Developmental genoarchitectonics as a key tool to interpret the mature anatomy of the chondrichthyan hypothalamus according to the prosomeric model.

The hypothalamus is a key vertebrate brain region involved in survival and physiological functions. Understanding hypothalamic organization and evolution is important to deciphering many aspects of vertebrate biology. Recent comparative studies based on gene expression patterns have proposed the existence of hypothalamic histogenetic domains (paraventricular, TPa/PPa; subparaventricular, TSPa/PSPa; tuberal, Tu/RTu; perimamillary, PM/PRM; and mamillary, MM/RM), revealing conserved evolutionary trends. To shed light on the functional relevance of these histogenetic domains, this work aims to interpret the location of developed cell groups according to the prosomeric model in the hypothalamus of the catshark Scyliorhinus canicula, a representative of Chondrichthyans (the sister group of Osteichthyes, at the base of the gnathostome lineage). To this end, we review in detail the expression patterns of ScOtp, ScDlx2, and ScPitx2, as well as Pax6-immunoreactivity in embryos at stage 32, when the morphology of the adult catshark hypothalamus is already organized. We also propose homologies with mammals when possible. This study provides a comprehensive tool to better understand previous and novel data on hypothalamic development and evolution.

Identifying Amygdala-Like Territories in Scyliorhinus canicula (Chondrichthyan): Evidence for a Pallial Amygdala.

To identify the putative amygdalar complex in cartilaginous fishes, our first step was to obtain evidence that supports the existence of a pallial amygdala in the catshark Scyliorhinus canicula, at present the prevailing chondrichthyan model in comparative neurobiology and developmental biology. To this end, we analyzed the organization of the lateral walls of the telencephalic hemispheres of adults, juveniles, and early prehatching embryos by immunohistochemistry against tyrosine hydroxylase (TH), somatostatin (SOM), Pax6, serotonin (5HT), substance P (SP), and Met-enkephalin (MetEnk), calbindin-28k (CB), and calretinin (CR), and by in situ hybridization against regulatory genes such as Tbr1, Lhx9, Emx1, and Dlx2. Our data were integrated with those available from the literature related to the secondary olfactory projections in this shark species. We have characterized two possible amygdalar territories. One, which may represent a ventropallial component, was identified by its chemical signature (moderate density of Pax6-ir cells, scarce TH-ir and SOM-ir cells, and absence of CR-ir and CB-ir cells) and gene expressions (Tbr1 and Lhx9 expressions in an Emx1 negative domain, as the ventral pallium of amniotes). It is perhaps comparable to the lateral amygdala of amphibians and the pallial amygdala of teleosts. The second was a territory related to the pallial-subpallial boundary with abundant Pax6-ir and CR-ir cells, and 5HT-ir, SP-ir, and MetEnk-ir fibers capping dorsally the area superficialis basalis. This olfactory-related region at the neighborhood of the pallial-subpallial boundary may represent a subpallial amygdala subdivision that possibly contains migrated cells of ventropallial origin.

The Shark Basal Hypothalamus: Molecular Prosomeric Subdivisions and Evolutionary Trends.

The hypothalamus is a key integrative center of the vertebrate brain. To better understand its ancestral morphological organization and evolution, we previously analyzed the segmental organization of alar subdivisions in the catshark Scyliorhinus canicula, a cartilaginous fish and thus a basal representative of gnathostomes (jawed vertebrates). With the same aim, we deepen here in the segmental organization of the catshark basal hypothalamus by revisiting previous data on ScOtp, ScDlx2/5, ScNkx2.1, ScShh expression and Shh immunoreactivity jointly with new data on ScLhx5, ScEmx2, ScLmx1b, ScPitx2, ScPitx3a, ScFoxa1, ScFoxa2 and ScNeurog2 expression and proliferating cell nuclear antigen (PCNA) immunoreactivity. Our study reveals a complex genoarchitecture for chondrichthyan basal hypothalamus on which a total of 21 microdomains were identified. Six belong to the basal acroterminal region, the rostral-most point of the basal neural tube; seven are described in the tuberal region (Tu/RTu); four in the perimamillar region (PM/PRM) and four in the mamillar one (MM/RM). Interestingly, the same set of genes does not necessarily describe the same microdomains in mice, which in part contributes to explain how forebrain diversity is achieved. This study stresses the importance of analyzing data from basal vertebrates to better understand forebrain diversity and hypothalamic evolution.

The Ciliary Margin Zone of the Mammalian Retina Generates Retinal Ganglion Cells.

The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. Because Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2-/- mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity.

The Shark Alar Hypothalamus: Molecular Characterization of Prosomeric Subdivisions and Evolutionary Trends.

The hypothalamus is an important physiologic center of the vertebrate brain involved in the elaboration of individual and species survival responses. To better understand the ancestral organization of the alar hypothalamus we revisit previous data on ScOtp, ScDlx2/5, ScTbr1, ScNkx2.1 expression and Pax6 immunoreactivity jointly with new data on ScNeurog2, ScLhx9, ScLhx5, and ScNkx2.8 expression, in addition to immunoreactivity to serotonin (5-HT) and doublecortin (DCX) in the catshark Scyliorhinus canicula, a key species for this purpose since cartilaginous fishes are basal representatives of gnathostomes (jawed vertebrates). Our study revealed a complex genoarchitecture for the chondrichthyan alar hypothalamus. We identified terminal (rostral) and peduncular (caudal) subdivisions in the prosomeric paraventricular and subparaventricular areas (TPa/PPa and TSPa/PSPa, respectively) evidenced by the expression pattern of developmental genes like ScLhx5 (TPa) and immunoreactivity against Pax6 (PSPa) and 5-HT (PPa and PSPa). Dorso-ventral subdivisions were only evidenced in the SPa (SPaD, SPaV; respectively) by means of Pax6 and ScNkx2.8 (respectively). Interestingly, ScNkx2.8 expression overlaps over the alar-basal boundary, as Nkx2.2 does in other vertebrates. Our results reveal evidences for the existence of different groups of tangentially migrated cells expressing ScOtp, Pax6, and ScDlx2. The genoarchitectonic comparative analysis suggests alternative interpretations of the rostral-most alar plate in prosomeric terms and reveals a conserved molecular background for the vertebrate alar hypothalamus likely acquired before/during the agnathan-gnathostome transition, on which Otp, Pax6, Lhx5, and Neurog2 are expressed in the Pa while Dlx and Nkx2.2/Nkx2.8 are expressed in the SPa.

Prosomeric organization of the hypothalamus in an elasmobranch, the catshark Scyliorhinus canicula.

The hypothalamus has been a central topic in neuroanatomy because of its important physiological functions, but its mature organization remains elusive. Deciphering its embryonic and adult organization is crucial in an evolutionary approach of the organization of the vertebrate forebrain. Here we studied the molecular organization of the hypothalamus and neighboring telencephalic domains in a cartilaginous fish, the catshark, Scyliorhinus canicula, focusing on ScFoxg1a, ScShh, ScNkx2.1, ScDlx2/5, ScOtp, and ScTbr1 expression profiles and on the identification α-acetylated-tubulin-immunoreactive (ir), TH-ir, 5-HT-ir, and GFAP-ir structures by means of immunohistochemistry. Analysis of the results within the updated prosomeric model framework support the existence of alar and basal histogenetic compartments in the hypothalamus similar to those described in the mouse, suggesting the ancestrality of these subdivisions in jawed vertebrates. These data provide new insights into hypothalamic organization in cartilaginous fishes and highlight the generality of key features of the prosomeric model in jawed vertebrates.

Mechanisms of endoderm formation in a cartilaginous fish reveal ancestral and homoplastic traits in jawed vertebrates.

In order to gain insight into the impact of yolk increase on endoderm development, we have analyzed the mechanisms of endoderm formation in the catshark S. canicula, a species exhibiting telolecithal eggs and a distinct yolk sac. We show that in this species, endoderm markers are expressed in two distinct tissues, the deep mesenchyme, a mesenchymal population of deep blastomeres lying beneath the epithelial-like superficial layer, already specified at early blastula stages, and the involuting mesendoderm layer, which appears at the blastoderm posterior margin at the onset of gastrulation. Formation of the deep mesenchyme involves cell internalizations from the superficial layer prior to gastrulation, by a movement suggestive of ingressions. These cell movements were observed not only at the posterior margin, where massive internalizations take place prior to the start of involution, but also in the center of the blastoderm, where internalizations of single cells prevail. Like the adjacent involuting mesendoderm, the posterior deep mesenchyme expresses anterior mesendoderm markers under the control of Nodal/activin signaling. Comparisons across vertebrates support the conclusion that endoderm is specified in two distinct temporal phases in the catshark as in all major osteichthyan lineages, in line with an ancient origin of a biphasic mode of endoderm specification in gnathostomes. They also highlight unexpected similarities with amniotes, such as the occurrence of cell ingressions from the superficial layer prior to gastrulation. These similarities may correspond to homoplastic traits fixed separately in amniotes and chondrichthyans and related to the increase in egg yolk mass.

Contributions of developmental studies in the dogfish Scyliorhinus canicula to the brain anatomy of elasmobranchs: insights on the basal ganglia.

The basic anatomy of the elasmobranch brain has been previously established after studying the organization of the different subdivisions in the adult brain. However, despite the relatively abundant immunohistochemical and hodologic studies performed in different species of sharks and skates, the organization of some brain subdivisions remains unclear. The present study focuses on some brain regions in which subdivisions established on the basis of anatomical data in adults remain controversial, such as the subpallium, mainly the striatal subdivision. Taking advantage of the great potential of the lesser spotted dogfish, Scyliorhinus canicula, as a model for developmental studies, we have characterized the subpallium throughout development and postembryonic stages by analyzing the distribution of immunomarkers for GABA, catecholamines, and neuropeptides, such as substance P. Moreover, we have analyzed the expression pattern of regulatory genes involved in the regionalization of the telencephalon, such as Dlx2, Nkx2.1, and Shh, and followed their derivatives throughout development in relation to the distribution of such neurochemical markers. For further characterization, we have also analyzed the patterns of innervation of the subpallium after applying tract-tracing techniques. Our observations may shed light on postulate equivalences of regions and nuclei among elasmobranchs and support homologies with other vertebrates.

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway.

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.

Pax6 expression during retinogenesis in sharks: comparison with markers of cell proliferation and neuronal differentiation.

Pax6 is a highly conserved transcription factor that appears involved in the entire process of retinogenesis, including maintenance of proliferation of retinal progenitors and differentiation of particular neuron fates. To gain insight into the retinogenesis in fish, we study the dynamics of Pax6 expression in the developing and mature retina of two sharks that inhabit in particular environments, and compare it with the dynamics of a marker of cell proliferation (proliferating cell nuclear antigen, PCNA) and markers of neuronal differentiation, such as glutamic acid decarboxylase (GAD), calretinin (CR), tyrosine-hydroxylase, and serotonin (5-HT). Our results reveal that Pax6 is expressed in PCNA-immunoreactive cells within the nonlayered retina, suggesting a role for Pax6 in proliferating progenitors. Pax6 expression decays as development proceeds and eventually remains in some postmitotic cells, which points to additional roles of Pax6 following neurogenesis. Double immunofluorescence reveals Pax6/CR colocalization in the ganglion cell layer, Pax6/5-HT in the inner part of the inner nuclear layer (INLi), and Pax6/GAD in the INLi and horizontal cell layer. Our results suggest that Pax6 may contribute to neuron diversification in the neural retina.

Regionalization of the shark hindbrain: a survey of an ancestral organization.

Cartilaginous fishes (chondrichthyans) represent an ancient radiation of vertebrates currently considered the sister group of the group of gnathostomes with a bony skeleton that gave rise to land vertebrates. This out-group position makes chondrichthyans essential in assessing the ancestral organization of the brain of jawed vertebrates. To gain knowledge about hindbrain evolution we have studied its development in a shark, the lesser spotted dogfish Scyliorhinus canicula by analyzing the expression of some developmental genes and the origin and distribution of specific neuronal populations, which may help to identify hindbrain subdivisions and boundaries and the topology of specific cell groups. We have characterized three developmental periods that will serve as a framework to compare the development of different neuronal systems and may represent a suitable tool for comparing the absolute chronology of development among vertebrates. The expression patterns of Pax6, Wnt8, and HoxA2 genes in early embryos of S. canicula showed close correspondence to what has been described in other vertebrates and helped to identify the anterior rhombomeres. Also in these early embryos, the combination of Pax6 with protein markers of migrating neuroblasts (DCX) and early differentiating neurons (general: HuC/D; neuron type specific: GAD, the GABA synthesizing enzyme) revealed the organization of S. canicula hindbrain in both transverse segmental units corresponding to visible rhombomeres and longitudinal columns. Later in development, when the interrhombomeric boundaries fade away, accurate information about S. canicula hindbrain subdivisions was achieved by comparing the expression patterns of Pax6 and GAD, serotonin (serotoninergic neurons), tyrosine hydroxylase (catecholaminergic neurons), choline acetyltransferase (cholinergic neurons), and calretinin (a calcium-binding protein). The patterns observed revealed many topological correspondences with other vertebrates and led to reconsideration of the current view of the elasmobranch hindbrain segmentation as peculiar among vertebrates.

Calretinin immunoreactivity in the developing retina of sharks: comparison with cell proliferation and GABAergic system markers.

The calcium-binding protein calretinin (CR) has been widely used as a marker of neuronal differentiation. In the present study we analyzed the distribution of CR-immunoreactive (CR-ir) elements in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus). We compared the distribution of CR with that of a proliferation marker (the proliferating cell nuclear antigen, PCNA) in order to investigate the time course of CR expression during retinogenesis and explored the relationship between CR and glutamic acid decarboxylase (GAD), the synthesizing enzyme of the gamma-aminobutyric acid (GABA), which has been reported to play a role in shark retinogenesis. The earliest CR immunoreactivity was concurrently observed in subsets of: a) ganglion cells in the ganglion cell layer; b) displaced ganglion cells in the inner plexiform layer and inner part of the inner nuclear layer (INLi); c) amacrine cells in the INLi, and d) horizontal cells. This pattern of CR distribution is established in the developing retina from early stage 32, long after the appearance of a layered retinal organization in the inner retina, and coinciding with photoreceptor maturation in the outer retina. We also demonstrated that CR is expressed in postmitotic cells long after they have exited the cell cycle and in a subset of GABAergic horizontal cells. Overall our results provide insights into the differentiation patterns in the elasmobranch retina and supply further comparative data on the development of CR distribution in the retina of vertebrates. This study may help in understanding the possible involvement of CR in aspects of retinal morphogenesis.

Patterns of cell proliferation and rod photoreceptor differentiation in shark retinas.

We studied the pattern of cell proliferation and its relation with photoreceptor differentiation in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus). Cell proliferation was studied with antibodies raised against proliferating cell nuclear antigen (PCNA) and phospho-histone-H3, and early photoreceptor differentiation with an antibody raised against rod opsin. As regards the spatiotemporal distribution of PCNA-immunoreactive cells, our results reveal a gradual loss of PCNA that coincides in a spatiotemporal sequence with the gradient of layer maturation. The presence of a peripheral growth zone containing pure-proliferating retinal progenitors (the ciliary marginal zone) in the adult retina matches with the general pattern observed in other groups of gnathostomous fishes. However, in the shark retina the generation of new cells is not restricted to the ciliary marginal zone but also occurs in retinal areas that contain differentiated cells: (1) in a transition zone that lies between the pure-proliferating ciliary marginal zone and the central (layered) retina; (2) in the differentiating central area up to prehatching embryos where large amounts of PCNA-positive cells were observed even in the inner and outer nuclear layers; (3) and in the retinal pigment epithelium of prehatching embryos. Rod opsin immunoreactivity was observed in both species when the outer plexiform layer begins to be recognized in the central retina and, as we previously observed in trout, coincided temporally with the weakening in PCNA labelling.

