Lens Vesicle Research Articles

Abl kinases regulate FGF signaling independent of Crk phosphorylation to prevent Peters anomaly.

Peters anomaly, the most common cause of congenital corneal opacity, stems from corneal-lenticular adhesion. Despite numerous identified mutations, a cohesive molecular framework of the disease's etiology remains elusive. Here, we identified Abl kinases as pivotal regulators of FGF signaling, as genetic ablation of Abl kinases restores lens induction even in the absence of FGF signaling. Intriguingly, both Abl kinase deficiency and increased FGF-Ras activity result in Peters anomaly independent of ERK signaling, which can be rescued by allelic deletion of Abl substrate, Crk. However, contrary to the prevailing belief that Abl kinases regulate Crk proteins by direct phosphorylation, mutations at Abl kinase phosphorylation sites on Crk and CrkL did not yield any observable effects. Instead, our findings reveal that Abl kinases phosphorylate Ptpn12, which in turn inhibits p130Cas phosphorylation and Crk recruitment, crucial for Rho GTPases activation and cytoskeletal dynamics. Consequently, Abl kinase deficiency reduces actomyosin contractility within the lens vesicle and genetically interacts with RhoA inhibition. Conversely, Rac1 deletion mitigates Peters anomaly in models with aberrant FGF, Abl kinase and RhoA signaling. Our results demonstrate that Abl kinases regulate FGF signaling to balance RhoA and Rac1 activity via the Ptpn12-p130Cas pathway, suggesting that targeting tension-mediated lens vesicle separation could be a therapeutic strategy for Peters anomaly.

Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development.

Fibroblast growth factor (FGF) signaling elicits multiple downstream pathways, most notably the Ras/MAPK cascade facilitated by the adaptor protein Grb2. However, the mechanism by which Grb2 is recruited to the FGF signaling complex remains unresolved. Here we showed that genetic ablation of FGF signaling prevented lens induction by disrupting transcriptional regulation and actin cytoskeletal arrangements, which could be reproduced by deleting the juxtamembrane region of the FGF receptor and rescued by Kras activation. Conversely, mutations affecting the Frs2-binding site on the FGF receptor or the deletion of Frs2 and Shp2 primarily impact later stages of lens vesicle development involving lens fiber cell differentiation. Our study further revealed that the loss of Grb2 abolished MAPK signaling, resulting in a profound arrest of lens development. However, disrupting the Grb2 binding site on Shp2 or abrogating Shp2 phosphatase activity only modestly influenced FGF signaling, whereas mutating the presumed Shp2 dephosphorylation site on Grb2 did not impede MAPK signaling in lens development, indicating that Shp2 is only partially responsible for Grb2 recruitment. In contrast, we observed that FGF signaling is required for the phosphorylation of the Grb2-binding sites on Shc1 and the deletion of Shc1 exacerbates the lens vesicle defect caused by Frs2 and Shp2 deletion. These results reveal that Shc1 collaborates with Frs2 and Shp2 to target Grb2 in FGF signaling.

Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

The RhoGAP-myosin Myo9b regulates ocular lens pit morphogenesis.

During eye development the lens placode invaginates to form the lens pit. Further bending of lens epithelium and separation from ectoderm leads eventually to a spherical lens vesicle with enclosed extracellular fluid. Changes in epithelial morphology involve the actin cytoskeleton and its regulators. The myosin Myo9b is simultaneously an actin-based motor and Rho GTPase-activating protein that regulates actin cytoskeleton organization. Myo9b-deficient adult mice and embryos were analyzed for eye malformations and alterations in lens development. Myo9b-deficient mice showed a high incidence of microphthalmia and cataracts with occasional blepharitis. Formation of the lens vesicle during embryonic lens development was disordered in virtually all embryos. Lens placode invagination was less deep and gave rise to a conical structure instead of a spherical pit. At later stages either no lens vesicle was formed or a significantly smaller one that was not enclosed by the optic cup. Expression of the cell fate marker Pax6 was not altered. Staining of adherens junctions and F-actin was most intense at the tip of conical invaginations, suggesting that mechanical forces are not properly coordinated between epithelial cells that form the pit. Myo9b is a critical regulator of ocular lens vesicle morphogenesis during eye development.

