Bone Morphogenetic Protein Signaling Is Essential for Terminal Differentiation of the Intestinal Secretory Cell Lineage

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1 GASTROENTEROLOGY 2007;133: 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, and NATHALIE PERREAULT* *Canadian Institutes of Health Research Team on Digestive Epithelium, Département d Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Quebec, Canada; and the Molecular Developmental Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina See editorial on page Background & Aims: Bone morphogenetic proteins (Bmps) are morphogens known to play key roles in gastrointestinal development and pathology. Most Bmps are produced primarily by the mesenchymal compartment and activate their signaling pathways following a paracrine or autocrine route. The aim of this study was to investigate the role of epithelial Bmp signaling in intestinal morphogenesis and maintenance of adult epithelial cell functions. Methods: With the use of tissue-specific gene ablation, we generated mice lacking the Bmp receptor type IA (Bmpr1a) exclusively in the intestinal epithelium. Bmpr1a mutant and control mice were sacrificed for histology, immunofluorescence, Western blot analysis, electron microscopy, and quantitative polymerase chain reaction. Results: As well as showing increased proliferation and altered intestinal epithelial morphology, Bmpr1a mutant mice revealed that epithelial Bmp signaling is associated with impaired terminal differentiation of cells from the secretory lineage but not with the determination of cell fate. Loss of Bmp signaling exclusively in the epithelial compartment is not sufficient for the initiation of the de novo crypt phenomenon associated with juvenile polyposis syndrome. Conclusions: Epithelial Bmp signaling plays an important role in the terminal differentiation of the intestinal secretory cell lineage but not in de novo crypt formation. These findings emphasize the importance of delineating the contribution of the stroma vs the epithelium in gastrointestinal physiology and pathology. The adult intestinal mucosa is composed of undifferentiated pluripotent stem cells as well as undifferentiated daughter cells located in the lower portion of the intestinal crypt and differentiated functional epithelial cells found along the villus. The commitment of the daughter cell to differentiate is acquired in the upper third of the crypt where the cell loses its ability to divide. 1,2 There are 4 types of terminally differentiated intestinal cells derived from stem cells that can be divided into 2 categories. First are the absorptive cells, which play a role in the absorption of nutrients, and, second, cells of the secretory lineage including the mucin-secreting goblet cells, the hormone-secreting enteroendocrine cells, and the antimicrobial peptide secreting Paneth cells. 1,2 Such cellular dynamics and specification of cell fate requires a complex and precise regulation of gene expression along the anterior/posterior and crypt villus axes. 1,2 The defined expression of certain genes at a specific time or at a precise location requires a specialized microenvironment that depends on a variety of growth factors, cell cell interactions, and epithelial mesenchymal interactions. 3 5 Recent studies have identified the Hedgehog, platelet-derived growth factor, and bone morphogenetic protein (Bmp) signaling pathways as key mediators in crypt and villus morphogenesis as well as maintenance of intestinal stem cells. 4,6 8 The Bmps are morphogens belonging to the transforming growth factor superfamily. Bmps play active roles in many cellular functions in adult animals, 9 signaling through the serine/threonine kinase receptor subtypes I and II. The main Bmps found in the small intestine, Bmp2 and Bmp4, 5 perform their signaling through the type IA receptor (Bmpr1a). 9 Recently, 3 research groups generated mice with impaired Bmp signaling affecting the epithelial and stromal compartments of the gut simultaneously. 4,7,8 All showed the phenomenon of de novo crypt in the intestinal mucosa because the intestinal epithelium of these mice resembles the mucosa of patients with juvenile polyposis syndrome. This shows a significant contribution of both epithelial and mesenchymal Bmp signaling in intestinal pathology. Interestingly, a recent study by Kim et al 10 revealed that the selective loss of Smad4 in the T cell, a prominent stromal component, led to epithelial tumors throughout the gastro- Abbreviations used in this paper: Bmps, bone morphogenetic proteins; Bmpr1a, bone morphogenetic protein receptor type IA; BrdU, bromodeoxyuridine; Ngn3, Neurogenin by the AGA Institute /07/$32.00 doi: /j.gastro

2 888 AUCLAIR ET AL GASTROENTEROLOGY Vol. 133, No. 3 intestinal tract, whereas its epithelial-specific deletion did not. Deletion of Smad4 in T cells suggests that loss of Smad4-dependent signaling in cells within the epithelial microenvironment has an important role in the evolution of juvenile polyposis syndrome. However, a specific role and the molecular relevance of excluding the Bmp pathway from one particular cell compartment over the other in gastrointestinal maintenance of adult epithelial cell functions remain unknown. This study shows that the loss of Bmp signaling exclusively in the intestinal epithelium has a dramatic consequence on epithelial proliferation and terminal differentiation and maturation of cells from the secretory lineage, but not on the initiation of, de novo crypts. Materials and Methods Animals SVEV-Bmpr1a loxp/loxp mice were provided by Dr. Mishina and the C57BL/6 12.4KbVilCre transgenic line was provided by Dr. Gumucio. Both mutations were genotyped following protocols already published. 11,12 Tissue Collection, RNA Extraction, and Gene Expression Analysis Total RNA was isolated and processed using the Totally RNA extraction kit (Ambion, Austin, TX). Reverse-transcription polymerase chain reaction analysis was performed using AMV-RT (Roche Diagnostic, Laval, Quebec, Canada) following the manufacturer s protocol. Quantitative real-time polymerase chain reaction was performed using the Quantitech SYBR Green Kit from Qiagen (Mississauga, Ontario, Canada) with the Roche Diagnostic Light Cycler. Primer sequences are available on request. Tissue Preparation and Histologic Staining Jejunum were fixed in 4% paraformaldehyde overnight at 4 C, then dehydrated and embedded in paraffin. Alcian blue and H&E stainings were performed as previously described. 13 Bromodeoxyuridine Incorporation, Immunofluorescence, and In Situ Hybridization Mice were injected with 10 L of bromodeoxyuridine (BrdU) (Invitrogen, Burlington, Ontario, Canada) per gram of body weight 90 minutes before death. Immunofluorescence staining was performed as previously described. 13 The following antibodies were used: anti-brdu (1:50; Roche Diagnostic), anti-lysozyme (1:2; DakoCytomation, Mississauga, Ontario, Canada), anti phosphorylated-smad (1:200; Cell Signaling, Boston, MA), anti chromogranin A (1:2; Zymed), anti intestinal fatty acid binding protein (1:5000; kindly provided by Dr. Gordon), anti-bmpr1a (1:50; Abgent, San Diego, CA), anti activated-caspase3 (1:1500; Cell Signaling), and fluorescein isothiocyanate conjugated anti-rabbit or mouse immunoglobulin G (1:200; Vector, Burlington, Ontario, Canada). In situ hybridization was performed according to the Wilkinson 14 procedure, modified by Lantz et al, 15 and sucrase-isomaltase riboprobes were synthesized as previously described. 16 Protein Extraction and Western Blot Analysis Total protein was isolated from the jejunum as described previously. 3 Nuclear proteins were isolated using the ProteoExtract Subcellular Proteome Extraction kit (EMD Biosciences, San Diego, CA) following the manufacturer s protocol. Fifty (total) or 15 (nuclear) micrograms of protein were separated as described previously. 3 The following antibodies were used and incubated overnight at 4 C: anti c-myc (1:200), anti cyclin D1/D2 (1: 1000), anti-actin (1:5000), anti histone H1 (1:1000), antirabbit or goat horseradish peroxidase (1:3000) (all from Santa Cruz Biotech, Santa Cruz, CA), and anti -catenin (1:2500) (Cell Signaling). Quantification and Statistical Tests Images of alcian blue labeled goblet cells and chromogranin A stained enteroendocrine cells were acquired and positive cells were counted per crypt/villus axis. Nuclei were counted using 4=,6-diamidino-2-phenylindole. Variation of crypt number and crypt fission was assessed by counting the total number of crypts, villi, and crypt fissions in a blind manner. Paneth cells were quantified with lysozyme immunofluorescence. Proliferation was measured by counting BrdU-labeled cells per crypt. Statistics were calculated using the Mann Whitney or the Student 2-tailed t test. All statistical analyses were performed using Systat10 (Systat software Inc., San Jose, CA). Results Loss of Epithelial Intestinal Bmp Signaling Increases Epithelial Proliferation But Is Insufficient to Initiate De Novo Crypts To determine the specific contribution of epithelial Bmp signaling on intestinal physiology and cell function, we used the tissue-specific gene deletion of Bmpr1a using the Cre-loxP system. To delete the Bmpr1a gene exclusively in the epithelium, we crossed Bmpr1a loxp/loxp mice with a Villin-Cre transgenic line, which directs Cre expression in the epithelium of the small intestine and colon, but not in the mesenchymal compartment. 11,12 Villin-Cre;Bmpr1a loxp/loxp mice survived to at least 1 year of age and grew normally. Bmpr1a immunofluorescence of control mice (Figure 1A) revealed a strong expression in the upper portion of the crypt unit and villus, with weaker staining in cells at the bottom of the crypt (Figure 1E). Bmpr1a expression was lost exclusively in the intestinal epithelium of the

