Extracellular ADP regulates lesion-induced in vivo cell proliferation and death in the zebrafish retina

Regeneration and growth that occur in the adult teleost retina by neurogenesis have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. In this report, we demonstrate that endogenous purinergic signals regulate cell proliferation induced by a cytotoxic injury of the adult zebrafish retina which mainly damages inner retinal layers. Particularly, we found that ADP but not ATP or adenosine significantly enhanced cell division as assessed by 5-bromo-2'-deoxyuridine incorporation following injury, during the degenerative and proliferative phase of the regeneration process. This effect of ADP occurs via P2Y1 metabotropic receptors as shown by intra-ocular injection of selective antagonists. Additionally, we describe a role for purinergic signals in regulating cell death induced by injury. Scavenging of extracellular nucleotides significantly increased cell death principally seen in the inner retinal layers. This effect is partially reproduced by blocking P2Y1 receptors suggesting a neuroprotective function for ADP, which is derived from extracellular ATP probably released by dying cells as a consequence of the ouabain treatment. This study demonstrates a crucial role for ADP as a paracrine signal in the repair of retinal tissue following injury.     

Fgf signaling is required for photoreceptor maintenance in the adult zebrafish retina

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.     

A moving wave patterns the cone photoreceptor mosaic array in the zebrafish retina

In this paper, we describe the embryonic origin and patterning of the planar mosaic array of cone photoreceptor spectral subtypes in the zebrafish retina. A discussion of possible molecular mechanisms that might generate the cone mosaic array considers but discards a model that accounts for formation of neuronal mosaics in the inner retina and discusses limitations of mathematical simulations that reproduce the zebrafish cone mosaic pattern. The formation and organization of photoreceptors in the ommatidia of the compound eye of Drosophila is compared with similar features in the developing zebrafish cone mosaic, and a model is proposed that invokes spatiotemporally coordinated cell-cell interactions among cone progenitors to determine the identity and positioning of cone spectral subtypes.     

STAT1 activation regulates proliferation and differentiation of renal progenitors

We have shown previously that activation of STAT1 contributes to the pathogenesis of Wilms tumor. This neoplasm caricatures metanephric development and is believed to originate from embryonic renal mesenchymal progenitors that lose their ability to undergo mesenchymal–epithelial transition (MET). Therefore, we hypothesized that STAT1 is also activated and functional during metanephric development. Here we have demonstrated that both STAT1 and STAT3 are activated during normal development of the embryonic kidney. Furthermore, activation of STAT1 stimulated the proliferation of metanephric mesenchymal cells, but it prevented MET and tubulogenesis induced by leukemia inhibitory factor, which preferentially activates STAT3. Consistent with its negative regulation of metanephric mesenchymal differentiation, inhibition of STAT1 activation with protein kinase CK2 inhibitor TBB or RNAi-mediated knockdown of STAT1 promoted differentiation of metanephric progenitors and abolished the effect of cytokine-induced STAT1 activation in these cells. Additionally, a cell-permeable peptide that inhibits STAT1-mediated transactivation by targeting the STAT1 N-domain also blocked cytokine-induced STAT1-dependent proliferation in metanephric progenitors and promoted LIF-induced MET and tubulogenesis. Finally, the STAT1 peptide inhibitor caused the down regulation of survival/anti-apoptotic factors, Mcl-1 and Hsp-27, and induced apoptosis in renal tumor cells with constitutively active STAT1, indicating that STAT1 is required for these cells to survive. These findings show that both metanephric progenitors and renal tumor cells utilize a STAT1-dependent mechanism for growth or survival.     

Angiopoietin-like 3 regulates hepatocyte proliferation and lipid metabolism in zebrafish

Loss-of-function mutations in angiopoietin-like 3 (ANGPTL3) cause familial hypobetalipoproteinemia type 2 (FHBL2) in humans. ANGPTL3 belongs to the angiopoietin-like family, the vascular endothelial growth factor family that is structurally similar to angiopoietins and is known for a regulator of lipid and glucose metabolism, although it is unclear how mutations in ANGPTL3 lead to defect in liver development in the vertebrates. We report here that angptl3 is primarily expressed in the zebrafish developing liver and that morpholino (MO) knockdown of Angptl3 reduces the size of the developing liver, which is caused by suppression of cell proliferation, but not by enhancement of apoptosis. However, MO knockdown of Angptl3 did not alter angiogenesis in the developing liver. Additionally, disruption of zebrafish Angptl3 elicits the hypocholesterolemia phenotype that is characteristic of FHBL2 in humans. Together, our findings propose a novel role for Angptl3 in liver cell proliferation and maintenance during zebrafish embryogenesis. Finally, angptl3 morphants will serve as a good model for understanding the pathophysiology of FHBL2.     

