Duplicated Myosin V Genes in Teleosts Show Evolutionary Rate Variations among the Motor and Cargo-Binding Domains.

We analyzed evolutionary rates of conserved, duplicated myosin V (myo5) genes in nine teleost species to examine the outcomes of duplication events. Syntenic analysis and ancestral chromosome mapping suggest one tandem gene duplication event leading to the appearance of myo5a and myo5c, two rounds of whole genome duplication for vertebrates, and an additional round of whole genome duplication for teleosts account for the presence and location of the myo5 genes and their duplicates in teleosts and other vertebrates and the timing of the duplication events. Phylogenetic analyses reveal a previously unidentified myo5 clade that we refer to now as myo5bb. Analysis using dN/dS rate comparisons revealed large regions within duplicated myo5 genes that are highly conserved. Codons identified in other studies as encoding functionally important portions of the Myo5a and Myo5b proteins are shown to be highly conserved within the newly identified myo5bb clade and in other myo5 duplicates. As much as 30% of 319 codons encoding the cargo-binding domain in the myo5aa genes are conserved in all three codon positions in nine teleost species. For the myo5bb cargo-binding domain, 6.6% of 336 codons have zero substitutions in all nine teleost species. Using molecular evolution assays, we identify the myo5bb branch as being subject to evolutionary rate variation with the cargo-binding domain, having 20% of the sites under positive selection and the motor domain having 8% of its sites under positive selection. The high number of invariant codons coupled with relatively high dN/dS values in the region of the myo5 genes encoding the ATP-binding domain suggests the encoded proteins retain function and may have acquired novel functions associated with changes to the cargo-binding domain.

An ENU mutagenesis screen in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in Colobomas.

PURPOSE: To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. METHODS: A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. RESULTS: A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta(1), identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2(uta1) mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2(uta1) mutant embryos. CONCLUSIONS: We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas.

Developmental expression of muscarinic receptors in the eyes of zebrafish.

In previous work, we have shown that light-adaptive pigment granule dispersion can be induced in vitro by treating retinal pigment epithelium (RPE) isolated from bluegill retina with acetylcholine or its analog carbachol and that these agents act through muscarinic receptors to induce pigment granule dispersion. RPE is a monolayer of tissue found between the neural retina and the choroid. In fish, RPE has long apical projections enmeshed with the distal part of photoreceptors, reaching down to the level of their nuclei. The RPE disperses melanin pigment granules into the apical projections to shield light-sensitive photoreceptor outer segments from photobleaching when fish are under bright-light conditions. During development, RPE begin to respond to light at 5days post-fertilization, raising the question of whether responsiveness is correlated to receptor expression. Here, we isolate, clone and sequence chrm-odd receptor genes in zebrafish, characterize them phylogenetically and observe their expression in the eyes of the zebrafish at different developmental stages using RT-PCR and immunofluorescence microscopy. We find that zebrafish express six unique chrm-odd receptor subtypes: chrm1a, chrm1b, chrm3a, chrm3b, chrm5a and chrm5b - and these receptors are differentially expressed during development. Our phylogenetic analysis confirms the assignments of chrm1b and chrm5b, isolated here, as well as other muscarinic receptor genes and their duplicates and suggests previously described muscarinic receptors may need to be reclassified. Differences between the expression patterns of ostensibly duplicated genes raise the possibility that subtle differences between the duplicates may enable refined regulation of specific developmental events.

Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens.

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.

The vacuolar-ATPase complex regulates retinoblast proliferation and survival, photoreceptor morphogenesis, and pigmentation in the zebrafish eye.

PURPOSE: The vacuolar (v)-ATPase complex is a key regulator of the acidification of endosomes, lysosomes, and the luminal compartments of several cell types, tissues, and organs; however, little is know about the in vivo function of the v-ATPase complex or its roles during eye development. This study was conducted to characterize ocular defects in five zebrafish mutants in which core components of the v-ATPase complex were affected (atp6v1h, atp6v1f, atp6v1e1, atp6v0c, and atp6v0d1), as well as a sixth mutant in which a v-ATPase associated protein (atp6ap1) was affected. METHODS: v-ATPase mutant zebrafish were characterized by histologic, molecular, and ultrastructural analyses. RESULTS: v-ATPase mutant zebrafish were oculocutaneous albinos and presented with defects in the formation and/or survival of melanosomes and with malformations in the retinal pigmented epithelium (RPE) that compromised melanosome distribution. They were microphthalmic, and BrdU incorporation assays indicated that retinoblast cell cycle exit and sustained proliferation in the ciliary marginal zone (CMZ) were compromised. v-ATPase mutants also possessed elevated levels of apoptotic neurons within their retinas and brains. Photoreceptor outer segment morphology was abnormal in the mutant eye with rosette structures forming adjacent to the affected regions of the RPE. Ultrastructural analyses indicate that RPE cells in v-ATPase mutants possess numerous membrane-bounded vacuoles containing undigested outer segment material. In situ hybridization analyses localized v-ATPase subunit transcripts within the RPE. CONCLUSIONS: These results demonstrate that the v-ATPase complex plays several critical roles during vertebrate eye development and maintenance, and they suggest that defects in v-ATPase complex function could possibly underlie human ocular disorders that affect the RPE.

