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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.


Yki-dependent regulation of Dilp8 adjusts organ growth and limits developmental instability.(a,c,e,g) JNK-dependent regulation of dilp8 expression is not affected in dilp8-PFΔ123 mutants. (b,d,f,h) represent higher magnification images of the area shown in the dashed lines. Wing discs were dissected from the indicated genotypes and stained for Dilp8. (i) Mutation of the three Sd-binding sites in the dilp8 promoter by gene editing induces FA. Bar histograms shows FA indices (FAi) of left and right wing areas measured on individuals of the indicated genotypes. ***P<0.0001, F-test for unequal distributions.
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f4: Yki-dependent regulation of Dilp8 adjusts organ growth and limits developmental instability.(a,c,e,g) JNK-dependent regulation of dilp8 expression is not affected in dilp8-PFΔ123 mutants. (b,d,f,h) represent higher magnification images of the area shown in the dashed lines. Wing discs were dissected from the indicated genotypes and stained for Dilp8. (i) Mutation of the three Sd-binding sites in the dilp8 promoter by gene editing induces FA. Bar histograms shows FA indices (FAi) of left and right wing areas measured on individuals of the indicated genotypes. ***P<0.0001, F-test for unequal distributions.

Mentions: We ultimately wanted to know whether the Yki-dependent regulation of Dilp8 function is central to the mechanism allowing organ growth coordination. To test the biological relevance of the HRE in the dilp8 promoter, we carried out gene editing using a combination of CRISPR-induced double-strand breaks and ends-out homologous recombination24 to replace the endogenous dilp8-PF with either dilp8-PFΔ123 or a control dilp8-PF at the dilp8 locus (see Supplementary Figs 2 and 3 and see the ‘Methods' section). As expected, Yki-dependent induction of dilp8 expression was efficiently suppressed in flies carrying mutations in the HRE (dilp8-PFΔ123; Fig. 4a–d). By contrast, JNK-dependent regulation of dilp8 expression was not compromised in this genetic background (Fig. 4e–h). We observed that dilp8-PFΔ123 flies exhibit increased levels of FA, although flies carrying a replacement with the control dilp8-PF do not (Fig. 4i, Supplementary Fig. 4). Finally, wing size distribution is neither modified in dilp8-PFΔ123 flies, nor in dilp8KO/KO flies compared with control animals (Supplementary Fig. 5). This suggests that the FA phenotype is not due to a disruption of a general size-control mechanism operating independently in each disc. Overall, our data demonstrate that Yki-dependent regulation of dilp8 expression plays a critical role in limiting developmental variability. It also contrasts with our observation that hep75 mutant flies do not exhibit elevated FA (Supplementary Fig. 6). These results are consistent with a central role of Yki in coordinating organ growth and limiting developmental variability through its effect on dilp8 expression.


The Hippo signalling pathway coordinates organ growth and limits developmental variability by controlling dilp8 expression
Yki-dependent regulation of Dilp8 adjusts organ growth and limits developmental instability.(a,c,e,g) JNK-dependent regulation of dilp8 expression is not affected in dilp8-PFΔ123 mutants. (b,d,f,h) represent higher magnification images of the area shown in the dashed lines. Wing discs were dissected from the indicated genotypes and stained for Dilp8. (i) Mutation of the three Sd-binding sites in the dilp8 promoter by gene editing induces FA. Bar histograms shows FA indices (FAi) of left and right wing areas measured on individuals of the indicated genotypes. ***P<0.0001, F-test for unequal distributions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121414&req=5

f4: Yki-dependent regulation of Dilp8 adjusts organ growth and limits developmental instability.(a,c,e,g) JNK-dependent regulation of dilp8 expression is not affected in dilp8-PFΔ123 mutants. (b,d,f,h) represent higher magnification images of the area shown in the dashed lines. Wing discs were dissected from the indicated genotypes and stained for Dilp8. (i) Mutation of the three Sd-binding sites in the dilp8 promoter by gene editing induces FA. Bar histograms shows FA indices (FAi) of left and right wing areas measured on individuals of the indicated genotypes. ***P<0.0001, F-test for unequal distributions.
Mentions: We ultimately wanted to know whether the Yki-dependent regulation of Dilp8 function is central to the mechanism allowing organ growth coordination. To test the biological relevance of the HRE in the dilp8 promoter, we carried out gene editing using a combination of CRISPR-induced double-strand breaks and ends-out homologous recombination24 to replace the endogenous dilp8-PF with either dilp8-PFΔ123 or a control dilp8-PF at the dilp8 locus (see Supplementary Figs 2 and 3 and see the ‘Methods' section). As expected, Yki-dependent induction of dilp8 expression was efficiently suppressed in flies carrying mutations in the HRE (dilp8-PFΔ123; Fig. 4a–d). By contrast, JNK-dependent regulation of dilp8 expression was not compromised in this genetic background (Fig. 4e–h). We observed that dilp8-PFΔ123 flies exhibit increased levels of FA, although flies carrying a replacement with the control dilp8-PF do not (Fig. 4i, Supplementary Fig. 4). Finally, wing size distribution is neither modified in dilp8-PFΔ123 flies, nor in dilp8KO/KO flies compared with control animals (Supplementary Fig. 5). This suggests that the FA phenotype is not due to a disruption of a general size-control mechanism operating independently in each disc. Overall, our data demonstrate that Yki-dependent regulation of dilp8 expression plays a critical role in limiting developmental variability. It also contrasts with our observation that hep75 mutant flies do not exhibit elevated FA (Supplementary Fig. 6). These results are consistent with a central role of Yki in coordinating organ growth and limiting developmental variability through its effect on 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.