Doublecortin in the Fish Visual System, a Specific Protein of Maturing Neurons.

Doublecortin (DCX) is a microtubule associated protein, essential for correct central nervous system development and lamination in the mammalian cortex. It has been demonstrated to be expressed in developing-but not in mature-neurons. The teleost visual system is an ideal model to study mechanisms of adult neurogenesis due to its continuous life-long growth. Here, we report immunohistochemical, in silico, and western blot analysis to detect the DCX protein in the visual system of teleost fish. We clearly determined the expression of DCX in newly generated cells in the retina of the cichlid fish Astatotilapia burtoni, but not in the cyprinid fish Danio rerio. Here, we show that DCX is not associated with migrating cells but could be related to axonal growth. This work brings to light the high conservation of DCX sequences between different evolutionary groups, which make it an ideal marker for maturing neurons in various species. The results from different techniques corroborate the absence of DCX expression in zebrafish. In A. burtoni, DCX is very useful for identifying new neurons in the transition zone of the retina. In addition, this marker can be applied to follow axons from maturing neurons through the neural fiber layer, optic nerve head, and optic nerve.

Cultures of glial cells from optic nerve of two adult teleost fish: Astatotilapia burtoni and Danio rerio.

BACKGROUND: In vitro studies are very useful to increase the knowledge of different cell types and could be the key to understand cell metabolism and function. Fish optic nerves (ON) can recover visual functions by reestablishing its structure and reconnecting the axons of ganglion cells. This is because fish show spontaneous regeneration of the central nervous system which does not occur in mammals. In addition, several studies have indicated that glial cells of ON have different properties in comparison to the glial cells from brain or retina. Consequently, providing an in vitro tool will be highly beneficial to increase the knowledge of these cells. NEW METHOD: We developed a cell culture protocol to isolate glial cells from ON of two teleost fish species, Danio rerio and Astatotilapia burtoni. RESULTS: The optimized protocol allowed us to obtain ON cells and brain-derived cells from adult teleost fish. These cells were characterized as glial cells and their proprieties in vitro were analyzed.Comparison with Existing Method(s): Although it is striking that ON glial cells show peculiarities, their study in vitro has been limited by the only published protocol going back to the 1990s. Our protocol makes glial cells of different fish species available for experiments and studies to increase the understanding of these glial cell types. CONCLUSIONS: This validated and effective in vitro tool increases the possibilities on studies of glial cells from fish ON which implies a reduction in animal experimentation.

Sox2 expression in the visual system of two teleost species.

The visual system of teleost fish shows growth and regeneration capacities during the entire animal's life. Thus, the visual system of adult fish serves as a model for studying neurogenesis in the vertebrate central nervous system (CNS). Our study focused on the expression pattern of Sox2 in the fish visual system. Sox2 is a transcription factor known for its function in keeping stem cell properties, and as a regulator of cell fate during development, especially in the visual system. We used two different fish species: Astatotilapia burtoni and Danio rerio. In the visual system of fish, we identified Sox2 positive cells in the stem cell niche in the peripheral retina, in Müller cells and amacrine cells in the differentiated retina, and glial cells in the optic nerve (ON). We did not observe hardly any Sox2 expression in the optic nerve head (ONH). In the ON, Sox2 positive glial cells were lining the fascicles of new axons. Taking together, the broad spectrum of Sox2 expression indicates that this protein has different functions in the CNS of adult vertebrates. The results suggest that Sox2 has functions associated with the pathway of new axons from the retina. To understand the variety of cell types and subtypes and their plasticity potential in the visual system of fish will be essential to comprehend the growing and regenerating CNS in adult vertebrates.

Effects of retinoic acid exposure during zebrafish retinogenesis.

