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The Hippo signalling pathway coordinates organ growth and limits developmental variability by controlling dilp8 expression

View Article: PubMed Central - PubMed

ABSTRACT

Coordination of organ growth during development is required to generate fit individuals with fixed proportions. We recently identified Drosophila Dilp8 as a key hormone in coupling organ growth with animal maturation. In addition, dilp8 mutant flies exhibit elevated fluctuating asymmetry (FA) demonstrating a function for Dilp8 in ensuring developmental stability. The signals regulating Dilp8 activity during normal development are not yet known. Here, we show that the transcriptional co-activators of the Hippo (Hpo) pathway, Yorkie (Yki, YAP/TAZ) and its DNA-binding partner Scalloped (Sd), directly regulate dilp8 expression through a Hpo-responsive element (HRE) in the dilp8 promoter. We further demonstrate that mutation of the HRE by genome-editing results in animals with increased FA, thereby mimicking full dilp8 loss of function. Therefore, our results indicate that growth coordination of organs is connected to their growth status through a feedback loop involving Hpo and Dilp8 signalling pathways.

No MeSH data available.


Related in: MedlinePlus

Yki directly regulates dilp8 expression through a HRE in the dilp8 promoter.(a) Schematic of the dilp8 promoter region and the HRE harbouring the three putative Sd-binding sites (indicated as green squares). The dilp8 promoter fragments used to study Yki-dependent regulation of dilp8 expression in vivo are shown. In dilp8-PFΔ123, mutations of the three putative Sd-binding sites are indicated as red squares. (b) DNA pull-down experiments show that binding of Sd to the dilp8-PF is mediated by the three Sd-binding sites. The indicated DNA fragments were incubated with lysates from S2 cells transfected with Sd-Flag. Band intensities represents the average of three independent experiments: for diap-PF: 3.8±0.7 (positive control), for act: 1±0.2 (negative control), for dilp8-PF: 5.1±1.0, and for dilp8-PF Δ123: 1.9±0.4. (c) Luciferase assay showing that Yki/Sd activate gene expression through the HRE in the dilp8-PF. S2 cells were transfected with Yki and Sd. The ability of Yki/Sd to induce gene expression from the indicated promoter fragments was measured (triplicate samples, error bars represent s.e.m.). (d–s) Yki induces dilp8 transcription through the HRE in vivo. Wing imaginal discs carrying GFP-labelled Yki-expressing clones were dissected from transgenic flies carrying the indicated dilp8 promoter fragments fused to the LacZ-encoding sequence. The full dilp8 promoter and dilp8-PF, but not dilp8-intron1 and dilp8-PFΔ123, induces lacZ expression as detected by β-gal staining (d,h,l,p in red) in the GFP-labelled yki-overexpressing clones (e,i,m,n in green). In each condition, yki overexpression leads to elevated levels of endogenous Dilp8 protein (f,j,n,r in white).
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f3: Yki directly regulates dilp8 expression through a HRE in the dilp8 promoter.(a) Schematic of the dilp8 promoter region and the HRE harbouring the three putative Sd-binding sites (indicated as green squares). The dilp8 promoter fragments used to study Yki-dependent regulation of dilp8 expression in vivo are shown. In dilp8-PFΔ123, mutations of the three putative Sd-binding sites are indicated as red squares. (b) DNA pull-down experiments show that binding of Sd to the dilp8-PF is mediated by the three Sd-binding sites. The indicated DNA fragments were incubated with lysates from S2 cells transfected with Sd-Flag. Band intensities represents the average of three independent experiments: for diap-PF: 3.8±0.7 (positive control), for act: 1±0.2 (negative control), for dilp8-PF: 5.1±1.0, and for dilp8-PF Δ123: 1.9±0.4. (c) Luciferase assay showing that Yki/Sd activate gene expression through the HRE in the dilp8-PF. S2 cells were transfected with Yki and Sd. The ability of Yki/Sd to induce gene expression from the indicated promoter fragments was measured (triplicate samples, error bars represent s.e.m.). (d–s) Yki induces dilp8 transcription through the HRE in vivo. Wing imaginal discs carrying GFP-labelled Yki-expressing clones were dissected from transgenic flies carrying the indicated dilp8 promoter fragments fused to the LacZ-encoding sequence. The full dilp8 promoter and dilp8-PF, but not dilp8-intron1 and dilp8-PFΔ123, induces lacZ expression as detected by β-gal staining (d,h,l,p in red) in the GFP-labelled yki-overexpressing clones (e,i,m,n in green). In each condition, yki overexpression leads to elevated levels of endogenous Dilp8 protein (f,j,n,r in white).

