Assessing the Roles of Potential Notch Signaling Components in Instructive and Permissive Pathways with Two Drosophila Pericardial Reporters.

The highly conserved Notch signaling pathway brings about the transcriptional activation of target genes via either instructive or permissive mechanisms that depend on the identity of the specific target gene. As additional components of the Notch signaling pathway are identified, assessing whether each of these components are utilized exclusively by one of these mechanisms (and if so, which), or by both, becomes increasingly important. Using RNA interference-mediated knockdowns of the Notch component to be tested, reporters for two Notch-activated pericardial genes in Drosophila melanogaster, immunohistochemistry, and fluorescence microscopy, we describe a method to determine the type of signaling mechanism-instructive, permissive, or both-to which a particular Notch pathway component contributes.

The Drosophila Forkhead/Fox transcription factor Jumeau mediates specific cardiac progenitor cell divisions by regulating expression of the kinesin Nebbish.

Forkhead (Fkh/Fox) domain transcription factors (TFs) mediate multiple cardiogenic processes in both mammals and Drosophila. We showed previously that the Drosophila Fox gene jumeau (jumu) controls three categories of cardiac progenitor cell division-asymmetric, symmetric, and cell division at an earlier stage-by regulating Polo kinase activity, and mediates the latter two categories in concert with the TF Myb. Those observations raised the question of whether other jumu-regulated genes also mediate all three categories of cardiac progenitor cell division or a subset thereof. By comparing microarray-based expression profiles of wild-type and jumu loss-of-function mesodermal cells, we identified nebbish (neb), a kinesin-encoding gene activated by jumu. Phenotypic analysis shows that neb is required for only two categories of jumu-regulated cardiac progenitor cell division: symmetric and cell division at an earlier stage. Synergistic genetic interactions between neb, jumu, Myb, and polo and the rescue of jumu mutations by ectopic cardiac mesoderm-specific expression of neb demonstrate that neb is an integral component of a jumu-regulated subnetwork mediating cardiac progenitor cell divisions. Our results emphasize the central role of Fox TFs in cardiogenesis and illustrate how a single TF can utilize different combinations of other regulators and downstream effectors to control distinct developmental processes.

Three distinct mechanisms, Notch instructive, permissive, and independent, regulate the expression of two different pericardial genes to specify cardiac cell subtypes.

The development of a complex organ involves the specification and differentiation of diverse cell types constituting that organ. Two major cell subtypes, contractile cardial cells (CCs) and nephrocytic pericardial cells (PCs), comprise the Drosophila heart. Binding sites for Suppressor of Hairless [Su(H)], an integral transcription factor in the Notch signaling pathway, are enriched in the enhancers of PC-specific genes. Here we show three distinct mechanisms regulating the expression of two different PC-specific genes, Holes in muscle (Him), and Zn finger homeodomain 1 (zfh1). Him transcription is activated in PCs in a permissive manner by Notch signaling: in the absence of Notch signaling, Su(H) forms a repressor complex with co-repressors and binds to the Him enhancer, repressing its transcription; upon alleviation of this repression by Notch signaling, Him transcription is activated. In contrast, zfh1 is transcribed by a Notch-instructive mechanism in most PCs, where mere alleviation of repression by preventing the binding of Su(H)-co-repressor complex is not sufficient to activate transcription. Our results suggest that upon activation of Notch signaling, the Notch intracellular domain associates with Su(H) to form an activator complex that binds to the zfh1 enhancer, and that this activator complex is necessary for bringing about zfh1 transcription in these PCs. Finally, a third, Notch-independent mechanism activates zfh1 transcription in the remaining, even skipped-expressing, PCs. Collectively, our data show how the same feature, enrichment of Su(H) binding sites in PC-specific gene enhancers, is utilized by two very distinct mechanisms, one permissive, the other instructive, to contribute to the same overall goal: the specification and differentiation of a cardiac cell subtype by activation of the pericardial gene program. Furthermore, our results demonstrate that the zfh1 enhancer drives expression in two different domains using distinct Notch-instructive and Notch-independent mechanisms.

Conserved signaling mechanisms in Drosophila heart development.

Signal transduction through multiple distinct pathways regulates and orchestrates the numerous biological processes comprising heart development. This review outlines the roles of the FGFR, EGFR, Wnt, BMP, Notch, Hedgehog, Slit/Robo, and other signaling pathways during four sequential phases of Drosophila cardiogenesis-mesoderm migration, cardiac mesoderm establishment, differentiation of the cardiac mesoderm into distinct cardiac cell types, and morphogenesis of the heart and its lumen based on the proper positioning and cell shape changes of these differentiated cardiac cells-and illustrates how these same cardiogenic roles are conserved in vertebrates. Mechanisms bringing about the regulation and combinatorial integration of these diverse signaling pathways in Drosophila are also described. This synopsis of our present state of knowledge of conserved signaling pathways in Drosophila cardiogenesis and the means by which it was acquired should facilitate our understanding of and investigations into related processes in vertebrates. Developmental Dynamics 246:641-656, 2017. © 2017 Wiley Periodicals, Inc.

Two Forkhead transcription factors regulate cardiac progenitor specification by controlling the expression of receptors of the fibroblast growth factor and Wnt signaling pathways.

Cardiogenesis involves the coordinated regulation of multiple biological processes by a finite set of transcription factors (TFs). Here, we show that the Forkhead TFs Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu), which govern cardiac progenitor cell divisions by regulating Polo kinase activity, play an additional, mutually redundant role in specifying the cardiac mesoderm (CM) as eliminating the functions of both Forkhead genes in the same Drosophila embryo results in defective hearts with missing hemisegments. This process is mediated by the Forkhead TFs regulating the fibroblast growth factor receptor Heartless (Htl) and the Wnt receptor Frizzled (Fz): CHES-1-like and jumu exhibit synergistic genetic interactions with htl and fz in CM specification, thereby implying that they function through the same genetic pathways, and transcriptionally activate the expression of both receptor-encoding genes. Furthermore, ectopic overexpression of either htl or fz in the mesoderm partially rescues the defective CM specification phenotype in embryos lacking both Forkhead genes. Together, these data emphasize the functional redundancy that leads to robustness in the cardiac progenitor specification process, and illustrate the pleiotropic functions of Forkhead TFs in different aspects of cardiogenesis.

Machine learning classification of cell-specific cardiac enhancers uncovers developmental subnetworks regulating progenitor cell division and cell fate specification.

The Drosophila heart is composed of two distinct cell types, the contractile cardial cells (CCs) and the surrounding non-muscle pericardial cells (PCs), development of which is regulated by a network of conserved signaling molecules and transcription factors (TFs). Here, we used machine learning with array-based chromatin immunoprecipitation (ChIP) data and TF sequence motifs to computationally classify cell type-specific cardiac enhancers. Extensive testing of predicted enhancers at single-cell resolution revealed the added value of ChIP data for modeling cell type-specific activities. Furthermore, clustering the top-scoring classifier sequence features identified novel cardiac and cell type-specific regulatory motifs. For example, we found that the Myb motif learned by the classifier is crucial for CC activity, and the Myb TF acts in concert with two forkhead domain TFs and Polo kinase to regulate cardiac progenitor cell divisions. In addition, differential motif enrichment and cis-trans genetic studies revealed that the Notch signaling pathway TF Suppressor of Hairless [Su(H)] discriminates PC from CC enhancer activities. Collectively, these studies elucidate molecular pathways used in the regulatory decisions for proliferation and differentiation of cardiac progenitor cells, implicate Su(H) in regulating cell fate decisions of these progenitors, and document the utility of enhancer modeling in uncovering developmental regulatory subnetworks.

Two forkhead transcription factors regulate the division of cardiac progenitor cells by a Polo-dependent pathway.

The development of a complex organ requires the specification of appropriate numbers of each of its constituent cell types, as well as their proper differentiation and correct positioning relative to each other. During Drosophila cardiogenesis, all three of these processes are controlled by jumeau (jumu) and Checkpoint suppressor homologue (CHES-1-like), two genes encoding forkhead transcription factors that we discovered utilizing an integrated genetic, genomic, and computational strategy for identifying genes expressed in the developing Drosophila heart. Both jumu and CHES-1-like are required during asymmetric cell division for the derivation of two distinct cardiac cell types from their mutual precursor and in symmetric cell divisions that produce yet a third type of heart cell. jumu and CHES-1-like control the division of cardiac progenitors by regulating the activity of Polo, a kinase involved in multiple steps of mitosis. This pathway demonstrates how transcription factors integrate diverse developmental processes during organogenesis.

Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors.

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs - including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) - to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, our results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression.

Sex comes in from the cold: the integration of sex and pattern.

There has recently been a revolution in our understanding of how the Drosophila sex-determination hierarchy generates somatic sexual dimorphism. Most significantly, the sex hierarchy has been shown to modulate the activities of well-known signaling molecules (FGF, Wnt and TGF beta proteins) and transcription factors (BAB and DAC) to direct various sex-specific aspects of growth and differentiation. As some of the genes encoding these proteins are also the targets of Hox gene action, these and other findings are revealing the levels at which the sex determination and Hox patterning pathways are integrated to control growth, morphogenesis and differentiation.

Sex-specific deployment of FGF signaling in Drosophila recruits mesodermal cells into the male genital imaginal disc.

A central issue in developmental biology is how the deployment of generic signaling proteins produces diverse specific outcomes. We show that Drosophila FGF is used, only in males, to recruit mesodermal cells expressing its receptor to become part of the genital imaginal disc. Male-specific deployment of FGF signaling is controlled by the sex determination regulatory gene doublesex. The recruited mesodermal cells become epithelial and differentiate into parts of the internal genitalia. Our results provide exceptions to two basic tenets of imaginal disc biology-that imaginal disc cells are derived from the embryonic ectoderm and belong to either an anterior or posterior compartment. The recruited mesodermal cells migrate into the disc late in development and are neither anterior nor posterior.