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Mesenchyme-epithelial interactions in gastrointestinal development and tumorigenesis

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Janghee Woo
Abstract:
Non-cell autonomous control from mesenchymal cells profoundly affects fundamental biological processes in development and tumorigenesis. The following chapters will investigate how a single mesenchymal transcriptional factor, Barx1, can drive mouse gut development and how MMP9 in mouse intestinal mesenchyme can alter subsequent intestinal tumorigenesis. Early in gastrointestinal development, the homeobox gene Barx1 directs gut endoderm to differentiate to stomach epithelial cells through Wnt antagonism. Expression of the homeobox gene Barx1 is confined to the stomach mesenchyme and the levels drop steadily after ∼E12 until it is barely detectable in older embryos. To extend our understanding of an inductive role for Barx1 in development, I investigated an unanticipated role of Barx1 in development of the proximal (thoracic) foregut. As explained in Chapter 2, I identified severe tracheo-esophageal defects in Barx1-/- embryos and established the role of Barx1 as a negative regulator of Wnt signaling necessary for specification of esophageal epithelium. In Chapter 3, I question a specific regulation mechanism for Barx1 expression in mouse stomach mesenchyme: miRNA-associated regulation. Depletion of the miRNA-processing enzyme Dicer in cultured stomach mesenchyme and conditional Dicer gene deletion in mice significantly increased Barx1 levels, disrupted stomach and intestine development. Computational and experimental studies identified miR-7a and miR-203 repress Barx1 expression in stomach mesenchymal cells and its function in inducing gastric epithelium. Subepithelial myofibroblasts (SEMFs) reside just below the basement membrane in the intestinal lamina propria, adjacent to epithelial cells. In Chapter 4, I hypothesized that SEMFs can differentially regulate epithelial growth depending on their position along the rostral-caudal axis (small intestine versus colon). Murine ileal SEMFs enhance epithelial proliferation through secretion of MMP9. MMP9 regulates proliferation of intestinal progenitors and tumorigenesis in ApcMin mice. Extracellular MMP9 activates epithelial integrin and RTKs through laminin cleavage. Stromal MMP9, highly expressed in 16% of 642 human CRC samples, correlated with tumoral CpG island methylation and absence of KRAS mutations Properties of tumors expressing stromal MMP9 and associated growth signal activation hence identify non-cell autonomous mechanisms that may bypass or complement common CRC mutations early in intestinal tumorigenesis.

Table of Contents Chapter 1: Introduction 1 I.Mesenchyme-epithelial interactions in development and tumorigenesis 2 II. Mesenchymal transcriptional factor, Barxl in gastrointestinal development 5 III. Intestinal subepithelial myofibroblasts (ISEMFs) in intestinal physiology and tumorigenesis 11 IV. Conclusions 16 References 18 Chapter 2: Barxl-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation 25 Summary 27 Introduction 28 Results 32 Discussion 43 Experimental procedures 46 References 48 Chapter 3: Regulation of mouse stomach development and Barxl expression by specific microRNAs 51 Summary 53 Introduction 53 Results and Discussion 55 Conclusions 70 Experimental procedures 71 References 76 Chapter 4: Stroma-derived matrix metalloproteinase 9 (MMP9) drives early growth of colorectal tumors through integrin and ERBB receptor signaling 80 ix

Summary 82 Introduction 83 Results 88 Discussion 112 Experimental procedures 116 References 128 Chapter 5: Summary and discussion 137 I. Barxl and foregut development 138 II. Barxl and gastrointestinal development 140 III. Intestinal mesenchyme and tumorigenesis 145 References 152 Appendix 155 x

Chapter 1 Introduction

I. Mesenchyme-epithelial interactions in development and tumorigenesis Development: Mesenchymal cells underlying digestive and other epithelia play critical roles in development and tumorigenesis. Tissue recombination experiments (Ishizuya-Oka and Mizuno, 1984; Kedinger et al., 1986) in the early 1980's confirmed that the source of mesenchyme is a crucial determinant of gut patterning. Exposure of chick embryonic stomach endoderm to intestinal mesenchyme induces intestinal epithelium, indicating that the mensenchyme carries instructive information that can reprogram undifferentiated endoderm (Ishizuya-Oka and Mizuno, 1984). Likewise, when postnatal subepithelial myofibroblast (SEMF) cultures from rat small intestine were associated with chick embryonic gizzard endoderm, the latter went on to develop heterotopic intestinal features (Haffen et al., 1983). Furthermore, associations between colon endoderm and small intestinal mesenchyme resulted in a typical small intestinal morphology and expression of small intestine-specific genes (Cunha et al., 1985; Duluc et al., 1994). Indeed, only intestinal mesenchyme or cultured myofibroblasts can induce differentiation of cultured intestinal endodermal and crypt cells, indicating that intestinal mesenchyme and subepithelial myofibroblasts enable morphogenesis and cytodifferentiation of both adult crypt and embryonic progenitor cells (Kedinger et al., 1986; Stallmach et al., 1989). 2

Furthermore, the unspecified E7 mouse endoderm receives instructive signals, including fibroblast growth factors, and subsequently develops through its anteroposterior axis (Wells and Melton, 1999). Certain segments of foregut respond to signals secreted from adjacent heart or notochord cells that permit liver and pancreas differentiation, respectively (Jung et al., 1999; Rossi et al., 2001; Wells and Melton, 1999). Several transcription factors that are exclusively expressed in mesenchyme direct and specify endodermal fate. For example, the stomach mesenchymal transcription factor Barxl (Kim et al., 2005) specifies gastric epithelia identity through inhibition of transient Wnt signaling. FoxM (Perreault et al., 2005) and Foxf 1/2 (Ormestad et al., 2006), winged helix transcription factors expressed in the mesenchyme of the gastrointestinal tract, regulate proliferation of intestinal epithelial cells in mouse embryos as well as adult stages through BMP and Wnt signaling. Tumohgenesis: Extending the above paradigm, several studies demonstrate the powerful effects of mesenchyme/stroma on tumor initiation and progression. Genetic modification of human stromal cells before implantation of normal human mammary epithelium results in the outgrowth of benign and malignant lesion (Kuperwasser et al., 2004). Stromal over-expression of FGF10 results in prostate epithelial hyper-proliferation, which correlates with up-regulation of the androgen receptor and activation of the AKT pathway (Memarzadeh et al., 2007). A non- tumorigenic human prostatic epithelial cell line can be permanently transformed by cancer-associated fibroblasts or rat urogenital sinus mesenchyme (Ohuchida 3

et al., 2004). Senescent human fibroblasts promote the proliferation and the tumorigenesis of mutant epithelial cells, and radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interaction. (HGF-cMet) (Krtolica et al., 2001). Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF1/CXCL12 secretion (Orimo et al., 2005). /v/7-dependent neurofibromas may require a microenvironment containing Nf1+I~ and c-kit- dependent bone marrow (Yang et al., 2008). Hedgehog signaling from tumor stroma regulates tumorigenesis in Hh ligand-expressing carcinomas (Yauch et al., 2008). Deletion of vascular endothelial growth factors in myeloid cells accelerates mammary tumorigenesis (Stockmann et al., 2008). Smad4-deficient intestinal tumors recruit CCR1+ myeloid cells that promote invasion through matrix metalloproteinases MMP2 and MMP9. Mesenchymal stem cells within tumor stroma promote breast cancer metastases through interactions between chemokine ligands and receptors CCL5 and CCR5 (Karnoub et al., 2007). Mesenchymal signals in mouse intestinal tumorigenesis have been investigated in mouse models. For example, inhibition of BMP signaling in the intestinal epithelium by transgenic expression of Noggin results in the formation of numerous ectopic crypt units that lie perpendicular to the crypt-villus axis and ultimately form adenomas (Haramis et al., 2004). Conditional inactivation of Bmprla in the mouse small intestine disturbs gut epithelial regeneration, with expansion of stem and progenitor cell populations and eventual polyposis, 4

