Saulius Sumanas, Ph.D. - US grants

DESCRIPTION (provided by applicant): Arterial and venous (A-V) patterning is critical for the establishment of functional embryonic and adult vasculature. Proper A-V differentiation is critical for the formation of functional vessels during tissue repair processes such as wound healing and is associated with a number of pathological conditions including A-V malformations and tumor induced angiogenesis. Therefore understanding the mechanisms of A-V differentiation will lead to new treatments in tumorigenesis, wound repair and multiple vascular disorders. While it is difficult to study A-V patterning in mammalian systems, transparent zebrafish embryos are easily accessible for observation and experimental manipulations. During formation of the major vessels, vascular endothelial progenitor cells (EPCs) assume arterial or venous identity prior to the initiation of circulation. However, it is not understood how EPCs choose among the arterial or venous fates if they are all exposed to the same signaling molecules such as Vegf and Hh. We have found that the expression timing of an evolutionarily conserved master regulator of vasculogenesis Etsrp / Etv2 is one of the critical factors in A-V differentiation. Furthermore, arterial and venous progenitors may originate at different spatial locations, the inner and the outer lines and thus experience different Vegf concentrations. We hypothesize that the arterial- venous fate of EPCs is determined by the combination of etsrp-dependent timing of Vegf receptor flk1 and hyaluronan (HA) receptor stabilin 2 (stab2) expresion and their spatial location within Vegf gradient. To demonstrate that the timing of etsrp expression affects A-V differentiation, photoactivatable morpholinos will be used to inhibit etsrp function at different developmental stages in zebrafish embryos. Fate mapping and time- lapse imaging will be performed to characterize the cell movements and to determine the arterial-venous fates of the inner and outer lines of EPCs. To determine if Vegf and Hh gradients play a role in the activation of Notch signaling and arterial differentiation within the inner line angioblasts, overexpression and loss of function approaches will be used to inhibit Vegf, Hh and Notch signaling combined with lineage tracing to determine the A-V cell fates. To determine if Etsrp downstream target Stab2 functions as a receptor for HA and induces activation of Notch signaling during A-V differentiation, A-V defects in Stab2 and HA synthase Has2 knockdown embryos will be investigated. It will be analyzed if HA-Stab2 signaling leads to Tyr-phosphorylation of Stab2 and ERK phosphorylation, resulting in Notch pathway activation and arterial marker expression. Upon completion of this study, we will have identified the molecular mechanism how the timing of etsrp expression and Vegf gradient lead to the differential expression of arterial and venous genes. The acquired knowledge will be critical in our understanding of molecular mechanisms of A-V differentiation and will have an impact on developing treatments for multiple diseases and pathological conditions related to vasculature formation which include A-V malformations, diabetic retinopathy, wound healing and tumorigenesis.! PUBLIC HEALTH RELEVANCE: The proposed project will utilize a zebrafish model system to investigate molecular mechanisms of arterial-venous specification. It will determine how the timing and location of endothelial cell specification translate into differential arterial and venous cell fates. It will investigate how different inputs from transcription factors such as Etsrp / Etv2 and morphogens such as Vegf are integrated to induce or inhibit expression of arterial or venous specific genes. The acquired knowledge will be critical in our understanding of fundamental mechanisms of vascular differentiation, which will lead to new treatments in tumorigenesis, wound repair and multiple vascular disorders.

DESCRIPTION (provided by applicant): Arterial and venous (A-V) patterning is critical for the establishment of functional embryonic and adult vasculature. Proper A-V differentiation is critical for the formation of functional vessels during tissue repair processes such as wound healing and is associated with a number of pathological conditions including A-V malformations and tumor induced angiogenesis. Therefore understanding the mechanisms of A-V differentiation will lead to new treatments in tumorigenesis, wound repair and multiple vascular disorders. While it is difficult to study A-V patterning in mammalian systems, transparent zebrafish embryos are easily accessible for observation and experimental manipulations. During formation of the major vessels, vascular endothelial progenitor cells (EPCs) assume arterial or venous identity prior to the initiation of circulation. However, it is not understood how EPCs choose among the arterial or venous fates if they are all exposed to the same signaling molecules such as Vegf and Hh. We have found that the expression timing of an evolutionarily conserved master regulator of vasculogenesis Etsrp / Etv2 is one of the critical factors in A-V differentiation. Furthermore, arterial and venous progenitors may originate at different spatial locations, the inner and the outer lines and thus experience different Vegf concentrations. We hypothesize that the arterial- venous fate of EPCs is determined by the combination of etsrp-dependent timing of Vegf receptor flk1 and hyaluronan (HA) receptor stabilin 2 (stab2) expresion and their spatial location within Vegf gradient. To demonstrate that the timing of etsrp expression affects A-V differentiation, photoactivatable morpholinos will be used to inhibit etsrp function at different developmental stages in zebrafish embryos. Fate mapping and time- lapse imaging will be performed to characterize the cell movements and to determine the arterial-venous fates of the inner and outer lines of EPCs. To determine if Vegf and Hh gradients play a role in the activation of Notch signaling and arterial differentiation within the inner line angioblasts, overexpression and loss of function approaches will be used to inhibit Vegf, Hh and Notch signaling combined with lineage tracing to determine the A-V cell fates. To determine if Etsrp downstream target Stab2 functions as a receptor for HA and induces activation of Notch signaling during A-V differentiation, A-V defects in Stab2 and HA synthase Has2 knockdown embryos will be investigated. It will be analyzed if HA-Stab2 signaling leads to Tyr-phosphorylation of Stab2 and ERK phosphorylation, resulting in Notch pathway activation and arterial marker expression. Upon completion of this study, we will have identified the molecular mechanism how the timing of etsrp expression and Vegf gradient lead to the differential expression of arterial and venous genes. The acquired knowledge will be critical in our understanding of molecular mechanisms of A-V differentiation and will have an impact on developing treatments for multiple diseases and pathological conditions related to vasculature formation which include A-V malformations, diabetic retinopathy, wound healing and tumorigenesis.! PUBLIC HEALTH RELEVANCE: The proposed project will utilize a zebrafish model system to investigate molecular mechanisms of arterial-venous specification. It will determine how the timing and location of endothelial cell specification translate into differential arterial and venous cell fates. It will investigate how different inputs from transcription factors such as Etsrp / Etv2 and morphogens such as Vegf are integrated to induce or inhibit expression of arterial or venous specific genes. The acquired knowledge will be critical in our understanding of fundamental mechanisms of vascular differentiation, which will lead to new treatments in tumorigenesis, wound repair and multiple vascular disorders.

Project Summary Different types of immune deficiencies and cancers of the immune system which include lymphoma, different types of leukemia, multiple myeloma and others affect millions of people including children each year in the United States. Treatment options are often limited and may include risky procedures such as hematopoietic stem cell (HSC) transplantation. Ultimately, ability to differentiate hematopoietic progenitors in vitro into selected blood cell lineages including HSCs would enable more effective therapies for many different immune deficiencies and cancers. However, our knowledge of molecular mechanisms that govern hematopoiesis and immune cell differentiation is still limited. While it is difficult to study immune cell development in mammalian embryos, zebrafish has emerged as a highly advantageous system for embryonic studies. Transparent embryos are easily accessible for experimental manipulations and observations. Molecular mechanisms that regulate hematopoiesis and immune cell development are highly conserved between zebrafish and mammalian embryos. Here we have identified a novel hematopoietic site in zebrafish embryos which contains a previously unrecognized group of putative hematopoietic progenitors, pronephros associated cells (PACs). PACs are recognized by expression of transcription factors Etv2 and Scl, two known hematopoietic regulators. Our preliminary data argue that PACs can contribute to immune lineages such as macrophages and can translocate into the vasculature. However, the full lineage potential of these cells, their functional role and molecular pathways regulating their development are not known. We hypothesize that PACs represent a previously unknown type of multipotent hematopoietic progenitor cells, and contribute to immune cell lineages. The following specific aims are proposed: 1) Determine if PACs are multipotent hematopoietic progenitors and identify their lineage contributions; 2) Determine the functional role of PACs in hematopoiesis and immune system development and characterize their transcriptional profile. Lineage tracing and fate-mapping of PACs will be performed using time-lapse imaging and cell labeling approaches in zebrafish embryos to determine contribution of PACs to different hematopoietic lineages. Cell ablation will be used to deplete the PAC population, followed by the analysis of hematopoietic defects. FACS sorting approach followed by RNA-Seq will be used to define the transcriptional profile of PACs. Data obtained in this proposal will answer the key questions regarding the lineage contributions and functional role of PACs. We expect that our research will ultimately identify an alternative pathway that regulates hematopoiesis including myeloid and lymphoid progenitor development, and will lead to novel therapeutic approaches that may generate functional myeloid and lymphoid progenitors in a variety of blood and immune disorders and cancers.

