DEV01876 Supplementary Material

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• Supplemental Figure 1 - Fig. S1. Fgf8 is not necessary for EB placode neurogenesis. Homozygous fgf8 (ace) mutant embryos (right) and their wildtype sibs (left) were processed for in situ hybridization with ngn1, phox2a and phox2b at 36 hpf and immunolabeling with Hu antibody at 80 hpf. All panels show lateral views. Anterior is left. ngn1, phox2a, phox2b, and Hu expression is present in all epibranchial placodes and ganglia. Note a shift in the phox2b expression domains marking glossopharyngeal and small vagal ganglia. As Fgf8 is required for induction of the otic vesicle as well as segmentation of the branchial arches, we suspect these differences in EB neuron distribution may be indirect. Scale bars, 50 mm. Abbreviations: al-anterior lateral line ganglion; e-eye; f-facial placode or ganglion; g-glossopharyngeal placode or ganglion; pl-posterior lateral line placode or ganglion; t-trigeminal ganglion; v-vagal placode or ganglion.

• Supplemental Figure 2 - Fig. S2. Chondrogenic neural crest is not required for early EB placode neurogenesis. Embryos injected with morpholino directed against foxd3 (right) and uninjected controls (left) were processed for in situ hybridization with dlx2 (26 hpf) and ngn1 (36 hpf) and immunolabeling with Hu antibody (80 hpf). All panels show lateral views. Anterior is left. dlx2 expression in the posteriormost, chondrogenic neural crest stream is strongly reduced (arrows). However, ngn1 expression is normal at 36 hpf in foxd3 morphants, indicating that chondrogenic neural crest is not required for early EB placode neurogenesis. In contrast, Hu antibody staining at 80 hpf revealed that various cranial ganglia, including the trigeminal, lateral line, and EB ganglia, were reduced in size and many Hu-positive neurons failed to coalesce into distinct ganglia, indicating a late requirement for Foxd3 during cranial ganglia development. Scale bars, 50 mm. Abbreviations are as in Supplemental Fig. 1.