LRH-1 mitigates intestinal inflammatory disease by maintaining epithelial homeostasis and cell survival

Epithelial dysfunction and crypt destruction are defining features of inflammatory bowel disease (IBD). However, current IBD therapies targeting epithelial dysfunction are lacking. The nuclear receptor LRH-1 (NR5A2) is expressed in intestinal epithelium and thought to contribute to epithelial renewal. Here we show that LRH-1 maintains intestinal epithelial health and protects against inflammatory damage. Knocking out LRH-1 in murine intestinal organoids reduces Notch signaling, increases crypt cell death, distorts the cellular composition of the epithelium, and weakens the epithelial barrier. Human LRH-1 (hLRH-1) rescues epithelial integrity and when overexpressed, mitigates inflammatory damage in murine and human intestinal organoids, including those derived from IBD patients. Finally, hLRH-1 greatly reduces disease severity in T-cell-mediated murine colitis. Together with the failure of a ligand-incompetent hLRH-1 mutant to protect against TNFα-damage, these findings provide compelling evidence that hLRH-1 mediates epithelial homeostasis and is an attractive target for intestinal disease.

I nflammatory bowel disease (IBD) is a chronic disorder that is characterized by bouts of intense gastrointestinal inflammation, ultimately resulting in destruction of the epithelial lining of the gut 1 . Although defects in genes expressed in the gut epithelium have been associated with IBD 2,3 , the contribution of the epithelium to this disease remains understudied, particularly in comparison to the intensive interrogation of the immune component. However, the recent establishment of mouse and human intestinal organoids has provided an excellent experimental platform to explore intrinsic epithelial defects in patients and mouse models with disease 4,5 .
An important regulatory factor for intestinal epithelia is Liver Receptor Homolog 1 (LRH-1, NR5A2). This nuclear receptor has been shown to be expressed in intestinal crypts, where intestinal stem cells (ISCs) reside 6 , and where it contributes to epithelial renewal by potentiating WNT/β-catenin signaling [6][7][8] . Recent GWAS meta-analyses of IBD patients found a significant association between LRH-1 and IBD 9,10 . Animal studies using heterozygous (Lrh-1 +/− ) or conditional knockout (Lrh-1 fl/fl ; VilCreERT2) did not note any apparent epithelial defects at baseline, but did report a defect in epithelial proliferation and susceptibility to colitis 6,11 . Interestingly, the elimination of LRH-1 in mouse intestine and human colon cancer cell lines resulted in decreased glucocorticoid production [12][13][14] , which has the potential to lead to the kind of increased intestinal inflammation observed in mouse models of colitis 11,13 . This atypical nuclear receptor contains a well-ordered hormone-binding pocket, which binds signaling phospholipids including phosphoinositides [15][16][17][18] . However, structural and biochemical studies have revealed major differences between the human and mouse orthologs; hLRH-1 manifests a greater ligand-binding dependency 15,17,19 .
Here we investigate the physiological and pathophysiological function of hLRH-1 in the intestinal epithelium. Using humanized mouse intestinal organoids, a humanized in vivo IBD model, and human intestinal organoids, we uncover an essential role for LRH-1 in intestinal epithelial homeostasis and cell survival, which mitigates inflammatory injury. Our data rationalize efforts required to target this nuclear receptor for the treatment of IBD.

Results
LRH-1 maintains epithelial integrity and viability. In order to investigate the role of LRH-1 in gut epithelia, LRH-1 expression and the effects of its deletion were determined in mouse intestinal organoids. Similar to prior in vivo studies 6 , mLrh-1 was found in the crypt domain of intestinal organoids, but was also detected at lower levels in the villus domain (Fig. 1a). Using Lrh-1 fl/fl ;Vil-CreERT2 (Lrh1 IEC-KO ) mice, intestinal organoids were generated following conditional and acute deletion of mLRH-1 (Fig. 1b). Consistent with the proposed role for LRH-1 in Wnt/β−cateninregulated cell growth 6 , deletion of mLRH-1 increased cell death and lowered organoid viability in a modified MTT reduction assay 20 , compared to control organoids from Lrh1 fl/fl mice (Fig. 1c).
Transcriptional profiling of Lrh1 fl/fl and Lrh1 IEC-KO intestinal organoids revealed significant gene changes in cell survival and apoptosis pathways (Fig. 1d, e), suggesting a role for LRH-1 in intestinal epithelial homeostasis and viability. Consistent with this notion, a marked increase in activated Caspase 3 (Casp-3) was observed in the intestinal crypt domain of Lrh1 IEC-KO organoids, which was further exacerbated by TNFα (Fig. 1f, g). As expected, given the documented role of LRH-1 in intestinal epithelial proliferation 6,21 , Lrh1 IEC-KO intestinal organoids exhibited decreased cell proliferation measured by 5-ethynyl-2deoxyuridine (EdU) incorporation (Supplementary Figure 1).
Because epithelial damage is a major contributor to chronic inflammatory disease in IBD 22 , we investigated whether loss of LRH-1 compromises the epithelial barrier. Indeed, significant failure of the epithelial barrier was observed in Lrh1 IEC-KO intestinal organoids using a vital dye exclusion assay (Fig. 1h). Together, these data support an essential role for LRH-1 in epithelial viability and resilience.
LRH-1 affects crypt survival and differentiation via Notch. Notch expression in the intestinal crypt preserves LGR5 + stem cells while restricting secretory lineages and is critical for ISC survival 23,24 . We then asked if the observed crypt cell death in Lrh1 IEC-KO organoids might arise from impairment in Notch signaling. Indeed, both Notch1 transcripts and protein levels were diminished in Lrh1 IEC-KO organoids (Fig. 2a, b). Because Notch is also a key factor in epithelial differentiation 23,24 , cell numbers and markers for Paneth, goblet, and enteroendocrine cells (EECs) were assessed after loss of LRH-1. As expected, lowered Notch signaling in Lrh1 IEC-KO organoids resulted in downregulation of the stem cell markers Lgr5 and Olfm4, while leading to upregulation of Lys and Muc-2; two respective markers for secretory Paneth and goblet cells (Fig. 2b). The number of goblet cells doubled in Lrh1 IEC-KO intestines, and Paneth cells were visibly expanded in intestinal crypts (Fig. 2c, d). Surprisingly, rather than observing an expansion of EECs, as previously described with Notch inhibition 23,24 , the number of enterochromaffin cells, a representative sub-population of EEC cells, was significantly reduced in Lrh1 IEC-KO intestine (Fig. 2c, d), as were levels of EECspecific transcripts (Fig. 1d). Collectively, these data imply that LRH-1 is necessary for maintenance of Notch signaling and cell survival and for proper allotment of intestinal epithelial cell types.
Human LRH-1 prevents intestinal crypt death and TNFα injury. We next asked whether restoring or overexpressing LRH-1 might strengthen epithelial resilience to an inflammatory challenge. Human LRH-1 (hLRH-1), rather than mLRH-1, was chosen because it displays greater ligand-dependent activation and is the relevant isoform in human disease. As illustrated in Fig. 3a, hLRH-1, unlike mLRH-1, lacks the salt bridge at the mouth of the ligand-binding pocket and requires a positive charge to stabilize this domain 15,25 . This role is fulfilled by the phosphate in the polar head group of its phospholipid ligand. Expression of hLRH-1 in mouse intestinal organoids was achieved by an AAV8-mediated infection protocol, which was optimized using AAV8-GFP. This method resulted in rapid and efficient gene expression (Fig. 3b) that persisted for the life of the epithelial cell (Supplementary Figure 2) and permitted dosing that could either match or exceed endogenous levels of mLRH-1 (Fig. 3c, left). Nuclear expression of hLRH-1 was detected throughout the crypt and villus zones (Fig. 3b).
Human LRH-1 expression was able to fully rescue crypt cell death and maintain viability in Lrh1 IEC-KO organoids even upon challenge by TNFα (Fig. 3d-f). To ascertain if ligand binding is necessary for hLRH-1-mediated rescue, we next attempted to salvage organoid viability with the well-characterized ligandbinding-defective variant of hLRH-1 (PM; Fig. 3a) 26,27 . Bulky hydrophobic residues were modeled in the binding pocket to impede ligand uptake without effecting protein integrity, as previously demonstrated in cultured cell lines 26 . Indeed, the human PM variant is stably expressed in intestinal organoids (Fig. 3c, right), and despite the fact that the hPM retains modest transcriptional activity 26,27 , it failed to rescue TNFα-induced cell death in Lrh1 IEC-KO intestinal organoids (Fig. 3f, light blue bar).
