Cartilage, muscle and neural crest defects in nls. (A-D) Antimyosin immunostaining of muscles in whole-mounted wild-type (A,C) and nls (B,D) embryos at 3.5 days. As shown in lateral view (A,B), in wild types, both dorsal and ventral muscles of the mandibular and hyoid arches are present, but shortened in nls, which is clearer for the ventral muscles in ventral view (compare C with D). In branchial arches both dorsal pharyngeal wall muscles and the transverse ventral muscles (black asterisks) are absent in nls (white asterisk). (E,F) Alcian Blue staining of cartilages in whole-mounted wild type (E) and nls mutants (F) at 120 hpf, ventral views. Wild types form five ceratobranchial elements (asterisks), and in nls all but the first are deleted, as are the small hypobranchial and basibranchial cartilages in these segments at the midline. Note the absence of the pectoral fin skeleton (pec). (G,H) Immunostaining of branchial pouches with Zn8 antibody. Pouches 3 and 4 are absent in nls. Whole-mount in situ hybridisation of wild-type (I-L) and nls embryos (I′-L′′. (I,I′) Dorsolateral views of dlx2 expression in three streams of migrating cranial neural crest in both wild type and nls (m, mandibular; h, hyoid; b, branchial stream). (J,J′) Lateral views at 40 hpf showing dlx2 expression in arch primordia. Expression in the branchial arches is lost in nls. (K,K′) Lateral views at 32 hpf showing TUNEL staining of apoptotic cells in the lens and trigeminal ganglion of wild-type embryos (arrowhead), as well as in migrating neural crest cells in the branchial arches in nls mutants (K′, arrow). (L,L′) Dorsal views at 24 hpf, showing Acridine Orange staining of apoptotic cells in the anterior end of the notochord in nls (L′, arrows). ah and ao, dorsal hyoideal muscles; cb, ceratobranchial; ima, intermandibularis anterior; imp, intermandibularis posterior; ih, interhyoideus; h, heart; hh, hyohyoideus; lap and do, dorsal mandibular muscles; sh, sternohyoideus; pec, pectoral fin.
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