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Files in this Data Supplement:
Fig. S1. Pax2a is expressed in EB placodes and is required for EB placode development. (A) Expression analyses of pax2a at 18, 20, 24 and 31.5 hpf. Whole-mount panels are lateral views of the embryos; transverse sections are at the level of the facial placode and glossopharyngeal placodes. pax2a expression begins in the presumptive facial placode at 18 hpf (open arrow). At 20-24 hpf, its expression extended caudally to include presumptive glossopharyngeal and vagal placodes. Between 28 and 31.5 hpf, pax2a expression was maintained in the individual placodes and internalized neuroblasts. Transverse sections at 24 hpf revealed the presence of pax2a mRNA (arrowheads) in the thickened columnar epithelium (the extent of the thickened epithelium is marked by arrows), a hallmark of cranial placodes. Interestingly, the facial domain of pax2a expression was restricted to the ventral part of the epithelium, indicating that the EB placodes are restricted to the ventral part of the competent ectoderm. (B) Zebrafish embryos were collected at 36 and 50 hpf, and processed for immunolabeling with Pax2 (red) and Hu (green) antibody. Pax2a protein (arrowheads) was downregulated in the differentiated Hu-positive EB neurons. (C) Pax2a activity is required during EB ganglia development. The zebrafish pax2a mutant no isthmus (noi) displayed a reduction in EB neurons. (D) Foxi1 activity is required for pax2a expression. Notice that pax2a message is absent in the hsy/foxi1-mutant embryos. a, acoustic ganglion; al, anterior lateral line ganglion; f, facial placode or ganglion; g, glossopharyngeal placode or ganglion; mhb, midhindbrain boundary; nt, neural tube; ov, otic vesicle; pl, posterior lateral line ganglion; t, trigeminal ganglion; v, vagal placode or ganglion. Scale bars: 50 μm.
Fig. S2. Fgf signaling is active in EB placode at the time of induction. Expression analyses of fgfr1, fgfr2, erm and pea3 at 13-14 hpf. Whole-mount panels (left) are dorsal views of the embryos; transverse sections (right) are at the level of r2-r4. Notice expression of these markers just lateral to the neural tube (arrowheads). Transverse sections revealed ectodermal expression of fgfr1, erm, and pea3 (arrows). Scale bars: 50 μm.
Fig. S3. Mesodermal fgf3 and fgf8 expression is absent in MZoep mutants. MZoep mutants and age-matched controls were analyzed for fgf3 and fgf8 expression at 15 hpf. All panels show dorsal views. Mesodermal expression (arrowheads) of fgf3 and fgf8 was absent (bottom panels), whereas CNS expression was largely unaffected in MZoep mutants. mhb, midhindbrain boundary; r4, rhombomere4. Scale bar: 50 μm.
Fig. S4. fgf3+8 morphants phenocopy fgf3/8 double mutant phenotype. Zebrafish embryos injected with a fgf3 and fgf8 morpholino alone or together and uninjected controls were collected at 36 hpf and processed for in situ hybridization with phox2b riboprobe. All panels show lateral views. Notice that injection of the suboptimal amounts of fgf3-MO alone causes only a slight reduction in the EB ganglia. However, injection of the same amount of fgf3-MO together with fgf8-MO causes complete absence of the EB ganglia. Also notice that the locus coeruleus is completely eliminated in fgf3+8 morphants, allowing the efficiency of injections to be monitored. a, acoustic ganglion; al, anterior lateral line ganglion; f, facial placode or ganglion; g, glossopharyngeal placode or ganglion; mhb, midhindbrain boundary; nt, neural tube; ov, otic vesicle; pl, posterior lateral line ganglion; t, trigeminal ganglion; v, vagal placode or ganglion; lc, locus coeruleus; nc, vagal neural crest. Scale bars, 50 μm.
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