Influence of survival, promotion, and growth on pattern formation in zebrafish skin.
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ABSTRACT:
SUBMITTER: Konow C
PROVIDER: S-EPMC8110552 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundZebrafish display a striped skin pattern on their body; two types of connexins, namely, Connexin39.4 (Cx39.4) and Connexin41.8 (Cx41.8), are involved in stripe pattern formation. Herein, we investigated the role of the C-terminal (CT) domains of Cx39.4 and Cx41.8 in vivo and in vitro.MethodsTo investigate the role of CT domains in vivo, we established transgenic zebrafish lines expressing the CT-domain-modified connexin series in pigmented cells and observed skin patterns in fish. To investigate the role of the CT domains in vitro, we expressed the CT-domain modified connexin series in Neuro-2a (N2a) cells and calculated the plaque formation frequency.ResultsThe overexpression of Cx39.4 lacking a CT domain produced skin patterns similar to that produced by full-length Cx39.4 in the cx39.4 -/- mutant and in cx39.4 and cx41.8 double-knockout mutant zebrafish. Fluorescence-protein-fused CT-domain-modified Cx39.4 formed gap junction plaques between N2a cells. The overexpression of CT-truncated Cx41.8 rescued the mutant phenotype in the cx41.8 -/- mutant but did not function in the double knockout zebrafish. Fluorescence-protein-fused CT-truncated Cx41.8 hardly formed plaques between N2a cells without Cx39.4 but formed gap junction plaques when co-expressed with Cx39.4.ConclusionsThe CT domain of Cx39.4 is not required for protein function, at least in the pigment cells of zebrafish. However, the need for the CT domain of Cx41.8 depends on Cx39.4 expression.General significanceThese results provide evidence for the interactions between Cx39.4 and Cx41.8 in pigment cells of zebrafish and suggest that at least one connexin must have a CT domain.
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Project description:Gap junctions allow the direct and bidirectional transfer of small molecules between cells. Polyamine sensitivity, which has been observed for a certain gap junction in vitro, confers rectification property to gap junction. Here we report that the polyamine sensitivity of gap junctions in vivo is crucial for skin pattern formation in zebrafish. Transgenic experiments have revealed that several connexin genes were able to rescue the spot phenotype of mutant zebrafish. Mutational analyses of the N-terminal region of connexins revealed that the ExxxE motif, a hypothetical polyamine-binding site, was important for connexin's role in pattern formation. Ectopic expression of spermidine/spermine N(1)-acetyltransferase (SSAT), a polyamine metabolic enzyme, also caused stripe pattern changes, which further indicates that the polyamine sensitivity of gap junctions is crucial. This is the first report to show that polyamine sensitivity has a physiologically relevant function and is related to skin pattern formation in animals.
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Project description:Random pattern skin flap transplantation is frequently applied in plastic and reconstructive surgery, but the distal part of skin flaps often suffers necrosis due to ischemia. Astragaloside IV (AS-IV), a natural saponin purified from Astragalus membranaceus, may have beneficial functions for flap survival. In this study, rats were divided into a control group and an AS-IV treatment group, and underwent surgery using a modified "McFarlane flap" model. After intragastric administration of vehicle control or AS-IV for their respective groups, flap survival area and water content were measured 7 days after surgery. Flap tissue was separated to test protein expressions related to angiogenesis, inflammation, oxidative stress and autophagy via western blot, immunohistochemistry, and immunofluorescence. Results showed that AS-IV improved flap survival area and reduced tissue edema. AS-IV also increased mean vessel densities and upregulated levels of VEGF protein, both of which indicate increased angiogenesis. Furthermore, AS-IV depressed leukocyte infiltration, decreased expressions of inflammatory proteins TNF-α, IL1β and IL6, increased SOD activity, decreased MDA content, and stimulated autophagy. Overall, our results suggest that AS-IV promotes skin flap survival via inducing angiogenesis, depressing inflammation and dampening oxidative stress; it also activates autophagy, which may be an underlying mechanism for oxidative stress depression.
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Project description:The development of the pigmentation pattern in zebrafish is a tightly regulated process that depends on both the self-organizing properties of pigment cells and extrinsic cues from other tissues. Many of the known mutations that alter the pattern act cell-autonomously in pigment cells, and our knowledge about external regulators is limited. Here, we describe novel zebrafish mau mutants, which encompass several dominant missense mutations in Aquaporin 3a (Aqp3a) that lead to broken stripes and short fins. A loss-of-function aqp3a allele, generated by CRISPR-Cas9, has no phenotypic consequences, demonstrating that Aqp3a is dispensable for normal development. Strikingly, the pigment cells from dominant mau mutants are capable of forming a wild-type pattern when developing in a wild-type environment, but the surrounding tissues in the mutants influence pigment cell behaviour and interfere with the patterning process. The mutated amino acid residues in the dominant alleles line the pore surface of Aqp3a and influence pore permeability. These results demonstrate an important effect of the tissue environment on pigment cell behaviour and, thereby, on pattern formation.
| S-EPMC5482984 | biostudies-literature
Project description:Pattern formation is a key aspect of development. Adult zebrafish exhibit a striking striped pattern generated through the self-organisation of three different chromatophores. Numerous investigations have revealed a multitude of individual cell-cell interactions important for this self-organisation, but it has remained unclear whether these known biological rules were sufficient to explain pattern formation. To test this, we present an individual-based mathematical model incorporating all the important cell-types and known interactions. The model qualitatively and quantitatively reproduces wild type and mutant pigment pattern development. We use it to resolve a number of outstanding biological uncertainties, including the roles of domain growth and the initial iridophore stripe, and to generate hypotheses about the functions of leopard. We conclude that our rule-set is sufficient to recapitulate wild-type and mutant patterns. Our work now leads the way for further in silico exploration of the developmental and evolutionary implications of this pigment patterning system.
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Project description:Transforming growth factor beta1 (TGFbeta1) expression is elevated by tumor promoters in the mouse skin, but its role in tumor promotion has not been well defined. To investigate this, we have compared TGFbeta1+/+ and +/- mice in a two-stage skin chemical carcinogenesis protocol. Surprisingly, TGFbeta1+/- mice had fewer number and incidence of benign papillomas, reduced epidermal and tumor cell proliferation and reduced epidermal TGFbeta1 and nuclear p-Smad2 localization in response to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) compared with TGFbeta1+/+ mice. Maximal TPA activation of protein kinase C (PKCalpha) as measured by activity assays and activation of target genes and induction of cornified envelopes correlated with TGFbeta1 gene dosage in keratinocytes and addition of exogenous TGFbeta1 restored the cornification defect in TGFbeta1+/- keratinocytes. Similarly, inhibition of ALK5-suppressed TPA-mediated PKCalpha activation suggesting that physiological levels of TGFbeta1 are required for maximal activation of PKC-dependent mitogenic responses. Paradoxically, the TPA-induced inflammatory response was greater in TGFbeta1+/- skin, but TGFbeta1+/+ papillomas had more tumor infiltrating myeloperoxidase-positive cells and pro-inflammatory gene expression was elevated in v-ras(Ha)-transduced TGFbeta1+/+ but not TGFbeta1+/- keratinocytes. Thus, ras activation switches TGFbeta1 to a pro-inflammatory cytokine. Despite this differential proliferative and inflammatory response to TPA and enhanced papilloma formation in the TGFbeta1+/+ mice, the frequency of malignant conversion was reduced compared with TGFbeta1+/- mice. Therefore, TGFbeta1 promotes benign tumors by modifying tumor promoter-induced cell proliferation and inflammation but retains a suppressive function for malignant conversion.
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Project description:Polarity establishment, the spontaneous generation of asymmetric molecular distributions, is a crucial component of many cellular functions. Saccharomyces cerevisiae (yeast) undergoes directed growth during budding and mating, and is an ideal model organism for studying polarization. In yeast and many other cell types, the Rho GTPase Cdc42 is the key molecular player in polarity establishment. During yeast polarization, multiple patches of Cdc42 initially form, then resolve into a single front. Because polarization relies on strong positive feedback, it is likely that the amplification of molecular-level fluctuations underlies the generation of multiple nascent patches. In the absence of spatial cues, these fluctuations may be key to driving polarization. Here we used particle-based simulations to investigate the role of stochastic effects in a Turing-type model of yeast polarity establishment. In the model, reactions take place either between two molecules on the membrane, or between a cytosolic and a membrane-bound molecule. Thus, we developed a computational platform that explicitly simulates molecules at and near the cell membrane, and implicitly handles molecules away from the membrane. To evaluate stochastic effects, we compared particle simulations to deterministic reaction-diffusion equation simulations. Defining macroscopic rate constants that are consistent with the microscopic parameters for this system is challenging, because diffusion occurs in two dimensions and particles exchange between the membrane and cytoplasm. We address this problem by empirically estimating macroscopic rate constants from appropriately designed particle-based simulations. Ultimately, we find that stochastic fluctuations speed polarity establishment and permit polarization in parameter regions predicted to be Turing stable. These effects can operate at Cdc42 abundances expected of yeast cells, and promote polarization on timescales consistent with experimental results. To our knowledge, our work represents the first particle-based simulations of a model for yeast polarization that is based on a Turing mechanism.
| S-EPMC5864077 | biostudies-literature
Project description:Colour patterns of adult fish are composed of several different types of pigment cells distributing in the skin during juvenile development. The zebrafish, Danio rerio, displays a striking pattern of dark stripes of melanophores interspersed with light stripes of xanthophores. A third cell type, silvery iridophores, contributes to both stripes and plays a crucial role in adult pigment pattern formation. Several mutants deficient in iridophore development display similar adult phenotypes with reduced numbers of melanophores and defects in stripe formation. This indicates a supporting role of iridophores for melanophore development and maintenance. One of these mutants, rose (rse), encodes the Endothelin receptor b1a. Here we describe a new mutant in zebrafish, karneol (kar), which has a phenotype similar to weak alleles of rse with a reduction in iridophore numbers and defects of adult pigment patterning. We show that, unlike rse, kar is not required in iridophores. The gene defective in the kar mutant codes for an endothelin-converting enzyme, Ece2, which activates endothelin ligands by proteolytic cleavage. By morpholino-mediated knockdown, we identify Endothelin 3b (Edn3b) as the ligand for endothelin receptor signalling in larval iridophores. Thus, Endothelin signalling is involved in iridophore development, proliferation and stripe morphogenesis in larvae as well as adult zebrafish. In mammals the pathway is required for melanocyte development; therefore, our results indicate a previously unrecognized close evolutionary relationship between iridophores in zebrafish and melanocytes in mammals.
| S-EPMC4058085 | biostudies-literature
Project description:Color patterns are prominent features of many animals and are of high evolutionary relevance. In basal vertebrates, color patterns are composed of specialized pigment cells that arrange in multilayered mosaics in the skin. Zebrafish (Danio rerio), the preeminent model system for vertebrate color pattern formation, allows genetic screens as powerful approaches to identify novel functions in a complex biological system. Adult zebrafish display a series of blue and golden horizontal stripes, composed of black melanophores, silvery or blue iridophores, and yellow xanthophores. This stereotyped pattern is generated by self-organization involving direct cell contacts between all three types of pigment cells mediated by integral membrane proteins [1-5]. Here, we show that neuropeptide signaling impairs the striped pattern in a global manner. Mutations in the genes coding either for galanin receptor 1A (npm/galr1A) or for its ligand galanin (galn) result in fewer stripes, a pale appearance, and the mixing of cell types, thus resembling mutants with thyroid hypertrophy [6]. Zebrafish chimeras obtained by transplantations of npm/galr1A mutant blastula cells indicate that mutant pigment cells of all three types can contribute to a normal striped pattern in the appropriate host. However, loss of galr1A expression in a specific region of the brain is sufficient to cause the mutant phenotype in an otherwise wild-type fish. Increased thyroid hormone levels in mutant fish suggest that galanin signaling through Galr1A in the pituitary is an upstream regulator of the thyroid hormone pathway, which in turn promotes precise interactions of pigment cells during color pattern formation.
| S-EPMC6971688 | biostudies-literature
Project description:Animal skin pigment patterns are excellent models to study the mechanism of biological self-organization. Theoretical approaches developed mathematical models of pigment patterning and molecular genetics have brought progress; however, the responsible cellular mechanism is not fully understood. One long unsolved controversy is whether the patterning information is autonomously determined by melanocytes or nonautonomously determined from the environment. Here, we transplanted purified melanocytes and demonstrated that melanocytes could form periodic pigment patterns cell autonomously. Results of heterospecific transplantation among quail strains are consistent with this finding. Further, we observe that developing melanocytes directly connect with each other via filopodia to form a network in culture and in vivo. This melanocyte network is reminiscent of zebrafish pigment cell networks, where connexin is implicated in stripe formation via genetic studies. Indeed, we found connexin40 (cx40) present on developing melanocytes in birds. Stripe patterns can form in quail skin explant cultures. Several calcium channel modulators can enhance or suppress pigmentation globally, but a gap junction inhibitor can change stripe patterning. Most interestingly, in ovo, misexpression of dominant negative cx40 expands the black region, while overexpression of cx40 expands the yellow region. Subsequently, melanocytes instruct adjacent dermal cells to express agouti signaling protein (ASIP), the regulatory factor for pigment switching, which promotes pheomelanin production. Thus, we demonstrate Japanese quail melanocytes have an autonomous periodic patterning role during body pigment stripe formation. We also show dermal agouti stripes and how the coupling of melanocytes with dermal cells may confer stable and distinct pigment stripe patterns.
| S-EPMC6452743 | biostudies-literature