Terminal differentiation of villus-tip enterocytes is governed by distinct members of Tgfb superfamily

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgf. Here we show that individual Bmp ligands and Tgf drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed mainly from the centre to the upper part of the villus, and it activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus-tip mesenchymal cells, and it influences the adhesive properties of villus-tip epithelial cells and the expression of antimicrobial peptides. Hence, Bmp2 promotes the terminal enterocytic differentiation at the villus-tip. Additionally, Tgf induces an epithelial gene expression programs similar to that triggered by Bmp2. The inhibition of Bmp receptor type I in vivo and using intestinal organoids lacking Smad4 revealed that Bmp2-driven villus-tip program is activated by a canonical Smad-dependent mechanism. Finally, we established an organoid cultivation system that enriches for villus-tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Altogether our data suggest that not only Bmp gradient, but also the activity of individual Bmp drives specific enterocytic programs. Intestinal crypts were initially seeded in ENR medium (without any Bmp/Tgfb). After 48 h the debris was removed: old medium was removed and matrigel domes containing organoids were broken with 1 ml plastic tip with cut-off tip and transferred into 15 ml tube containing 10 ml of ice-cold PBS. The crypts were incubated 10 min on ice, sedimented and then upper 8 ml of PBS containing cell debris was sucked out and replaced by fresh PBS. Organoids were centrifuged (300 g, 4 °C, 5 min) and put into fresh matrigel domes. Recombinant Bmps (500 ng/ml each) were added for 24. Mesenchymal cells from duodenum of PdgfraH2BeGFP mouse were isolated as described previously8,9. The duodenal tissue was harvested, opened longitudinaly and rinsed. The tissue was gently rocked for 30 minitues at room temperature in Gentle Cell Dissociation Reagent to remove epithelial cell. After the detachment of epithelium remaining pieces were digested for 35 minutes in DMEM supplelemented with 1 mg/ml collagenase D and 0,3 mg/ml dispase in thermomixer at 37°C with gentle shaking (60 rpm in horizonally oriented 50ml Falcon tube). Afterwards cells were filtered through 70m cell strainer and seeded. 24 h after seeding non-adherent cells were washed out. The cells were cultivated in MesenCult medium. The medium was changed 3 times a week. The mesenchymal cells were immortalised by transduction of (HPV16)-E6 gene using retroviral particles. For the retroviral production PlatinumE cells were transfected with pMXs-EF1-Puro retroviral construct containing (HPV16)-E6 insert re-cloned from p1322HPV-16-E6 vector. Lipofectamine 3000 and OptiMEM medium were used according to manufacturers protocol. Transfection medium was changed for cultivation medium 6 h after transfection. Viral particles were produced for 72 h. Retroviral partciles were concentrated overnight at 4 °C by RetroX concentrator and used for infection of mesenchymal cells by spinoculation (30 °C, 450 g ,1 h). 72 hours after the spinoculation the medium with viral particles was changed and cells were cells were selected with puromycin (1 μg/ml) and further expanded. The immortalized cells were sorted based on internal eGFP fluorescence intensity to eGFPlow eGFPhigh and further separatelly expanded. Occassionally,the cells were retreated with puromycin. The cells were passaged with TripleExpres solution. Total RNA was isolated using RNeasy Micro Kit (Qiagen 74004).Sequencing libraries were prepared from total RNA using the KAPA mRNA Hyperprep Kit, followed by size distribution analysis in the Agilent 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent). Libraries were sequenced in two runs of the Illumina NextSeq 500 instrument (Illumina, USA) using a 75nt single-end configuration. The complete processing of isolated RNA was done by Genomics and Bioinformatics core facility at Institute of Molecular Genetics of the Czech Academy of Sciences.