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Windpipe controls Drosophila intestinal homeostasis by regulating JAK/STAT pathway via promoting receptor endocytosis and lysosomal degradation.

Ren W, Zhang Y, Li M, Wu L, Wang G, Baeg GH, You J, Li Z, Lin X - PLoS Genet. (2015)

Bottom Line: Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions.Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation.Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

No MeSH data available.


Related in: MedlinePlus

Wdp expression is positively regulated by JAK/STAT signaling in Drosophila intestines.(A) ChIP analysis was performed to monitor the binding of STAT92E to wdp genomic regions with STAT92E antibody using adult intestines expressing Upd and STAT92E under the esgts driver for 10 days at 29°C. The localization of four putative STAT92E-binding sites (BS1-4) is indicated by a black square frame. The square boxes with green, red or blue colors represent putative STAT92E binding sites localized in wdp genomic region with 2, 3 or 4 spacers respectively. (B) wdp mRNA expression was obviously increased in esgts>upd intestines at 29°C for 7 days using RT-qPCR quantification. Mean ± SD are shown. **p<0.01. (C) The relative activity of the indicated luciferase vectors, which contain different putative STAT92E binding sites (BS1-4) from wdp genomic regions, upon addition of Upd expressing cells. Mean ± SD are shown. *p<0.1, **p<0.01. (D and D’) Wdp (red, by Wdp) is ubiquitously expressed in both small progenitor cells and large nuclei ECs in control midguts at 29°C for 7 days. (E and E’) Wdp expression (red, by Wdp) was significantly increased around the GFP+ clusters (arrowheads) in esgts >upd, STAT midguts at 29°C for 7 days. (F and F’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down Dome at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. (G and G’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down STAT at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. Blue indicates DAPI staining. Scale bars, 20μm.
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pgen.1005180.g001: Wdp expression is positively regulated by JAK/STAT signaling in Drosophila intestines.(A) ChIP analysis was performed to monitor the binding of STAT92E to wdp genomic regions with STAT92E antibody using adult intestines expressing Upd and STAT92E under the esgts driver for 10 days at 29°C. The localization of four putative STAT92E-binding sites (BS1-4) is indicated by a black square frame. The square boxes with green, red or blue colors represent putative STAT92E binding sites localized in wdp genomic region with 2, 3 or 4 spacers respectively. (B) wdp mRNA expression was obviously increased in esgts>upd intestines at 29°C for 7 days using RT-qPCR quantification. Mean ± SD are shown. **p<0.01. (C) The relative activity of the indicated luciferase vectors, which contain different putative STAT92E binding sites (BS1-4) from wdp genomic regions, upon addition of Upd expressing cells. Mean ± SD are shown. *p<0.1, **p<0.01. (D and D’) Wdp (red, by Wdp) is ubiquitously expressed in both small progenitor cells and large nuclei ECs in control midguts at 29°C for 7 days. (E and E’) Wdp expression (red, by Wdp) was significantly increased around the GFP+ clusters (arrowheads) in esgts >upd, STAT midguts at 29°C for 7 days. (F and F’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down Dome at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. (G and G’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down STAT at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. Blue indicates DAPI staining. Scale bars, 20μm.

Mentions: From the candidate genes, wdp, a gene previously shown to be highly expressed in the developing trachea [47], was identified. Wdp was ranked in the top 10% through our bioinformatics analysis of ChIP results. At least 3 significant peaks (p<0.01) containing conserved STAT92E binding sites (TTCN3/4GAA) [15] were found in the 5’ UTR and genomic region of wdp (Fig 1A). Moreover, wdp mRNA levels as determined by RT-qPCR were increased in response to ectopic JAK/STAT signaling in esgts>upd intestines (Fig 1B). To further analyze the transcriptional regulation of wdp by JAK/STAT signaling, we identified four potential STAT92E binding sites (BS1-, BS2-, BS3- and BS4), and generated luciferase reporters that contain these potential binding sites. With the addition of Upd expressing S2 cells, the luciferase activity in cells transfected with BS2-, BS3- or BS4- luciferase constructs was obviously increased (Fig 1C). These results suggest that the expression of Wdp might be regulated by JAK/STAT signaling through BS2, BS3 and BS4 binding sites.


Windpipe controls Drosophila intestinal homeostasis by regulating JAK/STAT pathway via promoting receptor endocytosis and lysosomal degradation.

