The pancreas is an organ playing an essential role as a dual exocrine and endocrine gland (Johansson and Grapin-Botton,2002; Habener et al.,2005). The endocrine function relies on the islets of Langerhans, which are clusters of cells separated from the branched exocrine tree and dispersed in the pancreas. Islets are composed of five endocrine cell types, each secreting a specific hormone (Johansson and Grapin-Botton,2002; Habener et al.,2005; Lyttle et al.,2008). The clustered organization is essential for the metabolic function of the islets, notably synchronized and synergistic secretion of insulin by beta cells coupled by gap-junctions (Bosco et al.,1989; Bavamian et al.,2007). Pancreatic islets are polyclonal (Deltour et al.,1991; Desgraz and Herrera,2009) and form through the aggregation of endocrine cells originating from different locations. These cells can then proliferate, especially in the perinatal and early postnatal period to expand islet mass. Because all pancreatic endocrine cells arise from epithelial progenitors during embryonic development, they must delaminate from the epithelium, migrate in the mesenchyme, and aggregate into clusters. There are some evidences suggesting that newly generated endocrine cells delaminate by achieving an epithelial-to-mesenchymal transition (EMT; Chiang and Melton,2003; Rukstalis and Habener,2007; Cole et al.,2009). EMT is a process by which an epithelial cell becomes mesenchymal and which involves profound phenotypic changes that include the loss of cell–cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties (Thiery et al.,2009; Acloque et al.,2009). Achieving a successful EMT induces the cell to exit the epithelium, a process commonly referred to as delamination. After embryonic day (E) 14.5 in mouse embryos, it was reported that endocrine cells exhibit low E-cadherin expression and up-regulate N-cadherin, although it is unclear when this switch occurs (Hutton et al.,1993; Johansson et al.,2010). Moreover, Snail2 (previously known as Slug), a zinc finger transcription factor that promotes EMT, was shown to be expressed in the pancreas (Rukstalis and Habener,2007). After delamination, endocrine cells migrate in the mesenchyme. Several genes have been shown to play a role in this process such as integrins, Rac1 and matrix metalloproteases (Miralles et al.,1998; Cirulli et al.,2000; Miettinen et al.,2000; Yebra et al.,2003; Perez et al.,2005; Greiner et al.,2009). Finally, endocrine cells aggregate into clusters. Cadherin function is required for β-cell aggregation (Dahl et al.,1996). Indeed, expression of a dominant negative E-cadherin construct, inhibiting the function of all cadherins, resulted in perturbed islets aggregation. N-cadherin was recently shown to be dispensable for endocrine cells aggregation, suggesting that this function depends on E-cadherin or redundant activities of N- and E-cadherin (Johansson et al.,2010). However, the role of E-cadherin in delamination has not been investigated (Dahl et al.,1996).
During development, pancreatic endocrine cells derive from pancreas progenitors expressing Pdx1 (Gu et al.,2002) and Sox9 (Piper et al.,2002; Seymour et al.,2007), which then transiently express Neurogenin 3, a protein that is necessary for endocrine cell differentiation (Apelqvist et al.,1999; Gradwohl et al.,2000; Schwitzgebel et al.,2000; Grapin-Botton et al.,2001; Gu et al.,2002; Johansson et al.,2007; Desgraz and Herrera,2009). Similarly, neurons differentiate upon expression of Ngn1, 2, or 3 (Sommer et al.,1996; Ma et al.,1999; Hand et al.,2005; Ge et al.,2006). In the cortex, Ngn2 also promotes the migration of neurons through long distances to reach their final position and form the typical layered organization (Hand et al.,2005; Ge et al.,2006). In this tissue, although the binding of Ngn2 to DNA is required for inducing differentiation it is not necessary for migration (Hand et al.,2005; Ge et al.,2006). The migration program is activated as the RhoA GTPase, a regulator of actin dynamics, is repressed. The repression of RhoA by Ngn2 appears to rely on the displacement of the coactivator CREBS binding protein (CBP) from the RhoA promoter to that of NeuroD or other direct differentiation targets of Ngn2. We investigated whether Ngn3 similarly controls endocrine cell movement in the pancreas.
To better understand whether and how Ngn3 controls delamination of pancreatic endocrine cells, we analyzed in detail the different steps of pancreatic endocrine cell delamination. We found that Ngn3-positive endocrine progenitor cells lose apical contact, a feature previously reported as an early event in EMT. Subsequently, they exhibit all the features of classical EMT. Using the chicken as a model system, we showed that Ngn3 is sufficient to induce this EMT and that similar mechanisms occur in the mouse pancreas, under the control of Ngn3. Chick pancreas development is very similar to mice and humans although the ratios between insulin-positive and glucagon-positive cells are different in the three species (Dieterlen-Lievre and Beaupain,1974; Rawdon and Larsson,2000). Finally, we show that Ngn3 indirectly controls Snail2 expression, which likely leads to a subsequent EMT in endocrine cells.