Use of vivo-morpholinos for gene knockdown in the postnatal shark retina.

Work in the catshark Scyliorhinus canicula has shown that the evolutionary origin of postnatal neurogenesis in vertebrates is earlier than previously thought. Thus, the catshark can serve as a model of interest to understand postnatal neurogenic processes and their evolution in vertebrates. One of the best characterized neurogenic niches of the catshark CNS is found in the peripheral region of the retina. Unfortunately, the lack of genetic tools in sharks limits the possibilities to deepen in the study of genes involved in the neurogenic process. Here, we report a method for gene knockdown in the juvenile catshark retina based on the use of Vivo-Morpholinos. To establish the method, we designed Vivo-Morpholinos against the proliferation marker PCNA. We first evaluated the possible toxicity of 3 different intraocular administration regimes. After this optimization step, we show that a single intraocular injection of the PCNA Vivo-Morpholino decreases the expression of PCNA in the peripheral retina, which leads to reduced mitotic activity in this region. This method will help in deciphering the role of other genes potentially involved in postnatal neurogenesis in this animal model.

Embryonic nutritional hyperglycemia decreases cell proliferation in the zebrafish retina.

Diabetic retinopathy (DR) is one of the leading causes of blindness in the world. While there is a major focus on the study of juvenile/adult DR, the effects of hyperglycemia during early retinal development are less well studied. Recent studies in embryonic zebrafish models of nutritional hyperglycemia (high-glucose exposure) have revealed that hyperglycemia leads to decreased cell numbers of mature retinal cell types, which has been related to a modest increase in apoptotic cell death and altered cell differentiation. However, how embryonic hyperglycemia impacts cell proliferation in developing retinas still remains unknown. Here, we exposed zebrafish embryos to 50 mM glucose from 10 h postfertilization (hpf) to 5 days postfertilization (dpf). First, we confirmed that hyperglycemia increases apoptotic death and decreases the rod and Müller glia population in the retina of 5-dpf zebrafish. Interestingly, the increase in cell death was mainly observed in the ciliary marginal zone (CMZ), where most of the proliferating cells are located. To analyze the impact of hyperglycemia in cell proliferation, mitotic activity was first quantified using pH3 immunolabeling, which revealed a significant decrease in mitotic cells in the retina (mainly in the CMZ) at 5 dpf. A significant decrease in cell proliferation in the outer nuclear and ganglion cell layers of the central retina in hyperglycemic animals was also detected using the proliferation marker PCNA. Overall, our results show that nutritional hyperglycemia decreases cellular proliferation in the developing retina, which could significantly contribute to the decline in the number of mature retinal cells.

Timing and Distribution of Mitotic Activity in the Retina During Precocial and Altricial Modes of Avian Development.

During development of the vertebrate retina, mitotic activity is defined as apical when is located at the external surface of the neuroepithelium or as non-apical when is found in more internal regions. Apical mitoses give rise to all retinal cell types. Non-apical mitoses are linked to committed horizontal cell precursors that subsequently migrate vitreo-sclerally, reaching their final position in the outer surface of the inner nuclear layer, where they differentiate. Previous studies have suggested differences in the timing of retinal maturation between altricial and precocial bird species. In the present study we analyze qualitatively and quantitatively the mitotic activity in the developing retina of an altricial (zebra finch, Taeniopygia guttata) and a precocial (Japanese quail, Coturnix coturnix) bird species. We found that pHisH3-immunoreactive apical and non-apical mitoses were abundant in the T. guttata retina at the hatching stage. In contrast, pHisH3 immunoreactivity almost disappeared from the quail retina at the embryonic day 10 (E10). Furthermore, we also found that the onset of the appearance of non-apical mitoses occurred at later stages in the altricial bird species than in the precocial one. The disappearance of apical mitoses and the spatiotemporal distribution of non-apical mitoses followed central to peripheral and dorsal to ventral gradients, similar to gradients of cell differentiation described in the retina of birds. Therefore, these results suggest that retinal neurogenesis is active at the hatching stage in T. guttata, and that horizontal cell differentiation is delayed in the altricial bird species compared to the precocial one. Together, this study reveals important insights into the timing differences that regulate bird retinal maturation and provides a better understanding of the evolution of avian altriciality and precociality.

