Cross species analysis of Prominin reveals a conserved cellular role in invertebrate and vertebrate photoreceptor cells

The two fundamental types of photoreceptor cells have evolved unique structures to expand the apical membrane to accommodate the phototransduction machinery, exemplified by the cilia-based outer segment of the vertebrate photoreceptor cell and the microvilli-based rhabdomere of the invertebrate photoreceptor. The morphogenesis of these compartments is integral for photoreceptor cell integrity and function. However, little is known about the elementary cellular and molecular mechanisms required to generate these compartments. Here we investigate whether a conserved cellular mechanism exists to create the phototransduction compartments by examining the functional role of a photoreceptor protein common to both rhabdomeric and ciliated photoreceptor cells, Prominin. First and foremost we demonstrate that the physiological role of Prominin is conserved between rhabdomeric and ciliated photoreceptor cells. Human Prominin1 is not only capable of rescuing the corresponding rhabdomeric Drosophila prominin mutation but also demonstrates a conserved genetic interaction with a second photoreceptor protein Eyes Shut. Furthermore, we demonstrate the Prominin homologs in vertebrate and invertebrate photoreceptors require the same structural features and post-translational modifications for function. Moreover, expression of mutant human Prominin1, associated with autosomal dominant retinal degeneration, in rhabdomeric photoreceptor cells disrupts morphogenesis in ways paralleling retinal degeneration seen in ciliated photoreceptors. Taken together, our results suggest the existence of an ancestral Prominin-directed cellular mechanism to create and model the apical membranes of the two fundamental types of photoreceptor cells into their respective phototransduction compartments.     

Selective loss of RPGRIP1-dependent ciliary targeting of NPHP4, RPGR and SDCCAG8 underlies the degeneration of photoreceptor neurons

The retinitis pigmentosa GTPase regulator (RPGR) and nephrocystin-4 (NPHP4) comprise two key partners of the assembly complex of the RPGR-interacting protein 1 (RPGRIP1). Mutations in RPGR and NPHP4 are linked to severe multisystemic diseases with strong retinal involvement of photoreceptor neurons, whereas those in RPGRIP1 cause the fulminant photoreceptor dystrophy, Leber congenital amaurosis (LCA). Further, mutations in Rpgrip1 and Nphp4 suppress the elaboration of the outer segment compartment of photoreceptor neurons by elusive mechanisms, the understanding of which has critical implications in uncovering the pathogenesis of syndromic retinal dystrophies. Here we show RPGRIP1 localizes to the photoreceptor connecting cilium (CC) distally to the centriole/basal body marker, centrin-2 and the ciliary marker, acetylated-α-tubulin. NPHP4 abuts proximally RPGRIP1, RPGR and the serologically defined colon cancer antigen-8 (SDCCAG8), a protein thought to partake in the RPGRIP1 interactome and implicated also in retinal–renal ciliopathies. Ultrastructurally, RPGRIP1 localizes exclusively throughout the photoreceptor CC and Rpgrip1^nmf247 photoreceptors present shorter cilia with a ruffled membrane. Strikingly, Rpgrip1^nmf247 mice without RPGRIP1 expression lack NPHP4 and RPGR in photoreceptor cilia, whereas the SDCCAG8 and acetylated-α-tubulin ciliary localizations are strongly decreased, even though the NPHP4 and SDCCAG8 expression levels are unaffected and those of acetylated-α-tubulin and γ-tubulin are upregulated. Further, RPGRIP1 loss in photoreceptors shifts the subcellular partitioning of SDCCAG8 and NPHP4 to the membrane fraction associated to the endoplasmic reticulum. Conversely, the ciliary localization of these proteins is unaffected in glomeruli or tubular kidney cells of Rpgrip1^nmf247, but NPHP4 is downregulated developmentally and selectively in kidney cortex. Hence, RPGRIP1 presents cell type-dependent pathological effects crucial to the ciliary targeting and subcellular partitioning of NPHP4, RPGR and SDCCAG8, and acetylation of ciliary α-tubulin or its ciliary targeting, selectively in photoreceptors, but not kidney cells, and these pathological effects underlie photoreceptor degeneration and LCA.     

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background  

Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.     

The homodimeric kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment development

Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.     

The polarity protein Pard3 is required for centrosome positioning during neurulation

Microtubules are essential regulators of cell polarity, architecture and motility. The organization of the microtubule network is context-specific. In non-polarized cells, microtubules are anchored to the centrosome and form radial arrays. In most epithelial cells, microtubules are noncentrosomal, align along the apico-basal axis and the centrosome templates a cilium. It follows that cells undergoing mesenchyme-to-epithelium transitions must reorganize their microtubule network extensively, yet little is understood about how this process is orchestrated. In particular, the pathways regulating the apical positioning of the centrosome are unknown, a central question given the role of cilia in fluid propulsion, sensation and signaling. In zebrafish, neural progenitors undergo progressive epithelialization during neurulation, and thus provide a convenient in vivo cellular context in which to address this question. We demonstrate here that the microtubule cytoskeleton gradually transitions from a radial to linear organization during neurulation and that microtubules function in conjunction with the polarity protein Pard3 to mediate centrosome positioning. Pard3 depletion results in hydrocephalus, a defect often associated with abnormal cerebrospinal fluid flow that has been linked to cilia defects. These findings thus bring to focus cellular events occurring during neurulation and reveal novel molecular mechanisms implicated in centrosome positioning.     

The zebrafish Pard3 ortholog is required for separation of the eye fields and retinal lamination

The vertebrate retina develops from a sheet of neuroepithelial cells. Because adherens and tight junctions are critical for epithelial and neuronal differentiation in a variety of eukaryotic systems, we examined the role of Par-3, a PDZ scaffold protein that is critical in cellular membrane junction formation. We cloned the zebrafish Par-3 ortholog (pard3), which encodes two Pard3 proteins (150 and 180 kDa) that differ in their carboxyl-terminus. Immunohistochemistry revealed that Pard3 localized to the apical region of the retinal and brain neuroepithelium, partially overlapping the adherens junction-associated actin bundles. After retinal lamination, the Pard3 protein was restricted to the outer limiting membrane and the outer and inner plexiform layers in the retina. Reducing Pard3 expression with antisense morpholinos caused loss of the retinal pigmented epithelia, disruption of retinal lamination, and cell death in the ventral diencephalon, which resulted in cyclopia. Overexpressing Pard3 by injection of wild-type pard3 mRNA resulted in cyclopia and eyeless embryos. Thus, Pard3 plays a critical role in the origination and separation of zebrafish eye fields and retinal lamination.     

Rhodopsin transport in the membrane of the connecting cilium of mammalian photoreceptor cells

The transport of the photopigment rhodopsin from the inner segment to the photosensitive outer segment of vertebrate photoreceptor cells has been one of the main remaining mysteries in photoreceptor cell biology. Because of the lack of any direct evidence for the pathway through the photoreceptor cilium, alternative extracellular pathways have been proposed. Our primary aim in the present study was to resolve rhodopsin trafficking from the inner to the outer segment. We demonstrate, predominantly by high-sensitive immunoelectron microscopy, that rhodopsin is also densely packed in the membrane of the photoreceptor connecting cilium. Present prominent labeling of rhodopsin in the ciliary membrane provides the first striking evidence that rhodopsin is translocated from the inner segment to the outer segment of wild type photoreceptors via the ciliary membrane. At the ciliary membrane rhodopsin co-localizes with the unconventional myosin VIIa, the product of human Usher syndrome 1B gene. Furthermore, axonemal actin was identified in the photoreceptor cilium, which is spatially co-localized with myosin VIIa and opsin. This actin cytoskeleton of the cilium may provide the structural bases for myosin VIIa-linked ciliary trafficking of membrane components, including rhodopsin.     

Primary cilia in retinal pigment epithelium development and diseases

Retinal pigment epithelium (RPE) is a highly polarized epithelial monolayer lying between the photoreceptor layer and the Bruch membrane. It is essential for vision through participating in many critical activities, including phagocytosis of photoreceptor outer segments, recycling the visual cycle-related compounds, forming a barrier to control the transport of nutrients, ions, and water, and the removal of waste. Primary cilia are conservatively present in almost all the vertebrate cells and acts as a sensory organelle to control tissue development and homeostasis maintenance. Numerous studies reveal that abnormalities in RPE lead to various retinal diseases, such as age-related macular degeneration and diabetic macular oedema, but the mechanism of primary cilia in these physiological and pathological activities remains to be elucidated. Herein, we summarize the functions of primary cilia in the RPE development and the mutations of ciliary genes identified in RPE-related diseases. By highlighting the significance of primary cilia in regulating the physiological and pathological processes of RPE, we aim to provide novel insights for the treatment of RPE-related retinal diseases.     

The pineal gland in wild-type and two zebrafish mutants with retinal defects

Light and transmission electron microscopy were used to characterize the ultrastructural features of the pineal glands of wild-type and two mutant zebrafish strains that have retinal defects. Particular attention was given to the pineal photoreceptors. Photoreceptors in the pineal gland appear quite similar to retinal cone photoreceptors, having many of the same structural characteristics including outer segment disk membranes often confluent with the plasma membrane, calycal processes surrounding the outer segments, and classic connecting cilia. The pineal photoreceptor terminals differ from photoreceptor terminals in the retina in that they have short synaptic ribbons and make dyad synapses which may or may not be invaginated. Pineal photoreceptors in two zebrafish mutants with abnormal retinal photoreceptors were also studied. Pineal photoreceptors in the niezerka (nie) mutant degenerate, as they do in the retina, indicating that pineal and retinal photoreceptors share at least some genes. However, the synaptic terminals of no optokinetic response c (nrc) pineal photoreceptors are normal, suggesting that this mutation is specific to the retina.     

NINL and DZANK1 Co-function in Vesicle Transport and Are Essential for Photoreceptor Development in Zebrafish

Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.     

Morphology of the pineal organ in the salamander Ensatina eschscholtzi

The pineal organ of Ensatina eschscholtzi, a terrestrial and secretive species of salamander of the family Plethodontidae, is a photoreceptive structure lying on the dorsal surface of the diencephalon. The pineal is flattened with a broad lumen and consists of three cell types: photoreceptors, supportive cells, and neurons. Pineal photoreceptors are typical vertebrate photoreceptors and possess outer segment formations which, however, are frequently contorted and disorganized. Sloughing of apical portions of outer segments and vesiculation along the lateral edges of outer segment membrane disks are consistently observed and presumed to represent mechanisms of outer segment membrane recycling. Photoreceptors have basal processes which synapse with neural dendrites. Synapses between photoreceptor basal processes are occasionally observed. All synapses are characterized by synaptic ribbon structures of variable number, size, and configuration. Dense-core vesicles are occasionally observed mingled with clear synaptic vesicles within photoreceptor basal processes. Supportive cells within the pineal function in phagocytosis and recycling of shed outer segment membrane material, and neurons are localized at the lateral margins of the organ. The latter send axons into the ipsilateral side of the dorsal diencephalon. The pineal organ of Ensatina shows marked variation in overall size (cell total), cell type proportions, absolute neuron number, and ratio of photoreceptor number to neuron number for individual pineals. None of these morphological parameters is correlated with body size, sex, or season, and it is assumed that such variability represents significant variation in photosensory capabilities. It is suggested that the pineal organ of Ensatina is a partially degenerate photoreceptive structure.     

Ultrastructure of epidermal eyespots of Microstomum lineare (Turbellaria,macrostomida)

Summary The eyespots of Microstomum lineare were studied by electron microscopy, light microscopy, and fluorescence microscopy. Each eyespot consists of two ciliary photoreceptor cells shielded by pigment cells and additional sensory cells. The photoreceptor cells are characterized by a distal intracellular cavity lined with 50–100 interwoven cilia. The other sensory cells are of two ultrastructurally different types, one with long cilia predominating and the other with balloonlike cilia. The pigment cells, which envelop processes of the sensory cells, contain pigment vacuoles varying in size and content and give a bright red fluorescence by the Falck-Hillarp method. The eyespots are suggested to perform a dual function as photoreceptors and chemoreceptors. The evolutionary significance of ciliary photoreceptors in Turbellaria is discussed.     

Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors.

Methodology/Principal Findings

Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc''s gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors.

Conclusions/Significance

In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

Cephalochordate melanopsin: evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells

Animal photoreceptor cells can be classified into two distinct types, depending on whether the photopigment is borne on the membrane of a modified cilium (ciliary type) or apical microvilli (rhabdomeric type) [1]. Ciliary photoreceptors are well known as vertebrate rods and cones and are also found in several invertebrates. The rhabdomeric photoreceptor, in contrast, is a predominant type of invertebrate visual cell, but morphologically identifiable rhabdomeric photoreceptors have never been found in vertebrates. It is hypothesized that the rhabdomeric photoreceptor cell had evolved to be the photosensitive retinal ganglion cell for the vertebrate circadian photoentrainment [2, 3 and 4] owing to the fact that some molecules involved in cell differentiation are common among them [5]. We focused on the cephalochordate amphioxus because it is the closest living invertebrate to the vertebrates, and interestingly, it has rhabdomeric photoreceptor cells for putative nonvisual functions [6]. Here, we show that the amphioxus homolog of melanopsin [7, 8 and 9], the circadian photopigment in the photosensitive retinal ganglion cells of vertebrates, is expressed in the rhabdomeric photoreceptor cells of the amphioxus and that its biochemical and photochemical properties, not just its primary structure, are considerably similar to those of the visual rhodopsins in the rhabdomeric photoreceptor cells of higher invertebrates. The cephalochordate rhabdomeric photoreceptor represents an evolutionary link between the invertebrate visual photoreceptor and the vertebrate circadian photoreceptor.     

Eye evolution at high resolution: The neuron as a unit of homology

Based on differences in morphology, photoreceptor-type usage and lens composition it has been proposed that complex eyes have evolved independently many times. The remarkable observation that different eye types rely on a conserved network of genes (including Pax6/eyeless) for their formation has led to the revised proposal that disparate complex eye types have evolved from a shared and simpler prototype. Did this ancestral eye already contain the neural circuitry required for image processing? And what were the evolutionary events that led to the formation of complex visual systems, such as those found in vertebrates and insects? The recent identification of unexpected cell-type homologies between neurons in the vertebrate and Drosophila visual systems has led to two proposed models for the evolution of complex visual systems from a simple prototype. The first, as an extension of the finding that the neurons of the vertebrate retina share homologies with both insect (rhabdomeric) and vertebrate (ciliary) photoreceptor cell types, suggests that the vertebrate retina is a composite structure, made up of neurons that have evolved from two spatially separate ancestral photoreceptor populations. The second model, based largely on the conserved role for the Vsx homeobox genes in photoreceptor-target neuron development, suggests that the last common ancestor of vertebrates and flies already possessed a relatively sophisticated visual system that contained a mixture of rhabdomeric and ciliary photoreceptors as well as their first- and second-order target neurons. The vertebrate retina and fly visual system would have subsequently evolved by elaborating on this ancestral neural circuit. Here we present evidence for these two cell-type homology-based models and discuss their implications.     

Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase–activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone–rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca²⁺GCAPs but also elevates this isozyme''s resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S⁺). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S⁺ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca²⁺ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone–rod dystrophy 6 pathology.     

Vertebrate features revealed in the rudimentary eye of the Pacific hagfish (Eptatretus stoutii)

Hagfish eyes are markedly basic compared to the eyes of other vertebrates, lacking a pigmented epithelium, a lens and a retinal architecture built of three cell layers: the photoreceptors, interneurons and ganglion cells. Concomitant with hagfish belonging to the earliest-branching vertebrate group (the jawless Agnathans), this lack of derived characters has prompted competing interpretations that hagfish eyes represent either a transitional form in the early evolution of vertebrate vision, or a regression from a previously elaborate organ. Here, we show the hagfish retina is not extensively degenerating during its ontogeny, but instead grows throughout life via a recognizable PAX6+ ciliary marginal zone. The retina has a distinct layer of photoreceptor cells that appear to homogeneously express a single opsin of the RH1 rod opsin class. The epithelium that encompasses these photoreceptors is striking because it lacks the melanin pigment that is universally associated with animal vision; notwithstanding, we suggest this epithelium is a homologue of gnathosome retinal pigment epithelium (RPE) based on its robust expression of RPE65 and its engulfment of photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in its wiring, despite lacking a morphologically distinct layer of interneurons: multiple populations of cells exist in the hagfish inner retina and subsets of these express markers of vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine interpretations of early vertebrate visual system evolution.     

oko meduzy and related crumbs genes are determinants of apical cell features in the vertebrate embryo

BACKGROUND: Polarity is an essential attribute of most eukaryotic cells. One of the most prominent features of cell polarity in many tissues is the subdivision of cell membrane into apical and basolateral compartments by a belt of cell junctions. The proper formation of this subdivision is of key importance. In sensory cells, for example, the apical membrane compartment differentiates specialized structures responsible for the detection of visual, auditory, and olfactory stimuli. In other tissues, apical specializations are responsible for the propagation of fluid flow. Despite its importance, the role of genetic determinants of apico-basal polarity in vertebrate embryogenesis remains poorly investigated. RESULTS: We show that zebrafish oko meduzy (ome) locus encodes a crumbs gene homolog, essential for the proper apico-basal polarity of neural tube epithelia. Two ome paralogs, crb2b and crb3a, promote the formation of apical cell features: photoreceptor inner segments and cilia in renal and auditory systems. The motility of cilia is defective following the impairment of crb2b function. Apical surface defects in ome- and crb2b-deficient animals are associated with profound disorganization of neuronal architecture and with the formation of pronephric cysts, respectively. Unexpectedly, despite differences in their structure and expression patterns, crumbs genes are, at least partially, functionally interchangeable. CONCLUSIONS: ome and related crumbs genes are necessary for the formation of gross morphological features in several organs, including the CNS and the renal system. On the cellular level, crumbs genes regulate the formation of both ciliary and nonciliary apical membrane compartment.     

The fine structure of the lens and photoreceptors of Nereis virens

Summary The cell membrane in the distal region of the photoreceptors of Nereis virens is formed into large numbers of tubular microvilli which interdigitate with those of neighbouring photoreceptors. No traces of cilia or basal bodies have been found, but a prominent axial filament with a repeat pattern of 660 Å extends from the upper to the lower parts of the cell. Large numbers of mitochondria are found in the outer segment of the photoreceptors around the bases of the microvilli. As it passes through the pigment layer the photoreceptor is invaded by finger-like intrusions of the pigment cells which serve to prevent light entering the eye from behind. The lens consists of fine filaments or rod-shaped bodies secreted by the pigment cells, which pass to the lens up narrow pillars between the outer segments of the photoreceptors. Attention is drawn to the evolutionary parallel between the shadow reflex of Branchiomma and the off responses of the photoreceptors in the distal retina of Pecten, both animals being exceptional within their phyla in having photoreceptors of the ciliary type.