In vivo analysis of hyaloid vasculature morphogenesis in zebrafish: A role for the lens in maturation and maintenance of the hyaloid

Two vascular networks nourish the embryonic eye as it develops – the hyaloid vasculature, located at the anterior of the eye between the retina and lens, and the choroidal vasculature, located at the posterior of the eye, surrounding the optic cup. Little is known about hyaloid development and morphogenesis, however. To begin to identify the morphogenetic underpinnings of hyaloid formation, we utilized in vivo time-lapse confocal imaging to characterize morphogenesis of the zebrafish hyaloid through 5 days post fertilization (dpf). Our data segregate hyaloid formation into three distinct morphogenetic stages: Stage I: arrival of hyaloid cells at the lens and formation of the hyaloid loop; Stage II: formation of a branched hyaloid network; Stage III: refinement of the hyaloid network. Utilizing fixed and dissected tissues, distinct Stage II and Stage III aspects of hyaloid formation were quantified over time. Combining in vivo imaging with microangiography, we demonstrate that the hyaloid system becomes fully enclosed by 5 dpf. To begin to identify the molecular and cellular mechanisms underlying hyaloid morphogenesis, we identified a recessive mutation in the mab21l2 gene, and in a subset of mab21l2 mutants the lens does not form. Utilizing these “lens-less” mutants, we determined whether the lens was required for hyaloid morphogenesis. Our data demonstrate that the lens is not required for Stage I of hyaloid formation; however, Stages II and III of hyaloid formation are disrupted in the absence of a lens, supporting a role for the lens in hyaloid maturation and maintenance. Taken together, this study provides a foundation on which the cellular, molecular and embryologic mechanisms underlying hyaloid morphogenesis can be elucidated.     

Proper patterning of the optic fissure requires the sequential activity of BMP7 and SHH

The optic disc develops at the interface between optic stalk and retina, and enables both the exit of visual fibres and the entrance of mesenchymal cells that will form the hyaloid artery. In spite of the importance of the optic disc for eye function, little is known about the mechanisms that control its development. Here, we show that in mouse embryos, retinal fissure precursors can be recognised by the expression of netrin 1 and the overlapping distribution of both optic stalk (Pax2, Vax1) and ventral neural retina markers (Vax2, Raldh3). We also show that in the absence of Bmp7, fissure formation is not initiated. This absence is associated with a reduced cell proliferation and apoptosis in the proximoventral quadrant of the optic cup, lack of the hyaloid artery, optic nerve aplasia, and intra-retinal misrouting of RGC axons. BMP7 addition to organotypic cultures of optic vesicles from Bmp7-/- embryos rescues Pax2 expression in the ventral region, while follistatin, a BMP7 antagonist, prevents it in early, but not in late, optic vesicle cultures from wild-type embryos. The presence of Pax2-positive cells in late optic cup is instead abolished by interfering with Shh signalling. Furthermore, SHH addition re-establishes Pax2 expression in late optic cups derived from ocular retardation (or) embryos, where optic disc development is impaired owing to the near absence of SHH-producing RGC. Collectively, these data indicate that BMP7 is required for retinal fissure formation and that its activity is needed, before SHH signalling, for the generation of PAX2-positive cells at the optic disc.     

Pax6 heterozygous eyes show defects in chamber angle differentiation that are associated with a wide spectrum of other anterior eye segment abnormalities