suggesting that BMP signaling controls intestinal stem cells (He et al., 2004). However, no study of intestinal tumors has demonstrated that ligands such as BMP4 from mesenchymal cells directly affects tumorigenesis. It is also unknown whether mesenchymal cells are actively involved in tumorigenesis because all studies to date have utilized an indirect approach through antagonism or genetic ablation of signal receptors in the epithelial compartment. II. Mesenchymal transcriptional factor, Band in gastrointestinal development During mouse embryonic development, the Band homeobox gene is expressed in the mesenchymal cells of molar teeth and stomach. During early stages of molar development, Barxl has an instructive role, directing the as yet undetermined ectomesenchymal cells in the proximal region of the jaws to follow a multicuspid tooth developmental pathway (Miletich et al., 2005). Band is first expressed in the predental neural crest derived mesenchyme (ectomesenchyme) in the jaw primordia, in a very restricted group of cells from which molar tooth germs will form. During subsequent development, Barxl continues to be expressed in molar dental mesenchyme and its derivatives but is not expressed during incisor development. The function of Band during molar tooth development was investigated by ectopically expressing Band in presumptive incisor tooth mesenchyme. This was achieved either by interfering with the regulation of Band gene expression (Tucker et al., 1998) or more directly by electroporating a Band expression construct. In each case the result was 5

consistent, namely that molar-like teeth developed in place of incisors (Miletich et al., 2005). Therefore, Band is implicated to have an inductive role in directing predental ectomesenchymal cells to undergo morphogenesis leading to multicuspid tooth development. Early in gastrointestinal development, expression of the homeobox gene Band is confined to the stomach mesenchyme (Kim et al., 2005). Barxl was identified as a factor that is expressed abundantly, transiently and selectively in the mesenchyme and mesothelium of the developing stomach. Band levels in the stomach peak at E13.5 and its levels became untraceable in the neonatal or adult stomach. E12 stomach endoderm co-culture with Barxl siRNA-treated mesenchyme led to loss of gastric epithelial markers and expression of intestine- specific transcripts. Enforced expression of Barxl in E12 intestinal mesenchyme attenuated intestine-specific transcripts in co-cultured intestine-derived endoderm and increases expression of the gastric markers. Band''' embryonic stomach revealed reduced stomach size, fusiform shape, fundic dysmorphogenesis and pyloric sphincter agenesis with evidence of posteriorization in the rostral Gl endoderm at E12.5. Moreover, Kim and et al. found that Barxl functions predominantly to regulate sFRP1 and sFRP2, genes that reduce local Wnt activity. Barxl deficiency in stomach mesenchymal cells reduced cellular levels of sFRP1 and sFRP2 mRNA. A Barxl-induced increase in local sFRP levels limits the extent of canonical Wnt signaling to allow stomach-specific epithelial cell differentiation. These signals act on endoderm to direct stomach epithelial 6

differentiation and distinguish the target cells from an alternative intestinal fate (Kim etal., 2005). As Band expression is exclusively mesenchymal (Kim et al., 2005; Tissier-Seta et al., 1995), the mucosal anomalies in Band'1' mice most likely originate from mesenchymal perturbations. Further mouse genetic experiments using Band'1' ;TOPGALTg confirmed that Band'1' embryos showed prominent (3-gal activity throughout the proximal foregut endoderm, a region characterized as a posteriorized stomach (Kim et al., 2007). It provided another line of evidence that Barxl functions in part to attenuate Wnt signaling in developing stomach endoderm. In addition, Barxl'1' spleen failed to appear in the usual position, apposed to the greater curvature of the stomach; instead, it was markedly hypoplastic and embedded within the dorsal pancreas. Barxl expression is reported in a columnar cell layer termed the splanchnic mesodermal plate, which is likely to correspond to the future spleen capsule, contiguous with the mesogastrium (Hecksher-Sorensen et al., 2004). Mesothelial Band expression is limited to the region surrounding the stomach, spleen and caudal surface of the liver, and does not extend into the mesenteric lining of intestinal loops. Unlike the stomach endoderm, (3-gal activity was not detected at any point in the developing spleen in TOPGALTg or Band''';TOPGA\J9 embryos. Among genes involved in spleen development and specification, Wt1 mRNA is significantly reduced in Band'1' mesothelium, whereas Band mRNA expression is preserved in the embryonic stomach and mesothelium of Wt1 mutants. Further investigations 7

B t u D Figure 1.1 Schematic representation of normal foregut endodermal morphogenesis and esophageal atresia (EA)/tracheoesophageal fistula (TEF) (Que etal., 2006) 8

suggested Barxl may specify spleen development in part through a Wnt- independent manner through Wt1 in the dorsal mesothelium (Kim et al., 2007). Kim et al. (Kim et al., 2007) found radial asymmetry with a squamous mucosa on one surface and a cuboidal epithelium on the other in proximal Band'1' foregut. Ultra-structural analysis also highlighted many cells lining the Band'1' proximal foregut extended numerous apical cilia, a characteristic feature of a respiratory epithelium. Recent studies showed Wnt/p catenin signaling in foregut endoderm specifies respiratory epithelial fate from undifferentiated foregut (Goss et al., 2009; Harris-Johnson et al., 2009). Barxl can inhibit Wnt/p catenin signaling in stomach endoderm through sFRP1 and sFRP2 in the mesenchyme (Kim et al., 2005). These observations led us to investigate the role of Barxl in foregut development to address abnormal foregut specification in Band'1' embryos and aberrant Wnt/|5 catenin signaling in the foregut, which will be discussed in Chapter 2. The primitive gut develops from the fusion of definitive endoderm with the splanchnic mesoderm, first appearing as a fold on the ventral surface of unturned mouse embryos. By the end of embryonic day (E) 9 (Fig 1.1 A), the anterior gut tube forms the foregut diverticulum, which originates from a small groove in the ventral midline endoderm and will differentiate into most of the oral cavity, pharynx, esophagus, and respiratory tract. Between E9.5 and E10 (Fig 1.1B), the 9

anterior foregut narrows to form the prospective esophagus, coinciding with appearance on the ventral aspect of the oropharyngeal floor of the laryngo tracheal groove, which extends to form the trachea (Kaufman, 1992; Que et al., 2006). While mesenchymal signals direct outgrowth of lung buds, the cells from the lung buds (green) will give rise to the bronchi and distal respiratory tree at the later stage. Reciprocal interactions between the endoderm and its adjoining mesenchyme direct patterning, morphogenesis, and maturation of these foregut- derived tissues (Kim et al., 2005; Morrisey and Hogan; Que et al., 2009). Tracheo-esophageal fistula and esophageal atresia (EA/TEF), congenital defects that occur in 1 per 2,000 to 4,000 live human births, often in association with other digestive tract anomalies (Brunner and van Bokhoven, 2005), reflect errors in patterning and morphogenesis of the anterior foregut tube (Fig 1.1D and E). The results from Band'1' embryos may be relevant to molecular defects in the tracheo-esophageal fistula and also suggest a new and unexpected role for Barxl in these processes and its mechanism of regional, non-cell autonomous inhibition of canonical Wnt signaling. How Barxl expression is regulated in gastrointestinal development remains unclear. Earlier studies showed BMP4 may regulate Band expression during tooth development. BMP4 was shown to inhibit expression of the Band and to restrict expression to the proximal, presumptive molar mesenchyme of mouse embryos at E10. The inhibition of BMP signaling early in mandible development by the action of exogenous Noggin protein resulted in ectopic Band expression 10