Effects of Zinc Oxide Nanoparticles on Neural Tube Development in Early Chicken Embryos.

In recent years, Zinc oxide nanoparticles (ZnO-NPs) have been widely used as a mineral filter in cosmetics. Therefore, the possible exposure of pregnant women to ZnO-NPs is gradually increasing. Thus, we aimed to investigate the effect of ZnO-NPs on neural tube development in early chicken embryos. Fifty pathogen-free fertilized eggs were initially incubated for thirty hours. The eggs were divided into 5 groups. In the control group (C) the egg's apex was opened and closed without any administration. In the distilled water group (DW), 10 microliters of distilled water were injected into the sub-blastodermic area. ZnO-NP suspensions were prepared in distilled water and injected sub-blastodermically into the low, medium and high dose ZnO-NP groups (10 mg/kg, 30 mg/kg, and 50 mg/kg, respectively). Incubation was completed in 72 hours, and embryological and neural tube development was evaluated histologically with a light microscope. Embryos in all groups were evaluated according to the Hamburger-Hamilton (HH) staging. It was observed that the staging progressed by the developmental process between 68-72 hours, which is equivalent to the 19-20th stage of HH. Differentiated otic vesicle, optic cup, lens vesicle, pharynx, and Rathke\'s pouch were all observed in embryo sections. Both forebrain and hindbrain vesicles were easily distinguished in the sections by cranial flexion. Neural tube closure defect was not detected in any of the groups. In our observations, ZnO-NPs did not affect neural tube development at the applied dose ranges. We believe that additional studies with higher doses using a higher number of subjects will help clarify the conflicting data in the literature.

PECULIARITIES OF THE ORBIT MORPHOGENESIS AT AN EARLY PERIOD OF HUMAN ONTOGENESIS

The aim: To clarify the sources and determine the chronological sequence of the germs of anatomical structures of the orbit at the early period of human ontogenesis. Materials and methods: Using a complex of methods of morphological examination (morphometry, microscopy, three-dimensional computer reconstruction and statistical analysis) 30 series of consecutive histological sections of human embryos and prefetuses aged from 3 till 8 weeks od IUD (3.0-30.0 mm parietal-coccygeal length (PCL)) were studied. Results: At the 4rd week of IUD the orbital region is represented by the place of close contact of the neuroectoderm of the optic vesicle with the adjacent integumentary ectoderm,as well as the mesenchyme surrounding this place of contact. In embryos of 4.0 mm PCL, the rudiment of the optic stalk is observed as a result of the transformation of the junction of the area of the eye rudiment with the brain. Conclusions: 1. The rudiments of the organ of vision (lens placodes) appear in the 4rd week of IUD. At the 5th week of IUD as a result of gradual intussusception of crystalline placodes into the adjacent mesenchyme, lens pit are formed, and then - lens vesicles. 2. Rudiments of the extraocular muscles (except for the inferior oblique muscle) was detected at the end of the 5th week of IUD. The rudiment of the inferior oblique muscle develops from a single mesodermal islet located in the mesenchyme medially and below the eyeball. Simultaneously with the rudiments of the extraokular muscles, the trochlear and abductor nerves are develop and ingrown into the orbit. 3. The development of blood vessels of the orbit occurs from two sources - from the islands of local angiogenesis, which begins in the 5th week of IUD, and from extraorganic vessels, which can be traced in the form of a vascular network at the end of the 6th week of IUD. The combination of both sources is observed at the end of the 7th week of IUD. 4. The embryonic period of ontogenesis is the first critical period in the development of the human orbit, due to the formation of the muscles, nerve and vascular structures.

Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia

AbstractDuring an ENU (N‐ethyl‐N‐nitrosourea) mutagenesis screen, we observed a dominant small‐eye mutant mouse with viable homozygotes (gene symbol Aey69). We determined linkage to mouse chromosome 3 between the markers D3Mit188 and D3Mit11; sequencing demonstrated a 358A‐>C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71T‐>C (Val24Ala) mutation in the Gja8 gene. Analysis of eye development in the homozygous mutants documented an altered lens development starting from the lens vesicle stage including decreasing expression of crystallins and of lens‐specific transcription factors PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by BRN3 and OTX2. These changes in the retina happen in parallel with apoptotic processes in the lens vesicle. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. At postnatal stages, all principal retinal cell types are present, when the overgrowing retina fills the entire bulbus. Morpholino‐mediated knock‐down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations (cyclopia to microphthalmia). The wild‐type Hist2h3c1 mRNA can rescue the morpholino‐induced defects corroborating its specific function in lens development. The data highlight the importance of Hist2h3c1 (encoding a canonical H3.2 variant) in the formation of lens and retina.

The role of Wnt/β‐catenin signaling pathway in lens and retina development

AbstractWnt/β‐catenin signaling pathway functions by regulating the amount of the transcriptional co‐activator β‐catenin that controls key developmental gene expression programs. However, β‐catenin is known to have a dual function in cells. Apart from its key role as a nuclear effector of Wnt/β‐catenin signaling pathway (transcriptional function), β‐catenin is an integral structural component of cadherin‐based adherens junctions (cytoskeletal function). The essential role of β‐catenin during development is demonstrated by the finding that β‐catenin knockout mice fail to develop beyond embryonic day E6.5. Tissue‐specific deletion of β‐catenin gene in lens and neural retina resulted in a number of ocular phenotypes such as formation of ectopic lenses, failure to form lens vesicle, altered fiber cell differentiation and disruption of typical laminated structure of neural retina. In order to distinguish the phenotypes resulting from cytoskeletal or transcriptional function of β‐catenin we used mouse strain, in which the endogenous β‐catenin is replaced by mutant form of the protein, retaining cytoskeletal but lacking transcriptional function. We found that mice deficient for transcriptionally competent β‐catenin manifest abnormal development of lens epithelium and ciliary marginal zone of the neural retina. Our data unequivocally identify specific roles of canonical Wnt signaling for lens and neural retina development.

Verification and spatial mapping of TRPV1 and TRPV4 expression in the embryonic and adult mouse lens

The transient receptor protein vanilloid channels, TRPV1 and TRPV4, have recently been shown to be mechanosensors in the ocular lens that act to transduce physical changes in lens volume and internal hydrostatic pressure into the activation of signalling pathways in lens epithelial cells. These pathways in turn regulate ion and water transport to ensure that the optical properties of the lens remain constant. Despite the functional evidence that implicate the roles of TRPV1 and TRPV4 in the lens, their respective cellular expression patterns in the different regions of the lens has to date not been fully characterised. Using Western blotting we have confirmed that TRPV1 and TRPV4 are expressed throughout all regions (epithelium, outer cortex, inner cortex/core) of the adult mouse lens. Subsequent immunolabeling of lens cryosections confirmed that TRPV1 and TRPV4 are expressed throughout all regions of the lens, but revealed differentiation-dependent differences in the subcellular expression of the two channels in the different regions. In the epithelium and outer cortex, intense TRPV1 and TRPV4 labeling was predominately associated with the cytoplasm. In a discrete zone in the inner cortex, labeling for both proteins was greatly diminished, but could be enhanced by incubating sections with the detergent Triton X-100 to reveal TRPV1 and TRPV4 labelling that was associated with the membrane. This suggests that in this region of the lens there is a potential interacting protein that masks the binding of the TRPV1 and TRPV4 antibodies to their respective epitopes in the lens inner cortex. In the core of the lens, which contains the embryonic nucleus, TRPV1 and TRPV4 labelling was associated exclusively with fibre cell membranes. This labelling in the lens core of the adult mouse lens appeared to originate in early development as a similar membrane labelling was observed at embryonic day 10 (E10) of the cells in the lens vesicle that subsequently forms the embryonic nucleus in the adult lens. During subsequent stages of embryonic development TRPV1 and TRPV4 remained membranous in the inner cortex and core, while showing labelling that was associated with the cytoplasm in the superficial outer cortical region. The extent of cytoplasmic labelling for TRPV4, but not TRPV1, in this cortical region could however be dynamically regulated by cutting the zonules that normally attach the lens to the ciliary body. We have shown an early onset and continuous expression of TRPV1 and TRPV4 across all lens regions, and that TRPV4 can be dynamically trafficked into the membranes of differentiating fibre cells, results that suggests that these mechanosensitive channels may also be functionally active in lens fibre cells.

