Thursday, November 21, 2024
11/21/2024
Project Summary During embryonic development, extracellular proteins, known as morphogens, play a key role in conveying positional information and establishing cell fates. While theoretical descriptions have posited plausible models for how morphogens might shape embryonic development, until recently, quantitative experimental data have been lacking. The Nodal pathway is essential for early development, and is necessary for stabilization of the epiblast, specification of the mesendoderm, and formation of the left-right axis. Nodal and its inhibitors Lefty1/2 have long served as a model for tissue patterning by signaling activators and their cognate inhibitors, with several studies suggesting that the Nodal-Lefty system generates self-organizing patterns via a Turing mechanism. Unlike other TGFβ family members, Nodal has been found to signal as an obligatory heterodimer with a cofactor (GDF1/3), and signaling requires a coreceptor (Cripto) in receiving cells in addition to the standard receptor complex. There is some evidence for long-range dispersal of Nodal, particularly from overexpression studies, and from studies of left-right axis specification, while recent studies of germ layer patterning in Zebrafish have suggested that it functions at fairly short range over only a few cell tiers. We have taken advantage of CRISPR editing in human pluripotent stem cells (hPSCs) to tag the Nodal gene with a fluorescent protein allowing us to visualize it at endogenous concentrations for the first time. Using a self-organizing hPSC system that forms patterns of different germ layers along the radial axis of an hPSC colony, we demonstrated that, in this context, Nodal signaling is primarily autocrine and juxtacrine, signaling only to the immediate neighbors of producing cells. This short-range signaling activity spreads via a relay mechanism in which Nodal producing cells signal to their neighbors which causes them to transcribe Nodal protein and signal to the next layer of cells. This mode of relay signaling has received much less attention than Turing mechanisms, even though it may operate in a number of contexts in which signaling is short range. Here, we will build upon these tools and findings to perform quantitative experiments and mathematical modeling to understand how the system of Nodal, and its inhibitors, cofactors, and coreceptor functions in patterning. We will pursue three aims. (1) Understand how the relay signaling system of Nodal and Lefty1/2 establishes dynamic patterns of signaling activity and cell fate (2) Understand the role of cofactors and coreceptors in ligand dispersal and patterning. (3) Uncover how crosstalk between Wnt and Nodal regulates signaling and cell fate. Together our studies will quantitatively probe how a paradigmatic signaling system shapes fate patterns in the embryo during early development. As our experiments are performed in human cells, our findings will shed important light on the role of these pathways in patterning defects, as well as provide a foundation for manipulating signaling to created patterned tissues for regenerative medicine.
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