Genomic Regulatory Networks and Animal Development

The synthesis of gene expression data and cis-regulatory analysis permits the elucidation of genomic regulatory networks. These networks provide a direct visualization of the functional interconnections among the regulatory genes and signaling components leading to cell-specific patterns of gene activity. Complex developmental processes are thereby illuminated in ways not revealed by the conventional analysis of individual genes. In this review, we describe emerging networks in several different model systems, and compare them with the gene regulatory network that controls dorsoventral patterning of the Drosophila embryo. The synthesis of gene expression data and cis-regulatory analysis permits the elucidation of genomic regulatory networks. These networks provide a direct visualization of the functional interconnections among the regulatory genes and signaling components leading to cell-specific patterns of gene activity. Complex developmental processes are thereby illuminated in ways not revealed by the conventional analysis of individual genes. In this review, we describe emerging networks in several different model systems, and compare them with the gene regulatory network that controls dorsoventral patterning of the Drosophila embryo. The recent sequencing of diverse animal genomes has facilitated the systematic examination of gene expression patterns using whole-genome methods. These methods, primarily microarray techniques, have identified most or all of the genes engaged in specific developmental processes. The further characterization of genes encoding transcription factors and cell signaling components provides the foundation for elucidating comprehensive gene networks (Levine and Davidson, 2005Levine M. Davidson E.H. Gene regulatory networks for development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4936-4942Crossref PubMed Scopus (473) Google Scholar). Once DNA binding sequences are determined for the transcription factors, bioinformatics methods can identify the enhancers that determine where and when the genes within the network are expressed (Markstein and Levine, 2002Markstein M. Levine M. Decoding cis-regulatory DNAs in the Drosophila genome.Curr. Opin. Genet. Dev. 2002; 12: 601-606Crossref PubMed Scopus (0) Google Scholar). Circuit diagrams can be created that organize the various inputs and outputs from each gene within a network (Longabaugh et al., 2005Longabaugh W.J. Davidson E.H. Bolouri H. Computational representation of developmental genetic regulatory networks.Dev. Biol. 2005; 283: 1-16Crossref PubMed Scopus (160) Google Scholar). Analysis of the circuit diagrams provides testable predictions and new insights into complex developmental processes (Davidson, 2001Davidson E.H. Genomic Regulatory Systems: Development and Evolution. Academic Press, San Diego, CA2001Google Scholar). An increasing number of gene regulatory networks are being determined as whole-genome studies become more prevalent. The first such network was determined for endomesoderm specification in the pregastrular sea urchin embryo, Strongylocentrotus purpuratus (Davidson et al., 2002Davidson E.H. Rast J.P. Oliveri P. Ransick A. Calestani C. Yuh C.H. Minokawa T. Amore G. Hinman V. Arenas-Mena C. et al.A genomic regulatory network for development.Science. 2002; 295: 1669-1678Crossref PubMed Scopus (1127) Google Scholar; reviewed by Oliveri and Davidson, 2004Oliveri P. Davidson E.H. Gene regulatory network controlling embryonic specification in the sea urchin.Curr. Opin. Genet. Dev. 2004; 14: 351-360Crossref PubMed Scopus (0) Google Scholar). More recent studies have led to the elucidation of networks underlying mesoderm specification in the frog Xenopus laevis (reviewed by Koide et al., 2005Koide T. Hayata T. Cho K.W. Xenopus as a model system to study transcriptional regulatory networks.Proc. Natl. Acad. Sci. USA. 2005; 102: 4943-4948Crossref PubMed Scopus (0) Google Scholar), dorsoventral patterning of the Drosophila embryo (reviewed by Stathopoulos and Levine, 2004Stathopoulos A. Levine M. Whole-genome analysis of Drosophila gastrulation.Curr. Opin. Genet. Dev. 2004; 14: 477-484Crossref PubMed Scopus (52) Google Scholar), vulva differentiation in Caenorhabditis elegans (Inoue et al., 2005Inoue T. Wang M. Ririe T.O. Fernandes J.S. Sternberg P.W. Transcriptional network underlying Caenorhabditis elegans vulval development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4972-4977Crossref PubMed Scopus (0) Google Scholar), B cell differentiation in the mammalian immune system (Singh et al., 2005Singh H. Medina K.L. Pongubala J.M. Contingent gene regulatory networks and B cell fate specification.Proc. Natl. Acad. Sci. USA. 2005; 102: 4949-4953Crossref PubMed Scopus (157) Google Scholar), and segmentation of the Drosophila embryo (Ochoa-Espinosa et al., 2005Ochoa-Espinosa A. Yucel G. Kaplan L. Pare A. Pura N. Oberstein A. Papatsenko D. Small S. The role of binding site cluster strength in Bicoid-dependent patterning in Drosophila.Proc. Natl. Acad. Sci. USA. 2005; 102: 4960-4965Crossref PubMed Scopus (128) Google Scholar). Comparisons of these networks identify common strategies (e.g., Koide et al., 2005Koide T. Hayata T. Cho K.W. Xenopus as a model system to study transcriptional regulatory networks.Proc. Natl. Acad. Sci. USA. 2005; 102: 4943-4948Crossref PubMed Scopus (0) Google Scholar, Levine and Davidson, 2005Levine M. Davidson E.H. Gene regulatory networks for development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4936-4942Crossref PubMed Scopus (473) Google Scholar). For example, in sea urchins and frogs, signaling pathways are active at even the earliest points of development to establish differential patterns of gene activity. However, in Drosophila, gene expression is established using mechanisms that do not require cell signaling because the embryo is a syncytium and cell boundaries have not yet been established. Similarities between all three systems are seen at later stages, when feedback loops are extensively used to “lock down” differentiation states established by transient signals. As more gene network models become available, comparisons among an even broader spectrum of developmental processes will provide additional insights into mechanisms of tissue differentiation and organogenesis. Here, we review the methods used to determine the dorsoventral patterning network in Drosophila, and then describe how similar strategies can be used in emergent systems, including the differentiation of the C-lineage and pharynx in C. elegans (Baugh et al., 2005aBaugh L.R. Hill A.A. Claggett J.M. Hill-Harfe K. Wen J.C. Slonim D.K. Brown E.L. Hunter C.P. The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo.Development. 2005; 132: 1843-1854Crossref PubMed Scopus (86) Google Scholar, Gaudet et al., 2004Gaudet J. Muttumu S. Horner M. Mango S.E. Whole-genome analysis of temporal gene expression during foregut development.PLoS Biol. 2004; 2: e352Crossref PubMed Scopus (0) Google Scholar), eye lens differentiation in mice (Mu et al., 2005Mu X. Fu X. Sun H. Beremand P.D. Thomas T.L. Klein W.H. A gene network downstream of transcription factor Math5 regulates retinal progenitor cell competence and ganglion cell fate.Dev. Biol. 2005; 280: 467-481Crossref PubMed Scopus (100) Google Scholar), and notochord differentiation in the sea squirt Ciona intestinalis (Kusakabe, 2005Kusakabe T. Decoding cis-regulatory systems in ascidians.Zoolog. Sci. 2005; 22: 129-146Crossref PubMed Scopus (0) Google Scholar). We discuss common strategies employed by these emerging networks and the dorsoventral patterning network. Our aim is to demonstrate that it is possible to formulate gene networks from a variety of systems, providing interesting insights to help guide future experimental approaches. Even the process of creating a provisional network stimulates fresh approaches to old problems. Dorsoventral patterning in the Drosophila embryo has been studied for many years. Even before the genome was sequenced, genetic approaches identified more than ten genes that affect patterning along the dorsal-ventral axis. When the Drosophila genome became available, whole-genome methods permitted the identification of additional components of the network, thereby facilitating efforts to understand how the network controls patterning and cell movements in the embryo (Figure 1). Drosophila gastrulation is initiated by the maternal transcription factor Dorsal, which is distributed in a broad nuclear gradient in the precellular embryo (reviewed in Anderson, 1998Anderson K.V. Pinning down positional information: dorsal-ventral polarity in the Drosophila embryo.Cell. 1998; 95: 439-442Abstract Full Text Full Text PDF PubMed Google Scholar, Roth, 2003Roth S. The origin of dorsoventral polarity in Drosophila.