Molecular Mechanisms of Arterial-Venous Differentiation in Zebrafish

描述（由申请人提供）：动脉和静脉（A-V）模式对于建立功能性胚胎和成人血管系统至关重要。适当的A-V分化对于组织修复过程（例如伤口愈合）期间功能性血管的形成至关重要，并且与许多病理状况（包括A-V畸形和肿瘤诱导的血管生成）相关。因此，了解A-V分化的机制将导致肿瘤发生，创伤修复和多种血管疾病的新治疗。 虽然很难在哺乳动物系统中研究A-V模式，但透明的斑马鱼胚胎很容易进行观察和实验操作。在大血管形成过程中，血管内皮祖细胞（EPCs）在循环开始之前具有动脉或静脉的身份。然而，目前尚不清楚如果EPCs都暴露于相同的信号分子如VEGF和Hh，它们如何在动脉或静脉命运中进行选择。我们已经发现，在进化上保守的主调节血管生成Etsrp /Etv 2的表达时机是在A-V分化的关键因素之一。此外，动脉和静脉祖细胞可能起源于不同的空间位置，内部和外部线，因此经历不同的VEGF浓度。我们假设EPCs的动脉-静脉命运是由VEGF受体flk 1和透明质酸（HA）受体stabilin 2（stab 2）表达的etsrp依赖性时序及其在VEGF梯度内的空间位置决定的。为了证明etsrp表达的时机影响A-V分化，将使用可光活化的吗啉代来抑制斑马鱼胚胎中不同发育阶段的etsrp功能。将进行命运绘图和延时成像以表征细胞运动并确定EPC的内部和外部线的动脉-静脉命运。为了确定Vegf和Hh梯度是否在内层成血管细胞内的Notch信号传导和动脉分化的激活中起作用，将使用过表达和功能丧失方法来抑制Vegf、Hh和Notch信号传导，并结合谱系追踪来确定A-V细胞命运。为了确定Etsrp下游靶标Stab 2是否作为HA的受体起作用并在A-V分化期间诱导Notch信号传导的激活，将研究Stab 2和HA合酶Has 2敲低胚胎中的A-V缺陷。将分析HA-Stab 2信号传导是否导致Stab 2的Tyr-磷酸化和ERK磷酸化，从而导致Notch途径激活和动脉标志物表达。 完成本研究后，我们将确定etsrp表达的时间和VEGF梯度如何导致动脉和静脉基因差异表达的分子机制。所获得的知识将是至关重要的，在我们的A-V分化的分子机制的理解，并将对开发治疗多种疾病和与血管形成相关的病理条件，包括A-V畸形，糖尿病视网膜病变，伤口愈合和肿瘤发生的影响。 公共卫生相关性：拟议的项目将利用斑马鱼模型系统来研究动脉-静脉特化的分子机制。它将确定内皮细胞特化的时间和位置如何转化为不同的动脉和静脉细胞命运。它将研究来自转录因子如Etsrp /Etv 2和形态发生因子如Vegf的不同输入如何整合以诱导或抑制动脉或静脉特异性基因的表达。所获得的知识将是至关重要的，在我们的血管分化的基本机制的理解，这将导致新的治疗肿瘤发生，伤口修复和多种血管疾病。

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.