All three neural crest-derived chromatophore cell types are affected by enz mutations.
Dorsal views of 54 hpf (A–E) and 82 hpf (F–J) wild-type (A, F) and enz mutant (B–E, G–J) embryos. (A, A inset) Wild-type melanophores are stellate and darkly pigmented at 54 hpf. (B–E) In contrast, enz^b431 (B), enz^os7 (C), enz^os15 (D) and enz^os18 (E) mutant embryos have small, punctate melanophores at this stage (compare insets in A and B). Yellow xanthophores, observed dorsally in the head of wild-type embryos (arrowhead in A) appear to be absent in enz homozygotes (B–E, arrowheads). (F–J) Iridescent iridiphores are also reduced in size in enz mutants (G–J, arrowheads) compared to wild-type siblings (F, arrowheads) at 82 hpf.

enz larvae and adults are undersized compared to wild-type siblings.
Lateral (A, B) and dorsal (C, D) views of 7 dpf wild-type (A, C) and enz (B, D) larvae. (A) Wild-type zebrafish develop swim bladders between 4 and 6 dpf (arrowhead). (B) The vast majority of enz homozygous larvae do not develop swim bladders (arrowhead). Some melanophores recover in size and morphology in enz homozygotes (D) compared to wild-type siblings (C). (E–L) Those enz homozygous larvae that do develop swim bladders survive, but are runted compared to wild-type siblings (stage-matched wild-type and enz mutant larvae shown at the same magnification). Lateral (E, F) and dorsal (G–J) views of wild-type (E, G, I) and enz mutant (F, H, J) larvae at 21 dpf (E–H) and 30 dpf (I, J). Melanophores continue to be paler in enz mutant larva than in wild-type siblings through at least 30 dpf (see arrowheads in G–J). (K, L) Lateral views of 119 dpf wild-type (K) and enz homozygous (L) adults. Normal overall morphology and pigmentation of enz mutant adults, as well as nascent fin stripe formation (L, arrowheads), suggests generalized growth retardation in enz mutants compared to wild-type siblings.

enz selectively affects chromatophores among neural crest derivatives.
Wild-type (A, C, E, G) and enz (B, D, F, H) mutant embryos. Craniofacial cartilages revealed with alcian blue staining are normal at 5 dpf (A, B: ventral view). Cervical sympathetic neurons, which express TH immunoreactivity (arrowheads in C, D), are indistinguishable between wild-type (C) and enz mutant (D) embryos at 7 dpf. (E–H) Hu-positive neurons of the dorsal root ganglia (E, F, arrowheads) and the enteric nervous system (arrowheads in G, H) also appear normal in enz mutant embryos (F, H) compared to wild-type siblings (E, G) at 7 dpf.

The numbers and distribution of chromatophore precursors appear normal in enz homozygotes at 24 hpf.
Lateral views of 24 hpf wild-type (A, C, E, G) and enz mutant (B, D, F, H) embryos. Early neural crest cells (crestin; A, B), xanthoblasts (xdh; C, D), melanoblasts (dct; E, F) and all chromatophore precursors (ednrb1; G, H) are all qualitatively normal at this stage.

Melanophore cell morphology changes in enz mutant embryos.
(A, B) At 27 hpf, wild-type melanophores are large, stellate and well-pigmented (A). enz melanophores are also large and stellate at this stage, but are pale compared to wildtype (B). (C, D) By 31 hpf, enz melanophore begin to transition to a punctate morphology (D), while wild-type melanophores remain large with many processes (C). (E, F) Wild-type (E) and enz mutant (F) embryos at 34.5 hpf. The morphological transition of melanophores in enz homozygotes continues in a rostro-caudal wave and is complete by approximately 48 hpf. (G–J) At 36 hpf, melanosomes are distributed throughout the cytoplasm of wild-type melanophores, reflecting the stellate morphology of these cells (G). (H) dct mRNA (red) is likewise distributed in the extensive processes of wild-type cells (arrowheads). Punctate distribution of melanosomes (I) and dct mRNA (J, red) in enz^os18 mutant melanophores is identical at 36 hpf reflecting cell shape change.

Xanthophores are qualitatively reduced in number and size in enz mutants.
(A, B) fms expression in 48 hpf wild-type (A) and enz mutant (B) embryos. Qualitatively reduced fms expression suggests that fewer xanthophores are present in enz mutants than in wild-type siblings at this stage (arrowheads in A and B). (C, D) Methylene blue-stained xanthophores appear much larger in wild-type (C) embryos than in enz mutants (D) at 3 dpf.

enz acts cell autonomously with respect to melanophore development.
(A) Nomarski image of a 28 hpf enz mutant host that has received cells from a wild-type donor (anterior to the left). Several large, dark melanophores (arrowheads) are present in addition to pale melanophores (arrows) normally observed in enz mutant embryos at this stage (see also Figure 5B). (B) High magnification of a wild-type cell (boxed area in (A) that formed a dark melanophore in the mutant environment. (C) Melanophore in (B) viewed under a rhodamine filter. (D) Wild-type cells form dark, stellate melanophores in 48 hpf gol hosts. (E) enz cells give rise to punctate melanophores in 48 hpf gol hosts.

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