Molecular Mechanisms of Arterial-Venous Differentiation in Zebrafish

描述(由申请人提供)：动脉和静脉(A-V)模式对于建立有功能的胚胎和成人血管系统至关重要。在创伤愈合等组织修复过程中，适当的动静脉分化对功能性血管的形成至关重要，并与许多病理情况有关，包括动静脉畸形和肿瘤诱导的血管生成。因此，了解动静脉分化的机制将为肿瘤的发生、创面修复和多血管疾病提供新的治疗方法。虽然很难研究哺乳动物系统中的动静脉模式，但透明的斑马鱼胚胎很容易进行观察和实验操作。在主要血管的形成过程中，血管内皮祖细胞(EPC)在开始循环之前具有动脉或静脉特性。然而，如果内皮祖细胞都暴露在相同的信号分子(如血管内皮生长因子和HH)下，它们如何在动脉或静脉命运中选择，目前还不清楚。我们发现，进化上保守的血管生成主调控因子Etsrp/ETV2的表达时机是动静脉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，即内线和外线，因此经历了不同的血管内皮生长因子浓度。我们假设血管内皮祖细胞的动静脉命运是由依赖etsrp的血管内皮生长因子受体flk1和透明质酸(HA)受体稳定蛋白2(Stab2)表达的时间及其在血管内皮生长因子梯度中的空间位置共同决定的。为了证明etsrp表达的时机影响A-V的分化，将在斑马鱼胚胎的不同发育阶段使用可光激活的吗啉来抑制etsrp的功能。将进行命运图和时间推移成像，以表征细胞的运动，并确定内皮祖细胞内线和外线的动静脉命运。为了确定VEGF和HH梯度是否在内线血管母细胞内Notch信号的激活和动脉分化中发挥作用，将采用过表达和功能丧失的方法抑制VEGF、HH和Notch信号，并结合谱系追踪来确定A-V细胞的命运。为了确定Etsrp下游靶基因Stab2是否作为HA的受体，并在A-V分化过程中诱导Notch信号的激活，我们将研究Stab2和HA合成酶Has2基因敲除胚胎中的A-V缺陷。将分析HA-Stab2信号是否导致Stab2酪氨酸磷酸化和ERK磷酸化，从而导致Notch通路激活和动脉标志物表达。在这项研究完成后，我们将确定ETSRP表达的时机和血管内皮生长因子梯度如何导致动脉和静脉基因差异表达的分子机制。所获得的知识将对我们理解动静脉分化的分子机制至关重要，并将对开发多种疾病的治疗方法和与血管形成相关的病理条件产生影响，这些疾病包括动静脉畸形、糖尿病视网膜病变、伤口愈合和肿瘤发生。 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静脉规范的分子机制。它将决定内皮细胞规格的时间和位置如何转化为动脉和静脉细胞的不同命运。它将研究转录因子(如Etsrp/ETV2)和形态因子(如血管内皮生长因子)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。所获得的知识将对我们理解血管分化的基本机制至关重要，这将导致肿瘤发生、伤口修复和多发性血管疾病的新治疗。

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.