Deconstructing evolution of adult phenotypes: genetic analyses of kit reveal homology and evolutionary novelty during adult pigment pattern development of Danio fishes.

The cellular bases for evolutionary changes in adult form remain largely unknown. Pigment patterns of Danio fishes are a convenient system for studying these issues because of their diversity and accessibility and because one species, the zebrafish D. rerio, is a model organism for biomedical research. Previous studies have shown that in zebrafish, stripes form by migration and differentiation of distinct populations of melanophores: early metamorphic (EM) melanophores arise widely dispersed and then migrate into stripes, whereas late metamorphic (LM) melanophores arise already within stripes. EM melanophores require the kit receptor tyrosine kinase, as kit mutants lack these cells but retain LM melanophores, which form a residual stripe pattern. To see if similar cell populations and genetic requirements are present in other species, we examined D. albolineatus, which has relatively few, nearly uniform melanophores. We isolated a D. albolineatus kit mutant and asked whether residual, LM melanophores develop in this species, as in D. rerio. We found that kit mutant D. albolineatus lack EM melanophores, yet retain LM melanophores. Histological analyses further show that kit functions during a late step in metamorphic melanophore development in both species. Interestingly, kit mutant D. albolineatus develop a striped melanophore pattern similar to kit mutant D. rerio, revealing latent stripe-forming potential in this species, despite its normally uniform pattern. Comparisons of wild types and kit mutants of the two species further show that species differences in pigment pattern reflect: (1) changes in the behavior of kit-dependent EM melanophores that arise in a dispersed pattern and then migrate into stripes in D. rerio, but fail to migrate in D. albolineatus; and (2) a change in the number of kit-independent LM melanophores that arise already in stripes and are numerous in D. rerio, but few in D. albolineatus. Our results show how genetic analyses of a species closely related to a biomedical model organism can reveal both conservatism and innovation in developmental mechanisms underlying evolutionary changes in adult form.

Histological preparation of embryonic and adult zebrafish eyes.

INTRODUCTIONThis protocol describes the histological preparation of embryonic and adult zebrafish eyes. The methods described here can be easily adapted for use on other zebrafish tissues.

Evolutionary diversification of pigment pattern in Danio fishes: differential fms dependence and stripe loss in D. albolineatus.

The developmental bases for species differences in adult phenotypes remain largely unknown. An emerging system for studying such variation is the adult pigment pattern expressed by Danio fishes. These patterns result from several classes of pigment cells including black melanophores and yellow xanthophores, which differentiate during metamorphosis from latent stem cells of presumptive neural crest origin. In the zebrafish D. rerio, alternating light and dark horizontal stripes develop, in part, owing to interactions between melanophores and cells of the xanthophore lineage that depend on the fms receptor tyrosine kinase; zebrafish fms mutants lack xanthophores and have disrupted melanophore stripes. By contrast, the closely related species D. albolineatus exhibits a uniform pattern of melanophores, and previous interspecific complementation tests identified fms as a potential contributor to this difference between species. Here, we survey additional species and demonstrate marked variation in the fms-dependence of hybrid pigment patterns, suggesting interspecific variation in the fms pathway or fms requirements during pigment pattern formation. We next examine the cellular bases for the evolutionary loss of stripes in D. albolineatus and test the simplest model to explain this transformation, a loss of fms activity in D. albolineatus relative to D. rerio. Within D. albolineatus, we demonstrate increased rates of melanophore death and decreased melanophore migration, different from wild-type D. rerio but similar to fms mutant D. rerio. Yet, we also find persistent fms expression in D. albolineatus and enhanced xanthophore development compared with wild-type D. rerio, and in stark contrast to fms mutant D. rerio. These findings exclude the simplest model in which stripe loss in D. albolineatus results from a loss of fms-dependent xanthophores and their interactions with melanophores. Rather, our results suggest an alternative model in which evolutionary changes in pigment cell interactions themselves have contributed to stripe loss, and we test this model by manipulating melanophore numbers in interspecific hybrids. Together, these data suggest evolutionary changes in the fms pathway or fms requirements, and identify changes in cellular interactions as a likely mechanism of evolutionary change in Danio pigment patterns.

Pigment pattern evolution by differential deployment of neural crest and post-embryonic melanophore lineages in Danio fishes.

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.