3115 – THE COLLAGEN-MODIFYING LYSYL HYDROXYLASE, PLOD2, IS REQUIRED FOR HEMATOPOIETIC STEM CELL SPECIFICATION

Abstract

Hematopoietic stem cells (HSCs) continuously replenish the hematopoietic system and form the basis of transplant therapies for hematologic and autoimmune diseases. In vertebrates, HSCs are developmentally specified from a transient population of specialized endothelial cells known as hemogenic endothelium (HE) located in the floor of the dorsal aorta. Under the influence of intrinsic and extrinsic differentiation cues, HE cells lose their endothelial fate and acquire hematopoietic identity through an endothelial-to-hematopoietic transition (EHT). Decades of intensive study have identified key peptide signals required for HSC specification, but a complete understanding remains elusive. Extracellular matrix (ECM) is a major component of the HSC inductive environment able both to signal directly and also regulate display of known signaling factors, but few or no studies have directly addressed the role of ECM in HSC specification. Collagen, the most abundant protein in vertebrates, forms the structural backbone of ECM and its maturation into stiffened fibers is regulated by Plod2 (Lysyl Hydroxylase 2), which catalyzes hydroxylation of key collagen lysines as a precondition to fibril crosslinking. Plod2 thus regulates ECM stability, permeability, and stiffness, and has previous roles described in malignant epithelial-to-mesenchymal transition (EMT) and metastasis. We have performed in vivo expression and functional studies in the zebrafish model. Our data show that plod2 is expressed in tissues adjacent to the dorsal aorta at the time of EHT, while plod2 loss-of-function by targeted deletion or antisense abrogates emergence of HSCs in the AGM. We are currently extending our findings to mammals. Our studies identify ECM architecture and its enzymatic regulation as central to the establishment of definitive hematopoiesis. Hematopoietic stem cells (HSCs) continuously replenish the hematopoietic system and form the basis of transplant therapies for hematologic and autoimmune diseases. In vertebrates, HSCs are developmentally specified from a transient population of specialized endothelial cells known as hemogenic endothelium (HE) located in the floor of the dorsal aorta. Under the influence of intrinsic and extrinsic differentiation cues, HE cells lose their endothelial fate and acquire hematopoietic identity through an endothelial-to-hematopoietic transition (EHT). Decades of intensive study have identified key peptide signals required for HSC specification, but a complete understanding remains elusive. Extracellular matrix (ECM) is a major component of the HSC inductive environment able both to signal directly and also regulate display of known signaling factors, but few or no studies have directly addressed the role of ECM in HSC specification. Collagen, the most abundant protein in vertebrates, forms the structural backbone of ECM and its maturation into stiffened fibers is regulated by Plod2 (Lysyl Hydroxylase 2), which catalyzes hydroxylation of key collagen lysines as a precondition to fibril crosslinking. Plod2 thus regulates ECM stability, permeability, and stiffness, and has previous roles described in malignant epithelial-to-mesenchymal transition (EMT) and metastasis. We have performed in vivo expression and functional studies in the zebrafish model. Our data show that plod2 is expressed in tissues adjacent to the dorsal aorta at the time of EHT, while plod2 loss-of-function by targeted deletion or antisense abrogates emergence of HSCs in the AGM. We are currently extending our findings to mammals. Our studies identify ECM architecture and its enzymatic regulation as central to the establishment of definitive hematopoiesis.