B_creck tor vocates, Received: 19 September 2021 | Revised: 4 December 2021 | Accepted: 10 December 2021 Dor ioattilepasi73 ORIGINAL ATicte SE wie CIC-c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila Jinping Huang* | Xiao Sheng* | Zhangpeng Zhuo" | Danging Xiao" | Kun Wu | Gang Wan? | Haiyang Chen?@ cuangdong Province Key Laboratory of Phamaceutical Functional Genes Key Laboratory of Gene Engineering ofthe Ministry of Education, State Key Laboratory oF Biocon Schoo! of Lie Sciences, Sun Yat-sen Univesity, Guangenou, Guangdong, Cina Laboratory ot Metabollem ana Aging Research Netionl Clini Reseach Center fr Grits, West Clns Hott Slenusn Unherty. Chen, Sinus china Comespondence Haiyang Chen, Labersteryaf Metzboism | Abstract sn zing Recearch, Nationa iia , ane Aang Research, National Cel Objectives: Adult stem cells uphold a delicate balance between quiescent and ac Cha spat Suen Uns tive states, which is crucial for tissue homeostasis. Whereas many signalling path Cheng 100% Sencha. Cengnycio4n Serwon.cnra | ways that regulate epithelial stem cells hae been reported, many regulators remain ‘Gang Wan. Guangdong Province Key Se Laberatoryof Pharmaceutical Functor | Materials and Methods: Fes were used to generate tssue-specitic gene knock Gees ey at ene van, | _ dom and gene Knockout. GRT-PCR was used to assess the relative mRNA levels Stxerey Laborer Bena, sect | _Immunoffuorescence was used to determine protein localization and expression pat of te Scenes, Sun ataen Urea. of fe senes Snatsennverst, | terns. Clonal analyses were use to observe the phenotype. RNAseq was used to Ema cremyszascued cn 60 screen downstream mechanisms. sonlaonaton Results: Here, we report a member of the chloride channel family Cc, which fs Nitra ay ad asec rogram specifically expressed in Drosophila intestinal stem/progenitor clls and regulates of china, Grantaara Number: i : eeirencce coca soorexoiosa01; | testinal stem cel (ISC) proliferation under physiological conditions and upon tissue ‘ational Natural Sclence Foundation of damage. Mechanistically, we found that the ISC loss induced by the depletion of CIC-< eee ee eee aa” | _ in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway. ‘21671254, 22070798 and 9174920; ‘Gunngdong Netra Scence Funds for Conclusie fings reveal an ISC-specific function of CIC-c in regulating stem Distinguish Youre Scholar, Grant Distnases Youre choi, ark cell maintenance and proliferation, thereby providing new insights into the functional CGuzngdong Science and Teennology links among the chloride channel family, ISC proliferation and tissue homeostasis. Department, Grant/Award Numoer 202081212060031; Bosc and Appled asi Research Foundation of Guangdong. GGrant/Sward Number: 2019A1515210746 ur fi Huang and Sheng shouldbe coridered it fist autoc “Tis ean open acces article under the terme of the Creative Commons Attlodton icsne, which porns ure, clerioulon and reproduction in any med, provided the orginal works propery cited '© 2021 The Author. Cel Proliferation Published by John Wey & Sons Lt CalProlferation 20225513178, ‘wilyonneltray.com/Joumalepr | 4.0f 47 htpssdotorg/tO an Lop. 13173207 wiry HUANG era 1 | INTRODUCTION In most adult somatic organs, resident adult stem cells, whose high proliferation and differentiation abilities compensate for cell loss, are responsible for both tissue homeostasis and organ functions throughout the lifespan of the organism. Compensation of cell loss is especially important intssues with a high turnover rate such as, the intestinal epithelium. Intestinal stem cell (SCs) undergo cel vision and differentiation to rapidly replenish damaged cells, thereby maintaining intestine integrity during physiological turnover orn re sponse to damage.*® Thus, characterizing the regulators and signal ling pathways that control stem cell activity and maintain the eitical, balance between cell generation and degeneration has been one of the main focuses of developmental biology and regenerative med: icine, However, how cel-fate transitions occur and how signaling pathways and transcriptional networks control these coordinated celular changes remain largely unexplored “The Drosophila melanogaster intestine shares many similarities with its human counterpart and thus has emerged as 2 power {ul system to study the role of intestinal stem cells in adult tissue homeostasis and regeneration because of its relatively simple and wellcharacterized stem cell lineage and tractable genetic manipu lation? Drosophila ISCs are smal diploid cells scattered along the basement membrane of the midgut epithelium and specifically ex press the transcription factor Escargot (Esp) and Notch ligand Delta (O02 The ISCs divide asymmetrically to generate new stem cells, and ether transient post-mitotic progenitor cells named enteroblasts, (EBs) or enteroendoctine mother cells (EMCS). EBs progressively cifferentiate into polyploid enterocytes (ECs), which are respons ble for the absorption of nutrients in the midgut. The other type of differentiated cells in the Drosophila intestine is enteroendocrine cells (EES), These cells emerge from EMCs, which express markers of both ISCs (Ese) and EES (Prospero). high level of Notch signal ling dives ISCs to produce ECs, whereas 2 low level dives them to produce EEs (Figure 14)**** Under homeostatic conditions, the SCs in Drosophila midgut are largely quiescent. The numberof SCs and progenitor cells inthe midgut is relatively small and remain sta ble. However, these ISCs promptly undergo proliferation and di ferentiation when the tissue is injured." This cellutar response Is essential for maintaining the epithelial homeostasis since a failure {0 replace lost cells may compromise tissue function and overpro duction of cells may lead to cancer. Under both homeostatic and stressed conditions, ISC divisions are modulated by numerous regu- lators, such as Sox21a, GATAe and Hairless," and signalling path- ways, such as the EGER, Wat, mTOR and JAK/STAT pathways ‘to maintain the critical balance between cell generation and degen- eration. However, how these signalling pathways are regulated and integrated by which is unclear, (Chloride channe!-3(CIC-3)isa member of the voltage-denendent Chloride channel CIC family, which functions in cell volume mainte- nance, cell excitability, lysosomal acidification ion homeostasis and CI” transport across the cell membrane.