Mengqing Xiang - US grants

DESCRIPTION (Adapted from applicant's abstract): The proposal is concerned with studies designed to analyze both in vitro and in vivo, the function of Brn-3b, a POU-domain transcription factor, in RGC development, and to explore underlying mechanisms for RGC degeneration under pathological conditions using the brn-3b gene locus as a molecular tool. First, transcriptional properties and structure/function relationships of Brnb-3b will be analyzed using biochemical approaches including gel mobility shift, methylation interference foot printing, and co-transfection transcription assays. These in vitro studies will provide initial information on how Brn-3b may specifically control its target gene expression in RGC development. Secondly, various histochemical and immunochemical approaches will be utilized to investigated whether Brn-3b is activated in dividing retinoblasts, and to compare cell proliferation and death between Brn-3b (+/+) and (-/-) retinas. These in vivo studies are expected to gain crucial information regarding the timing and mode of Brn-3b function during RGC development. Thirdly, Brb-3b will be replaced by homologous recombination in mice with the alkaline phosphatase reporter to examine the projections of axons, or with bcl-2 gene to study rescue of apoptosis in Brn-3b-expressing RGCs. The resulting data will help in understanding the function of Brn-3b in RGCs, and may also shed light on degeneration of RGCs in pathological conditions. Together, the proposed studies on Brn-3b function will provide fundamental insights into the molecular mechanism that control mammalian retinal development. In the long term, the information should help better understand the mechanisms leading to certain blinding disorders, and also may provide new directions for treatment modalities.

DESCRIPTION (provided by applicant): A central issue in developmental neurobiology is understanding the molecular regulatory networks that control neuronal specification, differentiation and maintenance. A number of homeodomain transcription factors have been shown to be key molecular players in the control of neurogenesis. We have demonstrated that the POU homeodomain transcription factors Brn3a and Brn3b play essential roles during sensorineural development. The Barhl1 homeodomain factor is selectively expressed in the inner ear sensorineural epithelium as well as in the central nervous system (CNS), suggesting that it may also play an important role in sensorineural development and CNS development. This proposal aims to address the fundamental mechanisms governing vertebrate neural development, using homeodomain-containing transcription factors as models for our analyses. The studies outlined in this proposal are designed to provide integrated approaches for understanding the molecular and cellular mechanisms by which homeodomain transcription factors control sensonneural development and CNS development. Three specific aims will be pursued. First, various developmental defects will be analyzed in the spiral, vestibular and geniculate ganglia of single and compound Brn3a and Brn3b knockout mice. The goal is to understand in vivo the developmental and cellular processes that Brn3a and/or Brn3b regulate during development of the facial-stato-acoustic ganglion. Second, to identify Brn3a binding proteins that may modulate Brn3a selection and activation of target genes during sensorineural development, novel Brn3a protein partners will be isolated using a yeast two-hybrid screening approach. Their functional relevance will be investigated by studying their effects on DNA-binding, transcriptional property and subcellular localization of Brn3a, and by examining their developmental expression patterns. Third, to understand in vivo the roles that Barhl1 plays during vertebrate inner ear and CNS development, we will generate and characterize Barhl1 knockout mice and analyze the biological consequences of forced expression of Barhl1 in the chick cerebellum. Together, these proposed studies will provide important insights into the in vivo biological activities of Brn3 and Barhl1 transcription factors as well as general insights into the molecular mechanisms that govern mammalian neurogenesis and neurological disorders.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this application is to understand the molecular bases that regulate the determination, differentiation and maintenance of different retinal cell types. A number of transcription factors have been shown to play a key role during vertebrate retinogenesis. Among them is the POU domain transcription factor Brn3b which is critically required for the development of retinal ganglion cells. The Barhl2 homeodomain transcription factor may also play an important role in retinal development as it is expressed in ganglion cells as well as in neurons within the inner nuclear layer of the retina. [unreadable] [unreadable] The studies outlined in this application are designed to provide integrated approaches to address the fundamental mechanisms governing vertebrate retinal development, using the Brn3b and Barhl2 transcription factors as models for our analyses. Three specific aims will be pursued: i) to investigate the mechanisms by which Bm3b controls the differentiation and survival of retinal ganglion cells. These studies aim to identify Brn3b downstream genes by a "candidate gene" approach as well as the microarray technology followed by tests of their biological significance; ii) to identify and functionally characterize proteins interacting with Brn3b. We aim to identify Brn3b binding proteins that may regulate the selection and activation of its target genes during retinal development. Novel protein partners for Bm3b will be isolated using a yeast two-hybrid screening approach. Their functional relevance will be investigated by studying their effects on DNA-binding and transcriptional properties of Brn3b, and by examining their expression patterns and effects on Brn3b-mediated retinal ganglion cell differentiation; iii) to study in vivo the role of Barhl2 gene during retinal development. Two complementary approaches will be employed to analyze the in vivo functions of Barhl2 during retinogenesis. One is a loss-of-function approach involving targeted gene disruption to produce mice deficient for Barhl2. The other is a "gain-of-function" approach involving retrovirus-mediated overexpression of Barhl2 in the chick and mouse retina. Together, these proposed studies will provide important insights into the molecular regulatory networks that govern mammalian retinal development and may provide the foundation for better understanding and treatment of certain retinal disorders.