in the distal, presumptive incisor mesenchyme and a transformation of tooth identity from incisor to molar (Tucker et al., 1998). However, the precise temporo-spatial control over Band expression in gastrointestinal tract has not been fully understood. I will address this question, at least in part, in Chapter 3 to identify the roles of micro RNAs (miRNAs) in developing stomach mesenchyme and their Barxl regulation. III. Intestinal subepithelial myofibroblasts (ISEMFs) Principal biological processes such as cell fate specification, morphogenesis, proliferation, differentiation, cell migration and apoptosis necessitate cell-to-cell interactions. These interactions take place by contact of cells with each other or with the extracellular matrix (ECM) or through the response to soluble mediators. Although myofibroblasts were recognized morphologically a century ago, a growing body of evidence indicates that the myofibroblasts play an important role in the regulation of these fundamental processes (Powell et al., 2005). ISEMFs reside in a subepithelial location throughout the gastrointestinal tract from esophagus to anus and in the gallbladder and pancreas (Kaye et al., 1968; Komuro and Hashimoto, 1990). ISEMFs exist as a syncytium that extends throughout the lamina propria of the gut, merging with the pericytes surrounding the blood vessels (Joyce et al., 1987). In the crypts, the myofibroblasts appear to be scaphoid and oval and resemble shingles on a roof (Naftalin and Pedley, 1999). They are attached to each other by gap junctions and adherens junctions 11

throughout the syncytium. In the upper regions of the colonic crypts and in the small intestinal villi, the ISEMF take on a stellate morphology (Powell et al., 1999). The basal lamina consists of numerous fenestrations, and cell processes of the myofibroblasts or the epithelium can extend through these fenestrae (Komuro and Hashimoto, 1990). The myofibroblasts also contribute to a subepithelial sheet of reticular fibers that also contains foramina through which lymphocytes and macrophages cross over (Toyoda et al., 1997). Just below colonic epithelia, the reticular sheet is called the collagen table. Outgrowths of the myofibroblasts continue through this table and adjoin the basal lamina under the surface epithelial cells with foot processes. Therefore, the subepithelial space appears to have two fenestrated barriers: the basal lamina and the subepithelial reticular sheet in the small intestine (or collagen table in the colon), both of which are formed by connective tissue fibrils secreted by the myofibroblasts or materially contributed by the myofibroblasts themselves (Powell et al., 1999). Myofibroblasts may be defined morphologically and immunologically through identification of expressed cytoskeletal proteins. Indicated as "myo", myofibroblasts are smooth-muscle-like fibroblasts, which react to a-SM actin. ISEMF can be further distinguished from smooth muscle of the muscularis mucosae, which is also a-SM actin positive, but negative for desmin expression. Therefore, both ISEMF and muscularis mucosae stain for a-SM actin, but the 12

ISEMF is negative for desmin and positive for vimentin. On the contrary, the smooth muscle cell is positive for desmin and negative for vimentin (Pujuguet et al., 1996). It becomes clear that ISEMFs profoundly affect intestinal epithelial development and differentiation. Fibroblasts having inductive properties on epithelial differentiation and proliferation have been successfully isolated and cultured from the gut lamina propria (Fritsch et al., 1997). The cultured intestinal fibroblasts of rat intestine were as potent as embryonic intestinal mesenchyme to differentiate undifferentiated embryonic endoderm to intestinal endoderm expressing brush- border enzymes like sucrase and maltase (Haffen et al., 1983). Human T84 intestinal epithelial cells formed unorganized cell clusters within the gels, but when given fibroblast support, the T84 cell colonies organized into luminal formations, and basement membranes including laminin were well deposited. The cells in the columnar single cell-layer luminal formations were differentiated, showing microvilli, up-regulated alkaline phosphatase brush border activity, and mucin profiles typical for small intestine (Halttunen et al., 1996). In addition, primary adult human colonic subepithelial myofibroblasts induced migration, morphological and cytological differentiation in T84 colonic epithelial cells. (McKaigetal., 1999). Of interest, SEMF cell lines from different levels of rat gut (proximal jejunum, distal ileum and colon) display regional characteristics, including differential 13

expression of transforming growth factor (TGF)-(3 and hepatocyte growth factor (HGF) (Plateroti et al., 1998). Distal ileal SEMFs show higher TGF-B expression than cells from the large intestine and in the rat, HGF expression is highest in colonic SEMFs. The specific transcriptional profiles of SEMFs from different regions of the Gl tract or in different pathological conditions are maintained through culture passages (Adegboyega et al., 2002). Previous discussion illustrated how fibroblasts play a significant role in tumor initiation, progression and metastasis (Bhowmick et al., 2004). Intestinal myofibroblasts have been shown to actively participate in neoplastic process. ISEMFs are the primary mesenchymal component that reacts in colon adenoma and carcinoma as well as familial adenomatous polyposis (FAP) (Powell et al., 1999; Sappino et al., 1989). ISEMFs appear to be responsible for the desmoplastic reactions seen in many gastrointestinal tumors (Tiitta et al., 1994). Myofibroblasts were significantly associated with adenomas and well- differentiated adenocarcinomas, but less with poorly differentiated carcinomas (Yao and Tsuneyoshi, 1993). It has been proposed that tumor-associated myofibroblasts play a role in the invasion and metastasis of colorectal tumor cells. In colon carcinomas, this epithelial-mesenchymal association is physically disrupted, leading to the production of an abnormal, type IV collagen defective, basement membrane and generates the environment for cancer cells to invade the basement membrane (Martin et al., 1996). SEMFs may alter cell-to-cell adhesion protein expression and promote invasiveness of cancer cells (Dimanche-Boitrel et al., 1994). Perhaps, various secreted molecules and 14

cytokines from ISEMFs may be largely responsible for mesenchyme-associated tumorigenesis (Dignass et al., 1994; McKaig et al., 1999; Vermeulen et al.). Recently, Vermeulen et al. found myofibroblast-secreted factors including HGF enhance Wnt signaling activity and restored stem cell-like activity in colon canner cells (Vermeulen et al.). Myofibroblasts also appear to be the primary mesenchymal element found in Peutz-Jeghers syndrome (Kinzler and Vogelstein, 1998) and hamartomatous polyps seen in juvenile polyposis coli (Howe et al., 1998). Vogelstein and Kinzler classified juvenile polyposis syndrome (JPS) and hamartomatous polyps from ulcerative colitis (UC) as a group with "Landscaper defects". Patients with JPS and UC develop hamartomatous polyps in which the proliferating defective population of cells appears to be derived from the stroma. Consequently, the epithelial cells associated with the polyps are more likely to undergo neoplastic transformation, as a result of an abnormal microenvironment. Likewise, the initially normal epithelial cells associated with the inflammatory process of UC are at increased risk of neoplastic transformation (Kinzler and Vogelstein, 1998). Of interest, the stromal cells, but not the epithelial cells, of most hamartomas from JPS patients contain a clonal genetic alteration (Jacoby et al., 1997), whereas clonal genetic alterations have been demonstrated in epithelial cells, but not stromal cells, of the polyps arising in patients with familial adenomatous polyposis (FAP) (Boland et al., 1995). 15

ISEMFs play fundamental roles in intestinal development, inflammation and tumorigenesis. Of particular interest, small intestinal SEMFs induced intestine- lineage differentiation in gastric endoderm, whereas other fibroblasts from skin, lung and muscle failed to induce heterotypic differentiation (Stallmach et al., 1989). The underlying molecular mechanism remains unclear, but it is clear that ISEMFs have an inductive ability to specify endodermal fate in developmental process as embryonic stomach mesenchyme shown in Kim et al. (Kim et al., 2005). The role of myofibroblasts in tumorigenesis has been implicated in many types of cancers. Particularly, in colon cancer, myofibroblasts not only participate in neoplastic process, but appear to be the source of genetic alteration (Jacoby et al., 1997), eventually leading to epithelial transformation. Given developmental and tumorigenic implications of ISEMFs, I will try to address the following questions in Chapter 4: a difference between the mouse small and large intestinal SEMFs in supporting growth of intestinal epithelial cells and the underlying mechanism of interactions between ISEMFs and intestinal epithelial cells. IV. Conclusions Cellular processes and molecular mechanisms of embryonic development are recognized to correlate with those in cancer, and a growing body of evidence highlights various signaling, transcriptional, and metabolic pathways that are shared between embryonic development and malignant tumorigenesis. A systematic approach with gene expression profiles from colon cancers and 16

SAGE from developmental gut identified, for genes that are overexpressed in colon cancer, 8% to 19% likelihood that they were expressed transiently during gut epithelial morphogenesis in development (Hu and Shivdasani, 2005). Not only do the molecular mechanisms appear common, but the cellular interactions and behaviors also share common characteristics in developmental process and tumor progression. For example, as was learned more recently, during specific steps of embryogenesis and organ development the cells within certain epithelia appear to be plastic and thus able to move back and forth between epithelial and mesenchymal states via the processes of EMT and MET (Lee et al., 2006). The EMT process can be found in the transformed epithelia that later undergo the EMTs enabling invasion and metastasis (Kalluri and Weinberg, 2009). Many developmental processes and tumorigenic processes including EMTs require cell-to-cell interactions. Mesenchyme has been identified as the source of triggering the cellular process by providing various secreted factors or direct cellular contacts (Bhowmick et al., 2004; Kim et al., 2005; Morrisey and Hogan). Therefore, understanding the cellular processes and molecular mechanisms underlying mesenchyme-epithelial interactions in development and tumorigenesis has become particularly of interest and the rest of chapters will address these questions regarding mesenchymal roles in various biological processes: mesenchymal transcription factor, Barxl-mediated regulation in foregut development, precise expression control over Barxl in gastrointestinal development, and stromal MMP9 in intestinal tumorigenesis. 17

Full document contains 186 pages
Abstract: Non-cell autonomous control from mesenchymal cells profoundly affects fundamental biological processes in development and tumorigenesis. The following chapters will investigate how a single mesenchymal transcriptional factor, Barx1, can drive mouse gut development and how MMP9 in mouse intestinal mesenchyme can alter subsequent intestinal tumorigenesis. Early in gastrointestinal development, the homeobox gene Barx1 directs gut endoderm to differentiate to stomach epithelial cells through Wnt antagonism. Expression of the homeobox gene Barx1 is confined to the stomach mesenchyme and the levels drop steadily after ∼E12 until it is barely detectable in older embryos. To extend our understanding of an inductive role for Barx1 in development, I investigated an unanticipated role of Barx1 in development of the proximal (thoracic) foregut. As explained in Chapter 2, I identified severe tracheo-esophageal defects in Barx1-/- embryos and established the role of Barx1 as a negative regulator of Wnt signaling necessary for specification of esophageal epithelium. In Chapter 3, I question a specific regulation mechanism for Barx1 expression in mouse stomach mesenchyme: miRNA-associated regulation. Depletion of the miRNA-processing enzyme Dicer in cultured stomach mesenchyme and conditional Dicer gene deletion in mice significantly increased Barx1 levels, disrupted stomach and intestine development. Computational and experimental studies identified miR-7a and miR-203 repress Barx1 expression in stomach mesenchymal cells and its function in inducing gastric epithelium. Subepithelial myofibroblasts (SEMFs) reside just below the basement membrane in the intestinal lamina propria, adjacent to epithelial cells. In Chapter 4, I hypothesized that SEMFs can differentially regulate epithelial growth depending on their position along the rostral-caudal axis (small intestine versus colon). Murine ileal SEMFs enhance epithelial proliferation through secretion of MMP9. MMP9 regulates proliferation of intestinal progenitors and tumorigenesis in ApcMin mice. Extracellular MMP9 activates epithelial integrin and RTKs through laminin cleavage. Stromal MMP9, highly expressed in 16% of 642 human CRC samples, correlated with tumoral CpG island methylation and absence of KRAS mutations Properties of tumors expressing stromal MMP9 and associated growth signal activation hence identify non-cell autonomous mechanisms that may bypass or complement common CRC mutations early in intestinal tumorigenesis.