The expression of SNRNP200 in zebrafish. Whole mount in situ hybridization revealed the expression of the Snrnp200 gene of zebrafish at the two-cell stage (A), 50%-epiboly (B), 8-somite stage (C), 1pdf (D), and 2pdf (E). In situ hybridization (ISH) of zebrafish retinal cryosections exhibit its expression is enriched at the ciliary marginal zone (CMZ) at 3dpf (F) and 5dpf (G).

Specification of melanocytes, iridophores and xanthophores is delayed in tfec mutants.(A-F) pnp4a expression in Ib(sp) requires Tfec. At 24 hpf, the number of dorsally located, and of partially fate restricted, migrating chromatoblasts (arrows) expressing pnp4a is reduced in tfec mutants (A,B). Expression overlying the eye is also affected (A,B, insets). This reduction is still prominent at 30 hpf (C,D), when staining is also visibly affected in the lateral patches. pnp4a expression is undetectable in differentiated iridophore positions (E, asterisks) in tfec mutants at 48 hpf (F). pnp4a+ iridophore lineage cells overlying the RPE are noticeably reduced in tfec mutants compared to WT siblings (A-F, insets). (G-L) Generation of tfec-positive Ib(sp) from late Cbls requires Tfec. At 24 hpf, tfec mutants present with anterior expansion of the tfec+ premigratory NC domain (G,H, arrowheads) and lack of medially migrating, Ib(sp) progenitors (arrows). At 36 hpf, the number of dorsal and migrating tfec+ Ib(df) (arrows), as well as those located in the lateral patches is reduced in mutants, compared to WT or heterozygous siblings (I,J). At 48 hpf, a reduced number of cells (asterisks) express tfec along the dorsal stripe of mutant embryos, compared to WT or heterozygous siblings (K,L). tfec mutant embryos were distinguishable after whole-mount in situ hybridisation by the lack of RPE melanisation (I-L, insets). tfec+ iridophore lineage cells overlying the RPE are noticeably reduced in tfec mutants compared to WT siblings (G-L, insets). (M-R) Mb(sp) generation from the late Cbl is delayed in tfec mutants. At 24 hpf, mitfa marker expression is restricted to an increased number of premigratory Cbls (arrowheads) and is undetectable in migrating Mb(sp) (arrows) in tfec mutants (M,N). At 30 hpf, the numbers and distributions of mitfa-positive late Cbls (arrowheads) are indistinguishable between tfec mutants and WT or heterozygous siblings (O,P), but the delay in Mb(sp) migration (arrows) remains distinguishable towards the tail. tfec mutant embryos lack RPE melanisation (O,P, insets). At 48 hpf, mitfa expression in mature melanocytes (asterisks) is indistinguishable between tfec mutants and WT or heterozygous siblings (Q,R). (S, T) tfec mutants lack ltk expression in premigratory late Cbls (arrowhead), in migrating Ib(sp) (arrow) and in iridoblasts of the eye (insets). (U-X) Xanthoblast (Xbl(sp)) specification from Cbls is delayed, as indicated by examinations of two lineage markers, gch2 (U,V) and aox5 (W,X). At 24 hpf, laterally migrating Xbl(sp) (arrows) are restricted to more anterior regions and precursors located in the head are reduced in tfec mutants (V,X) compared to their WT siblings (U,W). All tfec mutant panels show the tfecba6 allele except (V,X) which show the tfecvc60 allele. LP, lateral patches; m, melanisation. (A-J,M-X): lateral views. (K-L, insets of Q-R): dorsal views. Head towards the left. Scale bars: 100 μm.

Sulf2b is expressed within Shh-producing cells whereas Sulf2a is restricted to neural progenitors. Here and in all subsequent panels, side views of embryos (a,c,e,g,k,l) are at the level of the trunk spinal cord, anterior to the left and dorsal to the top, and spinal cord transverse sections (b,d,f,h,i,j) are shown dorsal to the top. Developmental stages are indicated in each panel. (a–j) Detection of sulf2b (a–d) and sulf2a (e–j) mRNAs by whole-mount in situ hybridisation. Images are representative examples of at least five embryos. Grey outlines (b,d,f,h,i,j) label the spinal cord edge. Brackets in a,c,e,g indicate the position of the spinal cord. Dotted lines in b,d,f,h) delineate Shh-producing cells, i.e. the medial floor plate at 24 hpf and the medial floor plate + lateral floor plat at 36 hpf. Note that sulf2a mRNA is detected in neural progenitors but not in medial and lateral floor plate cells. (k-l) Double detection of sulf2a (red) and shh (green) mRNAs by fluorescent in situ hybridisation, showing that sulf2a expression does not overlap the shh-expressing domain (white brackets).

Sema3fa is required to refine and limit retinal vessel branching in the larval eye. Live imaging of vessels (white) in Tg(kdrl:mCherry) WT (A,C,E,E’) and sema3faca304 heterozygous (G,G’), and homozygous (B,D,F,F’) eyes during retinal intraocular vascular development acquired by confocal microscopy. (A, B) In lateral views of WT (A) and sema3faca304 homozygous embryos, the hyaloid artery (endothelial cells labeled by mCherry) contacts the lens (asterisk) of the retina by 24 hpf (N = 1 WT n = 3; sema3faca304 n = 4). (C, D) Ventral views of WT (C: N = 1, n = 4) and sema3faca304 (D: N = 1, n = 4) eyes reveal that the hyaloid artery (arrowhead) forms a vascular plexus network around the lens by 72 hpf. Also evident is the nasal ciliary artery (arrows). (E–G) The WT intraocular hyaloid vessel (E’ is a magnified view of the boxed area in E) undergoes remodeling between 4-5 dpf to simplify the network (Hartsock et al., 2014). At 4 dpf, the numbers of vessel crossovers (arrows) are increased in sema3fa heterozygous (G,G’: N = 1, n = 2/2) and homozygous (F,F’: N = 4, n = 12/12) mutant embryos as compared to WT siblings (E, E’: N = 4, n = 10/11 show refinement). Scale bar: 100 µm; 50 µm for E’–G’.