On the Development of Pigment Patterns in Amphibians

SYNOPSIS. Pigmentary mutants in amphibians provide importantvehicles for studying basic problems in development. Some ofthese mutants exert influences on the tissue environment inwhich the chromatophores differentiate and others involve theexpression of pigment in specific types of pigment cells. Melanophoresare the best known of all chromatophores, and albinism has beenmuch studied. Genes controlling its expression may operate atdifferent levels. Some are involved in the production of tyrosinase,whileothers affect the melanosomal matrix. In contrast, melanoidmutants are characterized by an overproduction of eumelanin,usually through the differentiation of an excessive number ofmelanophores. Melanoid mutants of the axolotl also exhibit a great diminutionin xanthophore and iridophore number, and the same seems tobe true of some melanoid-like mutants of leopard frogs. Thepteridine pigments of xanthophores and purines of iridophoresare often products of xanthine dehydrogenase (XDH) activity,and it is suspected that the expression of the melanoid phenotypemay result from a genetic defect involving this enzyme. Thisis supported by experiments involving the administration ofan XDH inhibitor, allopurinol, to normal larvae. This inhibitorresults in the production of partial phenocopies of the melanoidcondition. Blue mutants involving either partial or completediminution of xanthophore and iridophore pigments may also bebased upon deficient XDH activity.     

Pigment cell differentiation: the relationship between pterin content, allopurinol treatment, and the melanoid gene in axolotls

The effects of allopurinol (an inhibitor of the enzyme xanthine dehydrogenase (XDH] and the melanoid gene on pigment cell differentiation in the axolotl were examined by analyzing pigment components of the xanthophore (pterins). Pterin contents of skin extracts (70% ethanol) from wild type, allopurinol-treated and melanoid axolotls were determined by thin layer chromatography (TLC) and fluorometric scanning of TLC plates. Heights of peaks produced were used as a quantitative measure for pterin content. Results reveal that melanoid animals contain significantly reduced amounts of all seven pterins examined as compared with wild type animals. Allopurinol-treated animals have reduced levels of four pterins (xanthopterin, isoxanthopterin, biopterin and sepiapterin) as compared with the wild type. These findings suggest that the alterations in pterin biosynthetic pathways, either by drug-induced inhibition of XDH activity or by the melanoid gene, produce similar dramatic changes in pigment phenotype which are manifested by alterations in pigment cell differentiation.     

Observations on the pigmentation of the pigeon iris

There are three genetically controlled iris types found in the pigeon, two of which contain stromal pigment cells, the third lacks pigment cells. The yellow (gravel) and white (pearl) iris types have pigment cells that contain birefringent pigment granules (crystals) and are ultrastructurally similar to iridophores of poikilothermic vertebrates. Both these iris types contain guanine as a major "pigment" and, in addition, the yellow iris contains at least two yellow fluorescing pigments that are tentatively identified as pteridines. The pigment cells of the yellow and white irises are structurally identical differing only in the presence or absence of these yellow pigments. The stromal pigment cells of the white iris correspond in structure and pigment chemistry to classical iridophores although they lack strong irridescence and are therefore perhaps best considered leucophores. The pigment cells of the yellow iris can be considered "reflecting xanthophores" having the combined properties of both classical xanthophores and iridophore/leucophores.     

The Rosy Locus in Drosophila Melanogaster: Xanthine Dehydrogenase and Eye Pigments

The rosy gene in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. Xanthine dehydrogenase is not synthesized in the eye, but rather is transported there. The present report describes the ultrastructural localization of XDH in the Drosophila eye. Three lines of evidence are presented demonstrating that XDH is sequestered within specific vacuoles, the type II pigment granules. Histochemical and antibody staining of frozen sections, as well as thin layer chromatography studies of several adult genotypes serve to examine some of the factors and genic interactions that may be involved in transport of XDH, and in eye pigment formation. While a specific function for XDH in the synthesis of the red, pteridine eye pigments remains unknown, these studies present evidence that: (1) the incorporation of XDH into the pigment granules requires specific interaction between a normal XDH molecule and one or more transport proteins; (2) the structural integrity of the pigment granule itself is dependent upon the presence of a normal balance of eye pigments, a notion advanced earlier.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER: Further Evidence in Support of a CIS-Acting Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

