A molecular phenotype atlas of the zebrafish retina

The rasborine cyprinid Danio rerio (the zebrafish) has become a popular model of retinal function and development. Its value depends, in part, on validation of homologies with retinal cell populations of cyprinine cyprinids. This atlas provides raw and interpreted molecular phenotype data derived from computationally classified sets of small molecule signals from different cell types in the zebrafish retina: L-alanine, L-aspartate, L-glutamine, L-glutamate, glutathione, glycine, taurine and γ-aminobutyrate. This basis set yields an 8-dimensional signature for every retinal cell and formally establishes molecular signature homologies with retinal neurons, glia, epithelia and endothelia of other cyprinids. Zebrafish photoreceptor classes have been characterized previously: we now show their metabolic profiles to be identical to those of the corresponding photoreceptors in goldfish. The inner nuclear layer is partitioned into precise horizontal, bipolar and amacrine cell layers. The horizontal cell layer contains at least three and perhaps all four known classes of cyprinine horizontal cells. Homologues of cyprinid glutamatergic ON-center and OFF-center mixed rod-cone bipolar cells are present and it appears likely that all five classes are present in zebrafish. The cone bipolar cells defy simple analysis but comprise the largest fraction of bipolar cells, as in all cyprinids. Signature analysis reveals six molecular phenotypes in the bipolar cell cohort: most are superclasses. The amacrine cell layer is composed of ≈64% GABA+ and 35% glycine+ amacrine cells, with the remainder being sparse dopaminergic interplexiform cells and other rare unidentified neurons. These different amacrine cell types are completely distinct in the dark adapted retina, but light adapted retinas display weak leakage of GABA signals into many glycinergic amacrine cells, suggesting widespread heterocellular coupling. The composition of the zebrafish ganglion cell layer is metabolically indistinguishable from that in other cyprinids, and the signatures of glial and non-neuronal cells display strong homologies with those in mammals. As in most vertebrates, zebrafish Müller cells possess a high glutamine, low glutamate signature and contain the dominant pool of glutathione in the neural retina. The retinal pigmented epithelium shows a general mammalian signature but also has exceptional glutathione content (5–10 mM), perhaps required by the unusually high oxygen tensions of teleost retinas. The optic nerve and the marginal zone of the retina reveal characteristic metabolic specializations. The marginal zone is strongly laminated and its nascent neurons display their characteristic signatures before taking their place in the retina proper.     

Muscarinic signaling influences the patterning and phenotype of cholinergic amacrine cells in the developing chick retina

Background  

Many studies in the vertebrate retina have characterized the differentiation of amacrine cells as a homogenous class of neurons, but little is known about the genes and factors that regulate the development of distinct types of amacrine cells. Accordingly, the purpose of this study was to characterize the development of the cholinergic amacrine cells and identify factors that influence their development. Cholinergic amacrine cells in the embryonic chick retina were identified by using antibodies to choline acetyltransferase (ChAT).     

oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell-cell interactions of the retinal neuroepithelial sheet

Mutations of the oko meduzy (ome) locus cause drastic neuronal patterning defect in the zebrafish retina. The precise, stratified appearance of the wild-type retina is absent in the mutants. Despite the lack of lamination, at least seven retinal cell types differentiate in oko meduzy. The ome phenotype is already expressed in the retinal neuroepithelium affecting morphology of the neuroepithelial cells. Our experiments indicate that previously unknown cell-cell interactions are involved in development of the retinal neuroepithelial sheet. In genetically mosaic animals, cell-cell interactions are sufficient to rescue the phenotype of oko meduzy retinal neuroepithelial cells. These cell-cell interactions may play a critical role in the patterning events that lead to differentiation of distinct neuronal laminae in the vertebrate retina.     

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.     

Ca2+ signaling and early embryonic patterning during the blastula and gastrula periods of zebrafish and Xenopus development

It has been proposed that Ca(2+) signaling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern forming events during early vertebrate development [L.F. Jaffe, Organization of early development by calcium patterns, BioEssays 21 (1999) 657-667; M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11-21; S.E. Webb, A.L. Miller, Calcium signalling during embryonic development, Nat. Rev. Mol. Cell Biol. 4 (2003) 539-551]. With reference to the embryos of zebrafish (Danio rerio) and the frog, Xenopus laevis, we review the Ca(2+) signals reported during the Blastula and Gastrula Periods. This developmental window encompasses the major pattern forming events of epiboly, involution, and convergent extension, which result in the establishment of the basic germ layers and body axes [C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253-310]. Data will be presented to support the suggestion that propagating waves (both long and short range) of Ca(2+) release, followed by sequestration, may play a crucial role in: (1) Coordinating cell movements during these pattern forming events and (2) Contributing to the establishment of the basic embryonic axes, as well as (3) Helping to define the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen [E. Gilland, A.L. Miller, E. Karplus, R. Baker, S.E. Webb, Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation, Proc. Natl. Acad. Sci. USA 96 (1999) 157-161; J.B. Wallingford, A.J. Ewald, R.M. Harland, S.E. Fraser, Calcium signaling during convergent extension in Xenopus, Curr. Biol. 11 (2001) 652-661]. The various potential targets of these Ca(2+) transients will also be discussed, as well as how they might integrate with other known pattern forming pathways known to modulate early developmental events (such as the Wnt/Ca(2+)pathway; [T.A. Westfall, B. Hjertos, D.C. Slusarski, Requirement for intracellular calcium modulation in zebrafish dorsal-ventral patterning, Dev. Biol. 259 (2003) 380-391]).     

Gastrula organiser and embryonic patterning in the mouse

Embryonic patterning of the mouse during gastrulation and early organogenesis engenders the specification of anterior versus posterior structures and body laterality by the interaction of signalling and modulating activities. A group of cells in the mouse gastrula, characterised by the expression of a repertoire of "organiser" genes, acts as a source and the conduit for allocation of the axial mesoderm, floor plate and definitive endoderm. The organiser and its derivatives provide the antagonistic activity that modulates WNT and TGFbeta signalling. Recent findings show that the organiser activity is augmented by morphogenetic activity of the extraembryonic and embryonic endoderm, suggesting embryonic patterning is not solely the function of the organiser.     

Inductive patterning of the embryonic brain in Drosophila

In vertebrates (deuterostomes), brain patterning depends on signals from adjacent tissues. For example, holoprosencephaly, the most common brain anomaly in humans, results from defects in signaling between the embryonic prechordal plate (consisting of the dorsal foregut endoderm and mesoderm) and the brain. I have examined whether a similar mechanism of brain development occurs in the protostome Drosophila, and find that the foregut and mesoderm act to pattern the fly embryonic brain. When the foregut and mesoderm of Drosophila are ablated, brain patterning is disrupted. The loss of Hedgehog expressed in the foregut appears to mediate this effect, as it does in vertebrates. One mechanism whereby these defects occur is a disruption of normal apoptosis in the brain. These data argue that the last common ancestor of protostomes and deuterostomes had a prototype of the brains present in modern animals, and also suggest that the foregut and mesoderm contributed to the patterning of this 'proto-brain'. They also argue that the foreguts of protostomes and deuterostomes, which have traditionally been assigned to different germ layers, are actually homologous.     

The Polycomb group protein MEDEA controls cell proliferation and embryonic patterning in Arabidopsis

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Retinoic acid in the anteroposterior patterning of the zebrafish trunk

Retinoic acid has been linked to pattern formation in the vertebrate anteroposterior axis. This report describes the spatial and temporal distributions of both endogenous retinoic acid and retinoic acid synthase activity along the anteroposterior axis of neurulating zebrafish embryos, as detected by a transient transgenic assay and by a zymography bioassay. Both retinoic acid levels and synthase activity were found to be highest in anterior regions of the trunk at all of the stages which were analysed. The drug disulfiram inhibited retinoic acid synthase activity in the zebrafish trunk both in vitro and in vivo, and reduced retinoic acid levels in vivo. Disulfiram treatment of neurulating embryos resulted in larvae with hypertrophic wavy notochords, shortened spinal cords and deformed pectoral fins. The results support the hypothesis that retinoic acid plays a role in the coordination of axial patterning at the developing node/zone of involution, as well as in the subsequent development of anterior trunk structures such as the fins.     

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.     

In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina

Two-photon excitation microscopy was used to reconstruct cell divisions in living zebrafish embryonic retinas. Contrary to proposed models for vertebrate asymmetric divisions, no apico-basal cell divisions take place in the zebrafish retina during the generation of postmitotic neurons. However, a surprising shift in the orientation of cell division from central-peripheral to circumferential occurs within the plane of the ventricular surface. In the sonic you (syu) and lakritz (lak) mutants, the shift from central-peripheral to circumferential divisions is absent or delayed, correlating with the delay in neuronal differentiation and neurogenesis in these mutants. The reconstructions here show that mitotic cells always remain in contact with the opposite basal surface by means of a thin basal process that can be inherited asymmetrically.     

