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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 inhibits ISC proliferation and restricts the ISC overproliferation caused by ectopic JAK/STAT signaling.(A-C) Dl/Pros in MARCM control clones (A), MARCM clones of wdp1 cells (B) and MARCM clones of wdp1 cells with simultaneous Wdp expression (C). The overproliferation of ISC observed in wdp1 mutant MARCM clones (B) was rescued in the presence of transgenic wdp (C). (D) Quantification of clone size 6D ACI, including control, wdp1 mutant and wdp1 mutant while expressing transgenic wdp MARCM clones. Mean±SD are shown. n = 9 intestines. **p<0.01. (E-G) Brdu incorporation (Red, by Brdu) in MARCM control clones (E), wdp1 mutant clones (F) and wdp1 mutant clones with simultaneous Wdp expression (G). The increased Brdu incorporation in wdp1 mutant MARCM clones (F) was rescued with the simultaneous Wdp expression (G). (H) Quantification about the percentage of Brdu incorporation per clone with different genotypes. Mean±SD are shown. n = 6–9 intestines. **p<0.01. (I-P) Adult flies with control (I and M), wdp1 mutant (J and N) and wdp1 mutant while expressing transgenic Wdp (K and O) MARCM clones were treated with DSS at 29°C for 4 days. Under stress conditions, the number of Dl/Pros positive cells within wdp1 clones was also increased compared with controls (I and J). The overabundance of PH3 positive cells due to wdp depletion (M and N) was suppressed in the presence of transgenic wdp (O). (L) Quantification about the percentage of Dl+ and Pros+ cells per clone with indicated genotypes under DSS treatment. Mean±SD are shown. n = 8–10 intestines. (P) Quantification about the percentage of PH3+ cells per clone in intestines containing different MARCM clones under DSS treatment. Mean±SD are shown. n = 11–15 intestines. **p<0.01. (Q and R) The number of GFP+ cells was mildly increased upon knockdown of wdp using Su(H)GBE-lacZ; esgts driver (R) compared with controls (Q) at 29°C for 7 days. (S-V) Adult flies of esgts or esgts >wdp RNAi were treated with 3% DSS for 4 days at 29°C. Cross-section of midgut epithelium with the indicated genotypes was shown in T and V. Upon DSS treatment, the number of GFP+ clusters (S and U) as well as the thickness of midgut epithelium (T and V) from esgts >wdp RNAi midguts were significantly increased compared with controls. The intestinal lumen is indicated by white double-headed arrows and the intestinal wall by yellow brackets. (W-Z) The overproliferation of ISCs caused by upd expression (W and X) using the esgts driver was strikingly enhanced with simultaneous wdp knockdown (Y and Z). Cross-section of midgut epithelium with the indicated genotypes was shown in X and Z. (Z’) Quantification of midgut epithelium thickness (μm) with the indicated genotypes. Mean±SD are shown. n = 7–10 intestines. **p<0.01. Blue indicates DAPI staining. Scale bars, 20μm.
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pgen.1005180.g003: Wdp inhibits ISC proliferation and restricts the ISC overproliferation caused by ectopic JAK/STAT signaling.(A-C) Dl/Pros in MARCM control clones (A), MARCM clones of wdp1 cells (B) and MARCM clones of wdp1 cells with simultaneous Wdp expression (C). The overproliferation of ISC observed in wdp1 mutant MARCM clones (B) was rescued in the presence of transgenic wdp (C). (D) Quantification of clone size 6D ACI, including control, wdp1 mutant and wdp1 mutant while expressing transgenic wdp MARCM clones. Mean±SD are shown. n = 9 intestines. **p<0.01. (E-G) Brdu incorporation (Red, by Brdu) in MARCM control clones (E), wdp1 mutant clones (F) and wdp1 mutant clones with simultaneous Wdp expression (G). The increased Brdu incorporation in wdp1 mutant MARCM clones (F) was rescued with the simultaneous Wdp expression (G). (H) Quantification about the percentage of Brdu incorporation per clone with different genotypes. Mean±SD are shown. n = 6–9 intestines. **p<0.01. (I-P) Adult flies with control (I and M), wdp1 mutant (J and N) and wdp1 mutant while expressing transgenic Wdp (K and O) MARCM clones were treated with DSS at 29°C for 4 days. Under stress conditions, the number of Dl/Pros positive cells within wdp1 clones was also increased compared with controls (I and J). The overabundance of PH3 positive cells due to wdp depletion (M and N) was suppressed in the presence of transgenic wdp (O). (L) Quantification about the percentage of Dl+ and Pros+ cells per clone with indicated genotypes under DSS treatment. Mean±SD are shown. n = 8–10 intestines. (P) Quantification about the percentage of PH3+ cells per clone in intestines containing different MARCM clones under DSS treatment. Mean±SD are shown. n = 11–15 