Fig. 1

Fig. 1

Detection of developing iridophores using expression of tfec.

(A) Chromogenic WISH identifies tfec transcription in positions matching iridophore (iph) positions at 72 hpf. Images show the posterior trunk of the same individual, live and post-WISH processing. (B) tfec is expressed in sox10-positive NCCs (region in brackets) at 18 hpf, which have downregulated foxd3 but have not yet detectably activated early pigment lineage markers such as ltk, mitfa and pnp4a. (C) RNAscope reveals co-expression of tfec and ltk (arrows) in the anterior region of the posterior trunk (ARPT) during the timecourse of iridoblast specification, both in medially migrating cells at 24 hpf, and in dorsally located specified iridoblasts (ib(sp)) and iph at 30 hpf and 48 hpf, respectively. (C’) Nearly 100% of tfec+ cells co-express ltk during iridophore development (see S3 Table). Error bars indicate standard deviation between embryos. p-values were derived using a two-tailed, unpaired t-test. Using WISH at 24 hpf (D,E) tfec transcription is detectable in most or all cells of the multipotent premigratory NC domain (D, red line) and in a subset of cells of the posterior trunk located dorso-laterally to the spinal cord (D, vertical arrow) and in the medial pathway, between the somites and the notochord (D, horizontal arrow; Ei, arrows). Expression is only more weakly detectable in cells on the lateral pathway along the ARPT, between epidermal keratinocytes and somites (Eii, arrow). At 30 hpf (F,G) tfec is expressed in premigratory NCCs of the tail (red line) and in expected iridoblast positions, specifically dorsally located and medially migrating cells of the posterior trunk, the developing lateral patches and the eye (arrows). At 48 hpf (H) tfec is expressed along the dorsal, ventral and yolk sac stripes (vertical arrows), as well as in the lateral patches (arrowhead) and overlying the eye (horizontal arrow), in a pattern distinctive of differentiated iridophores. (I) RNAscope indicates presence of mitfa transcript in tfec-positive cells (arrows) migrating along the medial pathway in the ARPT at 24 hpf, but not in those located dorsally at 30 hpf, or at 48 hpf. In all three stages, mitfa+;tfec- cells are detectable (asterisks). (I’) Mean percentages of tfec+ cells co-expressing mitfa at each stage of development (refer to S3 Table). Error bars indicate the standard deviation between embryos. A two-tailed, unpaired t-test was used to derive indicated p-values. All panels show lateral views, except dorsal views in (A; D,F insets). Head towards the left. RNAscope panels: single focal planes shown. e, epidermis; ICM, intermediate cell mass; LPs, lateral patches; no, notochord; RL, reflected light. Scale bars: (A,B,D,E,F,G,H) 50 μm; (C,I) 20 μm.