Global Genomic Regulatory Code for the gastrula stage sea urchin embryo

California Institute Of Technology, Pasadena CA

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Abstract

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).