Project Summary Intracranial aneurysms (IA) are berry- or balloon-like defects in the wall of a major intracranial artery and are present in 1-2% of the population. They commonly result in subarachnoid hemorrhage, which leads to death in 30-40% of the patients. There is currently no effective therapy to treat SAH and only limited treatment options to prevent IA rupture. Both environmental and genetic factors have been attributed to the aneurysm formation; however, the genetic factors and their underlying mechanisms are still largely unknown. We have identified mutations in collagen COL22A1 as potential contributors to the development of IAs in human patients. In the Familial Intracranial Aneurysm study, led by our collaborators, whole exome sequencing resulted in identification of a single nucleotide polymorphism (SNP) in a highly conserved region of COL22A1 present in only affected family members. However, biological function of COL22A1 is currently not known, and it is not clear if the identified mutation is causative of aneurysms in humans. We propose to use a zebrafish model to determine the function of COL22A1 in maintaining vascular integrity and to identify potential therapeutic strategies that would lead to the prevention of aneurysm formation and rupture. The protein sequence of COL22A1 is highly conserved between humans and zebrafish, and the zebrafish have emerged as a highly advantageous model system for in vivo analysis of vascular function and disease mechanisms. Our preliminary data indicate that COL22A1 zebrafish mutants display increased susceptibility to hemorrhages and show abnormal vascular dilations comparable to aneurysms in human patients, while inducible expression of the human mutant SNP results in increased frequency of hemorrhages in zebrafish embryos. We hypothesize that COL22A1 is involved in regulating vascular integrity and permeability and that mutations in COL22A1 cause intracranial aneurysms. The following specific aims are proposed: 1) Determine the functional role of COL22A1 in the maintenance of vascular stability; 2) Determine if mutations in COL22A1 cause intracranial aneurysms; 3) Perform a chemical screen to discover drug candidates that suppress hemorrhages in COL22A1 mutant embryos. Zebrafish COL22A1 mutant embryos and adults will be analyzed for morphological and functional defects. The human mutation will be modeled in zebrafish by creating a knock-in allele using a CRISPR / Cas9 mediated homology-directed repair and analyzing it for IA related phenotypes. A chemical library screen will be performed using zebrafish COL22A1 mutants to identify candidate drugs that may compensate for the deficiency in COL22A1 function. The proposed project will identify the biological function of COL22A1 homolog in vivo. It will further determine if mutations in COL22A1 cause IAs, and identify drug candidates that can be used for IA treatments. Understanding genetic causes of aneurysms will enable screening to identify patients at risk and will promote development of new treatments that can prevent devastating consequences of intracranial hemorrhages.

Project Summary Multiple vascular diseases including hypertension, diabetes, atherosclerosis, and others are associated with the dysfunction of vascular endothelium. However, there are currently no effective methods to incorporate new endothelial cells into damaged vessels in vivo which could contribute to vascular regeneration and repair. New blood vessels form by two distinct mechanisms, vasculogenesis, which is differentiation of vascular endothelial cells de novo, and angiogenesis, formation of new vessels by branching from the existing vessels. It is currently thought that vasculogenesis is largely limited to the initial vascular network during embryogenesis, while the majority of the later vessels form by angiogenesis from the existing vasculature. While it is difficult to study vasculogenesis in the mammalian embryos, zebrafish has emerged as an advantageous model system to study vascular development. Molecular mechanisms that control vascular development are highly conserved between all vertebrates including zebrafish and humans. Here we have discovered a novel population of putative vascular progenitors in the zebrafish embryos. These cells show high expression of ETS transcription factor etv2, a known key regulator of vasculogenesis, and are located adjacent to the pronephros (pronephros-associated cells, PACs). Our preliminary data indicate that PACs are the major source of organ specific vasculature, and they contribute to the embryonic vasculature by a novel mechanism of cell intercalation into functional blood vessels. Our data further suggest that PACs are likely conserved in mammalian embryos. In addition, we have identified Junctional Adhesion Molecule Jam2b as one of key regulators required for PAC formation. We hypothesize that PACs are a novel group of multipotent vascular progenitors which provide important contribution for vascular growth. The following specific aims are proposed: 1) Define contribution of PACs to different types of blood vessels; 2) Identify functional role for PACs in vascular development; 3) Identify the role of Jam2b and other upstream regulators in the formation of PACs and vascular development. Lineage tracing approaches will be employed to determine contribution of PACs to different types of vessels in zebrafish embryos. PAC ablation and etv2 conditional inhibition strategies will be used to test the functional role of PACs in zebrafish, and their formation will also be investigated in murine embryos. The role of jam2b in PAC formation, its interaction with Vegf signaling pathway, and functional roles of other PAC-enriched genes will be analyzed in zebrafish. Data obtained in this proposal will answer the key questions regarding the identity and functional role of PACs, and are likely to uncover a novel mechanism of vascular growth. The mechanisms of vasculogenesis are highly conserved, and our preliminary data suggest that similar progenitors are also present in the mammalian embryos. Understanding the mechanism of how PACs form and incorporate into existing vasculature may lead to a new direction to repair damaged vessels for therapeutic purposes.