Order to Disorder Transition in Zebrafish Tailbud Cell Migration

项目摘要 集体细胞迁移是涉及发育和疾病的高度变化的过程。取决于 在上下文中，细胞运动的适当有序程度可以从几乎完全有序或 层流转变为非常无序或湍流。秩序是如何被控制的还没有被很好地理解。的 斑马鱼的尾芽为研究这个问题提供了一个独特的机会。进入尾芽的细胞 通过背内侧（DM）区在一个进行性的，有序的方式，直到他们到达尾芽的尖端。 在那里，中胚层形成的细胞经历EMT以进入祖细胞区（PZ）。PZ中的细胞运动是 非持久性的，以高度无序为特征。然而，在这个领域的细胞运动是 仍然是集体的，这表现为存在频繁的反向、瞬态、各向异性的左右通量 由瞬时的局部命令产生。最终，PZ细胞移动到有丝分裂前中胚层（PSM）， 细胞运动很少。体内和计算机模拟实验表明，有序和无序迁移 是产生正确成形的躯干和尾巴所必需的。DM的过度无序运动减少 细胞通过尾芽的流动导致短体轴的形成，而过度有序的细胞 PZ中的运动引起不适当地延长各向异性通量，形成稳定的细胞涡旋， 胚胎细胞向左右两侧的不对称分配和体轴弯曲。而Wnt、FGF和 已知BMP调节尾芽中的有序程度，但调节局部有序的机制仍然很差 明白这个项目将采取跨尺度的方法，从转录到细胞骨架极性， 组织来研究这个过程。单细胞RNA测序的初步分析 野生型胚胎和经历降低顺序（FGF或BMP）的处理的胚胎的scRNAseq 抑制）已经鉴定了受时间和信号调节的基因。在目标1中，scRNAseq结果 将通过荧光原位杂交进行验证。此外，具有过度顺序的胚胎的scRNAseq 还将进行由Wnt抑制诱导的免疫抑制。在目标2中，DM和DM之间细胞骨架极性的变化 野生型和Wnt、FGF和BMP抑制胚胎中的PZ将使用活荧光报告基因进行测定 通过共焦成像。在目标3中，关于PZ中局部秩序的原因的两个假设，作为细胞拥挤 将测试通过细胞将其极性与外部信号对齐而诱导的涌现特性。一是 管道将被构建以识别光片、时移显微镜数据中的局部顺序。然后相关 将使用分泌的GFP检测细胞外空间来测量局部秩序和细胞拥挤之间的关系。到 为了研究定向迁移的作用，将在TBX 16/mesogenin 1双突变体中定量顺序， 缺乏方向性。我们还将进行微扰实验来测试两个候选化学引诱物 sdf-1/cxcl 12和爱帕琳蛋白是从scRNAseq数据集鉴定的，因为它们能够在PZ中排列细胞运动。

Project Summary Collective cell migration is a highly varied process involved in development and disease. Depending on context, the appropriate degree of order in cell movements can range from a nearly completely ordered or laminar flow to a very disordered or turbulent flow. How order is controlled is not well understood. The zebrafish tailbud provides a unique opportunity to investigate this question. Cells entering the tailbud move through the dorsal medial (DM) zone in a processive, orderly fashion until they reach the tip of the tailbud. There mesoderm fated cells undergo an EMT to enter the progenitor zone (PZ). Cell movements in the PZ are non-persistent and characterized by a high degree of disorder. Nevertheless, cell movement in this domain is still collective as indicated by the presence of frequently reversing, transient, anisotropic left-right fluxes generated by transient, local order. Ultimately PZ cells move into the presomitic mesoderm (PSM) which has very little cell movement. In vivo and in silico experiments indicate that both orderly and disorderly migration are necessary to produce a properly formed trunk and tail. Excessively disordered motions in the DM decrease the flux of cells through the tailbud leading to the formation of a short body axis while excessively ordered cell motions in the PZ induce inappropriately prolonged anisotropic fluxes, the formation of stable cell vortices, asymmetric allotment of cells to the left or right side of the embryo and a bent body axis. While Wnt, FGF, and BMP are known to regulate the degree of order in the tailbud, mechanisms for tuning local order remain poorly understood. This project will take a cross-scale approach from the transcriptional to cytoskeletal polarity to tissue organization to investigate this process. Preliminary analyses of single cell RNA sequencing (scRNAseq) of wildtype embryos and embryos subject to treatments that decrease order (FGF or BMP inhibition) have identified genes subject to temporal and signaling regulation. In Aim 1, the scRNAseq results will be validated by fluorescent in situ hybridization. Additionally, scRNAseq of embryos with excessive order induced by Wnt inhibition will also be performed. In Aim 2, changes in cytoskeletal polarity between the DM and PZ in wildtype and Wnt, FGF, and BMP inhibited embryos will be assayed using live fluorescent reporters imaged by confocal. In Aim 3, two hypotheses as to the cause of local order in the PZ, as a cell crowding induced emergent property or by cells aligning their polarity with an external signal, will be tested. First, a pipeline will be constructed to identify local order in lightsheet, timelapse microscopy data. Then correlation between local order and cell crowding will be measured using a secreted GFP to detect extracellular space. To investigate the role of directed migration, order will be quantified in the tbx16/mesogenin1 double mutant which lacks directional bias. We will also perform perturbative experiments to test two candidate chemoattractants identified from the scRNAseq dataset, sdf-1/cxcl12 and apelin, for their ability to order cell motion in the PZ.