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Functional study of transcription factors during retinal development

by
Haisong Jiang
B.S., Shanghai JiaoTong University - 2000 Shanghai, China



Thesis Advisor: Mengqing Xiang, Ph.D.
Graduate Program in Molecular Genetics,
Microbiology & Immunology

CABM, Room 010
Piscataway

Wednesday, September 29, 2010
2:00 p.m.


Abstract

The mammalian retina is a laminated sensorineural epithelium composed of six classes of neurons that include the rod, cone, bipolar, horizontal, amacrine (AC), and ganglion cells (RGCs), and one type of glia, the Müller cells. The developmental processes are under the control of intrinsic and extrinsic factors. How these diverse cell types and subtypes arise during retinogenesis still remain largely to be defined at the molecular level. During my PH.D., I studied roles of Brn3b, Nr4a2 and Ptf1a during retinal development.

Firstly, we show that the previously unsuspected POU domain transcription factor Brn3b plays such a critical role. Loss of Brn3b function in mice leads to mis-specification of early RGC precursors as late-born RGC, amacrine and horizontal cells whereas misexpressed Brn3b suppresses non-RGC cell fates while promoting the RGC fate. Microarray profiling and other molecular analyses reveal that in RGC precursors Brn3b normally represses the expression of a network of retinogenic factor genes involved in fate commitment and differentiation of late-born RGC, amacrine, horizontal, and cone cells. Our data suggest that Brn3b specifies the RGC fate from multipotential precursors not only by promoting RGC differentiation but also by suppressing non-RGC differentiation programs as a safeguard mechanism.

Secondly, we show that Nr4a2 is also critically involved in the specification of AC subtype identity. During mouse retinogenesis, Nr4a2 is expressed in a subset of postmitotic GABAergic ACs and their precursors. Its targeted inactivation results in the loss of a subpopulation of GABAergic ACs that include all dopaminergic and p57Kip2+ neurons as well as a simultaneous increase of calbindin+ ACs. Misexpressed Nr4a2 can promote GABAergic AC differentiation and repress calbindin+ ACs, whereas its dominant-negative form has the ability to suppress the GABAergic AC fate. Moreover, the expression of Nr4a2 is positively regulated by Foxn4 and negatively controlled by Brn3b, two retinogenic factors previously shown to promote and suppress GABAergic ACs, respectively. These data suggest that Nr4a2 is both necessary and sufficient to confer AC precursors with the identity of a GABAergic AC phenotype, and that it may network with multiple other retinogenic factors to ensure proper specification and differentiation of AC neurotransmitter subtypes.

Finally, we showed misexpressed ptf1a can promote glycinergic, dopaminergic amacrine cells and horizontal cells fates but repressed other retina cell fates (ganglion, bipolar, muller and photoreceptor cells). Furthermore, Tcfap2 proteins were downregulated in the ptf1a null mutant mice. Knockdown of Tcfap2a and Tcfap2b can suppress differentiation of Amacrine cells. In the meanwhile, we also found forced expression of ptf1a in retinal progenitors activates the expression of Tcfap2b, which in turn promote glycinergic amacrine cell fates and suppress the differentiation of photoreceptor cells. Moreover, Ptf1a can direct bind to Tcfap2b promoter region. Thus, these data suggest that Tcfap2 proteins are the direct downsteam genes of Ptf1a in the differentiation of Amacrine cells.


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