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.     

Ontogeny of centers containing luteinizing hormone-releasing hormone in the brain of platyfish (Xiphophorus maculatus) as determined by immunocytochemistry

We have traced the development from birth to puberty of the ir-LHRH producing centers, the nucleus olfactoretinalis (NOR), nucleus praeopticus periventricularis (NPP) and nucleus lateralis tuberis pars posterior (NLT), in male sibling platyfish genetically determined by differing P alleles to mature at two different ages. In sexually immature fish of both genotypes ir-LHRH is first localized in the NOR in perikarya and neuronal fibers and in the NPP where it is limited to the fibers. At the onset of puberty in early maturing fish (11 weeks), ir-LHRH accumulates in perikarya and fibers of the NPP and NLT. Late maturing siblings first have ir-perikarya in the NPP and a few ir-fibers in the NLT at the same developmental stage but at a later age (25-26 weeks). Ir-perikarya are still not seen in the NLT in late-maturing fish even when maturation is complete. This pattern of a sequential development of the three ir-LHRH containing areas of the brain, which we refer to as a "cascade effect", is similar in early and late genotypes. It is directly correlated to stage of development and not to chronological age. It is clear that the appearance of LHRH producing brain centers precedes, and is presumably essential for, the completion of the development of pituitary gonadotrops and the subsequent maturation of the gonads. The functional significance of the NOR in puberty and reproduction and as a possible site of action for the P gene is discussed.     

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.     

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.     

Adenosine receptor expression in the adult zebrafish retina

Purinergic Signalling - Adenosine is an endogenous nucleoside in the central nervous system that acts on adenosine receptors. These are G protein-coupled receptors that have four known subtypes:...     

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.     

Differential calcium signaling by cone specific guanylate cyclase-activating proteins from the zebrafish retina

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca(2+)-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca(2+)-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca(2+) around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca(2+)/Mg(2+)-dependent conformational changes and in the Ca(2+)-dependent monomer-dimer equilibrium. Direct Ca(2+)-binding revealed that all zGCAPs bound at least three Ca(2+). The corresponding apparent affinity constants reflect binding of Ca(2+) with high (≤ 100 nM), medium (0.1-5 μM) and/or low (≥ 5 μM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca(2+)-sensor system in zebrafish rod and cone cells supporting a Ca(2+)-relay model of differential zGCAP operation in these cells.     

Formation of cone mosaic of zebrafish retina.

In the zebrafish retina, four types of cone photoreceptor cells (or cones) with different sensitive frequencies are arranged in a regular pattern, named "cone mosaic". A pair of small cones, one sensitive to red and the other sensitive to green, is in close contact and forms a "double cone". In addition, there are two kinds of single cones, sensitive to blue and to UV, respectively. We study characteristics of cell-differentiation rules that realize stable formation of cone mosaic. Assumptions are: undifferentiated cells are arranged in a regular square lattice, and they are one of the three types (B, U, and D cells). A D cell has two parts (G and R-parts) and takes one of the four directions. The cells change their cell type and orientation following a continuous-time Markovian chain. The state transtion occurs faster if it increases the stabilities of the focal cell, in which the stability is the sum of affinities with neighboring cells. After the transient period, the system may reach a stable pattern (pre-pattern). The pattern becomes fixed later when the cells are fully differentiated in which B cells, U cells, and D cells become blue-sensitive, UV-sensitive, and double cones, respectively. We search for the combinations of affinities between cell states that can generate the same cone mosaic patterns as in zerbrafish retina. Successful transition rules give (1) zero or small affinity with the pairs of cell states that are absent in the zebrafish cone mosaic (lambda(UR), lambda(BG)and the contact of two cells of the same type); (2) a large affinity between a part of D cells and a non-D cell (lambda(UG)and lambda(BR)); and (3) a positive affinity of an intermediate magnitude between two non-D cells (lambda(BU)) and between two parts of D cells (lambda(GR)). The latter should be of a magnitude of about 60-90% of the former. The time needed to form a regular pattern increases with the lattice size if all the cells start pre-pattern formation simultaneously. However, the convergence time is shortened considerably if the pre-pattern formation occurs only in a narrow band of morphogenetic cell layer that sweeps from one end of the lattice to the other.     

In vivo electroporation of morpholinos into the adult zebrafish retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection. In all cases, resident Müller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days. Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.     

