Regeneration In Zebrafish Research Articles

Retinal regeneration following OCT-guided laser injury in zebrafish.

Establish a focal injury/regeneration model in zebrafish using laser photocoagulation guided by optical coherence tomography (OCT). Adult zebrafish were imaged by OCT and confocal scanning laser ophthalmoscopy (cSLO) in room air through a contact lens. Using a beam combiner, 532-nm laser photocoagulation was applied using the OCT C-scan image for targeting. Laser spots of 42 to 47 mW were delivered to the retina. At multiple intervals post injury, fish were imaged using both OCT and cSLO to follow the progression of each lesion. Histologic sections and TUNEL staining were performed to monitor the injury response. Round lesions (26057 ± 621 μm(2)) localized to the outer retina were successfully applied. Laser application was visualized by real-time OCT and lesions were detectable by both OCT and cSLO in vivo. Lesion size increased 1 day post lesion then decreased in size. Histologic sections showed focal areas of damage localized primarily to the outer retina. By 3 weeks, the damaged areas had regenerated and a fully laminated structure was re-established. However, subtle changes can still be detected by OCT, cSLO imaging, and histology. Infrared darkfield imaging was more sensitive than OCT at revealing subtle changes in regenerated areas. Optical coherence tomography-guided laser photocoagulation is a useful tool for inducing localized lesions and studying retinal regeneration in zebrafish. This novel method will allow us to characterize the cellular and molecular changes that take place at the interface between normal and damaged tissue. Regeneration can be observed using high-resolution OCT and cSLO imaging in vivo.

Upregulation of leukemia inhibitory factor (LIF) during the early stage of optic nerve regeneration in zebrafish.

Fish retinal ganglion cells (RGCs) can regenerate their axons after optic nerve injury, whereas mammalian RGCs normally fail to do so. Interleukin 6 (IL-6)-type cytokines are involved in cell differentiation, proliferation, survival, and axon regrowth; thus, they may play a role in the regeneration of zebrafish RGCs after injury. In this study, we assessed the expression of IL-6-type cytokines and found that one of them, leukemia inhibitory factor (LIF), is upregulated in zebrafish RGCs at 3 days post-injury (dpi). We then demonstrated the activation of signal transducer and activator of transcription 3 (STAT3), a downstream target of LIF, at 3–5 dpi. To determine the function of LIF, we performed a LIF knockdown experiment using LIF-specific antisense morpholino oligonucleotides (LIF MOs). LIF MOs, which were introduced into zebrafish RGCs via a severed optic nerve, reduced the expression of LIF and abrogated the activation of STAT3 in RGCs after injury. These results suggest that upregulated LIF drives Janus kinase (Jak)/STAT3 signaling in zebrafish RGCs after nerve injury. In addition, the LIF knockdown impaired axon sprouting in retinal explant culture in vitro; reduced the expression of a regeneration-associated molecule, growth-associated protein 43 (GAP-43); and delayed functional recovery after optic nerve injury in vivo. In this study, we comprehensively demonstrate the beneficial role of LIF in optic nerve regeneration and functional recovery in adult zebrafish.

Hydrogen peroxide primes heart regeneration with a derepression mechanism.

While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H2O2 acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H2O2 (~30 μM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H2O2 formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H2O2 by catalase overexpression markedly impaired cardiac regeneration while exogenous H2O2 rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H2O2 is an essential and sufficient signal in this process. Mechanistically, elevated H2O2 destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H2O2 plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H2O2 potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.

Sensory hair cell regeneration in the zebrafish lateral line.

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish.

Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration.

A major issue in regenerative medicine is the control of progenitor cell mobilisation. Apoptosis has been reported as playing a role in cell plasticity, and it has been recently shown that apoptosis is necessary for organ and appendage regeneration. In this context, we explore its possible mode of action in progenitor cell recruitment during adult regeneration in zebrafish. Here, we show that apoptosis inhibition impairs blastema formation and nerve growth, both of which can be restored by exogenous adenosine acting through its A2B receptor. Moreover, adenosine increases the number of progenitor cells. Purinergic signalling is therefore an early and essential event in the pathway from lesion to blastema formation and provides new targets for manipulating cell plasticity in the adult.

Kidney injury and regeneration in zebrafish.

Renal tubule epithelial cells can regenerate in response to acute injury. Although this process remains poorly understood, it appears to involve the reactivation of pathways that are operative during embryonic kidney formation. A better understanding of renal regeneration may lead to the development of new therapies that can attenuate acute kidney injury or expedite recovery. The zebrafish is being used as a model to understand renal regeneration. In this review, we summarize the current knowledge on zebrafish kidney formation, describe methods for inducing acute injury, and focus on the unique capacity of the zebrafish adult kidney to undergo de novo nephron formation in response to damage.

