Fig. S2

Erbb3b and Adgra2 are required for the development of most skin-innervating DRG neurons, Related to Figure 1

(A) Lateral views of the larval spinal cord. Arrowheads, DRG cell bodies. Brackets, RB cell bodies. (B,C) Quantification of the total number of Tg(-17.6isl2b:GFP)-expressing DRG neurons (B) and dorsal root ganglia (C) per one side of the fish. n=12-15 fish per genotype. Black bars, mean ± SEM. *, p<0.01, Wilcoxon rank sum test. (D,E) Dorsolateral brightfield and fluorescent images of juvenile fish. Dashed boxes indicate regions of magnification. mitfaw2/w2 animals lack melanophores (Lister et al., 1999) and were used to aid visualization of the spinal cord. (F,G) Fluorescent images along the rostral spinal cord. Dashed boxes indicate regions of magnification. Arrowheads indicate the bilateral ganglia in F’’’ and unilateral ganglion in G’’’. (H) Images of multiple regions of lateral trunk skin from individual erbb3b-/- and sibling adults. Insets, regions imaged. Note the non-uniform skin innervation in erbb3b-/- animals. (I,J) Quantification of Tg(p2rx3a>mCherry) axon density in lateral trunk skin. Imaged regions indicated in panel H. n=5 fish per genotype, twelve separate 0.25 mm2 regions were quantified per fish. Black bars, mean ± SEM. *, p<0.01, Wilcoxon rank sum test. (K-N) Projections through the epidermis of scales isolated from adgra2-/- and sibling adults. Note the sparse innervation by NC-derived axons in the adgra2-/- epidermis. These remaining axons are likely not nociceptive, because they do not express the nociceptive marker Tg(p2rx3a>mCherry) (see Figure 1J). Based on the distribution and morphology of these remaining endings, they do not penetrate to superficial strata nor do they appear to innervate Merkel cells. Arrows in panels L and N indicate Schwann cell bodies. (O) Quantification of epidermal axon length based on tracings of the neural crest lineage reporter in the indicated genotypes. n=13-15 scales isolated from n=3 fish per genotype. Axon length is expressed as mm/0.1 mm2. Black bars, mean ± SEM. *, p<0.01, Wilcoxon rank sum test. Transgenes: (A) Tg(-17.6isl2b:GFP) (Pittman et al., 2008); (D,E) Tg(p2rx3a>mCherry); (F,G) Tg(-17.6isl2b:GFP) and Tg(p2rx3a>mCherry); (H) Tg(p2rx3a>mCherry); (L,N) Tg(-28.5Sox10:Cre);Tg(ubb:GswitchR) (red channel only). Staining: (K,M) axons (acTubulin) and nuclei (DAPI). Scale bars, 100 μm (A, F’’’, G’’’), 2 mm (D,E), 250 μm (F,G,H) and 25 μm (K-N).