Decline in Constitutive Proliferative Activity in the Zebrafish Retina with Ageing.

It is largely assumed that the teleost retina shows continuous and active proliferative and neurogenic activity throughout life. However, when delving into the teleost literature, one finds that assumptions about a highly active and continuous proliferation in the adult retina are based on studies in which proliferation was not quantified in a comparative way at the different life stages or was mainly studied in juveniles/young adults. Here, we performed a systematic and comparative study of the constitutive proliferative activity of the retina from early developing (2 days post-fertilisation) to aged (up to 3-4 years post-fertilisation) zebrafish. The mitotic activity and cell cycle progression were analysed by using immunofluorescence against pH3 and PCNA, respectively. We observed a decline in the cell proliferation in the retina with ageing despite the occurrence of a wave of secondary proliferation during sexual maturation. During this wave of secondary proliferation, the distribution of proliferating and mitotic cells changes from the inner to the outer nuclear layer in the central retina. Importantly, in aged zebrafish, there is a virtual disappearance of mitotic activity. Our results showing a decline in the proliferative activity of the zebrafish retina with ageing are of crucial importance since it is generally assumed that the fish retina has continuous proliferative activity throughout life.

Loss of Active Neurogenesis in the Adult Shark Retina.

Neurogenesis is the process by which progenitor cells generate new neurons. As development progresses neurogenesis becomes restricted to discrete neurogenic niches, where it persists during postnatal life. The retina of teleost fishes is thought to proliferate and produce new cells throughout life. Whether this capacity may be an ancestral characteristic of gnathostome vertebrates is completely unknown. Cartilaginous fishes occupy a key phylogenetic position to infer ancestral states fixed prior to the gnathostome radiation. Previous work from our group revealed that the juvenile retina of the catshark Scyliorhinus canicula, a cartilaginous fish, shows active proliferation and neurogenesis. Here, we compared the morphology and proliferative status of the retina in catshark juveniles and adults. Histological and immunohistochemical analyses revealed an important reduction in the size of the peripheral retina (where progenitor cells are mainly located), a decrease in the thickness of the inner nuclear layer (INL), an increase in the thickness of the inner plexiform layer and a decrease in the cell density in the INL and in the ganglion cell layer in adults. Contrary to what has been reported in teleost fish, mitotic activity in the catshark retina was virtually absent after sexual maturation. Based on these results, we carried out RNA-Sequencing (RNA-Seq) analyses comparing the retinal transcriptome of juveniles and adults, which revealed a statistically significant decrease in the expression of many genes involved in cell proliferation and neurogenesis in adult catsharks. Our RNA-Seq data provides an excellent resource to identify new signaling pathways controlling neurogenesis in the vertebrate retina.

Analysis of Programmed Cell Death and Senescence Markers in the Developing Retina of an Altricial Bird Species.

This study shows the distribution patterns of apoptotic cells and biomarkers of cellular senescence during the ontogeny of the retina in the zebra finch (T. guttata). Neurogenesis in this altricial bird species is intense in the retina at perinatal and post-hatching stages, as opposed to precocial bird species in which retinogenesis occurs entirely during the embryonic period. Various phases of programmed cell death (PCD) were distinguishable in the T. guttata visual system. These included areas of PCD in the central region of the neuroretina at the stages of optic cup morphogenesis, and in the sub-optic necrotic centers (St15-20). A small focus of early neural PCD was detected in the neuroblastic layer, dorsal to the optic nerve head, coinciding with the appearance of the first differentiated neuroblasts (St24-St25). There were sparse pyknotic bodies in the non-laminated retina between St26 and St37. An intense wave of neurotrophic PCD was detected in the laminated retina between St42 and P8, the last post-hatching stage included in the present study. PCD was absent from the photoreceptor layer. Phagocytic activity was also detected in Müller cells during the wave of neurotrophic PCD. With regard to the chronotopographical staining patterns of senescence biomarkers, there was strong parallelism between the SA-ß-GAL signal and p21 immunoreactivity in both the undifferentiated and the laminated retina, coinciding in the cell body of differentiated neurons. In contrast, no correlation was found between SA-ß-GAL activity and the distribution of TUNEL-positive cells in the developing tissue.