The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina

Long noncoding RNAs (lncRNAs) are emerging as regulators of many basic cellular pathways. Several lncRNAs are selectively expressed in the developing retina, although little is known about their functional role in this tissue. Vax2os1 is a retina-specific lncRNA whose expression is restricted to the mouse ventral retina. Here we demonstrate that spatiotemporal misexpression of Vax2os1 determines cell cycle alterations in photoreceptor progenitor cells. In particular, the overexpression of Vax2os1 in the developing early postnatal mouse retina causes an impaired cell cycle progression of photoreceptor progenitors toward their final committed fate and a consequent delay of their differentiation processes. At later developmental stages, this perturbation is accompanied by an increase of apoptotic events in the photoreceptor cell layer, in comparison with control retinas, without affecting the proper cell layering in the adult retina. Similar results are observed in mouse photoreceptor-derived 661W cells in which Vax2os1 overexpression results in an impairment of the cell cycle progression rate and cell differentiation. Based on these results, we conclude that Vax2os1 is involved in the control of cell cycle progression of photoreceptor progenitor cells in the ventral retina. Therefore, we propose Vax2os1 as the first example of lncRNA that acts as a cell cycle regulator in the mammalian retina during development.     

Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone

The amyloid precursor protein (APP) is a type I transmembrane protein of unknown physiological function. Its soluble secreted form (sAPP) shows similarities with growth factors and increases the in vitro proliferation of embryonic neural stem cells. As neurogenesis is an ongoing process in the adult mammalian brain, we have investigated a role for sAPP in adult neurogenesis. We show that the subventricular zone (SVZ) of the lateral ventricle, the largest neurogenic area of the adult brain, is a major sAPP binding site and that binding occurs on progenitor cells expressing the EGF receptor. These EGF-responsive cells can be cultured as neurospheres (NS). In vitro, EGF provokes soluble APP (sAPP) secretion by NS and anti-APP antibodies antagonize the EGF-induced NS proliferation. In vivo, sAPP infusions increase the number of EGF-responsive progenitors through their increased proliferation. Conversely, blocking sAPP secretion or downregulating APP synthesis decreases the proliferation of EGF-responsive cells, which leads to a reduction of the pool of progenitors. These results reveal a new function for sAPP as a regulator of SVZ progenitor proliferation in the adult central nervous system.     

Pattern formation in the zebrafish retina

During the past 15 years, the zebrafish has become established as a genetic model organism to study vertebrate development. It is particularly well suited for the analysis of the retina, and several genetic screens have yielded a large number of mutants affecting retinal development. Most of these mutants still await thorough analysis and molecular characterization, but work on a handful of genes has already generated interesting results that shed some light on patterning mechanisms employed in the vertebrate retina.     

N‐cadherin regulates the proliferation and differentiation of ventral midbrain dopaminergic progenitors

Adherens junction (AJ) between dopaminergic (DA) progenitors maintains the structure of ventricular zone and polarity of radial glia cells in the ventral midbrain (vMB) during embryonic development. However, it is unclear how loss of N‐cadherin might influence the integrity of the AJ and the process of DA neurogenesis. Here, we used conditional gene targeting approaches to perform the region‐specific removal of N‐cadherin in the neurogenic niche of DA neurons in the vMB. Removal of N‐cadherin in the vMB using Shh‐Cre disrupts the AJs of DA progenitors and radial glia processes in the vMB. Surprisingly, loss of N‐cadherin in the vMB leads to a significant expansion of DA progenitors, including those expressing Sox2, Ngn2, and Otx2. Cell cycle analyses reveal that the cell cycle exit in the progenitor cells is decreased in the mutants from E11.5 to E12.5. In addition, the efficiency of DA progenitors in differentiating into DA neurons is decreased from E10.5 to E12.5, leading to a marked reduction in the number of DA neurons at E11.5, E12.5, and E17.5. Loss of N‐cadherin leads to the diffuse distribution of β‐catenin proteins, which are a critical component of AJ and Wnt signaling, from the AJ throughout the entire cytoplasm in neuroepithelial cells, suggesting that canonical Wnt signaling might be activated in the DA progenitors in vMB. Taken together, these results support the notion that N‐cadherin regulates the proliferation of DA progenitors and the differentiation of DA neurons through canonical Wnt‐β‐catenin signaling in the vMB. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 518–529, 2013     

Organization of cardiac chamber progenitors in the zebrafish blastula

Organogenesis requires the specification of a variety of cell types and the organization of these cells into a particular three-dimensional configuration. The embryonic vertebrate heart is organized into two major chambers, the ventricle and atrium, each consisting of two tissue layers, the myocardium and endocardium. The cellular and molecular mechanisms responsible for the separation of ventricular and atrial lineages are not well understood. To test models of cardiac chamber specification, we generated a high-resolution fate map of cardiac chamber progenitors in the zebrafish embryo at 40% epiboly, a stage prior to the initiation of gastrulation. Our map reveals a distinct spatial organization of myocardial progenitors: ventricular myocardial progenitors are positioned closer to the margin and to the dorsal midline than are atrial myocardial progenitors. By contrast, ventricular and atrial endocardial progenitors are not spatially organized at this stage. The relative orientations of ventricular and atrial myocardial progenitors before and after gastrulation suggest orderly movements of these populations. Furthermore, the initial positions of myocardial progenitors at 40% epiboly indicate that signals residing at the embryonic margin could influence chamber fate assignment. Indeed, via fate mapping, we demonstrate that Nodal signaling promotes ventricular fate specification near the margin, thereby playing an important early role during myocardial patterning.     

Math3 and NeuroD regulate amacrine cell fate specification in the retina

The basic helix-loop-helix genes Math3 and NeuroD are expressed by differentiating amacrine cells, retinal interneurons. Previous studies have demonstrated that a normal number of amacrine cells is generated in mice lacking either Math3 or NEUROD: We have found that, in Math3-NeuroD double-mutant retina, amacrine cells are completely missing, while ganglion and Müller glial cells are increased in number. In the double-mutant retina, the cells that would normally differentiate into amacrine cells did not die but adopted the ganglion and glial cell fates. Misexpression studies using the developing retinal explant cultures showed that, although Math3 and NeuroD alone only promoted rod genesis, they significantly increased the population of amacrine cells when the homeobox gene Pax6 or Six3 was co-expressed. These results indicate that Math3 and NeuroD are essential, but not sufficient, for amacrine cell genesis, and that co-expression of the basic helix-loop-helix and homeobox genes is required for specification of the correct neuronal subtype.     

Genetics of photoreceptor development and function in zebrafish

The vertebrate photoreceptor is a cell of unique morphology and function. It is an exquisite light detector, both sensitive and adaptable. Several unusual morphological features facilitate photoreceptor function. Signal detection is accomplished by a specialized apical structure, the outer segment. There, the capture of light produces fluctuations in cell membrane potential, which are then transmitted to the downstream circuitry of the retina via a rare type of synaptic junction, the ribbon synapse. The development, maintenance and function of the vertebrate photoreceptor cell have been studied mainly in four model organisms, ranging from an amphibian to man. A teleost fish, the zebrafish, is an important recent addition to this group. Genetic screens in zebrafish have identified an impressive collection of photoreceptor cell mutants, including the absence or malformation of specific morphological features as well as functional abnormalities. These mutant strains are currently studied using both molecular and embryological tools and provide important insights into photoreceptor biology.     

Analysis of gene function in the zebrafish retina

Mutagenesis screens in zebrafish have uncovered several hundred mutant alleles affecting the development of the retina and established the zebrafish as one of the leading models of vertebrate eye development. In addition to forward genetic mutagenesis approaches, gene function in the zebrafish embryo is being studied using several reverse genetic techniques. Some of these rely on the overexpression of a gene product, others take advantage of antisense oligonucleotides to block function of selected loci. Here we describe these methods in the context of the developing eye.     

Mef2cb regulates late myocardial cell addition from a second heart field-like population of progenitors in zebrafish

Two populations of cells, termed the first and second heart field, drive heart growth during chick and mouse development. The zebrafish has become a powerful model for vertebrate heart development, partly due to the evolutionary conservation of developmental pathways in this process. Here we provide evidence that the zebrafish possesses a conserved homolog to the murine second heart field. We developed a photoconversion assay to observe and quantify the dynamic late addition of myocardial cells to the zebrafish arterial pole. We define an extra-cardiac region immediately posterior to the arterial pole, which we term the late ventricular region. The late ventricular region has cardiogenic properties, expressing myocardial markers such as vmhc and nkx2.5, but does not express a full complement of differentiated cardiomyocyte markers, lacking myl7 expression. We show that mef2cb, a zebrafish homolog of the mouse second heart field marker Mef2c, is expressed in the late ventricular region, and is necessary for late myocardial addition to the arterial pole. FGF signaling after heart cone formation is necessary for mef2cb expression, the establishment of the late ventricular region, and late myocardial addition to the arterial pole. Our study demonstrates that zebrafish heart growth shows more similarities to murine heart growth than previously thought. Further, as congenital heart disease is often associated with defects in second heart field development, the embryological and genetic advantages of the zebrafish model can be applied to study the vertebrate second heart field.     

Circadian rhythms in the retina of rats with photoreceptor degeneration

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.     

Multiple visual pigments in a photoreceptor of the salamander retina

Although a given retina typically contains several visual pigments, each formed from a retinal chromophore bound to a specific opsin protein, single photoreceptor cells have been thought to express only one type of opsin. This design maximizes a cell''s sensitivity to a particular wavelength band and facilitates wavelength discrimination in retinas that process color. We report electrophysiological evidence that the ultraviolet-sensitive cone of salamander violates this rule. This cell contains three different functional opsins. The three opsins could combine with the two different chromophores present in salamander retina to form six visual pigments. Whereas rods and other cones of salamander use both chromophores, they appear to express only one type of opsin per cell. In visual pigment absorption spectra, the bandwidth at half-maximal sensitivity increases as the pigment''s wavelength maximum decreases. However, the bandwidth of the UV-absorbing pigment deviates from this trend; it is narrow like that of a red-absorbing pigment. In addition, the UV-absorbing pigment has a high apparent photosensitivity when compared with that of red- and blue-absorbing pigments and rhodopsin. These properties suggest that the mechanisms responsible for spectrally tuning visual pigments separate two absorption bands as the wavelength of maximal sensitivity shifts from UV to long wavelengths.