Retinal development and opsin gene expression during the juvenile development in red spotted grouper (Epinephelus akaara).

To produce healthy and stable seed production, we need to obtain information and understand vision that affects behavior of red spotted grouper. We examined their expression and retinal development during the juvenile development. Short-wavelength sensitive opsin (SWS2), a cone photoreceptor, began to be expressed from lens and ear vesicle formation stage and its expression increased until 10 days after hatching (dah). In case of middle-wavelength sensitive opsin (MWS), its expression was detected at 3 dah and reached the highest level at 21 dah. The expression of long-wavelength sensitive opsin (LWS) was first observed from 3 dah and their expression decreased thereafter. Rhodopsin, a rod photoreceptor, was found to be expressed from 2 dah and its expression reached the highest level at 50 dah. The outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer began to differentiate at 2 dah, while choroid first appeared at 4 dah so that the eyes became black. These results indicate that the development of retina mostly completes around 4 dah. It seems that the development of the retina and the expression of the opsin genes are closely related to the behavior such as hunting prey, considering that the timing of the completion of the development of the retina, the timing of gene expression, and the timing of completion of yolk absorption are similar.

Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia

During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358 A-> C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71 T-> C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development.

The Effect of Zinc-Deficient Pregnant Albino Rats on the Retinal Development of their Embryos and Offspring: Light Microscopic and Immunohistochemical Study

AbstractBackground: Zinc is involved in many biochemical func-tions. Over 300 enzymes require zinc for their activation and nearly 2000 transcription factors require zinc for gene expres-sion. Zinc is also an effective antioxidant and anti-inflammatory agent. Nutritional deficiency of zinc may affect nearly 2 billion subjects in the developing world.Aim of Study: Because of the prevalence of malnutrition throughout the world this study was carried out to detect the possible effects of zinc-deficient pregnant albino rats on the retinal development of their embryos and offspring.Material and Methods: Female rats after mating were randomly divided into 2 equal groups: One control group received single intraperitoneal injection of distilled water on day 9 of gestation and one experimental group received intraperitoneal injections of 1.10 phenanthroline (zinc chelator purchased from Sigma Company), in a single dose of 30 mg/kg on day 9 of gestation. From the embryos and offspring of both control and treated mothers (at the following prenatal gestational days: 13, 15, 17, 19 and the following postnatal days: (newborn, 7 days, 15 days and 21 days), retinal specimens were processed for Haematoxylin & Eosin staining technique and immunohistochemical study using caspase-3 antibody was used to detect apoptosis in the postnatal age groups.Results: The light microscopic study of the 13 days zinc deficient embryo retina showed apparent shrinkage of the lens vesicle and the optic cup was well formed but it appeared to be shrunken. The retinal pigment epithelium (RPE) of 15th, 17th and 19th day old zinc-deficient embryos consistently contained apoptotic cells with vacuolated cytoplasm and rarified nuclei. The neuroblastic layer showed apparent de-crease in thickness and less mitotic features. The ganglion cell layer revealed numerous deeply stained pyknotic nuclei in the 19th gestational day. Retinae of the newborn, 7 days, 15 days, 21 days-old zinc deficient albino rats showed that the (RPE) contained some vacuolated cells with pyknotic nuclei. The organization of the retina was altered in zinc deficient rats. In addition, apparent reduction in the total retinal thickness and degenerative changes in some layers were observed. By the immunohistochemical study, there was intense positive caspase-3 reaction in all layers of the treated rat retina.Conclusion: This study revealed that zinc has a role in the developing rat retina and its deficiency causes cytoarchi-tectural and maturational changes in the retina of the albino rats. These histological changes may impair the visual functions of the retina; so it is recommended that further investigations should be carried out to determine the relevancy of deprivation of zinc as an etiologic factor in the development of retinal degeneration.

Identification of Novel Gata3 Distal Enhancers Active in Mouse Embryonic Lens.

