Many hematological malignancies require ablation of diseased cells and subsequent reestablishment of the hematopoietic compartment following transplantation of healthy multipotent hematopoietic stem cells (HSC). Unfortunately, many patients fail to receive a life saving HSC transplant due to an inability to find a HLA-matched donor, an issue more commonly affecting patients of minority or mixed ethnic backgrounds. This limitation would be overcome by the ability to generate HSCs in vitro. However, the development of protocols capable of generating functional HSCs for clinical use remains bottlenecked by safety concerns, low scalability and reproducibility. These protocols may be refined by identifying key factors driving HSC generation which occurs during embryonic development via an Endothelial-to-Hematopoietic-Transition (EHT). A critical balance of exogenous factors produced by the Aorta-Gonad-Mesonephros (AGM) microenvironment promote this hematopoietic transdifferentiation, but remain poorly understood.Our preliminary data indicates that cell extrinsic Sash1 signaling promotes EHT, and that loss of Sash1 impairs the formation of intra-aortic hematopoietic clusters (IAHCs) from which HSCs emerge. In this project we focused on characterizing how loss of Sash1 impairs EHT with the aim of improving our understanding of the extrinsic mechanisms that promote HSC generation for regenerative medicine. Using a combination of transcriptomics approaches we profiled endothelial, hematopoietic and Sash1-expressing cells from the AGMs of transgenic embryos lacking functional Sash1. Through this analysis, and functional follow-up with conditional knockout mice, we identified candidate Sash1-regulated factors within the mesenchymal niche that promote the early requisite stages of IAHC formation, including morphogenic changes and cell cycle activation.

Hematopoietic stem and progenitor cells (HSPCs) originate from an endothelial-to-hematopoietic transition (EHT) during embryogenesis. Characterization of early hemogenic endothelial (HE) cells is required to understand what drives hemogenic specification and to accurately define cells capable of undergoing EHT. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we define the early subpopulation of pre-HE cells based on both surface markers and transcriptomes. We identify the transcription factor Meis1 as an essential regulator of hemogenic cell specification in the embryo prior to Runx1 expression. Meis1 is expressed at the earliest stages of EHT and distinguishes pre-HE cells primed towards the hemogenic trajectory from the arterial endothelial cells that continue towards a vascular fate. Endothelial-specific deletion of Meis1 impairs the formation of functional Runx1-expressing HE which significantly impedes the emergence of pre-HSPC via EHT. Our findings implicate Meis1 in a critical fate-determining step for establishing EHT potential in endothelial cells.

The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1(-/-) mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with beta-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy.

Hematopoietic stem cells (HSCs) originate during embryogenesis and colonize the bone marrow, after expansion in the fetal liver, to sustain adult hematopoiesis. In vivo imaging studies have supported the emergence of HSCs form the ventral endothelium of the dorsal aorta in the aorta-gonado-mesonephros (AGM) region through a process termed endothelial-to-hematopoietic transition (EHT). Generation of HSC is a dynamic process requiring temporal and cell-specific changes within a subset of cells, the hemogenic endothelium, to drive a new cell fate. Although several pathways have been shown to play a critical role in this transition, the molecular programming driving EHT remains elusive. In this study, we aim to understand how the transcription factor Meis1 affects the emergence of HSC by taking advantage of various mouse models. Using a GFP reporter mouse, we mapped Meis1 expression in the developing dorsal aorta and showed that Meis1 expression enriches for the hemogenic endothelium compared to vascular endothelium. We combined these data with observations of endothelial-specific deletion of Meis1 in vivo, using VE-cadherin (VEC)-Cre recombinase, to gain a better understanding of the requirements for Meis1 during this dynamic developmental process. Conditional Meis1-deletion impaired the hematopoietic potential of the AGM endothelium based on specific gene expression and significantly reduce the emergence of cells with hematopoietic surface markers. Our ability to derived HSC in vitro is in its infancy and was largely influenced by studies of normal developmental processes. Therefore, understanding the regulatory network driving EHT remains essential to develop better strategies for HSC specification.

Sash1 acts as a scaffold molecule regulating signal transduction downstream of TLR4. By investigating its function in vivo, our lab showed that deletion of Sash1 in mice leads to developmental defects during embryogenesis, impacting primarily the lung and the hematopoietic system. We observe a significant reduction in hematopoietic stem and progenitor cells (HSPCs), both phenotypically and functionally, in the fetal liver of Sash1-knockout embryos compared to their wildtype littermates at embryonic day (E)14.5. Further investigation of this phenotype revealed that a deficiency is already present as early as E10.5, when the first HSPCs emerge from intra-aortic hematopoietic clusters in the dorsal aorta via a process termed endothelial-to-hematopoietic transition (EHT). Specifically, we found that fewer hematopoietic clusters are formed in the dorsal aorta of Sash1-knockout embryos which correlates with a reduced number of pre-HSCs based on surface markers and decreased expression of EHT-related genes. Interestingly, our data suggests that Sash1 plays a cell non-autonomous role, acting on an already specified hemogenic endothelium, to promote the progression of EHT. Therefore, we aim to understand how signaling via Sash1 stimulates definitive hematopoiesis in the early embryo. Developmental studies have largely guided the current effort to derive HSPC in vitro and further investigation of extrinsic signaling pathways involved may provide new insight to support cells undergoing EHT.