The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions.     

Immunolocalization of retinal-binding protein (RALBP)-like protein in compound eye and ocellus of drosophila melanogaster (Diptera : Drosophilidae)

A retinal-binding protein (RALBP)-like protein was found in Drosophila melanogaster (Diptera : Drosophilidae) visual system by immunoelectron microscopy using anti-squid RALBP antibody. Immunoblot analysis showed that RALBP-like protein has a molecular weight of about 120 kDa. It was detected mainly in the central cavity of compound eye ommatidia and in the interphotoreceptor space of the ocellus. The protein was localized in the cytoplasm of photoreceptor cells as well. However, these molecules were not detected in the cone cells, crystalline cone, and pigment cells. The molecular size and the localization of the Drosophila RALBP-like protein are similar to those of the vertebrate interphotoreceptor retinoid binding protein (IRBP). It is suggested that this protein may function as a Drosophila IRBP.     

Preferential adhesion maintains separation of ommatidia in the Drosophila eye

In the Drosophila eye, neighboring ommatidia are separated by inter-ommatidial cells (IOCs). How this ommatidial spacing emerges during eye development is not clear. Here we demonstrate that four adhesion molecules of the Irre cell recognition module (IRM) family play a redundant role in maintaining separation of ommatidia. The four IRM proteins are divided into two groups: Kirre and Rst are expressed in IOCs, and Hbs and Sns in primary pigment cells (1°s). Kirre binds Hbs and Sns in vivo and in vitro. Reducing activity of either Rst or Kirre alone had minimal effects on ommatidial spacing, but reducing both together led to direct ommatidium:ommatidium contact. A similar phenotype was also observed when reducing both Hbs and Sns. Consistent with the role of these factors in sorting ommatidia, mis-expression of Hbs plus Sns within a single IOC led to complete separation of the cell from neighboring ommatidia. Our results indicate mutual preferential adhesion between ommatidia and IOCs mediated by four IRM proteins is both necessary and sufficient to maintain separation of ommatidia.     

The Claudin Megatrachea Protein Complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.     

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesis

Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.

Methods

Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.

Results

PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.

Conclusions

In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.     

Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function

Septate junctions (SJs) in epithelial and neuronal cells play an important role in the formation and maintenance of charge and size selective barriers. They form the basis for the ensheathment of nerve fibers in Drosophila and for the attachment of myelin loops to axonal surface in vertebrates. The cell-adhesion molecules NRX IV/Caspr/Paranodin (NCP1), contactin and Neurofascin-155 (NF-155) are all present at the vertebrate axo-glial SJs. Mutational analyses have shown that vertebrate NCP1 and its Drosophila homolog, Neurexin IV (NRX IV) are required for the formation of SJs. In this study, we report the genetic, molecular and biochemical characterization of the Drosophila homolog of vertebrate contactin, CONT. Ultrastructural and dye-exclusion analyses of Cont mutant embryos show that CONT is required for organization of SJs and paracellular barrier function. We show that CONT, Neuroglian (NRG) (Drosophila homolog of NF-155) and NRX IV are interdependent for their SJ localization and these proteins form a tripartite complex. Hence, our data provide evidence that the organization of SJs is dependent on the interactions between these highly conserved cell-adhesion molecules.     

Unique and Overlapping Functions of Formins Frl and DAAM During Ommatidial Rotation and Neuronal Development in Drosophila

The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Interestingly, we also found that frl mutants exhibit an axon growth phenotype in the mushroom body, a center for olfactory learning in the Drosophila brain, which is also affected in a subset of PCP genes. Significantly, Frl cooperates with Cdc42 and another formin, DAAM, during mushroom body formation. This study thus suggests that different formins can cooperate or act independently in distinct tissues, likely integrating various signaling inputs with the regulation of the cytoskeleton. It furthermore highlights the importance and complexity of formin-dependent cytoskeletal regulation in multiple organs and developmental contexts.     

