The extensively branched structure of the mature pancreas is attained by continued growth and remodeling of the primitive tubular epithelium in both dorsal and ventral buds. Once the buds have been induced, they are critically dependent on interaction with the mesenchyme for continued morphogenesis and cytodifferentiation. Moreover, during tubulogenesis, cytodifferentiation is suppressed, which allows for the expansion of progenitor cell populations, and implies direct communication between the genetic programs controlling each process. Mouse explant studies showed that isolated pancreatic epithelium failed to develop acinar tissues in the absence of mesenchyme, and that secreted factors were likely the mediators of this development . In complementary studies in vivo, targeted ablation of pancreatic mesenchyme showed essential roles for epithelial-mesenchymal signaling during both early and late bud morphogenesis . FGF10 from pancreatic mesenchyme supports the proliferative expansion of the epithelium as well as the maintenance of undifferentiated pancreatic progenitor cells via induction of the Notch pathway [24,25]. Additional signals from the mesenchyme instruct the continued development of the buds. For instance, reciprocal EphB signaling between mesenchyme and epithelium is positively required for branching, growth, and cytodifferentiation , while unidentified signals from blood vessels restrain these processes . Even as these and other unknown signals regulate its growth, pancreatic mass is ultimately constrained by an intrinsic program established in the pancreatic progenitor domain . Finally, as the foregut grows and elongates, the developing ventral pancreatic tissues rotate along with the gut, and ultimately fuse with the dorsal bud to generate mature pancreatic architecture. Although congenital malformations arising from defective pancreatic bud fusion are relatively common, including annular pancreas and pancreas divisum, the genetic pathways underlying this process are not well understood .
Cytodifferentiation in the developing pancreas
As stated earlier, the early evaginating pancreatic buds are made up of progenitor cells that express Pdx1 and Ptf1a. These early pancreatic progenitor cells will induce the specification of multipotent progenitor cells (MPCs), which direct derivative cell populations toward a particular fate. The early developing pancreatic buds are marked by the appearance of cells with low-level digestive enzyme production and an initial wave of glucagon- and insulin-expressing cell types, a period referred to as the “primary transition” of pancreas formation. The term “secondary transition” is applied to the phase during mid-pancreas development that is marked by the differentiation of exocrine cells and the major wave of islet cell formation . The secondary transition is characterized by a dramatic increase in cells expressing acinar digestive enzymes, as well as a large increase in cells producing endocrine hormones including insulin, glucagon, ghrelin, somatostatin, or pancreatic polypeptide.
Preceding this major wave of differentiation, the secondary transition also encompasses the emergence of the MPCs and the establishment of the pre-acinar and bipotent duct/endocrine cell populations from which the differentiated exocrine, and endocrine or duct cells derive, respectively (Figure 4.2).
In recent years, a network of genes has been identified whose products specify the development of the different cell types (see Table 4.2). The importance of these genes has largely been identified by lineage tracing studies and targeted mutations in mouse models, and has led to two important concepts in pancreatic cytodifferentiation. First, the products of many of these genes function in a “cell-autonomous” manner, meaning that their expression level in a given cell type alters the fate and function of that cell. Second, misexpression of specific genes in magnitude or in a spatial (cell- or domain-specific) or temporal (time of development-specific) manner can redirect developing progenitor cells to cell fates they would otherwise not have adopted. With respect to the latter concept, the study of cell-autonomous factors has the potential to identify means through which other cell types might be converted to β cells for the treatment of different forms of diabetes.
Tip versus trunk domains
The stratification of the pancreatic epithelium and the resulting formation of microlumens is an essential morphological change that occurs during the primary transition. The subsequent remodeling of the ductal plexus and branching of the epithelium continues throughout embryonic pancreas development . Epithelial branching leads to the morphogenesis of different domains, which become most apparent at the beginning of the secondary transition. There is mounting evidence that MPCs exist within these emerging epithelial domains. In particular, Melton and colleagues proposed the identity of the MPCs as those cells expressing the factors Ptf1a, Pdx1, c-Myc, and Cpa1 . This concept stemmed from a genome-wide transcription factor analysis in mouse pancreas tissue at embryonic day (E) 14.5, whereby gene expression patterns were identified to segregate into particular domains of the developing pancreas. Specifically, patterns emerged that could be grouped into five domains: pan-epithelium, tip, trunk, mesenchyme, and vasculature. Genes discovered to be expressed in the tip domain later segregated into differentiated acinar cells, whereas genes expressed in the trunk domain were identified in the ducts or differentiated endocrine cells. Taken together, data from multiple studies suggest that MPCs residing in the tip domain will give rise to pre-acinar cells, destined to become exocrine tissue, and bipotent duct/endocrine cells that reside in the trunk of the branching epithelium (Figure 4.2).