*** Several diseases, in- _luding eystic fibrosis, neuronal cerca ipofuscinosis, allergic rhiitis and myocardial ischemnia,** have been discovered tobe associated vith aberrant expression of CIC-3, CIC-3 has been shown to actively participate in varlous cancers, facltating the aggressiveness and metastasis of various types of malignancies, including breast, cervi= ‘al, prostate, and nasopharyncal cancers and glioma tumours, Additionally, CIC-3 is expressed in human and mouse intestinal _p- ithelium** Genetic deletion of CIC3 increases the susceptibility of mice to DSS- or TNBS-induced experimental colts and thus pre- venting the intestinal recovery.% Although CIC-3 has important foles in cegulating the cel cycle, migration and apoptosis of tumour ‘els, its functions in intestinal stem cells ae largely unknown. This study found that CIC-c (the homologous gene of CIC3 in Drosophila) is speciticallyexpressedinthe ISCs and EBs of Drosoptila midguts. Inhibition of CICc expeession in ISCs could inhibit ther re newal. Adgitionally, CIC-¢ was found to regulate ISC proliferation ‘through the EGFR signaling pathway. This study not only uncovers 22 new function of the chloride chennel gene CIC-c in the modula- ton of ISC proliferation but also improves our understanding of ISC functions 2 | MATERIALS AND METHODS 2. | Drosophila lines and husbandry ‘The following fly lines were obtained from the Bloomington Drosophia stock centre (BDSC}: w'**, UAS-EGFR™ ([BSC# 9533), UUAS-stg-H (BDSC# 56562), UAS-rpe(BDSC# $822), UAS-RabS-GFP (BDSC# 43336), UAS-Rab7-GFP (BDSC# 42705), UAS-sSpi (BDSC# 58436), Di(3Ust 4 (BDSC# 1917) and CiG-c RNA (BDSC# 27034), UUAS- Kon (FlyORF # F002754) was obtained from the FIyORF FIGURE 1 CiCcis specifically expressedin stem and progenitor cells inthe intestinal epithelium of the adult Drosophila (A) Schematic lagram ofthe cell types and markers in the Drosophila midgut (B) Strategy of construction of Drasopila CIC-c-3 x HA knockin (C) Expression of CIC-c-3 x HA protein red in the sg-Gala” > GFP” cells ISCs and EBs; co-stained with GFP, green in the Ré region of the Drosophila midgut. (D) Expression of CIC-c-3 x HA protein (ed) in the ISCs labelled by Delta staining, green) inthe Ré region of the DDrosophiia midgut. € and F) Expression of CIC-c-3 x HA protein red) n the NRE-Gald” > GFP* cells (Bs; co-stained with GFP, green) Inthe R4 region of the Drosophila midgut. Yellow arrowhead indicates the NRE-Gala® > GFP* cells with small nucle! (newly formed EBS) (@ Expression of CIC¢-3 x HA protein ed) in the EEs (labelled by Prospero staining, green) in the R4 region ofthe Drosophila midgut (H Expression of CIC-¢3 x HA protein (red) in the Myo14-Gald! > GFP* cells (ECs: c-stained with GFP, geen) inthe Ré region of the Drosophiia midgut. EB, enteroblast; EC, enterocyte: ISC, intestinal stem cell, DAPI-stained nuclei are showin in blue. Scale bar represents 10ym i i i2) (EO storms Hi ERS <03- REP S'arm(UTR}— oc Qp=aRNA IC-c gonopr6 Samitasteypt 2. SamUTR) cic-c genome “#°MY) yy homologous recombination Stamm -Lastewon SHA: 5, AMNO=09-RFPMbSaMHUTRI— Goer: CCegHA _ GFPIHA CiC-6-HATMGB 5 Gl MyotA-Gold, w0-Gal80-, UAS-GFPI: CICoSHHA _GFPIMAN4or37 wiry HUANG era “The folowing fy fines were obtained from the Vienna Drosophila Resource Center (VDRC: cic RNAi (v103042), cic RNAI (7108805), CIC-c RNA\ 6485) and CiC-c RNA (v6466, ‘The following fly lines were kindly provided as gifts: esg-GFP, e35-Golf, UAS-lacZ tub-Gal4 and FTR2A lines by Dr. Allan Spradling; and es§-Gal4®, Myol4-Gal4®,ISC-Gala® and NRE-Gold® ines by Dr. Beniamin Ohistein. All the Drosophila lines used in this study are listed in Table St Flies were cultured in the standatd medium (50 g cornmeal, 18.75 g yeast, 80 g sucrose, 20 g alucose, 5 pagar and 30 mi propi- onic acid per 1 L of media) at 25°C and with 65% humidity on a 12h light/12 h dar cycte. Unless indicated otherwise, only females were used inthis study, 2.2 | Generation of the knockout, knock-in and transgenic fy lines To knock out Cc, the folowing two sgRNAs located in the fore part of the CDS region of CC-c were desianed using CRISPR Optimal Target Finder with the maximum stringency thttp//toos ycrspr. ‘molbio:visc.edu/targetFinder/), Each sgRNA was synthesized in vitro, ‘and then subcloned into the PMDIBT vector to acquire the U6 pro moter. Then, the US promoter and sgRNA were recombined into the 3ttB vector. This sgRNA construct was injected into the fly line yt) {248}; VK00037 (BDSC# 9752) to generate CIC-csgRNAaleles. The (IC-c SaRNA fly line was then crossed with the vas-Cas9 fly line to Cbiain the mutantalleles. Two independent mutant alleles were con firmed by sequencing the CIC locus and named CIC and CiC-<%, To generate CiC-c 3H knodein lines, two constructs were ger erated, one with two sgRNAs and the other with ahomologous recom bination sequence. The sgRNA construct was generated as mentioned above. To generate the homologous recombination construct, the 5! homologous arm (-1 KB upstream sequence before the termination codon), 3 x HA and the 3° homologous arm (-1 KB downstream se ‘quence after the termination codon) were inserted into the PASK vec ‘or. All the final vectors were verified by sequencing and injected by Fungene Biotechnology (Being, Cina). The two sgRNAS were used to generate DNA double-strand breaks. The 5! and 3° homologous arms were used for homologous recombination repai.3x PS-RFP was sed for screen, Allthe ssNA sequence uscd arc listed in Table S2, ‘To get UAS-CIC-c transgenic lines, the UAS-CIC-c expression vector was generated first. Full-length cDNA (RT-PCR from total mRNA of w#¥ flies) of CC-¢ was subcloned into the pEntry vector by using pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, CU101-02} and then sub-cloned into pTW vector by using LR recombination reaction. The primers ae listed in Table $3 2.3. | Clonal analysis Ce mutant ‘Mosaic Analysis with a Repressible Cell Marker’ (MARCM) clones were generated from the CIC-¢ mutant line via FLP/FRT-mediated mitotic recombination. FRTZA was recombined with CICc# to generate the FRT2A, CIC-cnull mutant line, which ‘was then crossed with the line yw, RsFLP, tub-Gl4, UAS-GFP/EM: tub-Gol80, FRT2A/TM6B to