Volume 130, Issue 4, Pages 1324-1332 (April 2006) Developmental Characteristics of Adapting Mouse Small Intestine Crypt Cells  Christopher R. Erwin, Marcus D. Jarboe, Maureen A. Sartor, Mario Medvedovic, Keith F. Stringer, Brad W. Warner, Michael D. Bates  Gastroenterology  Volume 130, Issue 4, Pages 1324-1332 (April 2006) DOI: 10.1053/j.gastro.2006.02.019 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 LCM of intestinal crypts. (A) Crypt enterocyte isolation by LCM. The morphology of the extracted crypts and adjacent cells is maintained during capture, showing that the correct population of cells is obtained. (B) RT-PCR of cell compartment–specific genes for cDNA made from total RNA isolated from LCM-extracted crypt cells, showing the specificity of the LCM procedure for crypts. Gastroenterology 2006 130, 1324-1332DOI: (10.1053/j.gastro.2006.02.019) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Comparison of genes differentially expressed in adult, developing, immature, and adapting crypt cell (SBR) small intestine. The Venn diagram shows the size of the circle for each group proportional to the number of genes in that group. The numbers of genes that overlap between groups or that are unique to a group are indicated. The adapting crypt cells have the highest proportion of unique genes (percentage for total genes in each group: 77% vs 57% for adult and 43% for immature). Gastroenterology 2006 130, 1324-1332DOI: (10.1053/j.gastro.2006.02.019) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Comparison of the encoded protein functions of genes highly expressed in adult, developing, and immature intestine and adapting intestinal crypt cells (SBR). Functional categories were assigned as described.13 Overall, the biological functions of the proteins encoded by about 70% of the cDNAs and expressed sequence tags (ESTs) could be identified based on public databases. Gastroenterology 2006 130, 1324-1332DOI: (10.1053/j.gastro.2006.02.019) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Hierarchical clustering of gene expression profiles. Comparisons of developing intestine at E16.5 and maturing intestine at P1 or P14; adult intestinal segments of duodenum (Duo), jejunum (Jej), and ileum; and samples from whole bowel adapting intestine (sham and SBR ileum) were made with the 6 biological repeats for the LCM isolated adapting crypts. This comparison was made using the list of 300 differentially expressed adapting crypt genes. Gastroenterology 2006 130, 1324-1332DOI: (10.1053/j.gastro.2006.02.019) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 RT-PCR time course study for selected genes. RT-PCR was performed (3 repeats per group) on cDNA prepared from RNA isolated from ileal crypts cells captured by LCM from mice 1, 2, or 3 days after sham or SBR operations (2–3 for each group). The same set of selected genes was used as in Table 3. The data are presented as SBR fold difference from sham levels (sham = 1) ± SEM. Gastroenterology 2006 130, 1324-1332DOI: (10.1053/j.gastro.2006.02.019) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Microscopic assessment of protein expression in intestinal crypts using anti-Cdx, anti-CD68, and anti-Pap antibodies. Under low magnification, (A) the crypts of SBR animals (arrows) are generally indistinguishable in sections stained with H&E from those of sham animals (although deeper crypts are found in SBR, data not shown). High magnification shows (B, 4′,6-diamidino-2-phenylindole stained) crypts of sham animals with low expression of (C, red signal; same tissue section as B, developed immunofluorescently) Cdx protein. In contrast, (D, 4′,6-diamidino-2-phenylindole stained) the crypts of SBR animals have increased expression of (E, red dots; same tissue section as D) Cdx protein in a nuclear distribution. Scattered CD68-positive cells are seen bordering the crypts of both (F) sham and (G) SBR animals in similar numbers (eg, top left of panels; combined 4′,6-diamidino-2-phenylindole/immunofluorescent images). There is a paucity of Pap expression in (H) untreated compared with (I) treated animals. (Original magnification: A, 100×; B–I, 400×.) Gastroenterology 2006 130, 1324-1332DOI: (10.1053/j.gastro.2006.02.019) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Prominent putative cis-regulatory elements in common adapting and developmental genes. (A) Relative position of overlapping promoter elements for 8 genes. The common name for each gene is given and the 500 base pairs upstream of the start site are presented. (B) Sequences of the overlapping putative cis-regulatory elements are indicated, with the gene common name and position presented. Gastroenterology 2006 130, 1324-1332DOI: (10.1053/j.gastro.2006.02.019) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions