Doctor of Philosophy

dissertationDevelopment of the neural retina is a complex process requiring step-wise induction of specified retinal progenitor cells (RPCs) from the neural ectoderm and their coordinated proliferative expansion and differentiation into the mature neurons and glia of the adult retina. The homeobox gene Vsx2 is expressed in RPCs and required for proper execution of this retinal program. In the absence of Vsx2 function, maintenance of retinal identity, RPC proliferation, and retinal neurogenesis are disrupted, with serious consequences on overall ocular development and visual function. Despite the obvious importance of Vsx2, an understanding of the molecular mechanisms by which Vsx2 regulates these processes is lacking and few direct targets have been identified. To further define the role of Vsx2, we sought to determine the relationship between Vsx2 and the extrinsic signaling pathways regulating RPC properties. Through the analysis of genetic chimeras and candidate signaling pathways, we evaluated the contribution of Vsx2 to the regulation of extrinsic signaling pathways involved in the cellular processes of retinal development. We find that Vsx2 mediates the response of RPCs to the signals driving retinal specification and maintenance, largely through cell autonomous repression of the RPE determinant Mitf. We also find that Vsx2-deficient RPCs exhibit a robust cell autonomous delay in the initiation of retinal neurogenesis, revealing an essential role for Vsx2 in the temporal regulation of neurogenic competence. In contrast, we find that regulation of RPC proliferation by Vsx2 involves significant cell nonautonomous iv contributions, suggesting an important role for Vsx2 in regulating the availability of retinal proliferation signals. Analysis of the retinal mitogen Sonic Hedgehog (Shh) and its signaling pathway in Vsx2-deficient retinas further supported this role, demonstrating that Vsx2 is required to ensure sufficient availability of Shh. We also describe a potential role for Vsx2 in the regulation of RPC responsiveness to Hedgehog (Hh) pathway stimulation. Taken together, the data presented in this dissertation demonstrate the requirement for Vsx2 in promoting both the reception and availability of the extrinsic signals necessary for the regulation of RPC properties, thereby ensuring the proper growth and differentiation of this important sensory tissue

Distinctive synaptic structural motifs link excitatory retinal interneurons to diverse postsynaptic partner types

Summary: Neurons make converging and diverging synaptic connections with distinct partner types. Whether synapses involving separate partners demonstrate similar or distinct structural motifs is not yet well understood. We thus used serial electron microscopy in mouse retina to map output synapses of cone bipolar cells (CBCs) and compare their structural arrangements across bipolar types and postsynaptic partners. Three presynaptic configurations emerge—single-ribbon, ribbonless, and multiribbon synapses. Each CBC type exploits these arrangements in a unique combination, a feature also found among rabbit ON CBCs. Though most synapses are dyads, monads and triads are also seen. Altogether, mouse CBCs exhibit at least six motifs, and each CBC type uses these in a stereotypic pattern. Moreover, synapses between CBCs and particular partner types appear biased toward certain motifs. Our observations reveal synaptic strategies that diversify the output within and across CBC types, potentially shaping the distinct functions of retinal microcircuits

Exploring aesthetics, design, and experience in the age of semiotic technology

nonPeerReviewe

The Dominating Role of N-Deacetylase/N-Sulfotransferase 1 in Forming Domain Structures in Heparan Sulfate*

Heparan sulfate (HS) is a highly sulfated polysaccharide participated in essential physiological functions from regulating cell growth to blood coagulation. HS contains sulfated domains known as N-S domains and low sulfate domains known as N-Ac domains. The distribution of the domain structures is likely governed by the action of glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here, we sought to determine the substrate specificity of NDST using model substrates and recombinant NDST protein. We discovered that NDST-1 carries out the modification in a highly ordered fashion. The enzyme sulfates the substrate from the nonreducing end toward the reducing end consecutively, leading to the product with a cluster of N-sulfo glucosamine residues. Furthermore, a preexisting N-sulfo glucosamine residue prevents the action of NDST-1 at the residues immediately located at the nonreducing end, allowing the formation of an N-Ac domain. Our results provide the long sought evidence for understanding the formation of sulfated versus nonsulfated domains in the HS isolated from cells and tissues. The study demonstrates the regulating role of NDST-1 in mapping the sulfation patterns of HS

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides*

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C5-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures