/eosin and the slides mounted with Eukitt. Other embryos were fixed in phosphate-buffered 6% glutaraldehyde and embedded in Epon as described (Franz 1990). Semithin sections were cut on a OMU3 Reichert microtome and stained with toluidine blue/pyronin red (Ito and Winchester 1963). Ultrathin sections of 70 nm were taken on an Ultracut E microtome (Reichert and Jung) and contrasted with lead citrate and uranyl acetate (Reynoids 1963). Sections were examined in a Philips EM 300 electron microscope.

Immunohistochemistry. Rabbit antiserum to desmin was purchased from Laboserv (FRG). Sections were incubated with primary antibody overnight and then treated with biotinylated sheep anti-rabbit Ig F(ab)2 (Dianova) and streptavidin-biotin-peroxidase complex (Dianova). The peroxidase activity was visualized by incubation in 0.05M Tris-BSS (pH7.6), 0.1% Triton X-100 containing 0.025% 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma) and 0.07% NiCI 2 and 7 gl per 100 ml HzO 2 (30%) for 10 rain at room temperature.

Results

Early defects in somites of Sp TM homozygotes The C M Z transgene has previously been s h o w n to be expressed at truncal levels o f e m b r y o s on day 10.5 o f gestation in spinal ganglia and the m y o t o m e cells o f

the d e r m o m y o t o m e ( K o t h a r y et al. 1991) (Fig. 1 a). In whole-stained n o r m a l embryos, the m y o t o m e cells can be seen as long spindle-shaped cells that are organised in several layers adjacent to the d e r m a t o m e and are oriented with their long axis parallel to the cranio-caudal axis o f the e m b r y o (Fig. I c). In n o r m a l embryos, lacZ activity is also detected in cells with the same p h e n o t y p e as the m y o t o m e cells, which apparently migrate ventrally f r o m the m y o t o m e s in a segmented pattern (Fig. I c, d). These are only observed in thoracic and l u m b a r segments (Fig. 1 a) and are tentatively identified as d e r m o m y o t o m e cells, which migrate ventrally to supply the ventral b o d y wall with m y o g e n i c cells. In whole-stained Splotch h o m o z y g o u s m u t a n t embryos, the m o s t obvious difference in lacZ expression f r o m the C M Z transgene was the absence o f lacZ activity in the sites o f the spinal ganglia, which are n o t f o r m e d in caudal segments o f this m u t a n t , and o f the cells extending ventrally f r o m the m y o t o m e s (Fig. I b, e). Moreover, in m o s t h o m o z y g o t e s , the lower thoracic and lumbar m y o t o m e s s h o w e d a peculiar disorganisation o f the m y o t o m e . The CMZ-expressing m y o t o m e cells were n o t oriented parallel to the longitudinal axis, but had an u n o r g a n i s e d appearance (Fig. 1 f). The spindle-shaped p h e n o t y p e o f the cells was the same as in the wildtype embryos. The n u m b e r o f m y o t o m e s as revealed by the expression o f lacZ f r o m the C M Z transgene was also appropriate, when c o m p a r e d to age-matched wildtype e m b r y o s at a given stage o f development.

Fig. la-f. Whole embryos stained for lacZ activity; embryos carrying the CMZ transgene on day 10.5 of gestation (a, b, e) and day 11.5 of gestation (c, d, f). a Wildtype embryo. Most of the myotomes, except for the occipital ones (black arrow) are covered by the intensely stained spinal ganglia (white arrowhead). In thoracic and lumbar segments, lacZ-positive cells have apparently migrated ventrally from the myotomes (black arrowhead). LacZ activity is also seen in the olfactory pit and several cranial ganglia (not indicated), b SplH/Sp TM embryo, lacZ activity in the myotomes can be observed in all segments (black arrow), because the spinal ganglia (white arrowhead) are not formed in thoracic and lumbar segments. Note the absence of the ventrally migrating cells in the mutant compared with the wildtype in a. e Wildtype embryo. Higher magnification of the thoracic area. LacZ activity demonstrates long spindle-shaped cells in the myotomes (black arrow) and the ventrally migrating population (black arrowhead). These cells are oriented parallel to the longitudinal axis of the embryo. Spinal ganglia, white arrowhead, d Wildtype embryo. Cross-sectioned slice of the lumbar region. The ventrally migrating cell population (black arrowhead) is continuous with the lacZ-positive cells in the myotome (black arrow), e Wildtype (left) and spln/sp TM embryo (right). The ventrally migrating lacZ-positive cell population (black arrowhead) is more condensed on day 10.5 of gestation than on day 11.5 (e). It is absent in the mutant embryo. In this mutant embryo the myotome cells (black arrow) in thoracic segments are arranged in a parallel longitudinal order, f SplH/Sp TM embryo. In this mutant embryo, the lacZ-positive myotome cells in lumbar segments (black arrows) are not organized in layers, but show some disorganisation of the myotome. The lumbar neural tube defect (spina bifida, Sb) of the mutant is lined with laeZ-positive cells at the epidermal/ neuroectodermal boundary

