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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.10.043 Byline: Smadar Ben-Tabou de-Leon, Eric H. Davidson Keywords: Mathematical modeling; Gene regulation in development; Dynamics; Logic functions Abstract: The dynamic process of cell fate specification is regulated by networks of regulatory genes. The architecture of the network defines the temporal order of specification events. To understand the dynamic control of the developmental process, the kinetics of mRNA and protein synthesis and the response of the cis-regulatory modules to transcription factor concentration must be considered. Here we review mathematical models for mRNA and protein synthesis kinetics which are based on experimental measurements of the rates of the relevant processes. The model comprises the response functions of cis-regulatory modules to their transcription factor inputs, by incorporating binding site occupancy and its dependence on biologically measurable quantities. We use this model to simulate gene expression, to distinguish between cis-regulatory execution of 'AND' and 'OR' logic functions, rationalize the oscillatory behavior of certain transcriptional auto-repressors and to show how linked subcircuits can be dealt with. Model simulations display the effects of mutation of binding sites, or perturbation of upstream gene expression. The model is a generally useful tool for understanding gene regulation and the dynamics of cell fate specification. Author Affiliation: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 7 June 2008; Revised 14 October 2008; Accepted 21 October 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.09.006 Byline: Veronica F. Hinman (a), Albert Nguyen (b), Eric H. Davidson (b) Keywords: Cis-regulatory evolution; Sea urchin; Asterina miniata; Gene regulatory network Abstract: The regulatory control of otx[beta]1/2 in the sea urchin Strongylocentrotus purpuratus and the sea star Asterina miniata provides an exceptional opportunity to determine the genomic basis of evolutionary change in gene regulatory network (GRN) architectures. Network perturbation analyses in both taxa show that Otx regulates the transcription factors gatae and krox/blimp1 and both of these transcription factors also feed back and regulate otx. The otx gene also autoregulates. This three way interaction is an example of a GRN kernel. It has been conserved for 500 million years since these two taxa last shared a common ancestor. Amid this high level of conservation we show here one significant regulatory change. Tbrain is required for correct otx[beta]1/2 expression in the sea star but not in the sea urchin. In sea urchin, tbrain is not co-expressed with otx[beta]1/2 and instead has an essential role in specification of the embryonic skeleton. Tbrain in these echinoderms is thus a perfect example of an orthologous gene co-opted for entirely different developmental processes. We isolate and test the sea star otx[beta]1/2 cis-regulatory module and demonstrate functional binding sites for each of the predicted inputs, including Tbrain. We compare it to the logic processing operating in the sea urchin otx[beta]1/2 cis-regulatory module and present an evolutionary scenario of the change in Tbrain dependence. Finally, inter-specific gene transfer experiments confirm this scenario and demonstrate evolution occurring at the level of sequence changes to the cis-regulatory module. Author Affiliation: (a) Department of Biological Sciences, Carnegie Mellon University, PA 15213, USA (b) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 28 March 2007; Revised 2 September 2007; Accepted 6 September 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.05.005 Byline: Pei Yun Lee, Jongmin Nam, Eric H. Davidson Keywords: Sea urchin; Gene regulation; GATA factors; cis-Regulatory analysis; gatae Abstract: The gatae gene of Strongylocentrotus purpuratus is orthologous to vertebrate gata-4,5,6 genes. This gene is expressed in the endomesoderm in the blastula and later the gut of the embryo, and is required for normal development. A gatae BAC containing a GFP reporter knocked into exon one of the gene was able to reproduce all aspects of endogenous gatae expression in the embryo. To identify putative gatae cis-regulatory modules we carried out an interspecific sequence conservation analysis with respect to a Lytechinus variegatus gatae BAC, which revealed 25 conserved non-coding sequence patches. These were individually tested in gene transfer experiments, and two modules capable of driving localized reporter expression in the embryo were identified. Module 10 produces early expression in mesoderm and endoderm cells up to the early gastrula stage, while module 24 generates late endodermal expression at gastrula and pluteus stages. Module 10 was then deleted from the gatae BAC by reciprocal recombination, resulting in total loss of reporter expression in the time frame in which it is normally active. Similar deletion of module 24 led to ubiquitous GFP expression in the gastrula and pluteus. These results show that Module 10 is uniquely necessary and sufficient to account for the early phase of gatae expression during endomesoderm specification. In addition, they imply a functional cis-regulatory module exclusion, whereby only a single module can associate with the basal promoter and drive gene expression at any given time. Author Affiliation: Division of