Transcription factors regulating pancreatic development

The mouse pancreas develops as evaginations from dorsal and ventral regions of the developing foregut destined to become duodenum [38]. Its developmental program is initiated by factors released by the mesoderm at E9.0. Later, the epithelium of the foregut undergoes branching morphogenesis into a ductal tree and results in the formation of two primordial pancreas organs consisting primarily of undifferentiated ductal epithelium. At E13.5 the gut rotates clockwise, bringing the two primordial pancreas buds together;

eventually, they fuse into a single organ. The exocrine pancreas differentiates from the ductal epithelium and afterwards acini are clearly visible. The endocrine cells appear from the very beginning of the epithelium and undergo extensive proliferation after the fusion of the dorsal and ventral buds, a stage often referred to as the second developmental transition. The islet cells then begin to organize into islet-like clusters, a process that continues maturation of the islets cells and still carries on until shortly after birth, this is the third developmental transition [39] (Fig. 3).

29

FIG. 3: Events of pancreatic organogenesis. Development of the dorsal rudiment only is depicted after E8.5. Yellow shading indicates the extent of Ipf1/Pdx1 expression and gray indicates the early-differentiated endocrine cell clusters. The double arrow indicates the orthogonal directions of cell division of islet versus acinar/ductal precursors. Adapted from [39].

30 Pdx1

The pancreas develops as evaginations from dorsal and ventral regions of the developing foregut that express the homeodomain transcription factor Pdx1 [40-43]. Pancreatic and duodenal homeobox 1 (Pdx1) is essential for the normal proliferation and differentiation of embryonic pancreatic precursors [40, 44, 45]. Epithelial cells in these buds proliferate and form branching ductules that differentiate and give rise to mature ductal, endocrine and acinar elements [39, 46-48]. Pdx1 is expressed early in all pancreatic progenitor cells and is later restricted to adult β-cells plus a fraction of δ-cells. Lineage tracing studies have shown that Pdx1-expressing cells at E8.5 are multipotent progenitors, giving rise to all pancreatic lineages [49, 50].

Pdx1 knockout mice display pancreatic agenesis [51]. Interestingly, in those knockout mice, some Insulin and Glucagon-expressing cells develop in the dorsal bud region. Mice heterozygous for Pdx1 have elevated blood glucose levels and impaired glucose tolerance [52]. Similarly, humans with heterozygous mutations in Ipf1/Pdx1 are highly predisposed to MODY (subtype 4) or adult-onset type 2 diabetes while homozygous mutations result in pancreatic aplasia [53, 54].

Interestingly, although the pancreatic epithelium of Pdx1 null-mice fails to develop, the surrounding mesenchyme grows and develops normally indicating that development of the pancreatic mesenchyme does not rely on the epithelium [55].

Pbx1

Pre B-cell leukemia transcription factor 1 (Pbx1) is expressed at E10.5 in mesenchymal and Pdx1-expressing epithelial cells of the developing pancreas. In the adult it is widely expressed in ductal, acinar and islet cells. Pbx1-/- mice exhibit agenesis of both the dorsal and ventral pancreas due to a strong (35-40%) reduction of epithelial cell proliferation suggesting that Pbx1 is essential for pancreatic epithelial cell proliferation [56]. Islet1 (isl1) and neurogenin3 (ngn3) two critical genes for the development of the endocrine cells of the pancreas are absent in Pbx1 mutant mice and accordingly no endocrine cells are present [56]. In addition to controlling the proliferation of pancreatic progenitor cells, Pbx1 seems to also play a role in the differentiation of endocrine cells.

31 Isl1

The LIM homeodomain protein Isl1 is expressed in the developingpancreas and the central nervous system [57, 58]. In adult animals,Isl1 is expressed in all of the hormone-producing cells of pancreaticislets, but its low level of expression in ß-cellsargues against it having an important function to regulate insulin gene transcription. Targeted disruption of the isl1 gene resultsin an early arrest of embryonic development on approximately E9.5 [46]. Isl1 is required for the formation of dorsal, butnot ventral, pancreatic mesenchyme. Mice in which Isl1 expressionis disrupted by gene knockout fail to develop a dorsal pancreas,but partially execute the differentiation of the ventral exocrine pancreas [59]. The dorsal pancreatic endoderm obtained fromIsl1-null mice and studied ex vivo responds to signals transmitted from co-cultured wild-type mesenchyme by differentiating into pancreatic tissue. These findings reinforce the critical requirement of mesenchymal signaling to the endoderm in allowing normal pancreatic development. The elucidation of additional signalingpathways that are critical for development of the pancreas requiresadditional investigation.

Ptf1 complex

Pancreas specific transcription factor-1 (Ptf1) is a trimeric transcription factor essential to the development of the pancreas and to the maintenance of the differentiated state of the adult exocrine pancreas. It is composed of three members of the basic helix-loop-helix (bHLH) family of proteins. The first member, p75, is essential for the nuclear localization of the complex [60]. The two other members, p64 and Ptf1a-p48 are the DNA-binding subunits [61]. Ptf1-p48 (Ptf1a) is expressed and restricted to all pancreatic progenitors being more specific than Pdx1 which in addition to the developing pancreas is also expressed in the caudal stomach and duodenum. The coexpression of both Pdx1 and Ptf1a is necessary and sufficient to trigger the pancreatic differentiation cascade.

During the second developmental transition (around E12.5) Ptf1a expression becomes restricted to developing acinar cells [62]. Functional binding sites for the Ptf1 complex have been found in the promoters of all acinar digestive enzymes examined [61, 63]. In Ptf1a-deficient embryos, pancreas development is severely impaired; exocrine progenitors are reprogrammed into a duodenal fate, there is no acini nor duct formation and endocrine cells were found to be relocated into the spleen [64].

32 Foxa1, Foxa2, Foxa3 (HNF3α, HNF3β, HNF3γ)

The Foxa group of the Forkhead family comprising Foxa1, Foxa2 and Foxa3 is essential for the regulation of hepatocyte-specific expression of several target genes [65]. They are expressed in embryonic endoderm, the germ layer that gives rise to the digestive system, and contribute to the specification of the pancreas and the regulation of glucose homoeostasis [66].

Foxa2 is expressed as early as E6.5 in the mesoderm and its expression is maintained high in the pancreas of adult mice. Foxa2 null-mice die at E10-11 [67, 68], but the formation of a dorsal pancreatic bulge is still possible [38]. Foxa2 null embryos do not survive long enough to study the role of Foxa2 in pancreatic development. In order to dissect the function of Foxa2 specifically in the pancreas, a β-cell-specific deletion was generated. These mice survived to birth and died around P10 from hypoglycemia. The specific β-cell knockout demonstrated islet disorganization, but cell lineages were present in appropriate proportions [69], indicating a possible role for Foxa2 in islet formation. Ablation of Foxa2 only after E14.5 results in an absence of α-cells [70], suggesting Foxa2 has a critical role in the regulation of the terminal differentiation steps and maturation of glucagon-producing α-cells.

The expression of Foxa1 initiates by E9 in the entire gut region including the liver primordium and the floorplate of the neural tube. The expression of Foxa1 is maintained in organs of endoderm lineage in the adult [71, 72]. Foxa1 null-mice die shortly after birth from hypoglycemia [73, 74]. The Foxa1 knockout mice have low levels of circulating plasma glucagon, but a normal pancreatic cell proportion [73, 74]. Foxa1 was demonstrated to activate the proglucagon gene promoter [73].

Foxa3 is first detected in the extra-embryonic endoderm before E8.0 [75] and more strongly in cells of the invaginating hindgut near E9.0. Foxa3 gene seems to plays a crucial role in maintenance of glucose homoeostasis in the adult [76]. Fox3 knockout mice are viable, with normal blood glucose levels [77].

33 Ngn3

Neurogenin3 (Ngn3), a basic helix-loop-helix (bHLH) transcription factor, expressed transiently in endocrine precursors [50, 78], is the earliest known marker for specific endocrine precursor cells [79, 80]. These Ngn3-expressing cells are a subset of the entire Pdx1+ endodermal epithelial cells, and they first appear at embryonic day E8.5, peak at E15.5 and are undetectable at birth. Ngn3 is never coexpressed with hormones or other markers of differentiated cells.

Ngn3 has been shown to regulate the expression of Beta2/NeuroD1 concurrently with Isl1 and Pax6 to give rise to endocrine cells during development and in adults [78, 79, 81-84]. As a consequence the pancreatic hormones are expressed, followed by switching off Ngn3 expression [83]. The specification of different islet cell types and the completion of the differentiationprocess require the activation of transcription factors thatare downstream of Ngn3 [85].

Mice with homozygous mutations in both ngn3 alleles lack differentiated endocrine cells but have nearly normal acini and ducts [79]. In addition to these major transcription factors several other transcription factors and signaling molecules play critical roles in pancreatic development and endocrine differentiation (for reviews see [39, 46, 47, 86-90]).