Cis-regulatory analysis of the sea urchin pigment cell gene polyketide synthase.

The Strongylocentrotus purpuratus polyketide synthase gene (SpPks) encodes an enzyme required for the biosynthesis of the larval pigment echinochrome. SpPks is expressed exclusively in pigment cells and their precursors starting at blastula stage. The 7th-9th cleavage Delta-Notch signaling, required for pigment cell development, positively regulates SpPks. In previous studies, the transcription factors glial cell missing (SpGcm), SpGatae and kruppel-like (SpKrl/z13) have been shown to positively regulate SpPks. To uncover the structure of the Gene Regulatory Network (GRN) regulating the specification and differentiation processes of pigment cells, we experimentally analyzed the putative SpPks cis-regulatory region. We established that the -1.5kb region is sufficient to recapitulate the correct spatial and temporal expression of SpPks. Predicted DNA-binding sites for SpGcm, SpGataE and SpKrl are located within this region. The mutagenesis of these DNA-binding sites indicated that SpGcm, SpGataE and SpKrl are direct positive regulators of SpPks. These results demonstrate that the sea urchin GRN for pigment cell development is quite shallow, which is typical of type I embryo development.

Expression of the tyrosinase-encoding gene in a colorless melanophore mutant of the medaka fish, Oryzias latipes.

In the medaka fish Oryzias latipes many mutants for body colors have been isolated. Among them, a colorless melanophore mutant b, carrying b alleles homozygously, has pigmented black eyes but orange-colored skin with amelanotic melanophores, suggesting the presence of a tissue-specific mechanism of melanin formation. To cast light on the molecular basis of the mechanism, we have cloned cDNAs for tyrosinase (Tyr), a key enzyme in melanin biosynthesis, from the wild-type (wt) fish. DNA sequence analysis revealed that all clones encode a protein of 540 amino acids, having five potential glycosylation sites and two copper-binding sites that are characteristic features of Tyr. Genomic DNA blot analysis disclosed that the Tyr gene is present as a single copy in the fish genome. Using a cDNA clone as a probe, RNA blot analysis was carried out. In the wt, the 2.2-kb Tyr mRNA was expressed in eyes and skin but not in liver, corresponding to tissue-specific melanin formation. In the b mutant, contrary to expectation, the mRNA was detected not only in eyes but also in amelanotic skin. Therefore, pigmentation of the skin controlled by b is not directly related to expression of the Tyr gene.

CK1 activates minus-end-directed transport of membrane organelles along microtubules.

Microtubule (MT)-based organelle transport is driven by MT motor proteins that move cargoes toward MT minus-ends clustered in the cell center (dyneins) or plus-ends extended to the periphery (kinesins). Cells are able to rapidly switch the direction of transport in response to external cues, but the signaling events that control switching remain poorly understood. Here, we examined the signaling mechanism responsible for the rapid activation of dynein-dependent MT minus-end-directed pigment granule movement in Xenopus melanophores (pigment aggregation). We found that, along with the previously identified protein phosphatase 2A (PP2A), pigment aggregation signaling also involved casein kinase 1ε (CK1ε), that both enzymes were bound to pigment granules, and that their activities were increased during pigment aggregation. Furthermore we found that CK1ε functioned downstream of PP2A in the pigment aggregation signaling pathway. Finally, we discovered that stimulation of pigment aggregation increased phosphorylation of dynein intermediate chain (DIC) and that this increase was partially suppressed by CK1ε inhibition. We propose that signal transduction during pigment aggregation involves successive activation of PP2A and CK1ε and CK1ε-dependent phosphorylation of DIC, which stimulates dynein motor activity and increases minus-end-directed runs of pigment granules.

A chemical genomic approach identifies matrix metalloproteinases as playing an essential and specific role in Xenopus melanophore migration.

To dissect the function of matrix metalloproteinases (MMPs) involved in cellular migration in vivo, we undertook both a forward chemical genomic screen and a functional approach to discover modulators of melanophore (pigment cell) migration in Xenopus laevis. We identified the 8-quinolinol derivative NSC 84093 as affecting melanophore migration in the developing embryo and have shown it to act as a MMP inhibitor. Potential targets of NSC 84093 investigated include MMP-14 and MMP-2. MMP-14 is expressed in migrating neural crest cells from which melanophores are derived. MMP-2 is expressed at the relevant time of development and in a pattern that suggests it contributes to melanophore migration. Morpholino-mediated knockdown of both MMPs demonstrates they play a key role in melanophore migration and partially phenocopy the effect of NSC 84093.

Expression and transmission of wild-type pigmentation in the skin of transgenic orange-colored variants of medaka (Oryzias latipes) bearing the gene for mouse tyrosinase.

Transgenic fish carrying a reconstructed mouse tyrosinase gene, mg-Tyrs-J, were produced by microinjecting the gene into the oocyte nucleus of an orange-colored variant of medaka (Oryzias latipes). Of 64 oocytes microinjected and subsequently inseminated, 13 embryos developed normally beyond hatching and three of them exhibited brown skin pigmentation in the adult as was commonly observed in the wild type of this species. Light and electron microscopic examination disclosed a ubiquitous distribution of typical melanophores in the skin of these transgenic fish. Judging from their population density and distribution pattern, it was presumed that melanogenesis in these fish was elicited in amelanotic melanophores that resided in the skin of the orange-colored fish of this variant. Immunofluorescence with use of the anti-mouse tyrosinase antiserum lacking reactivity to medaka tyrosinase clearly disclosed that the gene introduced was expressed in the melanophores of transgenic fish. Crosses of female transgenic fish and males from an orange-colored variant yielded offspring exhibiting wild-type or orange-colored pigmentation in a ratio of 1:1, thus implying that mg-Tyrs-J integrated into the medaka genome behaves like a dominant gene. Little melanogenesis was observed in xanthophores, leucophores and iridophores in transgenic fish, suggesting possible specificity in recognition of teleostean cell types (i.e., melanophores) by the regulatory region of the mouse tyrosinase gene.

Fish plasminogen activators: their identification and characterization.

Immunoblots of proteins extracted from the skin of a small viviparous fish (Xiphophorus) showed that a monoclonal antibody against human urokinase recognizes multiple molecular weight species of antigens. The immunoaffinity-purified antigens had serine-protease activity for the hydrolysis of a chromogenic substrate and could convert human plasminogen to plasmin in a manner similar to that for human urokinase in vitro. Two antigens with apparent molecular weights of 55 and 50 kilodaltons that had been purified on a fibrin-Celite column were separable on SDS-polyacrylamide gels and were characterized as major plasminogen activators on fibrin-agar indicator plates. The 125I-tryptic peptide maps of both antigens were similar to that of human urokinase; therefore, the fish activators and human urokinase are structurally and functionally related.

An association of melanophores appearing at metamorphosis as vehicles of asymmetric skin color formation with pigment anomalies developed under hatchery conditions in the Japanese flounder, Paralichthys olivaceus.

The mechanisms for asymmetric skin color formation in the Japanese flounder are studied with particular concerns to causes for pigment disorder (hypomelanosis) occurring under hatchery conditions. For an analysis of normal pigmentation, fish were raised with wild zooplanktons in an indoor hatchery, whilst for hypomelanosis, they were raised with Brazilian Artemia nauplii, a diet used in the hatcheries. Morphological observations, counting of melanophores, histochemical assay of DOPA-positive immature cells (melanoblasts), and radiometric estimation of tyrosinase activities in skins of developing larvae and juveniles indicate that 1) the structural plan for pigmentation in this species is bilaterally symmetric until metamorphosis, utilizing large-sized melanophores (hence larval melanophores) as main vehicles, and 2) an asymmetric coloration characteristic to metamorphosed juveniles is formed by an intensive development of smaller-sized melanophores (hence adult-type melanophores) appearing selectively in the ocular side at the later stages of metamorphosis and by an absence of it in the blind. These findings apparently indicate that 1) two types of melanophores occur in this species which differ with respect to morphological properties and developmental fate, and 2) selective differentiation of adult type melanophores in the ocular side of the body at or after metamorphosis is primarily responsible for an asymmetric coloration of its adult form. The similar assays on the fish fed with Artemia nauplii indicate that defective development of adult-type melanophores results in hypomelanosis in their ocular-sided skins, yielding a pigmentary pattern seen in the blind side of the metamorphosed juveniles with normal pigmentation.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosinases in Rana pipiens. Purification and physical properties.

