Fig. 5

gcm2 is required for craniofacial development and gill filament budding. (a–d) Light microscope images of 5 dpf embryos injected with the standard control morpholino (a, b) and MO1 (c, d). (a, b) Standard control morpholino injected embryo at 5 dpf displaying normal otolith, gastrointestinal and jaw development. Mis-matched control-injected embryos (n = 160) were indistinguishable from random-sequence morpholino or uninjected control-injected embryos. (b is enlarged image of boxed section in a). (c, d) gcm2 morphants display a failure of jaw protrusion (black arrow in c), hypoplastic intestine (white arrow in d), abnormal yolk absorption and underdeveloped otoliths (red arrow in d). The morphant phenotype was generated with an ATG targeted morpholino, MO1 (approximately 7.5 ng/embryo), at 98% frequency (n = 167). The phenotype was reproduced with an injection of two morpholino oligonucleotides combined (78% frequency, n = 84): a 5′ untranslated region targeted morpholino, MO2; and a second ATG targeted morpholino, MO3 (approximately 3.75 + 3.75 ng/embryo), which partially overlaps with the MO1 sequence. d is enlarged image of boxed section in c. (e–j) Morphants display specific defects in craniofacial cartilage development at 4 and 5 dpf. Four dpf uninjected and random-sequence morpholino-injected control embryos (n = 12) (e) stained with Alcian blue displayed wild-type craniofacial cartilages for this timepoint. gcm2 morphants (n = 67) displayed either “mild” defects in craniofacial cartilage development (29% of embryos scored) (f) or “severe” defects in craniofacial development (18% of embryos scored) (g). The mis-matched control morpholino failed to produce the phenotypes described (n = 27). Five dpf uninjected and random-sequence morpholino (n = 21) control embryos (h) stained with Alcian blue displayed wild-type craniofacial cartilages for this timepoint. gcm2 morphants (n = 79) displayed either “mild” defects in craniofacial cartilage development (31% of embryos scored) (i) or “severe” defects in craniofacial development (10% of embryos scored) (j). The mis-matched control morpholino failed to produce the phenotypes described (n = 35). (k, l) Reduced calcein staining in morphants. Calcein staining of the cleithrum at 4 dpf was invariable in uninjected controls (arrowhead in k) and in mis-matched morpholino injected controls (n = 43), but was absent in MO2 + MO3 injected morphants at 4 dpf (80% of embryos scored, n = 45) (l). (m, n) Normal rag1 expression in morphants. In situ hybridisation for rag1 at 76 hpf stained the developing thymus in uninjected controls (arrowhead in m) and in morphants (arrowhead in n). (o, p) Normal nkx2.1 expression in morphants. In situ hybridisation for nkx2.1 stained the developing thyroid at 44 hpf in uninjected controls (arrowhead in o) and in morphants (arrowhead in p). (q. t). Gill filament buds were absent or vastly reduced in morphants at 98 hpf. Gill filament buds were observed under DIC microscopy in uninjected controls at 98 hpf (n = 29) (q) but were absent or vastly reduced in morphants (n = 20/26 embryos for MO1 injected and n = 28/31 for MO2 + MO3 injected) (t). Buds are indicated by arrowheads, arches 3–6 are labelled, h = heart. (r, s, u, v). Gill filament buds expressing gcm2 were absent in gcm2 morphants. in situ hybridisation for gcm2 transcripts at 80 hpf in uninjected controls (r) showed the budding of gill filament buds as ruffled staining of arches 3–6 (arrowhead) (n = 20) which was absent or vastly reduced in morphants (n = 12/14 for MO1 injected, n = 33/40 for MO2 + MO3 injected) (u). in situ hybridisation for gcm2 transcripts at 98 hpf in uninjected controls (s) also showed the budding of gill filament buds (arrowhead) (n = 30) which was absent or vastly reduced in morphants (n = 16/26 MO1 injected, n = 48/50 MO2 + MO3 injected) (v). Five mismatched control injected embryos were indistinguishable from uninjected control embryos at 98 hpf (n = 22).