Ren W, Zhang Y, Li M, Wu L, Wang G, Baeg GH, You J, Li Z, Lin X - PLoS Genet. (2015)

Wdp expression is positively regulated by JAK/STAT signaling in Drosophila intestines.(A) ChIP analysis was performed to monitor the binding of STAT92E to wdp genomic regions with STAT92E antibody using adult intestines expressing Upd and STAT92E under the esgts driver for 10 days at 29°C. The localization of four putative STAT92E-binding sites (BS1-4) is indicated by a black square frame. The square boxes with green, red or blue colors represent putative STAT92E binding sites localized in wdp genomic region with 2, 3 or 4 spacers respectively. (B) wdp mRNA expression was obviously increased in esgts>upd intestines at 29°C for 7 days using RT-qPCR quantification. Mean ± SD are shown. **p<0.01. (C) The relative activity of the indicated luciferase vectors, which contain different putative STAT92E binding sites (BS1-4) from wdp genomic regions, upon addition of Upd expressing cells. Mean ± SD are shown. *p<0.1, **p<0.01. (D and D’) Wdp (red, by Wdp) is ubiquitously expressed in both small progenitor cells and large nuclei ECs in control midguts at 29°C for 7 days. (E and E’) Wdp expression (red, by Wdp) was significantly increased around the GFP+ clusters (arrowheads) in esgts >upd, STAT midguts at 29°C for 7 days. (F and F’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down Dome at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. (G and G’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down STAT at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. Blue indicates DAPI staining. Scale bars, 20μm.
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pgen.1005180.g001: Wdp expression is positively regulated by JAK/STAT signaling in Drosophila intestines.(A) ChIP analysis was performed to monitor the binding of STAT92E to wdp genomic regions with STAT92E antibody using adult intestines expressing Upd and STAT92E under the esgts driver for 10 days at 29°C. The localization of four putative STAT92E-binding sites (BS1-4) is indicated by a black square frame. The square boxes with green, red or blue colors represent putative STAT92E binding sites localized in wdp genomic region with 2, 3 or 4 spacers respectively. (B) wdp mRNA expression was obviously increased in esgts>upd intestines at 29°C for 7 days using RT-qPCR quantification. Mean ± SD are shown. **p<0.01. (C) The relative activity of the indicated luciferase vectors, which contain different putative STAT92E binding sites (BS1-4) from wdp genomic regions, upon addition of Upd expressing cells. Mean ± SD are shown. *p<0.1, **p<0.01. (D and D’) Wdp (red, by Wdp) is ubiquitously expressed in both small progenitor cells and large nuclei ECs in control midguts at 29°C for 7 days. (E and E’) Wdp expression (red, by Wdp) was significantly increased around the GFP+ clusters (arrowheads) in esgts >upd, STAT midguts at 29°C for 7 days. (F and F’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down Dome at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. (G and G’) Wdp expression (red, by Wdp) was reduced in the Flip-out clones (arrowheads) knocking down STAT at 29°C for 7 days. Square box is the enlarged image of the position labeled by yellow arrowhead. Blue indicates DAPI staining. Scale bars, 20μm.
Mentions: From the candidate genes, wdp, a gene previously shown to be highly expressed in the developing trachea [47], was identified. Wdp was ranked in the top 10% through our bioinformatics analysis of ChIP results. At least 3 significant peaks (p<0.01) containing conserved STAT92E binding sites (TTCN3/4GAA) [15] were found in the 5’ UTR and genomic region of wdp (Fig 1A). Moreover, wdp mRNA levels as determined by RT-qPCR were increased in response to ectopic JAK/STAT signaling in esgts>upd intestines (Fig 1B). To further analyze the transcriptional regulation of wdp by JAK/STAT signaling, we identified four potential STAT92E binding sites (BS1-, BS2-, BS3- and BS4), and generated luciferase reporters that contain these potential binding sites. With the addition of Upd expressing S2 cells, the luciferase activity in cells transfected with BS2-, BS3- or BS4- luciferase constructs was obviously increased (Fig 1C). These results suggest that the expression of Wdp might be regulated by JAK/STAT signaling through BS2, BS3 and BS4 binding sites.

Bottom Line: Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions.Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation.Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.

No MeSH data available.


Related in: MedlinePlus