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Fig. 1 a - f

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Fig. 2a-f. Skeletal muscle development in wildtype embryos (a, b, e) and SplH/Sp TM embryos (d, e, f) on day 13.5 of gestation. a, d Immunohistochemical detection of desmin in cross sections through the neck. There is no difference in the development of the cervical axial muscles between mutant and wildtype. Paraffin section, 6 p-m; x 75. N, neural tube; V, vertebral arc; C, common carotid artery, b, e Cross sections through the upper thorax. Paraffin, 5 p,m, haematoxylin/eosin; x 48. The axial muscles in the mu-

tant are slightly reduced. The shoulder muscles (arrowheads)originating from the scapula (S) are much less developed in the mutant. The musculus latissimus dorsi (L) appears reduced in size. R, rib. e, f Shoulder muscles, brachial muscles (B) and antebrachial muscles (A) are only represented by faint tissue condensations in the mutant embryo. H, humerus. Paraffin, 5 p-m, haematoxylin/eosin; • 48

Muscle formation in the head and the trunk of SpTM homozygotes on day 13.5 of gestation

is n o t d e t e c t a b l e after the d i s s o l u t i o n o f the d e r m o m y o t o m e , a n d o n l y r e a p p e a r s in skeletal muscle o n d a y 14 o f gestation. M u s c l e m o r p h o g e n e s i s was t h e r e f o r e s t u d ied h i s t o l o g i c a l l y in serial sections a n d i m m u n o h i s t o c h e m i c a l l y for d e s m i n expression. G r e a t care was t a k e n to select h o m o z y g o u s m u t a n t e m b r y o s t h a t h a d g r o w n to the s a m e size as their wild-

As Sp TM h o m o z y g o u s m u t a n t s die o n d a y 14 o f g e s t a t i o n , the latest stage o f m u s c l e d e v e l o p m e n t t h a t c o u l d be i n v e s t i g a t e d , was d a y 13.5 o f g e s t a t i o n . L a c Z e x p r e s s i o n f r o m the C M Z t r a n s g e n e in the d o r s a l axial muscles

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Fig. 3 a-d. Skeletal muscle development in the forearm of wiidtype (a, e) and SplH/SpTM embryos (b, d) on day 13.5 of gestation. a, b Epon, semithin sections, toluidin blue/pyronin red, x 112. e, d Immunohistochemical detection of desmin. Paraffin, 6 gin, x 75. a, b Individual extensor (E) and flexor (F) muscles have been formed in the wildtype, separated from one another by connective tissue (a). Two flexor anlagen can be detected in the mutant; they appear less condensed than in the wildtype. The extensor anlagen in the mutant are represented by a diffuse mesenchymai condensation. R, radius; U, ulna. e, d Wildtype embryos show individual extensor (E) and flexor (F) muscles, which express the muscle-specific protein desmin, whilst those of mutant embryos do not. R, radius; U, ulna

type littermates. Those homozygotes that showed both a lumbo-sacral and cranial neural tube defect were excluded f r o m this study, because they were usually smaller than the wildtype embryos or those homozygotes with spina bifida alone. Deviations f r o m the wildtype phenotype should therefore not be caused by a general retardation of development. In Splotch homozygotes on day 13.5 of gestation, the development of the extrinsic ocular muscles, the tongue and the facial muscles, which are derived f r o m the prechordal plate, the cranial somitomeres and occipital somites respectively (Hazelton 1970; N o d e n 1983, 1984; Jacob et al. 1984) was indistinguishable from those in wildtype littermates. The dorsal axial muscles at cervical and thoracic levels were formed and expressed desmin in the homozygous Splotch embryos (Fig. 2d), but their volume in cross sections appeared increasingly reduced along a rostro-caudal gradient (Fig. 2e). In lumbar sections, the dorsal axial muscles in the m u t a n t were represented by