Development of the Drosophila retina,a neurocrystalline lattice

Pattern formation in the Drosophila retina proceeds by the recruitment of cells, along a morphogenetic front, into a lattice. At the advancing front, marked by a dorso-ventral furrow in the eye imaginal disc, cells are organized into ommatidial precursors, each containing cells destined to become photoreceptors 2, 3, 4, 5, and 8. Behind the front, a mitotic wave produces photoreceptors 1, 6, and 7, plus the remaining cells needed to complete the ommatidia. During the third larval instar, the front sweeps anteriorly across the eye disc, leaving a highly ordered pattern in its wake. Preceding the dorso-ventral furrow is a groove that bisects the eye disc into dorsal and ventral halves and presumably plays a role in establishing the equatorial symmetry line. Cell lineage plays little role in pattern formation in the eye. Genetic mosaics show that the cells of each ommatidium are not derived from a single mother cell; the cells appear to be recruited at random at the morphogenetic front. Similarly, the mirror symmetry above and below the equator is not established by a clonal mechanism; a single clone can contribute cells to ommatidia on both sides of the equator.     

The emergence of order in the Drosophila pupal retina

During pupation, long-range order is imposed on the autonomously developing ommatidia which compose the Drosophila eye. To accomplish this, eight additional cell types arise: the primary, secondary, and tertiary pigment cells, and the four cells that form the bristle. These cells form an interweaving lattice between ommatidia. The lattice is refined when excess cells are removed to bring neighboring ommatidia into register. Recent evidence suggests that in larval development, local contacts direct cell fate. The same appears to be true during pupal development: the contacts a cell makes predict the cell type it will become. Cells which contact the anterior or posterior cone cells in an ommatidium invariably become primary pigment cells. Cells which contact primary pigment cells from different ommatidia become secondary and tertiary pigment cells. Bristle development is in several ways distinct from ommatidial development. The four cells of each bristle group appear to be immediate descendents of a single founder cell. During their early differentiation, they do not make stereotyped contacts with surrounding ommatidial cells, but do make particular contacts within the bristle group. And unlike the surrounding ommatidia, differentiation of the bristles radiates from the center of the eye to the edges. As cells are removed during two stages of programmed cell death, the bristles are brought into their final position. When all cells in the lattice have achieved their final position, a second stage of retinal development begins as structures specific to each cell type are produced. This paper follows these various stages of pupal development, and suggests how local cell-cell contacts may produce the cells needed for a functional retina.     

The blood-testis and blood-epididymis barriers are more than just their tight junctions

The terms blood-testis barrier (BTB) or blood-epididymis barrier (BEB), are often described as Sertoli cell-Sertoli cell tight junctions (TJs) or TJs between the epithelial cells in the epididymis, respectively. However, in reality, the BTB and BEB are much more complex than just the TJ. The focus of this minireview is to remind readers that the complete BTB and BEB are comprised of three components: anatomical, physiological, and immunological. The TJs form the anatomical (physical) barrier that restricts passage of molecules and cells from entering or exiting the lumen. The physiological barrier is comprised of transporters that regulate movement of substances in or out of the lumen, thus creating a microenvironment, which is critical for the proper development and maturation of germ cells. The immunological barrier limits access by the immune system and sequesters the majority of the autoantigenic germ cells. Combined with the overall immune-privilege of the testis, this suppresses detrimental immune responses against the autoantigenic germ cells. These three components on their own do not create a complete functional barrier; instead, it is the interaction between all three components that create a barrier of maximal competence.     

Blue and double-peaked green receptors depend on ommatidial type in the eye of the Japanese yellow swallowtail Papilio xuthus

The compound eye of the butterfly Papilio xuthus is composed of three spectrally distinct types of ommatidia. We investigated the blue and double-peaked green receptors that are encountered distally in type I and III ommatidia, by means of intracellular recordings, in vivo fluorescence microscopy, and histology. The blue receptors are R1 and/or R2 photoreceptors; they contain the same mRNA encoding the opsin of the blue-absorbing visual pigment. However, here we found that the sensitivity in the UV wavelength region strongly depends on the ommatidial type; the blue receptors in type I ommatidia have a distinctly depressed UV sensitivity, which is attributed to lateral filtering in the fused rhabdom. In the main, fronto-ventral part of the eye, the R3 and R4 photoreceptors of all ommatidia contain the same set of two mRNAs encoding the opsins of green-absorbing visual pigments, PxL1 and PxL2. The spectral sensitivities are double-peaked, but the UV sensitivity of the R3 and R4 photoreceptors in type I ommatidia appears to be reduced, similar to that of the co-localized blue receptors.     

灰茶尺蠖成虫复眼的超微结构及其明暗适应中的变化

【目的】明确灰茶尺蠖Ectropis grisescens成虫复眼的超微结构及其明暗适应中的变化,探究其调光机制。【方法】采用超景深显微镜测定了灰茶尺蠖成虫复眼的小眼数量、间角、直径和曲率半径等外部参数,并通过组织切片、光学显微镜和透射电子显微镜等技术观察了复眼的内部超微结构;通过光学显微镜观察了灰茶尺蠖成虫复眼在明暗环境中分别适应2 h后晶锥结构及色素颗粒的位置变化。【结果】灰茶尺蠖成虫复眼呈半球形,雌、雄虫单个复眼分别有2 502±105和3 123±78个小眼。小眼自远端至近端由角膜、晶锥、透明区构成的屈光层和由15个视网膜细胞构成的感光层组成。2个初级色素细胞包裹着晶锥,自角膜近端延伸至视网膜细胞核区的远端;每个小眼外围由6个次级色素细胞围绕,自角膜近端延伸至基膜;在透明区内14个视网膜细胞聚集成束(非感杆束),远端与晶锥束末端连接,在感光层内形成闭合型感杆束,延伸至第15个视网膜细胞(基部视网膜细胞)。在明暗适应时,灰茶尺蠖复眼的晶锥细胞间出现开闭,色素颗粒进行纵向位移,以适应外界的光强度的变化。【结论】灰茶尺蠖成虫复眼属于重叠像眼,感杆束为“14+1”模式;屏蔽色素颗粒的移...     

Morphogenesis and pattern formation in the retina of the crayfish Procambarus clarkii

Pattern formation and ommatidial differentiation in the crayfish retina were analyzed using confocal, light and electron microscopy. Optic primordia first appear in the embryo as round elevations covered by a surface epithelial layer. Retinal differentiation begins with a wave of mitotic activity that moves across this epithelium from lateral to medial. Ommatidial cell clusters are visible at the surface along a transition zone, which lies at the interface of the medial undifferentiated retina and the lateral patterned retina. This zone is 8–10 cells wide and composed of small uniform cell profiles. Lateral to the transition zone the initial ommatidial cell clusters form staggered rows across the surface. Each first row cluster contains eight retinula cells surrounded by four cone, two corneagenous and two distal pigment cells. Ommatidial clusters in the first nine rows show significant changes in their organization, which are visible at the surface of the retina. In row 10 the retinula cells recede from the surface and the cone cells close in above them creating a constant cell pattern at the surface. Rhabdome development begins distally and extends downward as the retinula cluster recedes from the surface. Movement of the retinula cells inward and enlargement of the cone and corneagenous cells at the surface creates a two-tiered organization characteristic of each ommatidium. Comparison of retinal pattern formation and differentiation in the crayfish with retinal morphogenesis in Drosophila and other insects show several similarities between the two arthropod groups.     

A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.     

Cofilin/ADF is required for retinal elongation and morphogenesis of the Drosophila rhabdomere

Drosophila photoreceptors undergo marked changes in their morphology during pupal development. These changes include a five-fold elongation of the retinal cell body and the morphogenesis of the rhabdomere, the light sensing structure of the cell. Here we show that twinstar (tsr), which encodes Drosophila cofilin/ADF (actin-depolymerizing factor), is required for both of these processes. In tsr mutants, the retina is shorter than normal, the result of a lack of retinal elongation. In addition, in a strong tsr mutant, the rhabdomere structure is disorganized and the microvilli are short and occasionally unraveled. In an intermediate tsr mutant, the rhabdomeres are not disorganized but have a wider than normal structure. The adherens junctions connecting photoreceptor cells to each other are also found to be wider than normal. We propose, and provide data supporting, that these wide rhabdomeres and adherens junctions are secondary events caused by the inhibition of retinal elongation. These results provide insight into the functions of the actin cytoskeleton during morphogenesis of the Drosophila eye.     

