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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 functions downstream of Upd but upstream of Hop.(A and A’) The activity and the expression regions of 10×STAT GFP were noticeably enhanced (arrow in A’) when hop3w was ectopically expressed using mirrorGal4 in the dorsal compartment marked by CD8-mRFP of early 3rd instar larva eye discs. D/V boundary is shown by the dotted line. All the eye discs shown here are oriented dorsal up, posterior right. (B and B’) Simultaneous expression of wdp was unable to suppress the increased 10×STAT GFP activity due to the hop3w overexpression in the dorsal compartment (arrow in B’). (C) Quantification about the ratio of 10×STAT GFP expression region (along the A-P axis) between dorsal and ventral part in early 3rd instar eye discs with indicated genotypes. Mean±SD are shown. n = 9–10 discs. (D-D’) Ectopic expression of hop3w using the esgts driver promotes ISC proliferation at 29°C for 7 days. (E-E’) Simultaneous expression of wdp cannot suppress overproliferation of ISC caused by ectopic hop3w expression using the esgts driver at 29°C for 7 days. (F) Quantification of the relative number of esgts>GFP cells with indicated genotypes. Mean±SD are shown. n = 12–15 intestines. (G) The increased activity of 10×STAT luciferase due to Upd expression can be suppressed by cotransfection of UAS-wdp. However, wdp overexpression can’t block the constitutive activation of JAK/STAT signaling caused by hop. The increased 10×STAT luciferase activity resulting from cotransfection of UAS-upd, dome and hop was obviously suppressed in the presence of wdp. Mean±SD are shown. *p<0.1, **p<0.01. (H) S2 cells transfected with UAS-dome-V5 alone or along with wdp were lysed and assessed for total Dome levels using V5 antibody. Blue indicates DAPI staining in D and E. Scale bars, 20μm.
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pgen.1005180.g005: Wdp functions downstream of Upd but upstream of Hop.(A and A’) The activity and the expression regions of 10×STAT GFP were noticeably enhanced (arrow in A’) when hop3w was ectopically expressed using mirrorGal4 in the dorsal compartment marked by CD8-mRFP of early 3rd instar larva eye discs. D/V boundary is shown by the dotted line. All the eye discs shown here are oriented dorsal up, posterior right. (B and B’) Simultaneous expression of wdp was unable to suppress the increased 10×STAT GFP activity due to the hop3w overexpression in the dorsal compartment (arrow in B’). (C) Quantification about the ratio of 10×STAT GFP expression region (along the A-P axis) between dorsal and ventral part in early 3rd instar eye discs with indicated genotypes. Mean±SD are shown. n = 9–10 discs. (D-D’) Ectopic expression of hop3w using the esgts driver promotes ISC proliferation at 29°C for 7 days. (E-E’) Simultaneous expression of wdp cannot suppress overproliferation of ISC caused by ectopic hop3w expression using the esgts driver at 29°C for 7 days. (F) Quantification of the relative number of esgts>GFP cells with indicated genotypes. Mean±SD are shown. n = 12–15 intestines. (G) The increased activity of 10×STAT luciferase due to Upd expression can be suppressed by cotransfection of UAS-wdp. However, wdp overexpression can’t block the constitutive activation of JAK/STAT signaling caused by hop. The increased 10×STAT luciferase activity resulting from cotransfection of UAS-upd, dome and hop was obviously suppressed in the presence of wdp. Mean±SD are shown. *p<0.1, **p<0.01. (H) S2 cells transfected with UAS-dome-V5 alone or along with wdp were lysed and assessed for total Dome levels using V5 antibody. Blue indicates DAPI staining in D and E. Scale bars, 20μm.

Mentions: To determine the level at which Wdp modulates JAK/STAT signaling, we examined the epistatic relationship between Wdp and the JAK/STAT pathway components. When hop was ectopically expressed using mirrorGal4 in eye discs, we observed increased JAK/STAT activity, as determined by an expanded expression region of 10×STAT GFP in the dorsal compartment which is marked by CD8-mRFP (Fig 5A and 5A’, arrow). Simultaneous expression of wdp failed to suppress the elevated JAK/STAT signaling caused by hop overexpression (Fig 5B, 5B’ and 5C), suggesting that Wdp acts upstream of Hop. Consistent with previous study [28], ectopic expression of Hop in midguts using the esgts driver led to weak expansion of esg positive cells (Fig 5D and 5D’). ISC over-proliferation caused by hop expression was not affected in the presence of wdp (Fig 5E, 5E’ and 5F). Thus, these epistatic experiments performed in both the midguts and eye discs placed Wdp upstream of Hop.


