Eric H. Davidson - US grants

This proposed research program is a direct extension of our earlier programs with the same fundamental goals: the investigation of reproduction, oogenesis and early embryonic development and the molecular mechanisms that control genetic activity. The recent developments in our ability to study both individual gene activity and the organization of gene regions at the sequence level will be utilized to study embryonic development in several animal systems with continued focus on the sea urchin. We wish to continue our studies of germline transformation of sea urchin embryos including the development of new transformation vectors carrying a variety of unmodified and modified gene regions. Since transformed individuals will survive to sexual maturity we expect to be able to examine hereditary as well as immediate effects during development, metamorphosis and maturation. We plan to clone and utilize genes that are expressed at specific times and in specific cell types. Using transformation and a variety of other approaches we wish to continue our examination of the mechanism of gene regulation and the role of chosen genes in development. We plan to continue our work on the overall structure, fate and function of maternal poly(A) RNA. We hope to determine whether particular classes of genes are constituitively transcribed into nuclear RNA. We plan to study the molecular mechanisms of initial determination in sea urchin micromeres. We plan to compare the patterns of transcription of specific genes in lampbrush chromosomes of Xenopus oocytes. We intend, through the integration of the many available methods for molecular examination of gene expression, to examine the ways in which the system of regulation establishes the processes of oogenesis and embryonic morphogenesis.

The overall objective of this Program Project is understanding the sequence organization of the animal genome. The Project unites efforts of five laboratories, and is directed into three Task Areas: I) Development of new technology for analysis of DNA sequence organization; II) Organization of specific genes and gene families; III) Repetitive sequences, transposable sequence elements, and evolution of the genome. The specific methods to be developed (Task Are I) include new procedures for two-dimensional electrophoresis of DNA fragments, for direct selection of cosmid and cDNA clones, for determining the reiteration frequency of repeated sequences, and for mapping repeat length and spacing in cloned DNA fragments. Among the specific genes and families to be studied (Task Area II) are the actin genes of echinoderms and Drosophila genes for several mammalian growth factors, genes for the opsin visual proteins of the Drosophila eye, and genes for the ion channels of mammalian nerve and muscle. Proposals focussed on genomic repeats, sequence element transposition, and evolution (Task Area III) include investigations of evolutionary transposition of repetitive sequences in echinoderm and human DNAs the origins of actin gene subfamilies in sea urchin evolution, and further sequence organization studies in cloned mammalian DNA fragments. This program Project operates in a highly cooperative manner in which enzymes required for manipulation of nucleic acids are prepared by a Core facility, and equipment, information, technology and results are freely shared among the participating laboratories.

The molecular mechanisms of early development in the sea urchin must be interpreted in terms of the embryonic cell lineage. The purpose of this investigation is to further explore the role of cell lineage relationships in the development of sea urchin embryos and larvae. A multiple approach is taken: 1) Building on the existing, partially completed database, some studies will address unsolved problems in the lineage of the S. purpuratus embryo by further defining the lineage and specification within the definitive vegetal plate, by determining more completely the fate of the eight small micromeres, and by describing the origins of the neuroblasts; 2) To describe lineage changes in regulative development, a group of experiments will compare normal lineage patterns with the lineage of cells in isolated animal caps treated with LiCl or fused with micromeres and will compare normal lineage patterns with those of meridional quarter embryos, and 3) To provide descriptions of lineage and specification in the morphogenesis of the juvenile echinoderm, the expression of histospecific marker genes in the rudiment of S. purpuratus larvae will be investigated by in situ hybridization. %%% A fundamental problem in developmental biology is how the various cell types found in an adult are derived from a single fertilized egg. This study will trace the cell fates of founder cells from very early divisions after fertilization to their ultimate form in a larvae. It will also explore the factors which determine the fate of those cells.

