Comparison of unc45a and unc45b mRNA expression at 48 hpf.

Wild type siblings (a,e,e′); unc45a^-/- (b,f,f′); unc45b^-/- (c,g,g′); and unc45b^-/-; unc45a^-/- (d,h,h′) mutants. unc45a expression is diffuse in the region of the brain and pharyngeal arches. unc45b mRNA expression from lateral (e–h) and dorsal (e′–h′) views, head to the left. Wild type siblings (e,e′) and unc45a^-/- (f,f′) mutants express unc45b in the extraocular (Eo), cardiac (Ca), trunk (Tr), and pectoral fin (Pf) muscles. Expression is either minimal or absent in the extraocular, cardiac, and pectoral fin muscles of the unc45b^-/- (g,g′) and unc45b^-/-; unc45a^-/- (h,h′) mutants while expression in the trunk musculature appears to up-regulated in these mutants. Apostrophes denote alternative views of the same embryo.

hsp90a mRNA is up-regulated in unc45b^-/- mutant embryos at 48 hpf.

In situ hybridization with anti-sense probes for hsp90a.1 (a,d,g,j), hsp90a.2 (b,e,h,k), and hsp90ab.1 (c,f,i,l) mRNA transcripts was performed on wild type siblings (a–c); unc45a^-/- (d–f); unc45b^-/- (g–i); and unc45b^-/-; unc45a^-/- (j–l) mutants. unc45b^-/- and unc45b^-/-; unc45a^-/- mutants displayed increased expression of hsp90a.1 (g,j) and hsp90a.2 (h,k), but not hsp90ab.1.

Whole-mount in situ hybridization of the myogenic regulatory factor myoD in cranial and trunk muscle precursors at 48 hpf.

Wild type siblings (a,a′); unc45a^-/- (b,b′); unc45b^-/- (c,c′); and unc45b^-/-; unc45a^-/- (d,d′) mutants. Compared to wild type siblings (a) myoD expression in the cranial muscle precursors is unchanged in the unc45a^-/- (b), unc45b^-/- (c), and unc45b^-/-; unc45a^-/- (d) mutants. unc45b^-/- (c′) and unc45b^-/-; unc45a^-/- (d′) mutants have increased expression of myoD in the trunk precursors compared to wild type siblings (a′) and unc45a^-/- mutants (b′). Primes denote alternative views of the same embryo. chv, constrictor hyoideus ventralis; im, intermandibularis; ir, inferior rectus; lr, lateral rectus; mr, medial rectus; pam, pharyngeal arch muscles; sh, sternohyoideus; so, superior oblique; sr, superior rectus.

Myosin localization in the craniofacial muscles of 4 dpf embryos.

Wild type siblings (a,a′); unc45a^-/- (b,b′); unc45b^-/- (c,c′); unc45b^-/-; unc45a^-/- (d,d′) mutants. Lateral (a-d) and ventral (a′–d′) views, head to the left. Expression is unaltered between wild type siblings (a,a′) and unc45a^-/- (b,b′), unc45b^-/- (c,c′) or unc45b^-/-; unc45a^-/- (d,d′) mutants. The sternohyoideus is displaced in unc45b^-/- (c,c′) and unc45b^-/-; unc45a^-/- (d,d′) embryos. Primes denote alternative views of the same embryo. ao, adductor operculi; am, adductor mandibulae; do, dilator operculi; dpw 1–5, dorsal pharyngeal wall; hh, hyohyoideus; ih, interhyoideus; ima, intermandibularis anterior; imp, intermandibularis posterior; lap, levator arcus palatini; pp, pterygoid process; sh, sternohyoideus.

Skeletal defects in unc45 mutants at 5 dpf.

Ventral views of Alcian Blue stained cartilages (a–d). Wild type siblings (a), unc45a^-/- (b), unc45b^-/- (c), and unc45b^-/-; unc45a^-/- (d) mutants. Wild type siblings and unc45a^-/- mutants (a,b) have robust cartilage staining whereas unc45b^-/- (c) and unc45b^-/-; unc45a^-/- (d) mutants exhibit decreased staining, improper angling of the ceratohyal cartilages, and shortening of the palatoquadrates and Meckel′s cartilage. The pectoral girdle (arrows) is missing or reduced in unc45b^/ (c) and unc45b^-/-; unc45a^-/- (d) embryos. cb, ceratobranchial; ch, ceratohyal; hs, hyosymplectic; mc, Meckel′s cartilage; pq, palatoquadrate. Small blue dots are an artifact of the fixation and staining process.

Morphology of 4 dpf unc45 mutants. Wild type siblings (a,a′); unc45a^-/- (b,b); unc45b^-/- (c,c′); and unc45b^-/-; unc45a^-/- (d,d′) mutants. Blood circulates through the hearts of wild type siblings (a,a′) and unc45a^-/- mutants (b,b′) but not in those of the unc45b^-/- mutants (c,c′,d,d′). Cardiac edema is most pronounced in the unc45b^-/- (c,c′) and unc45b^-/-; unc45a^-/- (d,d′) mutants and absent in wild type siblings (arrowheads). A fully inflated swim bladder is present only in wild type embryos, absent in unc45b^-/- and unc45b^-/-; unc45a^-/- mutants, and minimally inflated in unc45a^-/- mutants (arrows). Somite birefringency is reduced in unc45b^/- and unc45b^-/-; unc45a^-/- mutants compared to wild type and unc45a^-/- embryos. Apostrophes following a letter denote an increased magnification of the same embryo.

Pharyngeal arch formation and patterning of unc45 mutants is consistent with that of wild type siblings. Wild type siblings (a,e,i,m,q), unc45a^-/- (b,f,j,n,r), unc45b^-/- (c,g,k,o,s), and unc45b^-/-; unc45a^-/- (d,h,l,p,t) embryos. Expression of: hand2 at 30 hpf (a-d), dlx2a at 30 hpf (e-h), nkx2.3 at 36 hpf (i-l), nkx3.2 at 52 hpf (m-p), and sox9a at 48 hpf (q–t). Dorsal (a-d, e-h, i-l), ventral (m-p) and lateral (q-t) views. cb, ceratobranchial; p, pharyngeal arch. Numbers denote pharyngeal arch number.