a condensation of cells that did not express desmin (not shown). In the trunk, the segmentally derived intercostal muscles and the three layers of muscle in the abdominal wall were histologically normal and showed the same pattern of desmin expression in wildtype and Splotch m u t a n t embryos (Fig. 4a). In the cranial tail of h o m o z y g o u s mutants, where the neural tube did not close, a single pair of muscles could be identified lateral to the vertebrae (Fig. 4c). Cells in this muscle mass of the Splotch m u t a n t expressed desmin.

Development of the muscles in the limbs In wildtype embryos on day 13.5 of gestation, the individual muscle anlagen were developed (Figs. 2b, c and 3 a). The differentiated muscle tissue of these muscle anlagen could be detected by immunoreactivity for desmin

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Fig. 4a-d. Skeletal muscle development in Spln/Sp TM embryos on day 13.5 of gestation, a, e Immunhistochemical detection of desmin, Paraffin, 5 Ixm. b Haematoxylin/eosin staining, paraffin section, 5 gm. a The three muscles of the abdominal wall (arrowheads) develop normally in the Sp TM homozygous mutant. L, liver, b As in the foreleg, the muscle anlagen in the hindleg (arrowheads) are much reduced in the mutant embryo. F, femur. x 48. c The single lateral muscle anlage in the mutant tail shows desmin expression (star). The neural tube (NT) is not closed at this level, x 75. d The electronmicroscopic investigation of the muscle anlagen in the proximal hindleg reveals the scarce presence of myotubes that contain myofibrils (arrowhead). x ca 8,000

in the shoulder, brachial and antebrachial regions (Fig. 3c) as well as in the corresponding segments of the hindlimb (not shown). Electronmicroscopically, these muscle anlagen were composed of numerous m y o tubes that contained a b u n d a n t myofibrils (not shown). In Sp TM homozygotes, the shoulder muscles (M. serratus anterior, M. teres m a j o r et minor, M. subscapularis, M. supraspinatus, M. infraspinatus and M. latissimus dorsi) were m u c h reduced in size and only represented by sparse condensations of cells (Fig. 2 e, f). In the limbs of Sp 1H homozygotes, the formation of individual muscles had not progressed as far as in matched wildtype controls. In the forelimb, the premuscular masses of the extensors were represented by a diffuse condensation of cells and those of the flexors by two small muscle masses (Fig. 3b). Desmin expression was not detected at all in the forelimbs of Splotch h o m o zygous embryos (Fig. 3 d). In the hindlimb, muscle devel-

opment had progressed to form small muscular masses (Fig. 4b), in which myofibril formation could be detected electronmicroscopically in isolated myotubes (Fig. 4d). The cartilaginous anlagen of the limb bones developed normally as c o m p a r e d with wildtype controls (Figs. 2 f and 4b). Similarly, the differentiation of the f o r m of the hand- and footplate was not affected in homozygous m u t a n t embryos. Discussion

The Splotch mutation has previously been characterized as causing neural tube defects and defects of neural crestderived tissues in mice (Auerbach 1954; Franz 1989, 1990; Moase and Trasler 1989). In this report, we demonstrate that the Splotch mutation also interferes with the development of the limb muscles and the associated muscles of the shoulder. The dorsal axial muscles are