Mentions: Genome-wide CHIPseq analyses using anti-Yki antibodies recently identified a number of potential Yki target genes1723. Interestingly, these CHIPseq data identified a 600-bp promoter fragment localized 1.5 kb upstream of the coding region in the dilp8 locus. Close examination of this fragment reveals three potential Sd-binding sites (hereafter referred to as Hpo-responsive element (HRE))23, suggesting that dilp8 expression might be directly activated by a Yki/Sd heterodimer (see region map in Fig. 3a). To directly test this, we performed DNA pull-down assays by mixing lysates from cells expressing tagged Sd (Sd-Flag) and a 600-bp DNA fragment of the dilp8 promoter region centred around the HRE (dilp8 promoter fragment: dilp8-PF). A region in the diap promoter that is known to bind Sd (diap-PF) was used as positive control. We found that Sd-Flag binds the dilp8-PF and diap-PF with similar efficiency (Fig. 3b). Moreover, the binding of Sd-Flag to the dilp8-PF is abolished on targeted mutation of the three putative Sd-binding sites (dilp8-PFΔ123; Fig. 3b, Supplementary Fig. 1 and see the ‘Methods' section). To test the functional relevance of this binding assay, we analysed the potential of each of these fragments to promote transcription of the luciferase reporter gene in the presence of Sd and Yki. Consistent with the DNA pull-down result, both diap-PF and dilp8-PF, but not dilp8-PFΔ123, were able to activate transcription in the presence of Yki and Sd (Fig. 3c). To study the transcriptional regulation of dilp8 by Yki in vivo, we next generated transgenic fly lines carrying constructs harbouring either the full dilp8 promoter (dilp8-full-prom), dilp8-PF, dilp8-PFΔ123 or intron 1 of the dilp8 gene used as a negative control, all fused to the lacZ coding sequence (Fig. 3a). We found that both dilp8-full-prom and dilp8-PF were able to promote lacZ expression in Yki-overexpressing clones (Fig. 3d–o). Moreover, the ability of Yki to induce lacZ expression depends on the integrity of the HRE, since mutation of the three Sd-binding sites prevented all lacZ expression (Fig. 3p–s). Altogether these results are consistent with Yki-activating dilp8 gene expression through Sd bound to the HRE in the dilp8 promoter.


The Hippo signalling pathway coordinates organ growth and limits developmental variability by controlling dilp8 expression
Yki directly regulates dilp8 expression through a HRE in the dilp8 promoter.(a) Schematic of the dilp8 promoter region and the HRE harbouring the three putative Sd-binding sites (indicated as green squares). The dilp8 promoter fragments used to study Yki-dependent regulation of dilp8 expression in vivo are shown. In dilp8-PFΔ123, mutations of the three putative Sd-binding sites are indicated as red squares. (b) DNA pull-down experiments show that binding of Sd to the dilp8-PF is mediated by the three Sd-binding sites. The indicated DNA fragments were incubated with lysates from S2 cells transfected with Sd-Flag. Band intensities represents the average of three independent experiments: for diap-PF: 3.8±0.7 (positive control), for act: 1±0.2 (negative control), for dilp8-PF: 5.1±1.0, and for dilp8-PF Δ123: 1.9±0.4. (c) Luciferase assay showing that Yki/Sd activate gene expression through the HRE in the dilp8-PF. S2 cells were transfected with Yki and Sd. The ability of Yki/Sd to induce gene expression from the indicated promoter fragments was measured (triplicate samples, error bars represent s.e.m.). (d–s) Yki induces dilp8 transcription through the HRE in vivo. Wing imaginal discs carrying GFP-labelled Yki-expressing clones were dissected from transgenic flies carrying the indicated dilp8 promoter fragments fused to the LacZ-encoding sequence. The full dilp8 promoter and dilp8-PF, but not dilp8-intron1 and dilp8-PFΔ123, induces lacZ expression as detected by β-gal staining (d,h,l,p in red) in the GFP-labelled yki-overexpressing clones (e,i,m,n in green). In each condition, yki overexpression leads to elevated levels of endogenous Dilp8 protein (f,j,n,r in white).
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f3: Yki directly regulates dilp8 expression through a HRE in the dilp8 promoter.(a) Schematic of the dilp8 promoter region and the HRE harbouring the three putative Sd-binding sites (indicated as green squares). The dilp8 promoter fragments used to study Yki-dependent regulation of dilp8 expression in vivo are shown. In dilp8-PFΔ123, mutations of the three putative Sd-binding sites are indicated as red squares. (b) DNA pull-down experiments show that binding of Sd to the dilp8-PF is mediated by the three Sd-binding sites. The indicated DNA fragments were incubated with lysates from S2 cells transfected with Sd-Flag. Band intensities represents the average of three independent experiments: for diap-PF: 3.8±0.7 (positive control), for act: 1±0.2 (negative control), for dilp8-PF: 5.1±1.0, and for dilp8-PF Δ123: 1.9±0.4. (c) Luciferase assay showing that Yki/Sd activate gene expression through the HRE in the dilp8-PF. S2 cells were transfected with Yki and Sd. The ability of Yki/Sd to induce gene expression from the indicated promoter fragments was measured (triplicate samples, error bars represent s.e.m.). (d–s) Yki induces dilp8 transcription through the HRE in vivo. Wing imaginal discs carrying GFP-labelled Yki-expressing clones were dissected from transgenic flies carrying the indicated dilp8 promoter fragments fused to the LacZ-encoding sequence. The full dilp8 promoter and dilp8-PF, but not dilp8-intron1 and dilp8-PFΔ123, induces lacZ expression as detected by β-gal staining (d,h,l,p in red) in the GFP-labelled yki-overexpressing clones (e,i,m,n in green). In each condition, yki overexpression leads to elevated levels of endogenous Dilp8 protein (f,j,n,r in white).
Mentions: Genome-wide CHIPseq analyses using anti-Yki antibodies recently identified a number of potential Yki target genes1723. Interestingly, these CHIPseq data identified a 600-bp promoter fragment localized 1.5 kb upstream of the coding region in the dilp8 locus. Close examination of this fragment reveals three potential Sd-binding sites (hereafter referred to as Hpo-responsive element (HRE))23, suggesting that dilp8 expression might be directly activated by a Yki/Sd heterodimer (see region map in Fig. 3a). To directly test this, we performed DNA pull-down assays by mixing lysates from cells expressing tagged Sd (Sd-Flag) and a 600-bp DNA fragment of the dilp8 promoter region centred around the HRE (dilp8 promoter fragment: dilp8-PF). A region in the diap promoter that is known to bind Sd (diap-PF) was used as positive control. We found that Sd-Flag binds the dilp8-PF and diap-PF with similar efficiency (Fig. 3b). Moreover, the binding of Sd-Flag to the dilp8-PF is abolished on targeted mutation of the three putative Sd-binding sites (dilp8-PFΔ123; Fig. 3b, Supplementary Fig. 1 and see the ‘Methods' section). To test the functional relevance of this binding assay, we analysed the potential of each of these fragments to promote transcription of the luciferase reporter gene in the presence of Sd and Yki. Consistent with the DNA pull-down result, both diap-PF and dilp8-PF, but not dilp8-PFΔ123, were able to activate transcription in the presence of Yki and Sd (Fig. 3c). To study the transcriptional regulation of dilp8 by Yki in vivo, we next generated transgenic fly lines carrying constructs harbouring either the full dilp8 promoter (dilp8-full-prom), dilp8-PF, dilp8-PFΔ123 or intron 1 of the dilp8 gene used as a negative control, all fused to the lacZ coding sequence (Fig. 3a). We found that both dilp8-full-prom and dilp8-PF were able to promote lacZ expression in Yki-overexpressing clones (Fig. 3d–o). Moreover, the ability of Yki to induce lacZ expression depends on the integrity of the HRE, since mutation of the three Sd-binding sites prevented all lacZ expression (Fig. 3p–s). Altogether these results are consistent with Yki-activating dilp8 gene expression through Sd bound to the HRE in the dilp8 promoter.

View Article: PubMed Central - PubMed

ABSTRACT

Coordination of organ growth during development is required to generate fit individuals with fixed proportions. We recently identified Drosophila Dilp8 as a key hormone in coupling organ growth with animal maturation. In addition, dilp8 mutant flies exhibit elevated fluctuating asymmetry (FA) demonstrating a function for Dilp8 in ensuring developmental stability. The signals regulating Dilp8 activity during normal development are not yet known. Here, we show that the transcriptional co-activators of the Hippo (Hpo) pathway, Yorkie (Yki, YAP/TAZ) and its DNA-binding partner Scalloped (Sd), directly regulate dilp8 expression through a Hpo-responsive element (HRE) in the dilp8 promoter. We further demonstrate that mutation of the HRE by genome-editing results in animals with increased FA, thereby mimicking full dilp8 loss of function. Therefore, our results indicate that growth coordination of organs is connected to their growth status through a feedback loop involving Hpo and Dilp8 signalling pathways.

No MeSH data available.


Related in: MedlinePlus