Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. We demonstrate that Wingless (Wg) and Decapentaplegic (Dpp) confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated (dTCF, Mad, and Pointed-functioning in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted (Twist and Tinman) transcription factors on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development.

The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k (‘k-mers’). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org.

Apoptosis was induced in S-phase-arrested

( Jiang et al. 1991;Pan et al. 1991;Thisse et al. 1991;Ip et al. 1992). The early activities of these latter genes are essential for the specification of general mesodermal cell fate (Simpson 1983), whereas additional factors both intrinsic and extrinsic to this germ layer influence the subsequent differentiation of particular cells (Beer et al.

Mesodermal progenitors arise in theDuring animal development, a wide diversity of cellular identities must be specified within initially undifferentiated fields of cells. One solution to this problem is for a hierarchy of regulators to promote the progressive determination of cells, essentially carving out from the original field domains with increasingly restricted developmental potential. In such a mechanism, spatially localized factors first delineate a prepattern in which all cells are equally competent to adopt a particular identity (Stern 1954;Greenwald and Rubin 1992). The expression of additional regulatory molecules in cellular subsets within the prepatterned territory further limits the responses afforded particular cells. Precise refinement of the final pattern can be dictated by direct inhibitory interactions among neighboring cells (Greenwald and Rubin 1992;Simpson 1997). Although many details are known about the later pattern forming steps in a number of developmental systems, relatively little information is available for how early prepatterns are established (Greenwald and Rubin 1992;Kornfeld 1997;Simpson 1997;Vervoort et al. 1997).The Drosophila embryonic mesoderm provides an ideal system in which to investigate prepattern and pattern formation. The mesoderm arises from the ventral most cells of the blastoderm embryo under the influence of the zygotic genes, twist (twi) and snail (sna). Cells expressing these genes invaginate through the ventral furrow at gastrulation. Subsequently, the internalized mesodermal cells migrate dorsolaterally to form a uniform sheet beneath the ectoderm (Bate 1993;Leptin 1995), a process that is controlled by a fibroblast growth factor (FGF) receptor encoded by heartless (htl;Beiman et al. 1996;Gisselbrecht et al. 1996;Shishido et al. 1997;Michelson et al. 1998).

The evolution of transcriptional regulatory networks entails the expansion and diversification of transcription factor (TF) families. The forkhead family of TFs, defined by a highly conserved winged helix DNA-binding domain (DBD), has diverged into dozens of subfamilies in animals, fungi, and related protists. We have used a combination of maximum-likelihood phylogenetic inference and independent, comprehensive functional assays of DNA-binding capacity to explore the evolution of DNA-binding specificity within the forkhead family. We present converging evidence that similar alternative sequence preferences have arisen repeatedly and independently in the course of forkhead evolution. The vast majority of DNA-binding specificity changes we observed are not explained by alterations in the known DNA-contacting amino acid residues conferring specificity for canonical forkhead binding sites. Intriguingly, we have found forkhead DBDs that retain the ability to bind very specifically to two completely distinct DNA sequence motifs. We propose an alternate specificity-determining mechanism whereby conformational rearrangements of the DBD broaden the spectrum of sequence motifs that a TF can recognize. DNA-binding bispecificity suggests a previously undescribed source of modularity and flexibility in gene regulation and may play an important role in the evolution of transcriptional regulatory networks.transcription factor binding site motif | protein-DNA interactions T he regulation of gene expression by the interaction of sequence-specific transcription factors (TFs) with target sites (cis-regulatory elements) near their regulated genes is a central mechanism by which organisms interpret regulatory programs encoded in the genome to develop and interact with their environment. The emergence of new species has depended in part on the evolution of the network of interactions by which an organism's TFs control gene expression. Much attention has been paid to changes in cis-regulatory sequences over evolutionary time, because these changes can result in incremental modifications of organismal phenotypes without large-scale rewiring of transcriptional regulatory networks that would result from changes in TF DNA-binding specificity (1). Nevertheless, TFs and their DNA-binding specificities have changed over time (2). Gene duplication, followed by divergence of the resulting redundant TFs, has resulted in the emergence of families of paralogous TFs with diversified DNA-binding specificities and functions (3). Thus, identifying mechanisms by which related DNA-binding domains (DBDs) have acquired novel specificities is important for understanding TF evolution.The forkhead box (Fox) family of TFs spans a wide range of species and is one of the largest classes of TFs in humans. In metazoans, Fox proteins have vital roles in development of a variety of organ systems, metabolic homeostasis, and regulation of cell-cycle progression, and fungal Fox proteins are involved in cell-cycle progression and the expression of ribosomal proteins. ...

Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway--activated by both epidermal and fibroblast growth factor receptors--functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. We have investigated whether these opposing signals directly interact with each other, and we have examined how they are integrated by the responding cells to specify their unique fates. Our findings reveal that Ras and Notch do not function independently; rather, we have uncovered several modes of cross-talk between these pathways. Ras induces Notch, its ligand Delta, and the epidermal growth factor receptor antagonist, Argos. We show that Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells.

An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system.