Ptena, but not Ptenb, reduces regeneration after spinal cord injury in adult zebrafish

Based on the observation that the tumor suppressor gene PTEN (phosphatase and tensin homolog) reduces regeneration after spinal cord injury (SCI) as evidenced in the PTEN knockout mouse, we have investigated the function of Ptena and Ptenb, the two zebrafish homologs of mammalian PTEN, in adult zebrafish after spinal cord injury with the aim to assess the contribution of the two zebrafish genes to functional recovery in an animal species that spontaneously recovers from central nervous system injury. The inhibition of Ptena expression by antisense morpholino (MO) application improved spinal cord regeneration through 4 to 5weeks after injury. Retrograde tracing showed regrowth of axons from neurons of the regeneration-competent nucleus of the medial longitudinal fascicle in the brainstem in the Ptena MO-treated fish. Ptenb MO-treated fish recovered as well as control MO-treated fish at 4 and 5weeks after SCI, with their locomotion being similar to that of sham-injured and non-injured fish. The mRNA levels of Ptena were upregulated after SCI at the early stage after injury (12h and 6days) caudal to the lesion site, compared to the non-injured control, while the levels of Ptenb were upregulated only at 12h after injury. In situ hybridization experiments were in agreement with the qPCR measurements. At the protein level, Ptena was found to be expressed in spinal motoneurons and immature neurons. These results indicate that Ptena, but not Ptenb, inhibits regeneration in zebrafish, thus sharing this feature with PTEN in mammals. The fact that zebrafish regenerate better than mammals despite the inhibitory presence of Ptena is likely due to regeneration-conducive molecules that tip the balance from inhibition to enhancement. Interestingly, although Ptena and Ptenb have been shown to be functionally redundant in promoting the development of the fish larval central nervous system, they are not functionally redundant in the adult, suggesting that regeneration in fish is not predominantly due to the overall recapitulation of development.

Specific NuRD components are required for fin regeneration in zebrafish.

BackgroundEpimorphic regeneration of a missing appendage in fish and urodele amphibians involves the creation of a blastema, a heterogeneous pool of progenitor cells underneath the wound epidermis. Current evidence indicates that the blastema arises by dedifferentiation of stump tissues in the vicinity of the amputation. In response to tissue loss, silenced developmental programs are reactivated to form a near-perfect copy of the missing body part. However, the importance of chromatin regulation during epimorphic regeneration remains poorly understood.ResultsWe found that specific components of the Nucleosome Remodeling and Deacetylase complex (NuRD) are required for fin regeneration in zebrafish. Transcripts of the chromatin remodeler chd4a/Mi-2, the histone deacetylase hdac1/HDAC1/2, the retinoblastoma-binding protein rbb4/RBBP4/7, and the metastasis-associated antigen mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration, and these transcripts displayed a similar spatial and temporal expression patterns. In addition, chemical inhibition of Hdac1 and morpholino-mediated knockdown of chd4a, mta2, and rbb4 impaired regenerative outgrowth, resulting in reduction in blastema cell proliferation and in differentiation defects.ConclusionAltogether, our data suggest that specialized NuRD components are induced in the blastema during fin regeneration and are involved in blastema cell proliferation and redifferentiation of osteoblast precursor cells. These results provide in vivo evidence for the involvement of key epigenetic factors in the cellular reprogramming processes occurring during epimorphic regeneration in zebrafish.

Monensin Inhibits Canonical Wnt Signaling in Human Colorectal Cancer Cells and Suppresses Tumor Growth in Multiple Intestinal Neoplasia Mice

The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/β-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with β-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or β-catenin-induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of β-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line

Deafness caused by the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, nonmammalian animals (e.g., birds, amphibians, and fish) regenerate damaged hair cells. To understand better the reasons underpinning such disparities in regeneration among vertebrates, we set out to define at high resolution the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line. We performed RNA-Seq analyses on regenerating support cells purified by FACS. The resulting expression data were subjected to pathway enrichment analyses, and the differentially expressed genes were validated in vivo via whole-mount in situ hybridizations. We discovered that cell cycle regulators are expressed hours before the activation of Wnt/β-catenin signaling following hair cell death. We propose that Wnt/β-catenin signaling is not involved in regulating the onset of proliferation but governs proliferation at later stages of regeneration. In addition, and in marked contrast to mammals, our data clearly indicate that the Notch pathway is significantly down-regulated shortly after injury, thus uncovering a key difference between the zebrafish and mammalian responses to hair cell injury. Taken together, our findings lay the foundation for identifying differences in signaling pathway regulation that could be exploited as potential therapeutic targets to promote either sensory epithelium or hair cell regeneration in mammals.

