Host evolution shapes gut microbiome composition in Astyanax mexicanus.

The ecological and genetic changes that underlie the evolution of host-microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics, and evolutionary history on microbial community structure, we examined gut microbiomes of river- and three cave-adapted morphotypes of the Mexican tetra, Astyanax mexicanus, in their natural environments and under controlled laboratory conditions. Field-collected samples were dominated by very few taxa and showed considerable interindividual variation. We found that lab-reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish evolution shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrated that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure.

Metabolic reprogramming underlies cavefish muscular endurance despite loss of muscle mass and contractility.

Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefish Astyanax mexicanus, has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression of pparγ-the master regulator of adipogenesis-with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism.

Discovery and characterization of Hv1-type proton channels in reef-building corals.

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a distinct manner. The biophysical properties of these novel channels make them interesting model systems. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH-regulation processes and possible consequences of ocean acidification related to the function of these channels.

The dynamics of epidermal stratification during post-larval development in zebrafish.

BACKGROUND: The epidermis, as a defensive barrier, is a consistent trait throughout animal evolution. During post-larval development, the zebrafish epidermis thickens by stratification or addition of new cell layers. Epidermal basal stem cells, expressing the transcription factor p63, are known to be involved in this process. Zebrafish post-larval epidermal stratification is a tractable system to study how stem cells participate in organ growth. METHODS: We used immunohistochemistry, in combination with EdU cell proliferation detection, to study zebrafish epidermal stratification. For this procedure, we selected a window of post-larval stages (5-8 mm of standard length or SL, which normalizes age by size). Simultaneously, we used markers for asymmetric cell division and the Notch signaling pathway. RESULTS: We found that epidermal stratification is the consequence of several events, including changes in cell shape, active cell proliferation and asymmetrical cell divisions. We identified a subset of highly proliferative epidermal cells with reduced levels of p63, which differed from the basal stem cells with high levels of p63. Additionally, we described different mechanisms that participate in the stratification process, including the phosphorylation of p63, asymmetric cell division regulated by the Par3 and LGN proteins, and expression of Notch genes.

Adaptation to low parasite abundance affects immune investment and immunopathological responses of cavefish.

Reduced parasitic infection rates in the developed world are suspected to underlie the rising prevalence of autoimmune disorders. However, the long-term evolutionary consequences of decreased parasite exposure on an immune system are not well understood. We used the Mexican tetra Astyanax mexicanus to understand how loss of parasite diversity influences the evolutionary trajectory of the vertebrate immune system, by comparing river with cave morphotypes. Here, we present field data affirming a strong reduction in parasite diversity in the cave ecosystem, and show that cavefish immune cells display a more sensitive pro-inflammatory response towards bacterial endotoxins. Surprisingly, other innate cellular immune responses, such as phagocytosis, are drastically decreased in cavefish. Using two independent single-cell approaches, we identified a shift in the overall immune cell composition in cavefish as the underlying cellular mechanism, indicating strong differences in the immune investment strategy. While surface fish invest evenly into the innate and adaptive immune systems, cavefish shifted immune investment to the adaptive immune system, and here, mainly towards specific T-cell populations that promote homeostasis. Additionally, inflammatory responses and immunopathological phenotypes in visceral adipose tissue are drastically reduced in cavefish. Our data indicate that long-term adaptation to low parasite diversity coincides with a more sensitive immune system in cavefish, which is accompanied by a reduction in the immune cells that play a role in mediating the pro-inflammatory response.

Subterranean life: Behavior, metabolic, and some other adaptations of Astyanax cavefish.

The ability of fishes to adapt to any aquatic environment seems limitless. It is enthralling how new species keep appearing at the deep sea or in subterranean environments. There are close to 230 known species of cavefishes, still today the best-known cavefish is Astyanax mexicanus, a Characid that has become a model organism, and has been studied and scrutinized since 1936. There are two morphotypes for A. mexicanus, a surface fish and a cavefish. The surface fish lives in central and northeastern Mexico and south of the United States, while the cavefish is endemic to the "Sierra del Abra-Tanchipa region" in northeast Mexico. The extensive genetic and genomic analysis depicts a complex origin for Astyanax cavefish, with multiple cave invasions and persistent gene flow among cave populations. The surface founder population prevails in the same region where the caves are. In this review, we focus on both morphotype's main morphological and physiological differences, but mainly in recent discoveries about behavioral and metabolic adaptations for subterranean life. These traits may not be as obvious as the troglomorphic characteristics, but are key to understand how Astyanax cavefish thrives in this environment of perpetual darkness.