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 1317-1329Crossref PubMed Scopus (74) Google Scholar). A combination of classical genetic screens, subtractive hybridization methods, and microarray assays have identified ∼50 potential target genes that are regulated by different concentrations of the Dorsal gradient (e.g., Casal and Leptin, 1996Casal J. Leptin M. Identification of novel genes in Drosophila reveals the complex regulation of early gene activity in the mesoderm.Proc. Natl. Acad. Sci. USA. 1996; 93: 10327-10332Crossref PubMed Scopus (0) Google Scholar, Ray et al., 1991Ray R.P. Arora K. Nusslein-Volhard C. Gelbart W.M. The control of cell fate along the dorsal-ventral axis of the Drosophila embryo.Development. 1991; 113: 35-54Crossref PubMed Google Scholar, Simpson, 1983Simpson P. Maternal-zygotic gene interactions during formation of the dorsoventral pattern in Drosophila embryos.Genetics. 1983; 105: 615-632Crossref PubMed Google Scholar, Stathopoulos et al., 2002Stathopoulos A. Van Drenth M. Erives A. Markstein M. Levine M. Whole-genome analysis of dorsal-ventral patterning in the Drosophila embryo.Cell. 2002; 111: 687-701Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Roughly half of the Dorsal target genes encode predicted sequence-specific transcription factors, while the other half encode components of the FGF, EGF, and Dpp (TGFβ) cell signaling pathways (reviewed in Stathopoulos and Levine, 2004Stathopoulos A. Levine M. Whole-genome analysis of Drosophila gastrulation.Curr. Opin. Genet. Dev. 2004; 14: 477-484Crossref PubMed Scopus (52) Google Scholar). Recognition sequences have been determined for many of the encoded transcription factors, including Dorsal (GGGW4-5CCM), Twist (CACATGT), Snail (MMRCAWGT), and Schnürri (GRCGNCNNNNNGTCTG) (K. Senger and M.L., unpublished observations; Markstein et al., 2002Markstein M. Markstein P. Markstein V. Levine M.S. Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2002; 99: 763-768Crossref PubMed Scopus (293) Google Scholar, Pyrowolakis et al., 2004Pyrowolakis G. Hartmann B. Muller B. Basler K. Affolter M. A simple molecular complex mediates widespread BMP-induced repression during Drosophila development.Dev. Cell. 2004; 7: 229-240Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Senger et al., 2004Senger K. Armstrong G.W. Rowell W.J. Kwan J.M. Markstein M. Levine M. Immunity regulatory DNAs share common organizational features in Drosophila.Mol. Cell. 2004; 13: 19-32Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Clusters of these binding motifs were identified within extended genomic DNA intervals encompassing each of the 50 Dorsal target genes (Markstein et al., 2002Markstein M. Markstein P. Markstein V. Levine M.S. Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2002; 99: 763-768Crossref PubMed Scopus (293) Google Scholar, Papatsenko and Levine, 2005aPapatsenko D. Levine M. Computational identification of regulatory DNAs underlying animal development.Nat. Methods. 2005; 2: 529-534Crossref PubMed Scopus (0) Google Scholar, Stathopoulos et al., 2002Stathopoulos A. Van Drenth M. Erives A. Markstein M. Levine M. Whole-genome analysis of dorsal-ventral patterning in the Drosophila embryo.Cell. 2002; 111: 687-701Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Putative enhancers were predicted for the majority of the genes, and 18 were confirmed by direct experimentation (reviewed in Stathopoulos and Levine, 2004Stathopoulos A. Levine M. Whole-genome analysis of Drosophila gastrulation.Curr. Opin. Genet. Dev. 2004; 14: 477-484Crossref PubMed Scopus (52) Google Scholar). In these cases, small (<1 kb) genomic DNA fragments encompassing the binding clusters were attached to a lacZ reporter gene and assayed in transgenic embryos (e.g., see Figure 2). The resulting set of genes and enhancers controlling dorsoventral patterning of Drosophila embryos represents one of the most extensive collections available for any developmental process (Figure 1; Levine and Davidson, 2005Levine M. Davidson E.H. Gene regulatory networks for development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4936-4942Crossref PubMed Scopus (473) Google Scholar). The isolation of Dorsal target enhancers provided an opportunity to examine a basic problem in developmental biology: how does a morphogen gradient generate multiple thresholds of gene expression? Dorsal target enhancers fall into three basic categories (reviewed in Stathopoulos and Levine, 2002aStathopoulos A. Levine M. Dorsal gradient networks in the Drosophila embryo.Dev. Biol. 2002; 246: 57-67Crossref PubMed Scopus (144) Google Scholar). Type 1 enhancers are activated by high levels of the Dorsal gradient in the presumptive mesoderm. Twist is one of the first Type 1 genes that are activated by Dorsal (Jiang et al., 1991Jiang J. Kosman D. Ip Y.T. Levine M. The dorsal morphogen gradient regulates the mesoderm determinant twist in early Drosophila embryos.Genes Dev. 1991; 5: 1881-1891Crossref PubMed Google Scholar). The encoded Twist bHLH regulatory protein works in concert with Dorsal (Simpson, 1983Simpson P. Maternal-zygotic gene interactions during formation of the dorsoventral pattern in Drosophila embryos.Genetics. 1983; 105: 615-632Crossref PubMed Google Scholar) to regulate roughly half of all the Dorsal target enhancers. Most Type 1 enhancers contain a series of low-affinity Dorsal binding sites, and some also contain Twist sites. Such enhancers display an inverse relationship in the quality of Dorsal and Twist binding sites, whereby those containing high-quality Dorsal sites contain poor Twist sites, and vice versa. There is no obvious organization in the arrangement of the binding sites (e.g., CG12177; Figure 2A). Type 2 enhancers are activated by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm (e.g., rhomboid [rho]; Figure 2B). All of these enhancers contain optimal Dorsal and Twist binding sites, and at least one pair of sites exhibits tight linkage (<100 bp) and convergent orientation (Erives and Levine, 2004Erives A. Levine M. Coordinate enhancers share common organizational features in the Drosophila genome.Proc. Natl. Acad. Sci. USA. 2004; 101: 3851-3856Crossref PubMed Scopus (96) Google Scholar, Markstein et al., 2004Markstein M. Zinzen R. Markstein P. Yee K.P. Erives A. Stathopoulos A. Levine M. A regulatory code for neurogenic gene expression in the Drosophila embryo.Development. 2004; 131: 2387-2394Crossref PubMed Scopus (109) Google Scholar). This particular organization probably fosters cooperative DNA binding interactions between the Dorsal and Twist proteins (Jiang and Levine, 1993Jiang J. Levine M. Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the dorsal gradient morphogen.Cell. 1993; 72: 741-752Abstract Full Text PDF PubMed Scopus (242) Google Scholar). In addition, the Twist sites present in Type 2 enhancers also bind the Snail repressor, which recognizes the following consensus sequence: MMRCAWGT (K. Senger and M.L., unpublished observations; Ip et al., 1992Ip Y.T. Park R.E. Kosman D. Bier E. Levine M. The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo.Genes Dev. 1992; 6: 1728-1739Crossref PubMed Google Scholar). Approximately half of all Twist sites contain a 5′ C or A residue (M), and thereby also conform to the Snail consensus sequence. As a result, the Twist activator and Snail repressor directly compete for these shared sites, and Snail keeps the enhancers off in the ventral mesoderm. The Twist site in the CG12177 enhancer does not conform to the Snail consensus, and consequently, the gene is activated in the mesoderm. In regions where there are relatively high levels of Twist activator (as compared with Snail repressor), Twist binds the shared Twist/Snail recognition sequence within Type 2 enhancers and then facilitates binding of Dorsal to the neighboring linked site. In this respect, the core organization of Type 2 enhancers is reminiscent of the lambda switch (Markstein et al., 2004Markstein M. Zinzen R. Markstein P. Yee K.P. Erives A. Stathopoulos A. Levine M. A regulatory code for neurogenic gene expression in the Drosophila embryo.Development. 