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 functions downstream of Upd but upstream of Hop.(A and A’) The activity and the expression regions of 10×STAT GFP were noticeably enhanced (arrow in A’) when hop3w was ectopically expressed using mirrorGal4 in the dorsal compartment marked by CD8-mRFP of early 3rd instar larva eye discs. D/V boundary is shown by the dotted line. All the eye discs shown here are oriented dorsal up, posterior right. (B and B’) Simultaneous expression of wdp was unable to suppress the increased 10×STAT GFP activity due to the hop3w overexpression in the dorsal compartment (arrow in B’). (C) Quantification about the ratio of 10×STAT GFP expression region (along the A-P axis) between dorsal and ventral part in early 3rd instar eye discs with indicated genotypes. Mean±SD are shown. n = 9–10 discs. (D-D’) Ectopic expression of hop3w using the esgts driver promotes ISC proliferation at 29°C for 7 days. (E-E’) Simultaneous expression of wdp cannot suppress overproliferation of ISC caused by ectopic hop3w expression using the esgts driver at 29°C for 7 days. (F) Quantification of the relative number of esgts>GFP cells with indicated genotypes. Mean±SD are shown. n = 12–15 intestines. (G) The increased activity of 10×STAT luciferase due to Upd expression can be suppressed by cotransfection of UAS-wdp. However, wdp overexpression can’t block the constitutive activation of JAK/STAT signaling caused by hop. The increased 10×STAT luciferase activity resulting from cotransfection of UAS-upd, dome and hop was obviously suppressed in the presence of wdp. Mean±SD are shown. *p<0.1, **p<0.01. (H) S2 cells transfected with UAS-dome-V5 alone or along with wdp were lysed and assessed for total Dome levels using V5 antibody. Blue indicates DAPI staining in D and E. Scale bars, 20μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414558&req=5

pgen.1005180.g005: Wdp functions downstream of Upd but upstream of Hop.(A and A’) The activity and the expression regions of 10×STAT GFP were noticeably enhanced (arrow in A’) when hop3w was ectopically expressed using mirrorGal4 in the dorsal compartment marked by CD8-mRFP of early 3rd instar larva eye discs. D/V boundary is shown by the dotted line. All the eye discs shown here are oriented dorsal up, posterior right. (B and B’) Simultaneous expression of wdp was unable to suppress the increased 10×STAT GFP activity due to the hop3w overexpression in the dorsal compartment (arrow in B’). (C) Quantification about the ratio of 10×STAT GFP expression region (along the A-P axis) between dorsal and ventral part in early 3rd instar eye discs with indicated genotypes. Mean±SD are shown. n = 9–10 discs. (D-D’) Ectopic expression of hop3w using the esgts driver promotes ISC proliferation at 29°C for 7 days. (E-E’) Simultaneous expression of wdp cannot suppress overproliferation of ISC caused by ectopic hop3w expression using the esgts driver at 29°C for 7 days. (F) Quantification of the relative number of esgts>GFP cells with indicated genotypes. Mean±SD are shown. n = 12–15 intestines. (G) The increased activity of 10×STAT luciferase due to Upd expression can be suppressed by cotransfection of UAS-wdp. However, wdp overexpression can’t block the constitutive activation of JAK/STAT signaling caused by hop. The increased 10×STAT luciferase activity resulting from cotransfection of UAS-upd, dome and hop was obviously suppressed in the presence of wdp. Mean±SD are shown. *p<0.1, **p<0.01. (H) S2 cells transfected with UAS-dome-V5 alone or along with wdp were lysed and assessed for total Dome levels using V5 antibody. Blue indicates DAPI staining in D and E. Scale bars, 20μm.
Mentions: To determine the level at which Wdp modulates JAK/STAT signaling, we examined the epistatic relationship between Wdp and the JAK/STAT pathway components. When hop was ectopically expressed using mirrorGal4 in eye discs, we observed increased JAK/STAT activity, as determined by an expanded expression region of 10×STAT GFP in the dorsal compartment which is marked by CD8-mRFP (Fig 5A and 5A’, arrow). Simultaneous expression of wdp failed to suppress the elevated JAK/STAT signaling caused by hop overexpression (Fig 5B, 5B’ and 5C), suggesting that Wdp acts upstream of Hop. Consistent with previous study [28], ectopic expression of Hop in midguts using the esgts driver led to weak expansion of esg positive cells (Fig 5D and 5D’). ISC over-proliferation caused by hop expression was not affected in the presence of wdp (Fig 5E, 5E’ and 5F). Thus, these epistatic experiments performed in both the midguts and eye discs placed Wdp upstream of Hop.

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