Fig 1.
Stereotyped positions of cardiac cell subtypes.
Schematic diagram showing the relative positions of the nuclei of cardial cells (CCs) and three classes of pericardial cells (PCs): Tin-PCs, Odd-PCs, and Eve-PCs.
Fig 2.
Su(H) binding sites in the Him and zfh1 enhancers.
Both the Him enhancer (X:18,206,943..18,207,537) and the zfh1 enhancer (3R:30,762,904..30,763,833) are located 5´ to the endogenous genes (Drosophila melanogaster genome sequence release 6.29). The relative locations of the Su(H) binding sites on these enhancers are shown, as well as their wild-type sequences (uppercase). The nucleotide substitutions used to create binding site mutations that eliminate Su(H) binding are shown in red lowercase. Sequences for the wild-type and mutated enhancers are provided in S1–S4 Files.
Fig 3.
Su(H), Groucho, Hairless, and CtBP function as repressors for the Him enhancer in CCs.
(A-G´´´) lacZ reporter gene activity (β–galactosidase, green) driven by relevant Him enhancers in appropriate genotypes of stage 16 embryos. All CCs express Mef2 (red) while PCs are marked by Zfh1 (blue). Scale bar: 10 μm. (A-A´´´) The wild-type Him enhancer (HimWT) is active only in the Zfh1-expressing PCs. (B-B´´´) When Su(H) binding sites are mutated in the Him enhancer (HimSu(H)), the reporter is still active in Zfh1-expressing PCs but is also de-repressed in Mef2-expressing CCs (arrows). (C-C´´´) Knockdown of Su(H) with the dorsal mesoderm-targeted RNAi construct Su(H)HMS05748 driven by the TinD-GAL4 driver induces ectopic HimWT enhancer-driven β–galactosidase reporter activity in CCs (arrows). The fact that not all CCs express the same level of ectopic reporter activity likely reflects an incomplete RNAi knockdown of Su(H) levels in all cardiac cells. (D-D´´´) Similar ectopic HimWT enhancer-driven β–galactosidase reporter activity in CCs (arrows) is also induced by the TinD-GAL4 driven knockdown of Su(H) with a second RNAi construct, Su(H)HM05110. (E-G´´´) Dorsal mesoderm targeted knockdown of the co-repressors Groucho (E-E´´´), Hairless (F-F´´´), and CtBP (G-G´´´) by the TinD-GAL4 driven expression of the constructs groKK108953, HGD1458, and CtBPKK108401, respectively, also result in the ectopic induction of HimWT enhancer-driven β–galactosidase reporter activity in CCs (arrows) similar to that detected when either the Su(H) binding site is mutated in the enhancer (B-B´´´) or Su(H) is knocked down (C-D´´´). Sample sizes for these assays can be found in S1 Table.
Fig 4.
Su(H) functions as an activator for the zfh1 enhancer in a subset of PCs while Groucho, Hairless, and CtBP have no effect on zfh1 enhancer-driven expression.
(A-E) lacZ reporter gene activity (β–galactosidase, green) driven by relevant zfh1 enhancers in appropriate genotypes of stage 16 embryos. All CCs express Mef2 (red) while PCs are marked by Zfh1 (blue). Scale bar: 10 μm. (A-A´´´) The wild-type zfh1 enhancer (zfh1WT) drives lacZ reporter expression exclusively in all PCs. (B-B´´´) Mutagenesis of Su(H) binding sites in the zfh1 enhancer (zfh1Su(H)) abrogates reporter activity in Odd-PCs (arrows) and Tin-PCs (arrows) but not in Eve-PCs (arrowheads) compared to the wild-type enhancer (zfh1WT). The specific pericardial cell subtypes where expression was eliminated were initially identified based on their relative positions in the heart and subsequently confirmed (see Fig 5) by staining with PC subtype-specific antibodies. (C-E´´´) Depletion of Groucho (C-C´´´), Hairless (D-D´´´), or CtBP (E-E´´´) with cardiac mesoderm-targeted RNAi by the TinD-GAL4 driven expression of the constructs groKK108953, HGD1458, and CtBPKK108401, respectively, neither induced ectopic zfh1WT enhancer-driven reporter activity in CCs nor eliminated reporter activity in any PCs.
Fig 5.
Su(H) and Notch activate expression from the zfh1 enhancer in Odd-PCs and Tin-PCs.
(A-J´´´) lacZ reporter gene activity (β–galactosidase, green) driven by relevant zfh1 enhancers in appropriate genotypes of stage 16 embryos. Scale bar: 10 μm. All CCs express Mef2 (red) while in (A-D´´´) Odd-PCs are marked by Odd (blue), and in (E-J´´´) Eve-PCs are marked by Eve (blue). (A-A´´´) The wild-type zfh1 enhancer (zfh1WT) is active in all PCs including the Odd-PCs (arrows), the ventrally located Tin-PCs (asterisks), and Eve-PCs (arrowheads). (B-B´´´) When Su(H) binding sites are mutated in the zfh1 enhancer (zfh1Su(H)), reporter activity is only observed in Eve-PCs (arrowheads) and abrogated in Odd-PCs (arrows). The Eve-PC at the bottom of the panel is located dorsally above and masking an Odd-PC. Note that reporter expression is not detected in any ventral Tin-PCs either. (C-D´´´) Depletion of Su(H) with cardiac mesoderm-targeted RNAi using the constructs Su(H)HMS05748 (C-C´´´) and Su(H)HM05110 (D-D´´´) driven by the TinD-GAL4 driver also maintains zfh1WT enhancer-driven β-galactosidase reporter activity in Eve-PCs (arrowheads) and almost completely eliminates reporter expression in Odd-PCs (arrows) and Tin-PCs. The observation that some extremely faint reporter expression is detected in a few Tin-PCs and Odd-PCs likely reflects an incomplete RNAi knockdown of Su(H) levels in all heart cells. (E-E´´´) The wild-type zfh1 enhancer (zfh1WT) is active in all PCs including the Eve-PCs (arrowheads), Odd-PCs (arrows), and Tin-PCs (asterisks). (F-F´´´) When Su(H) binding sites are mutated in the zfh1 enhancer (zfh1Su(H)), the reporter is only expressed in Eve-PCs (arrowheads). (G-H´´´) Depletion of Su(H) with cardiac mesoderm-targeted RNAi using the constructs Su(H)HMS05748 (G-G´´´) and Su(H)HM05110 (H-H´´´) driven by the TinD-GAL4 driver also eliminates zfh1WT enhancer-driven β-galactosidase reporter activity in all PCs other than the Eve-PCs (arrowheads). (I-J´´´) Knockdown of Notch with cardiac mesoderm-targeted RNAi using the constructs NHMS00001 (I-I´´´) and NHMS00009 (J-J´´´) driven by the TinD-GAL4 driver also eliminates zfh1WT enhancer-driven reporter activity in all PCs other than the Eve-PCs (arrowheads).
Fig 6.
Constitutive Notch signaling activates ectopic transcription from both the Him and zfh1 enhancers in CCs.
(A-F´´´) lacZ reporter gene activity (β–galactosidase, green) driven by wild-type Him and zfh1 enhancers in appropriate genotypes of stage 16 embryos. All CCs express Mef2 (red) while PCs are marked by Zfh1 (blue). Scale bar: 10 μm. (A-A´´´) The wild-type Him enhancer (HimWT) is active only in the Zfh1-expressing PCs. (B-B´´´) Overexpression of Nicd in all cells of the cardiac mesoderm by the early TinD-GAL4 driver leads to the Mef2-expressing CCs exhibiting ectopic HimWT enhancer-driven β–galactosidase reporter activity (arrows). The reduced number of CCs is a known consequence of the inhibition of cardiac progenitor specification by Notch signaling at an earlier phase of development [41–43]. (C-C´´´) Overexpression of Nicd in all cells of the heart by the later Hand-GAL4 driver leads to some weakly Mef2-expressing CCs expressing both ectopic HimWT enhancer-driven β–galactosidase reporter activity and ectopic Zfh1 protein (arrowheads) while other weakly Mef2-expressing CCs exhibit just HimWT enhancer-driven reporter activity (arrows). (D-D´´´) The wild-type zfh1 enhancer (zfh1WT) is also active only in the Zfh1-expressing PCs. (E-E´´´) Overexpression of Nicd in all cells of the cardiac mesoderm by the early TinD-GAL4 driver leads to the Mef2-expressing CCs exhibiting ectopic zfh1WT enhancer-driven β–galactosidase reporter activity (arrows). As in (B-B´´´), the inhibition of cardiac progenitor specification by ectopic Notch signaling results in a reduced number of CCs. (E-E´´´) Overexpression of Nicd in all cells of the heart by the later Hand-GAL4 driver also leads to both ectopic zfh1WT enhancer-driven reporter activity and ectopic Zfh1 protein in the weakly Mef2-expressing CCs (arrowheads).
Fig 7.
Schematic of Notch signaling pathway regulation of gene expression decisions between PCs and CCs via permissive and instructive mechanisms.
Modes of regulation essential for the activation or repression of target genes are shown as solid green or red arrows, respectively, while those that are insufficient are shown as dashed arrows. (A, E) In cardial cells, PC genes such as Him with Notch-permissive PC enhancers are repressed by the Su(H)-co-repressor complex (A), while those with Notch-instructive PC enhancers like zfh1 are prevented from being transcribed by the absence of the required Nicd-Su(H) activator complex (E). The Delta ligand expressed by CCs activates the Notch receptor in neighboring PCs, with the resulting cleaved Nicd fragment associating with Su(H) to form a Nicd-Su(H) complex that outcompetes the Su(H)-co-repressor complex for Su(H) binding sites in the enhancers. For the Notch-permissive PC enhancers, the consequent displacement of repressor complex binding in PCs is sufficient to initiate transcription due to the presence of other local TF activators (A); the Notch-instructive PC enhancers are necessarily activated by the Nicd-Su(H) complex in PCs (E). (B, F) Mutating the Su(H) binding sites in either the Notch-permissive or the Notch-instructive PC enhancers prevents the Su(H)-co-repressor complex from binding to the enhancers in CCs and the Nicd-Su(H) activator complex from binding in PCs. For Notch-permissive PC enhancers, the resulting alleviation of repression is sufficient to ectopically transcribe the associated gene in CCs (B); however, it is not adequate for the Notch-instructive PC enhancers which require the binding of the Nicd-Su(H) activator complex to initiate transcription (F). The Su(H) site mutations in the Notch-instructive PC enhancers, however, also preclude the Nicd-Su(H) activator complex from binding to the enhancer in PCs and thus prevent the target gene from being transcribed there (F); despite the inability of the Nicd-Su(H) activator complex to bind, the absence of repression is sufficient to drive expression of target genes in PCs from the Notch-permissive PC enhancers (B). (C, G) Depleting the levels of the Su(H) TF prevents the Su(H)-co-repressor complex from forming and binding to the enhancers in CCs and the Nicd-Su(H) activator complex from forming and binding to the enhancers in PCs. The absence of the repressor complex is sufficient to initiate ectopic transcription of the associated gene for Notch-permissive PC enhancers in CCs (C), while the absence of the Nicd-Su(H) complex prevents transcription of the associated gene in both CCs and PCs for Notch-instructive PC enhancers (G). (D) Depleting the levels of a co-repressor prevents the Su(H)-co-repressor complex from forming and binding to the enhancers in CCs. The absence of the repressor complex thus initiates ectopic transcription of the associated gene for Notch-permissive PC enhancers in CCs. (H) Depleting the levels of Notch prevents the Nicd-Su(H) activator complex from forming and binding to the enhancers in PCs. The absence of the activator complex thus prevents transcription of the associated gene for Notch-instructive PC enhancers in both CCs and PCs.
Fig 8.
Model for the regulation of the zfh1 enhancer by a Notch-instructive mechanism in CCs, Odd-PCs, and Tin-PCs, and by a Notch-independent mechanism in Eve-PCs.
Modes of regulation essential for the transcriptional activation of zfh1 are shown as solid green arrows, while those that are insufficient are shown as dashed arrows. (A) For wild-type embryos, the absence of both the Nicd-Su(H) activator complex and the Eve-PC-specific TF binding to the zfh1 enhancer in cardial cells prevents zfh1 from being transcribed. The Delta ligand expressed by CCs activates Notch receptor in neighboring PCs, with the resulting cleaved Nicd fragment associating with Su(H) and outcompeting the co-repressor in Odd-PCs and Tin-PCs. In the Odd-PCs and Tin-PCs, the binding of the Nicd-Su(H) activator complex to the zfh1 enhancer initiates transcription. In the Eve-PCs, the binding of the Eve-PC-specific TF to the zfh1 enhancer is sufficient to activate transcription. (B) Mutating the Su(H) binding sites prevents the Nicd-Su(H) activator complex from binding to zfh1 enhancer in all CCs and PCs. In Odd-PCs and Tin-PCs which also lack the Eve-specific TF, the absence of binding of either of these activating complexes/TFs prevents zfh1 transcription. In contrast, in Eve-PCs, the Eve-PC-specific TF is present, binds to the zfh1 enhancer, and is sufficient to activate transcription. (C) Depleting the levels of the Su(H) TF prevents the Nicd-Su(H) activator complex from forming and binding to the enhancers in all PCs. In Odd-PCs and Tin-PCs, its absence and a lack of the Eve-PC-specific TF prevents zfh1 transcription. In Eve-PCs, the binding of the Eve-PC-specific TF to the zfh1 enhancer is again sufficient to initiate transcription. (D) Depleting the levels of Notch protein also prevents the Nicd-Su(H) activator complex from forming and binding to the enhancers in all PCs. As in (C), the absence of both the Nicd-Su(H) complex and the Eve-PC-specific TF prevents zfh1 transcription in Odd-PCs and Tin-PCs. But in Eve-PCs, where the Eve-PC-specific TF is present and able to bind to the zfh1 enhancer, transcription is activated.