Stem cells and their niche in homeostasis/regeneration and disease

The Minisymposium ”Stem Cells and Their Niche in Homeostasis/Regeneration and Disease” was aimed at highlighting the importance of cell biology aspects in understanding stem cell biology. Tudorita (Doina) Tumbar (Cornell University) started the session by summarizing two models of stem cell behavior during normal homeostasis. Stem cells can divide asymmetrically to generate a stem cell and a more differentiated cell. Conversely, she showed that hair follicle stem cells behave as a population: some differentiate and eventually die, while others self-renew by symmetric divisions. During hair follicle stem cell G0 quiescence, the transcription factor Runx1 drives a reversible step of differentiation to progenitors. The latter succumb irreversibly to rapid proliferation and terminal differentiation upon activation of tissue signals. Pantelis Rompolas (Valentica Greco's Lab, Yale University) reported evidence for a direct link between a specific niche location and cell fate, using in vivo imaging to trace single hair-follicle stem cells long term in live mice. Furthermore, by utilizing laser ablation, he showed that hair follicle stem cells may be dispensable and that other epithelial populations can be reprogrammed to support hair regeneration by entering the niche. Gage Crump (University of Southern California) presented evidence for a transfating of chondrocytes to the osteoblasts that rebuild bone during jawbone regeneration in zebrafish. Such transfating is not observed during development of the same bone, challenging the notion that adult regeneration of an organ simply recapitulates its development. He also presented evidence for a novel role of Ihh signaling in this process and for a similar process in mammalian bone regeneration. Heike Kroeger (Jonathan Lin's Lab, University of California, San Diego) presented a study that links unfolded protein response signaling to hESC differentiation and maturation via the function of ATF6. hESC activates the ATF6 signaling during hESC differentiation through the FGF2 signaling cascade. ATF6 signaling leads to dramatic expansion of the endoplasmic reticulum (ER) and enhances hESC differentiation. These findings provide a new mechanism underlying hESC differentiation that involves ATF6’s induction and activation of ER functions. Daniela Malide (National Institutes of Health) reported the use of combinatorial 5 fluorescent proteins to mark hematopoietic stem and progenitor cells that allowed in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allowed noninvasive fate mapping of multicolored hematopoietic stem/progenitor cell–derived cells in intact nonhematopoietic tissues for extensive periods of time following transplantation, demonstrating clearly delineated clones progressing from multicolored to monochromatic over time. Abby Gerhold (Paul Maddox's Lab, University of North Carolina, Chapel Hill) reported in vivo live imaging of Caenorhabditis elegans germ-line stem cells to investigate how mitotic progression is influenced by the physiological context. She provided evidence that 1) the duration of chromosome congression is reduced during developmental expansion of the stem cell pool, and 2) dietary restriction induces a spindle assembly checkpoint–dependent delay in anaphase onset, suggesting the basic mitotic process is affected by physiological factors. Yukiko Yamashita (University of Michigan, Ann Arbor) discussed her group's recent identification of Klp10A kinesin as the first stem cell–specific centrosomal component in the Drosophila male germ line. Klp10A seems to specifically regulate the length of stem cells’ mother centrosomes. She presented additional data suggesting Klp10A regulates stem cell behavior through the regulation of nanos mRNA.

Der Zebrabärbling als Modell für die Regeneration von Nervengewebe

In contrast to mammals where the formation of new neurons in the adult is very limited, teleost fish like the zebrafish form new neurons abundantly and have a high regenerative potential to repair lesions of the central nervous system. These news neurons will integrate into the existing neuronal networks. The challenge will be to unravel the molecular mechanisms of nervous tissue regeneration in zebrafish in the hope to employ this knowledge for development of new therapies for human patients.

Jak/Stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq.

