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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 regulates dilp8 transcription through Sd.Wing imaginal disc carrying RFP-labelled yki-expressing clones (a–d), GFP-labelled wt clones (e,f), GFP-labelled yki-expressing clones (g,h), GFP-labelled yki+sd RNAi clones (i,j), or GFP-negative clones mutant for ex (k,l), hpo (m,n) or wts (o,p), all dissected 116 h AED and stained for Dilp8 (c,f,h,j,l,n,p). In b, dilp8 expression is followed by GFP using the dilp8-GFP insertion allele (see the ‘Genotypes' section under the ‘Methods' section).
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f2: Yki regulates dilp8 transcription through Sd.Wing imaginal disc carrying RFP-labelled yki-expressing clones (a–d), GFP-labelled wt clones (e,f), GFP-labelled yki-expressing clones (g,h), GFP-labelled yki+sd RNAi clones (i,j), or GFP-negative clones mutant for ex (k,l), hpo (m,n) or wts (o,p), all dissected 116 h AED and stained for Dilp8 (c,f,h,j,l,n,p). In b, dilp8 expression is followed by GFP using the dilp8-GFP insertion allele (see the ‘Genotypes' section under the ‘Methods' section).

Mentions: In addition to JNK signalling, we identified the Hpo pathway as an important regulator of dilp8 expression. The Hpo pathway is an important regulator of organ growth and is thought to play a central role in organ size assessment1314. The core kinase module of the Hpo pathway includes the Hpo (Mst1/2 in humans) and Warts/Lats kinases, which suppress activation of the transcriptional co-activator Yorkie (Yki; YAP/TAZ in humans). When the Hpo pathway is inactive, Yki and its DNA-binding partner Scalloped (Sd) activate target genes and promote organ growth151617181920. We observed that reducing levels of the transcriptional co-activators Yki or Sd efficiently rescues the developmental delay in rn>avl RNAi animals and normalizes dilp8 transcript levels (Fig. 1a,b). Given the substantial evidence that crosstalk takes place between the Hpo and JNK signalling pathways2122, we tested whether Yki can regulate dilp8 expression independently of JNK signalling. Indeed, we found that dilp8 expression is still significantly upregulated by Yki overexpression in flies that are mutant for the JNK kinase Hemipterous (Hep; Fig. 1c–i). We next tested whether overexpression of yki is sufficient to activate dilp8 transcription. Using an enhanced green fluorescent protein trap inserted in the first intron of the dilp8 gene as a reporter for native dilp8 expression, we could observe increased levels of enhanced green fluorescent protein in yki-overexpressing clones (Fig. 2a,b). Consistent with this, Dilp8 protein levels were also elevated in these clones (Fig. 2c,d). In agreement with Yki-regulating gene expression through association with its co-activator Sd, we found that reducing Sd levels in Yki-overexpressing clones abolished the Yki-dependent upregulation of Dilp8 (Fig. 2e–j). We next examined dilp8 levels in clones carrying mutations in genes encoding upstream components of the Hpo pathway including expanded (ex), hpo and warts (wts). As expected, reducing the activity of upstream Hpo pathway members, which induces Yki activation, also increases Dilp8 protein levels (Fig. 2k–p).


The Hippo signalling pathway coordinates organ growth and limits developmental variability by controlling dilp8 expression
Yki regulates dilp8 transcription through Sd.Wing imaginal disc carrying RFP-labelled yki-expressing clones (a–d), GFP-labelled wt clones (e,f), GFP-labelled yki-expressing clones (g,h), GFP-labelled yki+sd RNAi clones (i,j), or GFP-negative clones mutant for ex (k,l), hpo (m,n) or wts (o,p), all dissected 116 h AED and stained for Dilp8 (c,f,h,j,l,n,p). In b, dilp8 expression is followed by GFP using the dilp8-GFP insertion allele (see the ‘Genotypes' section under the ‘Methods' section).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Yki regulates dilp8 transcription through Sd.Wing imaginal disc carrying RFP-labelled yki-expressing clones (a–d), GFP-labelled wt clones (e,f), GFP-labelled yki-expressing clones (g,h), GFP-labelled yki+sd RNAi clones (i,j), or GFP-negative clones mutant for ex (k,l), hpo (m,n) or wts (o,p), all dissected 116 h AED and stained for Dilp8 (c,f,h,j,l,n,p). In b, dilp8 expression is followed by GFP using the dilp8-GFP insertion allele (see the ‘Genotypes' section under the ‘Methods' section).
Mentions: In addition to JNK signalling, we identified the Hpo pathway as an important regulator of dilp8 expression. The Hpo pathway is an important regulator of organ growth and is thought to play a central role in organ size assessment1314. The core kinase module of the Hpo pathway includes the Hpo (Mst1/2 in humans) and Warts/Lats kinases, which suppress activation of the transcriptional co-activator Yorkie (Yki; YAP/TAZ in humans). When the Hpo pathway is inactive, Yki and its DNA-binding partner Scalloped (Sd) activate target genes and promote organ growth151617181920. We observed that reducing levels of the transcriptional co-activators Yki or Sd efficiently rescues the developmental delay in rn>avl RNAi animals and normalizes dilp8 transcript levels (Fig. 1a,b). Given the substantial evidence that crosstalk takes place between the Hpo and JNK signalling pathways2122, we tested whether Yki can regulate dilp8 expression independently of JNK signalling. Indeed, we found that dilp8 expression is still significantly upregulated by Yki overexpression in flies that are mutant for the JNK kinase Hemipterous (Hep; Fig. 1c–i). We next tested whether overexpression of yki is sufficient to activate dilp8 transcription. Using an enhanced green fluorescent protein trap inserted in the first intron of the dilp8 gene as a reporter for native dilp8 expression, we could observe increased levels of enhanced green fluorescent protein in yki-overexpressing clones (Fig. 2a,b). Consistent with this, Dilp8 protein levels were also elevated in these clones (Fig. 2c,d). In agreement with Yki-regulating gene expression through association with its co-activator Sd, we found that reducing Sd levels in Yki-overexpressing clones abolished the Yki-dependent upregulation of Dilp8 (Fig. 2e–j). We next examined dilp8 levels in clones carrying mutations in genes encoding upstream components of the Hpo pathway including expanded (ex), hpo and warts (wts). As expected, reducing the activity of upstream Hpo pathway members, which induces Yki activation, also increases Dilp8 protein levels (Fig. 2k–p).

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.