Bharesh K. Chauhan

Balanced Rac1 and RhoA activities regulate cell shape and drive invagination morphogenesis in epithelia

Epithelial bending is a central feature of morphogenesis in animals. Here we show that mutual antagonism by the small Rho GTPases Rac1 and RhoA determines cell shape, tissue curvature, and invagination activity in the model epithelium of the developing mouse lens. The epithelial cells of the invaginating lens placode normally elongate and change from a cylindrical to an apically constricted, conical shape. RhoA mutant lens placode cells are both longer and less apically constricted than control cells, thereby reducing epithelial curvature and invagination. By contrast, Rac1 mutant lens placode cells are shorter and more apically restricted than controls, resulting in increased epithelial curvature and precocious lens vesicle closure. Quantification of RhoA- and Rac1-dependent pathway markers over the apical–basal axis of lens pit cells showed that in RhoA mutant epithelial cells there was a Rac1 pathway gain of function and vice versa. These findings suggest that mutual antagonism produces balanced activities of RhoA-generated apical constriction and Rac1-dependent cell elongation that controls cell shape and thus curvature of the invaginating epithelium. The ubiquity of the Rho family GTPases suggests that these mechanisms are likely to apply generally where epithelial morphogenesis occurs.

Loss of RhoA in neural progenitor cells causes the disruption of adherens junctions and hyperproliferation

The organization of neural progenitors in the developing mammalian neuroepithelium is marked by cadherin-based adherens junctions. Whereas RhoA, a founding member of the small Rho GTPase family, has been shown to play important roles in epithelial adherens junctions, its physiological roles in neural development remain uncertain due to the lack of specific loss-of-function studies. Here, we show that RhoA protein accumulates at adherens junctions in the developing mouse brain and colocalizes to the cadherin–catenin complex. Conditional deletion of RhoA in midbrain and forebrain neural progenitors using Wnt1-Cre and Foxg1-Cre mice, respectively, disrupts apical adherens junctions and causes massive dysplasia of the brain. Furthermore, RhoA-deficient neural progenitor cells exhibit accelerated proliferation, reduction of cell- cycle exit, and increased expression of downstream target genes of the hedgehog pathway. Consequently, both lines of conditional RhoA-deficient embryos exhibit expansion of neural progenitor cells and exencephaly-like protrusions. These results demonstrate a critical role of RhoA in the maintenance of apical adherens junctions and the regulation of neural progenitor proliferation in the developing mammalian brain.

A Trio-RhoA-Shroom3 pathway is required for apical constriction and epithelial invagination.

Epithelial invagination is a common feature of embryogenesis. An example of invagination morphogenesis occurs during development of the early eye when the lens placode forms the lens pit. This morphogenesis is accompanied by a columnar-to-conical cell shape change (apical constriction or AC) and is known to be dependent on the cytoskeletal protein Shroom3. Because Shroom3-induced AC can be Rock1/2 dependent, we hypothesized that during lens invagination, RhoA, Rock and a RhoA guanine nucleotide exchange factor (RhoA-GEF) would also be required. In this study, we show that Rock activity is required for lens pit invagination and that RhoA activity is required for Shroom3-induced AC. We demonstrate that RhoA, when activated and targeted apically, is sufficient to induce AC and that RhoA plays a key role in Shroom3 apical localization. Furthermore, we identify Trio as a RhoA-GEF required for Shroom3-dependent AC in MDCK cells and in the lens pit. Collectively, these data indicate that a Trio-RhoA-Shroom3 pathway is required for AC during lens pit invagination.

Cdc42- and IRSp53-dependent contractile filopodia tether presumptive lens and retina to coordinate epithelial invagination

The vertebrate lens provides an excellent model with which to study the mechanisms required for epithelial invagination. In the mouse, the lens forms from the head surface ectoderm. A domain of ectoderm first thickens to form the lens placode and then invaginates to form the lens pit. The epithelium of the lens placode remains in close apposition to the epithelium of the presumptive retina as these structures undergo a coordinated invagination. Here, we show that F-actin-rich basal filopodia that link adjacent presumptive lens and retinal epithelia function as physical tethers that coordinate invagination. The filopodia, most of which originate in the presumptive lens, form at E9.5 when presumptive lens and retinal epithelia first come into close contact, and have retracted by E11.5 when invagination is complete. At E10.5 - the lens pit stage - there is approximately one filopodium per epithelial cell. Formation of filopodia is dependent on the Rho family GTPase Cdc42 and the Cdc42 effector IRSp53 (Baiap2). Loss of filopodia results in reduced lens pit invagination. Pharmacological manipulation of the actin-myosin contraction pathway showed that the filopodia can respond rapidly in length to change inter-epithelial distance. These data suggest that the lens-retina inter-epithelial filopodia are a fine-tuning mechanism to assist in lens pit invagination by transmitting the forces between presumptive lens and retina. Although invagination of the archenteron in sea urchins and dorsal closure in Drosophila are known to be partly dependent on filopodia, this mechanism of morphogenesis has not previously been identified in vertebrates.

Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover.

Epithelial morphogenesis: the mouse eye as a model system.

