Ravi Munjaal

Gravitaxis in Drosophila melanogaster: a forward genetic screen

Perception of the earths gravitational force is essential for most forms of animal life. However, little is known of the molecular mechanisms and neuronal circuitry underlying gravitational responses. A forward genetic screen using Drosophila melanogaster that provides insight into these characteristics is described here. Vertical choice mazes combined with additional behavioral assays were used to identify mutants specifically affected in gravitaxic responses. Twenty‐three mutants were selected for molecular analysis. As a result, 18 candidate genes are now implicated in the gravitaxic behavior of flies. Many of these genes have orthologs across the animal kingdom, while some are more specific to Drosophila and invertebrates. One gene (yuri) located close to a known locus for gravitaxis has been the subject of more extensive analysis including confirmation by transgenic rescue.

Genetics of Graviperception in Animals

Gravity is a constant stimulus for life on Earth and most organisms have evolved structures to sense gravitational force and incorporate its influence into their behavioral repertoire. Here we focus attention on animals and their diverse structures for perceiving and responding to the gravitational vector-one of the few static reference stimuli for any mobile organism. We discuss vertebrate, arthropod, and nematode models from the perspective of the role that genetics is playing in our understanding of graviperception in each system. We describe the key sensory structures in each class of organism and present what is known about the genetic control of development of these structures and the molecular signaling pathways operating in the mature organs. We also discuss the role of large genetic screens in identifying specific genes with roles in mechanosensation and graviperception.

Expression of pro‐ and anti‐angiogenic factors during the formation of the periocular vasculature and development of the avian cornea

Background: During embryonic development, endothelial precursor cells (angioblasts) migrate relatively long distances to form the primary vascular plexus. The migratory behavior of angioblasts and localization of the primitive blood vessels is tightly regulated by pro‐angiogenic and anti‐angiogenic factors encountered in the embryonic environment. Despite the importance of corneal avascularity to proper vision, it is not known when avascularity is established in the developing cornea and how pro‐ and anti‐angiogenic factors regulate this process. Results and Discussion: Using Tg(tie1:H2B:eYFP) transgenic quail embryos to visualize fluorescently labeled angioblasts, we show that the presumptive cornea remains avascular despite the invasion of cells from the periocular region where migratory angioblasts reside and form the primary vasculature. Semiquantitative reverse transcriptase polymerase chain reaction analysis and spatiotemporal examination of gene expression revealed that pro‐ and anti‐angiogenic factors were expressed in patterns indicating their potential roles in angioblast guidance. Conclusions: Our findings show for the first time that chick corneal avascularity is established and maintained during development as the periocular vasculature forms. We also identify potential candidate pro‐ and anti‐angiogenic factors that may play crucial roles during vascular patterning in the anterior eye. Developmental Dynamics 242:738–751, 2013.

Expression of CXCL12 and CXCL14 during eye development in chick and mouse.

Vertebrate eye development is a complex multistep process coordinated by signals from the lens, optic cup and periocular mesenchyme. Although chemokines are increasingly being recognized as key players in cell migration, proliferation, and differentiation during embryonic development, their potential role during eye development has not been examined. In this study, we demonstrate by section in situ hybridization that CXCL12 and CXCL14 are expressed during ocular development. CXCL12 is expressed in the periocular mesenchyme, ocular blood vessels, retina, and eyelid mesenchyme, and its expression pattern is conserved between chick and mouse in most tissues. Expression of CXCL14 is localized in the ocular ectoderm, limbal epithelium, scleral papillae, eyelid mesenchyme, corneal keratocytes, hair follicles, and retina, and it was only conserved in the upper eyelid ectoderm of chick and mouse. The unique and non-overlapping patterns of CXCL12 and CXCL14 expression in ocular tissues suggest that these two chemokines may interact and have important functions in cell proliferation, differentiation and migration during eye development.

Distinct Roles for Neuropilin1 and Neuropilin2 during Mouse Corneal Innervation

Trigeminal sensory innervation of the cornea is critical for protection and synthesis of neuropeptides required for normal vision. Little is known about axon guidance during mammalian corneal innervation. In contrast to the chick where a pericorneal nerve ring forms via Npn/Sema signaling, mouse corneal axons project directly into the presumptive cornea without initial formation of an analogous nerve ring. Here we show that during development of the mouse cornea, Npn1 is strongly expressed by the trigeminal ganglion whereas Npn2 is expressed at low levels. At the same time Sema3A and Sema3F are expressed in distinct patterns in the ocular tissues. Npn1sema−/− mutant corneas become precociously and aberrantly innervated by nerve bundles that project further into the corneal stroma. In contrast, stromal innervation was not affected in Npn2−/− mutants. The corneal epithelium was prematurely innervated in both Npn1sema−/− and Npn2−/− mutants. These defects were exacerbated in Npn1sema−/−;Npn2−/− double mutants, which in addition showed ectopic innervation of the region between the optic cup and lens vesicle. Collectively, our data show that Sema3A/Npn1 and Sema3F/Npn2 signaling play distinct roles and both are required for proper innervation of the mouse cornea.

Knockdown of CXCL14 disrupts neurovascular patterning during ocular development.

The C-X-C motif ligand 14 (CXCL14) is a recently discovered chemokine that is highly conserved in vertebrates and expressed in various embryonic and adult tissues. CXCL14 signaling has been implicated to function as an antiangiogenic and anticancer agent in adults. However, its function during development is unknown. We previously identified novel expression of CXCL14 mRNA in various ocular tissues during development. Here, we show that CXCL14 protein is expressed in the anterior eye at a critical time during neurovascular development and in the retina during neurogenesis. We report that RCAS-mediated knockdown of CXCL14 causes severe neural defects in the eye including precocious and excessive innervation of the cornea and iris. Absence of CXCL14 results in the malformation of the neural retina and misprojection of the retinal ganglion neurons. The ocular neural defects may be due to loss of CXCL12 modulation since recombinant CXCL14 diminishes CXCL12-induced axon growth in vitro. Furthermore, we show that knockdown of CXCL14 causes neovascularization of the cornea. Altogether, our results show for the first time that CXCL14 plays a critical role in modulating neurogenesis and inhibiting ectopic vascularization of the cornea during ocular development.