3 September 2007 BMP SIGNALING IN INTESTINAL CELL LINEAGE 889 crypt-villus axis in Villin-Cre;Bmpr1aloxP/loxP mice but not in the mesenchymal cells of the lamina propria (Figure 1B and F). Loss of Bmp signaling exclusively in the intestinal epithelium was assessed by immunofluorescence of phosphorylated-smad Phosphorylation and nuclear translocation of these Bmp effectors are features of an active Bmp signaling pathway.9 Phosphorylated-Smad immunostaining in control mice is observed mainly in villus epithelial cells, as well as at the Figure 1. Loss of Bmp signaling cascade and histologic analysis of the intestinal architecture in VillinCre;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 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). crypt villus junction, and to a lesser extent at the bottom of the crypt near the stem and Paneth cell region (Figure 1C and G). Immunoreactivity to phosphorylated-smad is lost in Villin-Cre;Bmpr1aloxP/loxP mice (Figure 1D and H). The expression pattern of phosphorylated-smad is predominantly in regions of the crypt villus axis composed of differentiating and terminally differentiated cells, suggesting a possible role for Bmp in intestinal epithelial cytodifferentiation.

4 890 AUCLAIR ET AL GASTROENTEROLOGY Vol. 133, No. 3 Histologic analysis showed that the intestine of Villin- Cre;Bmpr1a loxp/loxp mice displayed abnormal epithelial morphology with elongated villi and multiplication of crypt units (Figure 1J). Statistical analysis of the number of crypt units over the number of villi shows a significant 1.37-fold increase of crypt units (Figure 1K) invillin-cre; Bmpr1a loxp/loxp mice, suggesting a role for Bmp signaling in crypt fission. A statistical analysis of the percentage of crypt fission found in mutant compared with control animals revealed a 4.62-fold increase in crypt fission in Villin-Cre;Bmpr1a loxp/loxp mice (Figure 1L). We noticed the absence of de novo crypts along the villus as well as polyp growth in Villin-Cre;Bmpr1a loxp/loxp mice, even in 1-yearold animals. The re-initiating of crypt morphogenesis in the villi associated with juvenile polyposis syndrome has been reported in other mouse models with impaired Bmp signaling. 4,7,8 These results suggest different roles for mesenchymal as opposed to epithelial Bmp signaling in crypt morphogenesis and epithelial cell function. We next analyzed whether proliferation and/or programmed cell death were altered in these mice. Proliferation assays in Villin-Cre;Bmpr1a loxp/loxp mice showed a significant increase in the number of proliferating cells, which was not confined to the bottom but was scattered throughout the crypt (Figure 2B). Statistical analysis revealed a significant increase in proliferative cells of fold in Bmpr1a mutant mice (Figure 2E). No difference in activated caspase-3 immunostaining was observed between control and mutant mice (data not shown). These results confirmed that the thickening of the intestinal mucosa seen in the Villin-Cre;Bmpr1a loxp/loxp mice was a direct outcome of deregulation of the proliferation program of the epithelial cells. Loss of Epithelial Bmp Signaling Does Not Affect the Wnt/ -Catenin Signaling Pathway To verify the involvement of the Wnt/ -catenin signaling pathway in the deregulation of epithelial cell proliferation in Villin-Cre;Bmpr1a loxp/loxp mice, we performed Western blot analysis with nuclear epithelial intestinal extracts. As shown in Figure 2F, loss of epithelial Bmp signaling does not affect -catenin nuclear expression, suggesting no increase in Wnt/ -catenin signaling activity. We then analyzed c-myc and cyclin D1 (Figure 2G) and showed that c-myc is not modulated, whereas cyclin D1 is up-regulated in Villin-Cre;Bmpr1a loxp/loxp mice. Cyclin D1 expression is up-regulated not only by the Wnt/ -catenin pathway, but also by many other signaling pathways such as the Ras/Erk/mitogene activated protein kinase pathway. 17 Figure 2. Epithelial Bmp signaling regulates epithelial intestinal proliferation without interfering with the Wnt/ -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;Bmpr1a loxp/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 -catenin was observed between the jejunum of the Villin-Cre;Bmpr1a loxp/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).

5 September 2007 BMP SIGNALING IN INTESTINAL CELL LINEAGE 891 Bmpr1a Mutant Mice Have Impaired Terminal Differentiation of the Secretory Cell Lineage To characterize changes in the differentiation and maturation of the 4 intestinal cell types in Villin-Cre; Bmpr1a loxp/loxp mice, we stained the mucosa with specific markers for each cell type. Goblet cells were stained with alcian blue (Figure 3A and B) and counted. No significant change was found between Bmpr1a mutant mice and control littermates (Figure 3K), however, the goblet cells were consistently smaller in the mutant mice, suggesting a lack of maturity of these cells. Indeed, smaller mucigen granules and reduction in size were observed in the mutant animals (Figure 3H). No significant change was observed in the expression of the goblet cell specific marker Muc2 between the mutant and control animals (Table 1). Comparative analysis by quantitative polymerase chain reaction of both Klf4 and Tff3, factors that regulate goblet cell terminal differentiation, 18,19 revealed a decrease in expression of 3.47-fold and 2.06-fold, respectively, in the mutant animals (Table 1). Hence, these data show that mice with impaired Bmp signaling show alterations in goblet cell terminal differentiation and maturity. Bmp signaling activity at the bottom of the crypt near the stem and Paneth cell regions (Figure 1G) has been associated exclusively with stem cell renewal. 8 Herein, we observed a more compact staining of lysozyme, a Paneth cell marker, in Bmpr1a mutant mice (Figure 3D) as compared with the control (Figure 3C), however, the location 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;Bmpr1a loxp/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 m (G, H, I, and J).

6 892 AUCLAIR ET AL GASTROENTEROLOGY Vol. 133, No. 3 Table 1. Gene Expression Changes in the Jejunum of Villin- Cre;Bmpr1a loxp/loxp Mice Gene description Gene symbol Fold P Cell determination genes Math 1 Math Hairy and enhancer of split 1 Hes 1.32 NS Enterocyte markers E74-like factor 3 Elf NS Sucrase-isomaltase SI 1.75 NS Enteroendocrine cell markers Neurogenin 3 Ngn Neurogenic differentiation 1 Neurod Chromogranin A Chga Paneth cell markers Cryptdin Defcr Lysozyme Lyz Matrix metallopeptidase 7 MMP Goblet cell markers Mucin 2 Muc NS Kruppel-like factor 4 Klf Trefoil factor 3 Tff Hedgehog signaling pathway Indian Hedgehog IHH 1.49 NS GLI-Kruppel family member 1 Gli NS Patched 1 Ptc 1.89 NS NOTE. Fold changes represent the ratio of mean expression values (control/mutant). Negative values indicate reduction in Villin-Cre; Bmpr1a loxp/loxp intestines. NS, nonsignificant fold change (Mann Whitney test). of these cells was not affected by the loss of epithelial Bmp signaling. Statistical analysis revealed no significant decrease in the number of Paneth cells in the mutant animals (Figure 3K), suggesting that the determination of Paneth precursor cells is not affected by the loss of epithelial Bmp signaling. Thus, we hypothesized that Paneth cells were not differentiated fully in the mutant mice. Indeed, a reduction in the amount of Paneth cell secretory granule content was noted in mutant mice (Figure 3J). Finally, a decrease of 2.28-fold in matrilysin, 4.8-fold in lysozyme, and 5.24-fold in cryptdin messenger RNA (mrna) expressions, all important markers for Paneth cell functionality and maturation (Table 1), 20,21 was observed in the mutant mice. Expression of Indian Hedgehog, which negatively regulates the differentiation of Paneth precursor cells into fully mature cells, 22 as well as its downstream targets Gli-1 and Patched-1, were not affected by the loss of epithelial Bmp signaling (Table 1). These results support that Paneth cell terminal differentiation as well as maintenance can be linked to the instructive interaction of epithelial Bmp signaling. We next analyzed whether enteroendocrine cells were affected by the loss of epithelial Bmp signaling. Immunostaining with chromogranin A revealed fewer positive cells in Villin-Cre;Bmpr1a loxp/loxp mice (Figure 3F). We found a significant decrease of 75% of enteroendocrine cells per crypt villus axis in the mutant mice (Figure 3K), strongly suggesting that the enteroendocrine precursor is affected by the loss of epithelial Bmp signaling. Accordingly, we noted a 2.04-fold decrease in chromogranin A mrna levels in the mutant animals (Table 1). Comparative analysis by quantitative polymerase chain reaction of proenteroendocrine factors, such as Neurogenin 3 (Ngn3) and BETA2/NeuroD1, 13,23 revealed a significant decrease of 2.25-fold and 3.33-fold, respectively, in the mutant animals (Table 1). Thus, loss of epithelial Bmp signaling caused a reduction in Ngn3 and BETA2/NeuroD expression, leading to improper specification and differentiation of enteroendocrine cells. Absorptive Cell Differentiation Is Not Affected by Loss of Bmp Signaling We then investigated whether absorptive cell differentiation also was affected in Bmpr1a mutant mice. Differentiated cells of the absorptive lineage express specific markers such as intestinal fatty acid binding protein and sucrase-isomaltase. 1 In situ hybridization for sucrase-isomaltase (Figure 4A and B) and immunostaining for intestinal fatty acid binding protein (Figure 4C and D) revealed no modulation in these markers. No ultrastructural modification was noted between absorptive cells from mutant or control mice (Figure 4E and F) and no significant modulation of sucrase-isomaltase mrna levels was observed (Table 1). Thus, lack of terminal differentiation in mice with intestinal epithelial impaired Bmp signaling is restricted to cells of the secretory lineage. Bmp Signaling Does Not Affect the Secretory Precursor Cells Given the central role of hairy enhancer of split-1 and Math1 in intestinal epithelial specification, 24 and that Bmpr1a mutant mice have impaired terminal differentiation of the secretory cell lineage, we analyzed their levels of expression. A slight 1.95-fold decrease of Math1 but no significant modulation of hairy enhancer of split mrna levels were noted in the mutant animals (Table 1). Hence, loss of epithelial Bmp signaling is not sufficient to directly affect the secretory precursor cells or to change the fate of cells from the secretory to the absorptive cell lineage. Discussion Intercellular communications are decisive for intestinal morphogenesis as well as for the establishment and functional maintenance of the crypt villus axis. 3 5 Studies have identified the Bmp signaling pathway as a possible key mediator in crypt and villus morphogenesis as well as maintenance of intestinal stem cells. 4,7,8 However, the specific roles and contributions of the Bmp signaling pathway on one particular cell compartment compared with another in the maintenance of adult epithelial intestinal cell function has not yet been delineated. The current study generated a murine model with

7 September 2007 BMP SIGNALING IN INTESTINAL CELL LINEAGE 893 impaired Bmp signaling exclusively in the epithelial compartment of the intestine. In this study, we provide evidence that epithelial intestinal Bmp signaling plays a critical role in the maintenance of epithelial architecture within the crypt villus axis. We showed that loss of epithelial Bmp signaling leads to elongated villi and multiplication of the crypt units. The latter observation suggests a possible role for Bmp signaling in crypt fission. The role of Bmp signaling in stem cell renewal and consequently on crypt fission already has been addressed in previous work. 8 We have found the same occurrence in our Villin-Cre;Bmpr1a loxp/loxp mice, suggesting that the regulation of the crypt fission found at the bottom of the crypt unit is associated with epithelial Bmp signaling. Interestingly, and in major contrast with previous studies, 4,7,8 there is an absence of the de novo crypt phenomenon as well as polyp growth in Villin-Cre; Bmpr1a loxp/loxp mice even in 1-year-old animals. The key difference between the present study and those referred to previously is that herein, the Bmp signaling pathway is affected exclusively in the intestinal epithelium, whereas in previous studies both the epithelial and mesenchymal compartments were affected. These results suggest that the Bmp signaling pathway in the mesenchyme plays a significant role in crypt morphogenesis and juvenile polyposis syndrome whereas epithelial signalization plays a role in cell proliferation, stem cell renewal, 8 and maintenance of epithelial cell differentiation. We have investigated how epithelial Bmp signaling can suppress or restrain epithelial cell proliferation. Unexpectedly, we showed that the loss of epithelial Bmp signaling does not lead to an increase in -catenin nuclear expression, suggesting no increase in Wnt/ -catenin signaling activity. These results are contradictory to those recently reported by He et al, 8 who reported an increase in nuclear -catenin levels associated with an activation of target genes involved in cell-cycle progression. This increase in nuclear -catenin appears to be the consequence of the inhibition of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity in the intestinal crypt epithelium of Mx1-Cre;Bmpr1a loxp/loxp mice, resulting in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the inhibition of Gsk3 activity. He et al suggested that Bmp signaling inhibits intestinal stem cell self-renewal through 4 Figure 4. Epithelial Bmp signaling does not affect differentiation of absorptive cells. In situ hybridization revealed no variation in sucraseisomaltase 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 m (E and F).

8 894 AUCLAIR ET AL GASTROENTEROLOGY Vol. 133, No. 3 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/ -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 al 8 ). 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/ -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. activation of PTEN, leading to suppression of Wnt/ catenin signaling. However, their conclusions have raised some controversy in this field of research. Bjerknes and Cheng 25 have shown that the phosphorylated form of PTEN (p-pten)-positive cells identified by He et al 8 as intestinal stem cells are more likely to be the postmitotic secreting cells frequently found at the crypt base. After our result, we analyzed 2 known target genes of the Wnt/ -catenin pathway in our Villin-Cre;Bmpr1a loxp/loxp mice, namely c-myc and cyclin D1. Interestingly, we showed that c-myc is not modulated, whereas cyclin D1 is up-regulated in Villin-Cre;Bmpr1a loxp/loxp mice. Of note, cyclin D1 expression is enhanced not only by the Wnt/ -catenin pathway but also by many other signaling pathways such as the Ras/Erk/mitogene activated protein kinase pathway, 17 which also controls intestinal epithelial crypt cell proliferation. 26,27 Hence, one could speculate that epithelial Bmp signaling may inhibit proliferation of intestinal epithelial cells by interfering with the activation of this pathway instead of the Wnt/ -catenin pathway. In addition, epithelial Bmp signaling may directly control intestinal cell-cycle progression by modulating gene expression of cell inhibitors such as p21 Cip/Waf, p27 Kip, and p57 as reported previously in colon cancer cells 28 and in human keratinocytes. 29 Because the Bmpr1a gene ablation in the study with the Mx1-Cre;Bmpr1a loxp/loxp mice was in both the epithelial and stromal compartments of the gut, we think it is possible that the activation of the Wnt/ -catenin pathway is associated with the loss of mesenchymal Bmp signaling. Such action of the mesenchyme on the epithelial Wnt/ -catenin pathway has been shown previously and contributes to gastrointestinal epithelial tumor initiation. 3 Our study revealed an important and novel function for epithelial intestinal Bmp signaling in the proper terminal differentiation of cells from the intestinal secretory lineage. Our data suggest that key regulators of goblet cell terminal differentiation such as Tff3 and Klf4 are down-regulated in the mutant mice. Interestingly, Klf4 has been shown to be a target of Bmp signaling and to play a role in the maintenance of the vascular smooth muscle cell phenotype. 30 Here, we show that epithelial loss of Bmp signaling results in a significant decrease of Klf4 mrna and protein levels (data not shown), which leads to an important reduction in terminal differentiation of intestinal goblet cells. Our analysis exposed a dramatic decrease of 75% of enteroendocrine cells per crypt villus axis in the jejunum of Bmpr1a mutant mice as well as a significant decrease in proenteroendocrine factors such as Ngn3 and BETA2/NeuroD. These results strongly suggest that the enteroendocrine precursor cell, which is derived from the secretory precursor, is affected by the loss of epithelial Bmp signaling. This could be

9 September 2007 BMP SIGNALING IN INTESTINAL CELL LINEAGE 895 explained partially by the reduction in Ngn3 because this proenteroendocrine factor is known to play a role in the proliferation of the endocrine precursor. 13 Our data show a previously unknown role for epithelial Bmp signaling in enteroendocrine cell determination, as well as final specification through its involvement with the proenteroendocrine factors Ngn3 and BETA2/NeuroD. The Bmp signaling activity found at the bottom of the crypts thus far has been associated with stem cell renewal. 8 The differentiated Paneth cells found at the bottom of the crypts do not arise from direct differentiation of nearby stem or progenitor cells, but arrive there by the downward migration of already committed cells of the Paneth phenotype. 2 Our study shows that Paneth cell terminal differentiation can be linked to the instructive interaction of Bmp signaling. Because we observed no modulation in the number of Paneth cells in the mutant animals, this strongly suggests that the determination of Paneth precursor cells is not affected by the loss of epithelial Bmp signaling. Interestingly, Indian Hedgehog, the main representative of the hedgehog pathway in the gut, negatively regulates differentiation of Paneth precursor cells into fully mature cells. 