Currently in its 98th consecutive year, the Embryology Course of the Marine Biological Laboratory at Woods Hole has played an important role in the lives of many American developmental biologists. The objective of the Course is to train predoctoral and postdoctoral students for research careers in developmental biology In a unique physical and intellectual environment not duplicated In the nation's universities, medical schools or research institutes. The Embryology Course offers a dally series of formal lectures followed by extended discussions, laboratory research experience, technical workshops and informal seminars over a six week period in the summer. The Course is directed toward predoctoral and postdoctoral students who are committed to research careers in the field of development. The Course addresses major current problems in development, then critically explores those problems through discussions and through laboratories in which advanced new techniques are presented. Outstanding students are chosen from an international applicant pool. The teaching faculty are senior scientists in the field. The Course consists of four modules, each of which is about 10 days long. Each module is staffed by an average of three resident instructors and two outside lecturers. The themes of the modules change from year to year as important developments in the field change. The Course introduces students to a large number of terrestrial and marine embryos including nematodes, insects, amphibians, fish, chick, echinoderms, ascidian, gastropod molluscs, and mice. Techniques include current molecular biological techniques, embryonic manipulation, microinjection and micromanipulation, microscopy and computer enhanced imaging technologies, and a number of immunochemical approaches. Students are challenged to formulate and test hypotheses in a well equipped research environment and under the guidance of experienced research faculty and assistants. The Embryology Course occupies a 10,000 sq. ft. modern laboratory suite in the Loeb Building which is the main teaching facility at the Marine Biological Laboratory. The Lab includes faculty and student laboratories, a marine animal room, constant temperature rooms, imaging lab, tissue culture room, microinjection and micromanipulation facilities, and an equipment room with state-of-the-art research equipment. An adjacent seminar room and lecture hall are used by the Course as well. The Marine Biological Laboratory provides support facilities for purchasing and clerical work, radiation maintenance and safety, confocal and electron microscopy, an animal collection facility, and one of the nation's finest biological research libraries.

In the sea urchin embryo there are five territories of invariant lineage - the prospective aboral ectoderm, the prospective oral ectoderm the vegetal plate, the skeletogenic mesenchyme and the small micromeres. In addition to a unique pattern of gene expression, these territories are defined by individual cell lineage histories and generation of distinct cell types. The lineages forming these territories are completely segregated from each other by the 6th cleavage and spatially restricted gene expression begins one or two divisions later. In all the territories except the skeletogenic mesenchyme, the founder cells that are the progenitors of each territory achieve unique patterns of gene expression by means of intracellular interactions with other cells. The following studies are designed to determine the lineage and fate of founder cells for specific cell types within territories; to investigate the specific functional significance of interactions with neighboring cells in establishing certain of the territories; and to test the role of major signal transduction systems in the specification of territories and cell types. The proposed experiments fall into the following categories: 1. Vegetal plate and neuronal lineage tracing with caged flourescein- dextran. 2. Cell lineage and cell interaction studied by means of cell ablation. 3. Molecular characterization of abnormal phenotypes derived from introduction of a foreign serotonin ligand- receptor system. 4. Developmental consequences of ectopic stimulation of the PI signal transduction system in specific blastomeres of known lineage (using the serotonin receptor). 5. The effects of elevated levels of second messengers in whole embryos and individual blastomeres. 6. Developmental effects of the expression of foreign receptor kinases in whole embryos and individual cells.

The focus of research in the three laboratories that constitute this Program Project is the molecular basis of regulatory flow in early embryonic development. The Program Project will utilize the highly developed sea urchin and ascidian model systems for this research. Major objective are to develop functional comprehension of cis-regulatory systems that control genes expressed spatially in the early embryo, using genes that operate at different levels of the gene regulatory network; to discern the overall architecture of this network; and to discern the molecular mechanisms by which maternal transcription factors are activated, and control is transformed from maternal to zygotic regulatory processes. The three Research Components are:(1) Davidson Component, "Gene Regulatory Mechanisms and the Control of Early Embryogenesis"; (2) Fraser Component, "In Vivo Imaging of Gene Regulatory Events in the Early Embryo"; (3) Levine Component "Gene Regulation in the Ascidian Ciona intestinalis." The major aims of the Davidson Component are cis-regulatory analysis of genes expressed differentially in the sea urchin embryo, and identification and characterization of the maternal and zygotic transcription factors which control this expression. The major focus of the Fraser Component is visualization of the state of cis-regulatory elements in vivo and spatial visualization by new imaging methods of modifications of maternal transcription factors, using experimental systems characterized in the Davidson Component. The major focus of the Levine Component will be cis-regulatory analysis of certain key regulatory genes expressed in ascidian embryos, which are also to be characterized in the sea urchin embryo in the Davidson Component, and interphyletic gene transfer experiments to be carried out collaboratively between the Davidson and Levine labs. A salient characteristic of the proposed research is the engagement of powerful new technologies, some entirely novel. All the Components. Will rely on two Research Core Facilities, viz the SUMS Facility at Caltech's Marine Laboratory, which will provide sea urchins, gametes and nuclear extracts from which transcription factors are purified; and the Microsequencing Facility, where partially purified transcription factors are sequenced at picomole levels. An Administrative Core will oversee management, budget, and interlaboratory communication, with respect to material, financial, and intellectual matters.

For many years our research program has been focused on the mechanisms of gene regulation in early sea urchin embryos, and this will continue to be its major thrust. We have developed and successfully exploited an excellent suite of technologies for complete structure/function analysis of embryonic cis-regulatory systems. Among these technologies are: a gene transfer procedure that permits expression on many groups of hundreds of embryos at a time, so that the spatial and temporal behavior of expression vectors bearing mutations or consisting in part of synthetic DNAs can be studied in great variety; a novel methodology for knocking our transcription factor activity under experimental genetic control; a methodology for cloning transcription factors after affinity purification; use of large-scale high density arrayed clone libraries; advanced imaging technology for digital recording of spatial expression of fluorescent receptors; and a complete armamentarium of lineage tracing, cell biological, microdissection and in situ procedures for assay of spatial gene expression. In the next period we intend to extend and deepen our mechanistic understanding of gene regulation in early development, by exploiting these methods to characterize functionally some additional cis-regulatory elements that lie in key positions in the genetic control network of the early embryo. We will complete our analysis of the complex Endo16 cis-regulatory system, identifying specific repressors and activators that are not yet characterized. Some additional differentially expressed, downstream genes isolated by differential screening of high density arrayed libraries will be subjected to similar analysis, to determined their cis-regulatory organization; and to identify positive and negative spatial regulatory interactions & elements that lie at the termini of signal transduction systems. A major effort will be directed to cis-regulatory analysis of the systems controlling early zygotic expression of regulatory genes encoding transcription factors. Among these will be factors already identified as controllers of the terminal genes we have already studied. Our overall objective is to understand the gene regulatory network that is set into operation as control is shifted from the maternal to the zygotic transcription regulatory apparatus in cleavage and blastula stages. We will utilize several new approaches to isolate gene batteries, i.e., sets of downstream genes that are controlled by the same set of transcription factors. Major efforts will be devoted to trying to understand the disposition and complexity of maternal transcription factors, the functions of which are known from cis-regulatory analysis of their target genes. Here we have three specific objectives; first, localization in the early embryo by conventional confocal immunocytology, and assessment of regional activity in respect to DNA target site binding by a microcapillary-based shift procedure that we developed; second, analysis of transcription factor variants by mass spectrometry and other methods; and third, localization of variants in mass-isolated cell fractions representing different territories of the early embryos. We are also planning to apply our gene regulatory approaches to the significance of cell contacts in the blastula wall. The synthesis and turnover rates of beta-catenin will be measured to determine whether control of its availability is maternal or zygotic in the cleavage-blastula-stage embryo, and if zygotic we will investigate its cis-regulatory system. We will also study the mechanistic basis on CyIIIa cytoskeletal actin gene down-regulation in disaggregated ectoderm cells, exploiting our complete understanding of the CyIIIa cis- regulatory system. An additional approach to functional significance in cis- regulatory systems is to use interphyletic gene transfer. Thus we will determine the locus of expression in sea urchin embryos of ascidian cis- regulatory systems that control early expression of key transcription factors in endoderm (GATA factors, some homeodomain factors, and forkhead factors); and in mesoderm (e.g., snail and not). Similarly we will provide the corresponding sea urchin cis-regulatory reporter systems for tests in ascidian embryos. Since transcription factors can be directly isolated from sea urchin extracts, this approach may provide molecular evidence of the upstream (often maternal) regulators that generate early zygotic regulatory gene expression in chordate embryos as well.

DESCRIPTION (Adapted from applicant's description): The thirteenth consecutive meeting of the sea urchin developmental biology research community, called Sea Urchin Developmental Biology XIII, will be held from September 28 to October 2, 2000. The meeting will commence with registration and a plenary session on the first evening and end with a plenary session on the last morning. A banquet and business meeting will occupy the last evening. The invitees include all those interested in the development of the sea urchin. The meeting is intended to encourage information exchange in the area of sea urchin development and closely related areas such as evolution of developmental mechanisms. Sessions covering all areas of development from gametogenesis to larval development and metamorphosis will be mounted as well as those concerning cell biology of sea urchin gametes and embryos, genomics and evolution.

This application is to support the distribution to the sea urchin developmental molecular and cell biology research community of high density arrayed filters (macroarrays) containing large cDNA libraries, that represent every stage of embryogenesis, several individual cell types, and various adult tissues. We will also create and distribute microarrays bearing genes of specific interest to the community. Both macro- and microarrays will be generated using the Genetix robot at Caltech. The cDNA libraries have already all been constructed and arrayed as part of the Strongylocentrotus puipurntus Genome Project. Macroarray filter distribution to selected laboratories has been initiated on a pilot scale, with extremely enthusiastic responses, as attested by letters within the Application. Quality control testing will be routinely carried out, and the service we intend to maintain will include recovery and shipping of any clones identified by users of the macroarrays on request. The arrayed libraries wffl be maintained in freezer banks in my laboratory. An important additional objective is the management of a continuously updated, publicly available web site data base in which information as to the identity of clones in the arrays is accumulated from all laboratories utilizing them. The ancillary research and development component of the proposed work includes the perfection of a powerful new methodology for differential subtractive hybridization on the macroarrays; measurement of hybridization kinetics on microarrays to establish useful parameters of sensitivity under diverse experimental situations; development of probes for use on both micro-and macroarrays that represent any given regulatory state in the sea urchin embryo; and completion of a custom made unique processing system for quantitative differential analysis of macroarray screens using complex probes.

DESCRIPTION (adapted from investigator's abstract): It is proposed to construct a computational model which precisely describes the genomic regulatory apparatus required for endomesoderm specification in sea urchin embryos. The model is to be couched in terms of specific, experimentally verifiable or falsifiable predictions that define necessary inputs and outputs of key cis regulatory elements. These are elements which control expression in time and space of genes encoding transcription factors and certain signaling components, which other evidence has identified as relevant genes required in the process of endomesoderm specification. A first stage model of this kind has been built. The investigators will carry out kinetic measurements required to ascertain logical interrelations within the relevant cis regulatory systems and other experiments designed to test architectural features of the computational model. These studies will examine the predicted effects of target site mutations on spatial expression of genes involved in endomesoderm specification, under specific conditions of perturbation. They will also generate a 3D digital construction of the embryo through time, using confocal imaging data, and impose on it the gene expression profiles predicted by the computational regulatory model. The model will ultimately constitute a quantitative computational analysis of a genomic regulatory network, defined at the DNA sequence level, the function of which is to organize a complex, major process in animal development.

0212869
Cameron


Understanding the details of how individual cis-regulatory elements function and how the gene regulatory networks they form control developmental process is crucial to understanding how the genome works, how development works, and how evolution works. An effective method of locating individual cis-regulatory elements is comparative sequence analysis conducted at appropriate divergence times to reveal conserved elements. Current results show that this approach may yield a more than 10-fold increase in rate of experimental cis-regulatory element discovery, compared to the most efficient "blind" search methods. However, general rules for carrying out such analyses are not yet known, and initial work has shown very different degrees of similarity between genomic regions surrounding different genes in a single pair of sea urchin species. These investigators propose to determine the rules for efficient cis-regulatory sequence prediction by interspecific sequence analysis. To this end they will analyze and then test by gene transfer putative cis-regulatory elements identified in the vicinity of about 20 different genes, using several different echinoderm species that display a range of phylogenetic relatedness. The sea urchin embryo is a model system of choice in which this method can be quickly and effectively explored: species separated by various known evolutionary distances are available; they have already established methods for comparative prediction of conserved cis-regulatory sequence for one sea urchin species pair and shown that the methods work; and there is a practical high throughput gene transfer technology available. These investigators will construct BAC libraries for 6-8 species including sea urchins, and for more distant comparisons, a sea star and a hemichordate. Sequence of BACs containing the set of genes to be studied will be obtained, and they will then identify putative cis-regulatory regions for the candidate genes selected, as indicated by interspecific sequence comparison at diverse distances. These DNA fragments will be tested for cis-regulatory capability by gene transfer. Not only will this approach reveal rules for computational cis-regulatory analysis, but it will also support the extension of the current repertoire of BAC libraries, improve computational tools, and generate more efficient laboratory methods for this essential research area.

This proposal seeks support for the Arrayed Gene Library Resource for the purple sea urchin, Strongylocentrotus purpuratus. This Resource is the sole provider of genomic materials for the whole genome sequencing effort in addition to its service to the cell and developmental research community. There are two parts to the Resource: a robotic arrayed library and a computational facility. The robotics facility generates, maintains and distributes arrayed library materials to the research commuinity. The new service aims are web pages to enhance the usefulness of the system. The pages will detail: 1) perturbing reagents, probes, and specific gene primers used in this system;2) expression and function of sea urchin transcription factors;3) links between sea urchin DMA sequences and resource plasmid clones; 4) implement distribution of specific software and documentation;and 5) list community specific information. In the research component we will provide cDNA libraries to inform the annotation of the genome. We plan to: 1) to produce a tissue-specific library from adult radial nerve;2) to produce a library containing adult skeletogenic genes;3) to construct a library containing differentiation genes for adult oral structures;4) to produce a library from the juvenile lantern for muscle genes;and 5) to determine the quality of existing adult tissue libraries We envision the Sea Urchin Genome Resource as part of a distributed system whose individual efforts make up a highly connected, useable whole genome resource. We act as a portal to bring the accumulated experience of experimental developmental biology to bioinformaticists. And we present the bioinformatics tools and databases of genomic sequence collections to the experimentalist. All of the individual goals detailed above are direct responses to this view.

In this component of the Program Project we propose to extend gene regulatory network (GRN) analysis to the whole of the sea urchin embryo from early cleavage to shortly before feeding begins. Specific perturbations of gene expression are known which knock out development of specific parts of the embryo, and it is proposed to use these to isolate sets of regulatory and other genes by high sensitivity differential array screening that are specifically required for: skeletogenesis; specification and differentiation of aboral ectoderm; specification and differentiation of the oral ectoderm, ciliated band and hood; specification and differentiation of the archenteron, hindgut and foregut/midgut. Spatial and temporal expression of cDNA clones thus isolated will be characterized. GRNs for these components of the embryo will be formulated on the basis of a large-scale perturbation analysis including all relevant genes, and the components of the GRNs will be interlinked by examination of the regulatory targets of the signaling interactions by which the domains of the embryo are functionally coordinated during development. Key nodes of the GRNs will be subjected to experimental challenge by cis-regulatory analysis. The Proposal also includes development of various specific new technologies, some by a collaborative effort with our partners in the Program Project.

DESCRIPTION (provided by applicant): This renewal application follows on our success in generating a large-scale gene regulatory network (GRN) for development of the endomesoderm of the sea urchin embryo. Progress on this GRN from the beginning depended on computational tools and approaches developed under the auspices of this grant, as well as on system-level experimental approaches. When we originally applied for this grant, the endomesoderm GRN map was in a preliminary state of coalescence; now it is a well developed, experimentally testable, defined logic map of the genomic source code for embryonic specification. As described in Progress Report, we built the mode of presentation and analysis used for the GRN in the course of this project; and we developed from scratch an extremely effective computational approach to interspecific sequence comparison for the purpose of identifying cis-regulatory elements. This latter method is in wide use, and in our lab has permitted rapid identification of many cis-regulatory elements, thereby permitting test, at the DNA level, of the inputs produced in the GRN. We now propose to carry out computational development of the GRN to an entirely new level. We will produce an interactive GRN model that will: (i) display causal interrelations at any level from territorial functionalities (zoom out) to specific inputs into given cis-regulatory elements (zoom in); (ii) provide access to the interaction map, via given genes, via given times in development, via given embryonic spatial domains; (iii) permit incorporation into the GRN of kinetic relationships, as measurements become available; (iv) represent cis-regulatory elements as combinations of logic functions; and (v)provide links to relevant genomic sequence and to experimental evidence on which functional GRN linkages have been proposed and authenticated at the cis-regulatory level. We also propose to model and test many kinetic predictions by means of time course measurements, using approaches similar to those of our recently developed gene cascade kinetic simulations, described also in Progress Report. In addition we intend to complete an input/output kinetic analysis of a specific cis-regulatory system, as a demonstration project for mathematical representation of cis-regulatory operations within the GRN. Finally, we propose to identify given GRN circuit elements, produce synthetic cis-regulatory constructs in which normal operation of these elements will be altered, and reintroduce them into the embryo to determine whether our understanding of the GRN is sufficient to permit reengineering of the embryonic process.

genetic library; biomedical resource; sea urchins; molecular biology information system; Internet; information dissemination; microarray technology; animal genetic material tag; subtraction hybridization; complementary DNA;

The major objective of this Renewal Program Project Application remains as stated in its predecessor, to determine "the molecular basis of regulatory information flow in early embryonic development." However, to a significant extent as a result of our work on this Program, the meaning of these words has been transformed, and this Renewal Application is focused on the opportunity now confronting us to solve the problem of regulatory interaction flow in the embryo by determining the gene regulatory network (GRN) that directs its development. The GRN consists largely of interactions mandated by genomic cis-regulatory sequences controlling genes that encode transcription factors and some signaling components. The initial inputs are maternal factors, and the terminal output in each spatial domain is activation of genes encoding differentiation proteins. In the Davidson Component of the Program, GRN analysis of embryogenesis in the sea urchin, Strongylocentrotus purpuratus will be extended to the whole of the embryo, and almost the whole of embryogenesis (from shortly after fertilization to about 2.5 days). The Fraser Component is focused on novel approaches to imaging the activity of GRNs, using sea urchin and ascidian embryos. In the Levine Component key linkages of the endomesoderm GRN of the sea urchin embryo will be sought in the ascidian embryo, and the possibility will be examined that there is a basal cis-regulatory code for this fundamental process in embryogenesis shared between chordates and echinoderms. The Administrative section will oversee management, budget, and interlaboratory coordination with respect to financial, material and intellectual matters. A Research Core Service Facility similar but not identical to the current one is also requested, the basic role of which is to provide embryos, eggs and nuclear extracts of S. purpuratus to the three Research Components.

Intellectual Merit
The sequence differences that have occurred in functionally well characterized cis-regulatory modules over the course of evolution will be examined in order to reveal the rules of change in this information-rich segment of the genome. There now exists a set of libraries from well known non-chordate deuterostome species and functionally characterized candidate genes which comprise one of the most comprehensive experimental platforms ever assembled to address these questions. Compared to the reference species Strongylocentrotus purpuratus, the six comparable species' divergence times span from 20 to 540 million years. Candidate genes have been previously analyzed by gene transfer in the reference species and experimental and computational tools have been developed to undertake these comparative studies. From 8 candidates will be gathered the sequences of putative cis-regulatory modules at varying evolutionary distances. These will be compared to gauge the quality and quantity of changes that have occurred and their function will be verified using gene transfer of large-insert recombinant reporter constructs in the reference species. Besides supporting a theory of cis-regulatory module sequence evolution, the rules that emerge from these mechanisms will provide better predictive methods for discovering cis-regulatory modules in genomes where little previous experimental data is available.
Broader Impacts
Understanding how cis-regulatory modules evolve lies at the nexus of several major questions in modern biology. The mechanisms operating here will help explain us how the genome works, how development works and how evolution works. The application of the principles learned here will be disseminated through the usual academic channels including journal publications, meeting presentations, graduate and undergradate teaching, and books.
The PI and Co-PI have a history of involving undergraduates in laboratory research through the Caltech Summer Undergraduate Research Fellowships (SURF), often funded by REU supplements to NSF grants. Additionally, the PI routinely serves on SURF ad hoc review boards. The training of graduate and post-doctoral personnel is an important component of the operation of the Davidson Laboratory, the Center for Computational Regulatory Genomics and the Genome Facility.

We propose to develop a communal resource of BAG GFP expression vectors of all transcription factors expressed significantlyin the embryo of the sea urchin, Strongylocentrotus purpuratus, the genomic sequence of which will go on line shortly. Critically, as shown by our laboratory, BAG GFP recombinants can be injected into eggs and with high efficiency, and they express accurately, capturing the entire cis-regulatory systems intact. This greatly accelerates detailed cis-regulatory analyses. These recombinants can also be used to introduce exogenous regulatory gene products in temporally and spatiallyrestricted patterns within the embryo and larva. Both of these applications will greatly enhance analysis of how the genomic control systems which direct expression of regulatory genes prescribe early development. Specifically, we propose to isolate BAG recombinants for all relevant transcription factors (-100-200) in which the gene of interest is centrally located within the clone, and then to use standard homologous recombination techniques to replace the first exon of the endogenous gene with the coding sequence of the GFP reporter. An ongoing project within our laboratory has already established the expression profiles of most of the pertinent genes. We will then employ our methods for high throughput production of BAC-GFP recombinants in exonl of each gene, and build and authenticate each construct. GFP expression directed by each BAG recombinant will be analyzed to confirm faithful recapitulation of endogenous gene expression. We will also make BAG recombinants for any other gene encoding any kind of protein, that scientists in the community may request. All information needed by other investigators to facilitate access and promote the use of these tools will be made available on the Sea Urchin Genome web site that is currently administrated by our laboratory. Numerous letters from outside investigators attesting interest in this facility are included as an Appendix to this application.

To understand evolution the buck stops at the DNA, since the characters that differ between species that have a common ancestor are heritably encoded in their different DNA genomes. The Davidson laboratory has designed experiments to determine what really happened in a specific case of evolution that can be analyzed experimentally at the DNA level. They argue that the most interesting evolutionary features are those that control development of the body plan and that while there is much hand-waving about evolution, there are few cases where one can put one''s finger directly on the sequences that cause specific differences. They also propose that these differences are almost always in the modular control sequences that specifically determine where, when and how intensely genes are expressed in response to cellular regulatory state. The Davidson laboratory will focus on developing sea urchins and sea stars, which had a common ancestor about a half billion years ago and whose developmental control systems include several cases where comparisons reveal the same genes doing very different things. This differential regulation results in two kinds of embryos that are, in some respects, quite distinct in form and function. The Davidson laboratory has developed the technology to uncover the differences that matter in these control systems, and to convert them into one another experimentally. Nuts and bolts knowledge of evolution is not only basic to understanding our own and all other animals'' biology at a mechanistic level, but will have a gigantic practical payoff. Evolution is our only natural laboratory for change in genomic control systems. To re-engineer these systems for therapeutic or other benign purposes, will require that we learn the rules of allowed and forbidden change in these systems. The Davidson laboratory has significant broader impacts on the developmental biology community by developing new approaches and technologies and for pioneering work on unraveling gene regulatory circuitry during development. The laboratory also has a strong record of student training and participates in the Caltech''s summer SURF program for undergraduates.

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).

Core B: SCIENTIFIC AND ADMINISTRATIVE COORDINATION CORE The objective of this Core Unit is to assure tight administrative coordination of the whole POI and to systematically organize continuing inter-component communication. The SAC Core will be overseen by the PI, and the lead Senior Administrator will be Jane Rigg. An Assistant Administrator is also on staff, and Caltech provides the necessary Grants Management and Accounting services. These same arrangements, in particular the administrative operations of Jane Rigg, have functioned successfully throughout the 9 years of the current POI. Eariier she administered many large and small projects, including Foundation and Program Projects, dating back to 1971.

DESCRIPTION (provided by applicant): This Application addresses broad Challenge Area (08), Genomics, and Specific Challenge Topic 08-HG-101 "Technology and resources for high throughput functional analysis of functional elements in genomic sequences". A two year project is proposed to expand, to exploit, to extend, and to apply on a demonstration basis a completely novel approach to high throughput cis-regulatory analysis. We have very recently developed the initial component of this new technology as a tool for rapid validation of sea urchin embryo gene regulatory network models, and demonstrated its efficacy in facilitating the discovery and in measuring the quantitative activity of previously unknown cis-regulatory modules with a throughput of up to 100x that of traditional methods. The essential principle of this approach is use of sequence-tagged "barcoded" vectors which can be introduced together in large number in a single experiment and de-convolved later. But there remain many additional spinoffs and additional developments to be brought to practice, and the Challenge Grant program offers the opportunity to mount a "crash program" and bring these opportunities on line in the immediate future. In addition, the methods we have so far developed measure quantitative cis-regulatory output and not spatial activity. We propose additional technological developments to generate higher quality spatial expression data than obtainable by any other means and a high throughput method of recovering large sets of cis-regulatory modules operating in any given spatial regulatory state. The specific aims of this proposal include adapting the sequence tag method to NanoString technology to permit simultaneous assessment of activity of >100 different cis-regulatory modules;demonstrate the use of this method to obtain temporal output profiles of large numbers of cis-regulatory modules simultaneously;develop a very high accuracy method of determining spatial expression profiles of unknown cis-regulatory modules by use of NanoString measurements;and tune for general use a high throughput technology for isolating all cis-regulatory modules of a large unknown set which operate in a given time-space domain of the organism. Two additional comments are important: first, there is no a priori reason why these technologies should not be transferrable to any other system in which gene transfer by direct DNA injection is utilized;and second, in order to accomplish these objectives we shall have to build a new research subgroup. This will require hiring additional personnel, and in this respect both the scientific and organizational aspects of the proposal synergize with the objectives of the A.R.R.A. NIH initiative. This work is about finding the causal lines of control that determine how fundamental life processes are executed according to the instructions encoded in the genomic regulatory system. The most powerful approach to general solutions to complex disease states requires solid understanding of their control circuitry. Our practice must get beyond struggling to ameliorate effects rather than altering causes. This research shows the way to discovery of structure and function in causal genomic control systems.

The objective of Core A is indicated by its title. Its functions are defined as the provision of technologies, services and research materials used by multiple Research Components, which are difficult or expensive to generate, which can be provided by a central source, and indeed would be ridiculously wasteful for each lab to provide for itself. The emphasis here is on high technology aspects, and materials that require special facilities or know-how to generate.

DESCRIPTION (provided by applicant): All developmental, morphogenetic, and differentiation functions of animal cells are executed by large, specifically deployed batteries and cassettes of downstream protein coding genes. A major success of bioscience in recent years has been system level elucidation of the upstream gene regulatory networks (GRNs) that control pattern formation and determine development of the body plan. GRNs consist of genes encoding transcription factors and signaling molecules, plus the transcriptional linkages among these genes, and they determine the regulatory state of every cell at every point in developmental time. Therefore they include the control inputs into all the downstream genes that do the work of the cell. But an enormously important gap in understanding now separates the upstream GRNs that we are beginning to learn about from the downstream effectors of cell function: What is the actual control circuitry that determines the deployment of these downstream genes? How, exactly, are GRNs causally connected to cellular functions of differentiation and morphogenesis? In principle, given knowledge of the upstream GRN and various newly available technologies, this gap can be closed, and the problem solved at a system level, and this is the particular object of the present proposal. We will choose three cell types of the developing sea urchin embryo, each of general interest. Knowledge of the upstream GRNs of this embryo is more advanced than for any other system. The target cell types are the immune cells of the embryo, where the downstream effector genes encode a great variety of innate immunity proteins;gastrulating endoderm cells, where the downstream genes mediate gastrular invagination;and the totipotent set-aside cells of the embryonic coelomic pouches that in larval stage produce the adult body plan, where the downstream effector cells include those that maintain totipotency. Innate immunity, gastrular invagination, and totipotent cell lineages are all pan-bilaterian features. The approach, briefly, will be isolation of each cell type using specific regulatory gene expression and FACS;deep transcriptome sequencing;bioinformatic prediction;and validation of causal regulatory connections to specifically expressed genes by high throughput cis- regulatory analysis. This project will provide the first global upstream-downstream GRN for any developing system. It will inform as to basic principles by which downstream gene cassettes are organized. It will also serve as a technological demonstration project for similar approaches to mammalian systems. PUBLIC HEALTH RELEVANCE: This work is about finding the causal lines of control that determine how fundamental life processes are executed according to the instructions encoded in the genomic regulatory system. The most powerful approach to general solutions to complex disease states requires solid understanding of their control circuitry. Our practice must get beyond struggling to ameliorate effects rather than altering causes. This research shows the way to discovery of structure and function in causal genomic control systems.

This INSPIRE award is partially funded by the Evolution of Developmental Mechanisms Program in the Division of Integrated Organismal Systems in the Biology Directorate and by the Sedimentary Geology & Paleobiology Program in the Division of Earth Sciences in the Geology Directorate.

All contemporary animal forms, including ourselves, are the result of evolution from prior forms, as demonstrated unequivocally by the fossil record. But observation of fossils cannot inform as to how changes in body plan occurred; that question can only be answered experimentally. This interdisciplinary INSPIRE project capitalizes on a unique opportunity to study the same set of divergent characters from an evolutionary structural vantage point, using fossils; and from a developmental molecular biology vantage point studying processes in living descendants of the fossil lineages. Echinoderms have left a superb fossil record stretching back more than half a billion years because of their easily fossilized skeletons, some aspects of which will be studied with 3-D digital high energy X-ray technology for this project. The developmental processes which generate the specific skeletal structures which have arisen in given branches of echinoderm evolution can now also be accessed experimentally in the laboratory. This project has the unique opportunity of providing an explanation for what actually happened in evolution, in the specific terms of changes in genetically controlled process for building bodily structure. This is among the major, fundamental questions in bioscience. If successful the outcome will indeed transform this area of biology, by demonstrating a new powerful approach to understanding, in which is combined, sophisticated observation of deep time remains, with sophisticated experimental and synthetic exploration of living descendants. The project illustrates the potential power of interdisciplinary marriage between traditionally separate areas. It will require new ways of thought and in the process, joint training and research experiences will be instituted for involved graduate students in the Division of Biology at the California Institute of Technology and in the Department of Earth Sciences at the University of Southern California. This type of multidisciplinary cross-training will produce a new breed of evolutionary scientists who will pioneer further advances in understanding the mechanisms of evolution.

The objective of Core A is indicated by its title. Its functions are defined as the provision of technologies, services and research materials commonly used by the individual Research Project components. These are services or materials which are difficult or expensive to generate, which can be provided by a central source, and indeed would be ridiculously wasteful for each lab to provide for itself. The emphasis here is on high technology aspects, and materials that require special facilities or know-how to produce.

The proposed work falls into three general areas. The Initial objective is to extend solved gene regulatory networks (GRNs) controlling sea urchin embryonic specification, up to gastrula stage, to encompass the whole of the embryo. This will require de novo solution for one remaining complex domain of the embryo. Following this we will build a predictive digital computational model of regulatory specification throughout the embryo from a few h after fertilization to 30h, including all interactions between domains, using the approach and software recently applied to the endomesodermal half of the embryo. A second objective is to utilize synthetic re-engineering to ascertain the logic processing functions and to answer other questions about the meaning of particular network subcircuit designs encountered in the sea urchin endomesoderm GRN. Specifically we will target double negative gate circuitry, feedback circuitry, and also redeploy differentiation gene batteries. These studies will be carried out in the context of the developing embryo, rather than in isolated

The objecfive of this Core Unit is to assure tight administrative coordinafion of the whole Program Project and to organize systematically continuing inter-laboratory communication.