Zebrafish Fin Research Articles

Proteome profile of zebrafish caudal fin based on one‐dimensional gel electrophoresis LCMS/MS and two‐dimensional gel electrophoresis MALDI MS/MS analysis

Zebrafish (Danio rerio) is the widely used vertebrate model animal for understanding the complexity of development and disease process. Zebrafish has been also extensively used in understanding the mechanism of regeneration for its extensive capability of regenerating fins and other tissues. We have analyzed the proteome profile of zebrafish caudal fin in its native state based on one-dimensional gel electrophoresis LCMS/MS and two-dimensional gel electrophoresis MS/MS analyses. A total of 417 proteins were identified as zebrafish fin tissue specific, which includes 397 proteins identified based on one-dimensional gel electrophoresis LCMS/MS analysis and 101 proteins identified based on two-dimensional gel electrophoresis MALDI MS/MS. The proteins mapped to the zebrafish fin tissue were shown to be involved in various biological activities related to development, apoptosis, signaling and metabolic process. Focal adhesion, regulation of actin cytoskeleton, cancer-related pathways, mitogen-activated protein kinase signaling, antigen processing and presentation, and proteasome are some of the important pathways associated with the identified proteome data set of the zebrafish fin.

UV-induced fin damage in zebrafish as a system for evaluating the chemopreventive potential of broccoli and cauliflower extracts

This study applied broccoli and cauliflower extracts (whole, floret, and stem) to zebrafish larvae in parallel to receive 100 mJ/cm2 of UVB six times, and recorded their fin malformation phenotypes. Chemopreventive effects of each group, including UVB, whole-, floret-, and stem-extracts of broccoli and cauliflower on fin development were evaluated using Kaplan-Meier analysis, log-rank test, and Cox proportional hazards regression. Results showed that (1) zebrafish fins in the UVB + whole broccoli extract group are 6.20~9.32-times more likely to return to normal fins than ones in the UVB only group, but fins in the UVB + whole cauliflower extract group are only 5.13~11.10-times more likely to recover, indicated that whole broccoli and cauliflower extract had similar chemopreventive ability on fin development; and (2) the broccoli stem has the highest antioxidant capacity among other groups. In conclusion, zebrafish can be used as a system for evaluating the efficacy of other UVB protective compounds.

Clonal analyses reveal roles of organ founding stem cells, melanocyte stem cells and melanoblasts in establishment, growth and regeneration of the adult zebrafish fin

In vertebrates, the adult form emerges from the embryo by mobilization of precursors or adult stem cells. What different cell types these precursors give rise to, how many precursors establish the tissue or organ, and how they divide to establish and maintain the adult form remain largely unknown. We use the pigment pattern of the adult zebrafish fin, with a variety of clonal and lineage analyses, to address these issues. Early embryonic labeling with lineage-marker-bearing transposons shows that all classes of fin melanocytes (ontogenetic, regeneration and kit-independent melanocytes) and xanthophores arise from the same melanocyte-producing founding stem cells (mFSCs), whereas iridophores arise from distinct precursors. Additionally, these experiments show that, on average, six and nine mFSCs colonize the caudal and anal fin primordia, and daughters of different mFSCs always intercalate to form the adult pattern. Labeled clones are arrayed along the proximal-distal axis of the fin, and melanocyte time-of-differentiation lineage assays show that although most of the pigment pattern growth is at the distal edge of the fin, significant growth also occurs proximally. This suggests that leading edge melanocyte stem cells (MSCs) divide both asymmetrically to generate new melanocytes, and symmetrically to expand the MSCs and leave quiescent MSCs in their wake. Clonal labeling in adult stages confirms this and reveals different contributions of MSCs and transient melanoblasts during growth. These analyses build a comprehensive picture for how MSCs are established and grow to form the pigment stripes of the adult zebrafish fins.

Dermoskeleton morphogenesis in zebrafish fins

Zebrafish fins have a proximal skeleton of endochondral bones and a distal skeleton of dermal bones. Recent experimental and genetic studies are discovering mechanisms to control fin skeleton morphogenesis. Whereas the endochondral skeleton has been extensively studied, the formation of the dermal skeleton requires further revision. The shape of the dermal skeleton of the fin is generated in its distal growing margin and along a proximal growing domain. In these positions, dermoskeletal fin morphogenesis can be explained by intertissue interactions and the function of several genetic pathways. These pathways regulate patterning, size, and cell differentiation along three axes. Finally, a common genetic control of late development, regeneration, and tissue homeostasis of the fin dermoskeleton is currently being analyzed. These pathways may be responsible for the similar shape obtained after each morphogenetic process. This provides an interesting conceptual framework for future studies on this topic. Developmental Dynamics 239:2779–2794, 2010. © 2010 Wiley-Liss, Inc.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach

BackgroundDespite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV).ResultsTwo-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36).ConclusionsThe genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

TRANSLATIONAL IMPACT

The regeneration of damaged organs may eventually be achieved with stem cell-mediated therapy. In some models, adult progenitor cells are carefully directed to regenerate patterned, functional tissue structures through complex signaling pathways. Many of the pathways that promote regeneration also contribute to cancer formation. The selective ability to induce regeneration of tissue without causing cancer requires more knowledge of the signals that mediate these two processes. Ras is an important regulator of cell growth and is mutated in tumors of many cancer patients. Ras regulation of tissue regeneration is not well understood, which impedes the potential for stem cell therapy for regenerative medicine.Zebrafish are able to regenerate amputated fins that have a normal striped pattern. The ability of the stripe-forming melanocytes to faithfully regenerate in the correct position during the complex process of fin regeneration provides a useful model to dissect the pathways that direct stem cells. Little is known about the participation of Ras in zebrafish fin regeneration.When a zebrafish loses its fin, it replaces the lost bone and soft tissues, and reestablishes its characteristic pigment stripes from precursor melanocytes. To determine the effect of Ras on regeneration, the authors use a fin amputation and regeneration zebrafish model in which they manipulate Ras activity during fin regeneration. Elevated Ras activity causes dose-dependent expansion of pigmented melanocytes and melanocyte precursors. Active Ras also rescues pigmentation during fin regeneration in normally unpigmented mutants that lack functional c-Kit, which is a proto-oncogenic receptor tyrosine kinase that is predicted to be an upstream regulator of Ras. These findings position Ras as a key downstream mediator of c-Kit signaling during fin regeneration and demonstrate that Ras activity supports melanocyte precursor proliferation in the zebrafish fin during regeneration.This work demonstrates a central role for Ras in the zebrafish fin pigmentation model of regeneration. During Ras-induced hyperpigmentation, melanocytes occupy the correct niche within the fin rays and then resolve normally into stripes after experimental activation of Ras is discontinued. It will be interesting to develop models to analyze how progenitor proliferation is controlled in other cell lineages, and to determine how cell specialization affects the process of fin regeneration. Since Ras is important to both regeneration and cancer formation, it should be useful to identify the molecular events that prevent melanocyte hypercellularization in fin regeneration. Understanding the roles of Ras and other signaling molecules in stem and progenitor cell regulation should promote the potential use of stem cells in the therapeutic regeneration of human tissues.

Zebrafish ProVEGF-C expression, proteolytic processing and inhibitory effect of unprocessed ProVEGF-C during fin regeneration.

BackgroundIn zebrafish, vascular endothelial growth factor-C precursor (proVEGF-C) processing occurs within the dibasic motif HSIIRR214 suggesting the involvement of one or more basic amino acid-specific proprotein convertases (PCs) in this process. In the present study, we examined zebrafish proVEGF-C expression and processing and the effect of unprocessed proVEGF-C on caudal fin regeneration.Methodology/Principal FindingsCell transfection assays revealed that the cleavage of proVEGF-C, mainly mediated by the proprotein convertases Furin and PC5 and to a less degree by PACE4 and PC7, is abolished by PCs inhibitors or by mutation of its cleavage site (HSIIRR214 into HSIISS214). In vitro, unprocessed proVEGF-C failed to activate its signaling proteins Akt and ERK and to induce cell proliferation. In vivo, following caudal fin amputation, the induction of VEGF-C, Furin and PC5 expression occurs as early as 2 days post-amputation (dpa) with a maximum levels at 4–7 dpa. Using immunofluorescence staining we localized high expression of VEGF-C and the convertases Furin and PC5 surrounding the apical growth zone of the regenerating fin. While expression of wild-type proVEGF-C in this area had no effect, unprocessed proVEGF-C inhibited fin regeneration.Conclusions/SignificancesTaken together, these data indicate that zebrafish fin regeneration is associated with up-regulation of VEGF-C and the convertases Furin and PC5 and highlight the inhibitory effect of unprocessed proVEGF-C on fin regeneration.