FishNet: an online database of zebrafish anatomy

Background

Embryos of taxonomically different vertebrates are thought to pass through a stage in which they resemble one another morphologically. This "vertebrate phylotypic stage" may represent the basic vertebrate body plan that was established in the common ancestor of vertebrates. However, much controversy remains about when the phylotypic stage appears, and whether it even exists. To overcome the limitations of studies based on morphological comparison, we explored a comprehensive quantitative method for defining the constrained stage using expressed sequence tag (EST) data, gene ontologies (GO), and available genomes of various animals. If strong developmental constraints occur during the phylotypic stage of vertebrate embryos, then genes conserved among vertebrates would be highly expressed at this stage.

Results

We established a novel method for evaluating the ancestral nature of mouse embryonic stages that does not depend on comparative morphology. The numerical "ancestor index" revealed that the mouse indeed has a highly conserved embryonic period at embryonic day 8.0–8.5, the time of appearance of the pharyngeal arch and somites. During this period, the mouse prominently expresses GO-determined developmental genes shared among vertebrates. Similar analyses revealed the existence of a bilaterian-related period, during which GO-determined developmental genes shared among bilaterians are markedly expressed at the cleavage-to-gastrulation period. The genes associated with the phylotypic stage identified by our method are essential in embryogenesis.

Conclusion

Our results demonstrate that the mid-embryonic stage of the mouse is indeed highly constrained, supporting the existence of the phylotypic stage. Furthermore, this candidate stage is preceded by a putative bilaterian ancestor-related period. These results not only support the developmental hourglass model, but also highlight the hierarchical aspect of embryogenesis proposed by von Baer. Identification of conserved stages and tissues by this method in various animals would be a powerful tool to examine the phylotypic stage hypothesis, and to understand which kinds of developmental events and gene sets are evolutionarily constrained and how they limit the possible variations of animal basic body plans.     

FishNet: an online database of zebrafish anatomy

Background  

Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish.     

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.     

A comparison of the size of photoreceptor cells from the retina of the domestic pig as determined by light and scanning electron microscopy

While gathering data on the visual pigments of numerous species, many light micrographs have been taken of the photoreceptor cells containing the visual pigment after spectral recordings have been made from these cells. These micrographs are used to view the cells and obtain measurements of their size. Usually the morphology of the photoreceptor cells is retained adequately so that such measurements can be taken. Occasionally it is necessary to partially fix the retinal tissue which aids in the maintenance of photoreceptor morphology while allowing visual pigment spectral recordings to be made. We have found that primate retinal tissue, as well as some other mammalian tissue, disintegrates rapidly. Although partial fixation allows spectral recordings to be made before the micrographs are taken, the treatment does not always adequately preserve cell morphology for quality micrographs to be obtained. In these cases, visual pigment recordings are made from pieces of unfixed and partially fixed retinal tissue; an additional piece of the same retinal material is well-fixed, embedded and thick-sectioned for light microscopy.Preparing retinal material for sectioning is lengthy and time consuming so an alternative tissue preparation technique was sought. Material can be processed for the scanning electron microscope (SEM) more rapidly than for sectioning, however severe tissue shrinkage occurs during this process. It was found that although shrinkage does occur in the retinal tissue prepared for SEM, the relative proportions of the photoreceptor cells are maintained extremely well. Using the critical point drying method (CPD) pig retinal tissue was prepared for SEM. Scanning electron micrographs of the pig photoreceptors were taken for cell measurement. Since these micrographs could be made at higher magnification than is available by light microscopy, a more detailed view of the pig photoreceptor cells was obtained. Cell measurements made from the light and the scanning electron micrographs indicate that an approximate shrinkage of 50% occurs in the SEM prepared material.     

Onset and time course of apoptosis in the developing zebrafish retina

In mammalian development, apoptosis spreads over the retina in consecutive waves and induces a remarkable amount of cell loss. No evidence for such consecutive waves has been revealed in the fish retina so far. As the zebrafish is of growing importance as a model for retinal development and for degenerative retinal diseases, we examined the onset and time course of apoptosis in the developing zebrafish retina and in adult fish. We found that apoptosis peaked in the ganglion cell layer (GCL) and inner nuclear layer (INL) in early developmental stages (3-4 days post-fertilization; dpf) followed by a second, but clearly smaller wave at 6-7dpf. Apoptosis in the outer nuclear layer (ONL) started at 5dpf and peaked at 7dpf. This late-onset high peak of apoptosis of photoreceptors is different from that of all other species examined to date. With 1.09% of cells in the GCL and 1.10% in the ONL being apoptotic, the rate of apoptosis in the developing zebrafish retina was conspicuously lower than that observed in other vertebrates (up to 50% in GCL). During development (2-21dpf), apoptotic waves were most obvious in the central retina, whereas in the periphery near the marginal zone (MZ), apoptosis was much lower; in adult animals, practically no apoptosis was present in the central retina but it still occurred near the MZ. Our data show that the onset and time course of apoptosis in the GCL and INL of the zebrafish is comparable with other vertebrates; however, the amount of apoptosis is clearly reduced. Thus, apoptosis in the zebrafish retina may serve more as a mechanism for the fine tuning of the retinal neuronal network after mitotic waves during development or in remaining mitotic areas than as a mechanism for eliminating large numbers of excess cells.     