obtain hsFLP, tub-Gol4, UAS-GFP/+; tub- Gol80, FRT2A/ FRT2A, CIC-c lies. The fies were raised at 25°C, and S-day-old adult flies post-eclosion were subjected to Lh heat shock ‘twice in a water bath at 37°C. Afterwards, they were kept at 25°C and then dissected and observed at the indicated days after clonal induction (ACD, 24 | RNA-sequencing (RNA-seq) and data analysis The detailed process was described previously The Drosophila ‘adult midguts (RI-R5) were first cissected in phosphate-butfered saline (PBS) on ice. They were then immediately placed in a ~80°C freezes, Total RNA was isolated using the isothiocyanate-aleohal Phenyi-chloroform method, Whole RNA previous sequencing was cartied out on the NovaSeq 6000 platform (lumina) by Besry Genomics Corporation. The quality control was performed using Fast QC 0.11.8) The read length of the raw RNA-seq data was 150 bp. All the reads were aligned to the Drosophila reference genome (Ensembl BDGPS release-8%). The aligned-read sam files were then con- verted to bam files and sorted using SAMtools. DESeq2 (v1.22.2) ‘was used to determine the gene exoression profiles of samples. P-value < 0.05 following Benjamini and Hochberg correction for multiple hypothesis testing was considered to indicate differential gene expression, 25 | Cellsorting and RT-qPCR (One hundred to two hundred midguts were dissected in cold di- ‘ethyl pyrocarbonate (DEPC)-treated water PBS (0.1% final solu- tion of DEPC is added to 1x PBS) and incubated with 0.1% trypsin for 1h at 29°C, during which the sample was softly mixed every 15 min by pipetting and inverting several times. Dissociated sam ples were collected after centrifugating at 4°C, 40x g for 20 min, {and resuspended in DEPC-PBS. The suspension was filtered with 40-um filters. The filtered cellsare then sorted using a FACS Avia sorter [BD Biosciences) and collected into 0.5 ml DEPC-PBS. GFP* callin the midgut of fly strains carrying eg-Gald"> UAS-GFP fluo rescent markers were used to sort intestinal progenitor cell opu- lation, and the midguts of w! flies were set. as fluorescence gate. Total gut RNA was extracted from dissected midguts by using the Trizol reagent (Invitrogen). This RNA (1 yg) was used to gener- ate cDNA via reverse transcription, and the cDNA was subjected ‘to quantitative polymerase chain reaction (qPCRIin a QuantStudio 5 System (Thermo Fisher Scientific). The 2&7 method was Used to calculate the expression values. The relative expression ‘was normalized to that of Rp49. All the primers used are listed in Table $3 i i iHUANG Era aa wicey Lt” 2.6 | Immunofluorescence microscopy Adult midguts were dissected in PBS and then fixed with 4% Paraformaldehyde for 2h, followed by washing three times (10 min each) with PBS containing 0.1% Tween-20 (PBST). The midguts were then blocked in 0.1% BSA for 30 min at room tem- perature, followed by washing with PBST, and then incubated overnight at 4°C with primary antibodies diluted in PBST. After washing three times with PBST, the tissues were incubated with secondary antibodies and DAPI for 2 hat room temperature fol lowed by the same washing steps above. The sources and dilt tions of the primary and secondary antibodies used are listed in Table 82 Leica TCS-SPB confocal microscope was used to acquire all the immunoflvorescence images. The Leica Application Suite X LAS X), ‘Adobe Photoshop c-2020 and Adobe Illustrator cc2020 were used toassemnble the images. 2.7. | Bleomycin (BLM), dextran sodium sulphate (Ds) and paraquat (PQ) treatment A chromatography paper was cut into 4 x 6 cm strips and satu rated with 25 ug/ml BLM (Aladdin, B107423), 5 (wt/vol DSS(MP Biomedicals, CAS: 9011-18-1, 36-50 KD) or 5 mM PQ (Aladin, (M0676!) dissolved in 5% (wt/vol) sucrose, After being starved in empty vials for 1h, fies were transferred into vials with the BLM, DSS or PQ solution-saturated chromatography paper with ‘5% sucrose, After 24 h (for BLM or PQ) or 48 h (for DSS) of treat- iment, the flies were transferred tothe standard fy food, with the dally renewal ofthe food. Sucrose-only (5%, wt/vol) was used as a control 2.8 | TUNELassay | Adult midguts were dissected in PBS and fixed with 45 paraform: aldehyde for 2h, folowed by washing with PBST. Apoptosis was, assessed using the Apoptag Kit (Millipore) according tothe manu facturers instructions. 2.9 | EdUincorporation assay For EdU labelling, a chromatography paper was cut into 4 x 6 cm strips and saturated with 5% sucrose and 100 uM EdU (Baseclick) After being starved in empty vias for 1h, fies were transferred into Vals with the EaU solution-saturated chromatography paper for 24h. The guts ofthe flies were then dissected in PBS and fixed with 4% paraformaldehyde for 2 h, Edu incorporation was evaluated fo lowing the manufacturer's instructions. The immunostaining proc dure has previously been described. 240 | Detection of reactive oxygen species (ROS) by using dihydroethidium (DHE) Adult midguts were dissected in PBS and then incubated in 30 yM DHE (Invitrogen) for 5 min in the dark at room temperature, The sults were then washed twice with PBS, mounted and immediately ‘examined using a confocal microscope. 2.41 | Assessment of necrosis by using propidium iodide Adult midguts were dissected in PBS and then stained with 1.5 yM propidium iodide (Pl; Invitrogen) at room temperature for 45 min, ‘The quts were then fived with 4% formaldehyde for 20 min, ‘washed three times with PBST, rinsed twice with PBS, mounted in \Vectashield with DAPI and examined using a confocal microscope, 2.12 | Fluorescence intensity statistics Immunofluorescence images were analysed via confocal micros- ‘copy, and the fluorescence intensity statistics inthe region of nter- est [ROI] were calculated using Imagel. The detailed process was described previously. Mean fluorescence intensity = Integrated density of the backe ‘round region (or cell/Area ofthe background region (or cell) Relative mean fluorescence intensity =Mean fluorescence inten sity of the cell-Mean fluorescence intensity ofthe background region Relative fluorescence intensity = Integrated density of the eell- Mean fluorescence intensity ofthe background region x Area of the call 2.13 | Quantification and statistical analysis | For all the experiments, the data were processed using GraphPad Prism and presented as average = SD. Statistical significance was determined using the unpaired two-tailed Student's Hest unless ‘otherwise stated In the Figure legencs. For al the tests, p < 0.05 ‘was considered to indicate statistical significance. 3 | RESULTS 34 | CIC-cis specifically expressed in the stem and progenitor cells of the adult Drosophila intestinal epithelium ‘Toinvestigate the possible roe of CiC-in the regulation of intestinal stem calls, a CIGe3xHA knockin ly ine was generated (Figure 4B). We used the conditional temperature-sensitive driver esg-Gald i i isora wiry HUANG era (esg-Gala” to express the dsRNA construct (BDSC # 27034) against CIC-c and observed that the HA signal was strongly reduced (Figure SIA-C), We detected CIC-c expression in RI-R5 regions of the adult Drosophila midgut (Figure S1D). Using spectic cell markers to istinguish the cell-specific expression pattern of the endogenous CIC protein, we found that CiCc is specially expressed in esg- positive cells (Figure 10). By using esg-Gol4, UAS-GFP; Gal80" and [NRE-Gal80(ISC-Gal4* flies, we found that CiC-cis expressed in SCs, (Figure SAE) Adeitionally, costaining the CIC¢3 x HA midguts For the Di protein and HA tag confirmed that CIC-c is expressed in SCs (Figure 1D). Next, we found that CIC-c is expressed in some newly formed EBs with small nuclei (Figure 1E, yellow atroviheac), whereas no CIC expression was detected in mature EBs with large nucle (Figure 1F), These results imply that the expression of CiC-cls gradu ally lost during the ctferentiation from EBs to ECs. As predictee, 10 CIC+c expression was detected in mature ECs (Figure 1H) or EES, (Figure 1G) Taken together, these results indicate that CIC's spe ificaly expressed inthe stem/progenitor cells ofthe Drosophila in testinal epithelium. 3.2 | CIC-cis required for ISC proliferation in the Drosophila midguts Since the expression of CIC is uniquely restricted to ISCs and some newly formed progenitor cells, we frst depleted CiGc in SCs and EBs via esg-Gald driven RNA interference (RNAI fines CIC NAM (BDSC# 27034), CIC RNAI? (VORC v6445) and CiCe NAIF? (VDRC v6466). The CIC-c mRNA levels in these three RNA lines were measured via RT-GPCR (Figure S24). Knocking down CIC-cin the esg* cell for 7 days at 29°C significantly decreased the number of esg* cells (Figure 2A-C, Figure S2C-E). To further de termine in which cell types CIC:c exerts its functions, we knocked down CIC-cin either ISCs or EBs by using the folowing celspecific Gal4 drivers: ISC-Golé* for ISC knockdown: and NRE-Gal4, Gal80 (NRE-Gald*) for EB knockcown, We found that depletion of Cie in ISCs decreases the number of ISC* cell (Figure 20-F), whereas ‘no effects on the number of EBs in depletion of CiC- in NRE* cells, (Figure S2F-H). Additionally, depletion of CICcin EBs did not affect the number of esg” cells (Figure S2I-K), We also tested the effects of overexpression of CC-c on SCs. Overexpression of CC did not significantly promote ISC proliferation Figure S2I-N), These results suggest that CIC-c might act cell- autonomously in ISCs to regulate |SC proliferation. Next, Cc mutantalleles were generated using CRISPR/Cas9 to validate the CIC-c RNAi phenotypes. We designed a seRNA target. in the first exon of CIC-c and isolated two independent lines, each cartying one of the mutant alleles CIC and CIC. By sequencing the CIC-clocus, both ines were confirmed tobe mutantasmanybase deletions occurred after position .392 in exon 1 thus introducing a premature termination codon into this exon in both lines (Figure 2G), No progeny was obtained when these ines were crossed with the corresponding deficiency line Df @L) sted, in which the entire CIC-c locus is deleted (Figure S26). These results suggest that CIGc* and A1C-e are null or loss-oF function alleles. To observe the phenotypes of the CIC-e mutants. we used ‘the MARCM system in which both the control and CIC-c mutant clones were marked by GFP expression. The GFP-marked clones ‘were analysed! atthe Indicated time points after ACI. The control ‘clones showed normal growth at days 4, 7,11 and 14 (Figure 2H- K.P, and Figure $20), whereas the growth of the CIC-¢ mutant clones was inhibited (Figure 2L-P). This result suggests that the CCiC-¢ mutant clones have a restricted capacity to divide and gen- erate progeny. Taken together, these results strongly suggest acell-autonomous role for CIC-cin regulating ISC proliferation. 3.3 | CIC-cis required for midgut regeneration Intestinal stem cells confer ahigh regenerative capacity tothe intes tinal epithelium.”* They undergo rapid cell dvision and dferentia- tion to replenish damaged calls, thereby maintaining tissue integrity and the proper numberof differentiated cells under the homeostatic ‘conditions or in response to stress.” CIC-c has been shown to be Involved in ISC proliferation during homeostasis. Therefore, we uth lized the CIC-c-3xHA reporter line to visualize the expression level, ‘of CIC in exg" cells in injured midguts. The abundance of CICe ‘considerably increased in BLM (causes DNA breaks and genomic instability), DSS (@ model for experimental inflammatory bowel disease}- or PQ (induces oxidative stres}-injured midgut epithelium (Figure 3A-E], Treatment ofthe Drosophila intestinal tract with BLM, DSS or PQ caused intestinal damage and induced ISC hyperprolif- ‘eration as indicated bythe increase in the numbers of eg* and pH3* cell Figure 3F-.N.0}, However, the numbers of esg* and pH3* cells in the intestines of the fies with depleted CiC-c did not increase wien these flies were subjected to these injuries (Figure 3J-O) “These results suggest that CIC- is essential for inducing ISC prolit- eration in response to damage to the intestinal epithelium. 