Mechanisms of cell fate specification by glycosylation

The National Institute of General Medical Sciences (NIGMS) awarded Dartmouth College $2,252,479 under the Biomedical Research and Research Training program (CFDA 93.859) effective September 1, 2025, through July 31, 2029. This R35 Maximizing Investigators' Research Award funds fundamental research investigating the mechanisms by which protein glycosylation regulates cell fate specification. The research employs zebrafish embryo models and loss-of-function studies to elucidate how glycosylation controls the functions of cell fate regulators and determines cellular identity choices across four distinct cell types: arterial endothelial cells, venous endothelial cells, lymphatic endothelial cells, and hematopoietic stem/progenitor cells.

The project deliverables include detailed mechanistic findings on how five N-glycoproteins regulate cell fate transformation, particularly the conversion of arterial endothelial cells into hematopoietic stem/progenitor cells, along with functional analyses of glycan composition in dictating cell identity decisions. Research outputs will advance fundamental understanding of cell fate specification and provide foundational knowledge applicable to regenerative medicine and tissue repair applications. The research will be conducted at Dartmouth College's facilities in Hanover, New Hampshire.

Abstract

Cell fate is determined through a complex hierarchy of events that require tight regulation of signaling pathways. Misspecification of cell fate can have detrimental effects on downstream developmental events, leading to tissue mispatterning and disease. Key principles of cell fate specification remain unknown. Defining the mechanisms that provide spatial and temporal precision to gene regulatory networks is crucial for understanding how cell identity is established and can be leveraged to generate desired cell types for the repair of damaged or diseased tissues. In previous work, the PI has identified protein glycosylation as an unexplored mechanism that regulates cell fate. The long-term goal of the PI's research program is to exploit the intrinsic plasticity of endothelial cells to define the fundamental principles by which glycosylation determines the identities of differentiated cells. The proposed R35 project will investigate two parallel cell fate decisions, representing four distinct cell types: artery, vein, lymphatic, and hematopoietic stem/progenitor cells. IT will harness the optical transparency of zebrafish embryos to make highly quantitative measurements of these processes as they unfold naturally in vivo. IT will also leverage a first-of-its-kind list of glycoprotein candidates and its glycosylation sites that are misglycosylated in a cell fate mutant. The overall objectives of this study are: 1) to decipher how glycosylation regulates the functions of cell fate regulators; and 2) to determine how glycan composition dictates the choice of cell identities. In project 1, loss-of-function studies in zebrafish will reveal how five n-glycoproteins regulate the cell fate transformation of arterial endothelial cells into hematopoietic stem/progenitor cells. Additionally, missense mutations that prevent glycan attachment to these candidate proteins will be generated to assess the requirement for glycosylation in their fate-determining functions. Project 2 will use a cell fate mutant as a tool to test how glycosylation regulates the transformation of venous endothelial cells into lymphatic endothelial cell progenitors. The experimental approaches will identify the glycosylation enzymes and glycan repertoire required for this important fate decision, ultimately demonstrating that discrete glycans repertoires instruct endothelial cell identity. This research is innovative in that IT seeks to apply state-of-the art microscopy, genetic, and glycomic technologies to characterize glycosylation-dependent mechanisms of specification for multiple clinically relevant cell types in vivo. Using this process to compare cell fate decisions from a common lineage provides a rare opportunity to define the universal and cell-specific mechanisms of glycosylation in cell fate regulation. The proposed work is highly significant as the results have the potential to reveal new design principles of cell fate specification, and thereby inform new strategies of cellular reprogramming, via glycoengineering, for therapeutic interventions.