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Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development. Keywords: Amphibian egg, Tyrosinase, Pigment cell, Melanin, Oxygen

How transcriptional gene networks operate during development and how they have emerged during evolution are two fundamental and interconnected questions in the evo-devo field (Davidson in The regulatory genome: gene regulatory networks in development and evolution. Academic Press, Amsterdam, 2006; Carroll in Cell 134(1):25–36, 2008). In this chapter we discuss the origin of the vertebrate eye from a common ancestor and its gene regulatory network (GRN). In an attempt to shed light on the evolutionary history of the vertebrate eye, photoreceptive structures present in our chordate sister groups cephalochordates (lancelets) and urochordates (tunicates) will be examined. Additionally, we summarize the still fragmentary information on the specification of visual organs in these chordate groups., This work was supported by grants BFU2011-22916, BFU2014-53765-P, and P11-CVI-7256 to JRMM.

The neural crest is a vertebrate specific, developmentally transient population of pluripotent stem cells capable of crossing germ layer boundaries and differentiating into a multitude of different tissues. In Zebrafish, one of these cell types, the pigment cells of the body, first appear and differentiate in early embryos contributing to a stereotypical larval pigmentation pattern composed of melanophores, xanthophores and iridophores. At juvenile stages the larval pigmentation pattern undergoes a rapid transformation with the appearance of large numbers of newly arriving pigment cells that develop into the adult striped pattern of zebrafish skin. These melanophores arise from an undifferentiated cell population. Although ontogenetically derived from the neural crest, their exact source in juvenile and adult fish has remained unclear. Here I present the source of the late appearing melanophores of the adult pattern to be derived from a stem cell population, set aside early in development at the future site of the dorsal root ganglia as part of the peripheral nervous system. The melanophore progenitors use the spinal nerves as migratory routes leading to the hypodermis at the periphery. Using mutants, morpholino and small molecule inhibition, I dissect the specific roles of ErbB and Kit signaling in the establishment of the melanophore stem cells and the role of dorsal root ganglia as their niche. By combining blastula transplantation and live imaging techniques I have identified melanophore progenitor cells during regeneration becoming active at the site of the dorsal root ganglia. I have followed their lineage until melanization, thereby confirming their specific fate. Furthermore, I show the requirement of these melanophore stem cells for the adult pigment cell population. Continuing along with melanophore maturation and differentiation, I have positionally cloned the zebrafish albino mutant and present mutations in slc45a2 encoding a solute carrier protein as causing the phenotype. In humans, mutations in SLC45A2 lead to oculocutaneous albinism type IV and single nucleotide polymorphisms (SNPs) at this locus are associated with skin color variation, but little is known about the function of SLC45A2 in melanization. I present evidence for a role of Slc45a2 in pH and ionic homeostasis inside melanosomes and show how variation of these conditions affects the key melanin producing enzyme, tyrosinase. I also show that mutations in sandy/tyrosinase lead to a destruction of melanosomes with toxic effects to neighboring tissues. Taken together these results provide insight into the large amount of variation in SLC45A2 across many animals as opposed to the relative low variation associated with TYROSINASE in humans. Die Neuralleiste besteht aus einer Population von pluripotenten Stammzellen, die als embryonale Anlage bei der Entwicklung von Wirbeltieren entstehen. Zellen der Neuralleiste sind in der Lage, sich zu einer Vielzahl von Zelltypen zu differenzieren, wie unter anderem zu Pigmentzellen. In Zebrafischen erscheinen Pigmentzellen zuerst während der Embryonalentwicklung, und bilden ein typisches larvales Farbmuster aus. Später, während des jugendlichen Stadiums, entstehen eine Anzahl von neuen Pigmentzellen, die das Farbmusters des adulten Fisches bilden. Die Melanophoren dieses Musters entstammen einer undifferenzierten Zellpopulation, die von der Neuralleiste abgeleitet ist. Jedoch war ihr genauer Ursprung im jugendlichen und adulten Fisch bisher unbekannt. Ich beschreibe hier, dass die Vorläuferzellen der adulten Melanophoren einer Stammzellpopulation entstammen, die bereits früh während der Entwicklung segmental neben den Ganglien (Dorsal Root Ganglion, DRG) des peripheren Nervensystems angelegt wird. Nach ihrer Aktivierung wandern die Melanophorenstammzellen dann entlang der Spinalnerven in die Haut des Fisches hinein. Durch Analyse verschiedener Zebrafischmutanten, Injektionen von Morpholinos oder Nutzung von Hemmstoffen konnte ich die spezifischen Rollen der ErbB-und Kit-Signalwege bei der Anlage dieser Stammzellen am DRG und die Rolle des DRGs als Stammzellnische aufgliedern. Durch Transplantationsexperimente von Blastulazellen kombiniert mit Zeitrafferfilmen konnte ich die Aktivierung der Melanophorenstammzellen am DRG beobachten, ihre Entwicklung bis zum Zeitpunkt der Melanisierung verfolgen und somit ihre spezifische Herkunft nachweisen. Desweiteren zeige ich, dass diese Melanophorenstammzellen die adulten Melanophoren hervorbringen. Im Zusammenhang mit meiner Analyse der Differenzierung von Melanophoren habe ich die albino Zebrafischmutation positional kloniert und zeige, dass Mutationen im Gen slc45a2, welches ein Transmembran-Transportprotein kodiert, den albino Phänotyp verursachen. Im Menschen führen Mutationen in SLC45A2 zu okulokutanem Albinismus Typ IV. Polymorphismen an diesem Genlocus sind mit unterschiedlichen Hautfarben assoziiert, jedoch ist sehr wenig über die genaue Funktion von SLC45A2 im Melanisierungsprozess bekannt. Ich zeige, dass Slc45a2 möglicherweise eine Rolle bei der Kontrolle der pH- und ionischen Homöostase innerhalb der Melanosomen spielt, und demonstriere, wie eine Veränderung der Bedingungen ein Schlüsselenzym des Melaninstoffwechsels, Tyrosinase, beeinflussen. Weiterhin zeige ich, dass Mutationen in sandy/tyrosinase zur Zerstörung von Melanosomen und damit einhergehenden toxischen Effekten auf das umliegende Gewebe führen. Diese Ergebnisse erklären das Auftreten der vielfältigen bekannten Variationen in SLC45A2 in vielen Tierarten, im Gegensatz zu der relativ seltenen Variation im TYROSINASE Gen.

We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position-1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment.© 2013 Elsevier Ltd. 116 96 108 Cited By :15

The glial cells missing (gcm) regulatory gene of the sea urchin Strongylocentrotus purpuratus is first expressed in veg2 daughter cells as the genomic target of late cleavage stage Delta-Notch signaling from the skeletogenic mesoderm precursors. Gcm is required in veg2 progeny during late cleavages for the early phase of pigment cell precursor specification. Here we report on a later acting cis-regulatory module that assumes control of gcm expression by the early mesenchyme blastula stage and maintains it through pigment cell differentiation and dispersal. Cis-perturbation analyses reveal that the two critical elements within this late module are consensus matches to Gcm and Six1 binding sites. Significantly, six1 mRNA localizes to gcm+cells from the mesenchyme blastula stage onwards. Trans-perturbations with anti-sense morpholinos reveal a co-dependency between six1 and gcm. Six1 mRNA levels fall sharply after Gcm is depleted, while depleting Six1 leads to significant reductions in output of endogenous gcm or modular-reporters. These results support the conclusion gcm and six1 comprise a positive intergenic feedback loop in the mesodermal GRN. This often employed cross regulatory GRN feature here ensures self-sustaining gcm output in a cohort of fully specified pigment cell precursors at a relatively early developmental stage.

Pigment cells of the zebrafish, Danio rerio, offer an exceptionally tractable system for studying the genetic and cellular bases of cell fate decisions. In the zebrafish, neural crest cells generate three types of pigment cells during embryogenesis: yellow xanthophores, iridescent iridophores and black melanophores. In this study, we present evidence for a model whereby melanophores and iridophores descend from a common precursor whose fate is regulated by an interplay between the transcription factors Mitf and Foxd3. Loss of mitfa, a key regulator of melanophore development, resulted in supernumerary ectopic iridophores while loss of foxd3, a mitfa repressor, resulted in fewer iridophores. Double mutants showed a restoration of iridophores, suggesting that one of Foxd3's roles is to suppress mitfa to promote iridophore development. Foxd3 co-localized with pnp4a, a novel marker of early iridophore development, and was necessary for its expression. A considerable overlap was found between iridoblast and melanoblast markers but not xanthoblast markers, which resolved as cells began to differentiate. Cell lineage analyses using the photoconvertible marker, EosFP, revealed that both melanophores and iridophores develop from a mitfa+ precursor. Taken together, our data reveal a Foxd3/mitfa transcriptional switch that governs whether a bi-potent pigment precursor will attain either an iridophore or a melanophore fate.

International audience; Whole-genome duplications (WGDs) have occurred repeatedly in the vertebrate lineage, but their evolutionary significance for phenotypic evolution remains elusive. Here, we have investigated the impact of the fish-specific genome duplication (FSGD) on the evolution of pigmentation pathways in teleost fishes. Pigmentation and color patterning are among the most diverse traits in teleosts, and their pigmentary system is the most complex of all vertebrate groups. Using a comparative genomic approach including phylogenetic and synteny analyses, the evolution of 128 vertebrate pigmentation genes in five teleost genomes following the FSGD has been reconstructed. We show that pigmentation genes have been preferentially retained in duplicate after the FSGD, so that teleosts have 30% more pigmentation genes compared with tetrapods. This is significantly higher than genome-wide estimates of FSGD gene duplicate retention in teleosts. Large parts of the melanocyte regulatory network have been retained in two copies after the FSGD. Duplicated pigmentation genes follow general evolutionary patterns such as the preservation of protein complex stoichiometries and the overrepresentation of developmental genes among retained duplicates. These results suggest that the FSGD has made an important contribution to the evolution of teleost-specific features of pigmentation, which include novel pigment cell types or the division of existing pigment cell types into distinct subtypes. Furthermore, we have observed species-specific differences in duplicate retention and evolution that might contribute to pigmentary diversity among teleosts. Our study therefore strongly supports the hypothesis that WGDs have promoted the increase of complexity and diversity during vertebrate phenotypic evolution.

Eine Kopplung eines Homöobox Gens, Xi-X, von 15 cM mit den platyspezifischen Pigmentzellloci (PSPL) bzw. dem Tu-Komplex im Xiphophorus Melanom-Modell konnte gefunden werden. Ein Xi-X Transkript von 1,5 kb charakterisiert die Zellen der Grundfärbung (Mikromelanophoren, Xanthophoren) und die undifferenzierten Zellstadien. Ein anderes Transkript von 2,3 kb ist für die Musterpigmentzellen charakteristisch (Makromelanophoren, Xanthoerythrophoren, Erythrophoren). Die Zellen der Zelllinie DRLI sind vermutlich als Makropigmentzellen determiniert. Die unstimulierten Zellen produzieren Xi-X-Transkripte von 1,5 und 2,3 kb, eine retinsäure-stimulierte Differenzierung wird von einem Verlust des 1,5 kb Transkriptes begleitet. Schon frühe Blastenstadien von PSPL-kodierten Zellen sind vermutlich determiniert, durch ein 2,3 kb Xi-X-Transkript zu erkennen, Makropigmentzellen zu bilden. Das 1,5 kb Transkript ist in den Blastenstadien dieser Zellen anwesend und geht während der weiteren Differenzierung verloren. Die Frage wird gestellt, ob die Synthese des 2,3 kb Xi-X Transkriptes vom Gen RDiff in den RDiff-regulierten Pigmentzellmusterungen Sd und Sp abhängig sein könnte. Eine atypische Vererbung der PS- und HS-Xi-X-Fragmente konnte in den Bastarden beobachtet werden (8,6% der 70 einzelnd untersuchten Bastarde). Die Kopplungsgruppe Tu-Komplex fördert die Instabilität, die durch die Kopplungsgruppe RDiff unterbunden wird. Eine eventuelle Zuordnung von RDiff als ein Gen der Zellzyklus-Überwachung wird diskutiert. Ein möglicher Zusammenhang zwischen einer restriktiven Poly(GTCT/GACA)-Organisation bei X. maculatus, im Gegensatz zu X. helleri, und einer gut regulierten Expression des Tu-Komplexes wird angesprochen. Die Möglichkeit besteht, dass gewisse Aspekte der Geschlechtsbestimmung (X. helleri Männchen, die männliche und weibliche Heterogametie bei X. maculatus) durch die isolierte Genregion Xi-X widergespiegelt wird. Die Homologie von Xi-X zum Gen pdx-1 ist hoch (zum Zebrafisch 82% in einer Region von 259 nt), aber das abweichende Expressionsmuster der beiden Gene spricht dafür, dass Xi-X ein neues Mitglied der Xlox-Familie ist. Xi-X könnte bei Xiphophorus zusätzlich zum Gen pdx-1 ein zweites Xlox-Gen eines der ParaHox-Cluster repräsentieren, das möglicherweise nur in einem begrenzten Zweig der Fischevolution erhalten geblieben ist. Xi-X wird in der Leber (0,8 kb), Skelettmuskulatur (2,3 kb) und Herzen (0,8 kb) exprimiert. In der Leber steht die Expression von zwei verschiedenen Transkripten gleicher Länge, möglicherweise in entgegengesetzten Richtungen transkribiert, in einem quantitativ reziproken Verhältnis zu den Alimentationsvorgängen. 7 weitere Homöoboxen konnten isoliert werden und sind vermutlich Bestandteile von zwei Hox-Clustern, Xiox-1.1/1.2/1.3 und Xiox-2.1/2.2/2.3/(3.1), vorwiegend der paralogen Gruppen 5-7 zugehörend. A localization of a homeobox gene, Xi-X, on the sex chromosomes of Xiphophorus was found. The distance to the platy-specific pigmentcell-loci (PSPL) is 15cM. The gene is expressed in the pigment cells of the background coloration (micromelanophores, xanthophores; 1,5 kb transcript), another Xi-X transcript (2,3 kb) is characteristic for the pigment cells of the art/population-specific color patterns (macromelanophores, xanthoerythrophores, erythrophores). The cells of the cell line DrLi are probably determined as patterning macropigmentcells. The cells produce both Xi-X transcripts of 1,5 kb and 2,3 kb. Differentiation induced by retinoic acid was accomponied by disappearance of the 1,5 kb transcript. Early differentiation stages of PSPL-coded cells are probably determined to build macropigmentcells, recognized through a transcript of 2,3 kb. The transcript of 1,5 kb is present only in the early stages of these cells. This work discusses the possibility, that the synthesis of the 2,3 kb Xi-X transcript in the RDiff -regulated pigment cell patterns Sd and Sp is dependent on the gene RDiff. An atypical inheritance of the platy-specific and swordtail-specific Xi-X-restriction fragments in the backcross generations could be observed (8,6% of 70 fishes). The linkage group Tu-complex (PSPL) enhances the instability what is prevented by the linkage group RDiff. A potential role of RDiff as a checkpoint gene is discussed. A possible correlation between the restrictive poly(GTCT/GATA)-organization in X. maculatus, in contrast to the individual organisation in X. helleri, and a good regulated expression of the Tu-complex is discussed. Certain aspects of the sex determination (X. helleri male, the male and female heterogamety in X. maculatus) are maybe reflected in the investigated Xi-X-region. The homology of Xi-X to the gene pdx-1 is high (to pdx-1 of Danio rerio 82% in 259 nt), but a different expression pattern of the two genes suggests that Xi-X is a new member of the ParaHox-genes, a second Xlox-gene with presence only in a limited branch of the fish-evolution. Xi-X is also expressed in the liver (0,8 kb), heart (0,8 kb) and sceletal muscle (2,3 kb). In the liver the relative expression of two transcripts of the same length, probably transcribed in different directions, are quantitative inverse in digestion processes. 7 further homeobox genes was isolated and are probably parts of two Hox-clusters, Xiox-1.1/1.2/1.3 and Xiox-2.1/2.2/2.3/(3.1), most of the genes belonging to the paralogous groups 5-7.

Nonmuscle myosin-II is a motor protein that drives cell movement and changes in cell shape during tissue and organ development. This study has determined he dynamic changes in myosin-II distribution during Drosophila compound eye morphogenesis. In photoreceptor neurons, myosin-II is undetectable at the apical domain throughout the first half of pupal life, at which time this membrane domain is involuted into the epithelium and progresses toward the retinal floor. Myosin-II is deployed at the apical surface at about 60% of pupal development, once the developing rhabdomeres reach the retinal floor. Subsequently, myosin-II becomes restricted to two stripes at the sides of the developing rhabdomere, adopting its final position within the visual cells R1-6; here, myosin-II is associated with a set of actin filaments that extend alongside the rhabdomeres. At the midpupal stage, myosin-II is also incorporated into stress-fiber-like arrays within the basal endfeet of the pigment cells that then change their shape. This spatiotemporal pattern of myosin- II localization and the morphological defects observed in the eyes of a myosin-II mutant suggest that the myosin-II/F- actin system is involved in the alignment of the rhabdomeres within the retina and in the flattening of the retinal floor. The observation that the myosin-II/F-actin arrays are incomplete or disorganized in R7/R8 and in rhodopsin-1-null R1-6 suggests further that the establishment and stability of this cytoskeletal system depend on rhodopsin-1 expression. (C) 2004 Elsevier Inc. All rights reserved

Recent reports show that components of the extracellular matrix function as cell survival factors through the suppression of apoptosis (programmed cell death). In this report we show that attachment to fibronectin suppresses apoptosis of normal human fetal and neonatal melanocytes in vitro and that prevention of attachment to underlying matrix or attachment to poly-L-lysine is a potent inducer of apoptosis in melanocytes. A role for the beta1-integrin family in mediating cell survival signals was shown by the ability of beta1-blocking antibodies to enhance apoptosis in melanocytes attached to fibronectin, and by the ability of anti-beta1 antibodies immobilized on solid supports to suppress apoptosis in melanocytes. Cytochalasin D reversed the effect of fibronectin on the suppression of apoptosis in melanocytes, suggesting that an intact cytoskeleton is required for transduction of survival signals. A human metastatic melanoma cell line, SKMEL28, was resistant to apoptosis when grown in suspension or on poly-L-lysine, even after 4 d in culture in the absence of exogenous growth factors. These results suggest that fibronectin suppresses apoptosis in normal human melanocytes through an integrin-dependent pathway and that significant differences in the control of anchorage-dependent regulation of apoptosis exist in melanocytes and melanoma cells.

Recently, we demonstrated the expression of rhodopsin in the tail fin of the Xenopus tadpole, in which photosensitive melanophores exist (Miyashita et al , The photoreceptor molecules in Xenopus tadpole tail fin, in which melanophores exist. Zool Sci 18:671–674, 2001). The presence of opsin molecules in pigment cells of lower vertebrates raises the possibility that pigment cells in animal skin function as photosensors generally. To explore this possibility in higher vertebrates, we tried to detect photoreception molecules in mammalian melanocytes. We extracted total RNA from Melan a2, a cell line of immortal murine melanocyte, which is derived from C57BL mice. The DNA sequence obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was homologous to the corresponding portion of the sequence of ocular rhodopsin of mice. Western blotting and fluorescent immunocytochemistry showed the existence of the opsin protein in the melanocytes. Another cell line, EL4, which is derived from lymphoma of C57BL/6N, scarcely expresses opsin mRNA, as judged by RT-PCR. Thus expression of the opsin gene is not ubiquitous among immortal cell lines. Detection of rhodopsin mRNA in murine tissues of C57BL/6N by RT-PCR showed its presence in the eye and skin but not in the liver. The role of the opsin molecule in melanocyte is not known at present, but this will provide additional insight into photoreception systems in animal skin.