The Role of Twisted Gastrulation in the Extracellular Regulation of Bone Morphogenetic Protein Signalling

Abstract

With fundamental roles throughout development and homeostasis, the Bone Morphogenic Protein (BMP) signalling pathway is tightly regulated. In the extracellular environment, specific protein-protein interactions achieve precise control of signalling during developmental patterning. A well-studied paradigm is the embryonic dorsoventral patterning of Drosophila melanogaster and here Twisted gastrulation (Tsg), a conserved modulator of BMP signalling, displays both agonistic and antagonistic effects. Tsg exerts its effect through interaction with BMP ligands and the Chordin family of BMP antagonists, yet the nature of these interactions is unknown. This project aims to investigate how Tsg regulates BMP signalling in the extracellular space through specific protein interactions. Here, cell-based assays validate a proposed mode of interaction between Tsg and BMP ligands by mutation of a single key residue, Ile40, in the Tsg N-terminal domain. To study Tsg action in vivo, a CRISPR-based genome engineering strategy is used to generate an efficient reintegration system at the tsg locus in Drosophila. Introducing the same Ile40 point mutation in vivo significantly disrupts dorsoventral patterning, demonstrating conservation of the Tsg-BMP interaction and its necessity in developmental patterning. Furthermore, computational structural modelling identifies the Chordin family binding epitope on Tsg which is validated by mutational binding studies in vitro. Finally, phylogenetic analysis uncovers an evolutionary divergent Tsg Tail sequence, truncation of which in vivo produces a novel phenotype revealing the Tail is essential for full Tsg action. Uncovering the interactions between Tsg and its BMP and Chordin family binding partners furthers the understanding of fundamental developmental mechanisms. Moreover, these findings will be relevant to the diverse set of contexts in which Tsg acts, to improve the understanding of associated diseases and aid design of potential therapeutics.

Date of Award	14 Mar 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Clair Baldock (Supervisor) & Hilary Ashe (Supervisor)