The development of the chamber angle was studied in the eyes of heterozygous Pax6(lacZ/+) mutant mice (Nature 387 (1997) 406). Mutations in PAX6 cause aniridia, a condition that is frequently associated with glaucoma, a blinding disease that may be associated with chamber angle defects. Mesenchymal cells were seen in the chamber angle at P1-P5. In wild-type mice, these cells differentiated into typical trabecular meshwork (TM) cells next to Schlemm's canal. In Pax6(lacZ/+) mice, TM cells remained undifferentiated and Schlemm's canal was absent. From P1 to P4, staining for beta-galactosidase and immunoreactivity for Pax6 were observed in chamber angle mesenchyme, but were absent later. Cultured murine TM cells expressed Pax6. The defects in chamber angle and TM differentiation were associated with a wide spectrum of other anterior eye defects, which included various degrees of iris hypoplasia and corneal haze, isolated iridocorneal adhesions and atypical coloboma, and a vascularized cornea in all adult animals. A third of the animals showed Peters' anomaly including corneal opacity and iridocorneal adhesions. The separation of the lens from the cornea was incomplete, and epithelial layers of lens and cornea were continuous. Pax6 activity is directly required for differentiation of the chamber angle. Variations in phenotype of Pax6(lacZ/+) mice appear not to involve direct dominant-negative or dose-dependent effects.     

The genetic control of eye development and its implications for the evolution of the various eye-types

Mutations in the Pax 6 homologs of mammals and insects prevent eye development and targeted expression of both mammal and insect Pax 6 homologs is capable of inducing functional ectopic eyes. Supported by RNA interference experiments in planarians and nemerteans, these findings indicate that Pax 6 is a universal master control gene for eye morphogenesis. Since all metazoan eyes use rhodopsin as a photoreceptor molecule and the same master control gene for eye development, we postulate a monophyletic origin of the various eye types. The finding of well developed eyes in jellyfish which essentially lack a brain, leads us to propose that the eye as a sensory organ evolved before the brain which is an information processing organ. The finding of highly developed eyes with a lens, vitreous body, stacked membranes like a retina and shielding pigment in unicellular dinoflagellates, raises the possibility that the prototypic eyes might have been acquired from symbionts.     

The orchestration of mammalian tissue morphogenesis through a series of coherent feed-forward loops

Tissue morphogenesis requires intricate temporal and spatial control of gene expression that is executed through specific gene regulatory networks (GRNs). GRNs are comprised from individual subcircuits of different levels of complexity. An important question is to elucidate the mutual relationship between those genes encoding DNA-binding factors that trigger the subcircuit with those that play major "later" roles during terminal differentiation via expression of specific genes that constitute the phenotype of individual tissues. The ocular lens is a classical model system to study tissue morphogenesis. Pax6 is essential for both lens placode formation and subsequent stages of lens morphogenesis, whereas c-Maf controls terminal differentiation of lens fibers, including regulation of crystallins, key lens structural proteins required for its transparency and refraction. Here, we show that Pax6 directly regulates c-Maf expression during lens development. A 1.3-kb c-Maf promoter with a 1.6-kb upstream enhancer (CR1) recapitulated the endogenous c-Maf expression pattern in lens and retinal pigmented epithelium. ChIP assays revealed binding of Pax6 and c-Maf to multiple regions of the c-Maf locus in lens chromatin. To predict functional Pax6-binding sites, nine novel variants of Pax6 DNA-binding motifs were identified and characterized. Two of these motifs predicted a pair of Pax6-binding sites in the CR1. Mutagenesis of these Pax6-binding sites inactivated transgenic expression in the lens but not in retinal pigmented epithelium. These data establish a novel regulatory role for Pax6 during lens development, link together the Pax6/c-Maf/crystallin regulatory network, and suggest a novel type of GRN subcircuit that controls a major part of embryonic lens development.     

Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis

Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P(1) (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p(1)(-/-) mice. Here we show that strong induction of S1P(1) expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P(1) results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p(1)(-/-) limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1 alpha and its response gene VEGF were induced in S1p(1)(-/-) limbs. However, aberrant regulation of HIF-1 alpha and VEGF were not observed in embryonic fibroblasts derived from S1p(1)(-/-) mice, suggesting a non-cell autonomous effect of S1P(1) on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P(1) null mice in vivo. These data suggest that the function of S1P(1) in the developing vasculature is essential for proper limb development.     

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.     

Persistent hyperplastic primary vitreous

Persistent hyperplastic primary vitreous (PHPV) is a common congenital developmental anomaly of the eye that results following failure of the embryological, primary vitreous and hyaloid vasculature to regress. It typically presents unilaterally without associated systemic findings. Although the etiology is assumed to be identical in each of its three variants, PHPV is still subclassified into three presentations. The purely anterior presentation of PHPV is also known as persistent tunica vasculosa lentis and persistent posterior fetal fibrovascular sheath of the lens. It occurs in eyes with pathology of the anterior segment. This form typically involves cataract, glaucoma and a retrolenticular membrane. The purely posterior presentation of PHPV is termed falciform retinal septum and ablatio falcicormis congentia. It occurs in eyes with abnormalities confined to the posterior segment such as retinal folds, vitreal stalk, vitreal membranes, macular abnormalities and optic disc abnormalities. A combination of anterior and posterior presentations is the most commonly seen clinical presentation. Case report. We present a case in which an 11-year old male was referred to our office for reevaluation of a large angle esotropia, strabismic and deprivational amblyopia and previously diagnosed PHPV, OD. Conclusion. Without treatment, PHPV can produce recurrent intraocular hemorrhage, secondary glaucoma and eventually require enucleation. Early surgical intervention is necessary to prevent progressive pathologic changes and to obtain the best visual results. Finally, while PHPV is a documented source of leukocoria, clinicians should be aware of differential diagnoses which involve the white pupil (congenital cataract, retinoblastoma, Norrie's disease, retinopathy of prematurity, retinal detachment and Coat's disease).     

Otx genes are required for tissue specification in the developing eye

Patterning of the vertebrate eye appears to be controlled by the mutual regulation and the progressive restriction of the expression domains of a number of genes initially co-expressed within the eye anlage. Previous data suggest that both Otx1 and Otx2 might contribute to the establishment of the different eye territories. Here, we have analysed the ocular phenotype of mice carrying different functional copies of Otx1 and Otx2 and we show that these genes are required in a dose-dependent manner for the normal development of the eye. Thus, all Otx1(-/-); Otx2(+/-) and 30% of Otx1(+/-); Otx2(+/-) genotypes presented consistent and profound ocular malformation, including lens, pigment epithelium, neural retina and optic stalk defects. During embryonic development, optic vesicle infolding was severely altered and the expression of pigment epithelium-specific genes, such as Mitf or tyrosinase, was lost. Lack of pigment epithelium specification was associated with an expansion of the prospective neural retina and optic stalk territories, as determined by the expression of Pax6, Six3 and Pax2. Later in development the presumptive pigment epithelium region acquired features of mature neural retina, including the generation of Islet1-positive neurones. Furthermore, in Otx1(-/-); Otx2(+/-) mice neural retina cell proliferation, cell differentiation and apoptotic cell death were also severely affected. Based on these findings we propose a model in which Otx gene products are required for the determination and differentiation of the pigment epithelium, co-operating with other eye patterning genes in the determination of the specialised tissues that will constitute the mature vertebrate eye.     

Effect of chemical stabilizers of hypoxia-inducible factors on early lung development

Low oxygen stimulates pulmonary vascular development and airway branching and involves hypoxia-inducible factor (HIF). HIF is stable and initiates expression of angiogenic factors under hypoxia, whereas normoxia triggers hydroxylation of the HIF-1alpha subunit by prolyl hydroxylases (PHDs) and subsequent degradation. Herein, we investigated whether chemical stabilization of HIF-1alpha under normoxic (20% O(2)) conditions would stimulate vascular growth and branching morphogenesis in early lung explants. Tie2-LacZ (endothelial LacZ marker) mice were used for visualization of the vasculature. Embryonic day 11.5 (E11.5) lung buds were dissected and cultured in 20% O(2) in the absence or presence of cobalt chloride (CoCl(2), a hypoxia mimetic), dimethyloxalylglycine (DMOG; a nonspecific inhibitor of PHDs), or desferrioxamine (DFO; an iron chelator). Vascularization was assessed by X-gal staining, and terminal buds were counted. The fine vascular network surrounding the developing lung buds seen in control explants disappeared in CoCl(2)- and DFO-treated explants. Also, epithelial branching was reduced in the explants treated with CoCl(2) and DFO. In contrast, DMOG inhibited branching but stimulated vascularization. Both DFO and DMOG increased nuclear HIF-1alpha protein levels, whereas CoCl(2) had no effect. Since HIF-1alpha induces VEGF expression, the effect of SU-5416, a potent VEGF receptor (VEGFR) blocker, on early lung development was also investigated. Inhibition of VEGFR2 signaling in explants maintained under hypoxic (2% O(2)) conditions completely abolished vascularization and slightly decreased epithelial branching. Taken together, the data suggest that DMOG stabilization of HIF-1alpha during early development leads to a hypervascular lung and that airway branching proceeds without the vasculature, albeit at a slower rate.     

