ROLE OF BMP AND ACTIVIN/NODALSIGNALING REPRESSION IN RETINALPROGENITOR CELL SPECIFICATION ANDCONE PHOTORECEPTOR GENESIS

Abstract

Eye formation begins with the specification of the eye field from the anteriorneural plate during the transition from gastrulation to neuralization. The morphogeneticgradients formed by BMP and Activin/Nodal ligands organize the body axis andregulate intracellular signals to bias the cell lineages.Therefore, to understand theextracellular signaling mechanisms governing eye field specification, we looked closerat the mechanisms responsible for neuralization. This project takes advantage of thedevelopmental accessibility of theXenopus laevisembryo to investigate the role ofBMP and Activin/Nodal signaling pathways during early eye development.Nogginis a secreted BMP antagonist and is sufficient to induce formation of ectopic retinaltissue. My results presented in Chapter 2 show that intracellular repression of BMP-Smad1/5/8 and Activin/Nodal-Smad2/3 signaling was sufficient to replicate the retina-promoting activity of Noggin and efficiently drive pluripotentXenopusprimitive ectodermto a retinal progenitor cell fate. Furthermore, my work presented in Chapter 3 suggeststhat repression of BMP and Activin/Nodal in the anterior neural plate allows the anteriorneural patterning transcription factor, Otx2, to directly activate expression of the earliesteye field transcription factor,tbx3. This study elucidates the initial step by which theanterior neural plate is patterned to specify the eye field. We next translated our findings to mouse embryonic stem cell culture (Chapter 4). We observed that mouse embryonic stemcells can be efficiently directed towards retinal progenitor cells by first converting themto an early primitive ectoderm-like state, followed by simultaneous repression of BMPand Activin/Nodal and activation of FGF signaling. After differentiation, we observedthat photoreceptor genesis is synchronized, resulting in 96% of aggregates expressingmarkers for cone photoreceptors after 9 days. This differentiation protocol is more efficientand quicker than previously published mouse stem cell protocols. Optimizing treatmentconditions resulted in up to 62% of cells expressing the cone/rod photoreceptor marker,CRX. This study was the first to show that photoreceptor-like cells can be generated frommouse pluripotent stem cells as efficiently as published human pluripotent stem cell reports,but with a shorter culture period. These studies will provide the basis of future work todetermine the underlying molecular mechanism driving retinal differentiation.