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.