1. Genomic Regulatory Networks and Animal Development
Angelike Stathopoulos, Michael Levine 
Developmental Cell 
Volume 9, Issue 4, Pages (October 2005)
DOI: /j.devcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 The Dorsal-Ventral Gene Regulatory Network in Drosophila
(A) Summary of dorsoventral patterning genes. The green box on the left shows the sequence of interactions leading to the localized activation of Toll signaling in ventral regions of early embryos. The diagram to the right shows 48 different genes that are engaged in dorsal-ventral patterning. The majority of these genes encode sequence-specific transcription factors or components of cell signaling pathways. The bottom of the diagram represents ventral regions—the presumptive mesoderm—while the top corresponds to dorsal regions. Top to bottom indicates progressively later stages of development, beginning with the onset of cellularization and culminating in the completion of mesoderm invagination.
(B) The diagram displays the functional interconnections among ∼30 zygotic genes engaged in the subdivision of the dorsoventral axis into the five basic embryonic tissues: mesoderm (twi, sna, NF-Yc, htl, hbr, mes3, phm, and WntD), mesectoderm (X, Notch, Su(H), m8, and sim), ventral neurogenic ectoderm (brk, vnd, rho, and vn), dorsal neurogenic ectoderm (ind, msh, sog, SoxN, and ths), and dorsal ectoderm (tld, zen, dpp, scw, and shn). Arrows indicate transcriptional activation, while bars at the end of connecting lines indicate transcriptional repression. For example, high levels of Dorsal (red) in ventral regions directly interact with the 5′ twi enhancer (indicated by horizontal bar) and the encoded Twist protein works together with Dorsal to activate the expression of the snail gene in the mesoderm. Dorsal and Twist also activate brk, vnd, rho, and vn, but the Snail repressor keeps these genes off in the ventral mesoderm.
(C) The diagram displays the localized activities of the FGF, EGF, and TGFβ signaling pathways in the mesoderm, mesectoderm, and dorsal ectoderm, respectively. The htl and hbr genes encode FGF signaling components, which are induced by an FGF8 signaling molecule encoded by the ths gene expressed in the dorsal ectoderm (purple dot represents activation of the Htl FGF receptor). FGF signaling is important for the activation of cardiac genes such as eve in the dorsal mesoderm of older embryos. sim encodes a transcription factor that activates a number of EGF signaling components in the mesectoderm, including spitz, star, and rho. The processed Spitz signaling molecule activates the EGF receptor, ultimately leading to the activation of target genes such as otd and argos in the mesectoderm. These genes may be activated by the ETS-containing transcription factor encoded by pnt. EGF signaling also stimulates the expression of neurogenic genes such as D (Dichaete) and ind, which are essential for the formation of lateral neuroblasts in the definitive nerve cord. The Dorsal gradient regulates a variety of genes required for restricted Dpp (TGFβ) signaling in the dorsal ectoderm, including brk, sog, tld, and zen. Sog inhibits Dpp signaling in the neurogenic ectoderm, while brk encodes a transcriptional repressor that keeps Dpp target genes—like shn (schnürri)—off in the neurogenic ectoderm. sog, tld, and tsg encode extracellular proteins that modulate Screw and Dpp signaling activity within the dorsal ectoderm (indicated by lines connecting to green dots, which represent the BMP signaling molecules encoded by screw and dpp). Dpp signaling leads to the activation of a variety of target genes in dorsal (hnt, race, and doc), dorsolateral (tup and ush), and lateral (tsg, pnr, and shn) regions of the dorsal ectoderm. (The figure is adapted from Levine and Davidson, 2005.)
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2 Differential Threshold Enhancers
The indicated enhancers were attached to a minimal eve-lacZ reporter gene and expressed in transgenic embryos (right panels).
(A) The CG12177 gene encodes a putative nucleoside hydrolase that is specifically expressed in the presumptive mesoderm of early embryos. A putative enhancer was identified in the 5′ flanking region based on clustering of Dorsal (GGG—CYS; orange) and Twist (CAYATGT; blue) binding sites. The sequence of the enhancer is shown, along with the putative Dorsal and Twist sites. A genomic DNA fragment encompassing the binding sites directs localized expression of the lacZ reporter in the ventral mesoderm where there are peak levels of the Dorsal gradient.
