The goal of this project is to clarify the mechanisms leading to the commitment, or determination, of particular regions of the embryo to form a tissue or organ. This proposal focuses on lens formation in the frog Xenopus. While a great deal of information has accrued about genes that are critical for organ formation, still very little is knon about the integrative mechanisms by which commitment occurs. In the case of the developing lens the long history of study, accessibility of presumptive lens ectoderm (PLE) at all stages, and the many technical advantages of the Xenopus system provide unique assets for addressing the question of embryonic determination. My laboratory has characterized the numerous steps in the lens commitment process and here are described experiments to distinguish the last steps in this process: specification and determination.
The first aim of the project is to address the specific cellular mechanisms leading to determination, initially by examining the hypothesis that inhibition of responsiveness to the Wnt signaling pathway is responsible for determination. Examination of gene expression by RNA-Seq during commitment has revealed a number of candidate genes that could be key regulators of this process; for example, down regulation of Oct91, the Xenopus homologue of the stem cell gene Oct4, will be examined as a potential regulatory step in the determination process.
The second aim i s the development of an epigenetic resource to greatly enhance our studies of gene regulation by undertaking a set of ChIP-Seq analyses to identify key chromatin marks (e.g. histone modifications) associated with active enhancers in the PLE at three stages in the commitment process. These analyses will provide a key resource for identifying putative enhancers in regulatory genes and to assess the temporal association of the appearance of histone marks with gene activation and repression, and with the commitment process.
The final aim i s to examine the spatial commitment of a particular region of ectoderm toward lens formation at the neural plate stage. Four genes (pax6, six3, mab21l1 and mab21l2) have expression in a small zone defining the PLE region at this very early stage. Identification and analysis of the regulatory elements controlling this highly regulated expression (using information generated from the second aim) will provide new insights about how the spatial definition of the lens is established. Overall, this work will provide key information for understanding the ontogeny of the visual system. In addition it bears on important questions in stem cell biology which depend on understanding of the intimately related, but poorly understood mechanisms of commitment during early development. In turn this work bears on related issues of regenerative medicine involving cell fate reprogramming, and human diseases involving genetic lesions in key developmental genes, e.g. Aniridia in the case of eye formation.
The goal of this project is to understand how tissues in the embryo become committed to their fates, in this project, how the embryonic lens is 'determined' in the frog Xenopus. This work bears on important questions in stem cell biology, which requires an understanding of how cells in the embryo first become committed to form particular tissues. In turn this work bears on related issues of regenerative medicine involving cell fate reprogramming. Finally, we now know that many genetically based diseases in children stem from genetic lesions that affect the genes involved in forming tissues properly, and the knowledge gained from understanding how these genes function will help clarify the basis for these diseases. For example, in the case of the lens of the eye, Aniridia, or absence of an iris, i a genetic disease caused by defects in one of the genes being intensively studied in the project proposed here.
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|Nakayama, Takuya; Fisher, Marilyn; Nakajima, Keisuke et al. (2015) Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients. Dev Biol 408:328-44|
|Nakayama, Takuya; Blitz, Ira L; Fish, Margaret B et al. (2014) Cas9-based genome editing in Xenopus tropicalis. Methods Enzymol 546:355-75|
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|Bhatia, Shipra; Bengani, Hemant; Fish, Margaret et al. (2013) Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia. Am J Hum Genet 93:1126-34|
|Nakayama, Takuya; Fish, Margaret B; Fisher, Marilyn et al. (2013) Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis 51:835-43|