Comparative transcriptome analysis of wild and lab populations of Astyanax mexicanus uncovers differential effects of environment and morphotype on gene expression.

Studying how different genotypes respond to environmental variation is essential to understand the genetic basis of adaptation. The Mexican tetra, Astyanax mexicanus, has cave and surface-dwelling morphotypes that have adapted to entirely different environments in the wild, and are now successfully maintained in lab conditions. While this has enabled the identification of genetic adaptations underlying a variety of physiological processes, few studies have directly compared morphotypes between lab-reared and natural populations. Such comparative approaches could help dissect the varying effects of environment and morphotype, and determine the extent to which phenomena observed in the lab are generalizable to conditions in the field. To this end, we take a transcriptomic approach to compare the Pachón cavefish and their surface fish counterparts in their natural habitats and the lab environment. We identify key changes in expression of genes implicated in metabolism and physiology between groups of fish, suggesting that morphotype (surface or cave) and environment (natural or lab) both alter gene expression. We find gene expression differences between cave and surface fish in their natural habitats are much larger than differences in expression between morphotypes in the lab environment. However, lab-raised cave and surface fish still exhibit numerous gene expression changes, supporting genetically encoded changes in livers of this species. From this, we conclude that a controlled laboratory environment may serve as an ideal setting to study the genetic underpinnings of metabolic and physiological differences between the cavefish and surface fish.

Octopus insularis as a new marine model for evolutionary developmental biology.

Octopuses are intriguing organisms that, together with squids and cuttlefishes, form the extant coleoid cephalopods. This group includes many species that can potentially be used as models in the fields of biomedicine, developmental biology, evolution, neuroscience and even for robotics research. The purpose of this work is to first present a simple method for maintaining Octopus insularis embryos under a laboratory setup. Second, we show that these embryos are suitable for detailed analyses of specific traits that appear during developmental stages, including the eyes, hearts, arms, suckers, chromatophores and Kölliker's organs. Similar complex traits between cephalopods and vertebrates such as the visual, cardiovascular, neural and pigmentation systems are generally considered to be a result of parallel evolution. We propose that O. insularis embryos could be used as a model for evolutionary developmental biology (or EvoDevo) research, where comparisons of the morphogenetic steps in the building of equivalent organs between cephalopods and known vertebrate model systems could shed light on evolutionary convergences and deep homologies.

The role of gene flow in rapid and repeated evolution of cave-related traits in Mexican tetra, Astyanax mexicanus.

Understanding the molecular basis of repeatedly evolved phenotypes can yield key insights into the evolutionary process. Quantifying gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture occurs more frequently between diverging lineages than previously thought. In this study, we resequenced 47 whole genomes of the Mexican tetra from three cave populations, two surface populations and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our data set. The two lineages likely diverged much more recently than previous mitochondrial estimates of 5-7 mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between ~161 and 191 k generations ago. The favoured demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified gene flow among all lineages sampled. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite gene flow with surface populations. The recency of these estimated divergence events suggests that selection may drive the evolution of cave-derived traits, as opposed to disuse and drift. Finally, we show that a key trogolomorphic phenotype QTL is enriched for genomic regions with low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. Our study shows that gene flow must be considered in studies of independent, repeated trait evolution.

Linking host morphology and symbiont performance in octocorals.

Octocorals represent an important group in reef communities throughout the tropical seas and, like scleractinian corals, they can be found in symbiosis with the dinoflagellate Symbiodinium. However, while there is extensive research on this symbiosis and its benefits in scleractinians, research on octocorals has focused so far mainly on the host without addressing their symbiosis. Here, we characterized and compared the photophysiological features of nine Caribbean octocoral species with different colony morphologies (sea fan, plumes, whips and rods) and related key morphological features with their respective symbiont photobiology. Colony features (branch shape and thickness), as well as micromorphological features (polyp size, density), were found to be significantly correlated with symbiont performance. Sea fans and plumes, with thinner branches and smaller polyps, favor higher metabolic rates, compared to sea rods with thicker branches and larger polyps. Daily integrated photosynthesis to respiration ratios > 1 indicated that the autotrophic contribution to organisms' energy demands was important in all species, but especially in sea whips. This information represents an important step towards a better understanding of octocoral physiology and its relationship to host morphology, and might also explain to some extent species distribution and susceptibility to environmental stress.

Salivary characteristics in patients with familial alzheimer's disease due to e280a mutation / Características salivares en personas con enfermedad de alzheimer familiar por la mutación e280a

ABSTRACT Introduction: Alzheimer's disease is a neurodegenerative disorder characterized by the loss of cognitive functions. The prevalence of this disease worldwide is high, and therefore it is important to have a better understanding of the oral health needs and conditions of individuals with this disorder. The present study was carried out in a population with E280A mutation for Alzheimer's disease. The goal was to describe the salivary characteristics of persons with early familial Alzheimer's disease, in order to detect changes in the oral microbiome that can guide the dental management of these patients. Methods: transversal study in 37 participants living in the Metropolitan Area of the city of Medellín, aged 53 ± 6 years in average, in different stages of the disease: mild: 8, moderate: 7, and severe: 22, and evaluated by neuropsychological tests. Salivary samples were collected, evaluating saliva secretion rate and saliva buffer capacity, and conducting microbial analysis of the species most commonly found in the mouth. Results: 45.9% of participants showed a decreased rate of stimulated salivary secretion; salivary buffer capacity was decreased in 83.87% of participants, with average pH values of 3.449 ± 0.89 after the Ericsson test. Buffer capacity was altered in participants with decreased secretion rate and in those with no alteration in salivary secretion rate. High levels of microbial growth were observed, mainly for Streptococcus mutans and Candida albicans. Conclusions: This study suggests that other factors besides the pharmacological ones, like age and disease severity, may affect the salivary rate flow in patients with early familial Alzheimer's disease.

RESUMEN Introducción: la enfermedad de Alzheimer es una alteración neurodegenerativa caracterizada por la pérdida de funciones cognitivas. Existe una alta prevalencia de esta enfermedad a nivel mundial, por lo que resulta oportuno tener una mayor comprensión de las necesidades y condiciones de salud bucal de los sujetos con este desorden. El presente estudio se llevó a cabo en una población con mutación E280A para la enfermedad de Alzheimer. El objetivo consistió en describir las características salivares de las personas con enfermedad de Alzheimer familiar precoz, con el fin de detectar cambios en el microbioma bucal que puedan orientar el manejo odontológico de estos pacientes. Métodos: estudio transversal en 37 participantes que habitan el Área Metropolitana de la ciudad de Medellín, con una edad promedio de 53 ± 6 años, en diferentes estadios de la enfermedad: leve: 8, moderada: 7 y grave: 22, evaluados mediante pruebas neuropsicológicas. Se tomaron muestras salivares, se evaluó la tasa de secreción salivar y la capacidad buffer de la saliva y se efectuó un análisis microbiano de las principales especies presentes en boca. Resultados: el 45,9% de los participantes presentaron una tasa disminuida de secreción salivar estimulada; la capacidad buffer salivar estuvo disminuida en el 83,87% de los participantes, con valores promedios de pH luego de la prueba de Ericsson de 3,449 ± 0,89. La capacidad buffer se encontró alterada tanto en los participantes con tasa de secreción disminuida como en aquellos con tasa de secreción salivar no alterada. Se observó alto crecimiento microbiano, principalmente de Streptococcus mutans y Candida albicans. Conclusiones: este estudio sugiere que pueden existir otros factores, además de los farmacológicos, que afectan la tasa de flujo salivar en los pacientes con enfermedad de Alzheimer familiar precoz, como la edad y la severidad de la enfermedad.

CTCF knockout reveals an essential role for this protein during the zebrafish development.

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

Transit-Amplifying Cells in the Fast Lane from Stem Cells towards Differentiation.

Stem cells have a high potential to impact regenerative medicine. However, stem cells in adult tissues often proliferate at very slow rates. During development, stem cells may change first to a pluripotent and highly proliferative state, known as transit-amplifying cells. Recent advances in the identification and isolation of these undifferentiated and fast-dividing cells could bring new alternatives for cell-based transplants. The skin epidermis has been the target of necessary research about transit-amplifying cells; this work has mainly been performed in mammalian cells, but further work is being pursued in other vertebrate models, such as zebrafish. In this review, we present some insights about the molecular repertoire regulating the transition from stem cells to transit-amplifying cells or playing a role in the transitioning to fully differentiated cells, including gene expression profiles, cell cycle regulation, and cellular asymmetrical events. We also discuss the potential use of this knowledge in effective progenitor cell-based transplants in the treatment of skin injuries and chronic disease.

Evolutionary Developmental Biology (Evo-Devo) Research in Latin America.

Famous for its blind cavefish and Darwin's finches, Latin America is home to some of the richest biodiversity hotspots of our planet. The Latin American fauna and flora inspired and captivated naturalists from the nineteenth and twentieth centuries, including such notable pioneers such as Fritz Müller, Florentino Ameghino, and Léon Croizat who made a significant contribution to the study of embryology and evolutionary thinking. But, what are the historical and present contributions of the Latin American scientific community to Evo-Devo? Here, we provide the first comprehensive overview of the Evo-Devo laboratories based in Latin America and describe current lines of research based on endemic species, focusing on body plans and patterning, systematics, physiology, computational modeling approaches, ecology, and domestication. Literature searches reveal that Evo-Devo in Latin America is still in its early days; while showing encouraging indicators of productivity, it has not stabilized yet, because it relies on few and sparsely distributed laboratories. Coping with the rapid changes in national scientific policies and contributing to solve social and health issues specific to each region are among the main challenges faced by Latin American researchers. The 2015 inaugural meeting of the Pan-American Society for Evolutionary Developmental Biology played a pivotal role in bringing together Latin American researchers eager to initiate and consolidate regional and worldwide collaborative networks. Such networks will undoubtedly advance research on the extremely high genetic and phenotypic biodiversity of Latin America, bound to be an almost infinite source of amazement and fascinating findings for the Evo-Devo community.

DNA Damage Assessment in Zebrafish Embryos Exposed to Monceren(®) 250 SC Fungicide Using the Alkaline Comet Assay.

Monceren 250 SC is a commercial fungicide with the active ingredient 1-(4-chlorobenzyl)-1-(cyclopentyl)-3-phenylurea, also known as pencycuron. This compound inhibits the growth of fungi as Rhizoctonia solani that invades potato, rice, and cotton or as Pellicularia spp, which contaminates lettuce and tomato crops. In this study, we assessed genotoxicity or DNA damage by the alkaline comet assay in zebrafish blastula-stage embryos exposed to 250 to 1250 µg/mL of the Monceren fungicide or to Bleomycin (0.25 µg/mL) used as a positive control. Additionally, survival and spontaneous movement were monitored in embryos after exposure to different concentrations of fungicide. DNA damage was evaluated using three genotoxicity parameters of the alkaline comet assay: tail length, tail moment, and tail intensity. We found that Monceren 250 SC fungicide induces DNA damage, as shown by significant increases in the three genotoxicity parameters in zebrafish embryos compared with control embryos nonexposed to Monceren. Tail intensity was the more accurate parameter to evaluate genotoxicity levels in zebrafish embryos. At 48 h after exposure to the fungicide, the survival rate of larvae-embryos was reduced to 40-45%. This study shows that the Monceren 250 SC fungicide exerts genotoxic effects in zebrafish during early stages of development.

Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience.

Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b) during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

Zebrafish scarb2a insertional mutant reveals a novel function for the Scarb2/Limp2 receptor in notochord development.

BACKGROUND: Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus-renal failure). The zebrafish insertional mutant scarb2a(hi1463Tg) has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates. RESULTS: There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di-leucine motif. We found that scarb2a(hi1463Tg) homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole-mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short-lived in the presumptive notochord and precedes the complete differentiation of the notochord. CONCLUSIONS: This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development.

Betaglycan knock-down causes embryonic angiogenesis defects in zebrafish.