Calretinin-immunoreactive systems in the cerebellum and cerebellum-related lateral-line medullary nuclei of an elasmobranch, Scyliorhinus canicula.

Calretinin immunohistochemistry was used to study the organization of some cerebellar structures and lateral line medullary nuclei of an elasmobranch, the lesser-spotted dogfish Scyliorhinus canicula. In the cerebellar molecular layer, stellate cells are strongly calretinin-immunoreactive (CR-ir). Perikarya and dendrites of Purkinje cells are contacted by numerous stellate cell small CR-ir boutons. Some Purkinje cell perikarya are contacted by CR-ir climbing fibers forming complex axo-somatic contacts. In the granular layer, numerous CR-ir mossy fibers exhibited large swellings. Notable differences in density and diameter of mossy fibers are observed between the auricles and cerebellar body. Thin beaded CR-ir fibers are also present in the granular layer of the body. The lateral line nuclei of the octavolateralis region are comprised of a molecular-like cerebellar crest that covers the dorsal (electroreceptive) and the medial octavolateralis nuclei (mechanoreceptive). The cerebellar crest exhibited numerous CR-ir stellate cells. In the dorsal octavolateralis nucleus, the presence of conspicuous CR-ir cells and neuropil closely associated to the region of primary fiber terminals distinguishes it clearly from the medial nucleus, revealing major differences between the electroreceptive and mechanoreceptive primary nuclei of elasmobranchs. Moreover, CR distribution in the dogfish cerebellum showed interesting differences with those reported in cerebella of other vertebrates, indicating a high variability of cerebellar CR expression in phylogeny.

Early development of GABAergic cells of the retina in sharks: an immunohistochemical study with GABA and GAD antibodies.

We studied the ontogeny and organization of GABAergic cells in the retina of two elasmobranches, the lesser-spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus) by using immunohistochemistry for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). Both antibodies revealed the same pattern of immunoreactivity and both species showed similar organization of GABAergic cells. GABAergic cells were first detected in neural retina of embryos at stage 26, which showed a neuroepithelial appearance without any layering. In stages 27-29 the retina showed similar organization but the number of neuroblastic GABAergic cells increased. When layering became apparent in the central retina (stage-30 embryos), GABAergic cells mainly appeared organized in the outer and inner retina, and GABAergic processes and fibres were seen in the primordial inner plexiform layer (IPL), optic fibre layer and optic nerve stalk. In stage-32 embryos, layering was completed in the central retina, where immunoreactivity appeared in perikarya of the horizontal cell layer, inner nuclear layer and ganglion cell layer, and in numerous processes coursing in the IPL, optic fibre layer and optic nerve. From stage 32 to hatching (stage 34), the layered retina extends from centre-to-periphery, recapitulating that observed in the central retina at earlier stages. In adults, GABA/GAD immunoreactivity disappears from the horizontal cell layer except in the marginal retina. Our results indicate that the source of GABA in the shark retina can be explained by its synthesis by GAD. Such synthesis precedes layering and synaptogenesis, thus supporting a developmental role for GABA in addition to act as neurotransmitter and neuromodulator.

The segmental organization of the developing shark brain based on neurochemical markers, with special attention to the prosencephalon.

Brain regionalization has been extensively studied in tetrapods, teleosts and cyclostomes. In contrast, it has not been investigated in elasmobranchs, despite their key phylogenetic position to understand brain evolution in jawed vertebrates. In this study we provide a schematic view of the segmental pattern of the developing shark brain based on mapping of the expression of Pax6 and neurochemical markers such as calretinin, tyrosine hydroxylase, serotonin, and glutamic acid decarboxylase. By correlating the cytoarchitectonic limits with the specific location of these markers, we identify transverse and longitudinal boundaries and domains, which suggest a segmental pattern, reminiscent of the one described in other vertebrates. Taken together, these data provide an initial scheme, which will be further tested and refined using a broader range of genetic markers involved in patterning and differentiation.