Metabolic differentiation in the embryonic retina

Unlike healthy adult tissues, cancers produce energy mainly by aerobic glycolysis instead of oxidative phosphorylation. This adaptation, called the Warburg effect, may be a feature of all dividing cells, both normal and cancerous, or it may be specific to cancers. It is not known whether, in a normally growing tissue during development, proliferating and postmitotic cells produce energy in fundamentally different ways. Here we show in the embryonic Xenopus retina in vivo, that dividing progenitor cells depend less on oxidative phosphorylation for ATP production than non-dividing differentiated cells, and instead use glycogen to fuel aerobic glycolysis. The transition from glycolysis to oxidative phosphorylation is connected to the cell differentiation process. Glycolysis is indispensable for progenitor proliferation and biosynthesis, even when it is not used for ATP production. These results suggest that the Warburg effect can be a feature of normal proliferation in vivo, and that the regulation of glycolysis and oxidative phosphorylation is critical for normal development.     

Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina

Cellular mechanisms underlying the precision by which neurons target their synaptic partners have largely been determined based on the study of projection neurons. By contrast, little is known about how interneurons establish their local connections in vivo. Here, we investigated how developing amacrine interneurons selectively innervate the appropriate region of the synaptic neuropil in the inner retina, the inner plexiform layer (IPL). Increases (ON) and decreases (OFF) in light intensity are processed by circuits that are structurally confined to separate ON and OFF synaptic sublaminae within the IPL. Using transgenic zebrafish in which the majority of amacrine cells express fluorescent protein, we determined that the earliest amacrine-derived neuritic plexus formed between two cell populations whose somata, at maturity, resided on opposite sides of this plexus. When we followed the behavior of individual amacrine cells over time, we discovered that they exhibited distinct patterns of structural dynamics at different stages of development. During cellular migration, amacrine cells exhibited an exuberant outgrowth of neurites that was undirected. Upon reaching the forming IPL, neurites extending towards the ganglion cell layer were relatively more stable. Importantly, when an arbor first formed, it preferentially ramified in either the inner or outer IPL corresponding to the future ON and OFF sublaminae, and maintained this stratification pattern. The specificity by which ON and OFF amacrine interneurons innervate their respective sublaminae in the IPL contrasts with that observed for projection neurons in the retina and elsewhere in the central nervous system.     

Stromal progenitors are important for patterning epithelial and mesenchymal cell types in the embryonic kidney

Growth and expansion of the embryonic kidney is driven in large part by continuous branching morphogenesis and nephron induction that occurs in a restricted domain beneath the renal capsule called the nephrogenic zone. Here, new ureteric bud branches and nephron aggregates form surrounded by a layer of cortical stromal cell progenitors. The boundaries and inductive activities of the nephrogenic zone are maintained as the kidney grows. As new ureteric bud branches and nephrogenic aggregates form, older generations of ureteric bud branches, renal vesicles and stromal progenitors are displaced from the nephrogenic zone and undergo further differentiation surrounded by medullary stroma, a different population of stromal cells. Recent studies suggest that cortical and medullary stromal progenitors may be an important source of signals that maintain outer and inner zones of differentiation in the embryonic kidney, and regulate distinct events important for differentiation of nephrons and the collecting duct system.     

BMP signaling and early embryonic patterning

Bone morphogenetic proteins (BMPs) play pleiotropic roles during embryonic development as well as throughout life. Recent genetic approaches especially using the mouse gene knockout system revealed that BMP signaling is greatly involved in early embryonic patterning, which is a dynamic event to establish three-dimensional polarities. The purpose of this review is to describe the diverse function of BMPs through different receptor signaling systems during embryonic patterning including gastrulation and establishment of the left-right asymmetry.     

A growth curve of cell numbers in the neural retina of embryonic chicks

The growth of cell numbers in a normal embryonic population of neural retinal cells is described. The numbers were estimated from a time shortly after the neural retina first becomes recognizable to a time when numbers of retinal cells have become steady. Cell numbers were estimated in preparations of an entire neural retina dispersed into a suspension of single nuclei which were then counted in a Coulter counter. The growth curve of the ln numbers of cells has three phases of growth: an exponential phase during which there is steady-state exponential growth, a differentiative phase during which cell proliferation ceases and an end phase when no further change in cell numbers can be detected. The variances of the ln numbers of cells were highest during the exponential phase. The variances decreased during the differentiative phase and were at their lowest during the end phase. For variances to decrease requires mechanisms which control the final numbers of cells in the neural retina very precisely. The implications of mechanisms which operate by controlling cell lineages are explored.