Zebrafish Leucocyte tyrosine kinase controls iridophore establishment, proliferation and survival.

The zebrafish striped pattern results from the interplay among three pigment cell types; black melanophores, yellow xanthophores and silvery iridophores, making it a valuable model to study pattern formation in vivo. It has been suggested that iridophore proliferation, dispersal and cell shape transitions play an important role during stripe formation; however, the underlying molecular mechanisms remain poorly understood. Using gain- and loss-of-function alleles of leucocyte tyrosine kinase (ltk) and a pharmacological inhibitor approach, we show that Ltk specifically regulates iridophore establishment, proliferation and survival. Mutants in shady/ltk lack iridophores and display an abnormal body stripe pattern. Moonstone mutants, ltk(mne) , display ectopic iridophores, suggesting hyperactivity of the mutant Ltk. The dominant ltk(mne) allele carries a missense mutation in a conserved position of the kinase domain that highly correlates with neuroblastomas in mammals. Chimeric analysis suggests a novel physiological role of Ltk in the regulation of iridophore proliferation by homotypic competition.

Tight Junction Protein 1a regulates pigment cell organisation during zebrafish colour patterning.

Zebrafish display a prominent pattern of alternating dark and light stripes generated by the precise positioning of pigment cells in the skin. This arrangement is the result of coordinated cell movements, cell shape changes, and the organisation of pigment cells during metamorphosis. Iridophores play a crucial part in this process by switching between the dense form of the light stripes and the loose form of the dark stripes. Adult schachbrett (sbr) mutants exhibit delayed changes in iridophore shape and organisation caused by truncations in Tight Junction Protein 1a (ZO-1a). In sbr mutants, the dark stripes are interrupted by dense iridophores invading as coherent sheets. Immuno-labelling and chimeric analyses indicate that Tjp1a is expressed in dense iridophores but down-regulated in the loose form. Tjp1a is a novel regulator of cell shape changes during colour pattern formation and the first cytoplasmic protein implicated in this process.

Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish.

Interactions between all three pigment cell types are required to form the stripe pattern of adult zebrafish (Danio rerio), but their molecular nature is poorly understood. Mutations in leopard (leo), encoding Connexin 41.8 (Cx41.8), a gap junction subunit, cause a phenotypic series of spotted patterns. A new dominant allele, leo(tK3), leads to a complete loss of the pattern, suggesting a dominant negative impact on another component of gap junctions. In a genetic screen, we identified this component as Cx39.4 (luchs). Loss-of-function alleles demonstrate that luchs is required for stripe formation in zebrafish; however, the fins are almost not affected. Double mutants and chimeras, which show that leo and luchs are only required in xanthophores and melanophores, but not in iridophores, suggest that both connexins form heteromeric gap junctions. The phenotypes indicate that these promote homotypic interactions between melanophores and xanthophores, respectively, and those cells instruct the patterning of the iridophores.

Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system.

The introduction of engineered site-specific DNA endonucleases has brought precise genome editing in many model organisms and human cells into the realm of possibility. In zebrafish, loss-of-function alleles have been successfully produced; however, germ line transmission of functional targeted knock-ins of protein tags or of SNP exchanges have not been reported. Here we show by phenotypic rescue that the CRISPR/Cas system can be used to target and repair a premature stop codon at the albino (alb) locus in zebrafish with high efficiency and precision. Using circular donor DNA containing CRISPR target sites we obtain close to 50% of larvae with precise homology-directed repair of the alb(b4) mutation, a small fraction of which transmitted the repaired allele in the germ line to the next generation (3/28 adult fish). The in vivo demonstration of germ line transmission of a precise SNP exchange in zebrafish underscores its suitability as a model for genetic research.

Genetic inducible fate mapping in adult mice using tamoxifen-dependent Cre recombinases.

The Cre/lox site-specific recombination system allows the control of gene activity in space and time in almost any tissue of the mouse. A major technical advance was the development of tamoxifen-dependent Cre recombinases, such as CreER(T2), that can be activated by administration of tamoxifen to the animal. This powerful tool greatly facilitates the study of gene functions and the generation of more realistic animal models of sporadic human diseases. Another important application of tamoxifen-dependent Cre recombinases is genetic inducible fate mapping (GIFM). In GIFM studies, the inducible Cre/lox system is used to genetically label a defined cell population at a selected time by irreversible activation of the expression of a Cre-responsive reporter transgene. Then, marked cells are detected at later time points to determine how the originally labeled progenitors contribute to specific structures and cell types during pre- and postnatal development. GIFM was initially applied during mouse embryogenesis, but is now increasingly used for cell lineage tracing in adult mice under physiological and pathophysiological conditions. Here we describe the design of GIFM experiments in adult mice as exemplified by CreER(T2)-assisted tracing of vascular smooth muscle cells during the development of atherosclerotic lesions. First, we give an overview of reporter transgenes available for genetic cell marking that are expressed from the Rosa26 locus, such as ß-galactosidase and fluorescent proteins. Then we present detailed protocols for the generation of experimental mice for GIFM studies, the induction of cell labeling by tamoxifen treatment, and the detection of marked cells in fixed and live tissues. Each section also provides a discussion of limitations and common pitfalls of GIFM experiments. Most of the protocols can be easily adapted to other developmental stages, cell types, Cre recombinases, and reporter transgenes and, thus, can be used as general guidelines for GIFM studies in mice.

A dominant mutation in tyrp1A leads to melanophore death in zebrafish.

Melanin biosynthesis in vertebrates depends on the function of three enzymes of the tyrosinase family, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct or Tyrp2). Tyrp1 might play an additional role in the survival and proliferation of melanocytes. Here, we describe a mutation in tyrp1A, one of the two tyrp1 paralogs in zebrafish, which causes melanophore death leading to a semi-dominant phenotype. The mutation, an Arg->Cys change in the amino-terminal part of the protein, is similar to mutations in humans and mice where they lead to blond hair (in melanesians) or dark hair with white bases, respectively. We demonstrate that the phenotype in zebrafish depends on the presence of the mutant protein and on melanin synthesis. Ultrastructural analysis shows that the melanosome morphology and pigment content are altered in the mutants. These structural changes might be the underlying cause for the observed cell death, which, surprisingly, does not result in patterning defects.

Endothelin signalling in iridophore development and stripe pattern formation of zebrafish.

Colour patterns of adult fish are composed of several different types of pigment cells distributing in the skin during juvenile development. The zebrafish, Danio rerio, displays a striking pattern of dark stripes of melanophores interspersed with light stripes of xanthophores. A third cell type, silvery iridophores, contributes to both stripes and plays a crucial role in adult pigment pattern formation. Several mutants deficient in iridophore development display similar adult phenotypes with reduced numbers of melanophores and defects in stripe formation. This indicates a supporting role of iridophores for melanophore development and maintenance. One of these mutants, rose (rse), encodes the Endothelin receptor b1a. Here we describe a new mutant in zebrafish, karneol (kar), which has a phenotype similar to weak alleles of rse with a reduction in iridophore numbers and defects of adult pigment patterning. We show that, unlike rse, kar is not required in iridophores. The gene defective in the kar mutant codes for an endothelin-converting enzyme, Ece2, which activates endothelin ligands by proteolytic cleavage. By morpholino-mediated knockdown, we identify Endothelin 3b (Edn3b) as the ligand for endothelin receptor signalling in larval iridophores. Thus, Endothelin signalling is involved in iridophore development, proliferation and stripe morphogenesis in larvae as well as adult zebrafish. In mammals the pathway is required for melanocyte development; therefore, our results indicate a previously unrecognized close evolutionary relationship between iridophores in zebrafish and melanocytes in mammals.

Erratum: transparent, a gene affecting stripe formation in Zebrafish, encodes the mitochondrial protein Mpv17 that is required for iridophore survival.

[This corrects the article on p. 703 in vol. 2.].

Iridophores and their interactions with other chromatophores are required for stripe formation in zebrafish.

Colour patterns of adult fish are produced by several types of pigment cells that distribute in the dermis during juvenile development. The zebrafish, Danio rerio, displays a striking pattern of dark stripes of melanophores interspersed by light stripes of xanthophores. Mutants lacking either cell type do not form proper stripes, indicating that interactions between these two chromatophore types are required for stripe formation. A third cell type, silvery iridophores, participates to render a shiny appearance to the pattern, but its role in stripe formation has been unclear. Mutations in rose (rse) or shady (shd) cause a lack or strong reduction of iridophores in adult fish; in addition, the melanophore number is drastically reduced and stripes are broken up into spots. We show that rse and shd are autonomously required in iridophores, as mutant melanophores form normal sized stripes when confronted with wild-type iridophores in chimeric animals. We describe stripe formation in mutants missing one or two of the three chromatophore types. None of the chromatophore types alone is able to create a pattern but residual stripe formation occurs with two cell types. Our analysis shows that iridophores promote and sustain melanophores. Furthermore, iridophores attract xanthophores, whereas xanthophores repel melanophores. We present a model for the interactions between the three chromatophore types underlying stripe formation. Stripe formation is initiated by iridophores appearing at the horizontal myoseptum, which serves as a morphological landmark for stripe orientation, but is subsequently a self-organising process.

transparent, a gene affecting stripe formation in Zebrafish, encodes the mitochondrial protein Mpv17 that is required for iridophore survival.