intestines. **p<0.01. (Q and R) The number of GFP+ cells was mildly increased upon knockdown of wdp using Su(H)GBE-lacZ; esgts driver (R) compared with controls (Q) at 29°C for 7 days. (S-V) Adult flies of esgts or esgts >wdp RNAi were treated with 3% DSS for 4 days at 29°C. Cross-section of midgut epithelium with the indicated genotypes was shown in T and V. Upon DSS treatment, the number of GFP+ clusters (S and U) as well as the thickness of midgut epithelium (T and V) from esgts >wdp RNAi midguts were significantly increased compared with controls. The intestinal lumen is indicated by white double-headed arrows and the intestinal wall by yellow brackets. (W-Z) The overproliferation of ISCs caused by upd expression (W and X) using the esgts driver was strikingly enhanced with simultaneous wdp knockdown (Y and Z). Cross-section of midgut epithelium with the indicated genotypes was shown in X and Z. (Z’) Quantification of midgut epithelium thickness (μm) with the indicated genotypes. Mean±SD are shown. n = 7–10 intestines. **p<0.01. Blue indicates DAPI staining. Scale bars, 20μm.

Mentions: We further examined whether Wdp is involved in regulating ISC activity. First, mosaic analysis with repressible cell marker (MARCM) approach was used to generate GFP positively marked clones for wdp1 mutants [55]. The control ISC clones contained an average of 7–8 cells per clone 6 days after clone induction (ACI) (Fig 3A, 3D and 3E). In contrast, the wdp1 mutant ISC clones contained up to 30 cells per clone 6d ACI (Fig 3B, 3D and 3F). Moreover, the number of Dl/Pros positive cells, which mark ISC/ee respectively, was increased in wdp1 mutant ISC clones compared with controls (Fig 3A and 3B). In addition, Brdu incorporation within wdp1 mutant clones was also enhanced (Fig 3E, 3F and 3H), suggesting that loss of Wdp led to the increased proliferation of ISCs.


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 inhibits ISC proliferation and restricts the ISC overproliferation caused by ectopic JAK/STAT signaling.(A-C) Dl/Pros in MARCM control clones (A), MARCM clones of wdp1 cells (B) and MARCM clones of wdp1 cells with simultaneous Wdp expression (C). The overproliferation of ISC observed in wdp1 mutant MARCM clones (B) was rescued in the presence of transgenic wdp (C). (D) Quantification of clone size 6D ACI, including control, wdp1 mutant and wdp1 mutant while expressing transgenic wdp MARCM clones. Mean±SD are shown. n = 9 intestines. **p<0.01. (E-G) Brdu incorporation (Red, by Brdu) in MARCM control clones (E), wdp1 mutant clones (F) and wdp1 mutant clones with simultaneous Wdp expression (G). The increased Brdu incorporation in wdp1 mutant MARCM clones (F) was rescued with the simultaneous Wdp expression (G). (H) Quantification about the percentage of Brdu incorporation per clone with different genotypes. Mean±SD are shown. n = 6–9 intestines. **p<0.01. (I-P) Adult flies with control (I and M), wdp1 mutant (J and N) and wdp1 mutant while expressing transgenic Wdp (K and O) MARCM clones were treated with DSS at 29°C for 4 days. Under stress conditions, the number of Dl/Pros positive cells within wdp1 clones was also increased compared with controls (I and J). The overabundance of PH3 positive cells due to wdp depletion (M and N) was suppressed in the presence of transgenic wdp (O). (L) Quantification about the percentage of Dl+ and Pros+ cells per clone with indicated genotypes under DSS treatment. Mean±SD are shown. n = 8–10 intestines. (P) Quantification about the percentage of PH3+ cells per clone in intestines containing different MARCM clones under DSS treatment. Mean±SD are shown. n = 11–15 intestines. **p<0.01. (Q and R) The number of GFP+ cells was mildly increased upon knockdown of wdp using Su(H)GBE-lacZ; esgts driver (R) compared with controls (Q) at 29°C for 7 days. (S-V) Adult flies of esgts or esgts >wdp RNAi were treated with 3% DSS for 4 days at 29°C. Cross-section of midgut epithelium with the indicated genotypes was shown in T and V. Upon DSS treatment, the number of GFP+ clusters (S and U) as well as the thickness of midgut epithelium (T and V) from esgts >wdp RNAi midguts were significantly increased compared with controls. The intestinal lumen is indicated by white double-headed arrows and the intestinal wall by yellow brackets. (W-Z) The overproliferation of ISCs caused by upd expression (W and X) using the esgts driver was strikingly enhanced with simultaneous wdp knockdown (Y and Z). Cross-section of midgut epithelium with the indicated genotypes was shown in X and Z. (Z’) Quantification of midgut epithelium thickness (μm) with the indicated genotypes. Mean±SD are shown. n = 7–10 intestines. **p<0.01. Blue indicates DAPI staining. Scale bars, 20μm.
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pgen.1005180.g003: Wdp inhibits ISC proliferation and restricts the ISC overproliferation caused by ectopic JAK/STAT signaling.(A-C) Dl/Pros in MARCM control clones (A), MARCM clones of wdp1 cells (B) and MARCM clones of wdp1 cells with simultaneous Wdp expression (C). The overproliferation of ISC observed in wdp1 mutant MARCM clones (B) was rescued in the presence of transgenic wdp (C). (D) Quantification of clone size 6D ACI, including control, wdp1 mutant and wdp1 mutant while expressing transgenic wdp MARCM clones. Mean±SD are shown. n = 9 intestines. **p<0.01. (E-G) Brdu incorporation (Red, by Brdu) in MARCM control clones (E), wdp1 mutant clones (F) and wdp1 mutant clones with simultaneous Wdp expression (G). The increased Brdu incorporation in wdp1 mutant MARCM clones (F) was rescued with the simultaneous Wdp expression (G). (H) Quantification about the percentage of Brdu incorporation per clone with different genotypes. Mean±SD are shown. n = 6–9 intestines. **p<0.01. (I-P) Adult flies with control (I and M), wdp1 mutant (J and N) and wdp1 mutant while expressing transgenic Wdp (K and O) MARCM clones were treated with DSS at 29°C for 4 days. Under stress conditions, the number of Dl/Pros positive cells within wdp1 clones was also increased compared with controls (I and J). The overabundance of PH3 positive cells due to wdp depletion (M and N) was suppressed in the presence of transgenic wdp (O). (L) Quantification about the percentage of Dl+ and Pros+ cells per clone with indicated genotypes under DSS treatment. Mean±SD are shown. n = 8–10 intestines. (P) Quantification about the percentage of PH3+ cells per clone in intestines containing different MARCM clones under DSS treatment. Mean±SD are shown. n = 11–15 intestines. **p<0.01. (Q and R) The number of GFP+ cells was mildly increased upon knockdown of wdp using Su(H)GBE-lacZ; esgts driver (R) compared with controls (Q) at 29°C for 7 days. (S-V) Adult flies of esgts or esgts >wdp RNAi were treated with 3% DSS for 4 days at 29°C. Cross-section of midgut epithelium with the indicated genotypes was shown in T and V. Upon DSS treatment, the number of GFP+ clusters (S and U) as well as the thickness of midgut epithelium (T and V) from esgts >wdp RNAi midguts were significantly increased compared with controls. The intestinal lumen is indicated by white double-headed arrows and the intestinal wall by yellow brackets. (W-Z) The overproliferation of ISCs caused by upd expression (W and X) using the esgts driver was strikingly enhanced with simultaneous wdp knockdown (Y and Z). Cross-section of midgut epithelium with the indicated genotypes was shown in X and Z. (Z’) Quantification of midgut epithelium thickness (μm) with the indicated genotypes. Mean±SD are shown. n = 7–10 intestines. **p<0.01. Blue indicates DAPI staining. Scale bars, 20μm.
Mentions: We further examined whether Wdp is involved in regulating ISC activity. First, mosaic analysis with repressible cell marker (MARCM) approach was used to generate GFP positively marked clones for wdp1 mutants [55]. The control ISC clones contained an average of 7–8 cells per clone 6 days after clone induction (ACI) (Fig 3A, 3D and 3E). In contrast, the wdp1 mutant ISC clones contained up to 30 cells per clone 6d ACI (Fig 3B, 3D and 3F). Moreover, the number of Dl/Pros positive cells, which mark ISC/ee respectively, was increased in wdp1 mutant ISC clones compared with controls (Fig 3A and 3B). In addition, Brdu incorporation within wdp1 mutant clones was also enhanced (Fig 3E, 3F and 3H), suggesting that loss of Wdp led to the increased proliferation of ISCs.

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