Low Doses of Ionizing Radiation Promote Tumor Growth and Metastasis by Enhancing Angiogenesis

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

Proteomics Profiling of Single Organs from Individual Adult Zebrafish

The model organism zebrafish (Danio rerio) is extensively utilized in studies of developmental biology but is also being investigated in the context of a growing list of human age-related diseases. To facilitate such studies, we here present protein expression patterns of adult zebrafish organs, including blood, brain, fin, heart, intestine, liver, and skeletal muscle. Protein extracts were prepared from the different organs of two zebrafish and analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry. Zebrafish tissue was digested directly after minimal fractionation and cleaned up (the shotgun approach). Proteins were identified using Mascot software. In total, 1394 proteins were identified of which 644 were nonredundant. Of these, 373 demonstrated an organ-specific expression pattern and 57 had not been shown on protein level before. These data emphasize the need for increased research at the protein level to facilitate the selection of candidate proteins for targeted quantification and to refine systematic genetic network analysis in vertebrate development, biology, and disease.

Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

Genetic Analysis of Fin Development in Zebrafish Identifies Furin and Hemicentin1 as Potential Novel Fraser Syndrome Disease Genes

Using forward genetics, we have identified the genes mutated in two classes of zebrafish fin mutants. The mutants of the first class are characterized by defects in embryonic fin morphogenesis, which are due to mutations in a Laminin subunit or an Integrin alpha receptor, respectively. The mutants of the second class display characteristic blistering underneath the basement membrane of the fin epidermis. Three of them are due to mutations in zebrafish orthologues of FRAS1, FREM1, or FREM2, large basement membrane protein encoding genes that are mutated in mouse bleb mutants and in human patients suffering from Fraser Syndrome, a rare congenital condition characterized by syndactyly and cryptophthalmos. Fin blistering in a fourth group of zebrafish mutants is caused by mutations in Hemicentin1 (Hmcn1), another large extracellular matrix protein the function of which in vertebrates was hitherto unknown. Our mutant and dose-dependent interaction data suggest a potential involvement of Hmcn1 in Fraser complex-dependent basement membrane anchorage. Furthermore, we present biochemical and genetic data suggesting a role for the proprotein convertase FurinA in zebrafish fin development and cell surface shedding of Fras1 and Frem2, thereby allowing proper localization of the proteins within the basement membrane of forming fins. Finally, we identify the extracellular matrix protein Fibrillin2 as an indispensable interaction partner of Hmcn1. Thus we have defined a series of zebrafish mutants modelling Fraser Syndrome and have identified several implicated novel genes that might help to further elucidate the mechanisms of basement membrane anchorage and of the disease's aetiology. In addition, the novel genes might prove helpful to unravel the molecular nature of thus far unresolved cases of the human disease.

Mapping amorphous calcium phosphate transformation into crystalline mineral from the cell to the bone in zebrafish fin rays

The continuously forming fin bony rays of zebrafish represent a simple bone model system in which mineralization is temporally and spatially resolved. The mineralized collagen fibrils of the fin bones are identical in structure to those found in all known bone materials. We study the continuous mineralization process within the tissue by using synchrotron microbeam x-ray diffraction and small-angle scattering, combined with cryo-scanning electron microscopy. The former provides information on the mineral phase and the mineral particles size and shape, whereas the latter allows high-resolution imaging of native hydrated tissues. The integration of the two techniques demonstrates that new mineral is delivered and deposited as packages of amorphous calcium phosphate nanospheres, which transform into platelets of crystalline apatite within the collagen matrix.

