Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration

The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.     

Zebrafish ProVEGF-C Expression,Proteolytic Processing and Inhibitory Effect of Unprocessed ProVEGF-C during Fin Regeneration

Background

In 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 Findings

Cell 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/Significances

Taken 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.     

Temperature-Sensitive Mutations That Cause Stage-Specific Defects in Zebrafish Fin Regeneration

When amputated, the fins of adult zebrafish rapidly regenerate the missing tissue. Fin regeneration proceeds through several stages, including wound healing, establishment of the wound epithelium, recruitment of the blastema from mesenchymal cells underlying the wound epithelium, and differentiation and outgrowth of the regenerate. We screened for temperature-sensitive mutations that affect the regeneration of the fin. Seven mutations were identified, including five that fail to regenerate their fins, one that causes slow growth during regeneration, and one that causes dysmorphic bumps or tumors to develop in the regenerating fin. reg5 mutants fail to regenerate their caudal fins, whereas reg6 mutants develop dysmorphic bumps in their regenerates at the restrictive temperature. Temperature-shift experiments indicate that reg5 and reg6 affect different stages of regeneration. The critical period for reg5 occurs during the early stages of regeneration before or during establishment of the blastema, resulting in defects in subsequent growth of the blastema and failure to differentiate bone-forming cells. The critical period for reg6 occurs after the onset of bone differentiation and during early stages of regenerative outgrowth. Both reg5 and reg6 also show temperature-sensitive defects in embryonic development or in ontogenetic outgrowth of the juvenile fin.     

LKB1 and Notch Pathways Interact and Control Biliary Morphogenesis

Background

LKB1 is an evolutionary conserved kinase implicated in a wide range of cellular functions including inhibition of cell proliferation, regulation of cell polarity and metabolism. When Lkb1 is inactivated in the liver, glucose homeostasis is perturbed, cellular polarity is affected and cholestasis develops. Cholestasis occurs as a result from deficient bile duct development, yet how LKB1 impacts on biliary morphogenesis is unknown.

Methodology/Principal Findings

We characterized the phenotype of mice in which deletion of the Lkb1 gene has been specifically targeted to the hepatoblasts. Our results confirmed that lack of LKB1 in the liver results in bile duct paucity leading to cholestasis. Immunostaining analysis at a prenatal stage showed that LKB1 is not required for differentiation of hepatoblasts to cholangiocyte precursors but promotes maturation of the primitive ductal structures to mature bile ducts. This phenotype is similar to that obtained upon inactivation of Notch signaling in the liver. We tested the hypothesis of a functional overlap between the LKB1 and Notch pathways by gene expression profiling of livers deficient in Lkb1 or in the Notch mediator RbpJκ and identified a mutual cross-talk between LKB1 and Notch signaling. In vitro experiments confirmed that Notch activity was deficient upon LKB1 loss.

Conclusion

LKB1 and Notch share a common genetic program in the liver, and regulate bile duct morphogenesis.     

edn1 and hand2 Interact in Early Regulation of Pharyngeal Arch Outgrowth during Zebrafish Development

Endothelin-1 (Edn1) signaling provides a critical input to development of the embryonic pharygneal arches and their skeletal derivatives, particularly the articulating joints and the ventral skeleton including the lower jaw. Previous work in zebrafish has mostly focused on the role of Edn1 in dorsal-ventral (DV) patterning, but Edn1 signaling must also regulate tissue size, for with severe loss of the pathway the ventral skeleton is not only mispatterned, but is also prominently hypoplastic – reduced in size. Here we use mutational analyses to show that in the early pharyngeal arches, ventral-specific edn1-mediated proliferation of neural crest derived cells is required for DV expansion and outgrowth, and that this positive regulation is counterbalanced by a negative one exerted through a pivotal, ventrally expressed Edn1-target gene, hand2. We also describe a new morphogenetic cell movement in the ventral first arch, sweeping cells anterior in the arch to the region where the lower jaw forms. This movement is negatively regulated by hand2 in an apparently edn1-independent fashion. These findings point to complexity of regulation by edn1 and hand2 at the earliest stages of pharyngeal arch development, in which control of growth and morphogenesis can be genetically separated.     

Igf Signaling is Required for Cardiomyocyte Proliferation during Zebrafish Heart Development and Regeneration

Unlike its mammalian counterpart, the adult zebrafish heart is able to fully regenerate after severe injury. One of the most important events during the regeneration process is cardiomyocyte proliferation, which results in the replacement of lost myocardium. Growth factors that induce cardiomyocyte proliferation during zebrafish heart regeneration remain to be identified. Signaling pathways important for heart development might be reutilized during heart regeneration. IGF2 was recently shown to be important for cardiomyocyte proliferation and heart growth during mid-gestation heart development in mice, although its role in heart regeneration is unknown. We found that expression of igf2b was upregulated during zebrafish heart regeneration. Following resection of the ventricle apex, igf2b expression was detected in the wound, endocardium and epicardium at a time that coincides with cardiomyocyte proliferation. Transgenic zebrafish embryos expressing a dominant negative form of Igf1 receptor (dn-Igf1r) had fewer cardiomyocytes and impaired heart development, as did embryos treated with an Igf1r inhibitor. Moreover, inhibition of Igf1r signaling blocked cardiomyocyte proliferation during heart development and regeneration. We found that Igf signaling is required for a subpopulation of cardiomyocytes marked by gata4:EGFP to contribute to the regenerating area. Our findings suggest that Igf signaling is important for heart development and myocardial regeneration in zebrafish.     

Zebrafish atoh1 genes: classic proneural activity in the inner ear and regulation by Fgf and Notch

Hair cells of the inner ear develop from an equivalence group marked by expression of the proneural gene Atoh1. In mouse, Atoh1 is necessary for hair cell differentiation, but its role in specifying the equivalence group (proneural function) has been questioned and little is known about its upstream activators. We have addressed these issues in zebrafish. Two zebrafish homologs, atoh1a and atoh1b, are together necessary for hair cell development. These genes crossregulate each other but are differentially required during distinct developmental periods, first in the preotic placode and later in the otic vesicle. Interactions with the Notch pathway confirm that atoh1 genes have early proneural function. Fgf3 and Fgf8 are upstream activators of atoh1 genes during both phases, and foxi1, pax8 and dlx genes regulate atoh1b in the preplacode. A model is presented in which zebrafish atoh1 genes operate in a complex network leading to hair cell development.     

NF-κB Signaling Negatively Regulates Osteoblast Dedifferentiation during Zebrafish Bone Regeneration

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BMP and Notch Signaling Pathways differentially regulate Cardiomyocyte Proliferation during Ventricle Regeneration

Adult mammalian hearts show limited capacity to proliferate after injury, while zebrafish are capable to completely regenerate injured hearts through the proliferation of spared cardiomyocytes. BMP and Notch signaling pathways have been implicated in cardiomyocyte proliferation during zebrafish heart regeneration. However, the molecular mechanism underneath this process as well as the interaction between these two pathways remains to be further explored. In this study we showed BMP signaling was activated after ventricle ablation and acted epistatic downstream of Notch signaling. Inhibition of both signaling pathways differentially influenced ventricle regeneration and cardiomyocyte proliferation, as revealed by time-lapse analysis using a cardiomyocyte-specific FUCCI (fluorescent ubiquitylation-based cell cycle indicator) system. Further experiments revealed that inhibition of BMP and Notch signaling led to cell-cycle arrest at different phases. Overall, our results shed light on the interaction between BMP and Notch signaling pathways and their functions in cardiomyocyte proliferation during cardiac regeneration.     

The Arginine Decarboxylase Pathways of Host and Pathogen Interact to Impact Inflammatory Pathways in the Lung

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.     

Cx43-Dependent Skeletal Phenotypes Are Mediated by Interactions between the Hapln1a-ECM and Sema3d during Fin Regeneration

Skeletal development is a tightly regulated process and requires proper communication between the cells for efficient exchange of information. Analysis of fin length mutants has revealed that the gap junction protein Connexin43 (Cx43) coordinates cell proliferation (growth) and joint formation (patterning) during zebrafish caudal fin regeneration. Previous studies have shown that the extra cellular matrix (ECM) protein Hyaluronan and Proteoglycan Link Protein1a (Hapln1a) is molecularly and functionally downstream of Cx43, and that hapln1a knockdown leads to reduction of the glycosaminoglycan hyaluronan. Here we find that the proteoglycan aggrecan is similarly reduced following Hapln1a knockdown. Notably, we demonstrate that both hyaluronan and aggrecan are required for growth and patterning. Moreover, we provide evidence that the Hapln1a-ECM stabilizes the secreted growth factor Semaphorin3d (Sema3d), which has been independently shown to mediate Cx43 dependent phenotypes during regeneration. Double knockdown of hapln1a and sema3d reveal synergistic interactions. Further, hapln1a knockdown phenotypes were rescued by Sema3d overexpression. Therefore, Hapln1a maintains the composition of specific components of the ECM, which appears to be required for the stabilization of at least one growth factor, Sema3d. We propose that the Hapln1a dependent ECM provides the required conditions for Sema3d stabilization and function. Interactions between the ECM and signaling molecules are complex and our study demonstrates the requirement for components of the Hapln1a-ECM for Sema3d signal transduction.     

