Bone Morphogenetic Protein Pathway Antagonism by Grem1 Regulates Epithelial Cell Fate in Intestinal Regeneration

Background & Aims In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. Methods A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatio-temporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. Results In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. Conclusions BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease.

For gene expression analysis by quantitative polymerase chain reaction (PCR), tissues and cells were lysed, and RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA).Organoids were first extracted from collagen matrix by incubating with collagenase VIII (0.6 mg/mL in medium; Sigma-Aldrich) for 5 minutes or from Matrigel by washing with ice-cold medium, before lysis.Then, 1 cm of distal colon (irradiation experiment) was homogenized using a handheld homogenizer (Polytron; Kinematica, New York, NY).RNA was treated with deoxyribonuclease (DNase) I (Thermo Fisher Scientific).Complementary DNA (cDNA) was generated using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).To roughly normalize samples within an experiment, the same amount of RNA was taken per sample to make cDNA.Quantitative reverse transcription-PCR (qRT-PCR) was performed on the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System using Fast Universal PCR Master Mix and TaqMan Gene Expression assays (Applied Biosystems, Foster City, CA), listed in Supplementary Table 1.

RNA Sequencing
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) with on-column DNase treatment.RNA concentrations and purity were assessed using a NanoDrop One (Thermo Fisher).Samples with A260/A280 ratio <2 underwent further DNase treatment using DNase I, Amplification Grade (18068015, Thermo Fisher).RNA concentrations were measured using the Qubit RNA HS Assay Kit (Q32855, Thermo Fisher).RNA quality was assessed on a 2100 Bioanalyser Instrument (Agilent, Santa Clara, CA).Library construction and sequencing were performed at the Oxford Genomics Centre.Total RNA quantity and integrity were assessed using the Quant-IT RiboGreen RNA Assay Kit (Invitrogen, Carlsbad, CA) and the Agilent Tapestation.Messenger RNA purification, double-stranded cDNA generation, and library construction were performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490; New England Biolabs, Ipswich, MA) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L; New England Biolabs) with in-house adapters and barcode tags (using dual indexing).The concentrations used to generate the multiplex pool were determined by a Quant-iT PicoGreen dsDNA Assay (P7589; Invitrogen).The final size distribution of the pool was determined using a TapeStation system (Agilent), and quantified using a Qubit assay (Thermo Fisher).Libraries were sequenced on the NovaSeq 6000 (Illumina) as 150base pair paired-end reads.

Immunohistochemistry Analysis
Sections were deparaffinized in xylene and rehydrated through graded alcohols to water.Antigen retrieval was done by pressure cooking in 10 mmol/L citrate buffer (pH 6.0) for 5 minutes for Ki67, followed by 2 minutes incubation in 0.5% Triton-X100 in PBS.Endogenous peroxidase activity was blocked by incubating in 3% hydrogen peroxidase (in water) for 30 minutes (for TdTomato staining, hydrogen peroxidase was diluted in methanol instead of water).
For chromogenic visualization, sections were incubated with avidin-biotin complex (ABC; Vector Laboratories, Burlingame, CA) for 30 minutes and stained using DAB solution (Vector Laboratories), after which they were counterstained with hematoxylin, dehydrated, and mounted.For IHC staining after chromogenic ISH, the ImmPRESS-AP polymer reagent and ImmPACT Vector Red AP kit (Vector Laboratories) were used to develop a red signal.In case fluorescent secondary antibodies were used, sections were incubated with 4 0 ,6-diamidino-2-phenylindole (DAPI; RNAscope Multiplex Fluorescent kit v2) for 1 minute, then mounted with ProLong Gold antifade mountant (Thermo Fisher).For other fluorescent visualizations, Tyramide SuperBoost kits (Alexa Fluor 594 or 647, Thermo Fisher) were used.Alcian blue staining was performed using the standard protocol.Briefly, slides were incubated in Alcian blue for 30 minutes and washed in water.They were then incubated in nuclear fast red solution (Sigma-Aldrich) for 5 minutes and washed in running tap water for 1 minute, followed by dehydration and mounting.
Primary antibodies were incubated for 60 minutes and detected using the BOND Polymer Refine Detection System (DS9800; Leica Biosystems, Buffalo Grove, IL) according to the manufacturer's instructions, substituting DAB for the Opal fluorophores, with a 10-minute incubation time and without the hematoxylin step.Antigen retrieval at 100oC for 20 minutes, in accordance with standard Leica protocol, with Epitope Retrieval (ER) Solution 1 or 2 was performed before each primary antibody was applied.Sections were then incubated for 10 minutes with spectral DAPI (FP1490, Akoya Biosciences) and the slides mounted with VECTASHIELD Vibrance Antifade Mounting Medium (H-1700-10; Vector Laboratories).Whole-slide scans and multispectral images (MSI) were obtained on the Akoya Biosciences Vectra Polaris.Batch analysis of the MSIs from each case was performed with the inForm 2.4.8 software provided.Finally, batched analyzed MSIs were fused in HALO (Indica Labs) to produce a spectrally unmixed reconstructed whole-tissue image, ready for analysis.

Whole-Mount Antibody Staining
Colon was sectioned into 2-to 3-cm pieces, washed in 0.1% PBS-Tween for 2 days, blocked in 10% donkey serum in PBS overnight at 4 C, and incubated with DAPI (10 mg/ mL stock) in 10% donkey serum in PBS for 3 days.The tissue was finally washed with PBS-Tween for 1 day before being imaged on a TCS SP5 confocal microscope (Leica)
Small intestinal organoids isolated from steady-state wild-type mice were mechanically chopped into small pieces using a small-orifice Pasteur's pipette and seeded in Matrigel (Corning) or collagen matrix.Collagen matrix was prepared on ice by adding 10Â PBS (to make 1Â PBS) and 1N NaOH (to make 25 mmol/L NaOH) to collagen I (rat tail; Thermo Fisher).Organoids were treated with ERW medium, ERW medium with GREM1, or ERW medium with different concentrations of recombinant mouse Bmp4 protein (R&D Systems).The organoids received new medium after 2 days and were imaged and harvested for RNA analysis after 4 days of treatment.