Retinal differentiation in an altricial bird species, Taeniopygia guttata: An immunohistochemical study.

The bird retina offers an excellent model to investigate the mechanisms that coordinate the morphogenesis, histogenesis, and differentiation of neuron and glial cells. Although these developmental features have been intensively studied in the chicken (Gallus gallus, Linnaeus 1758), a precocial bird species, little is known about retinogenesis in altricial birds. The purpose of this study was to examine the differentiation of retinal cells in the altricial zebra finch (Taeniopygia guttata, Vieillot, 1817) and compare the results with those from previous studies in G. gallus. By using immunohistochemical techniques, the first differentiated TUJ1-/Isl1-positive neuroblasts were detected in the vitreal surface of the neuroblastic layer at later incubation times in T. guttata than in G. gallus (108 h vs 55 h). The immunoreactivity of these early differentiation markers coincided temporo-spatially with the appearance of the first PCNA-negative nuclei. Furthermore, the first visinin-positive photoreceptors (132 h vs 120 h) and the first Prox-1-immunoreactive neuroblasts (embryonic day 7.25 (E7.25) vs E6.5) were also detected at later embryonic stages in the retina of T. guttata than in the retina of G. gallus. At E13, one day before hatching, abundant PCNA- and pHisH3-immunoreactivities were detected in the T. guttata retina, while proliferation was almost absent in the G. gallus retina at perinatal stages. Therefore, these results suggest that cell differentiation in the retina is delayed in the altricial bird compared to precocial birds. Furthermore, the T. guttata retina was not completely developed at hatching, and abundant mitotically active precursor cells of retinal neurons were found, suggesting that retinal neurogenesis was intense at perinatal stages.

Retinal histogenesis in an altricial avian species, the zebra finch (Taeniopygia guttata, Vieillot 1817).

Comparative developmental studies have shown that the retina of altricial fish and mammals is incompletely developed at birth, and that, during the first days of life, maturation proceeds rapidly. In contrast, precocial fish and mammals are born with fully differentiated retinas. Concerning birds, knowledge about retinal development is generally restricted to a single order of precocial birds, Galliformes, due to the fact that both the chicken and the Japanese quail are considered model systems. However, comparison of embryonic pre-hatchling retinal development between altricial and precocial birds has been poorly explored. The purpose of this study was to examine the morphogenesis and histogenesis of the retina in the altricial zebra finch (Taeniopygia guttata, Vieillot 1817) and compare the results with those from previous studies in the precocial chicken. Several maturational features (morphogenesis of the optic vesicle and optic cup, appearance of the first differentiated neurons, the period in which the non-apical cell divisions are observable, and the emergence of the plexiform layers) were found to occur at later stages in the zebra finch than in the chicken. At hatching, the retina of T. guttata showed the typical cytoarchitecture of the mature tissue, although features of immaturity were still observable, such as a ganglion cell layer containing many thick cells, very thin plexiform layers, and poorly developed photoreceptors. Moreover, abundant mitotic activity was detected in the entire retina, even in the regions where the layering was complete. The circumferential marginal zone was very prominent and showed abundant mitotic activity. The partially undifferentiated stage of maturation at hatching makes the T. guttata retina an appropriate model with which to study avian postnatal retinal neurogenesis.