Date of Degree

9-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Daniel Weinstein

Committee Members

Cathy Savage-Dunn

Nathalia Holtzman

Mitchell Goldfarb

Xiajun Li

Subject Categories

Developmental Biology

Keywords

Pitx1, Pitx2c, cement gland, Xenopus, ectoderm, BMP

Abstract

Embryos of the African clawed frog Xenopus laevis are widely used for the study of early vertebrate development. The cement gland, which secrets mucus to help tadpoles attach to solid supports and live in relative safety, has long been used as a model to study the interplay between cell signaling pathways and transcription factors. It has been proposed that an intermediate level of Bone Morphogenetic Protein (BMP) signaling is essential for cement gland formation. In addition, several transcription factors have been linked to cement gland development. Among them, the homeodomain proteins Pitx1 and the closely related Pitx2c can generate ectopic cement gland formation; however, the mechanisms underlying this process remain obscure. We report here, for the first time, a requirement for Pitx proteins in cement gland formation, in vivo: knockdown of both Pitx1 and Pitx2c inhibits endogenous cement gland formation. Pitx1 transcriptionally activates downstream target genes through both direct and indirect mechanisms, and functions as a transcriptional activator to inhibit BMP signaling. This inhibition, required for the expression of pitx genes, is partially mediated by Pitx1-dependent follistatin expression. Depletion of BMP proteins inhibits induction of cement gland markers by Pitx1; furthermore, we find that Pitx1 physically interacts with Smad1, an intracellular transducer of BMP signaling. We propose a model of cement gland formation in which Pitx1 limits local BMP signaling within the cement gland primordium, and recruits Smad1 to activate cement gland genes.

We also identify the homeobox gene goosecoid, expressed in the pharyngeal endoderm adjacent to the cement gland, as a target of Pitx1. We find that Pitx1 inhibits Activin activity and binds to Smad2/3, an intracellular transducer of Activin/TGFβ signaling. Our studies suggest that a transactivating complex including Pitx1 and Smad2/3 stimulates goosecoid transcription during the development of anterior endoderm.

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