Time course Analysis of Gene expression patterns in ZebrafIsh Eye during Optic Nerve Regeneration

It is well-established that neurons in the adult mammalian central nervous system (CNS) are terminally differentiated and, if injured, will be unable to regenerate their connections. In contrast to mammals, zebrafish and other teleosts display a robust neuroregenerative response. Following optic nerve crush (ONX), retinal ganglion cells (RGC) regrow their axons to synapse with topographically correct targets in the optic tectum, such that vision is restored in approximately 21 days. What accounts for these differences between teleostean and mammalian responses to neural injury is not fully understood. A time course analysis of global gene expression patterns in the zebrafish eye after ONX can help to elucidate cellular and molecular mechanisms that contribute to a successful neuroregeneration. To define different phases of regeneration after ONX, alpha tubulin 1 (tuba1) and growth-associated protein 43 (gap43), markers previously shown to correspond to morphophological events, were measured by real time quantitative PCR (qPCR). Microarray analysis was then performed at defined intervals (6 hours, 1, 4, 12, and 21 days) post-ONX and compared to SHAM. Results show that optic nerve damage induces multiple, phase-related transcriptional programs, with the maximum number of genes changed and highest fold-change occurring at 4 days. Several functional groups affected by optic nerve regeneration, including cell adhesion, apoptosis, cell cycle, energy metabolism, ion channel activity, and calcium signaling, were identified. Utilizing the whole eye allowed us to identify signaling contributions from the vitreous, immune and glial cells as well as the neural cells of the retina. Comparisons between our dataset and transcriptional profiles from other models of regeneration in zebrafish retina, heart and fin revealed a subset of commonly regulated transcripts, indicating shared mechanisms in different regenerating tissues. Knowledge of gene expression patterns in all components of the eye in a model of successful regeneration provides an entry point for functional analyses, and will help in devising hypotheses for testing normal and toxic regulatory factors.

Cx40.8, a Cx43-like protein, forms gap junction channels inefficiently and may require Cx43 for its association at the plasma membrane

In addition to having a Cx43 ortholog, the zebrafish genome also contains a Cx43-like gene, Cx40.8. Here, we investigate the expression of cx40.8 in zebrafish fins and the function of Cx40.8 in HeLa cells. We find that cx40.8 is present in the same population of dividing cells as cx43. Unlike Cx43, dye coupling assays suggest that Cx40.8 only inefficiently forms functional gap junction channels. However, co-transfection reveals that Cx40.8 can co-localize with Cx43 in gap junction plaques, and that the resulting plaques contain functional gap junction channels. Together, these data suggest the possibility that Cx40.8 may functionally interact with Cx43 to regulate cell proliferation in vivo. Structured summary MINT- 7266123: cx40.8 (genbank_protein_gi:68354404) and cx43 (uniprotkb: O57474) colocalize (MI: 0403) by fluorescence microscopy (MI: 0416)

09-P097 Apical fold morphogenesis in zebrafish fin; differences from tetrapod limb development

09-P097 Apical fold morphogenesis in zebrafish fin; differences from tetrapod limb development

A gain of function mutation causing skeletal overgrowth in the rapunzel mutant

Mechanisms that regulate the growth and form of the vertebrate skeleton are largely unknown. The zebrafish mutant rapunzel has heterozygous defects in bone development, resulting in skeletal overgrowth, thus identification of the genetic lesion underlying rapunzel might provide insight into the molecular basis of skeletogenesis. In this report, we demonstrate that the rapunzel mutant results from a missense mutation in the previously uncharacterized rpz gene. This conclusion is supported by genetic mapping, identification of a missense mutation in rapunzel(c14) in a highly conserved region of the rpz gene, and suppression of the rapunzel homozygous embryonic phenotype with morpholino knockdown of rpz. In addition, rpz transcripts are identified in regions correlating with the homozygous embryonic phenotype (head, pectoral fin buds, somites and fin fold). This report provides the first gene identification for a mutation affecting segment number in the zebrafish fin and development of both the fin ray (dermal) and the axial skeleton.

Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish

In a microarray-based gene profiling analysis of Müller glia-derived retinal stem cells in light-damaged retinas from adult zebrafish, we found that 2 genes required for regeneration of fin and heart tissues in zebrafish, hspd1 (heat shock 60-kDa protein 1) and mps1 (monopolar spindle 1), were up-regulated. Expression of both genes in the neurogenic Müller glia and progenitors was independently verified by quantitative reverse transcriptase PCR and in situ hybridization. Functional analysis of temperature-sensitive mutants of hspd1 and mps1 revealed that both are necessary for Müller glia-based cone photoreceptor regeneration in adult zebrafish retina. In the amputated fin, hspd1 is required for the induction of mesenchymal stem cells and blastema formation, whereas mps1 is required at a later step for rapid cell proliferation and outgrowth. This temporal sequence of hspd1 and mps1 function is conserved in the regenerating retina. Comparison of gene expression profiles from regenerating zebrafish retina, caudal fin, and heart muscle revealed additional candidate genes potentially implicated in injury-induced epimorphic regeneration in diverse zebrafish tissues.

Fgf and Sdf-1 Pathways Interact during Zebrafish Fin Regeneration

The chemokine stromal cell-derived factor-1 (SDF1) was originally identified as a pre-B cell stimulatory factor but has been recently implicated in several other key steps in differentiation and morphogenesis. In addition, SDF1 as well as FGF signalling pathways have recently been shown to be involved in the control of epimorphic regeneration. In this report, we address the question of a possible interaction between the two signalling pathways during adult fin regeneration in zebrafish. Using a combination of pharmaceutical and genetic tools, we show that during epimorphic regeneration, expression of sdf1, as well as of its cognate receptors, cxcr4a, cxcr4b and cxcr7 are controlled by FGF signalling. We further show that, Sdf1a negatively regulates the expression of fgf20a. Together, these results lead us to propose that: 1) the function of Fgf in blastema formation is, at least in part, relayed by the chemokine Sdf1a, and that 2) Sdf1 exerts negative feedback on the Fgf pathway, which contributes to a transient expression of Fgf20a downstream genes at the beginning of regeneration. However this feedback control can be bypassed since the Sdf1 null mutants regenerate their fin, though slower. Very few mutants for the regeneration process were isolated so far, illustrating the difficulty in identifying genes that are indispensable for regeneration. This observation supports the idea that the regeneration process involves a delicate balance between multiple pathways.

Maintenance of blastemal proliferation by functionally diverse epidermis in regenerating zebrafish fins

Appendage regeneration in salamanders and fish occurs through formation and maintenance of a mass of progenitor tissue called the blastema. A dedicated epidermis overlays the blastema and is required for its proliferation and patterning, yet this interaction is poorly understood. Here, we identified molecularly and functionally distinct compartments within the basal epidermal layer during zebrafish fin regeneration. Proximal epidermal subtypes express the transcription factor lef1 and the blastemal mitogen shh, while distal subtypes express the Fgf target gene pea3 and wnt5b, an inhibitor of blastemal proliferation. Ectopic overexpression of wnt5b reduced shh expression, while pharmacologic introduction of a Hh pathway agonist partially rescued blastemal proliferation during wnt5b overexpression. Loss- and gain-of-function approaches indicate that Fgf signaling promotes shh expression in proximal epidermis, while Fgf/Ras signaling restricts shh expression from distal epidermis through induction of pea3 expression and maintenance of wnt5b. Thus, the fin wound epidermis spatially confines Hh signaling through the activity of Fgf and Wnt pathways, impacting blastemal proliferation during regenerative outgrowth.

Heterochronic Shift in Hox-Mediated Activation of Sonic hedgehog Leads to Morphological Changes during Fin Development

We explored the molecular mechanisms of morphological transformations of vertebrate paired fin/limb evolution by comparative gene expression profiling and functional analyses. In this study, we focused on the temporal differences of the onset of Sonic hedgehog (Shh) expression in paired appendages among different vertebrates. In limb buds of chick and mouse, Shh expression is activated as soon as there is a morphological bud, concomitant with Hoxd10 expression. In dogfish (Scyliorhinus canicula), however, we found that Shh was transcribed late in fin development, concomitant with Hoxd13 expression. We utilized zebrafish as a model to determine whether quantitative changes in hox expression alter the timing of shh expression in pectoral fins of zebrafish embryos. We found that the temporal shift of Shh activity altered the size of endoskeletal elements in paired fins of zebrafish and dogfish. Thus, a threshold level of hox expression determines the onset of shh expression, and the subsequent heterochronic shift of Shh activity can affect the size of the fin endoskeleton. This process may have facilitated major morphological changes in paired appendages during vertebrate limb evolution.