159 also reduced in caudal segments, albeit to a lesser degree compared to the limb musculature, whilst the muscles of the neck, the muscles of the head and the ventral body wall are not visibly affected. The development of muscles in the trunk proceeds from cranial to caudal and from proximal to distal. The defect of limb muscle formation in Sp TM homozygotes could not be caused by a general retardation of development, because in this investigation mutant embryos were selected that had grown to the same crown-rump length as the wildtype controls. The development of the external aspect of the hand- and footplate, and the bony elements of the limbs were normal compared with wildtype controls. The cranio-caudal gradient of the myotomal disorganisation in Sp TM homozygotes corresponds well with the cranio-caudal gradient of the neural crest defect (Auerbach 1954; Franz 1990) and with the gradient of axial muscle dysgenesis (this report). This might either suggest a parallel independent feature of the Splotch mutant phenotype or an interdependence of neural crest emigration and the development of axial musculature. The observation that muscle differentiation in the tail is not retarded in homozygous mutants indicates, however, that there is no general delay in the cranio-caudal progression of development. Alterations of the neural tube in Splotch mutants could also not influence the development of the limb muscles, as the removal of the neural tube and notochord causes the breakdown of the somites and agenesis of the myotomal muscles, but does not inhibit the development of the limb muscles (Teillet and LeDouarin 1983; Rong et al. 1990). Occasionally, one finds among the offspring of Splotch mutants embryos with neural tube defect, but without neural crest defect. In such embryos, presumably Splotch heterozygotes, the development of the limb muscles proceeds normally (Franz, submitted). Phenotypic effects of the Splotch mutation on sclerotome-derived skeletal elements have not been detected, with the exception of the displacement of the anlagen of the vertebral arcs in the areas of the rachischisis, which may be secondary to the neural tube defect. It is now generally accepted that the limb muscles are formed by myoblasts that are derived from the somites, while the tendons and the muscle-associated connective tissue originate from the limb mesenchyme (Chevallier et al. 1977; Christ et al. 1977). The expression of the CMZ transgene in myotome cells of the mature somites shows that myotome cells of Sp IH homozygotes do differentiate in the somites and acquire a spindleshaped phenotype. The myotome in most mutants is, however, disorganised, because the myotome cells do not form a multi-layered, longitudinally oriented structure. The significance of this disorganisation of the myotome with respect to the dysgenesis of the axial muscles in Sp TM mutants is not clear. One might speculate that the disorganisation of the Splotch myotome reflects a disorganisation of the whole somite, and thus may result in a reduction of available precursors of muscle tissue. The myogenic precursors

produced would then be sufficient to supply the segmental circumference of the trunk, but not the limbs. This seems, however, unlikely, because the axial muscles at cervical levels are much less reduced than at lumbo-sacral levels, whilst a pronounced reduction of limb muscles is found in both the fore- and the hindleg. Also, if the absence of muscle formation in the limbs of the Splotch mutant were a supply problem from a common somitic precursor pool, one might expect to see exceptionally some higher degree of muscle formation at least in the proximal limb, which in fact is not observed. It has recently been shown that the myogenic precursor cells of the axial back muscles are derived from the medial half of the somite, while those migrating to the limb buds originate from the lateral half of the somite (Ordahl and LeDouarin 1992). On the other hand, it is suggested that the precursors of the ventral body wall originate from epithelial cell sheets that detach from the ventral bud of the mature dermomyotome (Christ et al. 1983). Considering the results presented in this report, it appears that the Splotch mutation: 1. Affects the myogenic derivatives of the medial half somite quantitatively along a rostro-caudal gradient 2. Does not interfere with the development of the dermomyotome-derived muscles of the ventral body wall 3. Most dramatically, impairs the development of the limb muscles and associated shoulder muscles, which originate from the lateral halves of the somites It has recently been shown, that Splotch mutant mice carry deletions in the homeodomain of the Pax-3 gene (Epstein et al. 1991). Pax-3 expression is found among other sites in the undifferentiated epithelial somites (Goulding et al. 1991). The muscles of both the ventral body wall and of the limbs originate from the lateral halves of the somites (Ordahl and LeDouarin 1992). The differences between the muscle morphogenesis in the ventral body wall and the limbs of Sp TM homozygotes are striking and may demonstrate a compartmentalisation of the lateral half of the undifferentiated somite, in which the compartments are differentially dependent on Pax-3 for their development. Pax-3 may thus be involved in the compartmentalisation process, or selectively support the survival of myogenic derivatives destined to populate the limbs. Expression of Pax-3 is also found in the limb bud, proximal and distal, on days 10 and 11 of gestation (Goulding et al. 1991). At this stage, it is unlikely that the expression in the limb bud marks migrating myogenic precursor cells (Platzer 1978), but rather labels resident mesenchymal cells. It has long been established that the resident mesenchymal cells in the limb buds determine the formation of the muscle pattern (Chevallier et al. 1977; Christ et al. 1978) and can even form muscle anlagen from non-myogenic cells (Grim and Wachtler 1991). Thus, an alternative explanation for the pronounced disturbance of limb muscle morphogenesis in Splotch mutants may be that Pax-3 expression in limb mesenchymal cells provides environmental factors for the migration and/or survival of migrating myogenic precursors in the limb.