The regenerative failure of mammalian optic axons is partly mediated by Socs3-dependent inhibition of Jak/Stat signaling (Smith et al., 2009, 2011). Whether Jak/Stat signaling is part of the normal regenerative response observed in animals that exhibit an intrinsic capacity for optic nerve regeneration, such as zebrafish, remains unknown. Nor is it known whether the repression of regenerative inhibitors, such as Socs3, contributes to the robust regenerative response of zebrafish to optic nerve damage. Here we report that Jak/Stat signaling stimulates optic nerve regeneration in zebrafish. We found that IL-6 family cytokines, acting via Gp130-coupled receptors, stimulate Jak/Stat3 signaling in retinal ganglion cells after optic nerve injury. Among these cytokines, we found that CNTF, IL-11, and Clcf1/Crlf1a can stimulate optic axon regrowth. Surprisingly, optic nerve injury stimulated the expression of Socs3 and Sfpq (splicing factor, proline/glutamine rich) that attenuate optic nerve regeneration. These proteins were induced in a Jak/Stat-dependent manner, stimulated each other's expression and suppressed the expression of regeneration-associated genes. In vivo, the injury-dependent induction of Socs3 and Sfpq inhibits optic nerve regeneration but does not block it. We identified a robust induction of multiple cytokine genes in zebrafish retinal ganglion cells that may contribute to their ability to overcome these inhibitory factors. These studies not only identified mechanisms underlying optic nerve regeneration in fish but also suggest new molecular targets for enhancing optic nerve regeneration in mammals.

Expression patterns of dnmt3aa, dnmt3ab, and dnmt4 during development and fin regeneration in zebrafish

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.

Notch signaling regulates cardiomyocyte proliferation during zebrafish heart regeneration

The human heart's failure to replace ischemia-damaged myocardium with regenerated muscle contributes significantly to the worldwide morbidity and mortality associated with coronary artery disease. Remarkably, certain vertebrate species, including the zebrafish, achieve complete regeneration of amputated or injured myocardium through the proliferation of spared cardiomyocytes. Nonetheless, the genetic and cellular determinants of natural cardiac regeneration remain incompletely characterized. Here, we report that cardiac regeneration in zebrafish relies on Notch signaling. Following amputation of the zebrafish ventricular apex, Notch receptor expression becomes activated specifically in the endocardium and epicardium, but not the myocardium. Using a dominant negative approach, we discovered that suppression of Notch signaling profoundly impairs cardiac regeneration and induces scar formation at the amputation site. We ruled out defects in endocardial activation, epicardial activation, and dedifferentiation of compact myocardial cells as causative for the regenerative failure. Furthermore, coronary endothelial tubes, which we lineage traced from preexisting endothelium in wild-type hearts, formed in the wound despite the myocardial regenerative failure. Quantification of myocardial proliferation in Notch-suppressed hearts revealed a significant decrease in cycling cardiomyocytes, an observation consistent with a noncell autonomous requirement for Notch signaling in cardiomyocyte proliferation. Unexpectedly, hyperactivation of Notch signaling also suppressed cardiomyocyte proliferation and heart regeneration. Taken together, our data uncover the exquisite sensitivity of regenerative cardiomyocyte proliferation to perturbations in Notch signaling.

Remodeling and dedifferentiation of adult cardiomyocytes during disease and regeneration.

Cardiomyocytes continuously generate the contractile force to circulate blood through the body. Imbalances in contractile performance or energy supply cause adaptive responses of the heart resulting in adverse rearrangement of regular structures, which in turn might lead to heart failure. At the cellular level, cardiomyocyte remodeling includes (1) restructuring of the contractile apparatus; (2) rearrangement of the cytoskeleton; and (3) changes in energy metabolism. Dedifferentiation represents a key feature of cardiomyocyte remodeling. It is characterized by reciprocal changes in the expression pattern of "mature" and "immature" cardiomyocyte-specific genes. Dedifferentiation may enable cardiomyocytes to cope with hypoxic stress by disassembly of the energy demanding contractile machinery and by reduction of the cellular energy demand. Dedifferentiation during myocardial repair might provide cardiomyocytes with additional plasticity, enabling survival under hypoxic conditions and increasing the propensity to enter the cell cycle. Although dedifferentiation of cardiomyocytes has been described during tissue regeneration in zebrafish and newts, little is known about corresponding mechanisms and regulatory circuits in mammals. The recent finding that the cytokine oncostatin M (OSM) is pivotal for cardiomyocyte dedifferentiation and exerts strong protective effects during myocardial infarction highlights the role of cytokines as potent stimulators of cardiac remodeling. Here, we summarize the current knowledge about transient dedifferentiation of cardiomyocytes in the context of myocardial remodeling, and propose a model for the role of OSM in this process.

Regeneration of Liver After Extreme Hepatocyte Loss Occurs Mainly via Biliary Transdifferentiation in Zebrafish

The liver has high regenerative capacity, but it is not clear whether most biliary cells (particularly larger cholangiocytes) transdifferentiate into hepatocytes in regenerating liver. We investigated how this process might contribute to liver regeneration in zebrafish. Zebrafish transgenic lines were generated using the standard I-SceI meganuclease transgenesis technique. Hepatocytes of the Tg(lfabp:mCherry-NTR)(cq2) animals were ablated by the administration of metronidazole. We investigated transdifferentiation of biliary cells to hepatocytes and expression of markers using whole mount antibody staining, fluorescent in situ hybridization, and Cre/loxP-based genetic lineage tracing analyses. The role of biliary cells in hepatocyte regeneration was explored using zebrafish larvae with defects in biliary cell development. After extreme loss of hepatocytes, nearly all the biliary cells steadily lost their tubular morphology, proliferated, and expressed hepatocyte-specific markers. Cre/loxP-based inducible lineage tracing showed that new hepatocytes mainly arose from transdifferentiation of biliary cells; this process required Notch signaling and, in turn, activation of Sox9b in cholangiocytes. Activation of early endoderm and hepatoblast markers in most of the cholangiocytes indicated that biliary transdifferentiation includes a step of dedifferentiation into a bipotential intermediate. Defects in development of biliary cells impaired hepatocyte regeneration. Using our zebrafish liver regeneration model, we found that biliary cells can transdifferentiate into hepatocytes and are the major contributors to hepatocyte regeneration after extreme hepatocyte loss.

Abstract 18015: Loss of Swi/snf-complex Subunit Heart of Stone Leads to Cardiac Hyperplasia in Zebrafish

Fish and also neonatal mice possess the capacity to regenerate their hearts after myocardial damage. The adult mammalian heart only has a very limited capacity to regenerate after cardiac injury. Myocardial regeneration in zebrafish and newborn mice involves the dedifferentiation and proliferation of resident cardiomyocytes to replace the injured tissue. Unfortunately, only little is known about the factors and signaling cascades that regulate this regenerative capacity. In a forward genetics ENU-screen in zebrafish, we recently isolated the mutant heart of stone (hos). Hearts of hos mutant embryos show a severe thickening of the ventricle with almost complete obliteration of the ventricle. Based on cell count assays using histological sections we find significantly increased cell numbers in hos mutant ventricles. Based on BrdU incorporation assays as well as H3P and PCNA stainings, we find that ventricular hyperplasia in hos is caused by significantly enhanced cardiomyocyte proliferation. By positional cloning we show a splice donor site mutation within the zebrafish hos gene. hos is a subunit of the SWI/SNF chromatin remodeling complex, which is known to be involved in transcriptional regulation. The identified hos mutation specifically leads to an integration of Intron7 resulting in a reading frame shift and thereby to premature termination of hos protein translation. Injection of wild-type hos mRNA in hos mutant embryos restores the wild-type, while injection of morpholinos directed against the splice donor site of Exon7 phenocopy the hos mutant phenotype. In addition, by generating hos-null zebrafish using morpholinos directed against the translational start site of hos, we are also able to induce ventricular hyperplasia, suggesting that the hos mutation results in the complete loss of hos function. Interestingly, in contrast to hos-deficient embryos, targeted knockdown of the zebrafish SWI/SNF-complex core component BRG1 leads to severe hypoplastic hearts, indicating that hos in zebrafish might play a repressive role on SWI/SNF complex function and the control of cardiomyocyte proliferation in vivo.

Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.

Local Dkk1 Crosstalk from Breeding Ornaments Impedes Regeneration of Injured Male Zebrafish Fins

Precise spatiotemporal regulation of signaling activators and inhibitors can help limit developmental crosstalk between neighboring tissues during morphogenesis, homeostasis, and regeneration. Here, we find that the secreted Wnt inhibitor Dkk1b is abundantly produced by dense regions of androgen-regulated epidermal tubercles (ETs) on the surfaces of adult male zebrafish pectoral fins. High-speed videos and amputation experiments reveal that pectoral fins and their ETs are used for male spawning. Formation and vigorous turnover of ETs involve Dkk1b induction and maintenance, whereas Dkk1b is typically restricted from the regeneration blastema after an amputation injury. When amputation occurs through a region containing ETs, a Dkk1b-enriched wound epidermis forms and blastema formation is disrupted, compromising regeneration. Thus, homeostatic signaling by key breeding ornaments can interfere with injury-activated tissue regeneration. Our findings help explain sexually dimorphic fin regeneration in zebrafish and have implications for how regenerative potential might decline as development progresses or during species evolution.