22 The investigation of Indian Hedgehog expression revealed no significant modulation for this gene in mice with impaired epithelial Bmp signaling. Hence, Bmp signaling affects terminal differentiation and maturation of Paneth cells by a different mechanism than either the hedgehog or Wnt pathways. However, the decrease in matrilysin expression, which is also an important regulator of Paneth cell terminal differentiation, 20 could provide some explanation for a possible mechanism for the impaired Paneth cell terminal differentiation. Thus, our results reveal a distinct role for Bmp signaling at the bottom of the crypt in the terminal differentiation of Paneth cells. The phenotypes observed in the Bmpr1a mutant may suggest a greater role for Bmp signaling in the specification of intestinal precursor cells. Still, the change seen in Math1 expression in animals with impaired epithelial Bmp signaling might not be sufficient to directly affect precursor cells or to change the fate of cells from the secretory to the absorptive cell lineage. These observations clearly show that the loss of epithelial Bmp signaling affects the differentiation potential of the precursor cells, preventing them from fully acquiring their mature and functional phenotype. In summary, we show that epithelial Bmp signaling actively plays a role in terminal differentiation and maturation of cells from the secretory lineage. 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10 896 AUCLAIR ET AL GASTROENTEROLOGY Vol. 133, No Varnat F, Heggeler BB, Grisel P, et al. PPARbeta/delta regulates paneth cell differentiation via controlling the hedgehog signaling pathway. Gastroenterology 2006;131: Mutoh H, Fung BP, Naya FJ, et al. The basic helix-loop-helix transcription factor BETA2/NeuroD is expressed in mammalian enteroendocrine cells and activates secretin gene expression. Proc Natl Acad Sci U S A 1997;94: Yang Q, Bermingham NA, Finegold MJ, et al. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science 2001;294: Bjerknes M, Cheng H. Re-examination of P-PTEN staining patterns in the intestinal crypt. Nat Genet 2005;37: Rivard N, Boucher MJ, Asselin C, et al. MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells. Am J Physiol 1999;277:C652 C Aliaga JC, Deschenes C, Beaulieu JF, et al. Requirement of the MAP kinase cascade for cell cycle progression and differentiation of human intestinal cells. Am J Physiol 1999;277:G631 G Beck SE, Jung BH, Del Rosario E, et al. BMP-induced growth suppression in colon cancer cells is mediated by p21(waf1) stabilization and modulated by RAS/ERK. Cell Signal 2007;19: Gosselet FP, Magnaldo T, Culerrier RM, et al. BMP2 and BMP6 control p57(kip2) expression and cell growth arrest/terminal differentiation in normal primary human epidermal keratinocytes. Cell Signal 2007;19: King KE, Iyemere VP, Weissberg PL, et al. Kruppel-like factor 4 (KLF4/GKLF) is a target of bone morphogenetic proteins and transforming growth factor beta 1 in the regulation of vascular smooth muscle cell phenotype. J Biol Chem 2003;278: Received September 21, Accepted May 17, Address requests for reprints to: Nathalie Perreault, Département d Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, e Ave Nord, Sherbrooke, Quebec, Canada, J1H 5N4. Nathalie.Perreault@ USherbrooke.ca; fax: (819) Supported by the Canadian Institutes of Health Research and by the Natural Sciences and Engineering Research Council of Canada ( ) (N.P.) and the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (Y.M.). N.P. is a scholar from the Fonds de la recherche en santé du Québec. N.R. is a recipient of a Canadian Research Chair in Signaling and Digestive Physiopathology. The authors thank Pierre Chailler for his contribution to Figure 5, Elizabeth Herring for critical reading of the manuscript, Dr. Gumucio for providing the 12.4KbVilCre transgenic line, Dr. Gordon for providing the intestinal fatty acid binding protein antibody, and Denis Martel for assistance with electron microscopy. B.A.A. and Y.D.B. contributed equally to this work.