Genetic determinants of hyaloid and retinal vasculature in zebrafish

Background

The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.

Results

We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.

Conclusion

Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.     

Interaction between pericytes and endothelial cells leads to formation of tight junction in hyaloid vessels

The hyaloid vessel is a transient vascular network that nourishes the lens and the primary vitreous in the early developmental periods. In hyaloid vessels devoid of the support of astrocytes, we demonstrate that tight junction proteins, zonula occludens-1 and occludin, are regularly expressed at the junction of endothelial cells. To figure out the factor influencing the formation of tight junctions in hyaloid vessels, we further progress to investigate the interactions between endothelial cells and pericytes, two representative constituent cells in hyaloid vessels. Interestingly, endothelial cells interact with pericytes in the early postnatal periods and the interaction between two cell types provokes the up-regulation of transforming growth factor β1. Further in vitro experiments demonstrate that transforming growth factor β1 induces the activation of Smad2 and Smad3 and the formation of tight junction proteins. Taken together, in hyaloid vessels, pericytes seem to regulate the formation of tight junctions by the interaction with endothelial cells even without the support of astrocytes. Additionally, we suggest that the hyaloid vessel is a valuable system that can be utilized for the investigation of cell-cell interaction in the formation of tight junctions in developing vasculatures.     

Eye formation in the absence of retina

Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx−/− embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of β-catenin expression in the head surface ectoderm. This suggests that β-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.     

A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development

Background

Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB) and blood-retinal (BRB) barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP) in blood plasma, as an endogenous tracer.

Results

The temporal establishment of the BBB and BRB was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. We also examined the expression of claudin-5, a component of tight junctions during the first 4 days of development. We observed that the BBB of zebrafish starts to develop by 3 dpf, with expression of claudin-5 in the central arteries preceding it at 2 dpf. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB.

Conclusion

Zebrafish embryos develop BBB and BRB function simultaneously by 3 dpf, which is regulated by tight junction proteins. The Tg(l-fabp:DBP-EGFP) zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model.     

Development and pathology of the hyaloid, choroidal and retinal vasculature

During embryogenesis, the development and differentiation of the eye requires the concomitant formation of the neural/glial elements along with a dense vascular network. The adult neural retina is supported by two distinct vascular systems, the proper retinal vessels and the choroidal vessels. The two beds differ not only in their pattern of embryonic differentiation, but also in their function in the adult organism. The retinal vasculature has barrier properties similar to those observed in the brain, whereas the choroidal vessels display a highly fenestrated phenotype. The hyaloid vasculature is a transient embryonic vascular bed which is complete at birth in mammals and regresses contemporaneously with the formation of the retinal vasculature. The dependence of the retina on its blood supply makes it highly vulnerable to any vascular changes and indeed ocular diseases, such as proliferative retinopathy, age-related macular degeneration and the hyperplastic primary vitreous, which are associated with abnormalities of the different vascular beds of the eye. A number of factors have been implicated in developmental and pathological changes in vessel formation and regression, including fibroblast growth factors, platelet-derived endothelial growth factor and vascular endothelial growth factor, among others. The purpose of this review is to describe and discuss new insights into the mechanisms and molecular cues involved in the development of the normal and pathological vascular systems of the eye. The characterization of the molecules and cell-cell interactions involved in the formation, stabilization and regression of new vessels has led to the identification of potential control points for therapeutic intervention.     

A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye

Previously we reported the novel observation that astrocytes ensheath the persistent hyaloid artery, both in the Nuc1 spontaneous mutant rat, and in human PFV (persistent fetal vasculature) disease (Developmental Dynamics 234:36-47, 2005). We now show that astrocytes isolated from both the optic nerve and retina of Nuc1 rats migrate faster than wild type astrocytes. Aquaporin 4 (AQP4), the major water channel in astrocytes, has been shown to be important in astrocyte migration. We demonstrate that AQP4 expression is elevated in the astrocytes in PFV conditions, and we hypothesize that this causes the cells to migrate abnormally into the vitreous where they ensheath the hyaloid artery. This abnormal association of astrocytes with the hyaloid artery may impede the normal macrophage-mediated remodeling and regression of the hyaloid system.     

Autophagy-induced regression of hyaloid vessels in early ocular development

The hyaloid vessel is a transient intraocular circulatory system that undergoes a complete regression as the retina becomes matured with retinal vascularization. If the complete involution of the hyaloid vessels failes, the pathological persistence of these vessels results in persistent hyperplastic primary vitreous (PHPV) associated with severe ocular pathologies. Unfortunately despite its clinical significance, cellular and molecular processes involved in hyaloid regression remain to be elucidated. Herein, we for the first time demonstrated that autophagy could contribute to the regression of hyaloid vessels in early developing retina. In developing retina, hyaloid vessel regression coincided with retinal vascular development; this occurred simultaneous with apoptotic and autophagic processes. Moreover, in vascular endothelial cells under hypoxic conditions, LC3-II conversion was detected along with caspase-3 activation. The autophagy inducer rapamycin induced autophagy-mediated cell death of vascular endothelial cells in a dose-dependent manner. Moreover, rapamycin significantly enhanced the involution of hyaloid vessels in the early developing eye. Therefore, our results suggest that the autophagy pathway would be involved in hyaloid regression that occurs during early ocular development. Furthermore, activation of the autophagy pathway could be considered for a therapeutic approach to PHPV.     

Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos

Ocular coloboma is a potentially blinding congenital eye malformation caused by failure of optic fissure closure during early embryogenesis. The optic fissure is a ventral groove that forms during optic cup morphogenesis, and through which hyaloid artery and vein enter and leave the developing eye, respectively. After hyaloid artery and vein formation, the optic fissure closes around them. The mechanisms underlying optic fissure closure are poorly understood, and whether and how this process is influenced by hyaloid vessel development is unknown. Here we show that a loss-of-function mutation in lmo2, a gene specifically required for hematopoiesis and vascular development, results in failure of optic fissure closure in zebrafish. Analysis of ocular blood vessels in lmo2 mutants reveals that some vessels are severely dilated, including the hyaloid vein. Remarkably, reducing vessel size leads to rescue of optic fissure phenotype. Our results reveal a new mechanism leading to coloboma, whereby malformed blood vessels interfere with eye morphogenesis.     

Modification of flag leaf senescence and yield characters in barley (Hordeum vulgare L.) by gibberellic acid and kinetin

Barley plants (Hordeum vulgare L.) received foliar applications of 10^?4 M gibberellic acid (GA₃) and Kinetin (KN) individually and in combination at one or more of three growth stages: flag leaf appearance (I), ear emergence (II), and the first stage of senescence initiation in the flag leaf (III). Both plant growth regulators (PGR) hastened onset of senescence when sprayed at Stage I and/or Stage II. Treatment at Stage III, either alone or in combination with treatments at the other stages, tended to postpone senescence. Yield components also showed stage-dependent response: Stage I treatment increased the formation of total and bearing tillers, and Stage III treatment improved grain number and weight. However, while GA₃ proved more effective than KN, the two together acted antagonistically.     

miR-204 Targeting of Ankrd13A Controls Both Mesenchymal Neural Crest and Lens Cell Migration

Loss of cell adhesion and enhancement of cell motility contribute to epithelial-to-mesenchymal transition during development. These processes are related to a) rearrangement of cell-cell and cell-substrate adhesion molecules; b) cross talk between extra-cellular matrix and internal cytoskeleton through focal adhesion molecules. Focal adhesions are stringently regulated transient structures implicated in cell adhesion, spreading and motility during tissue development. Importantly, despite the extensive elucidation of the molecular composition of focal adhesions, the complex regulation of their dynamics is largely unclear. Here, we demonstrate, using live-imaging in medaka, that the microRNA miR-204 promotes both mesenchymal neural crest and lens cell migration and elongation. Overexpression of miR-204 results in upregulated cell motility, while morpholino-mediated ablation of miR-204 activity causes abnormal lens morphogenesis and neural crest cell mislocalization. Using a variety of in vivo and in vitro approaches, we demonstrate that these actions are mediated by the direct targeting of the Ankrd13A gene, which in turn controls focal cell adhesion formation and distribution. Significantly, in vivo restoration of abnormally elevated levels of Ankrd13A resulting from miR-204 inactivation rescued the aberrant lens phenotype in medaka fish. These data uncover, for the first time in vivo, the role of a microRNA in developmental control of mesenchymal cell migration and highlight miR-204 as a “master regulator” of the molecular networks that regulate lens morphogenesis in vertebrates.     

Proteomic identification of activin receptor-like kinase-1 as a differentially expressed protein during hyaloid vascular system regression

The hyaloid vascular system (HVS) is a transient network of capillaries that nourishes the embryonic lens and the primary vitreous of the developing eye. We used proteomic analysis and immunohistochemical staining to identify activin receptor-like kinase-1 (ALK1), a type I receptor for transforming growth factor-beta1, during the HVS regression phase. In addition, we overexpressed ALK1 in corneas implanted with bFGF (basic fibroblast growth factor) pellets and observed that ALK1 overexpression resulted in inhibition of bFGF-induced corneal neovascularization in vivo. Our data suggest that ALK1 may play a role in HVS regression during ocular development.     