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Acknowledgements. The authors would like to thank Mrs. S. Schwartz for excellent technical assistance. T.F. is supported by the Deutsche Forschungsgemeinschaft, Grant Nr. Fr 604/3-1. R.K. is supported by a fellowship from the Medical Research Council of Canada.

References Auerbach R (1954) Analysis of the developmental defects of a lethal mutation in the house mouse. J Exp Zool 127:305 329 Chevallier A, Kieny M, Mauger A (1977) Limb-somite relationship: origin of the limb musculature. J Embryol Exp Morphol 41 : 277-280 Christ B, Jacob HJ, Jacob M (1977) Experimental analysis of the origin of the wing musculature in avian embryos. Anat Embryol 150:171-186 Christ B, Jakob HJ, Jakob M (1978) Zur Frage der regionalen Determination der friihembryonalen Muskelanlagen. Experimentelle Untersuchungen an Wachtel- und Htihnerembryonen. Verh Anat Ges 72:353-357 Christ B, Jacob M, Jacob HJ (1983) On the origin and development of the ventrolateral abdominal muscles in the avian embryo. Anat Embryol 166:87-101 Epstein DJ, Vekemans M, Gros P (1991) Splotch (SpZH), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3. Cell 67 : 767-774 Franz T (1989) Persistent truncus arteriosus in the Splotch mutant mouse. Anat Embryol 180:457-464 Franz T (1990) Defective ensheathment of motoric nerves in the Splotch mutant mouse. Acta Anat 138:246-253 Goulding MD, Chalepakis G, Deutsch U, Erselius J, Gruss P (1991) Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J 10:1136-1147 Grim M, Wachtler F (1991) Muscle morphogenesis in the absence of myogenic cells. Anat Embryol 183 : 67-70 Grim M, Halata Z, Franz T (1992) Schwann cells are not required for guidance of motor nerves in the hindlimb in Splotch mutant mouse embryos. Anat Embryol 186 : 311 318

Hazetton RD (1970) A radioautographic analysis of the migration and fate of cells derived from the occipital somites in the chick embryo with special references to the development of the hypoglossal musculature. J Exp Embryol Morphol 24:455~466 Ito S, Winchester RF (1963) The fine structure of the gastric mucosa of the bat. J Cell Biol 16:541-578 Jacob M, Jacob HJ, Wachtler F, Christ B (1984) Ontogeny of avian extrinsic ocular muscles. Cell Tissue Res 237:549-557 Kothary R, Barton SC, Franz T, Norris ML, Hettle S, Surani MAH (1991) Unusual cell specific expression of a major human cytomegalovirus immediate early gene promoter-lacZ hybrid gene in transgenic mouse embryos. Mech Dev 35:25-31 Moase CE, Trasler DG (1989) Spinal ganglia reduction in the Splotch-delayed mouse neural tube defect mutant. Teratology 40 : 6%75 Noden DM (1983) The embryonic origins of avian craniofacial muscles and associated connective tissues. Am J Anat 168:257276 Noden DM (1984) Craniofacial development: new views on old problems. Anat Rec 208 : 1-13 Ordahl CP, LeDouarin NM (1992) Two myogenic lineages in the developing somites. Development 114:339-353 Platzer AC (1978) The ultrastructure of normal myogenesis in the limb of the mouse. Anat Rec 190:639-658 Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208212 Rong P, Teillet M, Ziller C (1990) Differential influence of the neural primordium on muscle development in myotome and limb. In: LeDouarin N, Dieterlen-Lievre F, Smith J (eds) The avian model in developmental biology: from organism to genes. CNRS Paris, pp 153-164 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor NY, Cold Spring Harbor Laboratory Teillet M, LeDouarin NM (1983) Consequences of neural tube and notochord excision on the development of the peripheral nervous system in the chick embryo. Dev Biol 98:192-211 Yang X-M, Trasler DG (1991) Abnormalities of neural tube formation in pre-spina bifida Splotch-delayed mouse embryos. Teratology 43 : 643 657