Gene duplication of the zebrafish kit ligand and partitioning of melanocyte development functions to kit ligand a

The retention of particular genes after the whole genome duplication in zebrafish has given insights into how genes may evolve through partitioning of ancestral functions. We examine the partitioning of expression patterns and functions of two zebrafish kit ligands, kit ligand a (kitla) and kit ligand b (kitlb), and discuss their possible coevolution with the duplicated zebrafish kit receptors (kita and kitb). In situ hybridizations show that kitla mRNA is expressed in the trunk adjacent to the notochord in the middle of each somite during stages of melanocyte migration and later expressed in the skin, when the receptor is required for melanocyte survival. kitla is also expressed in other regions complementary to kita receptor expression, including the pineal gland, tail bud, and ear. In contrast, kitlb mRNA is expressed in brain ventricles, ear, and cardinal vein plexus, in regions generally not complementary to either zebrafish kit receptor ortholog. However, like kitla, kitlb is expressed in the skin during stages consistent with melanocyte survival. Thus, it appears that kita and kitla have maintained congruent expression patterns, while kitb and kitlb have evolved divergent expression patterns. We demonstrate the interaction of kita and kitla by morpholino knockdown analysis. kitla morphants, but not kitlb morphants, phenocopy the null allele of kita, with defects for both melanocyte migration and survival. Furthermore, kitla morpholino, but not kitlb morpholino, interacts genetically with a sensitized allele of kita, confirming that kitla is the functional ligand to kita. Last, we examine kitla overexpression in embryos, which results in hyperpigmentation caused by an increase in the number and size of melanocytes. This hyperpigmentation is dependent on kita function. We conclude that following genome duplication, kita and kitla have maintained their receptor-ligand relationship, coevolved complementary expression patterns, and that functional analysis reveals that most or all of the kita receptor's function in the embryo are promoted by its interaction with kitla. © 2007 Hultman et al

Mutations in gfpt1 and skiv2l2 Cause Distinct Stage-Specific Defects in Larval Melanocyte Regeneration in Zebrafish

The establishment of a single cell type regeneration paradigm in the zebrafish provides an opportunity to investigate the genetic mechanisms specific to regeneration processes. We previously demonstrated that regeneration melanocytes arise from cell division of the otherwise quiescent melanocyte precursors following larval melanocyte ablation with a small molecule, MoTP. The ease of ablating melanocytes by MoTP allows us to conduct a forward genetic screen for mechanisms specific to regeneration from such precursors or stem cells. Here, we reported the identification of two mutants, earthaj23e1 and juliej24e1 from a melanocyte ablation screen. Both mutants develop normal larval melanocytes, but upon melanocyte ablation, each mutation results in a distinct stage-specific defect in melanocyte regeneration. Positional cloning reveals that the earthaj23e1 mutation is a nonsense mutation in gfpt1 (glutamine:fructose-6-phosphate aminotransferase 1), the rate-limiting enzyme in glucosamine-6-phosphate biosynthesis. Our analyses reveal that a mutation in gfpt1 specifically affects melanocyte differentiation (marked by melanin production) at a late stage during regeneration and that gfpt1 acts cell autonomously in melanocytes to promote ontogenetic melanocyte darkening. We identified that the juliej24e1 mutation is a splice-site mutation in skiv2l2 (superkiller viralicidic activity 2-like 2), a predicted DEAD-box RNA helicase. Our in situ analysis reveals that the mutation in skiv2l2 causes defects in cell proliferation, suggesting that skiv2l2 plays a role in regulating melanoblast proliferation during early stages of melanocyte regeneration. This finding is consistent with previously described role for cell division during larval melanocyte regeneration. The analyses of these mutants reveal their stage-specific roles in melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process, but also in late stages of differentiation of the regenerating melanocyte. We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis, and those involved in larval or adult growth

On the use of cartographic projections in visualizing phylo-genetic tree space

Phylogenetic analysis is becoming an increasingly important tool for biological research. Applications include epidemiological studies, drug development, and evolutionary analysis. Phylogenetic search is a known NP-Hard problem. The size of the data sets which can be analyzed is limited by the exponential growth in the number of trees that must be considered as the problem size increases. A better understanding of the problem space could lead to better methods, which in turn could lead to the feasible analysis of more data sets. We present a definition of phylogenetic tree space and a visualization of this space that shows significant exploitable structure. This structure can be used to develop search methods capable of handling much larger data sets

Defects in ErbB-Dependent Establishment of Adult Melanocyte Stem Cells Reveal Independent Origins for Embryonic and Regeneration Melanocytes

Adult stem cells are responsible for maintaining and repairing tissues during the life of an organism. Tissue repair in humans, however, is limited compared to the regenerative capabilities of other vertebrates, such as the zebrafish (Danio rerio). An understanding of stem cell mechanisms, such as how they are established, their self-renewal properties, and their recruitment to produce new cells is therefore important for the application of regenerative medicine. We use larval melanocyte regeneration following treatment with the melanocytotoxic drug MoTP to investigate these mechanisms in Melanocyte Stem Cell (MSC) regulation. In this paper, we show that the receptor tyrosine kinase, erbb3b, is required for establishing the adult MSC responsible for regenerating the larval melanocyte population. Both the erbb3b mutant and wild-type fish treated with the ErbB inhibitor, AG1478, develop normal embryonic melanocytes but fail to regenerate melanocytes after MoTP-induced melanocyte ablation. By administering AG1478 at different time points, we show that ErbB signaling is only required for regeneration prior to MoTP treatment and before 48 hours of development, consistent with a role in establishing MSCs. We then show that overexpression of kitla, the Kit ligand, in transgenic larvae leads to recruitment of MSCs, resulting in overproliferation of melanocytes. Furthermore, kitla overexpression can rescue AG1478-blocked regeneration, suggesting that ErbB signaling is required to promote the progression and specification of the MSC from a pre–MSC state. This study provides evidence that ErbB signaling is required for the establishment of adult MSCs during embryonic development. That this requirement is not shared with the embryonic melanocytes suggests that embryonic melanocytes develop directly, without proceeding through the ErbB-dependent MSC. Moreover, the shared requirement of larval melanocyte regeneration and metamorphic melanocytes that develops at the larval-to-adult transition suggests that these post-embryonic melanocytes develop from the same adult MSC population. Lastly, that kitla overexpression can recruit the MSC to develop excess melanocytes raises the possibility that Kit signaling may be involved in MSC recruitment during regeneration

The Genetic Structure of the Swedish Population

Peer reviewe

Mapping genomic loci implicates genes and synaptic biology in schizophrenia

Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies

<i>kitla</i> Overexpression Causes Hyperpigmentation in Wild-type but Not in <i>kita^b5</i> Embryos

<div><p>(A) Wild-type larva.</p><p>(B) Larva injected with <i>kitla::JRed</i> fusion construct shows hyperpigmentation with melanocytes covering a larger area than wild-type.</p><p>(C) <i>kita</i> larva.</p><p>(D) <i>kitla::JRed</i> injected into <i>kit^b5</i> embryos results in <i>kit^b5</i> phenotype.</p><p>(E) <i>kitla::JRed</i> hyperpigmented larvae have more melanocytes on the dorsal stripe than do wild-type. When <i>kitla</i> expression vector is injected into <i>kita</i> embryos, there is no change in the number of melanocytes. All larvae at 6 dpf (<i>n</i> = 10).</p><p>Scale bars: 150 μm.</p></div

Phylogenetic Tree of <i>kitl</i> Protein Sequences

<p>The topology of the tree suggests that the <i>kitl</i> locus duplicated before the teleost radiation. <i>Dr, Danio rerio</i> (zebrafish), <i>Fr, Fugu rubripes</i> (fugu), <i>Ga, Gasterosteus aculeatus</i> (stickleback), <i>Ol, Oryzias latipes</i> (medaka), <i>Am, Ambystoma mexicanum</i> (axolotl), <i>Gg, Gallus gallus</i> (chick), <i>Xl, Xenopus laevis (Xenopus), Mm, Mus Musculus</i> (mouse), <i>Hs, Homo sapiens</i> (human)<i>.</i> Bootstrap values from 1,000 replicates are labeled.</p