Pancreas specification

Through cell movements during foregut morphogenesis the dorsal and ventral foregut epithelium are placed into very different environments. This fact already indicates that the extrinsic signals and genetic programs that promote pancreas development in the dorsal and ventral foregut are different. A further difference is the derivation of several organs from the ventral foregut, whereas the dorsal foregut gives rise exclusively to the dorsal pancreas. This suggests that dorsal pancreas formation is a matter of induction, whereas ventral pancreas needs to be segregated from multiple organ lines (reviewed in McCracken and Wells, 2012). Several studies confirmed these assumptions. In chick, Kim and colleagues demonstrated in 1997 that presumptive dorsal pancreatic endoderm when cultivated in isolation showed no pancreatic gene expression, whereas ventral pre-pancreatic endoderm does (Kim at al., 1997). Extensive studies in ectodermal, mesodermal and endodermal explants from Xenopus embryos supplied the evidence that the dorsal pancreatic foregut endoderm initially receives inductive signals from the mesoderm at the onset of gastrulation. Retinoic acid (RA) could be identified as such an inductive signal and the involvement of one or more further signals is assumed (RA-source indicated in Fig. 1.3 A (red)) (Chen at al., 2004; Pan et al., 2007).

During foregut morphogenesis the dorsal foregut epithelium has initially contact with the notochord and subsequently with the dorsal aorta. Both tissues have been shown to be sources of permissive signals for pancreas formation (Kim et al., 1997;

Lammert at al., 2001). In chicken, dorsal pancreas development could be prevented by the removal of the notochord at an early stage (Kim et al., 1997). However, the notochord is not able to induce pancreatic gene expression in early gastrula stage or non-pancreatic somite-stage endoderm (Wells and Melton, 2000), which is in accordance with the finding that pre-pancreatic foregut endoderm obtained the ability, at an earlier time, to react to permissive signals from the notochord. These permissive signals were identified as Activin-βB and FGF2 (Fig. 1.3 B). Hebrok and colleagues found them as secreted factors from the notochord that suppress the expression of Sonic hedgehog (Shh) in the pancreatic endoderm. Shh is broadly expressed in the endodermal epithelium but is excluded from pancreatic endoderm (Hebrok at al., 1998; Hebrok et al., 2000). The removal of the notochord leads to an ectopic Shh expression that inhibits dorsal pancreas development (Hebrok et al., 2000). It was shown that the repression of Shh in the dorsal endoderm induces

19 Pdx1 expression in a dose dependent manner (Hebrok at al., 1998). With continuing embryonal development, the notochord is displaced by fused dorsal aortae that further signals to the dorsal pre-pancreatic endoderm (Lammert at al., 2001). These signals are required for the maintenance of Pdx1 expression and for the induction of Ptf1a expression (Yoshitomi and Zaret, 2004). It is assumed that aortic endothelial signals in addition act indirectly on pancreatic endoderm as they promote the survival of dorsal mesenchyme (reviewed in Edlund, 2002). Dorsal mesenchyme secretes FGF10 that is required for the maintenance of Pdx1 and Ptf1a expression (Jacquemin et al., 2006).

The ventral foregut gets in proximity to two mesodermal derivatives, the cardiac mesoderm and septum transversum (Fig. 1.3 B). The posterior region of the ventral foregut was shown to give rise to the ventral pancreas and liver. Explant studies in zebrafish and mouse support the presumption of a common bi-potential cell population in the ventral foregut endoderm that differentiate into liver and ventral pancreas (Deutsch et al., 2001; Bort et al., 2006; Chung et al., 2008). In several studies, signaling pathways have been identified which are involved in the segregation of hepatic and pancreatic fate, most of them appear to repress pancreas formation and promote liver development. FGF-signaling from the cardiac mesoderm (Jung et al., 1999) and BMP-signaling from the septum transversum (Rossi et al., 2001) induce hepatic fate. Deutsch and colleagues found an expression of Pdx1 in cultures of isolated mouse ventral foregut endoderm, whereas the expression of liver marker Hhex required signals from the cardiac mesoderm (Deutsch et al., 2001). These findings suggest the pancreas as a default state of the ventral foregut. Furthermore, a BMP antagonist TGIFβ-induced factor 2 (TGIF2) was found to be expressed in the posterior ventral foregut counteracting the expression of Hhex (Spagnoli and Brivanlou, 2008). Moreover, the assumption of pancreas as a default state is supported by the presence of a pre-existing chromatin modification pattern that promotes pancreas development in the ventral foregut endoderm (Xu et al., 2011; Arnes and Sussel, 2015).

Independent of the way of specification, both dorsal and ventral pancreatic progenitor cell populations are characterized by the co-expression of Pdx1 and Ptf1a (Afelik et al., 2006). During this early phase of pancreatic development, also referred to as primary transition, the pancreatic progenitors proliferate (Pictet et al., 1972). Thus, pancreatic foregut epithelium evaginates and forms the dorsal and two ventral buds. Furthermore, the first Insulin-expressing cells in Xenopus and Glucagon-expressing cells in mouse can be detected. In mouse, the primary transition occurs between E9.5 and E13.5 (Jorgensen et al., 2007). The

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corresponding time period in Xenopus is from stage 28 to stage 38 (Horb and Slack, 2002).

Fig. 1.3 Fate maps of Xenopus endoderm from gastrula to early somite stage and overview of signals involved in pancreas specification

(A) Gastrula stage (left) and early somite stage (right) Xenopus embryos with indicated colored domains that correspond to fate-map studies. The arrows indicate the migration/folding direction of the foregut. (modified after Zorn and Wells, 2009)

(B) Overview of signals involved in pancreas specification in an early somite stage embryo.

In the dorsal foregut endoderm, permissive signals from the notochord (FGF2 and Activin-βB) repress Shh transcription in the pre-pancreatic epithelium and allow Pdx1 expression.

FGF- and BMP-signaling from the cardiac mesoderm/septum transversuim promote liver (Hhex) and inhibit pancreas fate (Pdx1) in the ventral foregut epithelium. (modified after McCracken and Wells, 2012)

21 Genetic lineage-tracing experiments in mice revealed that all pancreatic cell- subtypes arise from a common pool of multipotent progenitor cells co-expressing Pdx1 and Ptf1a (Gu et al., 2002; Kawaguchi et al., 2002; Burlison et al., 2008). In Xenopus, ectopic co-expression of Ptf1a and Pdx1 is sufficient to convert duodenal precursor cells into pancreatic tissue (Afelik et al., 2006). Furthermore, it was shown that constitutively active forms of Pdx1 or Ptf1a can push liver progenitors to a pancreatic fate (Horb et al., 2003, Jarikji et al., 2007). As soon as the ventral and dorsal pancreatic domains in the foregut epithelium are specified, a set of transcription factors becomes specifically expressed in these regions (Fig. 1.4 A).

Beside Pdx1 and Ptf1a, the earliest transcription factor that marks pancreatic progenitor cells is Sox9 (Lioubinski et al., 2003; Lee and Saint-Jaennet, 2003). In mice, Sox9 was shown to be essential for proliferation and maintenance of pancreatic progenitors (Seymour et al., 2007; Seymour, 2014). Moreover, during the proliferation and expansion of the pancreatic progenitor field, Sox9 regulates a set of additional transcription factors in pancreatic progenitors including Hnf1b (Lynn et al., 2007). Genetic studies in mice revealed that Sox9 acts in cooperation with Pdx1 for the induction of pancreatic fate and the repression of the intestinal lineage differentiation (Shih et al., 2015).