The Molecular Basis for Skeletal Patterning

在发育过程中，一个单一的细胞，即受精卵，就会产生一个完整的动物。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伤的组织非常重要，但由于问题非常复杂，这在很大程度上仍然是未知的。因为发育的机制在进化中被很好地保守，所以从研究海胆等简单生物的发育中可以学到很多东西。拟议的研究集中在海胆幼虫骨骼图案形成的机制上，该骨骼是由一种类型的细胞(中胚层细胞)产生的，对来自胚胎表面的其他细胞(外胚层细胞)的信号做出反应。这位PI之前的工作揭示了一些外胚层信号，这些信号充当骨骼图案的指令。PI现在建议确定这些信号中的四个如何改变中胚层细胞，以指导它们在胚胎中的运动。这项工作将发现单个细胞内所有RNA的序列，以了解这四种信号如何改变60个中胚层中每一个中胚层中的RNA和蛋白质。它还将识别和测试中胚层细胞中的受体蛋白对四种信号的反应。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、进行独立研究的本科生和攻读博士学位的研究生。这项拟议的研究集中在海胆发育过程中骨骼图案形成的机制。骨骼由初级间充质细胞(PMC)分泌，而图案信息包含在外胚层中，并被迁移的PMC感知。PI以前发现了许多新的外胚层线索来形成骨骼的图案，这里建议通过在对照胚胎和特定图案线索被抑制的胚胎中使用单个PMCs的单细胞mRNA测序来确定四种不同和保守的线索(硫酸蛋白多糖、5-HETE、血管内皮生长因子和Univin)如何调节PMCs内的基因表达，以促进它们的多样化。时间进程分析和扰动分析的综合结果将被整合到私营军保公司多样化的时间网络模型中；该网络将扩展先前确定的私营军保公司早期规格网络。新的多样化网络中的关键基因将使用敲除方法结合体外PMC迁移分析进行功能测试。一种新的方法将被用来修复体外迁移后的细胞，同时保持它们的空间位置，这将允许确定特定的PMC亚群在朝向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.