Leukocyte tyrosine kinase functions in pigment cell development

Abstract

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.

Figure 1. shady mutants form an allelic series with reduced iridophores.

A, B, D, E, G, H) Incident light images of 72 hpf embryos to show eye (A, D, G) and posterior trunk (B, E, H) iridophores (silver spots) in embryonic alleles (D, E, shd^ty9, weaker; G, H, shd^ty82, strong) and WT siblings (A, B). C, F, I) Adult viable alleles (F, shd^j9e2, weaker; I, shd^j9s1, stronger; phenotypic severity shown most clearly by eye iridophores (arrowheads)) and WT sibling (C). In this and subsequent figures, embryos are shown in lateral view, except where noted. DS, dorsal stripe; VS, ventral stripe; *, Lateral patch.

Figure 2. shady mapping and identification as LTK orthologue.

A) shady (shd) map position on LG17; numbers of recombinants in 1000 shd mutant embryos between the marker and shd are given. B) PAC contig in shady region showing gene locations. C) Injection of PAC3 DNA (middle panel) rescues iridophore phenotype (arrows) of shd^ty82 mutants (no iridophores, lower panel) towards WT (upper panel). D) Injection of ltk morpholino into WT embryo generates shd mutant phenocopies (mo) with much-reduced iridophores (white arrowheads) compared with uninjected sibling (WT) or those injected with control morpholino (not shown). E) Schematics of predicted structures Alk and Ltk proteins in human (Hs), fruit fly (Dm) and zebrafish (Dr). Both WT and 3 mutant variants of the zebrafish are shown. Domains indicated are MAM (blue), LDLa (green), Gly-rich (gold), transmembrane (purple) and tyrosine kinase (red). Proteins are not shown to scale. F) Sequence traces show nucleotide substitution 2415A>T (cDNA) in shd^ty82 (left). RFLP analysis (right) shows homozygosity for the 2415A>T variant (shown by sensitivity to NheI, generating 2 fragments (*)) in 16 shd^ty82 mutants; 14 WT siblings show only WT allele (NheI insensitive, arrowhead) or are heterozygotes. G) Predicted protein sequence comparison of part of tyrosine kinase domain to show intact catalytic loop (red), but partially deleted activation loop (turquoise), in adult viable shd^j9e2 allele due to skipping of exon 26. Sequences are compared with those of human insulin receptor (Hs InsR; A18657) and LTK (Hs LTK; P29376). H) Bayesian analysis of vertebrate ALK/LTK amino acid alignment, using alignment 3 (see Figure S3). Numbers above branches indicate support values for each. Maximum likehood analysis of the same alignment provides the same topology. Translations are of our cDNAs (clones 1 and 3) and other zebrafish genes found by BLAST (XM_686872, XM_001342889 and XM_687805). For accession numbers of other sequences, see Table S2. For details and for other phylogenies obtained, see Figure S3.

Figure 3. Expression pattern of zebrafish ltk in WT (A–D,F,H,J,K,M,O,Q,S,U,W–Y,AC) and shd^ty82 homozygous embryos (G,I,L,N,P,R,T,V,Z,AD) throughout embryonic development.

Stages indicated in hpf. A–C) ltk-expressing cells in vicinity of eye (lower arrows in A,B) and in premigratory trunk NC (upper arrow in B and C) and in notochord (n). D) Dorsal view of posterior trunk of WT embryo to show ltk expression in scattered cells in dorsolaterally-positioned subset of premigratory NCCs (arrows). E,J) WT embryo treated with phenylthiourea, illuminated with incident light to show iridophore pattern (E), then fixed and processed for ltk ISH (J); individual cells are numbered. F,G,K,L,Q,R) Dorsoventral spread of ltk-expression in WT eye (F,K,Q); cells remain dorsal to eye in shd^ty82 mutants (G,L,R). H,I,M,N,S,T,Y,Z,AC,AD) Cells in premigratory (arrow) and migratory (*) positions and in nascent dorsal and ventral stripes are prominent in WT (H,M,S,Y,AC), but almost absent from shd^ty82 mutants (I,N,T,Z,AD). O,P,U,V,AA,AB,AE,AF) ltk expression pattern closely resembles ednrb1 expression in WT iridophores (AA,AE), but both markers are absent in shd^ty82 homozygous embryos (P,V,AB,AF). W) Plastic section through eye. X) Transverse section of posterior trunk.