The Canoe protein is necessary in adherens junctions for development of ommatidial architecture in the Drosophila compound eye

Rhabdomeres of the Drosophila melanogaster canoemisl mutant ommatidia were twisted, branched, and often fused to each other. A considerable proportion of rhabdomeres were found to have fallen below the retinal basement membrane. Electron-microscopic observations of the mutant ommatidia revealed that microvilli, the subcellular structures composing the rhabdome, were normal. As was the case with partial loss-of-function mutations in the canoe locus, overexpression of the wild-type canoe transgene driven by the heat shock promoter or sevenless enhancer in the wild-type canoe background caused malformation of the rhabdomeres in the adult ommatidia. Immunolabeling of the Canoe protein in the pupal retinae showed that it was accumulated in adherens junctions in photoreceptor rhabdomeres at high concentrations, as well as in pigment cells, bristle cells, and the interjunctional region of photoreceptors at a lower level. In the canoe mutant ommatidia, the Canoe protein concentration was dramatically decreased in adherens junctions, while it was maintained at a level comparable with the wild-type flies in the interjunctional region. Since Canoe or its mammalian homolog AF-6 is known to bind to F-actin and Ras, we suggest the possibility that Canoe couples Ras signaling with cytoskeleton, thereby supporting the straight elongation of rhabdomeres required for development of a regular array of ommatidia.     

Ellipse mutations in the Drosophila homologue of the EGF receptor affect pattern formation, cell division, and cell death in eye imaginal discs.

Ellipse alleles are mutations of the EGF-receptor homologue that reduce the number of ommatidia in the eye imaginal disc. Cobalt sulfide staining, expression of hairy and scabrous proteins, and mosaic analysis indicated that Elp mutations affect ommatidial precluster formation in the morphogenetic furrow. BrdU incorporation studies suggest that cells diverted from precluster formation instead enter S-phase after the morphogenetic furrow. Genetic studies suggest that the DER has multiple functions during eye development and that several recessive hypomorphic alleles affect another aspect of DER function that is required after precluster formation. Elp mutations show genetic interactions with the neurogenic mutations Notch and Delta. The small number of ommatidia that differentiate in Elp/Elp are separated more than in wildtype and have been studied to investigate what aspects of ommatidium development are intrinsic to the ommatidium itself. It appears that each developing ommatidium cues the determination of photoreceptors, cone cells, and primary pigment cells, but that the secondary and tertiary pigment cells, and the mechanosensory bristles, can form independently. The normal rotation of ommatidia in the dorsal-ventral axis does not require the presence of the ommatidial array. A short-range signal from a nearby ommatidium is important for mitosis. Cells not close to an ommatidium do not go through mitosis and many die.     

Interphotoreceptor retinoid-binding protein (IRBP)

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retinal pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.     

The early stages of ommatidial development in the flour beetle Tribolium castaneum (Coleoptera; Tenebrionidae)

 Using electron microscopy, the first stages of ommatidial development in the flour beetle Tribolium castaneum were analysed in relation to the cellular architecture of the adult compound eye and were compared to the corresponding patterning process in the fruit fly Drosophila melanogaster. The ommatidia of the slightly horse-shoe shaped beetle compound eye contain six peripheral and two central retinula cells. The rhabdomere of the posteriorly located central photoreceptor cell is restricted to the distal half of the rhabdom whilst that of the anterior one is restricted to its proximal half. The development of the compound eye takes place in an external eye imaginal disc. Most stages of ommatidial development, as known from Drosophila, i.e. arc-like cell groups, five-cell clusters, immature eight-cell clusters and symmetrical eight-cell clusters, are very precisely conserved between the two species. Two major differences exist: 1. In Tribolium, the cone cell precursor cells synchronously join to the immature eight-cell cluster. As a consequence, the symmetrical eight-cell cluster immediately transforms into a four-cone-cell cluster. 2. The maturing ommatidia do not undergo rotation in Tribolium. Overall, no morphological indiation for an equator in the adult Tribolium compound eye could be found. Considering the strong evolutionary conservation of early ommatidial development, homology of photoreceptor cells of distantly related insects is proposed to be inferred from their ontogenetic origin. Received: 6 November 1995 / Accepted: 9 April 1996