Molecular Mechanisms of Arterial-Venous Differentiation in Zebrafish

描述（由申请人提供）：动脉和静脉（A-V）模式是建立功能性胚胎和成人血管系统的关键。在伤口愈合等组织修复过程中，适当的a - v分化对于功能性血管的形成至关重要，并且与许多病理条件有关，包括a - v畸形和肿瘤诱导的血管生成。因此，了解A-V分化的机制将为肿瘤发生、伤口修复和多种血管疾病提供新的治疗方法。虽然在哺乳动物系统中研究A-V模式很困难，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（EPCs）在循环开始之前就具有动脉或静脉的身份。然而，如果EPCs都暴露于相同的信号分子（如Vegf和Hh），它们是如何选择动脉或静脉命运的，目前尚不清楚。我们发现一个进化上保守的血管发生主调控因子Etsrp / Etv2的表达时间是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，内线和外线，因此具有不同的Vegf浓度。我们假设EPCs的动脉-静脉命运是由etsrp依赖的Vegf受体flk1和透明质酸（HA）受体稳定蛋白2 （stab2）表达的时间和它们在Vegf梯度中的空间位置共同决定的。为了证明etsrp表达的时间会影响A-V分化，我们将使用光激活的morpholinos来抑制斑马鱼胚胎不同发育阶段的etsrp功能。命运图谱和延时成像将用于描述细胞运动特征，并确定内皮细胞内线和外线的动静脉命运。为了确定Vegf和Hh梯度是否在内线成血管细胞的Notch信号激活和动脉分化中发挥作用，我们将采用过表达和功能丧失的方法来抑制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下游目标Stab2是否作为HA受体并在a - v分化过程中诱导Notch信号的激活，将研究Stab2和HA合酶Has2敲低胚胎中的a - v缺陷。我们将分析HA-Stab2信号传导是否导致Stab2的tyrr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。本研究完成后，我们将确定etsrp表达和Vegf梯度的时间如何导致动脉和静脉基因的差异表达的分子机制。获得的知识将对我们理解A-V分化的分子机制至关重要，并将对开发与血管形成相关的多种疾病和病理条件的治疗产生影响，包括A-V畸形、糖尿病视网膜病变、伤口愈合和肿瘤发生。

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.