Figure 4. shd mutants show elevated NCC apoptosis, but pigment cell numbers are not recipricolly elevated.

A) Graph shows mean±s.d. fragmenting GFP+ NCCs in trunk and tail of embryos from an incross of 7.2sox10;egfp, shd^ty82/− carriers. Embryos were sorted at 30–48 hpf for dying premigratory or medial pathway NCCs, then genotyped at c. 3 dpf by iridophore phenotype. Two-tailed t test shows highly significant differences (P<0.0001). B–D) Melanophore number (mean±s.d.) in trunk and tail dorsal stripe at 3 dpf (B) and total dct-positive melanoblast number in posterior trunk and tail (C) and gch-positive xanthoblast number on lateral pathway of posterior trunk and tail in one side (D) at 30 hpf are indistinguishable in shd mutants and WT siblings. Two-tailed t test shows no difference in all cases (p>0.05).

Figure 5. Iridophore phenotype and ltk expression patterns in sox10 mutants.

A, B) Iridophores (arrows) are prominent in 5 dpf WT (A), but almost absent in sox10^m618 (1 residual cell is seen here in the dorsal stripe)(B) and sox10^t3 (not shown) mutants. C–H) ltk expression (purple, arrows) patterns in WT (C, E, G) and sox10^t3 mutants (D, F, H); stages as shown. Arrows indicate ltk-expressing cells on eye and in premigratory NC (24 and 30 hpf) and in iridoblasts (48 hpf); insets show dorsal view of posterior trunk and tail. Arrowhead labels ltk-expressing cells of lateral patch.

Figure 6. Early NC markers are unaffected in sox10 mutants.

WT (A,C,E,G,I) and sox10^t3 mutants (B,D,F,H,J) showing foxd3 (A,B,E,F) or snail2 (C,D,G,H) or ltk expression at 24 (A–D) and 30 hpf (E–J).

Figure 7. Co-expression of sox10 in ltk-expressing cells in sox10 mutants.

Dorsal views of posterior trunk of 30 hpf sox10^m618 (C,D) and WT sibling (A,B) embryos double-labelled for sox10 (red) and ltk (purple, arrows). Autofluorescence from red sox10 signal shown in panels B and D.

Figure 8. Model for iridophore development from neural crest.

We propose that early NCCs are initially highly multipotent (indicated by the rainbow shading) and express Sox10, but not Ltk. During progressive fate restriction, a subset of partially-restricted, but still multipotent (fewer colours), cells are formed, marked by expression of Ltk; we propose that these cells' fates include multiple pigment cell-types (Other partially-restricted cell-types are expected to exist, but are not shown here for simplicity). Ltk signalling, acting together with Sox10 function, initiates iridoblast development (specification) in some cells (indicated by blue colour). In contrast cells adopting other fates, e.g. melanophores, extinguish Ltk expression, but may express other characteristic lineage-specific RTKs, e.g. Kit). Specified iridoblasts maintain Ltk expression as they differentiate into pigmented iridophores (blue). The early mutant phenotype precludes direct examination of the role of Ltk in these differentiating cells, although we favour cell proliferation as the likely late function. In ltk mutants (ltk ⁻), iridoblast fate specification fails. Cells survive for sometime (although Ltk expression is lost), before apoptosing; we cannot rule out the possibility that some precursors may transfate to other cell-types, although our data indicates most do not become melanoblasts or xanthoblasts, perhaps because of an intrinsic order of fate-specification in multipotent progenitors. In sox10 mutants (sox10 ⁻) fate specification is also prevented; since in this mutant fate specification to all other fates is also prevented, these ltk-expressing precursors accumulate in a premigratory position, before they also eventually die.