Tangentially migrating GABAergic cells of subpallial origin invade massively the pallium in developing sharks.

We studied the development of the GABAergic system in the telencephalon of the dogfish Scyliorhinus canicula using GABA and glutamate decarboxylase (GAD) immunocytochemistry. The earliest GABA-expressing cells appeared in the basal telencephalon (subpallium) of stage 24 embryos. Shortly after, the subpallium showed abundant GABA-expressing neuroblasts near the meningeal surface or migrating radially in the neuroepithelium. The limit between the GABA-expressing region and the remainder of the telencephalon (pallium) was sharp and coincides with the pallial/subpallial boundary. At stage 28, GABA-expressing cells with the morphology of tangentially migrating cells (showing a thick growth cone-like leading process) migrate from a dome-shaped protrusion of the lateral subpallium and extended laterally and rostrodorsally into the pallium following either a superficial route or coursing periventricularly. At later stages, abundant GABA-expressing cells were seen in various pallial regions and strings of GABA-expressing cells, possibly migrating, were also noted. The colonization of the dogfish pallium by GABA-expressing cells, originating from the subpallium, is strongly reminiscent of the palliopetal tangential migrations of GABA-expressing cells demonstrated in the telencephalon of mammals and follows similar routes. These results strongly suggest that tangential migrations of GABA-expressing cells appeared very early in vertebrate forebrain evolution.

Development of the cerebellar body in sharks: spatiotemporal relations of Pax6 expression, cell proliferation and differentiation.

We have studied the patterns of cell proliferation, regional organization and differentiation in the cerebellar body of embryos and juveniles of two shark species by immunohistochemistry with antibodies against proliferating cell nuclear antigen (PCNA), Pax6, reelin (RELN), GABA, glutamic acid decarboxylase (GAD) and calretinin (CR). The organization of Pax6-expressing cells was also studied by in situ hybridization. Our results reveal that a transient secondary matrix zone, the external germinal layer, is formed in sharks at early stages of cerebellar development and is the source of the earliest Pax6-expressing (granule) cells. Later in development, new granule Pax6-expressing cells arise from medial proliferation zones and accumulate medially in the granular eminences. The GABAergic components appear very early, and show clear regional differences. The medial proliferation zones remain active even in adults. Taken together, the proliferation and differentiation markers used in the present study highlight striking similarities during development between the cerebellar body of elasmobranchs and the cerebella of tetrapods. These results show the importance of elasmobranch models to reconstruct the evolutionary developmental history of the vertebrate cerebellum.

Morphogenesis in the retina of a slow-developing teleost: emergence of the GABAergic system in relation to cell proliferation and differentiation.

Gamma-aminobutyric acid (GABA) has been implicated in cell proliferation and differentiation during development. In the present study, immunohistochemical techniques were used to investigate the development of the GABAergic system in the retina of the trout and its relation to markers of differentiation [calretinin (CR), and tyrosine hydroxylase (TH)]. The expression of Pax6, an eye-patterning protein involved in the proliferation and emergence of specific retinal cell types, was also studied. Retinal layering was observed to begin centrally in prehatching embryos, as the first GABAergic cells appeared in the ganglion cell layer (GCL) and inner part of the inner nuclear layer (INL). At hatching, GABAergic cells were also observed in the horizontal cell layer (HCL). In alevins, GABAergic cells and processes spread laterally following retinal growth although they did not invade neuroblastic retinal regions. CR- and Pax6-immunoreactive (ir) cells were first seen in the GCL and the inner part of the INL, whereas sparse TH-ir cells appeared in the INL. In juveniles, GABAergic cells were observed in the GCL, inner part of the INL and HCL, whereas CR-ir cells spread to the outer part of the INL and HCL. A subset of CR-ir in the GCL and of Pax6-ir cells in the GCL and INL showed colocalization with GABAergic markers. This study provides further comparative knowledge about the development of GABAergic system of the retina in teleosts and shows differences and similarities with that reported in fast-developing species such as zebrafish, in which retinal expression of GABA was transient in some populations.