Clonal origins of cells in the pigmented retina of the zebrafish eye

Mosaic analysis has been used to study the clonal basis of the development of the pigmented retina of the zebrafish, Brachydanio rerio. Zebrafish embryos heterozygous for a recessive mutation at the gol-1 locus were exposed to gamma-irradiation at various developmental stages to create mosaic individuals consisting of wild-type pigmented cells and a clone of pigmentless (golden) cells in the eye. The contribution of individual embryonic cells to the pigmented retina was measured and the total number of cells in the embryo that contributed descendants to this tissue was determined. Until the 32-cell stage, almost every blastomere has some descendants that participate in the formation of the pigmented retina of the zebrafish. During subsequent cell divisions, up to the several thousand-cell stage, the number of ancestral cells is constant: approximately 40 cells are present that will give rise to progeny in the pigmented retina. Analysis of the size of clones in the pigmented retina indicates that the cells of this tissue do not arise through a rigid series of cell divisions originating in the early embryo. The findings that each cleavage stage cell contributes to the pigmented retina and yet the contribution of such cells is highly variable are consistent with the interpretation that clonal descendants of different blastomeres normally intermix extensively prior to formation of the pigmented retina.     

NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish

neuroD is a member of the family of proneural genes, which function to regulate the cell cycle, cell fate determination and cellular differentiation. In the retinas of larval and adult teleosts, neuroD is expressed in two populations of post-mitotic cells, a subset of amacrine cells and nascent cone photoreceptors, and proliferating cells in the lineages that give rise exclusively to rod and cone photoreceptors. Based on previous studies of NeuroD function in vitro and the cellular pattern of neuroD expression in the zebrafish retina, we hypothesized that within the mitotic photoreceptor lineages NeuroD selectively regulates aspects of the cell cycle. To test this hypothesis, gain and loss-of-function approaches were employed, relying on the inducible expression of a NeuroD^EGFP fusion protein and morpholino oligonucleotides to inhibit protein translation, respectively. Conditional expression of neuroD causes cells to withdraw from the cell cycle, upregulate the expression of the cell cycle inhibitors, p27 and p57, and downregulate the cell cycle progression factors, Cyclin B1, Cyclin D1, and Cyclin E2. In the absence of NeuroD, cells specific for the rod and cone photoreceptor lineage fail to exit the cell cycle, and the number of cells expressing Cyclin D1 is increased. When expression is ectopically induced in multipotent progenitors, neuroD promotes the genesis of rod photoreceptors and inhibits the genesis of Müller glia. These data show that in the teleost retina NeuroD plays a fundamental role in photoreceptor genesis by regulating mechanisms that promote rod and cone progenitors to withdraw from the cell cycle. This is the first in vivo demonstration in the retina of cell cycle regulation by NeuroD.     

Spectral inference reveals principal cone-integration rules of the zebrafish inner retina

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Sexual difference in LH-cells of the neonatal rats as revealed by immunocytochemistry

Summary Localization and number of pituitary LH-cells were studied in neonatal male and female rats (from the birth to 12th day) applying anti-HCG serum in immunoenzymological procedures. The cells increased in number with developing age after birth. The cells in males and females were equal in number until 4 days of age, whereas thereafter the increase of the cell number in females exceeded that in males. After birth, the cells are mainly concentrated ventrally, being ventro-lateral in the anterior region but converging into the medial-ventral area in the posterior part of the gland. Some dispersion in a dorsal direction is also noted in the latter region. At birth the cells begin to appear in the dorsal area in the anterior portion, as well as in the posterior portion, particularly in the area close to the intermediate lobe and in the zone adjacent to the residual lumen. This was particularly evident in females after 4 days of age. Thus it is concluded that in rats the sexual differences in the pituitary become apparent after the 4th day of postnatal life.