Loss of the mucosal barrier alters the progenitor cell niche via Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling

Abstract

The mucous barrier of our digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases such as Crohn's disease, colitis, and colon cancer, but mechanistic insights into these processes and diseases are limited. We have previously shown that loss of a conserved O-glycosyltransferase (PGANT4) in Drosophila results in aberrant secretion of components of the peritrophic/mucous membrane in the larval digestive tract. Here, we show that loss of PGANT4 disrupts the mucosal barrier, resulting in epithelial expression of the IL-6-like cytokine Upd3, leading to activation of JAK/STAT signaling, differentiation of cells that form the progenitor cell niche, and abnormal proliferation of progenitor cells. This niche disruption could be recapitulated by overexpressing upd3 and rescued by deleting upd3, highlighting a crucial role for this cytokine. Moreover, niche integrity and cell proliferation in pgant4-deficient animals could be rescued by overexpression of the conserved cargo receptor Tango1 and partially rescued by supplementation with exogenous mucins or treatment with antibiotics. Our findings help elucidate the paracrine signaling events activated by a compromised mucosal barrier and provide a novel in vivo screening platform for mucin mimetics and other strategies to treat diseases of the oral mucosa and digestive tract.

Keywords: Drosophila; JAK/STAT; O-glycosylation; Tango1; Upd3; glycosylation; interleukin 6 (IL-6); mucin; mucous membrane; mucus; niche; peritrophic membrane; progenitor cell.

Figure 1.

Loss of the peritrophic membrane causes epithelial cell apoptosis and progenitor cell proliferation. A, the peritrophic membrane (as detected by the lectin H. pomatia; cyan) that coats the epithelial cells of the WT (c135>VDRC60000) midgut is lost upon knockdown of pgant4 (c135>pgant4^RNAi). The epithelial cell layer of the digestive tract (outlined with actin; red) is disorganized upon loss of the peritrophic membrane. Magnified views of insets are shown below each image. Nuclear staining is shown in blue. Scale bars, 20 μm. B, loss of the peritrophic membrane (c135>pgant4^RNAi) results in increased epithelial apoptosis, as detected by the apoptosis marker, Dcp-1 (cyan). The epithelial cell layer is outlined by actin staining (red), and nuclei are white. White arrows indicate cells that are detached and/or undergoing apoptosis. Scale bars, 20 μm. C, loss of the peritrophic membrane (c135>pgant4^RNAi) results in increased cell proliferation throughout the anterior, middle, and posterior midgut regions relative to WT, as detected by EdU (green). Scale bars, 50 μm. D, phosphohistone H3-positive (PH3, green) proliferating cells are the AMPs that are detected by the Delta marker (Dl, red). ECs are those with the large nuclei (DNA, blue). Scale bar, 10 μm. E, loss of the peritrophic membrane results in up-regulation of antimicrobial gene expression. qPCR analysis of antimicrobial gene expression was performed on cDNA synthesized from RNA extracted from WT and c135>pgant4^RNAi third instar midguts. Values were normalized to rp49 and are plotted as -fold change in gene expression. Error bars, S.D. ***, p < 0.001.

Figure 2.

Loss of the peritrophic membrane alters the progenitor cell niche. A, diagram of PCs (green) wrapping AMPs (yellow) to form the progenitor cell niche. B, crescent-shaped PCs (as detected by the marker Su(H)-lacZ in red) are shown wrapping AMPs (small blue nuclei) in the WT (Su(H)GBE-lacZ;c135) third instar midgut. Upon loss of the peritrophic membrane (Su(H)GBE-lacZ;c135>pgant4^RNAi), PC shape is altered. AMP clusters (small nuclei without Su(H)-lacZ staining) are outlined with dotted white lines. Scale bars, 10 μm. C, Armadillo staining (Arm; white) outlines tight, distinct clusters of AMPs in the WT (c135>VDRC60000) midgut. Loss of the peritrophic membrane (c135>pgant4^RNAi) results in the loss of the small, tight clustering of AMPs that are no longer wrapped by PCs. Nuclear staining is shown in blue. Scale bars, 20 μm. D, PC fate is altered in the absence of the peritrophic membrane. Su(H)-positive PCs (red) do not express the EC marker, Pdm-1 (cyan), in the WT (Su(H)GBE-lacZ;c135) midgut. In the absence of the peritrophic membrane (Su(H)GBE-lacZ;c135>pgant4^RNAi), many Su(H)-positive PCs now also express Pdm-1. PCs are denoted by red arrows, and ECs are denoted by white arrows. Nuclear staining is shown in blue. Scale bars, 10 μm. E, quantitation of the percentage of doubly positive Su(H) and Pdm-1/singly positive Su(H) cells in a WT (Su(H)GBE-lacZ;c135) midgut and a midgut without the peritrophic membrane (Su(H)GBE-lacZ;c135>pgant4^RNAi). For each genotype, doubly positive Su(H) and Pdm-1 cells and singly positive Su(H) cells were counted in five third instar larval posterior midgut regions close to the junction between the midgut and hindgut as described under “Experimental procedures.” Data are presented as the percentage of doubly positive Su(H) and Pdm-1 cells over singly positive Su(H) cells. Each dot represents the data from one larva. Error bars, S.D. ***, p < 0.001.

Figure 3.

Loss of the peritrophic membrane activates JAK/STAT signaling in niche cells and progenitor cells. A, qPCR analysis shows a dramatic increase in expression of upd3 and its downstream target socs36E in flies without peritrophic membrane (c135>pgant4^RNAi) relative to WT (c135>VDRC60000). Error bars, S.D. ***, p < 0.001. B, RNA in situ hybridization to upd3 (red) reveals that it is expressed in ECs (large nuclei) in animals without peritrophic membrane (c135>pgant4^RNAi). No detectable expression of upd3 was seen in WT. Nuclear staining is shown in blue. Scale bars, 20 μm. C, JAK/STAT signaling (as detected by the reporter Stat92E-GFP; green) is seen in the PCs (but not the AMPs) of the progenitor cell niche in the WT (Stat92E-GFP, c135>VDRC60000) third instar midgut. Upon loss of the peritrophic membrane (Stat92E-GFP, c135>pgant4^RNAi), JAK/STAT signaling is greatly increased in PCs, which now have a rounded morphology. Additionally, JAK/STAT signaling is now seen in some clusters of AMPs (white circles and arrows). The red arrow shows an AMP cluster that is still wrapped by a PC and is not JAK/STAT-positive. Cells with large nuclei (DNA, blue) are ECs. Scale bars, 20 μm.

Figure 4.

Upd3 and JAK/STAT signaling are responsible for altered PC morphology and progenitor cell proliferation. A, overexpression of upd3 in ECs of WT larvae (Myo1A>UAS-upd3) resulted in the activation of JAK/STAT signaling as detected by increased expression of the downstream target socs36E. qPCR was performed by comparing gene expression between midguts of larvae overexpressing upd3 (Myo1A>UAS-upd3) with those without upd3 overexpression (Myo1A). Error bars, S.D. ***, p < 0.001. EC-based expression of upd3 also resulted in altered PC morphology (as detected by the PC marker Su(H)-lacZ; red) (B) and PC differentiation (as detected by the induction of expression of GFP in PCs that are still partially wrapping AMPs in Myo1A>UAS-upd3, UAS-GFP larvae) (C). Clusters of PCs and AMPs are outlined with a dotted white line for each genotype. Expression of GFP under the control of the EC-specific promoter Myo1A is seen in PCs (white arrows) in Myo1A>UAS-upd3, UAS-GFP larvae but not in the absence of upd3 overexpression (Myo1A>UAS-GFP). D, upd3 overexpression (Myo1A>UAS-upd3) also increased cell proliferation relative to the control (Myo1A). Error bars, S.D. **, p < 0.01. Scale bars, 10 μm.

Figure 5.

Deletion of upd3 in pgant4 RNAi larvae reduces socs36E expression and rescues the progenitor cell niche. A, qPCR analysis of gene expression in c135>pgant4^RNAi and Δupd3; c135>pgant4^RNAi third instar midguts. Values were normalized to rp49 and are plotted as -fold change in gene expression. Error bars, S.D. ***, p < 0.001. Niche cell morphology (B) and levels of JAK/STAT signaling (C) are restored in the pgant4 RNAi background upon deletion of upd3, as detected by the Stat92E-GFP reporter (green) (compare Stat92E-GFP, c135>pgant4^RNAi midguts with Δupd3; Stat92E-GFP, c135>pgant4^RNAi midguts). Nuclear staining (DNA) is shown in blue. Scale bars, 20 μm. D, deletion of upd3 in larvae without peritrophic membrane (Δupd3; c135>pgant4^RNAi) reduced cell proliferation within the midgut. EdU-positive cells were counted in five third instar larval midguts. Each dot represents one larva. Error bars, S.D. Bar, mean. ***, p < 0.001.

Figure 6.

Restoration of the peritrophic membrane rescues cell proliferation and JAK/STAT signaling. Overexpression of the cargo receptor, Tango1, in PR cells of c135>pgant4^RNAi third instar larvae (c135>pgant4^RNAi, tango1^OE) restores the peritrophic membrane (as detected by the lectin H. pomatia; cyan) (A), rescues AMP proliferation (as detected by EdU; green) (B), and decreases upd3 and socs36E expression levels to those of WT (c135>VDRC60000) (C). Actin (red) outlines the epithelial cell layer. Scale bars, 50 μm (A) and 20 μm (B). D, larvae deficient for pgant4 were fed either water (H₂O), CMC, Muc2, or Muc5AC. Muc2 was the only compound that reduced upd3 and socs36E expression in the c135>pgant4^RNAi background. Two additional independent trials are shown in Fig. S5. E, qPCR analysis of antimicrobial gene expression in WT (c135>VDRC60000) and peritrophic membrane-deficient larvae (c135>pgant4^RNAi) that were fed antibiotic-containing food. F, qPCR analysis reveals a decrease in upd3 and socs36E gene expression when c135>pgant4^RNAi larvae are raised on antibiotic-containing food. Values were normalized to rp49 and are plotted as -fold change in gene expression. Error bars, S.D. G, quantitation of proliferation (PH3-positive cells) within the midguts of WT larvae, c135>pgant4^RNAi larvae, and c135>pgant4^RNAi larvae raised on antibiotic-containing food. PH3-positive cells were counted in five third instar larval midguts. Each dot represents one larva. Error bars, S.D. Bar, mean. ***, p < 0.001; *, p < 0.05.

Figure 7.

Model depicting how loss of the peritrophic membrane (PM) affects epithelial cell integrity and the progenitor cell niche. In the absence of the peritrophic membrane (red), ECs are exposed to mechanical and microbial insults, resulting in damage and the up-regulation of the IL-6-like cytokine, Upd3. Upd3 from ECs increases JAK/STAT signaling in PCs, resulting in changes in PC morphology and fate and disruption of the progenitor cell niche. Once the niche is disrupted, AMPs are exposed to Upd3, resulting in activation of JAK/STAT signaling and abnormal cell proliferation.