Abstract
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.
Highlights
The remarkable cellular diversity observed in metazoan organs raises an important developmental question: how are the specification and differentiation of the many distinct cell types comprising such an organ achieved?The embryonic Drosophila heart provides a amenable system for addressing this question
Similar ectopic reporter activity in the cardial cells (CCs) mediated by the wild-type Holes in muscle (Him) enhancer (HimWT) is observed when Su(H) levels are depleted by RNA interference (RNAi) knockdowns driven by the cardiac mesoderm-specific TinD-GAL4 driver (Fig 3C–3D ́; S1 Table; S4 Fig) [3]
We found that RNAi-mediated individual knockdowns of gro, H, or C-terminal Binding Protein (CtBP) targeted to the cardiac mesoderm exhibits ectopic reporter activity by the HimWT enhancer in CCs similar that observed in the case of the HimSu(H) enhancer or the Su(H) RNAi (Fig 3E–3G ́; S1 Table), consistent with our hypothesis of Su(H) acting as part of a transcriptional repressor complex with Gro, H, and CtBP in the regulation of Him
Summary
The remarkable cellular diversity observed in metazoan organs raises an important developmental question: how are the specification and differentiation of the many distinct cell types comprising such an organ achieved?The embryonic Drosophila heart provides a amenable system for addressing this question. An organ that pumps hemolymph throughout the body cavity, the Drosophila heart consists of two groups of cells: an inner linear tube of paired contractile cardial cells (CCs) that express Myocyte enhancer factor 2 (Mef2), and an external sheath of nephrocytic pericardial cells (PCs) that express both Zn finger homeodomain 1 (zfh1) and Holes in muscle (Him) [1,2,3,4]. The PCs can be subdivided into additional cell subtypes based on their positions, the complexity of their individual gene expression programs, their morphology, and even the distinct cell lineages that generate them [5]. Stretches of DNA that are recognized and bound by particular combinations of sequence-specific DNA binding transcription factors (TFs), determine gene expression programs at the level of individual cells [10]. Further analysis of the enhancer of the PC-specific gene Him suggested that Su(H) forms a repressor complex that binds to this enhancer, with Notch signaling alleviating the repression in PCs to activate Him transcription [3]
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