In the skin of adult zebrafish, three pigment cell types arrange into alternating horizontal stripes, melanophores in dark stripes, xanthophores in light interstripes and iridophores in both stripes and interstripes. The analysis of mutants and regeneration studies revealed that this pattern depends on interactions between melanophores and xanthophores; however, the role of iridophores in this process is less understood. We describe the adult viable and fertile mutant transparent (tra), which shows a loss or strong reduction of iridophores throughout larval and adult stages. In addition, in adults only the number of melanophores is strongly reduced, and stripes break up into spots. Stripes in the fins are normal. By cell transplantations we show that tra acts cell-autonomously in iridophores, whereas the reduction in melanophores in the body occurs secondarily as a consequence of iridophore loss. We conclude that differentiated iridophores are required for the accumulation and maintenance of melanophores during pigment pattern formation. The tra mutant phenotype is caused by a small deletion in mpv17, an ubiquituously expressed gene whose protein product, like its mammalian and yeast homologs, localizes to mitochondria. Iridophore death might be the result of mitochondrial dysfunction, consistent with the mitochondrial DNA depletion syndrome observed in mammalian mpv17 mutants. The specificity of the tra phenotype is most likely due to redundancy after gene multiplication, making this mutant a valuable model to understand the molecular function of Mpv17 in mitochondria.

New tools for the identification of developmentally regulated enhancer regions in embryonic and adult zebrafish.

We have conducted a screen to identify developmentally regulated enhancers that drive tissue-specific Gal4 expression in zebrafish. We obtained 63 stable transgenic lines with expression patterns in embryonic or adult zebrafish. The use of a newly identified minimal promoter from the medaka edar locus resulted in a relatively unbiased set of expression patterns representing many tissue types derived from all germ layers. Subsequent detailed characterization of selected lines showed strong and reproducible Gal4-driven GFP expression in diverse tissues, including neurons from the central and peripheral nervous systems, pigment cells, erythrocytes, and peridermal cells. By screening adults for GFP expression, we also isolated lines expressed in tissues of the adult zebrafish, including scales, fin rays, and joints. The new and efficient minimal promoter and large number of transactivating driver-lines we identified will provide the zebrafish community with a useful resource for further enhancer trap screening, as well as precise investigation of tissue-specific processes in vivo.

Myosin-V regulates oskar mRNA localization in the Drosophila oocyte.

Intracellular mRNA localization is an effective mechanism for protein targeting leading to functional polarization of the cell. The mechanisms controlling mRNA localization and specifically how the actin and microtubule (MT) cytoskeletons cooperate in this process are not well understood. In Drosophila, Oskar protein accumulation at the posterior pole of the oocyte is required for embryonic development and is achieved by the transport of oskar mRNA and its exclusive translation at the posterior pole. oskar mRNA localization requires the activity of the MT-based motor Kinesin, as well as the formation of a transport-competent ribonucleoprotein (RNP) complex. Here, we show that didum, encoding the Drosophila actin-based motor Myosin-V, is a new posterior group gene that promotes posterior accumulation of Oskar. Myosin-V associates with the oskar mRNA transport complex preferentially at the oocyte cortex, revealing a short-range actomyosin-based mechanism that mediates the local entrapment of oskar at the posterior pole. Our results also show that Myosin-V interacts with Kinesin heavy chain and counterbalances Kinesin function, preventing ectopic accumulation of oskar in the cytoplasm. Our findings reveal that a balance of microtubule- and actin-based motor activities regulates oskar mRNA localization in the Drosophila oocyte.

An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster.

Large-scale screens for female-sterile mutations have revealed genes required maternally for establishment of the body axes in the Drosophila embryo. Although it is likely that the majority of components involved in axis formation have been identified by this approach, certain genes have escaped detection. This may be due to (1) incomplete saturation of the screens for female-sterile mutations and (2) genes with essential functions in zygotic development that mutate to lethality, precluding their identification as female-sterile mutations. To overcome these limitations, we performed a genetic mosaic screen aimed at identifying new maternal genes required for early embryonic patterning, including zygotically required ones. Using the Flp-FRT technique and a visible germline clone marker, we developed a system that allows efficient screening for maternal-effect phenotypes after only one generation of breeding, rather than after the three generations required for classic female-sterile screens. We identified 232 mutants showing various defects in embryonic pattern or morphogenesis. The mutants were ordered into 10 different phenotypic classes. A total of 174 mutants were assigned to 86 complementation groups with two alleles on average. Mutations in 45 complementation groups represent most previously known maternal genes, while 41 complementation groups represent new loci, including several involved in dorsoventral, anterior-posterior, and terminal patterning.