Biology, California Institute of Technology, 1200 E. California Blvd., Mail Code 156-29, Pasadena, CA 9112, USA Article History: Received 10 February 2007; Revised 1 May 2007; Accepted 4 May 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.03.033 Byline: Jongmin Nam (a), Yi-Hsien Su (a), Pei Yun Lee (a), Anthony J. Robertson (b), James A. Coffman (b), Eric H. Davidson (a) Keywords: Nodal; Oral ectoderm; Gene regulatory network; Community effect; TGF-beta; bZIP; SMAD; Sea urchin; Positive feedback regulation; Cis-regulatory analysis Abstract: Expression of the nodal gene initiates the gene regulatory network which establishes the transcriptional specification of the oral ectoderm in the sea urchin embryo. This gene encodes a TGF[beta] ligand, and in Strongylocentrotus purpuratus its transcription is activated in the presumptive oral ectoderm at about the 30-cell stage. Thereafter Nodal signaling occurs among all cells of the oral ectoderm territory, and nodal expression is required for expression of oral ectoderm regulatory genes. The cis-regulatory system of the nodal gene transduces anisotropically distributed cytoplasmic cues that distinguish the future oral and aboral domains of the early embryo. Here we establish the genomic basis for the initiation and maintenance of nodal gene expression in the oral ectoderm. Functional cis-regulatory control modules of the nodal gene were identified by interspecific sequence conservation. A 5' cis-regulatory module functions both to initiate expression of the nodal gene and to maintain its expression by means of feedback input from the Nodal signal transduction system. These functions are mediated respectively by target sites for bZIP transcription factors, and by SMAD target sites. At least one SMAD site is also needed for the initiation of expression. An intron module also contains SMAD sites which respond to Nodal feedback, and in addition acts to repress vegetal expression. These observations explain the main features of nodal expression in the oral ectoderm: since the activity of bZIP factors is redox sensitive, and the initial polarization of oral vs. aboral fate is manifested in a redox differential, the bZIP sites account for the activation of nodal on the oral side; and since the immediate early signal transduction response factors for Nodal are SMAD factors, the SMAD sites account for the feedback maintenance of nodal gene expression. Author Affiliation: (a) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA (b) Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA Article History: Received 8 February 2007; Revised 13 March 2007; Accepted 23 March 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.039 Byline: Meredith Howard-Ashby, Stefan C. Materna, C. Titus Brown, Lili Chen, R. Andrew Cameron, Eric H. Davidson Keywords: Transcription factor; Sea urchin; Homeobox; Development Abstract: A set of 96 homeobox transcription factors was identified in the Strongylocentrotus purpuratus genome using permissive blast searches with a large collection of authentic homeodomain sequences from mouse, human and fly. A phylogenetic tree was constructed to compare the sea urchin homeobox gene family to those of vertebrates, with the result that with the only a few exceptions, orthologs of all vertebrate homeodomain genes were uncovered by our search. QPCR time course measurements revealed that 65% of these genes are expressed within the first 48 h of development (late gastrula). For genes displaying sufficiently high levels of transcript during the first 24 h of development (late blastula), whole mount in situ hybridization was carried out up to 48 h to determine spatial patterns of expression. The results demonstrate that homeodomain transcription factors participate in multiple and diverse developmental functions, in that they are used at a range of time points and in every territory of the developing embryo. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 18 April 2006; Revised 4 August 2006; Accepted 16 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.033 Byline: Meredith Howard-Ashby, Stefan C. Materna, C. Titus Brown, Lili Chen, R. Andrew Cameron, Eric H. Davidson Keywords: Transcription factor; Sea urchin; Development; bHLH; Sox; Nuclear receptor; Basic zipper; smad Abstract: All genes encoding transcription factors of the bHLH, Nuclear Receptor, Basic Leucine Zipper, T-box, Smad, Sox, and other smaller families were identified in the Strongylocentrotus purpuratus genome by means of a permissive blast search of the genome using a database of known transcription factors. Phylogenetic trees were constructed for the major families, permitting a comparison of the regulatory protein repertoire of the sea urchin and other species. QPCR and whole mount in situ hybridization experiments revealed the temporal and spatial expression patterns of these genes during early development. These regulatory genes are initially expressed at a broad range of time points, and the large majority of genes of all families are expressed within the first 48 h of development. The observations suggest assignment of many regulatory genes to specific developmental sub-networks, including endomesodermal, oral, aboral, and apical. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 1 June 2006; Revised 5 August 2006; Accepted 16 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.09.031 Byline: Qiang Tu, C. Titus Brown, Eric H. Davidson, Paola Oliveri Keywords: Forkhead; Fkh; Fox; Sea urchin; Genome; Embryo Abstract: Transcription factors of the Forkhead (Fox) family have been identified in many metazoans, and play important roles in diverse biological processes. Here we define the set of fox genes present in the sea urchin genome, and survey their usage during development. This genome includes 22 fox genes, only three of which were previously known. Of the 23 fox gene subclasses identified in vertebrate genomes, the Strongylocentrotus purpuratus genome has orthologues of all but four (E, H, R and S). Phylogenetic analysis suggests that one S. purpuratus fox gene is equally related to foxA and foxB of vertebrates; this gene defines a new class. Two other genes appear to be specific to the sea urchin, with respect to the genomes so far sequenced. Fox genes orthologous with those of vertebrates but lacking in arthropod or nematode genomes may be deuterostome-specific (subclasses I, J1, J2, L1, M and Q1), while the majority are pan-bilaterian. All but one of the S. purpuratus fox genes (SpfoxQ1) are expressed during embryogenesis, most in a very specific temporal and spatial manner. The sea urchin fox genes clearly execute many different regulatory functions, and almost all of them participate in the process of embryonic development. Author Affiliation: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 31 May 2006; Revised 14 September 2006; Accepted 15 September 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.10.016 Byline: Meredith Howard-Ashby, Stefan C. Materna, C. Titus Brown, Qiang Tu, Paola Oliveri, R. Andrew Cameron, Eric H. Davidson Keywords: Regulome; Transcription factor usage; Indirect development Abstract: A global scan of transcription factor usage in the sea urchin embryo was carried out in the context of the Strongylocentrotus purpuratus genome sequencing project, and results from six individual studies are here considered. Transcript prevalence data were obtained for over 280 regulatory genes encoding sequence-specific transcription factors of every known family, but excluding genes encoding zinc finger proteins. This is a statistically inclusive proxy for the total 'regulome' of the sea urchin genome. Close to 80% of the regulome is expressed at significant levels by the late gastrula stage. Most regulatory genes must be used repeatedly for different functions as development progresses. An evolutionary implication is that animal complexity at the stage when the regulome first evolved was far simpler than even the last common bilaterian ancestor, and is thus of deep antiquity. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 5 October 2006; Revised 13 October 2006; Accepted 13 October 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.032 Byline: Stefan C. Materna, Meredith Howard-Ashby, Rachel F. Gray, Eric H. Davidson Keywords: Zinc finger; Zf-c2h2; Transcription factor; Genome; Sea urchin; Development Abstract: The C.sub.2H.sub.2 zinc finger is one of the most abundant protein domains and is thought to have been extensively replicated in diverse animal clades. Some well-studied proteins that contain this domain are transcriptional regulators. As part of an attempt to delineate all transcription factors encoded in the Strongylocentrotus purpuratus genome, we identified the C.sub.2H.sub.2 zinc finger genes indicated in the sequence, and examined their involvement in embryonic development. We found 377 zinc finger genes in the sea urchin genome, about half the number found in mice or humans. Their expression was measured by quantitative PCR. Up to the end of gastrulation less than a third of these genes is expressed, and about 75% of the expressed genes are maternal; both parameters distinguish these from all other classes of regulatory genes as measured in other studies. Spatial expression pattern was determined by whole mount in situ hybridization for 43 genes transcribed at a sufficient level, and localized expression was observed in diverse embryonic tissues. These genes may execute important regulatory functions in development. However, the functional meaning of the majority of this large gene family remains undefined. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 25 May 2006; Revised 4 August 2006; Accepted 16 August 2006

A study examines the fox genes found in the genome of sea urchin as well as its embryonic development. The transcription factors of the fox family is analyzed.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.016 Byline: Sagar Damle (a), Bridget Hanser (a)(b), Eric H. Davidson (a), Scott E. Fraser (a)(b)(c) Keywords: Confocal laser scanning microscopy; GFP; Sea urchin cis-regulation Abbreviations: CLSM, confocal laser scanning microscopy; GFP, green fluorescent protein; QPCR, quantitative real-time PCR; GRN, gene regulatory network; WMISH, whole mount in situ hybridization; hpf, hours postfertilization Abstract: Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells. Author Affiliation: (a) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA (b) Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA (c) Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 10 February 2006; Revised 8 June 2006; Accepted 9 June 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.05.037 Byline: Andrew Ransick, Eric H. Davidson Keywords: cis-regulation; Glial cells missing; Specification; Mesoderm; Pigment; Suppressor of Hairless; Notch; Transcriptional switch; Strongylocentrotus purpuratus Abstract: The glial cells missing regulatory gene of Strongylocentrotus purpuratus (spgcm) was proposed earlier to be the genomic target of Delta/Notch (D/N) signaling required for specification of the mesodermal precursors of pigment cells. Here, we show that microinjection of a spgcm antisense morpholino oligonucleotide results in larvae without pigment cells. Microinjection of an mRNA encoding a dominant negative form of Suppressor of Hairless (dn-Su(H)) results in reduced levels of spgcm mRNA, disruption of mesodermal founder cell specification and failure to produce pigment cells. These results confirm that this gene is required for pigment cell specification. Three cis-regulatory modules of the spgcm gene were identified, which when incorporated in a GFP expression construct recapitulate the early expression pattern of this gene. Spatial expression of this GFP expression construct is severely disrupted by co-expression of dn-Su(H) mRNA, confirming that spgcm is a direct target of canonical N signaling mediated through Su(H) inputs. cis-perturbation analysis by mutation of consensus Su(H) sites identified a conserved motif paired-site and a lone site in the middle module that function both to drive expression in SMC precursors which receive the Delta signal and to repress expression in ectopic locations which lack this signal. While these Su(H) target sites provide the cis-regulatory architecture with the core of an N signaling transcriptional response switch, both the on and off outputs from this module require additional inputs. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 25 April 2006; Accepted 25 May 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.021 Byline: Carolina B. Livi, Eric H. Davidson Keywords: Strongylocentrotus purpuratus; SET domain; PR domain; Cys2His2 zinc finger transcription factor; Endomesodermal specification; Gene regulatory network; Alternative splice forms Abstract: The blimp1/krox gene of Strongylocentrotus purpuratus, formerly krox1, encodes zinc finger transcription factors which play a central role in both early and late endomesoderm specification. Here we show that there are two alternative splice forms transcribed under the control of different regulatory regions. The blimp1/krox1b form was previously unknown, and is the form expressed during cleavage, beginning 6-9 h postfertilization. This form is required for the early events of endomesoderm specification. A different splice variant, blimp1/krox1a, is expressed only from gastrula stage onward. During cleavage stages the blimp1/krox gene is expressed in the large micromeres and veg2 descendents. Soon after, it is expressed in the ring of specified mesoderm cells at the vegetal pole of the blastula. Its expression is later restricted to the blastopore region and the posterior of the invaginating archenteron, and finally to the midgut and hindgut of the pluteus larva. The expression of blimp1/krox is dynamic, and involves several distinct spatial territories. A GFP recombinant BAC was created by substituting the GFP coding sequence for that of the second exon (1b), in order to distinguish the expression pattern of the early form from that of the late form. This construct closely mimics blimp1/krox1b expression during early stages of sea urchin development. To expand our knowledge of the downstream linkages of this gene, additional experiments were carried out using antisense morpholino oligos (MASO). We confirmed previously published data that blimp1/krox autoregulates its own expression, but discovered, surprisingly, that this gene represses rather than activates itself. This negative autoregulation is restricted to the mesodermal and probably skeletogenic territories during the blastula stage, as shown by in situ hybridization analysis of MASO injected embryos. The MASO perturbation analysis also revealed blimp1/krox inputs into other genes of the endomesoderm regulatory network. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 27 October 2005; Revised 15 February 2006; Accepted 16 February 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.024 Byline: Gabriele Amore, Eric H. Davidson Keywords: cis-Regulatory module; Gene regulation; Gene regulatory network; Gene transfer; Perturbation analysis Abstract: The Strongylocentrotus purpuratus cyclophilin1 gene (Sp-cyp1) is expressed exclusively in skeletogenic mesenchyme cells of the embryo, beginning in the micromere lineage of the early blastula stage and continuing after gastrulation during the syncytial deposition of the skeleton. This gene encodes a protein which is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. Sp-cyp1 is among the differentiation genes activated in the skeletogenic territory as a terminal function of the endomesodermal gene regulatory network. Network perturbation analysis had predicted the skeletogenic regulators Ets1 and Deadringer (Dri) to be its driver inputs. Here, we show that a 218-bp cis-regulatory DNA fragment recapitulates skeletogenic Sp-cyp1 expression; that elimination of either Ets1 or Dri inputs severely depresses the activity of expression constructs containing this DNA fragment; and that Ets1 and Dri target sites within the 218 bp fragment are required for normal expression. This indicates that the predicted inputs are direct. Other studies indicate that the same inputs are evidently necessary for expression of several other skeletogenic differentiation genes, and these genes probably constitute a skeletogenic gene battery, defined by its Ets plus Dri regulatory inputs. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 22 December 2005; Revised 3 February 2006; Accepted 16 February 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.09.047 Byline: Takuya Minokawa (a), Athula H. Wikramanayake (b), Eric H. Davidson (a) Keywords: wnt8; cis-regulation; Gene network; Sea urchin embryo Abstract: Expression of the wnt8 gene is the key transcriptional motivator of an intercellular signaling loop which drives endomesoderm specification forward early in sea urchin embryogenesis. This gene was predicted by network perturbation analysis to be activated by inputs from the blimp1/krox gene, itself expressed zygotically in the endomesoderm during cleavage; and by a Tcf1/[beta]-catenin input. The implication is that zygotic expression of wnt8 is stimulated in neighboring cells by its own gene product, since reception of the Wnt8 ligand causes [beta]-catenin nuclearization. Here, the modular cis-regulatory system of the wnt8 gene of Strongylocentrotus purpuratus was characterized functionally, and shown to respond to blockade of both Blimp1/Krox and Tcf1/[beta]-catenin inputs just as does the endogenous gene. The genomic target sites for these factors were demonstrated by mutation in one of the cis-regulatory modules. The Tcf1/[beta]-catenin and Blimp1/Krox inputs are both necessary for normal endomesodermal expression mediated by this cis-regulatory module; thus, the genomic regulatory code underlying the predicted signaling loop thus resides in the wnt8 cis-regulatory sequence. In a second regulatory region, which initiates expression in micromere and macromere descendant cells early in cleavage, Tcf1 sites act to repress ectopic transcription in prospective ectoderm cells. Author Affiliation: (a) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA (b) Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA Article History: Received 30 August 2005; Revised 21 September 2005; Accepted 23 September 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.04.023 Byline: William J.R. Longabaugh (a), Eric H. Davidson (b), Hamid Bolouri (a)(b) Abstract: Developmental genetic regulatory networks (GRNs) have unique architectural characteristics. They are typically large-scale, multi-layered, and organized in a nested, modular hierarchy of regulatory network kernels, function-specific building blocks, and structural gene batteries. They are also inherently multicellular and involve changing topological relationships among a growing number of cells. Reconstruction of developmental GRNs requires unique computational tools that support the above representational requirements. In addition, we argue that DNA-centered network modeling, separate descriptions of network organization and network behavior, and support for network documentation and annotation are essential requirements for computational modeling of developmental GRNs. Based on these observations, we have developed a freely available, platform-independent, open source software package (BioTapestry) which supports both the process of model construction and also model visualization, analysis, documentation, and dissemination. We provide an overview of the main features of BioTapestry. The BioTapestry software and additional documents are available from http://www.biotapestry.org. We recommend BioTapestry as the substrate for further co-development for and by the developmental biology community. Author Affiliation: (a) Institute for Systems Biology, Seattle, WA 98103-8904, USA (b) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

A specific prediction of our detailed cis-regulatory analysis of the Strongylocentrotus purpuratus (Sp) endo16 gene was that the later expression of this gene would be driven by a midgut-specific transcriptional regulator. We have now identified this factor and determined some of its functions. The cDNA sequence reveals it to be a POU domain factor related closely to the mammalian factors Brain-1, -2, and -4. The factor was termed SpBrn1/2/4 (henceforth Brn1/2/4). Quantitative measurements of transcript prevalence show that the gene is first activated in the 20-h blastula, but there remain only about 100 molecules of brn1/2/4 mRNA per embryo (only a few per endoderm cell) until an abrupt 10-fold increase occurs as gastrulation begins. Measured in the same embryos, the late rise in prevalence of endo16 transcripts follows that of brn1/2/4 transcripts. As predicted by the endo16 model, brn1/2/4 expression is confined perfectly to the midgut, coincident with the domain of endo16 expression. The kinetics of accumulation of these transcripts indicates that the switch into the late phase of endo16 expression occurs when the brn1/2/4 transcript level nears its plateau (2000 molecules mRNA per embryo), after which each endo16 gene produces about 1 mRNA every 2 min (about 380 molecules mRNA per min in the whole embryo). Arrest of Brn1/2/4 translation by MASO treatment blocks the late phase of endo16 expression and specifically abolishes expression of cis-regulatory Module B of endo16, while not affecting Module A, also as predicted. The brn1/2/4 gene lies downstream of the regulatory genes executing post-gastrular specification of the midgut, as shown by further gene expression perturbation experiments which provide an initial glimpse of the underlying network architecture.

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos. Keywords: cis-Regulatory element; Gene regulatory network; delta; Endomesoderm specification; Sea urchin

The Strongylocentrotus purpuratus hnf6 (Sphnf6) gene encodes a new member of the ONECUT family of transcription factors. The expression of hnf6 in the developing embryo is triphasic, and loss-of-function analysis shows that the Hnf6 protein is a transcription factor that has multiple distinct roles in sea urchin development, hnf6 is expressed maternally, and before gastrulation its transcripts are distributed globally. Early in development, its expression is required for the activation of PMC differentiation genes such as sm50, pm27, and msp130, but not for the activation of any known PMC regulatory genes, for example, alx, ets1, pmar1, or tbrain. Micromere transplantation experiments show that the gene is not involved in early micromere signaling. Early hnf6 expression is also required for expression of the mesodermal regulator gatac. The second known role of hnf6 is its participation after gastrulation in the oral ectoderm gene regulatory network (GRN), in which its expression is essential for the maintenance of the state of oral ectoderm specification. The third role is in the neurogenic ciliated band, which is foreshadowed exactly by a trapezoidal band of hnf6 expression at the border of the oral ectoderm and where it continues to be expressed through the end of embryogenesis. Neither oral ectoderm regulatory functions nor ciliated band formation occur normally in the absence of hnf6 expression. Keywords: Morpholino antisense oligonucleotide; Transcription factor; hnf6; ONECUT; Oral ectoderm; PMC; Sea urchin embryo

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2004.03.031 Byline: Meredith L. Howard, Eric H. Davidson Keywords: cis-Regulatory module; Gene regulatory network; Repression; Feedback loop Abstract: During development, an organism undergoes many rounds of pattern formation, generating ever-greater complexity with each ensuing round of cell division and specification. The instructions for executing this process are encoded in the cis-regulatory modules that direct the expression of developmental transcription factors and signaling molecules. Each transcription factor binding site within a cis-regulatory module contributes information about when, where, or how much a gene is turned on, and by dissecting the modules driving a given gene, all the inputs governing expression of the gene can be accurately identified. Furthermore, by mapping the output of each gene to the inputs of other genes, it is possible to reverse engineer developmental circuits and even whole networks. At this higher level of organization, common bilaterian strategies for specifying progenitor fields, locking down regulatory states, and driving development forward emerge. Article History: Received 20 January 2004; Revised 20 March 2004; Accepted 20 March 2004

An essential node in the gene regulatory network (GRN) for endomesoderm specification in the sea urchin embryo lies within the regulatory system of the otx gene. According to the predictions of the GRN, based on perturbation analysis and expression data, the [beta]1/2 transcription unit of this gene is engaged during specification in interactions with two other regulatory genes, krox and gatae. It is predicted to be driven into activity by the krox gene, to form a positively reinforcing functional loop with the gatae gene, and to respond to its own output as well. Here we isolate the relevant otx cis-regulatory element, and examine the specific input predictions of the GRN at the level of its genomic DNA sequence. This [beta]1/2-otx regulatory module performs the necessary functions, as shown by use of expression constructs. It requires gatae, otx, and krox inputs, as predicted, and it operates as an 'AND' logic processor in that removal of any one of these inputs essentially destroys activity. The necessary target sites were identified in the module sequence, and mutation of these sites was demonstrated to produce the same respective effects on construct expression as does blocking its regulatory inputs by treatment with morpholino anti-sense oligonucleotides. For spatial expression in the endoderm, one particular pair of Gata sites is essential and these function synergistically with an adjacent Otx site. We thus demonstrate directly the structure/function relationships of the genomic regulatory code, at this key node of the endomesoderm GRN. Keywords: Gene regulatory network; otx; krox; gatae; Endomesoderm specification; Sea urchin embryo

The Spdeadringer (Spdri) gene encodes an ARID-class transcription factor not previously known in sea urchin embryos. We show that Spdri is a key player in two separate developmental gene regulatory networks (GRNs). Spdri is expressed in a biphasic manner, first, after 12 h and until ingression in the skeletogenic descendants of the large micromeres; second, after about 20 h in the oral ectoderm, where its transcripts remain present at 30-50 mRNA molecules/cell far into development. In both territories, the periods of Spdri expression follow prior territorial specification events. The functional significance of each phase of expression was assessed by determining the effect of an [alpha]Spdri morpholino antisense oligonucleotide (MASO) on expression of 17 different mesodermal genes, 8 different oral ectoderm genes, and 18 other genes expressed specifically during endomesoderm specification. These effects were measured by quantitative PCR, supplemented by whole-mount in situ hybridization and morphological observations. Spdri is shown to act in the micromere descendants in the pathways that result in the expression of batteries of terminal skeletogenic genes. But, in the oral ectoderm, the same gene participates in the central GRN controlling oral ectoderm identity. Spdri is linked in the oral ectoderm GRN with several other genes encoding transcriptional regulators that are expressed specifically in various regions of the oral ectoderm. If its expression is blocked by treatment with [alpha]Spdri MASO, oral-specific features disappear and expression of the aboral ectoderm marker spec1 encompasses the whole of the ectoderm. In addition to disappearance of the oral ectoderm, morphological consequences of [alpha]Spdri MASO treatment include failure of spiculogenesis and of correct primary mesenchyme cell (pmc) patterning in the postgastrular embryo, and also failure of gastrulation. To further analyze these phenotypes, chimeric embryos were constructed consisting of two labeled micromeres combined with micromereless 4th cleavage host embryos; either the micromeres or the hosts contained [alpha]Spdri MASO. These experiments showed that, while Spdri expression is required autonomously for expression of skeletogenic genes prior to ingression, complete skeletogenesis also requires the expression of oral ectoderm patterning information. Presentation of this information on the oral side of the blastocoel in turn depends on Spdri expression in the oral ectoderm. Failure of gastrulation is not due to indirect interference with endomesodermal specification per se, since all endomesodermal genes tested function normally in [alpha]Spdri MASO embryos. Part of its cause is interference by [alpha]Spdri MASO with a late signaling function on the part of the micromere descendants that is needed to complete clearance of the Soxb1 repressor of gastrulation from the prospective endoderm, but in addition there is a nonautonomous oral ectoderm effect. Keywords: Gene regulatory network; Oral ectoderm; deadringer; Sea urchin embryo

pmarl is a transcription factor in the paired class homeodomain family that was identified and found to be transcribed in micromeres beginning at the fourth cleavage of sea urchin development [Dev. Biol. 246 (2002), 209]. Based on in situ data, molecular perturbation studies, and QPCR data, the recently published gene regulatory network (GRN) model for endomesoderm specification [Science 295 (2002) 1669; Dev. Biol. 246 (2002), 162] places pmarl early in the micromere specification pathway, and upstream of two important micromere induction signals. The goal of this study was to test these three predictions of the network model. A series of embryo chimeras were produced in which pmarl activity was perturbed in one cell that was transplanted to control hosts. At the fourth cleavage, micromeres bearing altered pmarl activity were combined with a normal micromereless host embryo. If [beta]-catenin signaling is blocked, the micromeres remain unspecified and are unable to signal to the host cells. When such [beta]-catenin-blocked micromeres also express Pmarl, all observed micromere functions are rescued. The rescue includes expression of the primary mesenchyme cell (PMC) differentiation program, expression and execution of the Delta signal to induce secondary mesoderm cell (SMC) specification in macromere progeny, and expression of the early endomesoderm induction signal necessary for full specification of the endoderm. Additionally, Pmarl expressed mosaically from inserted DNA constructs causes induction of ectopic Endo 16 in adjacent cells, demonstrating further that Pmarl controls expression of the early endomesoderm induction signal. Based on these experiments, Pmarl is an important transcription factor necessary for initiating the micromere specification program and for the expression of two inductive signals produced by micromeres. Each of the tests we describe supports the placement and function of Pmarl in the endomesoderm GRN model. Keywords: Gene regulatory network; Sox; Delta; [beta]-Catenin; Paired homeodomain

Steroid hormones trigger a wide variety of cell-specific responses during animal development, but the mechanisms by which these systemic signals specify either cell division, differentiation, morphogenesis or death remain uncertain. Here, we analyze the function of the steroid-regulated genes [beta]FTZ-F1, BR-C, E74A, and E93 during salivary gland programmed cell death. While mutations in the [beta]FTZ-F1, BR-C, E74A, and E93 genes prevent destruction of salivary glands, only [beta]FTZ-F1 is required for DNA fragmentation. Analyses of BR-C, E74A, and E93 loss-of-function mutants indicate that these genes regulate stage-specific transcription of the rpr, hid, ark, dronc, and crq cell death genes. Ectopic expression of [beta]FTZ-F1 is sufficient to trigger premature cell death of larval salivary glands and ectopic transcription of the rpr, dronc, and crq cell death genes that normally precedes salivary gland cell death. The E93 gene is necessary for ectopic salivary gland cell destruction, and ectopic rpr, dronc, and crq transcription, that is induced by expression of [beta]FTZ.F1. Together, these observations indicate that [beta]FTZ-F1 regulates the timing of hormone-induced cell responses, while E93 functions to specify programmed cell death. Key Words: steroid; ecdysone; programmed cell death; apoptosis; autophagy; development; metamorphosis; Drosophila.

Steroid hormones trigger dynamic tissue changes during animal development by activating cell proliferation, cell differentiation, and cell death. Here we characterize steroid regulation of changes in midgut structure during the onset of Drosophila metamorphosis. Following an increase in the steroid 20-hydroxyecdysone (ecdysone) at the end of larval development, future adult midgut epithelium is formed, and the larval midgut is rapidly destroyed. Mutations in the steroid-regulated genes BR-C and E93 differentially impact larval midgut cell death but do not affect the formation of adult midgut epithelia. In contrast, mutations in the ecdysone-regulated E74A and E74B genes do not appear to perturb midgut development during metamorphosis. Larval midgut cells possess vacuoles that contain cellular organelles, indicating that these cells die by autophagy. While mutations in the BR-C, E74, and E93 genes do not impact DNA degradation during this cell death, mutations in BR-C inhibit destruction of larval midgut structures, including the proventriculus and gastric caeca, and E93 mutants exhibit decreased formation of autophagic vacuoles. Dying midguts express the rpr, hid, ark, dronc, and crq cell death genes, suggesting that the core cell death machinery is involved in larval midgut cell death. The transcription of rpr, hid, and crq are altered in BR-C mutants, and E93 mutants possess altered transcription of the caspase dronc, providing a mechanism for the disruption of midgut cell death in these mutant animals. These studies indicate that ecdysone triggers a two-step hierarchy composed of steroid-induced regulatory genes and apoptosis genes that, in turn, regulate the autophagic death of midgut cells during development. Key Words: steroid; ecdysone; programmed cell death; apoptosis; autophagy; development; metamorphosis; Drosophila.

The evolutionary divergence of cnidarian and bilaterian lineages from their remote metazoan ancestor occurred at an unknown depth in time before the Cambrian, since crown group representatives of each are found in Lower Cambrian fossil assemblages. We report here a variety of putative embryonic, larval, and adult microfossils deriving from Precambrian phosphorite deposits of Southwest China, which may predate the Cambrian radiation by 25-45 million years. These are most probably of cnidarian affinity. Large numbers of fossilized early planula-like larvae were observed under the microscope in sections. Though several forms are represented, the majority display remarkable conformity, which is inconsistent with the alternative that they are artifactual mineral inclusions. Some of these fossils are preserved in such high resolution that individual cells can be discerned. We confirm in detail an earlier report of the presence in the same deposits of tabulates, an extinct crown group anthozoan form. Other sections reveal structures that most closely resemble sections of basal modern corals. A large number of fossils similar to modern hydrozoan gastrulae were also observed. These again displayed great morphological consistency. Though only a single example is available, a microscopic animal remarkably similar to a modern adult hydrozoan is also presented. Taken together, the new observations reported in this paper indicate the existence of a diverse and already differentiated cnidarian fauna, long before the Cambrian evolutionary event. It follows that at least stem group bilaterians must also have been present at this time. Key Words: cnidarian; fossil; Precambrian; embryo.

Gene network analysis requires computationally based models which represent the functional architecture of regulatory interactions, and which provide directly testable predictions. The type of model that is useful is constrained by the particular features of developmentally active cis-regulatory systems. These systems function by processing diverse regulatory inputs, generating novel regulatory outputs. A computational model which explicitly accommodates this basic concept was developed earlier for the cis-regulatory system of the endo16 gene of the sea urchin. This model represents the genetically mandated logic functions that the system executes, but also shows how time-varying kinetic inputs are processed in different circumstances into particular kinetic outputs. The same basic design features can be utilized to construct models that connect the large number of cis-regulatory elements constituting developmental gene networks. The ultimate aim of the network models discussed here is to represent the regulatory relationships among the genomic control systems of the genes in the network, and to state their functional meaning. The target site sequences of the cis-regulatory elements of these genes constitute the physical basis of the network architecture. Useful models for developmental regulatory networks must represent the genetic logic by which the system operates, but must also be capable of explaining the real time dynamics of cis-regulatory response as kinetic input and output data become available. Most importantly, however, such models must display in a direct and transparent manner fundamental network design features such as intra-and intercellular feedback circuitry; the sources of parallel inputs into each cis-regulatory element; gene battery organization; and use of repressive spatial inputs in specification and boundary formation. Successful network models lead to direct tests of key architectural features by targeted cis-regulatory analysis. Key Words: gene regulatory network; model; endo16 gene; regulatory genomics.