Extracellular matrix in vertebrate hematopoietic stem cell specification

(PLEASE KEEP IN WORD, DO NOT PDF) Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Hematopoietic stem cells (HSCs) are the self-renewing, tissue-specific stem cells in vertebrates that regenerate definitive hematopoietic lineages over the life of the individual. HSC transplant is a major treatment modality for leukemia and some non-malignant hematologic diseases including sickle cell disease, as well as a platform for delivery of gene therapy. Understanding the complete set of normal signals involved in embryonic specification of HSCs remains elusive. To identify missing inputs, we have been investigating the endogenous hematopoietic “specification niche” at the cellular, genetic, molecular, and architectural levels. HSCs emerge from specialized arterial “hemogenic endothelium” found in the aorta-gonads-mesonephros (AGM) region of the developing embryo. Transcriptional profiling has identified ECM genes as among the most highly expressed in hematopoietic niches at all stages of HSC ontogeny including during specification, but no studies have examined the normal organization of the ECM in the specification niche or addressed whether normal ECM organization and maturation is required for HSC specification. ECM comprises a complex and dynamic macromolecular network of proteins and proteoglycans that provides structure and can regulate cellular survival, proliferation, migration, and differentiation. Collagen is the major structural component of ECM. Our transcriptional profiling and network analysis of in vitro hematopoietic differentiation identified a collagen-modifying enzyme, procollagen-lysine, 2oxoglutarate 5-dioxygenase 2 (PLOD2), as a likely pivotal player in definitive hematopoietic emergence. We confirmed expression of Plod2 in the HSC specification niche of zebrafish and mouse and took advantage of antisense and genetic approaches to assess the effects of knockdown and loss-of-function during development. Our studies indicate Plod2 is required for HSC emergence in zebrafish and point to a conserved requirement in mammals. In the absence of Plod2, we observe diminished activity of Yap/Taz signaling, which is a mechanosensitive pathway responsive to ECM stiffness via integrin receptors. Our hypothesis is that ECM maturation/stiffening is an unrecognized input required for HSC specification. To test this hypothesis, we will interrogate ECM organization in the HSC specification niche, examine how PLOD2 contributes to niche architecture, and investigate a functional role for this enzyme in vertebrate HSC specification. Our studies are the first to examine a potential role for ECM in HSC specification in vivo.