Endocrine versus exocrine cell fate decision
It is in the early developing pancreatic domain, when progenitor cells are multipotent, that the endocrine versus exocrine decision is made. In the mouse, Ptf1a is located in the early pancreatic progenitor cells and over time becomes restricted to expression in the branching tips and then differentiated acinar cells . At the beginning of pancreas development the transcription factors Nkx6.1 and Nkx6.2 are co-expressed in the MPCs before becoming restricted and separated in their expression pattern. The Nkx6 factors and Ptf1a have been noted to function antagonistically in the decision between endocrine and exocrine cell fates, such that Nkx6 factors promote the endocrine decision whereas Ptf1a promotes the exocrine decision . The endocrine versus exocrine cell fate decision is also influenced by the level of expression of the transcription factor Neurogenin3 (Neurog3) in the progenitor cells. Specifically, a high level of Neurog3 is required for commitment to the endocrine fate . Moreover, Notch signaling is used in the trunk domain to subdivide this compartment between endocrine and ductal cells via a lateral inhibition mechanism. Neurog3 upregulates expression of the Notch ligand Delta-like 1 (Dll1) in endocrine progenitors, which activates the Notch pathway in neighboring cells thereby repressing their differentiation into endocrine cells.
The endocrine progenitor cell
The culmination of many studies has confirmed that in the developing mouse pancreas, the transcription factor that defines the endocrine progenitors is Neurog3. Neurog3-null mice exhibit absence of endocrine cells in the pancreas, and such mice develop neonatal diabetes and die shortly after birth . During mouse pancreas development a subset of hormone-expressing cells is observed as early as E9.5, whereas the major wave of endocrine differentiation occurs during the secondary transition. Lineage tracing experiments using genetically-engineered mouse reporter lines identified that, regardless of when the endocrine cell differentiates, all hormone-expressing cells are derived from cells that previously expressed Neurog3 [36,37].
The process of endocrine differentiation has also been linked to the morphological process of delamination of the progenitor cells from the pancreatic epithelium. Interestingly, the delamination of progenitor cells is initiated in the cells that express Neurog3 . Moreover, the subsequent differentiation into different endocrine cells types is influenced by the timing of Neurog3 expression. Specifically, altering the temporal expression of the gene encoding Neurog3 in the mouse influences the competence of progenitor cells to differentiate into the specific endocrine cell types, such that earlier expression produces almost exclusively α cells, whereas later expression produces varied ratios of all hormone-expressing cell types .
Previous models of pancreas development suggested that each Neurog3-expressing cell could give rise to any subsequent differentiated endocrine cell type. However, this perspective has been challenged by lineage tracing experiments using genetically-altered mice, which demonstrated that each Neurog3-expressing endocrine progenitor cell is in fact unipotent, and therefore destined to become a particular single-hormone expressing endocrine cell type  (Figure 4.2). The implication of this discovery is that the transcription factor “code” responsible for the differentiation of each hormone expressing cell type may be delineated before endocrine progenitors are specified.
Clearly, the expression of Neurog3 is of great significance to the development and differentiation of endocrine cells in the mouse. However, the effect of loss of this transcription factor in other species is not identical to the mouse. For example, in zebrafish Neurog3 is not observed in the pancreas . Homozygous mutations in Neurog3 have been identified in humans, resulting in congenital malabsorptive diarrhea and childhood-onset diabetes [42,43], but without congenital loss of pancreatic endocrine cells (as seen in the mouse). Nevertheless, the absence of enteroendocrine cells was noted in these individuals.
Other transcription factors are also expressed in the early endocrine cell population, and genetic deletion studies identified these factors to be crucial to endocrine cell differentiation. In particular, the transcription factor Islet1 (Isl1) is expressed in all mature, non-replicating islet cell types. Interestingly, Isl1 expression is also observed in the mesenchyme that surrounds the early dorsal pancreatic bud. The dorsal pancreatic mesenchyme does not form in Isl1-deficient mouse embryos, leading to a loss of exocrine differentiation in the dorsal pancreas; the pancreas is also devoid of all islets in these mice. Loss of the transcription factor Pax6 in the mouse leads to death shortly after birth. The pancreas of Pax6-deficient animals is devoid of α cells and has marked reductions in β, δ, and PP cells .Whereas a human mutation in the ISL1 gene has been identified in a patient with type 2 diabetes , no link to diabetes has been observed in humans with mutations in the PAX6 gene. Therefore similar to Neurog3, the functional importance between lower organisms and humans may not be completely conserved for genes involved in endocrine differentiation.