Endoderm is required for the neurogenesis and survival of EB placodes. Zebrafish embryos injected with a morpholino against cas (A,C,D, right) or ngn1 (B, right) and uninjected controls (A-D, left) were collected at various time points and processed for in-situ hybridization with ngn1, phox2a and phox2b riboprobes, immunolabeling with Zn-5 or Hu antibody, or TUNEL assay. All whole-mount panels show lateral views. Anterior is left. (A) Glossopharyngeal and small vagal placodes and ganglia are absent, while facial and large vagal placodes and ganglia are significantly reduced (arrows) after cas MO injection. Endodermal pouches are completely absent in cas morphants (Zn-5, white arrowheads). (B) phox2a and phox2b expression is absent in ngn1 morphants. phox2b-positive enteric neuronal precursors (arrow) were not affected by injection of ngn1-MO. (C) Transverse sections through the vagal placode revealed ectodermal expression of ngn1 (arrowheads), indicating that the remaining EB neurons in cas morphants derived from the placodes. (D) Endoderm is important for survival of the EB placode cells. Transverse section through the facial placode revealed TUNEL-positive cells in the cas morphants (arrowheads). Scale bars: 50 µm. a, acoustic ganglion; 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.
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