Angiogenesis is an essential requirement for embryonic development and adult homeostasis. Its deregulation is a key feature of numerous pathologies and many studies have shown that members of the transforming growth factor beta (TGF-ß) family of proteins play important roles in angiogenesis during development and disease. Betaglycan (BG), also known as TGF-ß receptor type III, is a TGF-ß coreceptor essential for mice embryonic development but its role in angiogenesis has not been described. We have cloned the cDNA encoding zebrafish BG, a TGF-ß-binding membrane proteoglycan that showed a dynamic expression pattern in zebrafish embryos, including the notochord and cells adjacent to developing vessels. Injection of antisense morpholinos decreased BG protein levels and morphant embryos exhibited impaired angiogenesis that was rescued by coinjection with rat BG mRNA. In vivo time-lapse microscopy revealed that BG deficiency differentially affected arterial and venous angiogenesis: morphants showed impaired pathfinding of intersegmental vessels migrating from dorsal aorta, while endothelial cells originating from the caudal vein displayed sprouting and migration defects. Our results reveal a new role for BG during embryonic angiogenesis in zebrafish, which has not been described in mammals and pose interesting questions about the molecular machinery regulating angiogenesis in different vertebrates. genesis 53:583-603, 2015. © 2015 Wiley Periodicals, Inc.

A stem cell proliferation burst forms new layers of P63 expressing suprabasal cells during zebrafish postembryonic epidermal development.

Organ growth during development is a highly regulated process with both temporal and spatial constraints. Epidermal stratification is essential for skin growth and development. Although the zebrafish has been well studied, it is not known when and how epidermal stratification occurs. This is because beyond the first five days of development our knowledge is currently limited. We found that epidermal stratification in zebrafish begins when the larvae reach a standard length (SL) of 6 mm at approximately 25 days of age. Over the next four days (from a SL of 6 to 9 mm), epidermis thickness increases almost four-fold. This represents a sudden increase in organ size, since for the previous 20 days of development, the epidermis has been only two layers thick. This pattern is different from that observed in mammals that undergo continuous stratification from E14.5-E18.5. To study how stem cell proliferation gives rise to the new epidermal layers, we used a combination of markers: one for cell proliferation (proliferating cell nuclear-antigen PCNA) and one for epidermal stem cells (P63 transcription factor). We identified, throughout the stratification process, two different waves of cell division. Initially, the most basal epidermal cells divided and generated a subset of suprabasal cells (possibly transient-amplifying cells); within the next several days, the basal cells stopped dividing, and the suprabasal cells began proliferation, giving rise to most of the cell types in the new layers. This part of the process is similar to what has been recently found during epidermal stratification in mammals.

The zebrafish mutants for the V-ATPase subunits d, ac45, E, H and c and their variable pigment dilution phenotype.

BACKGROUND: The V-ATPase is a proton pump that creates an acidic medium, necessary for lysosome function and vesicular traffic. It is also essential for several developmental processes. Many enzymes, like the V-ATPase, are assemblies of multiple subunits, in which each one performs a specific function required to achieve full activity. In the zebrafish V-ATPase 15 different subunits form this multimeric complex and mutations in any of these subunits induce hypopigmentation or pigment dilution phenotype. We have previously found variability in the pigment dilution phenotype among five of the V-ATPase zebrafish mutants. This work presents additional information about such differences and is an update from a previous report. FINDINGS: We describe the variable phenotype severity observed among zebrafish V-ATPase pigment dilution mutants studying mRNA expression levels from their corresponding genes. At the same time we carried out phylogenetic analysis for this genes. CONCLUSIONS: Based in the similarities between different pigment dilution mutants we suggest that there is an essential role for V-ATPases in melanosome biogenesis and melanocyte survival. Neither variable expression levels for the different V-ATPase subunits studied here or the presence of duplicated genes seems to account for the variable phenotype severity from this group of mutants. We believe there are some similarities between the pigment dilution phenotype from zebrafish V-ATPase insertional mutants and pigment mutants obtained in a chemical screening ("Tubingen pigmentation mutants"). As for some of these "Tubingen mutants" the mutated gene has not been found we suggest that mutations in V-ATPase genes may be inducing their defects.