Sonic hedgehog antagonists reduce size and alter patterning of the frog inner ear.

Sonic hedgehog (Shh) signaling plays a major role in vertebrate development, from regulation of proliferation to the patterning of various organs. In amniotes, Shh affects dorsoventral patterning in the inner ear but affects anteroposterior patterning in teleost ears. It remains unknown how altered function of Shh relates to morphogenetic changes that coincide with the evolution of limbs and novel auditory organs in the ear. In this study, we used the tetrapod, Xenopus laevis, to test how increasing concentrations of the Shh signal pathway antagonist, Vismodegib, affects ear development. Vismodegib treatment dose dependently alters the development of the ear, hypaxial muscle, and indirectly the Mauthner cell through its interaction with the inner ear afferents. Together, these phenotypes have an effect on escape response. The altered Mauthner cell likely contributes to the increased time to respond to a stimulus. In addition, the increased hypaxial muscle in the trunk likely contributes to the subtle change in animal C-start flexion angle. In the ear, Vismodegib treatment results in decreasing segregation between the gravistatic sensory epithelia as the concentration of Vismodegib increases. Furthermore, at higher doses, there is a loss of the horizontal canal but no enantiomorphic transformation, as in bony fish lacking Shh. Like in amniotes, Shh signaling in frogs affects dorsoventral patterning in the ear, suggesting that auditory sensory evolution in sarcopterygians/tetrapods evolved with a shift of Shh function in axis specification. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1385-1400, 2017.

Differential expression of endothelial nutrient transporters (MCT1 and GLUT1) in the developing eyes of mice.

The blood-brain barrier in the neonatal brain expresses the monocarboxylate transporter (MCT)-1 rather than the glucose transporter (GLUT)-1, due to the special energy supply during the suckling period. The hyaloid vascular system, consisting of the vasa hyaloidea propria and tunica vasculosa lentis, is a temporary vasculature present only during the early development of mammalian eyes and later regresses. Although the ocular vasculature manifests such a unique developmental process, no information is available concerning the expression of endothelial nutrient transporters in the developing eye. The present immunohistochemical study using whole mount preparations of murine eyes found that the hyaloid vascular system predominantly expressed GLUT1 in the endothelium, in contrast to the brain endothelium. Characteristically, the endothelium in peripheral regions of the neonatal hyaloid vessels displayed a mosaic pattern of MCT1-immunoreactive cells scattered within the GLUT1-expressing endothelium. The proper retinal vessels first developed by sprouting angiogenesis endowed with filopodia, which were absolutely free from the immunoreactivities of GLUT1 and MCT1. The remodeling retinal capillary networks and veins in the surface layer of the retina mainly expressed MCT1 until the weaning period. Immunostaining of MCT1 in the retina revealed fine radicular processes projecting from the endothelium, differing from the MCT1-immunonegative filopodia. These findings suggest that the expression of nutrient transporters in the ocular blood vessels is differentially regulated at a cellular level and that the neonatal eyes provide an interesting model for research on nutrient transporters in the endothelium.

Chloride intracellular channel 4 is critical for the epithelial morphogenesis of RPE cells and retinal attachment.

Retinal detachment is a sight-threatening condition. The molecular mechanism underlying the adhesion between the RPE and photoreceptors is poorly understood because the intimate interactions between these two cell types are impossible to model and study in vitro. In this article, we show that chloride intracellular channel 4 (CLIC4) is enriched at apical RPE microvilli, which are interdigitated with the photoreceptor outer segment. We used a novel plasmid-based transfection method to cell-autonomously suppress CLIC4 in RPE in situ. CLIC4 silenced RPE cells exhibited a significant loss of apical microvilli and basal infoldings, reduced retinal adhesion, and epithelial-mesenchymal transition. Ectopically expressing ezrin failed to rescue the morphological changes exerted by CLIC4 silencing. Neural retinas adjacent to the CLIC4-suppressed RPE cells display severe dysplasia. Finally, a high level of aquaporin 1 unexpectedly appeared at the apical surfaces of CLIC4-suppressed RPE cells, together with a concomitant loss of basal surface expression of monocarboxylate transporter MCT3. Our results suggested that CLIC4 plays an important role in RPE-photoreceptor adhesion, perhaps by modulating the activity of cell surface channels/transporters. We propose that these changes may be attributable to subretinal fluid accumulation in our novel retinal detachment animal model.

Microarray analysis of choroid/RPE gene expression in marmoset eyes undergoing changes in ocular growth and refraction.

PURPOSE: Visually guided ocular growth is facilitated by scleral extracellular matrix remodeling at the posterior pole of the eye. Coincident with scleral remodeling, significant changes in choroidal morphology, blood flow, and protein synthesis have been shown to occur in eyes undergoing ocular growth changes. The current study is designed to identify gene expression changes that may occur in the choroid/retinal pigment epithelium (RPE) of marmoset eyes during their compensation for hyperopic defocus as compared to eyes compensating for myopic defocus. METHODS: Total RNA was isolated from choroid/RPE from four common marmosets (Callithrix jacchus) undergoing binocular lens treatment using extended wear soft contact lenses of equal magnitude but opposite sign (+/-5 diopter [D]). After reverse transcription, cDNA was labeled and hybridized to a human oligonucleotide microarray and gene transcript expression profiles were determined. Real-time polymerase chain reaction (PCR) and western blot analysis were used to confirm genes and proteins of interest, respectively. RESULTS: Microarray analyses in choroid/RPE indicated 204 genes were significantly changed in minus lens-treated as compared with plus lens-treated eyes (p<0.05, Student's t-test). Differential choroid/RPE expression of protein tyrosine phosphatase, receptor type, B (PTPRB), transforming growth factor beta-induced (TGFBI), and basic fibroblast growth factor 2 (FGF-2) were confirmed by real-time PCR. TGFBIp was confirmed at the protein level by western blot analysis in marmoset and human cornea, choroid/RPE, and sclera. CONCLUSIONS: The present study demonstrated that significant gene expression changes occur in the marmoset choroid/RPE during visually guided ocular growth. The identification of novel candidate genes in choroid/RPE of marmoset eyes actively accelerating or decelerating their rates of ocular elongation may elucidate the choroidal response during the regulation of postnatal ocular growth and may lead to the identification of choroid/RPE signaling molecules that participate in scleral remodeling.

Developmental basis of nanophthalmos: MFRP Is required for both prenatal ocular growth and postnatal emmetropization.

BACKGROUND: Nanophthalmos is a genetic disorder characterized by very small, hyperopic eyes that are without gross structural defects. Recessive nanophthalmos is caused by severe mutations in the MFRP gene, which encodes a Frizzled-related transmembrane protein that is selectively expressed in the retinal pigment epithelium (RPE) and ciliary body. RESULTS: For two MFRP -/- adults, we have obtained records of refraction that begin in early childhood. At the age of 6 months, one patient's eyes already had a refractive error of +12.25 D, and over the next 20 years this slowly increased to +17.50 D. Adults homozygous for null mutations in MFRP have eyes with axial lengths shorter than those of normal newborns. Furthermore, the unusually high curvature of their corneas is consistent with eyes that had been smaller than normal during late fetal development. MFRP protein was first detected at 14 weeks of gestation, when it was restricted to the posterior pole RPE. By 20 weeks gestation, MFRP expression had spread laterally, and was found throughout the RPE. MFRP protein was detected in both posterior and lateral RPE of the adult eye. CONCLUSIONS: Embryonic function of the MFRP gene appears necessary for the eye to reach its full size at birth. Its onset of expression in the RPE during mid-gestation suggests that MFRP does not participate in early formation of the optic cup, and is consistent with a role in later growth and development of the eye. Patients without MFRP gene function exhibit no correction of refractive error during childhood, which suggests that this gene is essential for emmetropization, a complex process by which vision regulates axial growth of the eye.

Iris development in vertebrates; genetic and molecular considerations.

The iris plays a key role in visual function. It regulates the amount of light entering the eye and falling on the retina and also operates in focal adjustment of closer objects. The iris is involved in circulation of the aqueous humor and hence functions in regulation of intraocular pressure. Intriguingly, iris pigmented cells possess the ability to transdifferentiate into different ocular cell types of retinal pigmented epithelium, photoreceptors and lens cells. Thus, the iris is considered a potential source for cell-replacement therapies. During embryogenesis, the iris arises from both the optic cup and the periocular mesenchyme. Its interesting mode of development includes specification of the peripheral optic cup to a non-neuronal fate, migration of cells from the surrounding periocular mesenchyme and an atypical formation of smooth muscles from the neuroectoderm. This manner of development raises some interesting general topics concerning the early patterning of the neuroectoderm, the specification and differentiation of diverse cell types and the interactions between intrinsic and extrinsic factors in the process of organogenesis. In this review, we discuss iris anatomy and development, describe major pathologies of the iris and their molecular etiology and finally summarize the recent findings on genes and signaling pathways that are involved in iris development.

Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye.

In the developing eye of Drosophila, the EGFR and Notch pathways integrate in a sequential, followed by a combinatorial, manner in the specification of cone-cell fate. Here, we demonstrate that the specification of primary pigment cells requires the reiterative use of the sequential integration between the EGFR and Notch pathways to regulate the spatiotemporal expression of Delta in pupal cone cells. The Notch signal from the cone cells then functions in the direct specification of primary pigment-cell fate. EGFR requirement in this process occurs indirectly through the regulation of Delta expression. Combined with previous work, these data show that unique combinations of only two pathways--Notch and EGFR--can specify at least five different cell types within the Drosophila eye.

TAM receptor function in the retinal pigment epithelium.

The TAM receptor tyrosine kinase Mer is expressed by cells of the retinal pigment epithelium (RPE), and genetic studies have demonstrated that Mer is essential for RPE function. RPE cells that lack Mer exhibit a severely compromised ability to phagocytose the distal ends of photoreceptor (PR) outer segments, which leads to the complete postnatal degeneration of photoreceptors and to blindness. Although in vitro experiments have implicated Gas6 as the critical TAM ligand for this process, we find that Gas6 mutant mice have a histologically intact retina with no photoreceptor degeneration. We further find that, in addition to Mer, RPE cells also express another TAM receptor--Tyro 3--and that both of these receptors are instead activated independently by the Gas6-related ligand Protein S. This protein is also expressed by RPE cells. Finally, we demonstrate that loss of Mer function is accompanied by a substantial down-regulation in Tyro 3 as well. These observations indicate that both Mer and Tyro 3 act in mouse RPE cells and suggest that their biologically relevant ligand in these cells is Protein S.

p21 controls patterning but not homologous recombination in RPE development.

p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53.

Expression and localization of bestrophin during normal mouse development.

PURPOSE: Best macular dystrophy is caused by mutations in the VMD2 gene, which encodes the protein bestrophin. The purpose of this study was to determine the postnatal onset of expression of bestrophin mRNA and protein in the mouse retinal pigment epithelium (RPE). METHODS: Rabbit anti-mouse bestrophin polyclonal antisera designated Pab-003 was generated against a peptide derived from the C terminus of mouse bestrophin and characterized by Western blot and immunofluorescence staining of transfected cells. Expression of bestrophin mRNA during ocular development was studied with quantitative PCR. Bestrophin protein expression in the developing eye was observed by using immunohistochemistry. The onset of mouse phototransduction was determined by conventional electroretinography (ERG). RESULTS: Bestrophin mRNA was detected at embryonic day 15 in whole mouse eyes by RT-PCR. Real-time quantification of mouse bestrophin mRNA levels indicated that the highest levels of mRNA were present in the early postnatal period. In contrast, bestrophin in the RPE was first detected at postnatal day (P)10 by immunohistochemistry. Phototransduction, as determined by the presence of an ERG a-wave, was first observed at P10. CONCLUSIONS: The results of this study show that mouse bestrophin mRNA is present in the eye during embryogenesis and significantly precedes the onset of bestrophin protein expression at P10. The appearance of bestrophin in the basolateral plasma membrane of the RPE is coincident with the first detectable ERG a-wave. Because bestrophin is thought to play a role in generating the light peak, a late response of the ERG, these data support a temporal role for bestrophin in RPE responses to light. Furthermore, bestrophin protein appears to be a very late marker of RPE differentiation and to be subject to strong translational control.

Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6.

The transcription factors Pax2 and Pax6 are co-expressed in the entire optic vesicle (OV) prior and concomitant with the establishment of distinct neuroretinal, retinal, pigmented-epithelial and optic-stalk progenitor domains, suggesting redundant functions during retinal determination. Pax2; Pax6 compound mutants display a dose-dependent reduction in the expression of the melanocyte determinant Mitf, accompanied by transdifferentiation of retinal pigmented epithelium (RPE) into neuroretina (NR) in Pax2(-/-); Pax6(+/-) embryos, which strongly resembles the phenotype of Mitf-null mutants. In Pax2(-/-); Pax6(-/-) OVs Mitf fails to be expressed and NR markers occupy the area that usually represents the Mitf(+) RPE domain. Furthermore, both, Pax2 and Pax6 bind to and activate a MITF RPE-promoter element in vitro, whereas prolonged expression of Pax6 in the Pax2-positive optic stalk leads to ectopic Mitf expression and RPE differentiation in vivo. Together, these results demonstrate that the redundant activities of Pax2 and Pax6 direct the determination of RPE, potentially by directly controlling the expression of RPE determinants.

Expression and activation of STAT proteins during mouse retina development.

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis.

A novel function for Hedgehog signalling in retinal pigment epithelium differentiation.

Sonic hedgehog is involved in eye field separation along the proximodistal axis. We show that Hh signalling continues to be important in defining aspects of the proximodistal axis as the optic vesicle and optic cup mature. We show that two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. We show that this zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, we activated the pathway by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium.

Electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors.

Electron spin resonance (ESR) examinations of human retinal pigment epithelium melanosomes isolated from eyes of young and old donors were carried out. The examined ESR signal was a single line, which is characteristic for free radicals of eumelanin o-semiquinones. The content of free radicals related to melanosomes dry weight for samples from older donors (ages over 45 years) were higher than for sample from younger donors (between 14 and 22 years). Simultaneously, the content of free radicals calculated for one melanosome is constant and does not depend on age. The homogeneous broadening of the recorded ESR lines shows that there are no isolated spin packets in all investigated melanin samples. Slow spin-lattice (T1 approximately 10(-5) s) and fast spin-spin (T2 approximately 10(-8) s) relaxation processes occur in these samples. Saturation of the ESR lines at low microwave power was measured. High concentration of free radicals in melanosome samples was responsible for the fast spin-spin relaxation process.

Transplanted sheets of human retina and retinal pigment epithelium develop normally in nude rats.

This study investigated whether transplanted sheets of human fetal retina together with its retinal pigment epithelium (RPE) could develop and maintain their cytoarchitecture after long survival times. Transplant recipients were nine albino athymic nu/nu rats with a normal retina. The donor tissue was dissected from fetuses of 12-17 weeks gestational age. Transplants were analyzed at 5-12 months after surgery by light and electron microscopy, and immunohistochemistry with various antibodies specific for rhodopsin, S-antigen, transducin, neurofilament and synaptophysin. In 4 of 11 transplants, the RPE stayed as a monolayer sheet and supported the development of the retinal sheet with a normal lamination, including photoreceptor inner and outer segments. Cones and rods in the organized transplants were labeled with different photoreceptor markers. Inner and outer plexiform layers, containing cone pedicles and rods spherules, were immunoreactive for synaptophysin. As the recipients had a normal retina, transplant/host integration was not expected. However, at the transplant/host interface, there were sometimes areas without glial barriers, and neurofilament-containing processes could be observed crossing between transplant and host. In other, more disorganized transplants, the RPE cells were partially dispersed or clumped together in clusters. Such transplants developed photoreceptors in rosettes, often with inner and outer segments. In conclusion, sheets of human fetal retina transplanted together with its RPE to the subretinal space of nude rats can develop and maintain perfectly laminated transplants after long survival times, indicating the potential of applying cotransplantation to human patients with retinal diseases.

Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium.

PURPOSE: Identification of binding partners for ezrin, an actin-binding protein crucial for morphogenesis of apical microvilli and basolateral infoldings in RPE cells. METHODS: Rat eyes, rat primary RPE, the rat RPE-J cell line, and a clonal line of RPE-J cells transfected with human ezrin cDNA were analyzed by immunofluorescence microscopy and immunoblot. Immunofluorescence localization of two ezrin-binding proteins was performed in cryosections of rat eyes of various ages and in monolayers extracted with the detergent Triton X-100 and fixed in paraformaldehyde. The interaction of both proteins with ezrin and gluthathione-S-transferase (GST)-ezrin fusion proteins was analyzed by SDS-PAGE and immunoblot. RESULTS: Immunofluorescence microscopy of adult rat eyes detected a polarized distribution of ERM (ezrin, radixin, and moesin)-binding phosphoprotein of 50 kDa (EBP50) at the apical microvilli and synaptic-associated protein of 97 kDa (SAP97) at the basolateral surface of RPE cells, which overlapped with ezrin. These two PDZ (postsynaptic density protein [PSD-95]/disc large [DLG]-A/ZO-1) domain proteins had a similar polarized distribution and high resistance to detergent extractability, indicative of cytoskeletal association, both in primary cultures of rat RPE and in a clonal RPE-J cell line expressing high levels of transfected ezrin. RPE cell lysates from rat retinas of various postnatal ages revealed increasing levels of EBP50 and SAP97 compared with alphav integrin, a protein expressed at constant adult levels from birth. GST pull-down and immunoprecipitation experiments demonstrated a direct interaction between EBP50 and SAP97 and ezrin. CONCLUSIONS: The data indicate that EBP50 localizes at the apical microvilli, whereas SAP97 localizes at the basolateral surface of RPE cells, probably through a direct interaction with ezrin.

Autofluorescence of melanins induced by ultraviolet radiation and near ultraviolet light. A histochemical and biochemical study.

The induction of autofluorescence of melanins by UV radiation (330-380 nm) and near UV (400-440 nm) light (jointly called UV light) was studied in tissue sections using three commercially available mounting media. Only Immu-Mount (Shandon) was found suitable for this purpose. UV irradiation of melanins in sections mounted in this medium induced strong yellow autofluorescence irrespective of the type of the polymer (eumelanin, neuromelanin, pheomelanin and ochronotic pigment). The phenomenon of autofluorescence induction was also observed with isolated natural and in vitro prepared melanins. It was inhibited by anhydrous conditions, sodium azide and catalase. In parallel experiments, rapid degradation of melanins with an intermediate fluorescent stage was achieved in UV-irradiated sections mounted in media artificially enriched with hydrogen peroxide, or directly in aqueous solutions of H2O2, Na2O2 or HIO4. Oxidations not associated with UV light led to nonfluorogenic breakdown of melanins. These observations indicate that the common mechanism may be an oxidative attack resulting from a concerted action of hydrogen peroxide and UV light leading, through strongly fluorescent intermediates, to a complete bleaching and oxidative breakdown of melanin and melanin-like polymers. Reactive oxygen species (including ozone) are considered to be important reactants in these experiments. Lipopigments differ from melanin-like pigments by their primary autofluorescence, which mostly faded during continuous prolonged irradiation. The only regular exception was melanosis coli pigment, the autofluorescence of which was considerably augmented by UV irradiation. Our results demonstrate a novel type of fluorogen in autofluorescent pigment histochemistry. The implications of the results are discussed especially in the light of the possible presence of melanin-based fluorogens in lipopigments.

Specific Pax-6/microphthalmia transcription factor interactions involve their DNA-binding domains and inhibit transcriptional properties of both proteins.

Pax-6 and microphthalmia transcription factor (Mitf) are required for proper eye development. Pax-6, expressed in both the neuroretina and pigmented retina, has two DNA-binding domains: the paired domain and the homeodomain. Mice homozygous for Pax-6 mutations are anophthalmic. Mitf, a basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factor associated with the onset and maintenance of pigmentation, identifies the retinal pigmented epithelium during eye development. Loss of Mitf function results in the formation of an ectopic neuroretina at the expense of the dorsal retinal pigmented epithelium. In the present study, we investigated the interaction between Pax-6 and Mitf. In transient transfection-expression experiments, we found that transactivating effects of Pax-6 and Mitf on their respective target promoters were strongly inhibited by co-transfection of both transcription factors. This repression was due to direct protein/protein interactions involving both Pax-6 DNA-binding domains and the Mitf b-HLH-LZ domain. These results suggest that Pax-6/Mitf interactions may be critical for retinal pigmented epithelium development.

Fine structure and development of the retina of the grenadier anchovy Coilia nasus (Engraulididae, Clupeiformes).

A study of the morphogenesis of the grenadier anchovy retina was undertaken using light and electron microscopy. Five developmental stages from prelarvae 3 days after fertilization to adult fish were studied. In addition to the general morphology of the eye and retina, special emphasis was given to the development of the photoreceptors and pigment epithelium (PE). The earliest retinae showing structural features indicative of a functioning eye are pure cone retinae composed of rows of alternating long and short cones forming a transient, tesselated pattern. At this stage there is a conventional PE containing melanin. In older stages cone rows are separated by the newly formed rods and by PE wedges filled with diffusely reflecting guanine crystallites. The findings are compared with the retinae of other engraulidids and with the development of teleost retinae in general. Moreover, the observed structural changes are discussed with respect to the photic habitat conditions of these anadromous fish that move between coastal waters, estuary, and river.