Background: The tissue‐specific transcriptional programs during normal development require tight control by distal cis‐regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence‐specific DNA‐binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T‐cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown. Results: Here we describe the identification of a novel bipartite Gata3 lens‐specific enhancer located ∼18 kb upstream from its transcriptional start site. We also found that a 5‐kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP‐1, Ets‐, and Smad1/5‐binding sites as well as binding sites for lens‐associated DNA‐binding factors. Transient transfection studies of the promoter with the bipartite enhancer showed enhanced activation by BMP4 and FGF2. Conclusions: These studies identify a novel distal enhancer of Gata3 with high activity in lens and indicate that BMP and FGF signaling can up‐regulate expression of Gata3 in differentiating lens fiber cells through the identified Gata3 enhancer and promoter elements. Developmental Dynamics 247:1186–1198, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists

The Teratogenic Effects of Glyphosate Based Herbicide (GBH) on the Development of Chick Embryos

Objective: This study was conducted to investigate the teratogenic effects of GBH by using chick embryo as an animal model. Methods: The equal volume of 0.1 ml of 0.01%, 0.05%, 0.3%, and 0.5% w/v glyphosate solution were injected into yolk sacs of fertilized White Leghorn eggs at 21 h of incubation and repeated at the volume of 0.05 ml on the 3rd day of incubation. The embryos were observed for abnormalities on day 3, 6 and 10 of incubation. Results: The results showed that the mortality percentages increased as the concentration of glyphosate increased. Day 3 chick embryos showed retardation of development and several abnormalities, for instance, the irregular shape of the brain vesicles with an opening of anterior neuropore, small eye primordia with the optic cup and lens vesicle retardation, looser of the heart looping with dilated lumen, lesser number of branchial arches, absent of limb bud or tail fold. Day 6 chick embryos showed severe retardation of several organs. Microphthalmia, anophthalmia, ectopia cordis and ectopic viscerae were observed in day 6 chick embryos. On day 10, most embryos died earlier and living embryos showed normal external features but delayed ossifications which were significantly different from the control (P<0.05). Conclusion: Glyphosate was toxic to chick embryos which directly caused mortality and was also a powerful teratogen which caused growth retardation and malformations on day 3 and day 6 and skeletal alteration on day. The effects were predicted to occur in other embryos including humans. Pregnant women should avoid contamination with GBH especially in the first trimester of pregnancy.

Bi-allelic Loss-of-Function Variants in DNMBP Cause Infantile Cataracts

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C>T (p.Arg271∗) in large family F385 (nine affected individuals; LOD score = 5.18 at θ = 0), frameshiftdeletion c.2947_2948del (p.Asp983∗) in family F372 (two affected individuals), and frameshift variant c.2852_2855del (p.Thr951Metfs∗41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sif), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a ∼50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.

Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway.

ObjectiveTo investigate the influence of Msx2 conditional gene knockout during lens development in mice.MethodsLens-specific Msx2 knockout mice were generated using the Cre-loxP system. The eyes of Msx2 conditional knockout (Msx2CKO) and wild-type (Msx2WT) mice were examined during embryonic and early postnatal periods using histological, immunofluorescence, in situ hybridization, cell proliferation, apoptosis, and mRNA microarray analyses.ResultsMsx2CKO mice exhibited small lens formation and microphthalmia after birth, while Msx2CKO embryos exhibited a persistent lens stalk, small lens formation, and microphthalmia. Conditional deletion of Msx2 also led to an increased apoptosis rate, a significant reduction in FoxE3 expression, and an upregulation of Prox1 expression in the lens vesicle during the early embryonic period. Microarray comparison of Msx2CKO and Msx2WT lens transcriptomes identified a large number of differentially expressed genes. Real-time PCR showed that Casp8 and Casp3 expression was upregulated in Msx2CKO mice at post-natal day 1.ConclusionThe activation of apoptosis through the caspase-8/caspase-3 signaling pathway, together with the downregulation of FoxE3 expression, appeared to account for the smaller lens formation in Msx2CKO mice.

Apoptosis generates mechanical forces that close the lens vesicle in the chick embryo

During the initial stages of eye development, optic vesicles grow laterally outward from both sides of the forebrain and come into contact with the surrounding surface ectoderm (SE). Within the region of contact, these layers then thicken locally to create placodes and invaginate to form the optic cup (primitive retina) and lens vesicle (LV), respectively. This paper examines the biophysical mechanisms involved in LV formation, which consists of three phases: (1) lens placode formation; (2) invagination to create the lens pit (LP); and (3) closure to form a complete ellipsoidally shaped LV. Previous studies have suggested that extracellular matrix deposited between the SE and optic vesicle causes the lens placode to form by locally constraining expansion of the SE as it grows, while actomyosin contraction causes this structure to invaginate. Here, using computational modeling and experiments on chick embryos, we confirm that these mechanisms for Phases 1 and 2 are physically plausible. Our results also suggest, however, that they are not sufficient to close the LP during Phase 3. We postulate that apoptosis provides an additional mechanism by removing cells near the LP opening, thereby decreasing its circumference and generating tension that closes the LP. This hypothesis is supported by staining that shows a ring of cell death located around the LP opening during closure. Inhibiting apoptosis in cultured embryos using caspase inhibitors significantly reduced LP closure, and results from a finite-element model indicate that closure driven by cell death is plausible. Taken together, our results suggest an important mechanical role for apoptosis in lens development.

Unexpected ultrastructure of an eye in Spiralia: the larval ocelli of Procephalothrix oestrymnicus (Nemertea)

The evolution of eyes and their constituent photoreceptor cells in Metazoa in general and in Protostomia in particular remains unresolved with present morphological and developmental genetic data. This is mainly due to the lack of comprehensive ultrastructural data in some lineages, such as in the spiralian taxon Nemertea. The eyes of the derived Neonemertea possess rhabdomeric photoreceptor cells, considered typical of the protostome lineage. In the more basally branching palaeonemertean lineages, ultrastructural data on the eyes are wanting. Ultrastructural investigation of the eyes of the larva of the palaeonemertean Procephalothrix oestrymnicus reveals that, although in a similar position as the eyes of adult neonemertean species, the eyes in palaeonemertean larvae differ fundamentally from the expected protostomian type: They consist of one shading-pigment cell that forms a closed optical cavity embedded in the epidermis. Apart from basally distributed shading-pigment vesicles, the pigment cell apically possesses epidermal cilia and microvilli as well as sub-apical, tubular lens vesicles. Two ciliary photoreceptor cells project flattened ciliary membranes into the optical cavity formed by the pigment cell, whereas their basal portions are situated outside of the optical cavity, next to the shading-pigment cell. Although the structure of the eye in P. oestrymnicus is unparalleled in Nemertea, ciliary photoreceptor cells have been found in larval eyes of several other spiralian lineages. Occurrence of additional ciliary-type eyes in Spiralia deepens the doubts regarding the validity of the hypothesis of an exclusively rhabdomeric line of visual photoreceptor cell evolution in Protostomia.

Promoter-enhancer looping and shadow enhancers of the mouse αA-crystallin locus.

ABSTRACTGene regulation by enhancers is important for precise temporal and spatial gene expression. Enhancers can drive gene expression regardless of their location, orientation or distance from the promoter. Changes in chromatin conformation and chromatin looping occur to bring the promoter and enhancers into close proximity. αA-crystallin ranks among one of the most abundantly expressed genes and proteins in the mammalian lens. The αA-crystallin locus is characterized by a 16 kb chromatin domain marked by two distal enhancers, 5′ DCR1 and 3′ DCR3. Here we used chromatin conformation capture (3C) analysis and transgenic approaches to analyze temporal control of the mouse αA-crystallin gene. We find that DCR1 is necessary, but not sufficient alone to drive expression at E10.5 in the mouse lens pit. Chromatin looping revealed interaction between the promoter and the region 3′ to DCR1, identifying a novel enhancer region in the αA-crystallin locus. We determined that this novel enhancer region, DCR1S, recapitulates the temporal control by DCR1. Acting as shadow enhancers, DCR1 and DCR1S are able to control expression in the lens vesicle at E11.5. It remains to be elucidated however, which region of the αA-crystallin locus is responsible for expression in the lens pit at E10.5.

N-myc regulates growth and fiber cell differentiation in lens development

Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc-deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc-deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIβ. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis.