Cdx1 and Cdx2 expression during intestinal development Debra G. Silberg, Gary P. Swain, Eun Ran Suh, Peter G. Traber  Gastroenterology  Volume 119, Issue 4, Pages 961-971 (October 2000) DOI: 10.1053/gast.2000.18142 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Characterization of anti-Cdx2 antibody (1:200) by Western analysis of nuclear proteins. Protein (10 μg) from NIH 3T3 cell stable lines transfected with Cdx2 induced with IPTG and ColoDM cells showed a band at the expected size of 33–34 kilodaltons, whereas protein from NIH 3T3 cells transfected with vector alone and induced or Cdx2 uninduced did not bind the Cdx2 antibody. Gastroenterology 2000 119, 961-971DOI: (10.1053/gast.2000.18142) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Expression of Cdx1 and Cdx2 in embryonic intestine by immunohistochemistry (Cdx1 staining is brown [DAB], and Cdx2 staining is purple [NBT/BCIP]). (A–D) Blocking of anti–Cdx2-CANL antibody in mouse developing intestine 16.5 days pc. (A) Nuclear staining for Cdx2 was present in the proximal intestine when Cdx2 antibody was incubated with an irrelevant peptide. (B) Intestinal cells did not stain with antibody incubated with the CANL peptide used to generate the antibody. (C) Nuclei of the distal developing intestine did not stain with the Cdx2 antibody when incubated with the irrelevant peptide; only cytoplasmic and luminal staining was evident. (D) The staining seen in C did not block; therefore, the staining was nonspecific. (E–J) Expression at 13.5 days pc showed a clear difference in the pattern of expression of Cdx1 and Cdx2. (E and F) Expression of Cdx1 protein (brown) was detected at high levels in the nuclei of the endoderm in the distal developing intestine (G), in contrast to the very low levels in the proximal intestine, which was predominately cytoplasmic (F). (H–J) Expression of Cdx2 protein (blue) was in stark contrast to Cdx1 with intense nuclear expression in the proximal developing intestine (I) and no expression in the distal intestine (J). (K–P) Endoderm/epithelial transition at 14.5 days pc showed an increase in Cdx1 expression and maintenance of the proximal-to-distal gradient. (K–M) Cdx1 protein expression was predominately nuclear throughout the intestine, with more intense expression in the distal intestine (M) than the proximal intestine (L). (N–P) Expression of Cdx2 protein was at high levels throughout the proximal intestine (O) and decreased only at the most distal intestine (P) (in the area of most intense expression of Cdx1 [M]). (Q–T) The vertical distribution of Cdx1 is first seen at 15.5 days pc in the proximal developing intestine (Q) but not in the distal intestine (R). Cdx2 expression at 15.5 days pc is in both the crypt and villus in the proximal intestine (S) and is absent in the distal intestine (T). Original magnifications: A–D, F, G, I, J, Q–T, 400×; L, M, O, P, 200×; E, H, K, N, 40×. Gastroenterology 2000 119, 961-971DOI: (10.1053/gast.2000.18142) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 (A–X) Expression of Cdx1 and Cdx2 in postnatal (pn) tissue (Cdx1 staining is brown [DAB], and Cdx2 staining is purple [NBT/BCIP]). Immunohistochemistry of the intestine and colon from day 0 pn, birth (A–H), day 15 pn, suckling–weaning transition (I–P), and day 45 pn, adulthood (Q–X). Cdx1 expression (brown) in the proximal small intestine and jejunum at (A) day 0 pn, (I) day 15 pn, and (Q) day 45 pn was predominately localized in the nuclei of the intervillus region (day 0 pn) and crypt cells. In the distal small intestine and ileum, Cdx1 expression became progressively more localized to the crypts as the mice aged: (B) day 0 pn; (J) day 15 pn; (R) day 45 pn. Cdx1 was expressed in both the proximal (C, K, and S) and distal (D, L, and T) colon, with a gradient of expression forming in the adult (S and T) with a lower level of expression in the surface epithelium. Cdx2 expression (blue) in the proximal small intestine and jejunum at (E) day 0 pn, (M) day 15 pn, and (U) day 45 pn was heterogeneous with less expression at the tips of the villi and intervillus region (day 0 pn) and the base of the crypts. In the distal small intestine, where expression of Cdx2 was generally more intense and the villi were shorter, expression was throughout the villi with occasional nonexpressing cells and less intense expression in the intervillus region ([F] day 0 pn), or crypts ([N] day 15 pn and [V] day 45 pn). In the proximal colon ([G] day 0 pn, [O] day 15 pn, [W] day 45 p), Cdx2 protein was expressed with greater intensity toward the surface epithelium, with occasional nonexpressing cells. There was little Cdx2 expression in the distal colon at (H) day 0 pn or (P) day 15 pn; however, at day 45 pn (X), there was heterogeneous and weak expression in nuclei of the crypts, primarily toward the surface epithelium. (Y–BB) In situ hybridization of antisense probes to Cdx1 and Cdx2 messenger RNA (mRNA) in the adult ileum confirming the patterns seen in immunohistochemistry. (Y and Z) Cdx1 mRNA was localized predominantly in the crypts and (AA and BB) Cdx2 mRNA was expressed at relatively higher levels in the cells of the villi (Y and AA, hematoxylin; Z and BB, dark-field). Sense probes did not hybridize to the tissue sections (not shown). Original magnification: A–BB, 200×. Gastroenterology 2000 119, 961-971DOI: (10.1053/gast.2000.18142) Copyright © 2000 American Gastroenterological Association Terms and Conditions