Volume 133, Issue 3, Pages 887-896 (September 2007) Bone Morphogenetic Protein Signaling Is Essential for Terminal Differentiation of the Intestinal Secretory Cell Lineage  Benoit A. Auclair, Yannick D. Benoit, Nathalie Rivard, Yuji Mishina, Nathalie Perreault  Gastroenterology  Volume 133, Issue 3, Pages 887-896 (September 2007) DOI: 10.1053/j.gastro.2007.06.066 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Loss of Bmp signaling cascade and histologic analysis of the intestinal architecture in Villin-Cre;Bmpr1aloxP/loxP mice. Bmpr1a immunofluorescence in control mice revealed a (A) strong expression in the upper portion of the crypt and villus and a (E) weaker staining in the cells at the bottom of the crypt. (B and F) Expression was lost exclusively in the intestinal epithelial cells of the crypt–villus axis in Villin-Cre;Bmpr1aloxP/loxP mice. (A and B) Note the mesenchymal cells of the lamina propria express Bmpr1a (white arrowheads). Phosphorylated-Smad 1-5-8 immunostaining in control mice is observed mainly in (C) villus epithelial cells and at the crypt–villus junction, and to a lesser extent at the (G) bottom of the crypt near the stem and Paneth cell regions. The epithelial Bmp signaling cascade was lost in the (D and H) intestinal epithelium of Villin-Cre;Bmpr1aloxP/loxP mice, (D) but not in the mesenchymal cells of the lamina propria (white arrowheads). (J) H&E staining performed on the jejunum of mutant animals shows an important increase in the length of the villi and multiplication of the crypt units, but an absence of the de novo crypt phenomenon as well as polyp growth as compared with (I) controls. (J) Note the presence of crypt fission in the mutant animal (white arrow). (K) Crypt units and (L) crypt fission were counted from the jejunum of control (n = 8) and Villin-Cre;Bmpr1aloxP/loxP (n = 8) animals. The number of crypt units and fissions were increased significantly in the mutant (Mann–Whitney; P < .05). Magnification, 200× (A, B, C, D, I, and J) and 400× (E, F, G, and H). Gastroenterology 2007 133, 887-896DOI: (10.1053/j.gastro.2007.06.066) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Epithelial Bmp signaling regulates epithelial intestinal proliferation without interfering with the Wnt/β-catenin pathway. (A and C) Proliferating cells stained by BrdU incorporation are found at the bottom of the crypts in control animals. (B and D) Mutant mice display abnormal cell proliferation. BrdU-positive cells were counted from the jejunum of control (n = 3) and Villin-Cre;Bmpr1aloxP/loxP (n = 3) animals. (E) Statistical analysis of the number of positive BrdU cells revealed a significant increase in the mutant (Mann–Whitney; P < .05). No modulation of nuclear β-catenin was observed between the jejunum of the Villin-Cre;Bmpr1aloxP/loxP and control animals. (F) Histone H1 served as loading control for the nuclear extracts. An increase of cyclin D1 but no modulation of c-myc protein levels was observed in mutant compared with control animals. (G) Actin served as the loading control for the total protein extracts. Magnification, 200× (A and B) and 400× (C and D). Gastroenterology 2007 133, 887-896DOI: (10.1053/j.gastro.2007.06.066) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Epithelial Bmp signaling is not required for the determination of the secretory precursor cell but is necessary for proper differentiation of cells from the secretory lineage. Alcian blue staining revealed no variation in the number of goblet cells between the (A) control and (B) mutant animals. However, goblet cells were consistently smaller in the (H) mutant when compared with the (G) control as confirmed by electron microscopy (n = 4). Immunostaining with an anti-lysozyme antibody suggested a reduction in Paneth cell secretory granule content in (D) mutant compared with (C) control animals. Electron microscopy analysis showed a reduction in the number of secretory granules in (J) mutant compared with (I) control animals. Immunostaining with chromogranin A revealed that the number of chromogranin A–expressing cells was decreased in the (F) mutant intestinal epithelium when compared with the (E) controls. Goblet, Paneth, and enteroendocrine cells were counted from the jejunum of control and Villin-Cre;Bmpr1aloxP/loxP animals (n = 4). (K) Statistical analysis of the number of chromogranin A–positive cells revealed a significant decrease in mutant mice but no significant variation for goblet and Paneth cells (2-tailed Student t test; P < .05). Magnification, 200× (A, B, C, D, E, and F). Bar = 10 μm (G, H, I, and J). Gastroenterology 2007 133, 887-896DOI: (10.1053/j.gastro.2007.06.066) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Epithelial Bmp signaling does not affect differentiation of absorptive cells. In situ hybridization revealed no variation in sucrase-isomaltase mRNA expression between the (A) control and (B) mutant animals. No variations in the level of intestinal fatty acid binding protein staining were seen between the (C) control and (D) mutant mice. (E and F) Electron microscopy confirmed there was no difference in the size, polarization, or morphology of absorbent cells. Magnification, 200× (A, B, C, and D). Bar = 10 μm (E and F). Gastroenterology 2007 133, 887-896DOI: (10.1053/j.gastro.2007.06.066) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 (A) Comparative representation of phenotypes observed in mice with either total or epithelium-specific loss of intestinal Bmp signaling. The total loss of Bmp in the intestine leads to multiplication and fission of crypt units, increased epithelial proliferation, deregulation of the Wnt/β-catenin pathway, presence of de novo crypts in the villus and polyps (combined phenotypes from Madison et al,4 Haramis et al,7 and He et al8). The loss of Bmp signaling exclusively in the epithelium leads to increased length of villi, multiplication, and fission of crypt units; increased epithelial proliferation; and impaired terminal differentiation of cells from the secretory lineage. We found no evidence of de novo crypts and polyps as well as no modulation of the Wnt/β-catenin pathway. (B) Epithelial Bmp signaling action on the specification of intestinal secretory cell lineages. Epithelial Bmp signaling is not essential for maintenance or determination of the secretory precursor. It is needed for the proliferation of the enteroendocrine precursor cell through regulation of Ngn3 gene expression as well as their terminal differentiation given its effect on BETA2/Neurod1 gene expression. Epithelial Bmp signaling does not interfere with the maintenance or determination of the Paneth/goblet precursor cells but affects the gene expression necessary for their respective terminal differentiation, namely MMP7, Defcr, and Lyz for Paneth cells and Klf4 and Tff3 for goblet cells. ee, enteroendocrine; g, goblet; p, Paneth. Gastroenterology 2007 133, 887-896DOI: (10.1053/j.gastro.2007.06.066) Copyright © 2007 AGA Institute Terms and Conditions