Lens morphogenesis is dependent on Pax6‐mediated inhibition of the canonical Wnt/beta‐catenin signaling in the lens surface ectoderm

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens‐specific gene expression in the surface‐ectoderm. Supression of canonical Wnt/β‐catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down‐regulation of canonical Wnt signaling in the presumptive lens ectoderm. genesis 48:86–95, 2010. © 2009 Wiley‐Liss, Inc.     

Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye

Chimaeric mice were made by aggregating Pax6(-/-) and wild-type mouse embryos, in order to study the interaction between the optic vesicle and the prospective lens epithelium during early stages of eye development. Histological analysis of the distribution of homozygous mutant cells in the chimaeras showed that the cell-autonomous removal of Pax6(-/-) cells from the lens, shown previously at E12.5, is nearly complete by E9.5. Most mutant cells are eliminated from an area of facial epithelium wider than, but including, the developing lens placode. This result suggests a role for Pax6 in maintaining a region of the facial epithelium that has the tissue competence to undergo lens differentiation. Segregation of wild-type and Pax6(-/-) cells occurs in the optic vesicle at E9.5 and is most likely a result of different adhesive properties of wild-type and mutant cells. Also, proximo-distal specification of the optic vesicle (as assayed by the elimination of Pax6(-/-) cells distally), is disrupted in the presence of a high proportion of mutant cells. This suggests that Pax6 operates during the establishment of patterning along the proximo-distal axis of the vesicle. Examination of chimaeras with a high proportion of mutant cells showed that Pax6 is required in the optic vesicle for maintenance of contact with the overlying lens epithelium. This may explain why Pax6(-/-) optic vesicles are inefficient at inducing a lens placode. Contact is preferentially maintained when the lens epithelium is also wild-type. Together, these results demonstrate requirements for functional Pax6 in both the optic vesicle and surface epithelia in order to mediate the interactions between the two tissues during the earliest stages of eye development.     

Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6

We have studied the molecular basis of the Pax2 and Pax6 function in the establishment of visual system territories. Loss-of-function mutants have revealed crucial roles for Pax2 in the generation of the optic stalk and for Pax6 in the development of the optic cup. Ectopic expression of Pax6 in the optic stalk under control of Pax2 promoter elements resulted in a shift of the optic cup/optic stalk boundary indicated by the presence of retinal pigmented cells on the optic stalk. By studying mouse embryos at early developmental stages we detected an expansion of Pax2 expression domain in the Pax6(-/-) mutant and of Pax6 expression domain in the Pax2(-/-) embryo. These results suggest that the position of the optic cup/optic stalk boundary depends on Pax2 and Pax6 expression, hinting at a possible molecular interaction. Using gel shift experiments, we confirmed the presence of Pax2- and Pax6-binding sites on the retina enhancer of the Pax6 gene and on the Pax2 upstream control region, respectively. Co-transfection experiments revealed a reciprocal inhibition of Pax2 promoter/enhancer activity by Pax6 protein and vice versa. Based on our findings, we propose a model for Pax gene regulation that establishes the proper spatial regionalization of the mammalian visual system.     

Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.