Figure 1. Sox10:eGFP+/Ngn1:nRFP+ cells migrate and differentiate into microvillous neurons.
(A) Sox10:eGFP+ neural crest migrates dorsal to the eye and toward the olfactory region at 19 hpf as the olfactory placode is first apparent. (B)–(D) Time-lapse confocal microscopy of live embryos (Sox10:eGFP+; Ngn1:nRFP+) demonstrates that a subset of nasal cavity cells that express Ngn1:nRFP (B, boxed area arrowheads; z = 3.5 μm) ingress into the olfactory epithelium. (E)–(J) Selected time point and z-plane excerpts from full z stacks are shown from ∼29 hpf (C) to ∼33 hpf (D) and are 40' apart; z = 17.5 μm (consisting of five 3.5 μm slices). Arrowheads indicate two ingressing cells, one from the top and the other directly behind the olfactory epithelium. All ingressing cells express Ngn1:nRFP (F, inset arrowhead). Sox10:eGFP, green; Ngn1:nRFP, red. (K) At 53 hpf, all Sox10:eGFP+ cells in the olfactory epithelium are TRPC2:Venus+ microvillous neurons. (because eGFP signal bleeds into the Venus channel, overlap was confirmed via image processing; see Figure 1—figure supplement 1). Only a small number of microvillous neurons are not Sox10:eGFP+ (arrowheads). A single 3.5-μm-thick z-plane slice of the boxed area more clearly shows colocalization. Sox10:eGFP: green; TRPC2:Venus: red; Ngn1:nRFP: blue. e: eye; y: yolk; LOE: left olfactory epithelium; ROE: right olfactory epithelium. Orientation arrows: A: anterior; P: posterior; D: dorsal; V: ventral; L: lateral. Scale bars: 50 μm (A); 30 μm (B–D); 20 μm (E–K). See also Figure 1—figure supplements 1 and 2 and Videos 1 and 2.
Figure 1—figure supplement 1. Panel (K) from Figure 1 with Sox10:eGFP (green) channel removed (bottom) to better illustrate the large population of ciliated neurons present basally that are not Sox10:eGFP+ but are Ngn1:nRFP+ (blue).
Figure 1—figure supplement 2. (A) and (A') Sox10:eGFP+ microvillous neurons in fixed embryos stained with anti-GFP antibody colocalize with anti-HuC/D antibody staining at 53 hpf, confirming post-mitotic neuronal identity.