Retinoic acid (RA) is an important morphogen involved in retinal development. Perturbations in its levels cause retinal malformations such as microphthalmia. However, the cellular changes in the retina that lead to this phenotype are little known. We have used the zebrafish to analyse the effects of systemic high RA levels on retinogenesis. For this purpose we exposed zebrafish embryos to 0.1µM or 1µM RA from 24 to 48h post-fertilisation (hpf), the period which corresponds to the time of retinal neurogenesis and initial retinal cell differentiation. We did not find severe alterations in 0.1µM RA treated animals, but the exposure to 1µM RA significantly reduced retinal size upon treatment, and this microphthalmia persisted through larval development. We monitored histology and cell death and quantified both the proliferation rate and cell differentiation from 48hpf onwards, focusing on the retina and optic nerve of normal and 1µM treated animals. Retinal lamination and initial neurogenesis are not affected by RA exposure, but we found widespread apoptosis after RA treatment that could be the main cause of microphthalmia. Proliferating cells increased their number at 3days post-fertilisation (dpf) but decreased significantly at 5dpf maintaining the microphthalmic phenotype. Retinal cell differentiation was affected; some cell markers do not reach normal levels at larval stages and some cell types present an increased number compared to those of control animals. We also found the presence of young axons growing ectopically within the retina. Moreover although the optic axons leave the retina and form the optic chiasm they do not reach the optic tectum. The alterations observed in treated animals become more severe as larvae develop.

Immunocytochemical evidence of the localization of the Crumbs homologue 3 protein (CRB3) in the developing and mature mouse retina.

CRB3 (Crumbs homologue 3), a member of the CRB protein family (homologous to the Drosophila Crumbs), is expressed in different epithelium-derived cell types in mammals, where it seems to be involved in regulating the establishment and stability of tight junctions and in ciliogenesis. This protein has been also detected in the retina, but little is known about its localization and function in this tissue. Our goal here was to perform an in-depth study of the presence of CRB3 protein in the mouse retina and to analyze its expression during photoreceptor ciliogenesis and the establishment of the plexiform retinal layers. Double immunofluorescence experiments for CRB3 and well-known markers for the different retinal cell types were performed to study the localization of the CRB3 protein. According to our results, CRB3 is present from postnatal day 0 (P0) until adulthood in the mouse retina. It is localized in the inner segments (IS) of photoreceptor cells, especially concentrated in the area where the connecting cilium is located, in their synaptic terminals in the outer plexiform layer (OPL), and in sub-populations of amacrine and bipolar cells in the inner plexiform layer (IPL).

Characterization of Pax2 expression in the goldfish optic nerve head during retina regeneration.

The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2(+) astrocytes located in the ONH and S100(+) astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2(+) and S100(+) astrocytes have different responses. At 7 days after injury the number of Pax2(+) cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100(+) astrocytes in the retina surrounding the ONH and S100(+) processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100(+) astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2(+) cells increase. At the same time, S100(+)/GFAP(+)/GS(+) astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2(+)/PCNA(+) astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2(+) astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process.

Characterisation and differential expression during development of a duplicate Disabled-1 (Dab1) gene from zebrafish.

We identified a new duplicated Dab1 gene (drDab1b) spanning around 25kb of genomic DNA in zebrafish. Located in zebrafish chromosome 2, it is composed of 11 encoding exons and shows high sequence similarity to other Dab1 genes, including drDab1a, a zebrafish Dab1 gene previously characterised. drDab1b encodes by alternative splicing at least five different isoforms. Both drDab1a and drDab1b show differential gene expression levels in distinct adult tissues and during development. drDab1b is expressed in peripheral tissues (gills, heart, intestine, muscle), the immune system (blood, liver) and the central nervous system (CNS), whereas drDab1a is only expressed in gills, muscle and the CNS, suggesting a division of functions for two Dab1 genes in zebrafish adult tissues. RT-PCR analysis also reveals that both drDab1 genes show distinct developmental-specific expression patterns throughout development. drDab1b expression was higher than that of drDab1a, suggesting a major role of drDab1b in comparison with drDab1a during development and in different adult tissues. In addition, new putative Dab1 (a and/or b) from different teleost species were identified in silico and predicted protein products are compared with the previously characterised Dab1, demonstrating that the Dab1b group is more ancestral than their paralogue, the Dab1a group.

Prox1 expression in rod precursors and Müller cells.

The transcription factor Prox1 acts in rodent retinogenesis, at least in promoting cell cycle withdrawal and horizontal cell production. In the mature retina, this protein is detected at the inner nuclear layer of all vertebrate groups. We have made a neurochemical characterisation of Prox1(+) cell types in two different vertebrate groups: mammals and fish. As well as Prox1(+) horizontal cells, we have observed Prox1(+)/PKC-alpha(+) rod bipolar cells in mouse and cone ON and mixed b bipolar cells in goldfish. In mouse, only some CB(+) and CR(+) amacrine cells are Prox1(+) and the TH(+) and CR(+) amacrine cells are Prox1(-). However, in goldfish all CR(+) amacrine cells and TH(+) interplexiform cells are Prox1(+) and in the GCL displaced amacrine cells are also Prox1(+). Besides its expression in different interneuron subpopulations, we demonstrate, for the first time, the presence of Prox1 in the GS(+) and CRALBP(+) Müller cells in the retina of adult mammals and in developing and mature retina of fish. The presence of Prox1 in these cells appears to be related to survival or maintenance of their phenotype. We also demonstrate that in fish, where retinal formation persists into adulthood, Prox1 is expressed in dividing PCNA(+) cells at the peripheral growing zone, in rod progenitors at the inner and outer nuclear layers as well as in early progenitors during a retinal regeneration process after cryo-lesion of the peripheral growing zone. Therefore, Prox1 functions in vertebrate retinogenesis may be more complex than previously expected.

Differential expression and cellular localization of somatolactin-1 and -2 during early development in the gilthead sea bream.

The patterns of expression of the somatolactin 1 and 2 (SL1 and SL2) transcripts were studied during the early development of the gilthead sea bream (Sparus aurata). Gene expression of SL1 and SL2 were detected in embryos and in larvae, although both transcripts presented different levels of expression. The SL1 transcripts in contrast to the SL2 transcripts presented high expression levels in embryos and younger larvae. Moreover, the SL2 transcripts were slightly present or absence in embryonic stage and the newly hatched larvae, respectively. The differences in the expression levels of SL1 and SL2 in embryos and larvae may be due to the fact that two distinct genes express both isoforms of the protein. Thus, both SLs may play different physiological roles throughout development. Moreover, the hybridization signals for SL1- and SL2-mRNAs were detected in 4-day-old larvae. Both in larvae and adults the somatolactotroph cells co-expressed both transcripts of SL and were located bordering the neurohypophysis in the pars intermedia.

The degenerative and regenerative processes after the elimination of the proliferative peripheral retina of fish.

We have analyzed the modifications in the tench (Tinca tinca) retina after the complete cryo-elimination of the proliferative growing zone (PGZ), which participates in the continuous growth of the retina throughout the life of the fish. By using immunohistochemistry and electron microscopy we demonstrated that, after the lesion, degenerative and regenerative processes take place in the PGZ, in the ciliary zone, and in the transition zone located between the PGZ and the central retina. After 120 days postlesion, the PGZ was completely regenerated and its composition was similar to that of the control animals. Numerous proliferative PCNA-positive cells reappeared and new ganglion cells were formed. In the transition zone and the central retina numerous proliferative PCNA-positive cells also appeared. These are arranged, on occasion, as columnar units from the inner to the outer nuclear layer where the rod precursors and the progenitor cells, respectively, were located. The Müller cells, closely associated with these columnar units, appeared to use them as guides to migration during the regenerative process. Notably, modifications occurred in the ciliary zone, whose cells acquired similar characteristics to the PGZ cells. The ciliary zone cells, the Müller cells, the rod precursors, and the proliferative cells located in the inner nuclear layer appear to participate actively in the regeneration of the PGZ.

The glial design of a teleost optic nerve head supporting continuous growth.

This study demonstrates the peculiarities of the glial organization of the optic nerve head (ONH) of a fish, the tench (Tinca tinca), by using immunohistochemistry and electron microscopy. We employed antibodies specific for the macroglial cells: glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), and S100. We also used the N518 antibody to label the new ganglion cells' axons, which are continuously added to the fish retina, and the anti-proliferating cell nuclear antigen (PCNA) antibody to specifically locate dividing cells. We demonstrate a specific regional adaptation of the GS-S100-positive Müller cells' vitreal processes around the optic disc, strongly labeled with the anti-GFAP antibody. In direct contact with these Müller cells' vitreal processes, there are S100-positive astrocytes and S100-negative cells ultrastructurally identified as microglial cells. Moreover, a population of PCNA-positive cells, characterized as glioblasts, forms the limit between the retina and the optic nerve in a region homologous to the Kuhnt intermediary tissue of mammals. Finally, in the intraocular portion of the optic nerve there are differentiating oligodendrocytes arranged in rows. Both the glioblasts and the rows of developing cells could serve as a pool of glial elements for the continuous growth of the visual system.

Co-expression of somatolactin mRNAs in the pituitary gland of gilthead sea bream.

The expression of mRNAs of the two types of somatolactin which have been found, up to the present, in the pituitary of gilthead sea bream (Sparus aurata) have been analyzed by in situ hybridization (ISH). The method of non-radioactive ISH, which we optimize in this study, uses oligonucleotides labelled in the 5' end with biotin or dioxygenin as probes. This allows simple and double ISH of high specificity and sensitivity to be performed. The distinct somatolactin oligoprobes used present a strong signal fundamentally in the pars intermedia of the pituitary gland. For the first time we present cells that co-express both the forms of mRNA mentioned above. Moreover, after studying three groups of different sizes, produced by asynchronic growth of the gilthead sea bream in industrial cultivation, we did not find qualitative differences in the levels of expression of the somatolactin gene.

Flow cytometry measurement of the DNA contents of G0/G1 diploid cells from three different teleost fish species.

BACKGROUND: Although there is a lot information in the literature about genome size in fish, a high variability among data for the same species is reported, being mainly related to methodological aspects. Flow cytometry-based fluorescence measurements of intercalating dyes is the most attractive approach due to its precision, objectivity, high speed, and relative simplicity. METHODS: We analyze the DNA content of G0/G1 diploid nuclei of three teleost species (Carassius auratus, Tinca tinca, and Danio rerio) using flow cytometry. Forty-three animals were used and up to 50,000 retinal cells were analyzed per sample. Propidium iodide-associated fluorescence was assessed using a FACSCalibur flow cytometer. Standard human leukocytes were used as a reference. RESULTS: Our results show that C. auratus (3.584 +/- 0.058 pg per nucleus) and D. rerio (3.357 +/- 0.074 pg per nucleus) showed similar DNA contents per cell, whereas it was significantly lower (2.398 +/- 0.038 pg per nucleus) in T. tinca. Interestingly, a low intraspecies variability was observed, the coefficient of variation being 1.608%, 2.198%, and 1.573% for C. auratus, D. rerio, and T. tinca, respectively. CONCLUSIONS: The methodology used in this study provides an accurate and easy measurement of the genome size of a species.

Quantitative evaluation of the distribution of proliferating cells in the adult retina in three cyprinid species.

In the present study, a descriptive and quantitative analysis of all the proliferating cell populations present in the normal adult retina of three cyprinid species (goldfish, zebrafish, and tench) is reported. Evaluation of cell proliferation was performed in proliferating cell nuclear antigen (PCNA)-labeled tissue sections as well as in single-cell suspensions analyzed by flow cytometry. Our results show that the neural progenitors of the inner nuclear layer (INL) of cyprinids continue dividing in adulthood in uninjured retinas. These cells are probably related to the generation of rods in normal retinal growth, as well as in the production of any retinal cell type in regenerating processes. The distributions of both these cells and their presumptive progeny, the rod precursors, differ from one species to another, being homogeneous in zebrafish, displaced to the periphery in goldfish and to the temporal pole in tench. With regard to the cell apposition at the retinal periphery, it seems to be symmetrical in goldfish and zebrafish, based on a homogeneous extension of the peripheral growth zone (PGZ), but asymmetrical in tench, where it presents a significantly lower extension in the ventral retina. The flow cytometry analyses indicate that, overall, the proportion of proliferating cells is significantly greater in zebrafish retina despite the fact that body growth rate is lower in zebrafish than the other two species.