TGF-β Signaling Promotes Tissue Formation during Cardiac Valve Regeneration in Adult Zebrafish

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HMGB1 Contributes to Regeneration After Spinal Cord Injury in Adult Zebrafish

High mobility group box 1 (HMGB1, also called amphoterin) facilitates neurite outgrowth in early development, yet can exacerbate pathology and inhibit regeneration by inducing adverse neuroinflammation when released from dying cells, suggesting that HMGB1 plays a critical, yet undefined role in neuroregeneration. We explored whether HMGB1 contributes to recovery after complete spinal cord transection in adult zebrafish. Quantitative PCR and in situ hybridization revealed that HMGB1 mRNA levels decreased between 12 h to 11 days after spinal cord injury (SCI), then returned to basal levels by 21 days. Western blot and immunohistological analyses indicated that the time course of HMGB1 protein expression after SCI parallels that of mRNA. Immunofluorescence staining revealed that HMGB1 translocates from nuclei into the cytoplasm of spinal motoneurons at 4 and 12 h (acute stage) following SCI, then accumulates in the nuclei of motoneurons during the ensuing chronic stage (after 6 days following SCI). Immunohistology of transgenic zebrafish, expressing green fluorescent protein in blood vessels, showed enhanced HMGB1 expression in blood vessels in the vicinity of motoneurons. Application of anti-sense HMGB1 morpholinos inhibited locomotor recovery by 34 % and decreased axonal regeneration by 34 % compared to fish treated with a control morpholino. The present study shows that HMGB1 expression increases in both endothelial cells and motoneurons, suggesting that HMGB1 promotes recovery from SCI not only through enhancing neuroregeneration, but also by increasing angiogenesis. The inflammatory effects of HMGB1 are minimized through the decrease in HMGB1 expression during the acute stage.     

Unique and combinatorial functions of Fgf3 and Fgf8 during zebrafish forebrain development

Complex spatiotemporal expression patterns of fgf3 and fgf8 within the developing zebrafish forebrain suggest their involvement in its regionalisation and early development. These factors have unique and combinatorial roles during development of more posterior brain regions, and here we report similar findings for the developing forebrain. We show that Fgf8 and Fgf3 regulate different aspects of telencephalic development, and that Fgf3 alone is required for the expression of several telencephalic markers. Within the diencephalon, Fgf3 and Fgf8 act synergistically to pattern the ventral thalamus, and are implicated in the regulation of optic stalk formation, whereas loss of Fgf3 alone results in defects in ZLI development. Forebrain commissure formation was abnormal in the absence of either Fgf3 or Fgf8; however, most severe defects were observed in the absence of both. Defects were observed in patterning of both the midline territory, within which the commissures normally form, and neuronal populations, whose axons comprise the commissures. Analysis of embryos treated with an FGFR inhibitor suggests that continuous FGF signalling is required from gastrulation stages for normal forebrain patterning, and identifies additional requirements for FGFR activity.     

Transgenic Zebrafish for Studying Nervous System Development and Regeneration

1 tubulin gene expression is induced in the developing and regenerating CNS of vertebrates. Therefore, 1 tubulin gene expression may serve as a good probe for mechanisms underlying CNS development and regeneration. One approach to identify these mechanisms is to work backwards from the genome. This requires identification of 1 tubulin DNA sequences that mediate its developmental and regeneration-dependent expression pattern. Therefore, we generated transgenic zebrafish harboring a fragment of the 1 tubulin gene driving green fluorescent protein expression (GFP). In these fish, and similar to the endogenous gene, transgene expression was dramatically induced in the developing and regenerating nervous system. Although transgene expression generally declined during maturation of the nervous system, robust GFP expression was maintained in progenitor cells in the retinal periphery, lining brain ventricles and surrounding the central canal of the spinal cord. When these cells were cultured in vitro they divided and gave rise to new neurons. We also show that optic nerve crush in adult fish re-induced transgene expression in retinal ganglion cells. These studies identified a relatively small region of the 1 tubulin promoter that mediates its regulated expression pattern in developing and adult fish. This promoter will be extremely useful to investigators interested in targeting gene expression to the developing or regenerating nervous system. As adult transgenic fish maintain transgene expression in neural progenitors, these fish also provide a valuable resource of labeled adult neural progenitor cells that can be studied in vivo or in vitro. Finally, these fish should provide a unique in vivo system for investigating mechanisms mediating CNS development and regeneration.