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A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila.

Aikin R, Cervantes A, D'Angelo G, Ruel L, Lacas-Gervais S, Schaub S, Thérond P - PLoS ONE (2012)

Bottom Line: We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway.Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo.These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 7277, Inserm UMR 1091, Institut de Biologie Valrose (iBV), Centre de Biochimie, Nice, France.

ABSTRACT
Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

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In vivo validation of putative regulators of protein secretion using dsRNA transgenics.(A) Wg (green) and Hh (yellow) expression domains in the larval imaginal disc and the area patterned by Hh in the adult wing. (B) The wing phenotypes of adult flies expressing the indicated UAS-dsRNA under the control of en-Gal4. (C) Quantification of the wing intervein 3–4 domain. The intervein domain area for each wing was measured and normalized over total wing area. Results are shown as the mean percent reduction of the vein 3–4 domain relative to the control ± SD. “n” indicates the number of wings analyzed for each genotype.
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pone-0033665-g005: In vivo validation of putative regulators of protein secretion using dsRNA transgenics.(A) Wg (green) and Hh (yellow) expression domains in the larval imaginal disc and the area patterned by Hh in the adult wing. (B) The wing phenotypes of adult flies expressing the indicated UAS-dsRNA under the control of en-Gal4. (C) Quantification of the wing intervein 3–4 domain. The intervein domain area for each wing was measured and normalized over total wing area. Results are shown as the mean percent reduction of the vein 3–4 domain relative to the control ± SD. “n” indicates the number of wings analyzed for each genotype.

Mentions: To examine the role of these novel regulators of protein secretion in vivo, we employed transgenic Drosophila lines expressing particular RNA hairpins under the control of the UAS sequence, which allows for tissue-specific dsRNA expression. In the wing imaginal disc, the presumptive tissue of the adult wing, Hh is produced in the posterior compartment and patterns, after secretion, the anterior domain of the disc. This domain of the wing imaginal disc corresponds to the region between veins 3 and 4 of the adult wing (Figure 5A). Therefore, the area of the intervein space between veins 3 and 4 provides a readout of Hh activity. To assess the function of our novel candidates, en-Gal4 or hh-Gal4 drivers were used to direct the expression of the UAS-dsRNA specifically in Hh-producing cells (Figure 5A). The progeny were examined for reduction of the intervein space between veins 3–4 of the adult wing. As expected, the expression of a dsRNA against hh in the posterior compartment reduced the width of the Hh-patterned domain (Figure 5B and C). Using the same driver, the expression of dsRNA against our candidate genes caused a modest but significant reduction of this intervein area (Figure 5B and C, Figure S6, and Table S5) and no obvious accumulation of Hh level in posterior wing discs cells (data not shown). We also examined the possibility that overexpression of dsRNA might affect endogenous Hh protein levels by driving dsRNA expression in the dorsal compartment of the wing disc using apterous-Gal4 driver. Comparing Hh levels in the dorsal and ventral compartments demonstrated no decrease nor accumulation of Hh protein upon dsRNA expression against our candidates (Figure S7).


A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila.

Aikin R, Cervantes A, D'Angelo G, Ruel L, Lacas-Gervais S, Schaub S, Thérond P - PLoS ONE (2012)

In vivo validation of putative regulators of protein secretion using dsRNA transgenics.(A) Wg (green) and Hh (yellow) expression domains in the larval imaginal disc and the area patterned by Hh in the adult wing. (B) The wing phenotypes of adult flies expressing the indicated UAS-dsRNA under the control of en-Gal4. (C) Quantification of the wing intervein 3–4 domain. The intervein domain area for each wing was measured and normalized over total wing area. Results are shown as the mean percent reduction of the vein 3–4 domain relative to the control ± SD. “n” indicates the number of wings analyzed for each genotype.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303847&req=5

pone-0033665-g005: In vivo validation of putative regulators of protein secretion using dsRNA transgenics.(A) Wg (green) and Hh (yellow) expression domains in the larval imaginal disc and the area patterned by Hh in the adult wing. (B) The wing phenotypes of adult flies expressing the indicated UAS-dsRNA under the control of en-Gal4. (C) Quantification of the wing intervein 3–4 domain. The intervein domain area for each wing was measured and normalized over total wing area. Results are shown as the mean percent reduction of the vein 3–4 domain relative to the control ± SD. “n” indicates the number of wings analyzed for each genotype.
Mentions: To examine the role of these novel regulators of protein secretion in vivo, we employed transgenic Drosophila lines expressing particular RNA hairpins under the control of the UAS sequence, which allows for tissue-specific dsRNA expression. In the wing imaginal disc, the presumptive tissue of the adult wing, Hh is produced in the posterior compartment and patterns, after secretion, the anterior domain of the disc. This domain of the wing imaginal disc corresponds to the region between veins 3 and 4 of the adult wing (Figure 5A). Therefore, the area of the intervein space between veins 3 and 4 provides a readout of Hh activity. To assess the function of our novel candidates, en-Gal4 or hh-Gal4 drivers were used to direct the expression of the UAS-dsRNA specifically in Hh-producing cells (Figure 5A). The progeny were examined for reduction of the intervein space between veins 3–4 of the adult wing. As expected, the expression of a dsRNA against hh in the posterior compartment reduced the width of the Hh-patterned domain (Figure 5B and C). Using the same driver, the expression of dsRNA against our candidate genes caused a modest but significant reduction of this intervein area (Figure 5B and C, Figure S6, and Table S5) and no obvious accumulation of Hh level in posterior wing discs cells (data not shown). We also examined the possibility that overexpression of dsRNA might affect endogenous Hh protein levels by driving dsRNA expression in the dorsal compartment of the wing disc using apterous-Gal4 driver. Comparing Hh levels in the dorsal and ventral compartments demonstrated no decrease nor accumulation of Hh protein upon dsRNA expression against our candidates (Figure S7).

Bottom Line: We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway.Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo.These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR 7277, Inserm UMR 1091, Institut de Biologie Valrose (iBV), Centre de Biochimie, Nice, France.

ABSTRACT
Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

Show MeSH
Related in: MedlinePlus