The study of pancreatic development has gained significant momentum in the last few decades, as a direct consequence of the recognition that proper pancreatic growth and homeostasis is of critical medical importance. Diabetes is a chronic disease that results from either the autoimmune destruction of insulin-producing beta cells within pancreatic islets (Type I), or alternatively from ineffective use of insulin (Type II). This disease affects over 180 million people worldwide (www.who.int/en
). Understanding the ontogeny and differentiation of beta cells is likely to pave the way for regenerative therapies. Classical studies (Golosow and Grobstein, 1962
; Pictet et al., 1972
) and more recent work (Jorgensen et al., 2007
; Zhou et al., 2007
) have elucidated the landmarks of pancreatic development and identified key regulators of endocrine cell fate. However, while a few groundbreaking studies have significantly advanced our ability to drive stem cells towards the beta cell fate (D’Amour et al., 2005
; D’Amour et al., 2006
; Kroon et al., 2008
), much remains to be understood about the molecular pathways involved in differentiation of pancreatic cell lineages.
In mouse, the pancreas originates around embryonic day 9.5 (E9.5) from dorsal and ventral evaginations of the gut tube endoderm. These pancreatic ‘buds’ invade and grow into the visceral mesoderm. Shortly after E10.5, the gut tube rotates, resulting in the fusion of the dorsal and ventral pancreatic buds and the formation of a single organ. The pancreatic epithelium then begins to branch, initially extending short fingerlike lobules into the mesoderm. From E12.5 to birth, branching expands, creating a highly branched, three-dimensional organ that grows dramatically throughout gestation. During this time, the pancreatic epithelium undergoes a number of dynamic cellular changes, giving rise to a tree-like, tubular epithelial network. Along the trunk of the developing pancreatic tree, endocrine progenitors delaminate as individual cells from the endodermal epithelium. These progenitors migrate and coalesce into small islet-like clusters, which progressively join and proliferate into larger endocrine aggregates. Postnatally, these take on recognizable islet anatomy, consisting of a core of insulin-expressing β-cells, surrounded by a mantle of mostly α-cells (glucagon), but also δ-(somatostatin), ε-(ghrelin) and PP cells (pancreatic polypeptide) (Murtaugh and Melton, 2003
; Cleaver and Melton, 2004
; Jensen, 2004
During pancreas development, two temporal waves of endocrine differentiation have been identified and their initiation have been termed the ‘first’ and ‘secondary transitions’ (Pictet et al., 1972
). The first wave encompasses the generation of ‘early’ endocrine cells, between E9.5 and approximately E12.5. During this time, individual cells within the early pancreatic endoderm initiate expression of glucagon, with a smaller subset transiently co-expressing insulin, with glucagon, pancreatic polypeptide or peptide YY (Herrera et al., 1991
; Upchurch et al., 1994
). There is interesting controversy and uncertainty regarding the precise fate and potential function of these early endocrine cells. It has been reported that early glucagon- and insulin-expressing (and co-expressing) cells do not contribute to the mature endocrine pancreas (Herrera et al., 1998
; Herrera, 2000
). However lineage labeling, using indelible tracing of early endocrine cells with the Cre-Lox system, has demonstrated that at least some early endocrine cells (or their progenitors) can contribute to adult islets (Gu et al., 2002
). It therefore remains unclear how these early endocrine cells might contribute to either embryonic pancreatic function or later pancreas homeostasis. The second wave of endocrine differentiation, on the other hand, initiates around E12.5 (Pictet et al., 1972
), and results in the generation of single hormone-expressing cell types (β-cells, α-cells, δ-cells, ε-cells and PP cells). The second transition also encompasses a dramatic pancreatic transformation, characterized by rapid expansion and differentiation of exocrine and endocrine tissues. The branching epithelium reshapes itself into finer tubular ducts, with the tips of the branches differentiating into exocrine cells and endocrine progenitors delaminating along the central axis. Although initiating at E12.5, the cellular and morphological changes associated with this ‘second transition’ become most evident between E13.5–E14.5.
Recent work in the field of pancreas development suggests that different cascades of molecular regulation regulate these two temporally separate waves of islet endocrine cell generation. Pdx1 has been thought to regulate endocrine cells of the second transition, since only first transition endocrine cells can be found in Pdx1 null embryos (Jonsson et al., 1994
; Ahlgren et al., 1996
; Offield et al., 1996
). However, recent data demonstrates that although the early endocrine lineage does not require Pdx1 for specification, Pdx1 plays a role in its proper expansion and maintenance (Burlison et al., 2008
). In addition, some molecules are expressed differently during the two waves of pancreatic development. For example, the transcription factor HLXB9 is transiently expressed early in the pancreatic epithelium, but diminishes following the first transition. Later, however, it reinitiates expression in differentiated β-cells, coinciding with the onset of secondary transition (Li and Edlund, 2001
). During the second transition, the expression of many critical pancreatic transcription factors becomes differentially restricted to either endocrine or exocrine cell types. For instance, the transcription factors Pdx1, Nkx2.2, Nkx6.1 and Sox9, which are initially expressed widely in the pancreas, gradually become restricted to the endocrine lineage during this time, while Ptf1a becomes restricted to exocrine cells (Kim and MacDonald, 2002
). Even more strikingly, some factors only initiate expression after the secondary transition. For instance, it has been shown that the expression of the transcription factor MafA is specifically restricted to the endocrine cells of the second wave (Artner et al., 2007
), thus supporting the hypothesis that there exist distinct molecular cascades underlying the first and second wave of pancreatic endocrine differentiation.
One regulatory factor that is critical to endocrine differentiation is the bHLH transcription factor Ngn3 (Neurogenin3 or Neurog3). Ngn3 is transiently expressed in endocrine progenitor cells and it is never co-expressed with endocrine hormones in pancreatic islets (Schwitzgebel et al., 2000
). It therefore marks transitional endocrine progenitors. Genetic ablation of Ngn3
in mouse results in the complete failure of all pancreatic endocrine cell differentiation and neonatal lethality (Gradwohl et al., 2000
). Conversely, gain-of-function studies demonstrate that Ngn3 can induce differentiation of the four endocrine lineages (Apelqvist et al., 1999
; Schwitzgebel et al., 2000
; Grapin-Botton et al., 2001
). The complete failure of both early and late endocrine differentiation in Ngn3 null embryos suggests that NGN3 is required for endocrine cell specification, during both the first and secondary transition.
Recent work on Ngn3 has highlighted differences between early versus late embryonic endocrine cells. Using a transgenic temporally-controlled ‘addback’ system to express Ngn3
in an Ngn3
null background, Grappin-Botton and colleagues demonstrated that Ngn3
expression drives endocrine precursors to differentiate, however endodermal progenitors pass through different stages of ‘competence’, resulting in the differentiation of precursors down various lineages at different embryonic timepoints (Johansson et al., 2007
). Early embryonic endoderm displays the capacity to generate mostly glucagon-expessing cells, while later endoderm is able to generate the full range of endocrine cell types, including β cells. This suggests that there is likely to be temporal regulation of Ngn3
expression during different stages of pancreatic development.
Previous reports describe Ngn3 expression at various stages (Apelqvist et al., 1999
; Gradwohl et al., 2000
; Schwitzgebel et al., 2000
; van Eyll et al., 2006
; Murtaugh, 2007
; Burlison et al., 2008
), however, no detailed characterization currently exists that examines either Ngn3 expression initiation or maintenance throughout pancreatic development. Here, we present a detailed developmental profile of Ngn3
mRNA and protein expression throughout all stages of pancreatic budding and branching. Clarifying the temporal expression of Ngn3 expression, both during the first and secondary transition, will advance our understanding of both early and late islet endocrine cells.