Global Genomic Regulatory Code for the gastrula stage sea urchin embryo

我对这一部分的审查者所表现出的热情感到满意，因此，这一部分与本文件的原文几乎没有什么不同。然而，一切都是可以改进的，在审查员认为办法描述不够充分或提出了可能遇到的具体问题的地方增加了一些额外的澄清。具体地说，这些附录是：(1)我已经更明确地处理了在何时和何时控制基因扰动的问题 在需要的情况下，特别是在发育的早期阶段需要调控基因的情况下(#vi在S.A.I的程序性讨论中)。(2)我已经阐明了全球网络项目的概念性程序(在S.A.4的#1中进行了补充讨论)。 一位评论家评论说，当脊椎动物GRN与海胆GRN出现显著同源性时，用于研究它们的方法是“模糊的”。我想指出的是，文献中几乎唯一的系统的跨支GRN同源性研究(严格意义上)来自我们的作品，我在最近出版的《调控基因组》的第5章中对这一问题进行了批判性和详细的讨论： 发展和进化中的基因网络“(2006年)。 S.A.7现在专门致力于为关键基因空间表达的动态变化提供GRN解释，这些关键基因是McClay组件的S.A.I的主题。这一目标受到Smith和Davidson最近的工作(出版，2008b；附录；进度报告总结)的强烈推动，该工作进一步扩大了GRN作为对发展的解释的效力： 在这里，我们展示了基因组代码如何直接控制动态的、空间变化的WNT8和Notch信号模式。这项工作展示了在理解动态模式的因果关系的附加问题中应该遵循的方法。 一位评论家评论说，在Re S.A.S中，Bronner-Fraser组件和Davidson组件之间的整合可能会较少，这是明确针对这种相互作用的。在这方面，我要注意到最近的记录：玛丽安·布朗纳-弗雷泽出版的著作广泛证明，我们的GRN理论和实践对她的研究方向产生了密切影响： 她正在使用BioTapestry；她正在组织她的系统中的扰动分析，直接类似于我们的系统；她正在使用我们的许多方法；她正在像我们一样对关键基因进行顺式调控研究；等等。目前的计划将强调和加强这些相互作用，但关键是它们在科学上已经是真实和重要的。 图2已更新，因为自最初提交申请以来，GRN特别在内胚层区域生长；图9，临时外胚层GRN也是如此。 最后，由于最初的应用程序，我们有数据显示了NanoStringnCounter的出色性能，其中的一小部分现在将在第3C2节中进行说明。此外，在最初的申请提交后的几个月里，有7篇承认这笔赠款的论文出现在印刷品或新出版的印刷品中。其中许多都在申请书中讨论过，就像当时在新闻发布会上讨论的那样， 包括关于在c/S调控下阻断基因表达的新技术的论文(Smith等人，2008a)。此外，还有一篇论文描述了纳米串技术及其初步验证(Geiss等人，2008年)。然而，Smith等人(2008b)关于上述动态变化的空间模式的编程的论文具有特别重要的意义，并作为附录被包括在这里，同时还进行了简要的回顾 关于GRN电路(Davidson和Levine，2008)。

I am gratified at the level of enthusiasm expressed by reviewers of this Component, which is consequently little altered from the original in this submission. Everything can be improved however, and some additional clarifications have been added in places where Reviewers commented that the approaches were insufficiently described, or particular problems that might be encountered were raised. Specifically these addenda are: (1) I have dealt more explicitly with the problem of controlling perturbations of genes when and where desired, particularly for the not infrequent case that a regulatory gene is required at an earlier phase of development (# vi under procedural discussion of S.A.I). (2) I have clarified conceptual procedures for the global network project (additional discussion in # 1 of S.A.4). A reviewer commented that the methodology to be utilized in examining vertebrate GRNs as they emerge for significant homologies with sea urchin GRNs was "vague." I would like to note that virtually the only systematic trans-clade GRN homology studies (in the strict sense) in the literature have come from our works, and this matter is discussed critically and at length in Chapter 5 of my recent book "The Regulatory Genome: Gene Networks in Development and Evolution" (2006). S.A. 7 is now focused exclusively on providing GRN explanations for the dynamic changes in spatial expression of the key genes that are the subject of S.A.I of the McClay Component. This objective is motivated strongly by the very recent work of Smith and Davidson (In press, 2008b; Appendix; summarized in Progress Report), which has expanded even further the potency of GRNs as an explanation for development: here we show how the genomic code directly controls a dynamic, spatially changing pattern of Wnt8 and Notch signaling. This work shows the way to be followed in additional problems of causality in understanding dynamic patterning. A reviewer commented that there may be less integration between the Bronner-Fraser Component and the Davidson Component in re S.A.S, which is explicitly directed toward that interaction. I would like to note in this connection the recent track record: the published works of Marianne Bronner-Fraser display extensive evidence of the close influence our GRN theory and practice has had on her research orientation: she is using BioTapestry; she is organizing perturbation analyses in her system directly analogous to ours; she is using many of our methods; she is carrying out cis-regulatory studies of key genes just as do we; etc. The current plans will accentuate and intensify these interactions, but the point is that they are already scientifically real and important. Figure 2. has been updated as the GRN has grown particularly in the endoderm area since the original Application was submitted; and Fig.9, the provisional ectoderm GRN, likewise. Finally, since the original application, we have data showing the brilliant performance of the NanoString nCounter, a small sample of which is now illustrated in Section 3c2. Also in the way of progress, 7 papers acknowledging this Grant have appeared in print or are newly in Press in the months since the original Application was submitted. Many of these were discussed in that Application, as they were then In Press, including the paper on the new technology for blocking gene expression under c/s-regulatory control (Smith et al, 2008a). There is in addition a paper describing NanoString technology and its initial validation (Geiss et al, 2008). However, the paper of Smith et al (2008b) on programming of dynamically changing spatial patterns mentioned above is of particular importance and is included here as an Appendix, together with a brief review on GRN circuitry (Davidson and Levine, 2008).