Expressing hLRH-1 resulted in upregulation of known downstream targets including Shp, Cyp11a1, and Cyp11b1 as well as robust expression of new potential LRH-1 targets (Ctrb1 and Smcp), and mediators of cell survival and anti-inflammatory responses, including the decoy receptors Il1rn and Tnfrsf23 and the antiapoptotic factor Hmox1 ( Fig. 3g and Supplementary  Figures 3 and 4). Adding hLRH-1 to Lrh1 IEC-KO organoids also restored the integrity of the epithelial barrier, as demonstrated by a reduction of vital dye-positive organoids (Fig. 3h). Moreover, increasing the dosage of hLRH-1 in the presence of wild-type mLRH-1 ameliorated TNFα-induced cell death, suggesting that elevated LRH-1 activity protects against inflammatory damage (Fig. 3i, left). This effect extends to other epithelial insults, as overexpression of hLRH-1 also protected against damage by fluorouracil (5-FU), a common chemotherapeutic with intestinal toxicity (Fig. 3i, right). Taken together, these data demonstrate that hLRH-1 fully substitutes for mLRH-1 to restore cell survival and activate anti-inflammatory programs.
To confirm the survival role of LRH-1 in vivo, we used a humanized intestinal mouse model in which mLRH-1 is deleted and hLRH-1 expressed in an inducible Cre-dependent manner (referred to as hLRH1 IEC-Flex ). Despite the lower protein levels of hLRH-1 as compared to endogenous mLRH-1 in hLRH1 IEC-Flex organoids (Fig. 4a), expressing hLRH-1 reduced cell death by nearly 50% and restored Ctrb1 levels (Fig. 4b, c). These ex vivo results were confirmed in vivo by the near absence of cleaved Casp3 in intestinal crypts of the ileum (and to a lesser extent in villi) in hLRH1 IEC-Flex mice compared to Lrh1 IEC-KO (Fig. 4d).
Taken together, these data reveal that human LRH-1 can promote cell survival in murine intestinal epithelia lacking mLRH-1 and following challenge by TNFα.

LRH-1 protects human intestinal organoids from TNFα injury.
To determine whether the anti-inflammatory and prosurvival activity of LRH-1 translates to the human intestinal epithelium, human small intestinal organoids were derived from endoscopic biopsy samples of ileum from both Crohn disease patients and healthy individuals (Fig. 6a). Unlike murine small intestinal organoid cultures, human-derived organoids are maintained in a partially differentiated state, consisting of intestinal stem cells and partially differentiated Paneth cells, and undergo differentiation following WNT withdrawal 4 . Human Lrh1 is expressed at similar levels in both differentiated and undifferentiated human organoids (Supplementary Figure 5), and remains broadly distributed throughout the epithelium following differentiation, as confirmed by staining for secretory goblet (MUC2) and Paneth (LYZ) cells (Fig. 6b). This pattern closely matches the broad distribution of murine Lrh1 (Fig. 1a).
Increasing hLRH-1 dosage by AAV-mediated infection caused an upregulation of the LRH-1 target Ctrb1 (Fig. 6c). Importantly, in human organoids from both healthy individuals and Crohn disease patients, overexpression of hLRH-1 abrogated TNFαinduced cell death (Fig. 6d). Taking all the data in this study together, we conclude that LRH-1 plays an essential role in intestinal homeostasis and in ameliorating inflammation-induced injury in human intestinal epithelia.

Discussion
In this study, using multiple independent mouse and human ex vivo and in vivo intestinal models, we establish that the nuclear receptor LRH-1 (Nr5a2) has a crucial role in maintaining the intestinal epithelium. Acutely knocking out mLRH-1 resulted in decreased Notch signaling and increased cell death in the intestinal crypt. Importantly, humanization of the mouse intestinal epithelium by expression of hLRH-1 corrected these deficits. Moreover, overexpression of hLRH-1 in both mouse and human intestinal organoids imparted epithelial resistance to both TNFα, a major inflammatory cytokine in IBD, and 5-FU, a chemotherapeutic with intestinal toxicity. In the intact animal, expression of hLRH-1 ameliorated immune-mediated colitis. Using a viralmediated approach newly applied to intestinal organoids, we showed that efficient rescue by hLRH-1 is ligand dependent. These findings are important because they provide a compelling argument that drug targeting of LRH-1 could enhance resistance to inflammation and restore intestinal epithelial health in intestinal diseases such as IBD. Our study extends prior studies, reporting impaired cell renewal and enhanced chemical-induced colitis in heterozygous and conditional knockout mice, by demonstrating both a fundamental role for LRH-1 in the maintenance of epithelial viability and cell types, and the therapeutic potential of LRH-1 in intestinal disease. Further, we reveal that acute loss of mLRH-1 disrupts Notch expression, triggers increased apoptosis in the crypt and results in a breach in the epithelial barrier. Our data are consistent with the findings by Samuelson and colleagues that attenuating Notch signaling by genetic or pharmacological methods resulted in significant ISC apoptosis, crypt disruption, and expansion of secretory lineages. Interestingly, loss of LRH-1 not only recapitulates these findings, but also appears to compromise cells outside of the crypt base. We hypothesize that intestinal crypt apoptosis hinders renewal of the intestinal lining and exacerbates the immune inflammatory response 3 . In support of this idea, we demonstrate that loss of intestinal LRH-1 expression is associated with diminished animal survival and increased intestinal inflammation in the T-cell transfer model of colitis. Importantly, increased hLRH-1 has a clear beneficial impact on disease activity and colitis scores, consistent with our organoid models.
The ability to acutely knock out mLRH-1 and replenish with hLRH-1 in intestinal organoids has provided new insights into the identity and function of species-specific LRH-1 targets in the small intestine. Based on the rapid crypt cell death and spectrum of differentially expressed genes, cell survival is the most prominent pathway affected following acute loss of mLRH-1. Interestingly, despite the known species difference in ligand binding 15  upregulates anti-inflammatory genes. hLRH-1 enhances Il1rn and Tnfrsf23, which act as decoy receptors for circulating proinflammatory cytokines. Rdh9 (retinol dehydrogenase) was also upregulated threefold by hLRH-1; interestingly, this gene is also increased in the intestine of conventional as opposed to germ-free mice 28 . Ctrb1, encoding chymotrypsin, and Smcp appear to be two highly sensitive markers of LRH-1 activity that are robustly activated by hLRH-1 in both mice and human intestinal organoids. It is known that LRH-1 binds the proximal promoter of Ctrb1 29 , raising the possibility that fecal chymotrypsin may serve as a biomarker to assess and follow pharmacological manipulation of hLRH-1 activity in vivo. Finally, ex vivo and in vivo mouse models of enteritis, in which mLRH-1 is replaced with the human form that binds signaling phospholipids more efficiently 15,26,30 , will provide a valuable platform to test and probe the utility of any synthetic ligands. Our data suggest that LRH-1 may play a previously unappreciated role in epithelial cell differentiation in the intestine, in addition to ISC maintenance. Indeed, a similar role for LRH-1 has been reported in the pancreas 29,31 and recently in neural stem cells 32 . Interestingly, although we observe an increase in secretory Paneth and goblet cells, consistent with reduction of Notch signaling, loss of LRH-1 also leads to a significant drop in markers defining nearly all subclasses of EECs 33 . These data infer separate but positive roles for LRH-1 in Notch intestinal crypt signaling and EEC differentiation. Intriguingly, the latter effect may be regional, with the greatest loss of EEC cells following deletion of LRH-1 in the ileum and proximal colon (J.B. and H.I., unpublished data); both of which exhibit high LRH-1 expression 6 . An understanding of how and where LRH-1 promotes lineage commitment in the gastrointestinal tract remains to be determined.
An important unanswered question is whether increased LRH-1 expression drives unchecked proliferation and promotes dysplasia in the intestinal epithelium, as suggested previously. Indeed, an earlier study showed that LRH-1 haploinsufficiency reduced tumor burden in the APC MIN  . All values are normalized to five independent wells of untreated Lrh1 fl/fl enteroids, which is taken to be 0%. d Immunofluorescence of wild-type (Lrh1 fl/fl ), Lrh1 IEC-KO , and hLrh1 IEC-Flex ileum from adult male mice treated with two consecutive injections of tamoxifen. Staining for activated Casp3 (red) and CD44 (green), which marks intestinal epithelial crypt cells, is shown at lower (first column) and higher (second column) magnification. The appearance of apoptotic cells is indicated in the crypt region as well as the villus (white arrowheads and dashed white line) in Lrh1 IEC-KO ileum; some signal is also observed after expressing hLRH-1 in hLrh1 IEC-Flex . Scale bars = 50 μm. N = 2 per genotype. For panels b and c error bars are SEM with statistical analyses determined by Student's unpaired t test, two tailed with p values of *p = < 0.05, **p = < 0.01, and ****p = < 0.0001 promoters 8 , but this same study noted decreased Lrh1 in intestinal tumors. While our studies with the nonreplicating AAV vector preclude us from exploring the question of cell proliferation in infected intestinal organoids, we note that neither Cyclin D1 nor Cyclin E1 were changed after loss of mLRH-1 or expression of hLRH-1 (Supplementary Figure 4). The findings presented here leverage a new use for AAVdirected gene expression to rapidly manipulate small intestinal organoids. Positive features of AAV infection, versus classical lentiviral transduction, are the high infectivity, rapid onset of gene expression, and the ability to infect large organoid fragments. On the other hand, the nonreplicating nature of AAV restricts expression to the typical~7 days turnover for mouse intestinal organoids. Nonetheless, this system allows a rapid structure−function analysis by simultaneously knocking out and adding back variants, which we put to effective use in this study to show the ligand-dependency of hLRH-1 effects. As unique molecular signatures of intestinal epithelial subtypes continue to emerge 33,34 , engineering cell-specific promoters into the AAVsystem should allow a more granular functional assessment of individual intestinal epithelial cell types.
In summary, our study of human intestinal organoids, humanized murine intestinal organoids, and a humanized murine IBD model show that LRH-1 promotes normal intestinal epithelial homeostasis and can be leveraged protectively against intestinal inflammation.

Methods
Study approval. Animal studies were conducted in accordance with IACUC guidelines in strict accordance with the recommendations in the Guide for the Care Clinical DAI and colon colitis scores: To assess the clinical DAI body weight loss, diarrhea, guaiac-positive hematochezia, and appearance were monitored daily during the experiment. The DAI was determined according to a published scoring system 36 (Supplementary Table 1). For colon histological analysis, the colon was divided into three segments (proximal third, middle third, and distal third). Each segment was embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. Histological analysis was performed in the Cellular and Molecular Morphology Core of the Digestive Disease Center at Baylor College of Medicine. The sections were blindly scored using a standard histologic colitis score 37 . Three independent parameters were measured: severity of inflammation (0-3: none, slight, moderate, severe), depth of injury (0-3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0-4: none, basal one-third damaged, basal two-thirds damaged, only surface epithelium intact, entire crypt and epithelium lost). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1, 0-25%; ×2, 26-50%; ×3, 51-75%; ×4, 76-100%) averaged per colon. Human intestinal organoid culture. Endoscopic biopsy samples obtained from the terminal ileum were processed under a dissecting microscope to liberate intestinal crypts using EDTA chelation and mechanical disruption. Crypts were screened through a 100 μm filter, centrifuged, and suspended in ice-cold Matrigel. The suspension was plated on prewarmed cell culture plates. Following polymerization of Matrigel, propagation media (50% LWRN conditioned media from ATCC CRL-3276, supplemented with human EGF (Peprotech), A-83-01 (Tocris), SB202190 (Sigma), Gastrin (Sigma), Nicotinamide (Sigma), B27 (Life Technologies), N2 (Life Technologies), GlutaMax (Life Technologies), and HEPES (Sigma) in F12 Advanced DMEM (Life Technologies)) was added. For the first 48 h of culture, CHIR99021 (Stemgent) and thiazovin (Stemgent) were added to support stem cell growth. To induce differentiation, media was replaced with differentiation media (consisting of base culture media supplemented with 10% R-Spondin conditioned media, human EGF (Peprotech), human Noggin (Peprotech), A-83-01, Gastrin, NAC, B27, and N2). b Immunofluorescence for LRH-1 (green, top panels) in human intestinal organoid sections shows expression throughout the organoid with strongest expression occurring in the crypt domain (yellow dashed box and zoomed image, right). Differentiation markers for Paneth (Lyz, left) and goblet (Muc2, right) cells are shown below. Scale bar = 100 μm. c Expression of LRH-1 target gene Ctrb1 in human intestinal organoids is upregulated 72 h after infection with AAV-hLRH1 (blue) but not with control AAV (black). d Overexpression of hLRH-1 by AAV confers resistance to TNFα-mediated cell death. Human organoids from healthy donor and a Crohn disease patient were infected with AAV-hLRH (blue) or AAV-Control (black) (3.3×10 10 genome copies) for 72 h under differentiation conditions and then exposed to TNFα (20 ng/ml, 40 h). Data represent an N of at least three replicates with~50 organoids per well. For panels c and d error bars are SEM with statistical analyses determined by Student's unpaired t test, two tailed with p values of *p = < 0.05 columns (Zymo Research). DNAse-treated total RNA was used to generate cDNA using Superscript II (Invitrogen). Sybr green-based qPCR (Quanta) was performed on an Applied Biosystems Model 7900HT with primers as per Supplementary  Table 2. The ΔΔCt method was used for calculation of gene expression using Gapdh as reference. For RNA-Seq studies, RNA was isolated on Day 4 following AAV infection. Ovation RNA-Seq System V2 (NuGEN) was used to generate the cDNA library for sequencing on an Illumina HiSeq 4000. Data were analyzed using the GALAXY program suite 38 . Pathway analysis and annotations were performed with Ingenuity IPA (Qiagen) and Genecodis 39 , respectively. For colitis experiments, total RNA was isolated from snap-frozen colon tissues using Trizol Reagent (Invitrogen) and cDNA prepared with qScriptTM cDNA Synthesis Kit (Quanta Biosciences). Colonic gene expression was determined by qPCR using SYBR Green master (Kapa Biosystems Inc.). mRNA levels were normalized by the 36B4 gene expression. Prevalidated primers for qPCR were purchased from Qiagen (https:// www.qiagen.com/geneglobe/default.aspx).
Viability and proliferation assays. Murine intestinal organoids were plated in 10 μl Matrigel drops onto a prewarmed 96-well cell culture plate and following polymerization 100 μl prewarmed organoid growth media was added. Following Cre activation with 300 nM 4OH-tamoxifen, cultures were incubated with mTNFα for 40 h. Viability was assessed by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction assay as described 18 . Briefly, intestinal organoids were incubated with 500 μg/ml MTT for 2 h at 37°C. Media was aspirated and Matrigel dissolved in 2% SDS for 2 h at 37°C with shaking. MTT was then solubilized in DMSO and absorbance measured at OD 562 . To normalize for crypt seeding and background drop-out, data were normalized by resazurin where indicated in the text. Here, intestinal organoids were incubated with resazurin (10 μg/ml) for 6 h prior to administration of mTNFα. Media was removed and fluorescence measured (excitation 530 nm, emission 590 nm) and used to normalize MTT values 20 . Experiments were repeated a minimum of three times with five replicates per experiment. For human intestinal organoids, plates were set as above and organoids grown initially in propagation media for 24 h to establish organoids and then switched to differentiation media for the remainder of the experiment. Human TNFα was added after 72 h. Viability was determined by MTT 40 h after TNFα administration.
For proliferation studies, intestinal organoid cultures were incubated with EdU (10 μM) for 2 h and then fixed in 4% paraformaldehyde. Click-It chemistry was performed as per the manufacturer's recommendations on 5 μm cryosections (Life Technologies). For 5-FU experiment, organoids were incubated with 5-FU (5 μg/ml in DMSO; Millipore) for 24 h and viability determined as above.
Dextran exclusion assay. Intestinal organoids were exposed to mTNFα for 24 h and then incubated in 1 mg/ml Texas Red labeled dextran (average weight 10 kDa; Life Technologies) for 30 min. Following incubation, excess dye was removed by serial washes with PBS and the plate imaged immediately. Wells were scored for fraction of dye-retaining intestinal organoids. 30-50 intestinal organoids were seeded per well. Eight wells per experiment were scored for each condition. Opened intestinal organoids were excluded from analysis. Results were validated by a blinded, independent observer. AAV-directed gene expression. AAV viral particles expressing hLRH1 or GFP under direction of the thyroxine-binding globulin promoter were obtained from the University of Pennsylvania Viral Core. Intestinal organoids were isolated in cold PBS, pelleted at 1000 × g, and resuspended in ice-cold Matrigel. The mixture was added to chilled Eppendorf tubes containing virus on wet ice and then aliquoted immediately onto prewarmed cell culture plates. After Matrigel was set, organoid growth media was added.
Imaging. Live cell and intestinal organoid immunofluorescence imaging was performed on an Olympus IX51 microscope equipped with a DP71 imager. Mouse intestinal imaging was obtained on a Nikon Eclipse Ti equipped with a DS-Qi2 imager or an Olympus BX40 microscope with Magnafire imager.

Data availability
Deep sequencing data are archived under GEO accession number GSE116563. Reagents including mouse and organoid lines will be made available by request.