Early Lens Ablation Causes Dramatic Long-Term Effects on the Shape of Bones in the Craniofacial Skeleton of Astyanax mexicanus

The Mexican tetra, Astyanax mexicanus, exists as two morphs of a single species, a sighted surface morph and a blind cavefish. In addition to eye regression, cavefish have an increased number of taste buds, maxillary teeth and have an altered craniofacial skeleton compared to the sighted morph. We investigated the effect the lens has on the development of the surrounding skeleton, by ablating the lens at different time points during ontogeny. This unique long-term study sheds light on how early embryonic manipulations on the eye can affect the shape of the adult skull more than a year later, and the developmental window during which time these effects occur. The effects of lens ablation were analyzed by whole-mount bone staining, immunohistochemisty and landmark based morphometric analyzes. Our results indicate that lens ablation has the greatest impact on the skeleton when it is ablated at one day post fertilisation (dpf) compared to at four dpf. Morphometric analyzes indicate that there is a statistically significant difference in the shape of the supraorbital bone and suborbital bones four through six. These bones expand into the eye orbit exhibiting plasticity in their shape. Interestingly, the number of caudal teeth on the lower jaw is also affected by lens ablation. In contrast, the shape of the calvariae, the length of the mandible, and the number of mandibular taste buds are unaltered by lens removal. We demonstrate the plasticity of some craniofacial elements and the stability of others in the skull. Furthermore, this study highlights interactions present between sensory systems during early development and sheds light on the cavefish phenotype.     

The superficial vascular hyaloid system in the eye of the frogs,Rana temporaria and Rana esculenta

Summary The angioarchitecture of the superficial vascular hyaloid system (membrana vasculosa retinae) of the frog eye was studied by means of scanning electron microscopy of vascular corrosion casts. The terminal vessels form a single-layered sheath intimately adjacent to the vitreal side of the avascular retina. The hyaloid system is subdivided by the ventral venous trunk into three central areas: the dorsal, the temporo-ventral, and the naso-ventral area. Toward the ora serrata, the hyaloid system is bordered by an arterial ring, and by nasal and temporal venous branches forming more or less complete hemicircles. A vascular zone composed of several tongue-like sectors establishes an inter-connection between the peripheral vascular rings and the central areas of the fundus. The arterial blood is supplied from the arterial ring. The drainage of the hyaloid system is provided via two routes: (1) the Y-shaped ventral trunk collects blood from the central areas, (2) the two peripheral venous branches drain the tongue-like sectors. The vessels within the dorsal area follow preferentially a dorso-ventral meridional direction. This densely capillarized territory corresponds in localization to the area centralis retinae. The ultrastructure of microvessels of the hyaloid system is characterized by features typical for capillaries of the central nervous system.     

Vascular morphogenesis in the zebrafish embryo

During embryonic development, the vertebrate vasculature is undergoing vast growth and remodeling. Blood vessels can be formed by a wide spectrum of different morphogenetic mechanisms, such as budding, cord hollowing, cell hollowing, cell wrapping and intussusception. Here, we describe the vascular morphogenesis that occurs in the early zebrafish embryo. We discuss the diversity of morphogenetic mechanisms that contribute to vessel assembly, angiogenic sprouting and tube formation in different blood vessels and how some of these complex cell behaviors are regulated by molecular pathways.     

Sculpting the heart: Cellular mechanisms shaping valves and trabeculae

The transformation of the heart from a simple tube to a complex organ requires the orchestration of several morphogenetic processes. Two structures critical for cardiac function, the cardiac valves and the trabecular network, are formed through extensive tissue morphogenesis—endocardial cell migration, deadhesion and differentiation into fibroblast-like cells during valve formation, and cardiomyocyte delamination and apico-basal depolarization during trabeculation. Here, we review current knowledge of how these specialized structures acquire their shape by focusing on the underlying cellular behaviors and molecular mechanisms, highlighting findings from in vivo models and briefly discussing the recent advances in cardiac cell culture and organoids.     

The larval stages of the sea urchin, Strongylocentrotus purpuratus

The adult body plan of Strongylocentrotus purpuratus is established within the imaginal rudiment during the larval stages. To facilitate the study of these stages, we have defined a larval staging scheme, which consists of seven stages: Stage I, four-arm stage; Stage II, eight-arm stage; Stage III, vestibular invagination stage; Stage IV, rudiment initiation stage; Stage V, pentagonal disc stage; Stage VI, advanced rudiment stage; and Stage VI, tube-foot protrusion stage. Each stage is characterized by significant morphological features observed for the first time at that stage. This scheme is intended as a guide for determining the degree of larval development, and for identifying larval and adult structures. Larval anatomy was visualized using light and confocal microscopy as required on living material, whole mount fixed specimens, and serial sections. Antibody staining to localize specific gene products was also used. Detailed analysis of these data has furthered our understanding of the morphogenesis of the rudiment, and has suggested provocative questions regarding the molecular basis for these events. We intend this work to be of use to investigators studying gene expression and morphogenesis in postembryonic larvae.     

Clinical appearance of the hyaloid artery system in the newborn rhesus monkey.

Fifteen healthy newborn rhesus monkeys (Macaca mulatta) were given funduscopic examinations as part of a screening process before utilization in a research project. The hyaloid vasculature, a system that supplies blood to the embryonic ocular structures, was observed in each subject and was noted to regress clinically at two to three weeks of age. Although the phenomenon has been described morphologically in the literature, its clinical appearance has not been illustrated for the primate clinician.