(B) The rho gene encodes a membrane protease that processes the Spitz EGF ligand. It is specifically expressed in ventral regions of the neurogenic ectoderm. The rho enhancer is located in the 5′ flanking region and contains a series of Dorsal and Twist sites. The enhancer also contains several Snail repressor sites (MMRCAWGT; red) that inhibit expression in the ventral mesoderm. The rho enhancer directs a Type 2 lacZ expression pattern in transgenic embryos.
(C) The Type 3 sog gene contains an intronic enhancer with a series of optimal Dorsal binding sites. It also contains a composite Schnürri/Smad repression element, which might help define the dorsal border of gene expression (the element is highlighted in green). The sog enhancer directs a Type 3 lacZ expression pattern in transgenic embryos.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Repressors Delineate the Borders of Expression Domains along the Dorsoventral Axis
(A) Circuit diagram adapted from the one depicted in Figure 1 illustrating the interactions between these five repressors, sna, vnd, brk, ind, and shn. Sna protein restricts the expression of vnd, brk, and ind from the ventral-most region of the embryo. Vnd restricts the expression of ind from the ventrolateral region of the embryo. Brk and Shn presumably exhibit mutually repressive interactions that limit their expression patterns and also help shape the Dpp activity gradient. Additional repressors may also function to regulate dorsoventral patterning that is not depicted, such as the DNA binding protein that recognizes the A box within the ind enhancer (Stathopoulos and Levine, 2005).
(B) Shown is a lateral view of a cellular blastoderm embryo. In situ hybridization patterns using riboprobes are depicted for four of the five repressors known to control gene expression along the dorsoventral axis: sna (ventral regions in red), vnd (ventrolateral region in blue), ind (lateral region in red), and shn (dorsal region in green). Not depicted is the brk expression pattern which is similar to that of vnd.
(C) Feed-forward I. Dorsal activates twist expression, and the encoded Twist protein functions together with Dorsal to activate a variety of target genes. The broad Dorsal gradient produces a somewhat steeper Twist protein gradient that extends throughout the presumptive mesoderm and into ventral regions of the presumptive neurogenic ectoderm. Dorsal and Twist bind to a number of different target enhancers that mediate gene expression in the mesoderm (twi, sna, htl, hbr, and WntD), mesectoderm (sim), and ventral regions of the neurogenic ectoderm (brk, vnd, rh, and vn).
(D) Feed-forward II. The Dorsal gradient leads to localized expression of zen and dpp in the dorsal ectoderm. Dpp signaling leads to the localized activation of the Sax and Tkv receptors in dorsal regions of the dorsal ectoderm. The Smad transcription factors Mad and Medea are activated, and peak levels of these factors restrict zen expression to dorsal regions that will form the amnioserosa. Once Dpp signaling restricts zen, Mad/Medea work together with Zen to activate a variety of genes required for the differentiation and function of the amnioserosa, including hnt, race, doc, tup, and ush.
(E) Summary of the Dorsal-Sim-EGF signaling pathway. The Dorsal genomic regulatory network illustrates the relationship between the Dorsal gradient and the EGF signaling pathway active in ventral regions of the ventral nerve cord. Briefly, Dorsal activates Twist, which in turn induces Snail expression in the ventral mesoderm. Snail represses an unknown inhibitor of Notch signaling, thereby triggering activation of the Notch receptor in the single line of cells straddling the mesoderm. These cells express Sim, which in turn establishes the ventral midline of the nerve cord. Sim activates several components of the EGF signaling pathway: Spitz, Rho, and Star. This produces a localized source of the Spitz EGF ligand in the midline. High levels of EGF activate otd and other Type 1 target genes in the ventral midline. Low levels of EGF that diffuse into neighboring regions of the midline are thought to activate Type 2 target genes. However, the Dorsal network suggests that most or all Type 2 target genes are activated in the early embryo by lateral stripes of rho and vein expression in ventral regions of the neurogenic ectoderm. It is therefore possible that the Type 2 genes are merely maintained by the low levels of Spitz emanating from the midline. According to this model, EGF signaling is a simple on/off switch and does not function as a classical gradient morphogen.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
Proposed Structure of the Regulatory Network Specified by pal-1
A graph of predicted regulatory relationships based on the best candidate upstream activator for each gene as described in Baugh et al., (2005a) is presented. Temporal phase is indicated on the left. Lines with arrows represent cell-autonomous regulation by transcription factors and lines with dots represent regulation by signaling molecules. (The figure is from Baugh et al., 2005a.)