3.4 | Reducing CIC-cinhibits the proliferation of progenitor cells, but does not lead to apoptosis or necrosis Knocking down CiC:c in the exg* progenitor cells and clones de- creased the number of esg* cells ancl resulted in small clones re= spectivel, Apoptosisis 3 common reason for cll loss. To assess the possibilty that the esg* cells undergo apoptosis upon knocking down, ‘OC, the TUNEL assay was performed to detect apoptotic signals after 7 days of CiC-e RNAI. We did not observe. significant increase in the number of apoptotic esg* cells, compared withthe number in, the control flies (Figure 44,8,H), Forced expression of ror(an inducer ‘of apoptosis) exg* cells was used as. postive control Figure 4C.H)- “These results indicate thatthe cell loss caused by knocking down CIC“ in esg* cells nat due to an increase in apoptosis. i i i= ‘WILEY: esg-Gote> GEPMmAPI )UASiaez___ AD @)_CoaRNAD 1sc-Gaste> GEPIDAPI BC TaaC Ta (0 vasiacz 0) Cie RNAP TE) C1C-c (cG5284) x9, sgRNA wx MagcM 2a cepapy ()agaci 7aAc! HdACI (34g ACI ” fe se 22, cice FIGURE 2. CiC-cis required for ISC proliferation in the Drosophila midgut. (A and B) lmmunofluorescence images fro on ofthe midgut ofthe control is (A, es3-Gald"-driven UAS lacZ) of CIC-c-depleted flies (8, eg-Gald™driven CIC-c RNA 34) (©) Quantification of the number of cells in the midguts ofthe control and CIC-¢ RNAI groups, ROI: 2 x 10" yn area from the RS region ofthe Drosophila midgut. Gut a» 10; ROI > 20, (D and E) Immunofluorescence images from the RS region ofthe midgut ofthe control flies (D,ISC-Gala*-driven UAS-lecZ) or CIC-c-depleted flies (E, [5C-Gold® driven ClC-c RNAi", BOSC# 27034), GFP (grcen) identifi ISCs.F] Quantification of the numberof the GFP® cells in(D) and (E], ROK 2 x 10° um area from the R5 region ofthe Drosophila midgut Gut n = 10: ROI n > 30. (6) Schematic summary of the CIC-c mutant alleles generated using CRISPR-Cas9. (H-O) lmmunofluorescence analyses ofthe control (FRT2A, H-K) and CIC-c# mutant (L-O) MARCM clanes (green) in the midguts 4, 7,11 and 14¢ after clonal induction (ACI, [P) Quantification of the cell number per MARCM clane in H-O. Each dat corresponds to ane clone. Gut n = 10;clonen ISG, intestinal stem cell: MARCM, mosaic analysis with arepressible cell marker; RO, region of interest. DAPL-stained nucle are shown in blue, Scale bar represents 10 pm. Error bars represent SDs. Student's ttest, "7p < 0.0001“| woey mG y O12HA HDA contol ®)_aimia (pas 2 © paid «) ®) (cy o esgGali"> __GFPIDAPL ‘Sie sucrose ()sunie _pss2a ice RNAI * Fe oP gh het ae 0 at ath ct et it ak ee cath gah act aa ach aah Rca al cal alae Be ee eee tee FIGURE 8 tvidgt regeneration (A-D)Immunofturescence images from the RS region ofthe midgut showing th expression otein (red) upon Sucrase (A, BLM (6), D he fluorescenc Jor PQ(D) treat rt. (E) Quantiication intensi re midguts in (A-D). Each dot corresponds to one cell, Gut 25; celln 290. (Fl mmunafluorescence images rom the RS region of the midgut ofthe control les lesg-Galé”-driven UAS-lacZ) treated with sucrose (Fi, BLM (G), DSS (H) or PQ). U-Mi Immunofluorescence images from the RS region of the midgut of CIC-c-depleted flies (sg-Gal4-driven CiC-c RNA, BDSC# 27034) treated it J), BLM (K}, DSS (1) of PQ(M).(N) Quantification of the numberof the GFP* cell the midguts in (F-M). ROI:2 x 10* pnt are rom the RS resion of the midgut. Gut n= 10; ROI n 2 30, (0) Quantification ofthe number of the pH3" cells in the midguts in F-M. BLM Bleomycin; DAPI labels the nucleus (blue); DSS, Dextran Sodium Sulphate; PQ, Paraquat. ROI, region of interest. Seale bar represents 10 jm ror bats represent SDs. Student's t-test, ***p < 0.0001, Gut n> 2HUANG Era aa witey 22” Another possibility of cell death is necrosis. Therefore, we ine vestigated whether knocking down CIC-c causes necrosis in ISCs, Necross is characterized by early plasma-membrane rupture and accumulation of ROS, which can be assessed through PI staining and DHE staining respectively.°* We detected no increase in PI* or DHE signals after CiC-c depletion, compared withthe signals in the control (Figure 4D-G\l). Taken together, these results suggest that knocking down CiC-c does not induce apoptosis or necrosis in ICs. During homeostasis, the stability of 2 stem-cell pool requires continuous self-renewal of the stem cells, Inhibition oftheir pro Iteration results in the depletion of the stem-cell pool and loss of, stem cells. To determine whether CIC-c is required for the pro eration of ISCs, we performed EdU incorporation assay fa method to detect cell cycle) in esg-ga® > GFP flies after CIC-c RNAI and ‘uantified the number of EdU-positive cells per midgut, After 7d ‘of RNAi, the number of EdU* cells was lower in these flies than in the controls (Figure 4K-M), Furthermore, forced expression of the cell eycle regulator stg rescued the cell oss phenotype Induced by CIC-c depletion (Figure 4N-R and Figure S3A-E), These results, suggest that CIC-c is required in maintaining ISC profiferation and, the depletion of CiC-c suppressed ISC proliferation by inhibiting, the cell eycle. ‘The ditferentiaton of stem cell is another possible cause of the decline in the numberof stem cells, We thus investigated the effect of ClC-c depletion on ISC differentiation. The experimental results, showed that CIC¢ mutant didnot inhibit the formation of large nu clear ECs and pros* EEs (Faure SFG). Moreover, statistical data showed thatthe proportion of ECs in the CIC-e mutant clone was, Figher than that in the contral group (Figure S2H) “Therefore, we believed thatthe decrease in the numberof stem cells caused by the loss of CIC-c is due to the other fact that ISC could not maintain stemness thus undergoing differentiation 3.5 | Depletion of cic-c downregulates the members of the EGFR signalling in the Drosophila midgut To identity the mechanism whereby Cite regulates ISC prolifer tion, RNA-sea was performed on dissected midguts of the flies with (CUC-cdepleted in ess" cells esg-Galé* > CIC-c RNAI) and control les (esg-Gald > UAS-lacZ). The expression levels of esg and Di were sig- nificantly lower in the midguts with CiC-c-dealeted esg* calls than in the midguts of the control sroup (Figure 5B,C). This observation Js consistent with the role of CIC- in maintaining the stermness of SCs, Additionally, the results ofthe RNA-seq analyses showed that, a cluster of genes related to the EGFR signalling pathway (such as Este, Rho, Prt, SoxZta and £13210) was expressed at significantly lover levels in the CICc-depleted midguts than in the control mic guts (Figure SA and Figure S4A.B). Among these genes, Prt, Sox2ta and Ets21C have been reported to function downstream of the EGFR, signalling pathway and regulate ISC proliferation To confi these RNAcseq results, RT-gPCR analyses were par formed, The RT-qPCR results showed similar expression patterns to ‘those observed in the RNA-seq data (Figure SD-G). These findings strongly suggest that CICc regulates ISC proliferation through the EGFR signaling pathway. 3.6 | CiC-c regulates ISC proliferation through the EGFR signalling Previous studies have shown that the EGFR signalling pathway is, vitalin SC proliferation during homeostasis or stress-induced regen- ceration.°* The expression patter of the EGFR effector mitogen activated protein kinase (MAPK) in the midgut was examined by staining the midgut for d-ghosphorylated Erk (dpEr, the active {form of MAPK. Signals of hospho-Erk were detected in the midguts (of the control ies after 28 h of BLM treatment (Figure 68,C.E), but the CIC-c-depleted group showed an unchanged level of Erk activ ity In response to BLM treatment (Figure 68,D.F). Moreover, forced, ‘constitutive expression of the EGFR receptor (UAS-Eaft™) in ISCs was sufficient to induce byperprolferation along the whole gut epithelium (Figure 6&,H.N) The ISC proliferation defects observed In the CiCcdepleted flies were significantly restored by overex pressing Esfr™ (Figure 6F.G,,N) Consistently. the overexpression of Ef partially rescued the clonal growth inhibition caused by knocking out Cc (Figure 60-9), Moreover, activation of the EGFR, signalling by knocking down cic (an inhibitor of the EGFR signal ling pathway) suppressed the cell loss Induced by CIC-¢ depletion (Figure 6.GIKN). These data indicate that CICc regulates ISC proliferation through the EGFR signalling pathway. Notably, we ‘ound that overexpression of the secretory EGFR ligand spi and Krn ‘could not rescue the cell loss induced by the knockdown of CIC-c (Figure 4L-N and Figure S54-E). These results indicate that knock- ing down CICe might inhibit the transduction of EGFR signal from ‘extracellular to intracellular, thereby inhibiting the activation of the EGFR signaling pathway. 3.7 | Depletion of CIC-c inhibits the maturation of Rab5-labelled early endosomes and Rab7-labelled late endosomes ‘Our results indicated that CiC-c regulates the proliferation of ISCs through the EGFR signaling: however, how it affects this signalling pathway is unclear, Many studies have demonstrated that endocy- tosis affects the EGFR signaling. Ligand stimulation causes EGFR to internalize and be transported through the endacytic pathway. Therefore, endocytosis not only regulates the rate of EGFR deg- radation and circulation but also regulates the EGFR-mediated signal transduction **¥ By analysing our RNA‘sea data, we found that knocking down CIC-e downregulates several endocytosis- related genes, including Rab3, Rabex-5, Rab3-GEF, Rab26 and Rab40 (Figure 7A), These findings stronaly suggest that CIC-c might affect i i is00r17 witey HUANG era the vesicle transport system mediated by RAB family proteins. To test this hypothesis, RABS-labelled early endosomes and RAB7 labelled late endosomes were visualized in esg* cals, Under the ho rmeostatic conditions the RABS-GFP signal was remarkably lower in ClC-edepleted group thanin the contral (Figure 7B-D). There wes no citference in the level ofthe RAB7-GFP signal ater knocking down, CIC, compared with the control level (Figure 7E-G). These results, Indicate that CIC-c depletion affects the activity of early endosomes instead of late endosomes under homeostatic conditions. BLM treatment for 24 h considerably increased the RABS-GFP signal compared with the level in the untreated group (Figure 78,0.H.) However, knocking down CIC-c prevented this increase (Figure 71). Sila results were observed regarding the RAB7-GFP signal after 24h of BLM treatment (Figure 7K-M), These results indicate that, knocking down CiC-c might inhibit endocytosis 4 | DISCUSSION CIC is an intracellular CIC protein that functions as @ CI/H® ex changer. Reduced levels of CIC-Shave been detectedin patients with inflammatory bowel disease and mice subjected to DSS-induced co Tis" CIC-2 knockout mice are more sensitive to DSS-induced col. tis and show no signs of recovery after treatment.** However, how CIC-3 regulates intestinal function remains unclear. In this study, we showed in the midgut of adult Drosophila that CiC-e, the Dresophila, ortholog of CIC-3 is only expressedin the ISCs and. small subset of newly generated EBs but notin mature EBs that are about to differ entiat into ECs, CIC-¢ was not detected in terminally cifferentiated ECS or EEs either. Therefore, the expression of CIC-cin the midgut of Drosophila is stem cell-specific, suggesting that the regulatory effect (of CIC-3 on intestinal diseases may be related to autonomous regula tion of SC function Many studies have shown that CIC-3is involved in the regulation ‘ofthe cell cycle in many cancers.” Arecent study has shown that. in DU145 prostate cancer cells, CIC-3 aso acts asa signalling molecule ‘that directly interacts withthe stem cell factor SOX2, and then, the ‘two co-regulate the cell cycle Regulation of the cell cycles very Important for stem cell se-renewal. However, it still unknown, whether CIC-3isinvolvedin the regulation of adult stem cells. n this study, we showed that CIC-c is involved inthe regulation ofthe ISCs in adult Drosophila. Loss of CIC-in the Drosophila midgut ISCs leads to their loss and inhibits their proliferation, hindering Drosophila midgut ISCs from initiating regeneration through proliferation after an intestinal injury. Our study shows that CIC also plays an import= nt role in the maintenance of ISC stemness. The EGER signalling pathway plays an important role in regulat- ing the renewal and homeostasis of ISCs and s the most important signaling pathway in regulating the proliferation of midgut ISCs in Drosophila**** Under physiological conditions, bath ISC division and the stability of the midgut environment require the activity ‘of the EGFR signaling pathviay. Additionally, both compensatory ISC protferation and midgut regeneration after an intestinal injury require the activity ofthis pathway***4#-° However, the up- stream of the EGFR signalling in regulatory networks fs not fully un- ‘derstood. This study showed that CIC-c regulates ISC proliferation by regulating the EGFR signaling pathway. Knocking down CIC-c in ISCs inhibited the activation ofthis pathway, and the stem-cell, loss and proliferation inhibition caused by ClC-e deficiency were rescued by activating tis pathway. Many studies have proved that ‘endocytosis mediates the onset of EGFR signal attenuation; how- ‘ever, tive regulation of endocytosis on EGFR signal is very complex. not just the inhibition of EGER signal. Studies have shown thatthe adulation of endasome fusion, reflected by the changes in en- dosome number and size, could change the cstribution of p-EGFR between endosomes and the EGFR signaling activity of a cell could be regulated by changes in the fusion rte of endosomes, A mild == duction of homatypic early endosome fusions sufficient to modify cell fate and stop cancer cells proliferating and force them to dif: ferentiate instead.” Activated cell-surface EGFRs 2 and sorted atthe early endosome, The fate ofthe receptor has im- portant consequences for biological cell outputs, with the recycling, ternaized FIGURE 4 Reducing CIC c leads to a lss of progenitor cells due to decreased proliferation but not increased apoptosis or necrosis, (4-0) Immunofuorescence images from the RS region of the midgut ofthe negative-contol lies (A, esg-Gald"-driven UAS-lcZ}, CC- depleted flies (8, es-Gold”driven CIC-c RNAI) and positive-contral fies (C, s8-Gala*-driven UAS-rp) with TUNEL treatment. (D and Immunetluorescence images from the RS region of the midgut ofthe control les 9 255-Gal*-ctiven LAS-laeZ) and CiC-e-depleted flies (E, eg-Gala"driven CiC-c RNAI) with Pl treatment. White arrows indicate esg* cells. (F and G) Immunofluorescence mages from the BS region ofthe midgut ofthe control lies (F. xg-Gal® driven UAS-lacZ) and CIC-c-depleted flies (G, esg-Gal#*-driven CIC-cRNAD with DHE treatment. White arrows indicate esg* cells. (H) Quantification of the numberof the GFP* and TUNEL? cellsin the midgut in (A), (2) (oF (C), ROI: 2 10° rea from the RS region of the Drosophila midgut. Gut = 10; ROI n = 30. () Quantification ofthe number ofthe GFP* and PI cells in the midgut in (0) or (E). ROK: 2x 10 ym’ area from the R5 region of the Drosophila midgut, Gut » 10; ROI n = 30. ) Quantification of the numberof the GFP* and DHE* cells in the midgut in F) or (G). ROI: 2x 10° ym area from the RS region of the Drosophila midgut. Gutn 2 10; celln > 200. K and L Immunofluorescence images from the RS region of the midgut of the control flies (K. 38-Gola"-driven UAS-lacZ) and CIC-c-depleted flies (L,e5§-Gold" driven CIC-c RNA with EdU treatment. (M) Quantification of the number of the GFP” and EcU™ cells in the midgut in () or (U). ROL: 2 x 10* ym? area from the RS region ofthe Drosophila midgut. Gut a= 10: ROL 12220. (N-Q) Immunafluorescence analyses ofthe contro (N),CIC-c* null (0), stg-overexpressing P) and stq-averexpressing CiC-c*null(Q) MARCM clones (green in the midgut 7 days after clonal induction (AC) (R) Quantification ofthe cll number per MARCM clone in (N-Q). Each dot corresponds to one clone. Gut 210; clone n 2 30, DHE, dihydroethicium; MARCM, mosaic analysis with arepressibe cell marker: PI, propidium iodide; RO}, region of interest. DAPI-stained nuclei are shown in blue, Scale bar represents 10 ym in (A-G, K, L) and 25 ymin N-Q Error bars represent SDs. Student's e-est, *™p <(.0001. NS, non-significant (p > 0.05) i i iesg-Gald"> _GFPITUNELI A) UASJacz (A) )CIoeRNAL esg-Galte> __GFPIPL [GRATE] eso. caw _crPmHeN TC TAC 74 oy UAS-acz Cic-¢ RNAI CiC-¢ RNAI H) 0 0 rescence intercity cell RMFUVA.U) Ns. 29" and Pr celtROt ° esq" and TUNEL” cellRO! DHE Relative Mea UAS-neZ Cie RNAI ——— UASacz CIC-eRNAI UAS-rpr es-Galt> _GFPIESUN kK) UASIac2 «9 om AS IaeZ CiC-2 RNAI MARCM 2A _74ACI__cFPa N) Control Sig (R) 9” Bao 2 . 30 Poe gs oes" oe ye pathway favouring cll proliferation. Stress-activated , onised EGF mediated signaling EGFR trafficking and com : jecycled, thereby promoting cell survival and 1d survival? Therefore, inhibition of the EGER recy proliferation Early endosome damage results in the disruption way by endocytosis repressor may inhibit the activity of E:sort HUANG @Up Stab & 3 é og ® g ee 2 w erred i hee 10 2. —_ i, 3] é SF oso : 2° 3? ons } | 38 j me? j Bos 00 == j = . control §— CiC-¢ RNAI control — CIC-¢ RNAI control = CiC-c RNA 8, © © S 15: eee 1.2; shee 3 —— 3 10 =: 3 10. go Sa 0s ipo z Be o10 : Aon os : He d 3 os 3 os. Eos Bot Be oa ae conkol QO cote aD RA i FIGURE 5 Depletion of CIC-c downvegulates genes related to the EGFR signalling in the Drosophila midgut. (A) Voleana plats of differentially expressed genes between the CiC--depleted Drosophila (esg-Gal4 driven CIC-c RNAI) and contol (esg-Gal4"-driven UAS- lacZ) icguts. (2-G) Relative mRNA level of the genes related tothe EGFR signaling in the CIC-c-depleted Drosophila esg-Gold® driven (CIC-c RNAi) and control esg-Gol4-driven VAS-acZ) midgut. Error bars indicate the SD of three independent experiments, Student's t-test "p< 005, "p<0.01,"*™"p <0.0001 FIGURE 6 CiCcregulates the proliferation of intestinal stem cells through the EGFR signalling pathway. (A-D) Immuncfluorescence Images ofthe RS region of te midgut ofthe control lies (A, esg-Gald*-driven UAS-IacZ) with sucrose treatment, CI-c-depleted flies (B, 33-Gal® driven CIC-¢ RNAI) with sucrose treatment, contr lis (C, es9-Gald® driven UAS-lacZ) with BLM treatment, of CIC-c-depleted flies (D,e55-Gold* driven CiC-c RNAI with BLM treatment, stained for dark.) Quantification of the number of the dpErk.* cells in A-D. ROK: 3x 10" wm* area from the RS region of the Drosophila midgut. Gutn = 10: cell» 200. F-M) Immunofluorescence images from the RS region of the midgut of the control F, esg-Gald™ driven UAS-1acZ), CIC-c-depleted(G, esg-ald driven CIC-c RNAI, Eafr“*-overexpressing, ald driven UAS-Feh™), Egh™-overeupressing CIC-c-depleted ll, ex3-Gald® driven CIC-c RNAi with UAS-Egf™) cie- depleted), 55-Gol"-drven cic RNAI, [ec and CIC- 10: ROI n > 30.(0-R) Immunofluorescence analyses ofthe contral (0), CIC-c*-nul P, Egfr overexpressing (Q), and Eaf-overexpressing CiC-c nul (R) MARCM clones (green) in the midgut 7 days after clanal induction (ACI) (S| Quantification of the cell number per MARCM clone in (MP). Each dot corresponds to one clone. Gut n> 10; clone n » 30. ROI, region of interest: BLM, Bleomycin; MARCM, mosaic analysis with 2 repressible cell marker. DAPL-stained nuclei are shown in blue. Scale bar represents 10 um in (A-D, F=M) and 25 ym in (O-R)- Error bars represent SDs. Student's test, *"*p < 0.0001. NS, non-significant (p > 0.05)ae oe WL EY- es9-Galat> _ CFPISPEHK I (A)_UAS-JacZ 8) Clo-¢ RNAI © Sucrese LM e59-Gals™> _ GFPI fecra] OTE (©) UASIacZ = (S) CIC-ORNAL (i) UAS-Egi™ (JUAS-Egh™.CIC-cRNA) ° Pe radadah™ oe se sratalete’ SEEING a os wy ws WARCM 2A T6AGI GFE Cowal © ” B wo 3 30° * B to & ° pslocation of EGFR is endo nh 1 EGFR degradation, some re GFR can function as alsoshown that late endosomes have positive regulatory effects or and survival genes. Itcan GGFR signalling pathway. The pl4-MPI-MEKL also increase PCN) shance cellular prot located in the late endosomes, controls endoso: eration * Althous have proved thatw Pr 3 . 3 8 se spate enange) Cate RNA © ae az cou) ccna ©) = s =e — He) s gil = bee] ze OU i exo sccrge TT saoscrrown conta SCRA a i 3 8 Eo. : a? die se . soos sono TERT ST crPmAr conto) Dae RNAi Iss - He & 2 m0. 2g 2. ge 36 0 & J contol 16-¢ RNA Cic-edepietion ae serrenewal ast + Tissue Maintenance Increased Protterton and Repat IscHuaNGera. sso witey 222 FURE 7 Knocking down CIC inhibits the maturation of RabS labelled eatly endosomes and Rabi-labelled late endosomes. (A) Volcano plots of ifferentially exoressed genes between the CIC-c-depleted Drosophila es-Gald-driven CiC-c RNAi midguts and control Drosophia esg-Gal- driven UAS-acZ) midguts. @ and C) Imunofluerescence images from the RS region of the midgut ofthe control (B.e58-Gald-driven UAS-RABS-GFP) or CIC-c-depleted tes (C eg-Galé- driven UAS-RABS-GFP; CiC- RNAI) (0) Quantification ofthe RABS-GFP intensities in (8) and (C). Each dot corresponds to one cell Gutn 2 5:celln 250. (€ and F) Immunotlurescence images from the RS region of the midgut of the control [E, sg-Gald-drven UAS-RAB7-GFP) or CiC-cdepleted (F esg-Galt-driven UAS-RABT-GFP: CCC ANA) lis. (6) Quantification of the RAB7-GFP intensities in (€) and (F). Each dot corresponds to one cll. Gut n 2 5; can 2 50.(H and. 1 Immunofluorescence images from the RS region of the midgut ofthe contol (H, esg-Galt- driven UAS-RABS-GFP) or IC-c-depleted ¢-Gol-chven UAS-RABS-GFP: Cc RNAi is with BLM treatment. J) Quantification of the RABS-GFP intensties in (H) and) Each dot corresponds to one cel. Gutn 2 10; celln 2 50 (KandL) Immunetluorescence images from the RS region ofthe midgut of the control (K.e3g-Gald-criven UAS-RAB7-GFP) or CiC-c-depleted (L,esg-Galt-driven UAS-RAB7-GFP: CiC-¢ RNA) hes with BLM treatment. (M) Quantification of the RABT-GFP intensities i (K) and (U)Each dot corresponds to one cel Guta 210; celln 250. (N)A model presenting the rol of CIC in the canto of ISC proliferation. BLM, Blcomycin, DAPI-stained nucle! are shown in luc. Scale bar represents 10 um. Error bars represent SDs. Student’ test, "1p < 0.0001, NS, non-significant [p > 0.05) (of ERK signalling that is required to promote proliferation in vivo.* In addition, endocytic also affects signaling pathways that inter act with EGFR signalling pathway, such asthe JAK/STAT signaling pathway. Injury-induced JAK/STAT signalling promates cell prolt eration by activating EGFR signaling. Previous studies have shown, that blocking trafficking in distinct endosomal compartments leads to an inhibition of the JAK/STAT pathway, and the internalization and endocytic trafficking of activated Dome allows for compart rmentalized signaling to regulate subsets of Drosophila JAK/STAT transcriptional targets.” These results stronaly supports that the internalization and trafficking are both required for JAK/STAT ac tivity. Therefore, endocytosis may also affect the EGFR signaling pathway through indirect means. In summary, different stages of endocytosis inhibition can inhibit EGFR signalling activity in differ lent ways. Endocytosis is involved in the transduction of the EGFR, signal, and our experimental results showed that CIC-c deficiency inhibited endocytosis, These results suggest that CIC-c deficiency ‘may suppress the EGFR signalling by inhibiting endocytosis. How CIC-¢ deficiency inhibits endocytosis is still unclear, and thus, fur ther research is needed In conclusion this study revealed that CiC-c regulates the pro- liferation of Drosophila midgut ISCs by inducing the EGFR signaling, pathway (Figure 7N), thereby providing an important bass fo fu ther exploration ofthe regulatory role of CIC-cin adult stem cells ACKNOWLEDGEMENTS We thank BDSC, VDRC, Dr. Allan Spradling and Dr. Beniamin Oistein forthe fly strains, and DSHB forthe antibodies. This work as supported by the National Key Basic Research Program of China, (2020¥FA0803602 and 2018YFAOIOB301), the National Natural Science Foundation of China (31622031, 31671254 and 91749110, to HC) and the Guangdong Natural Science Funds for Distinguished "Young Scholar (20164030306037 to HC). This work was partly sup ported by the National Natural Science Foundation of China grants (2070798 to GW)and Basic and Applied Basic Research Foundation, ‘of Guangdong (2019151510744 to GW), and Guangdong Science and Technology Department (2020812206003). CONFLICT OF INTERESTS ‘The authors declare no competing interests AUTHOR CONTRIBUTIONS. JH and HC initiated the project and designed the research; JH, XS, ZZ, DX and KW performed the experiments, data collection an analyses. JH and XS prepared the Figures under the supervision of (GW and HC. JH and XS wrote the manuscript. GW and HC proof- read and gave advice. All the authors read and approved the final manuscript DATA AVAILABILITY STATEMENT Al the RNA-seq data of this study have been deposited in the Sequence Read Archive (SRA) under BioProject 1D PRINA7AS595, ‘The authors declare that all other data supporting the findings of this study are available within the article and its Supplementary Information (Figures 1-85, Tables S1-S3) or from the corresponding ‘author on reasonable request. orciD Haiyarg Chen © https:/orcid.org/0000-0001-8305-4060 REFERENCES 41. Guo Z, LucchettaE,RatetN, Onisten B, Maintenance ofthe alt Drocopa intestines reads lena to hameastasi. CurrOpin Genet Dev, 2016;80:61-86, 2. Miechell A, Berimon N- Evidence that stemcelisresie nthe adult Drosophila midgut epithelium, Nature. 2006:499(7075)475-479, 3. Onitein 8, Sprading A. The adult Dresepnila poste- ‘lor midgut is maintained by pluripotent stem cell. Nature 2006;499(7075)470-478, 4 Bteau B, Hechmuth CE, Jasper H. 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