Two enzymes with tyrosinase activity have been purified from the frog Rana pipiens. Both enzymes are isolated in an inactive form which can be activated with trypsin. Amino acid analysis, NH2-terminal amino acid determination (arginine for both proteins), and immunological evidence indicate that athe two enzymes are similar if not identical. They can be distinguished by their trypsin activation kinetics. Cell fractionation studies suggest that one form is found associated with the smooth endoplasmic reticulum whereas the other protein fraction is localized mainly within the premelanosomes. Sedimentation equilibrium studies demonstrate that both protein fractions are self-associating systmes. Ionic strength, temperature, and specific anion effects alter the equilibria of the associating systems. The monomeric molecule weight for both fractions is 30,000 and at low ionic strengths the predominant molecular weight species is the tetramer. The partial specific volume of each protein is 0.70.

A recombinant vaccinia virus infects Xenopus melanophores.

A recombinant vaccinia virus was employed to demonstrate infection of cultured Xenopus laevis melanophores. The recombinant virus contains one copy each of the Escherichia coli lac Z and human growth hormone genes under the transcriptional control of two separate viral promoters. Western blot analysis and in situ staining revealed the dependency of beta-galactosidase production in infected Xenopus cells on time and multiplicity of infection (MOI). Western blot analysis was used to demonstrate the production of a 65 kD vaccinia late protein and its variation over time and with MOI. When virus preparations from infected Xenopus cells were attempted, no amplification of virus was observed and only a minute portion of the original innoculum was recovered. We therefore propose an abortive infection of Xenopus pigment cells by vaccinia virus: The amphibian cells allow for the synthesis of viral proteins, but not for the efficient replication of competent virus. The findings have implications not only for our understanding of the virus/host interaction, but also for the efficient expression of exogenously introduced genes in cultured Xenopus melanophores.

Inhibition of adenylate cyclase activity in the goldfish melanophore is mediated by α2-adrenoceptors and a pertussis toxin-sensitive GTP-binding protein.

The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by α-adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·1⁻¹ 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3',5'-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol· 1⁻¹ and naphazoline at 1 µmol· 1⁻¹ inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3',5'-monophosphate in the cells in both the presence and the absence of isoproterenol, a beta-adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3',5'-monophosphate, but only when they were present at relatively high concentrations (above 100 µmol·1⁻¹). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3',5'-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 µg·m1⁻¹) for 15 h or treatment with 100 nmol·1⁻¹ yohimbine (an α2-adrenergic antagonist) inhibited the effects of the α-adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3',5'-monophosphate, but prazosin (an α1-adrenergic antagonist) at 100 nmol·1⁻¹ was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase,which occurs as a result of stimulation of a pathway that involves α2-adrenoceptors and a inhibitory GTP-binding protein.

L-NAME-induced dispersion of melanosomes in melanophores activates PKC, MEK and ERK1.

Melanosome movement represents a good model of cytoskeleton-mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nomega-nitro-L-arginine methyl ester (L-NAME) induced dispersion in melanophores pre-aggregated with melatonin. Activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) or calcium-dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal-regulated kinase (MEK)-ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of L-NAME-induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in L-NAME-dispersed melanophores. L-NAME also caused dispersion in latrunculin-B-treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the L-NAME-induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.