Morphogenesis is the developmental process by which tissues and organs acquire the shape that is critical to their function. Here, we review recent advances in our understanding of the mechanisms that drive morphogenesis in the developing eye. These investigations have shown that regulation of the actin cytoskeleton is central to shaping the presumptive lens and retinal epithelia that are the major components of the eye. Regulation of the actin cytoskeleton is mediated by Rho family GTPases, by signaling pathways and indirectly, by transcription factors that govern the expression of critical genes. Changes in the actin cytoskeleton can shape cells through the generation of filopodia (that, in the eye, connect adjacent epithelia) or through apical constriction, a process that produces a wedge-shaped cell. We have also learned that one tissue can influence the shape of an adjacent one, probably by direct force transmission, in a process we term inductive morphogenesis. Though these mechanisms of morphogenesis have been identified using the eye as a model system, they are likely to apply broadly where epithelia influence the shape of organs during development.

Subterranean mammals show convergent regression in ocular genes and enhancers, along with adaptation to tunneling

The underground environment imposes unique demands on life that have led subterranean species to evolve specialized traits, many of which evolved convergently. We studied convergence in evolutionary rate in subterranean mammals in order to associate phenotypic evolution with specific genetic regions. We identified a strong excess of vision- and skin-related genes that changed at accelerated rates in the subterranean environment due to relaxed constraint and adaptive evolution. We also demonstrate that ocular-specific transcriptional enhancers were convergently accelerated, whereas enhancers active outside the eye were not. Furthermore, several uncharacterized genes and regulatory sequences demonstrated convergence and thus constitute novel candidate sequences for congenital ocular disorders. The strong evidence of convergence in these species indicates that evolution in this environment is recurrent and predictable and can be used to gain insights into phenotype–genotype relationships.

Coenzyme Q10 in the Treatment of Corneal Edema in Kearns-Sayre: Is There an Application in Fuchs Endothelial Corneal Dystrophy?

Purpose: Corneal involvement in mitochondrial disease is seldom described. Kearns-Sayre syndrome (KSS) is a mitochondrial disorder characterized by retinitis pigmentosa, external ophthalmoplegia, and heart block. We report 2 patients with KSS with corneal lesions involving the endothelium, which improved with Coenzyme Q10 (CoQ10). Based on recent research regarding the role of dysfunctional oxidative metabolism in Fuchs Endothelial Corneal Dystrophy (FECD), we propose that mitochondrial diseases and FECD share a final pathway. Methods: A chart review was performed and a review of the literature was completed with a PubMed search using the terms “Kearns-Sayre Syndrome”, “mitochondria”, “endothelium”, “Fuchs endothelial corneal dystrophy”, and “cornea”. Results: There are 19 reports of corneal involvement in clinical phenotypes of mitochondrial disease. Nine of these 19 cases had findings consistent with KSS. Our patients with KSS had microcystic changes throughout the cornea and excrescences on the endothelial surface seen with ultrasound biomicroscopy, similar to the clinical findings in FECD. CoQ10 improved corneal disease in both children. CoQ10 deficiency has been reported in a variety of mitochondrial diseases, and efficacy of supplementation has been demonstrated. It may be beneficial in these patients because of its antioxidant properties and role in oxidative phosphorylation. Conclusions: The common deletion found in patients with KSS has recently been implicated in FECD, which has recently been shown to be a disease related to dysfunctional oxidative metabolism. Future research should explore the use of antioxidants, such as CoQ10 in patients with FECD.

Congenital aniridia: etiology, manifestations and management

ABSTRACT Congenital aniridia manifests as total or partial absence of the iris, caused most commonly by mutations in PAX6, FOXC1, PITX2, and CYP1B1. Recently two new genes, FOXD3 and TRIMM44, have also been implicated in isolated studies. We discuss the genotype-phenotype correlations for the main implicated genes. Classic aniridia is a panocular condition, which includes aniridia, cataract, corneal pannus, foveal, and optic nerve hypoplasia associated with mutations in the PAX6 gene. Classical aniridia is due to PAX6 mutations, while other genes contribute to aniridia-like phenotypes. We review the challenges involved in the management of aniridia and discuss various surgical interventions. The clinical importance of defining the genotype in cases of congenital aniridia has become acutely apparent with the advent of possible therapies for classical aniridia, which are discussed.

Isolated Sagittal Synostosis in a Boy with Craniofrontonasal Dysplasia and a Novel EFNB1 Mutation

Summary: Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder that shows greater severity in females and is largely attributed to mutations in EFNB1. A 7-year-old boy presented with hypertelorism, broad nasal root, midfacial hypoplasia, mandibular prognathia, ptosis, and scaphocephaly was clinically diagnosed with CFNS. Three-dimensional computed tomographic scans confirmed the isolated sagittal synostosis. His mother also showed clinical features of CFNS, but less severe. Genetic tests uncovered a novel C to T mutation at nucleotide 466 (c.466C>T) in exon 1 of EFNB1 for both. To the best of our knowledge, this is the only reported incident of CFNS in a male child exhibiting isolated sagittal synostosis.