The present report summarizes our recent progress in the genetic dissection of an elementary genetic unit in a higher organism, the rosy locus (ry:3--52.0) in Drosophila melanogaster. Pursuing the hypothesis that the rosy locus includes a noncoding control region, as well as a structural element coding for the xanthine dehydrogenase (XDH) peptide, experiments are described that characterize and map a rosy locus variant associated with much lower than normal levels of XDH activity. Experiments are described that fail to relate this phenotype to alteration in the structure of the XDH peptide, but clearly associate this character with variation in number of molecules of XDH per fly. Large-scale fine-structure recombination experiments locate the genetic basis for this variation in the number of molecules of XDH per fly to a site immediately to the left of the XDH structural element within a region previously designated as the XDH control element. Moreover, experiments clearly separate this "underproducer" variant site from a previously described "overproducer" site within the control region. Examination of enzyme activity in electrophoretic gels of appropriate heterozygous genotypes demonstrates the cis-acting nature of this variation in the number of molecules of XDH. A revision of the map of the rosy locus, structural and control elements is presented in the light of the additional mapping data now available.     

Allopurinol-induced melanism in the tiger salamander (Ambystoma tigrinum nebulosum).

The enzyme, xanthine dehydrogenase (XDH), has been examined in Ambystoma tigrinum nebulosum with respect to its role in pigmentation. It now seems probable that the melanoid gene (m) either codes directly for XDH or is somehow intimately connected with the normal function of this enzyme. Inhibition of XDH using the drug, allopurinol, results in animals which appear to be phenocopies of melanoid mutants as described for the Mexican axolotl (Ambystoma mexicanum). The effects of allopurinol in terms of specific pigmentary alterations were examined, and a new method for analyzing heterogeneous extracts of skin pigments (e.g., purines and pteridines) is presented. The significance of the link between XDH and melanism is discussed with emphasis on possible mechanisms of pigment induction and general applicability to biological systems.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER : Evidence for a Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

From a collection of electrophoretic variants of XDH obtained from laboratory strains and natural populations, a stock was isolated that was associated with much greater than normal levels of XDH activity. Preliminary recombination experiments demonstrated that this character maps to the rosy locus. While a series of observations failed to relate this phenotype to alteration in the structure of the XDH polypeptide, kinetic and immunological experiments did succeed in associating this character with variation in number of molecules of XDH/fly. Large scale fine structure recombination experiments locate the genetic basis for this variation in number of molecules of XDH/fly to a site very close to, but definitely outside of, the genetic boundaries of the XDH structural information. Observations are described which eliminate the possibility that we are dealing with a tandem duplication of the XDH structural element. Turning to a regulatory role for this genetic element located adjacent to the XDH structural information, a simple experiment is described which demonstrates that it functions as a "cis-acting" regulator of the XDH structural element.     

Genetic Limits of the Xanthine Dehydrogenase Structural Element within the Rosy Locus in DROSOPHILA MELANOGASTER

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.     

Streptomyces coelicolor A3(2) produces a new yellow pigment associated with the polyketide synthase Cpk

Streptomyces coelicolor A3(2) is an extensively studied model organism for the genetic studies of Streptomycetes - a genus known for the production of a vast number of bioactive compounds and complex regulatory networks controlling morphological differentiation and secondary metabolites production. We present the discovery of a presumptive product of the Cpk polyketide synthase. We have found that on the rich medium without glucose S. coelicolor A3(2) produces a yellow compound secreted into the medium. We have proved by complementation that production of the observed yellow pigment is dependent on cpk gene cluster previously described as cryptic type I polyketide synthase cluster. The pigment production depends on the medium composition, does not occur in the presence of glucose, and requires high density of spore suspension used for inoculation.     

Inductive effect of the eye tissues of adult clawed toads on the gastrula ectoderm

The inducing influence of adult eye tissues on the early gastrula ectoderm was studied in vitro. Both retina and pigment epithelium induced in the early gastrula ectoderm similar spectra of cell types, including nervous tissue, retina, pigment epithelium, lentoids, ectomesenchyme, and melanophores. It is suggested that the correspondence of these cell types with those arising at a spontaneous transdifferentiation of the isolated retina and pigment epithelium cells in vitro or at the induction of the early gastrula ectoderma by archencephalic endomesoderm during the normal development can be accounted for by that in these eye cells molecular determinants appeared as a result of induction and maintaina the stability of their differentiation and their potencies to transdifferentiation in vitro being reproduced during the lifetime of these cells.     

Effects of exogenous guanosine on chromatophore differentiation in the axolotl

Guanosine is shown to dramatically alter the pigment phenotype of axolotls by suppressing melanization and enhancing the biosynthesis and deposition of purine-derived pigments. Phenotypic changes caused by guanosine are manifested by altered chromatophore differentiation patterns such that few black pigment cells (melanophores) differentiate (and those that do are punctate and necrotic in appearance), whereas the development of yellow (xanthophore) and reflecting (iridophore) pigment cells is enhanced. Mechanisms for changes in chromatophore differentiation, and thus pattern formation, are discussed, including the possibility that pigment cells may undergo transdifferentiation in vivo.     

Activation of p59(Fyn) leads to melanocyte dedifferentiation by influencing MKP-1-regulated mitogen-activated protein kinase signaling.

Malignant melanoma is a cancer whose incidence is rising rapidly, but the mechanism by which normal melanocytes become malignant in vivo is still little understood. In the course of melanoma progression, a fraction of cells often becomes depigmented, which reflects the loss of the balance between mitogenic activities and differentiation in those pigment cells. A key factor involved in differentiation in pigment cells is mitogen-activated protein kinase (MAPK). However, because both activation and inhibition of MAPK signaling is known to correlate with differentiation, its function in pigment cells is still unclear. We investigated the role of MAPK signaling in pigment cells using the melanoma-inducing receptor tyrosine kinase Xmrk. Xmrk signaling in mouse melanocytes suppressed differentiation and induced a transformed phenotype. We found that this was based on sustained MAPK activation caused by low and transient expression of MAPK-phosphatase MKP-1. The Src kinase p59(Fyn) was thereby identified as being crucial for the receptor-mediated suppression of differentiation by down-regulating MKP-1 expression. Our findings reveal a novel mechanism of regulating the balance between differentiation and proliferation based on a Src kinase-modified MAPK activity. Moreover, they point to a new role for Src kinases in dedifferentiation and transformation of pigment cells.     

The use of expression profiling to study pigment cell biology and dysfunction

Regulation of gene expression is a fundamental process by which cells respond to both intracellular and extracellular signals. For a pigment cell, alterations in gene expression regulate the processes of cell migration, lineage restriction, differentiation, type of pigment produced, and progression from a normal pigment cell to that of melanoma. To date, the identification of genes involved in normal pigment cell development has been accomplished by the cloning of individual mutant alleles, a single gene at a time. Current advances in technology have now made it possible to use expression profile analysis to investigate, on a genomic scale, the process of pigment cell development and function. This review compares and contrasts the methods of subtractive suppressive polymerase chain reaction (PCR) and differential display with that of cDNA microarray analysis.     

Cloning and partial characterization of the xanthine dehydrogenase gene of Calliphora vicina, a distant relative of Drosophila melanogaster

In vitro enzymatic assays have shown that an enzyme with typical xanthine dehydrogenase (XDH) activities and electrophoretic mobility slightly different from that of Drosophila XDH is present in Calliphora tissues. A Calliphora genomic sequence has been isolated by low-stringency hybridization to the Drosophila rosy gene (XDH), and partially sequenced. This sequence has been shown to be unique, polymorphic, and it maps on chromosome I. Sequence comparisons provide compelling evidence that it belongs to the XDH gene of Calliphora. Interspecies transformation experiments, aimed at investigating functional as well as structural divergence of the XDH genes of Calliphora and Drosophila, are now possible.     

Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.     

Hereditary xanthinuria is not so rare disorder of purine metabolism

Hereditary xanthinuria (type I) is caused by an inherited deficiency of the xanthine oxidorectase (XDH/XO), and is characterized by very low concentration of uric acid in blood and urine and high concentration of urinary xanthine, leading to urolithiasis. Type II results from a combined deficiency of XDH/XO and aldehyde oxidase. Patients present with hematuria, renal colic, urolithiasis or even acute renal failure. Clinical symptoms are the same for both types. In a third type, clinically distinct, sulfite oxidase activity is missing as well as XDH/XO and aldehyde oxidase. The prevalence is not known, but about 150 cases have been described so far. Hypouricemia is sometimes overlooked, that´s why we have set up the diagnostic flowchart. This consists of a) evaluation of uric acid concentrations in serum and urine with exclusion of primary renal hypouricemia, b) estimation of urinary xanthine, c) allopurinol loading test, which enables to distinguish type I and II; and finally assay of xanthine oxidoreductase activity in plasma with molecular genetic analysis. Following this diagnostic procedure we were able to find first patients with hereditary xanthinuria in our Czech population. We have detected nine cases, which is one of the largest group worldwide. Four patients were asymptomatic. All had profound hypouricemia, which was the first sign and led to referral to our department. Urinary concentrations of xanthine were in the range of 170–598 mmol/mol creatinine (normal < 30 mmol/mol creatinine). Hereditary xanthinuria is still unrecognized disorder and subjects with unexplained hypouricemia need detailed purine metabolic investigation.     

Cell differentiation and malignancy

An understanding of the mechanism that controls growth and differentiation in normal cells would seem to be an essential requirement to elucidate the origin and reversibility of malignancy. For this approach I have mainly used normal and leukemic blood cells, and in most studies have used myeloid blood cells as a model system. Our development of systems for the in vitro cloning and clonal differentiation of normal blood cells made it possible to study the controls that regulate growth (multiplication) and differentiation of these normal cells and the changes in these controls in leukemia. Experiments with normal blood cell precursors have shown that normal cells require different proteins to induce growth and differentiation. We have also shown that in normal myeloid precursors, growth-inducing protein induces both growth and production of differentiation-inducing protein so this ensures the coupling between growth and differentiation that occurs in normal development. The origin of malignancy involves uncoupling of growth and differentiation. This can be produced by changes from inducible to constitutive expression of specific genes that result in asynchrony to the coordination required for the normal developmental program. Normal myeloid precursors require an external source of growth-inducing protein for growth, and we have identified different types of leukemic cells. Some no longer require and other constitutively produce their own growth-inducing protein. But addition of the normal differentiation-inducing protein to these malignant cells still induces their normal differentiation, and the mature cells are then no longer malignant. Genetic changes that produce blocks in the ability to be induced to differentiate by the normal inducer occur in the evolution of leukemia. But even these cells can be induced to differentiate by other compounds, including low doses of compounds now being used in cancer therapy, that induce the differentiation program by other pathways. This differentiation of leukemic cells has been obtained in vitro and in vivo, and our in vivo results indicate that this may be a useful approach to therapy. In some tumours, such as sarcomas, reversion from a malignant to a non-malignant phenotype can be a result of chromosome changes that suppress malignancy. But in myeloid leukemia, the stopping of growth in mature cells by induction of differentiation bypasses the genetic changes that produce the malignant phenotype. These conclusions can also be applied to other types of normal and malignant cells.     

Population Genetic Studies Revealed Local Adaptation in a High Gene-Flow Marine Fish,the Small Yellow Croaker (Larimichthys polyactis)

The genetic differentiation of many marine fish species is low. Yet local adaptation may be common in marine fish species as the vast and changing marine environment provides more chances for natural selection. Here, we used anonymous as well as known protein gene linked microsatellites and mitochondrial DNA to detect the population structure of the small yellow croaker (Larimichthys polyactis) in the Northwest Pacific marginal seas. Among these loci, we detected at least two microsatellites, anonymous H16 and HSP27 to be clearly under diversifying selection in outlier tests. Sequence cloning and analysis revealed that H16 was located in the intron of BAHCC1 gene. Landscape genetic analysis showed that H16 mutations were significantly associated with temperature, which further supported the diversifying selection at this locus. These marker types presented different patterns of population structure: (i) mitochondrial DNA phylogeny showed no evidence of genetic divergence and demonstrated only one glacial linage; (ii) population differentiation using putatively neutral microsatellites presented a pattern of high gene flow in the L. polyactis. In addition, several genetic barriers were identified; (iii) the population differentiation pattern revealed by loci under diversifying selection was rather different from that revealed by putatively neutral loci. The results above suggest local adaptation in the small yellow croaker. In summary, population genetic studies based on different marker types disentangle the effects of demographic history, migration, genetic drift and local adaptation on population structure and also provide valuable new insights for the design of management strategies in L. polyactis.     

Extension of the Limits of the Xdh Structural Element in DROSOPHILA MELANOGASTER

Experiments expanding the array of mutants affecting the xanthine dehydrogenase (XDH) structural element in Drosophila melanogaster are described. These include rosy eye color mutants which exhibit interallelic complementation, and mutants with normal eye color but lowered levels of XDH. Evidence is presented which argues that these are structural alterations in the enzyme. Recombination experiments were performed using these mutants as well as some electrophoretic variants. The two ends of the rosy locus are marked with mutant sites which are clearly structural in nature; the XDH structural element and the rosy null mutant map are completely concordant. A possible procedure to recover control element mutants is described.     

Recessive yellow in the Mongolian gerbil (Meriones unguiculatus)

A new autosomal recessive coat color mutant in the Mongolian gerbil (Meriones unguiculatus) is described: recessive yellow. On the dorsal side the mutant has a rich yellow to ginger color. Ventrally it shows the typical creamy white belly of a wild-type Mongolian gerbil. The dorsal yellow hairs have short black tips, and a light olive green base. A clear demarcation line between dorsal and ventral color is present. Crosses between recessive yellow animals and multiple homozygous recessive tester animals (a/a; c^chm/c^chm; g/g; p/p) resulted only in animals of an agouti (wild-type) phenotype, showing that the new allele is not allelic with any of the known coat color mutations in the Mongolian gerbil. Molecular studies showed that the new mutant is caused by a missence mutation at the extension (E) locus. On a non-agouti background (a/a; e/e) mutant animals look like a dark wild-type agouti. In contrast to wild-type agouti it shows yellow pigmentation and dark ticking at the ventral side, resulting in the absence of a demarcation line. Since black pigment is present in both the agouti and non-agouti variant (A/A; e/e and a/a; e/e), we conclude that recessive yellow in the Mongolian gerbil is non-epistatic to agouti. Additionally we describe a second mutation at the same locus leading to a similar phenotype, however without black pigment and diminishing yellow pigment during life. Fertility and viability of both new mutants are within normal range. The extension (E) gene is known to encode the melanocortin 1 receptor (MC1R). Interestingly, this is the only gene that is known to account for substantial variation in skin and hair color in humans. Many different mutations are known of which some are associated with higher skin cancer incidence.