The Molecular Basis for Skeletal Patterning

在发育过程中，单个细胞即受精卵发育成一个完整的动物。了解组织在发育过程中是如何形成和成形的，对于预防出生缺陷和了解如何修复受损组织非常重要，但由于问题非常复杂，这在很大程度上仍是未知的。因为潜在的发展机制在进化中得到了很好的保护，所以可以从研究简单生物（如海胆）的发展中学到很多东西。该研究的重点是海胆幼虫骨架形成的机制，它是由一种细胞（中胚层细胞）对胚胎表面其他细胞（外胚层细胞）发出的信号作出反应而产生的。该PI之前的工作揭示了许多外胚层信号，这些信号作为骨骼模式的指令。PI现在提议确定其中四个信号如何改变中胚层细胞，从而指导它们在胚胎内的运动。这项工作将发现单细胞内所有RNA的序列，以便了解这四种信号如何改变60个中胚层细胞中RNA和蛋白质的合成。它还将识别和测试中胚层细胞中受体蛋白对这四种信号的反应。除了在波士顿地区进行科学推广外，PI还将培训学生进行科学研究，包括暑期实习的女高中生、独立学习的本科生和攻读博士学位的研究生。本研究的重点是海胆发育过程中骨骼模式形成的机制。骨架由初代间充质细胞（PMCs）分泌，而模式信息包含在外胚层内，并由迁移的PMCs感知。PI之前发现了许多新的外胚层线索，这些线索对骨骼进行了模式化，这里提出通过对对照胚胎和特定模式线索被抑制的胚胎中的单个pmc进行单细胞mRNA测序，确定四种不同的和保守的线索（硫酸蛋白聚糖、5-HETEs、VEGF和Univin）如何调节pmc内的基因表达，以促进其多样化。时间过程和扰动分析的综合结果将整合到PMC多样化的时间网络模型中；该网络将扩展先前确定的pmc早期规范网络。新的多样化网络中的关键基因将使用敲低方法结合体外PMC迁移分析进行功能测试。一种新的方法将用于固定体外迁移后的细胞，同时保留它们的空间位置，这将允许确定特定PMC亚群在向特定线索或远离特定线索迁移后的行为。

During development, a single cell, the fertilized egg, gives rise to a complete animal. Understanding how tissues are formed and shaped during development is important for the prevention of birth defects and to understand how to repair wounded tissues, yet this remains largely unknown since the problem is very complex. Because the mechanisms underlying development are well-conserved in evolution, much can be learned from studying the development of simple organisms such as sea urchins. The proposed research focuses on the mechanisms underlying the patterning of the sea urchin larval skeleton, which is produced by one type of cell (mesodermal cells), reacting to signals from other cells on the surface of the embryo (ectodermal cells). Previous work by this PI uncovered a number of ectodermal signals that serve as instructions for the skeletal pattern. The PI now proposes to determine how four of those signals each change the mesodermal cells to direct their movement within the embryo. This work will discover the sequence of all the RNA inside single cells in order to understand how these four signals alter which RNA and proteins are made in each of the sixty mesodermal. It will also identify and test the response to the four signals by receptor proteins in the mesodermal cells. In addition to performing scientific outreach in the Boston area, the PI will train students in scientific research, including female high school students in summer internships, undergraduate students performing independent studies, and graduate students seeking Ph.D.s. The proposed research focuses on the mechanism underlying skeletal patterning during sea urchin development. The skeleton is secreted by primary mesenchymal cells (PMCs), while the patterning information is contained within the ectoderm, and sensed by the migrating PMCs. The PI previously identified numerous novel ectodermal cues that pattern the skeleton, and here proposes to determine how four distinct and conserved cues (sulfated proteoglycans, 5-HETEs, VEGF, and Univin) modulate gene expression within the PMCs to promote their diversification by using single-cell mRNA sequencing on individual PMCs in control embryos and in embryos in which specific patterning cues are inhibited. The combined results from time-course and perturbation analyses will be integrated into a temporal network model for PMC diversification; this network will extend the previously determined early specification network for PMCs. Key genes in the new diversification network will be functionally tested using knockdown approaches combined with in vitro PMC migration analyses. A novel approach will be used to fix cells following in vitro migration while preserving their spatial positions, which will allow determination of the behavior of specific PMC subsets following migration toward or away from specific cues.