Project Summary: The long-term objective of this proposal is to understand the molecular events that lead to the determination and differentiation of different retinal cell types. During mammalian retinogenesis, seven classes of cells are specified from multipotent progenitors by the action of various intrinsic and extrinsic factors. Recent molecular genetic studies involving loss-of-function and gain-of-function approaches have uncovered a number of transcription factors as pivotal intrinsic regulators of retinogenesis. These factors are found to act at different developmental processes to establish progenitor multipotency, define progenitor competence, determine cell fates, and/or specify cell types and subtypes. Therefore, transcription factors play key roles in controlling cell specification and differentiation during retinogenesis. Despite these important advances, however, the molecular targets and signaling events downstream from many transcription factors involved in retinal development still remain poorly understood. In this application, experiments are proposed that will focus on the molecular and developmental events regulated by the Foxn4 winged helix/forkhead transcription factor and its downstream molecules. Our previous studies have demonstrated a crucial role for Foxn4 in competence acquisition and/or fate commitment to amacrine and horizontal cells. However, it is unclear at present what are the genes and signaling events that act genetically downstream of Foxn4 to mediate its critical functions. To address these issues, four specific aims will be pursued: i) to analyze the role of Foxn4 in the specification of photoreceptor cells. Utilizing molecular, cellular and overexpression approaches, we aim to investigate in Foxn4 null retinas the expression profiles of photoreceptor and ganglion regulator genes, and the generation and distribution patterns of photoreceptor cells, as well as to examine the effect of misexpressed Foxn4 on the differentiation of photoreceptor cells;ii) to investigate Dll4-Notch signaling events during retinogenesis by overexpression analysis. We aim to employ an overexpression approach to determine the effect of Dll4 and its dominant-negative form on retinal cell specification and differentiation, and to investigate by chromatin immunoprecipitation and electrophoretic mobility shift assays whether Foxn4 has the ability to directly bind and activate the Dll4 promoter;iii) to study the biological function of Dll4 during retinogenesis by conditional gene targeting. We aim to generate and characterize retina-specific Dll4 knockout mice to identify the developmental and cellular processes that Dll4-Notch signaling regulates and establish a functional relationship between Dll4 and Foxn4 during mammalian retinogenesis;and iv) to map the fate of Foxn4- expressing retinal progenitors. Our goal is to determine the lineages of Foxn4-expressing retinal progenitors using the Cre-loxP fate-mapping strategy. The proposed studies together are expected to provide important insights into the genetic regulatory networks involved in retinal cell specification and differentiation.