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5
High-Affinity PHA-4 Sites Activate Pharyngeal Expression Earlier Than Low-Affinity Sites
Percent values indicate the percentage of transgenics exhibiting pharyngeal GFP expression. Dashed lines indicate the outline of the developing pharynx.
(A–C) A reporter construct with three copies of a high-affinity PHA-4 site (TGTTTGC) upstream of the Dpes-10 promoter reproducibly activates pharyngeal expression from the time of pharynx primordium formation (early) through embryogenesis.
(D–F) A reporter construct with three copies of a low-affinity PHA-4 site (TATTTGT) upstream of the Dpes-10 promoter activates pharyngeal expression from the time of attachment of the pharynx to the mouth (mid) through embryogenesis. (The figure is from Gaudet et al., 2004.)
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure. 6
A Model for the Gene Regulatory Network in Retinal Ganglion Cell Development
Relationships are based on microarray results (Green et al., 2003; Marquardt and Gruss, 2002; Mu et al., 2004, 2005). Genes are depicted such that the right side of the bent arrow for each gene signifies the gene product, and the left side signifies the transcriptional control region. Solid lines connecting genes indicate established upstream-downstream relationships and dashed lines suggest inferred relationships. Downward arrows signify gene activation and downward perpendicular lines signify gene repression. The connections are not necessarily meant to indicate direct target gene activation or repression. Connections stemming from the following regulators are shown: Notch, solid light blue; Pax6, solid green; Gli1, solid black; Math5, solid magenta; NRSF/REST, dashed red; Pou4f2 (Brn3b), solid blue; Isl1, Tle1, and Myt1, dashed black; Shh, solid turquoise; Gdf8, dashed magenta; Vegf, dashed green. Genes boxed in yellow represent regulators of general retinal competence; genes boxed in orange are proneural bHLH genes associated with established competence in retinal precursor cells (RPCs) for specific retinal cell fates; the light purple box represents the general repression of retinal ganglion cell (RGC) genes mediated by NRSF/REST; the light blue and dark blue boxes represent genes encoding RGC-specific upstream (light blue) and downstream (dark blue) transcription factors; genes boxed in green are those encoding proteins associated with RGC maturation and function; genes boxed in purple encode secreted signaling molecules. In some cases, only representative examples are shown in each box. This figure is from Mu et al. (2005), where a description of individual genes can be found.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7 Gene Regulatory Network Governing Notochord Differentiation
Notochord precursor cells are defined by the localized expression of Ci-Bra in the primary (A7.3 and A7.7) and secondary (B7.3) blastomeres (see red cells in diagram of 64-cell embryo in [B]).
(A) Provisional gene regulatory network for notochord differentiation. Macho-1 is a localized maternal determinant that is inherited by posterior vegetal blastomeres. It leads to the localized expression of ZicL, Snail, and Notch signaling in the secondary notochord lineage. β-catenin is selectively activated in anterior vegetal blastomeres. It leads to the localized expression of FoxA and FGF signaling in the primary notochord lineage. Ci-Bra leads to the activation of a variety of target genes required for the differentiation of the notochord, including collagen 2A1 (Coll2A1) and a divergent tropomyosin (Trop).
(B) The top part of the diagram shows drawings of progressively older embryos, oriented to display the vegetal regions. The localized muscle determinant, Macho-1, leads to the localized expression of the Ciona Snail repressor in the progenitor (B6.2) of the secondary notochord lineage. Snail keeps the Ci-Bra enhancer off in the tail muscles (purple blastomeres in the 64-cell embryo), and Notch signaling overcomes Snail repression in the secondary notochord cells (B7.3). Localized Ci-Bra in the primary and secondary notochord cells (red) leads to the activation of notochord differentiation genes such as Trop (or Ci-trop) (Di Gregorio and Levine, 1999). The tadpole on the right was electroporated with the Ci-trop notochord enhancer attached to a lacZ reporter gene. Staining is restricted to individual notochord cells in the tail. (The figure is adapted from Corbo et al., 2001.)
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions