Embryonic development and patterning of the stomach

through secretion of chemokines which induce the recruitment of lymphocytes to the side of inflammation (Bernardo and Fibbe 2013). Amongst others, these are e.g. TGFβ, HGF, prostaglandin-E2 (PGE-2) or programmed death-ligand 1 (PD-L1) activating immunosuppressive immune cells like regulatory T cells (Treg), macrophages or B cells (Di Nicola et al. 2002; Aggarwal and Pittenger 2005; Augello et al. 2005). Furthermore, in many tissues, stromal cells continuously express IL33 and IDO1 under homeostatic conditions to nourish Tregs and innate lymphoid cells (ILC) and hamper T cell proliferation (Buechler and Turley 2018). On the other hand, MSCs secrete pro-inflammatory chemokines and cytokines like interleukin (IL) 1β, IL6, IL7, IL8 or chemokine ligand 2 (CCL2) to attract and activate immune cells (Hoogduijn 2015;

Romieu-Mourez et al. 2009). Although MSCs do not function as classical immune cells, they are able to present antigens through major histocompatibility complex (MHC) class I molecules on their surface and are equipped with pattern recognition receptors like toll like receptors (TLRs) to recognize antigens from e.g. microbial pathogens (Hoogduijn 2015).

Although many general properties and characteristics of stromal cells are described and known as summarized above, a massive lack of knowledge exists regarding the distinct and precise role and function of tissue-specific stromal cells especially gastric stromal cells of the lamina propria in the communication with the adjacent epithelium.

1.2. Embryonic development and patterning of the stomach

The stomach derives from the posterior foregut of the endoderm in a multistep and complex process, which is still not fully understood. A highly conserved network of tightly regulated transcription factors and growth factors controls the organogenesis. Of note, the timing plays a crucial role in the whole organogenesis as the responsiveness and susceptibility of one cell population to a set of growth factors changes in the different developmental stages (Zorn and Wells 2009). The organ formation begins with the gastrulation in which the three germ layers mesoderm, endoderm, and ectoderm are formed (Figure 3A). In all vertebrate species, Nodal, a growth factor of the TGFβ

1.2 Embryonic development and patterning of the stomach

superfamily, is the driving force in the segregation of endoderm and mesoderm (Aoki et al. 2002; Ben-Haim et al. 2006; Hagos and Dougan 2007). The endoderm is further patterned into the foregut, midgut, and hindgut. This primitive gut tube is surrounded by mesoderm, the later mesenchyme, which is a source of growth factors that directly affect the developing epithelium and supports the anterior-posterior-axis patterning of the endoderm. The foregut develops into the esophagus, trachea, stomach, lung, thyroid, liver, biliary system and pancreas while the midgut forms the small intestine and the hindgut develops into the large intestine (Figure 3A). Through cell proliferation and differentiation during the fetal stage, finally, functional organs arise (Zorn and Wells 2009). The whole process of gastrulation and organ formation is tightly controlled and regulated through a complex and interacting network of growth factors including FGF, BMP, Wnt, Retinoic Acid (RA), Hedgehog and Notch (Figure 3B) (Dessimoz et al.

2006; Ameri et al. 2010; Tiso et al. 2002; Narita et al. 2000; Huelsken et al. 2000;

Bayha et al. 2009; Litingtung et al. 1998; Kikuchi et al. 2004). In the early stage of organ formation of the endoderm, active Wnt signaling and FGF10 together with BMP, originating from the adjacent mesoderm, drive intestinal patterning of the hindgut characterized by the expression of the transcription factor Cdx2 (Figure 3B) (Sherwood, Chen, and Melton 2009; Sherwood et al. 2011). The foregut expresses Sox2, a high mobility group-box gene transcription factor. BMP negatively regulates Sox2 and repression of BMP signaling during the early stages of stomach development is therefore essential (Figure 3B) (Rodriguez et al. 2010). Another important transcription factor in the stomach specification is BARX1, which is expressed by the mesenchyme adjacent to the nascent stomach, demonstrating the important endodermal-mesoderm interaction. BARX1 induces the expression of Wnt antagonists like secreted frizzle-related proteins (sFRP) leading to a regional inhibition of the Wnt pathway (Kim et al.

2007; Kim et al. 2005) to generate a sharp stomach-intestine boundary. The boundary of the stomach to other organs is also regulated by differential expression of transcription factors and gradients of growth factors (Kim and Shivdasani 2016).

1.2 Embryonic development and patterning of the stomach

Figure 3: The stomach originates from the posterior foregut of the endoderm. (A) During organogenesis, the stomach develops in a complex and tightly controlled process from the posterior foregut of the endoderm. (B) The endoderm patterning into foregut, midgut, and hindgut occurs during embryonic stage E8.5 in mice. Active Wnt, BMP and FGF signaling promote midgut and hindgut pattering characterized by the transcription factor expression Cdx2. Sox2 is expressed in the posterior foregut and the expression is repressed by BMP. RA is involved in the posterior patterning of the foregut, inducing the expression of the transcription factor Hnf1b. (C) A Wnt/β-Catenin gradient along the anterior-posterior axis of the posterior foregut promotes the further patterning of the stomach. Active Wnt/β-Catenin pathway stimulates the fundus specification in the stomach, while the inactivation of the Wnt pathway leads to antrum specification. Figure 3A modified from Zorn and Wells (2009), Figure 3B/C adapted from McCracken et al. (2017) (with permission from Annual Reviewes Inc. and Elsevier, conveyed through CCC).

After the initial stomach specification is completed, the regionalization, specification, and patterning of the stomach compartments into fundus, corpus, and antrum and the gland formation take place. The regionalization and patterning of the proximal-distal stomach axis are driven by strong epithelial-mesenchymal interactions. A set of differentially expressed transcription factors along this axis patterns the regional epithelium. The mouse stomach is differently structured than in humans. It additionally consists of a forestomach adjacent to the glandular stomach. The latter is similarly structured to the human stomach into fundus/corpus and antrum. In mice, high levels of Sox2 expression lead to forestomach specification, while a reduction in Sox2 provokes the expression of genes specific for the glandular stomach (Que et al. 2007). Further transcription factors which were described to be important in mice are GATA4 for

1.2 Embryonic development and patterning of the stomach during endoderm patterning (active Wnt would induce intestine development), an active Wnt pathway is necessary for the patterning of the stomach regions into fundus and antrum (Figure 3C). Furthermore, the deletion of the BMP receptor BMPR1A in foregut endoderm in mice had no effect on the stomach organogenesis, epithelial proliferation, and morphogenesis per se, but the loss of BMP signaling has an impact on gastric patterning, leading to anteriorization (Maloum et al. 2011). This again demonstrates the time-dependent effect of these pathways during organogenesis as also demonstrated by others (McCracken et al. 2017; Kim et al. 2005).

The epithelium of the early foregut and the future stomach shows a simple cuboidal shape, which needs to transit into the typical columnar morphology present in the stomach. In several steps, similar to the intestine, the primitive epithelial cell lining transforms into the complex glandular structure of the mature stomach (Grosse et al.

2011). In contrast to humans where the gland formation and differentiation of functional cell types occur in the fetal state, in mice, the maturation of the gastric glands and epithelial cells take place postnatal in the first weeks after birth (Stein et al. 1983;

Keeley and Samuelson 2010). Studies in chick embryos have revealed that an interplay of BMP, Wnt5a, sonic hedgehog (Shh) and Notch regulate the gland formation (McCracken and Wells 2017). For instance, Shin et al. (2006) have demonstrated that the inhibition of FGF10 in chick embryos impaired the gland formation. The overexpression of BMP2 in the underlying mesenchyme increased the number of gastric glands, while gland formation was inhibited by ectopic expression of the BMP antagonist Noggin (Narita et al. 2000).

In summary, sound knowledge about the mechanisms of the anterior-posterior and proximal-distal patterning in embryogenesis and morphogenesis exist gained from studies in animal models. A complex network of tightly regulated, interacting transcription factors and growth factors drive the process of organ formation. However, the understanding of the stomach specification in humans especially the patterning of

1.3 Signaling pathways