2004; 131: 2387-2394Crossref PubMed Scopus (109) Google Scholar, Ptashne, 2004Ptashne M. A Genetic Switch: Phage Lambda Revisited.Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2004Google Scholar). Type 3 enhancers are activated by the lowest levels of the Dorsal gradient, throughout the neurogenic ectoderm. The intronic short gastrulation (sog) enhancer contains a cluster of optimal Dorsal binding sites, and also contains a Schnürri (Shn) repressor site (Figure 2C; Markstein et al., 2002Markstein M. Markstein P. Markstein V. Levine M.S. Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2002; 99: 763-768Crossref PubMed Scopus (293) Google Scholar, Pyrowolakis et al., 2004Pyrowolakis G. Hartmann B. Muller B. Basler K. Affolter M. A simple molecular complex mediates widespread BMP-induced repression during Drosophila development.Dev. Cell. 2004; 7: 229-240Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). shn is expressed throughout the dorsal ectoderm, and may be directly repressed by the Dorsal gradient (Arora et al., 1995Arora K. Dai H. Kazuko S.G. Jamal J. O’Connor M.B. Letsou A. Warrior R. The Drosophila schnu¨rri gene acts in the Dpp/TGF β signaling pathway and encodes a transcription factor homologous to the human MBP family.Cell. 1995; 81: 781-790Abstract Full Text PDF PubMed Google Scholar, Grieder et al., 1995Grieder N.C. Nellen D. Burke R. Basler K. Affolter M. Schnu¨rri is required for Drosophila Dpp signaling and encodes a zinc finger protein similar to the mammalian transcription factor PRDII-BF1.Cell. 1995; 81: 791-800Abstract Full Text PDF PubMed Google Scholar). It is possible, but unproven, that Shn keeps Sog off in the dorsal ectoderm. It is conceivable that both activation and repression of Type 3 enhancers, which are readouts of the lowest levels of the Dorsal gradient, depend on unknown regulatory factors. In principle, such factors can be identified by computational searches for shared sequence motifs among coregulated enhancers. For example, the Type 3 sog and Neu4 (thisbe) enhancers contain a shared sequence motif, GCTGGYA, that might cooperate with the lowest levels of the Dorsal gradient to mediate activation (Stathopoulos et al., 2002Stathopoulos A. Van Drenth M. Erives A. Markstein M. Levine M. Whole-genome analysis of dorsal-ventral patterning in the Drosophila embryo.Cell. 2002; 111: 687-701Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The computational identification of shared sequence motifs has been used in the elucidation of the pharynx regulatory network in C. elegans, discussed below. The quality of individual Dorsal binding sites is an important determinant of the different threshold readouts of the gradient (Jiang and Levine, 1993Jiang J. Levine M. Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the dorsal gradient morphogen.Cell. 1993; 72: 741-752Abstract Full Text PDF PubMed Scopus (242) Google Scholar, Papatsenko and Levine, 2005bPapatsenko D. Levine M. Quantitative analysis of binding motifs mediating diverse spatial readouts of the Dorsal gradient in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2005; 102: 4966-4971Crossref PubMed Scopus (0) Google Scholar). Orthologous sequences were identified for each of the 18 enhancers in four divergent Drosophilids. The 72 enhancer sequences were characterized with respect to the number and quality of putative Dorsal binding sites. Each site was assigned a score based on its match to optimal Dorsal recognition sequences. A close correlation was observed between the quality of the best binding sites and the dorsal-ventral limits of gene expression. Enhancers with low-affinity sites tend to be activated in the ventral mesoderm in response to high levels of the Dorsal gradient, while enhancers with close matches to the optimal consensus sequence are activated by low levels of the gradient in the presumptive neurogenic ectoderm. These observations are consistent with a simple affinity threshold model, whereby the limits of gene expression depend on the in vivo occupancy of individual Dorsal binding sites (Jiang and Levine, 1993Jiang J. Levine M. Binding affinities and cooperative interactions with bHLH activators delimit threshold responses to the dorsal gradient morphogen.Cell. 1993; 72: 741-752Abstract Full Text PDF PubMed Scopus (242) Google Scholar). The combination of microarray assays, classical genetic screens, gene disruption assays, and enhancer analysis permits the synthesis of a genomic regulatory network for dorsal-ventral patterning (Davidson et al., 2002Davidson E.H. Rast J.P. Oliveri P. Ransick A. Calestani C. Yuh C.H. Minokawa T. Amore G. Hinman V. Arenas-Mena C. et al.A genomic regulatory network for development.Science. 2002; 295: 1669-1678Crossref PubMed Scopus (1127) Google Scholar, Levine and Davidson, 2005Levine M. Davidson E.H. Gene regulatory networks for development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4936-4942Crossref PubMed Scopus (473) Google Scholar). The network is represented as a circuit diagram showing the functional interactions among all of the known regulatory genes and cell signaling components engaged in dorsal-ventral patterning (Longabaugh et al., 2005Longabaugh W.J. Davidson E.H. Bolouri H. Computational representation of developmental genetic regulatory networks.Dev. Biol. 2005; 283: 1-16Crossref PubMed Scopus (160) Google Scholar). The identification of Dorsal target enhancers was essential for constructing the network because they directly integrate the activities of the sequence-specific activators and repressors (reviewed in Stathopoulos and Levine, 2004Stathopoulos A. Levine M. Whole-genome analysis of Drosophila gastrulation.Curr. Opin. Genet. Dev. 2004; 14: 477-484Crossref PubMed Scopus (52) Google Scholar). In addition, the enhancers serve as targets for cell signaling pathways, which ultimately influence the activities of specific transcription factors. For example, the activation of Notch signaling in the ventral-most cells of the neurogenic ectoderm leads to the activation of the ubiquitous Suppressor of Hairless (Su(H)) transcription factor, which binds to the single-minded (sim) enhancer and activates its expression (Markstein et al., 2004Markstein M. Zinzen R. Markstein P. Yee K.P. Erives A. Stathopoulos A. Levine M. A regulatory code for neurogenic gene expression in the Drosophila embryo.Development. 2004; 131: 2387-2394Crossref PubMed Scopus (109) Google Scholar, Morel and Schweisguth, 2000Morel V. Schweisguth F. Repression by suppressor of hairless and activation by Notch are required to define a single row of single-minded expressing cells in the Drosophila embryo.Genes Dev. 2000; 14: 377-388PubMed Google Scholar). Many features of this network are based on functional assays, including genetic analysis, gene dosage assays, and misexpression assays. For example, consider snail, which encodes a repressor that inhibits the expression of a variety of neurogenic genes (e.g., rho and sim) in the mesoderm. Mutant embryos homozygous for a null mutation in the sna gene exhibit expanded patterns of rho and sim expression in the mesoderm (Kosman et al., 1991Kosman D. Ip Y.T. Levine M. Arora K. Establishment of the mesoderm-neuroectoderm boundary in the Drosophila embryo.Science. 1991; 254: 118-122Crossref PubMed Google Scholar). Similarly, the misexpression of sna under the control of the even-skipped (eve) stripe 2 enhancer causes a gap in the sim and rho expression patterns in transgenic embryos (Cowden and Levine, 2002Cowden J. Levine M. The Snail repressor positions Notch signaling in the Drosophila embryo.Development. 2002; 129: 1785-1793Crossref PubMed Google Scholar). Point mutations in the Sna repressor sites contained within the rho enhancer cause a similar expansion of lacZ reporter gene expression in transgenic embryos (Ip et al., 1992Ip Y.T. Park R.E. Kosman D. Bier E. Levine M. The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo.Genes Dev. 1992; 6: 1728-1739Crossref PubMed Google Scholar). Additional perturbation tests have been done, including the creation of mutant embryos that contain broad anterior-posterior Dorsal or Twist gradients in place of the normal ventral-to-dorsal gradient (Huang et al., 1997Huang A.M. Rusch J. Levine M. An anteroposterior Dorsal gradient in the Drosophila embryo.Genes Dev. 1997; 11: 1963-1973Crossref PubMed Google Scholar, Stathopoulos and Levine, 2002bStathopoulos A. Levine M. Linear signaling in the Toll-Dorsal pathway of Drosophila: activated Pelle kinase specifies all threshold outputs of gene expression while the bHLH protein Twist specifies a subset.Development. 2002; 129: 3411-3419Crossref PubMed Google Scholar). These embryos exhibit novel patterns of gene expression reflecting autonomous responses of the different Dorsal target enhancers to appropriate concentrations of the ectopic Dorsal or Twist gradient. sna, sim, rho, intermediate neuroblasts defective (ind), and sog are expressed in increasingly broader patterns of expression across the anterior-posterior axis in response to diminishing levels of the Dorsal gradient, whereas the expression of only a subset of these genes is supported by the Twist gradient (Huang et al., 1997Huang A.M. Rusch J. Levine M. An anteroposterior Dorsal gradient in the Drosophila embryo.Genes Dev. 1997; 11: 1963-1973Crossref PubMed Google Scholar, Stathopoulos and Levine, 2002bStathopoulos A. Levine M. Linear signaling in the Toll-Dorsal pathway of Drosophila: activated Pelle kinase specifies all threshold outputs of gene expression while the bHLH protein Twist specifies a subset.Development. 2002; 129: 3411-3419Crossref PubMed Google Scholar). Together, all this information (gene expression information, genetic interactions, and enhancer analysis) is utilized to make connections between genes in order to construct a circuit diagram. What is the value of genomic regulatory networks? They reveal pathways of differentiation that can be precisely manipulated to generate different cell types, which is an area of intense current study due to the heightened interest in stem cell biology (e.g., Matthias and Rolink, 2005Matthias P. Rolink A.G. Transcriptional networks in developing and mature B cells.Nat. Rev. Immunol. 2005; 5: 497-508Crossref PubMed Scopus (166) Google Scholar, Shaywitz and Melton, 2005Shaywitz D.A. Melton D.A. The molecular biography of the cell.Cell. 2005; 120: 729-731Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Networks also provide the foundation for understanding the evolutionary diversification of related patterning processes (Carroll et al., 2001Carroll S.B. Grenier J.K. Weatherbee S.D. From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design. Blackwell Science, Malden, MA2001Google Scholar, Hinman et al., 2003Hinman V.F. Nguyen A.T. Cameron R.A. Davidson E.H. Developmental gene regulatory network architecture across 500 million years of echinoderm evolution.Proc. Natl. Acad. Sci. USA. 2003; 100: 13356-13361Crossref PubMed Scopus (0) Google Scholar, Levine and Davidson, 2005Levine M. Davidson E.H. Gene regulatory networks for development.Proc. Natl. Acad. Sci. USA. 2005; 102: 4936-4942Crossref PubMed Scopus (473) Google Scholar). Most importantly, networks reveal the underlying logic used to produce complexity, and thereby stimulate the formulation of new models and testable predictions. We consider a few examples of the logic used by the Dorsal gradient, including boundary repression, feed-forward mechanisms of gene activation, and the use of feedback loops to convert the transient Dorsal regulatory gradient into stable circuits of cellular differentiation. The Dorsal nuclear gradient is first established 90 min after fertilization, immediately after syncytial nuclei migrate to the periphery of the embryo (Roth et al., 1989Roth S. Stein D. Nusslein-Volhard C. A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo.Cell. 1989; 59: 1189-1202Abstract Full Text PDF PubMed Scopus (456) Google Scholar, Rushlow et al., 1989Rushlow C.A. Han K. Manley J.L. Levine M. The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila.Cell. 1989; 59: 1165-1177Abstract Full Text PDF PubM