Transcriptional Regulation in Pancreatic Development
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内分泌学杂志 2005年第3期
Laboratory of Molecular Endocrinology, Massachusetts General Hospital (J.F.H., M.K.T.), Howard Hughes Medical Institute (J.F.H.), Harvard Medical School (J.F.H., M.K.T.), Boston, Massachusetts 02114; and Novartis Institutes for BioMedical Research (D.M.K.), Cambridge, Massachusetts 02139
Address all correspondence and requests for reprints to: Dr. Joel F. Habener, Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, WEL320, Boston, Massachusetts 02114. E-mail: jhabener@partners.org.
Abstract
Considerable progress has been made in the understanding of the sequential activation of signal transduction pathways and the expression of transcription factors during pancreas development. Much of this understanding has been obtained by analyses of the phenotypes of mice in which the expression of key genes has been disrupted (knockout mice). Knockout of the genes for Pdx1, Hlxb9, Isl1, or Hex results in an arrest of pancreas development at a very early stage (embryonic d 8–9). Disruption of genes encoding components of the Notch signaling pathway, e.g. Hes1 or neurogenin-3, abrogates development of the endocrine pancreas (islets of Langerhans). Disruption of transcription factor genes expressed more downstream in the developmental cascade (Beta2/NeuroD, Pax4, NKx2.2, and Nkx6.1) curtails the formation of insulin-producing ?-cells. An understanding of the importance of transcription factor genes during pancreas development has provided insights into the pathogenesis of diabetes, in which the mass of insulin-producing ?-cells is reduced.
RECENT STUDIES OF the biology of pancreas development have shed new light on the cause of type 2 diabetes. The identification of transcription factors involved in regulation of the expression of key genes required in the developing and adult endocrine and exocrine pancreas has been provided by observing the phenotypic consequences of targeted disruptions of the genes (gene knockouts) encoding these factors in mice. Remarkably, almost without exception, disruption of these genes has resulted in phenotypes of impaired development of the pancreas and consequent diabetes. Furthermore, lessons learned from the gene knockouts in mice have been used to successfully identify mutations in several of the corresponding orthologous genes in individuals with familial monogenic type 2 diabetes.
In this minireview we attempt to encapsulate a rapidly growing body of knowledge that is providing insight into the genetic contributions to the development of diabetes. The thematic emphasis is on the impact that genetic polymorphisms or mutations in genes encoding transcription factors essential for pancreas development have on predisposition for diabetes. For more comprehensive, in-depth information on this topic, the reader is referred to several recent excellent reviews (1, 2, 3, 4, 5, 6, 7, 8, 9).
Anatomical and Morphological Development of the Pancreas
The pancreas consists of three main tissue cell types (in addition to vascular and stromal supporting tissues): the exocrine acinar tissue that produces digestive enzymes; the endocrine cells (islets of Langerhans) that produce the hormones involved in nutrient homeostasis, such as insulin and glucagon; and the elaborately branched ductal tree (10). The pancreas originates early in development [embryonic d 8.5 (e8.5) to e9.5 in the mouse] by the outcropping of two buds (ventral and dorsal) of cells from a specialized prepatterned endodermal epithelium located in the region of the foregut that is to become the duodenum (Fig. 1). By e10.5, the partially differentiated epithelium of the two buds undergoes branching morphogenesis into a ductal tree that by e12.5 results in the formation of two primordial pancreas organs consisting predominantly of an undifferentiated ductal epithelium (first developmental transition).
FIG. 1. Schematic diagram of pancreatic development in the mouse. On e8, prepatterned endodermal epithelium of the foregut forms dorsal (DP) and ventral (VP) buds by e9.5, which then develop into branching ducts and undifferentiated epithelium (e12.5; first development transition). Single endocrine cells are interspersed among the undifferentiated epithelium. The buds begin to differentiate into endocrine and exocrine cellular lineages by e14 and proliferate and expand extensively (second development transition). By e15, the dorsal and ventral pancreases rotate, fuse, and form a nearly fully developed pancreas by e19, containing the endocrine cells organized into isolated clusters that condense into the islets of Langerhans (third developmental transition). The third transition, consisting of maturation of endocrine cells and their acquisition of full nutrient responsiveness, continues for 2–3 wk after birth. The representative transcription factors expressed during the program of development are indicated in blue and are diagrammed in a simplified developmental cascade in Fig. 2. The approximate embryonic age (in days) is designated for each stage of development.
Between e13 and e14, the dorsal and ventral pancreata rotate and fuse into a single organ. During e14.5 and e15.5, the exocrine pancreas differentiates from the ductal epithelium; on e15.5, acini are clearly discernible from ducts. Endocrine cells are present from the very beginning of development (e9.5), but up until e14 they are arrayed as single cells within the ductal epithelium, after which they undergo extensive proliferation (second developmental transition). On e16, the endocrine cells begin to organize into islet-like clusters. The islets are not fully formed until shortly before birth on e18–e19 and undergo additional remodeling and maturation for 2–3 wk after birth (third developmental transition).
The endocrine cells of the pancreas arise from stem/progenitor cells located within the early (e9.5) gut endoderm (Fig. 1). Previous ideas that pancreatic endocrine cells are derived from the neural crest have been disproved by decisive quail-chick chimera experiments, and it is now established that they are of endodermal origin. It has been shown that isolated pancreatic endodermal-derived duct cells from embryonic rat pancreas can directly differentiate into hormone-expressing cells when cultured in the presence of gut mesenchyme (11). The earliest endocrine cells detected in the pancreatic anlage in the foregut (e9.5) express glucagon and members of the PP-fold family of hormones, peptide YY, pancreatic polypeptide, and neuropeptide Y.
Soon thereafter (e10–e10.5), the cells coexpress glucagon and insulin and then divide into distinct lineages that express glucagon (-cells) and insulin (?-cells). On about e14, somatostatin-expressing cells (-cells) arise. The precise temporal sequence of the expression of the hormones during pancreatic development remains somewhat controversial; the above description represents a consensus based on immunocytochemical examination of the mouse pancreas during embryonic development. Specific ablations of the -, ?-, and PP cell lineages have been accomplished by creating transgenic mice in which the cell-cidal diphtheria A toxin was targeted to the developing pancreas using the glucagon, insulin, and PP promoters (12). The targeted diphtheria A toxin driven by these respective promoters effectively ablated the -, ?-, and PP cell lineages, but only PP ablation alone impaired the formation of - and ?-cells, suggesting that PP-expressing cells, and not glucagon- or insulin-expressing cells, are precursors of the other endocrine cell types.
The signaling factors involved in specifying the segregation of the endocrine and exocrine compartments are of great interest. Studies of rat pancreatic rudiments grown in culture indicate that signals derived from the mesenchyme are either instructive or permissive for the determination of endocrine or exocrine cell lineages. Pancreas buds obtained from e12.5 rat embryos cultured in the presence of pancreatic mesenchyme develop into exocrine pancreatic tissue along with only a few immature insulin-producing endocrine cells. Buds cultured in the absence of pancreatic mesenchyme develop into endocrine tissue, and the development of exocrine tissue is retarded. Follistatin, present in e12.5 mesenchyme, mimics the effects of pancreatic mesenchyme to accelerate exocrine and limit endocrine differentiation (13).
Low levels of expression of endocrine and exocrine-specific genes are detectable for 2–3 d before development of the acinar cells characteristic of the differentiated pancreas (14, 15, 16) (first developmental transition). Segregation of insulin-expressing cells from amylase-expressing cells occurs on e15 (17). Levels of mRNAs encoding endocrine hormones and exocrine proteins increase markedly from e14 to e20 (16) by several thousand-fold (18) (second developmental transition) (Fig. 1).
The observation of a coordinate regulation of several genes expressed in the exocrine pancreas led to the identification of a complex of cell-specific DNA-binding proteins (PTF1) that activate the promoters of the several enzyme genes. PTF1 is detectable in acinar cells from e15 throughout adulthood and consists of a heterooligomer comprised of three subunits (19, 20). One of the three proteins, Ptf1a/P48, is an exocrine pancreatic-specific transcription factor of the basic helix-loop-helix (bHLH) type. Ptf1a RNA is detectable in the developing pancreas on e12 and was believed to be produced exclusively in the exocrine, and not the endocrine, pancreas (21). Mice nullizygous for the Ptfla/P48 gene die within hours of birth and have no detectable exocrine pancreatic tissue. Endocrine cells develop and migrate from the intestinal mesentery to colonize the spleen (22). A subsequent study showed that Ptfla is expressed in ductal, endocrine, and exocrine progenitors (23). Inactivation of Ptf1a switches pancreatic progenitors to intestinal epithelial progenitors. Transgenic expression of Pdx1 targeted by the Ptf1a promoter in Pdx1-null mice restores the development of pancreatic tissue. The Forkhead group of proteins, hepatic nuclear factor-3? (HNF3?; Foxa2) and Hnf3, binds to regulatory DNA sequences in the amylase promoter and contributes to exocrine-specific gene expression (24). Transcription of the amylase gene is stimulated by insulin, providing a possible mechanism for cross-talk between genes specific to the endocrine and exocrine compartments of the pancreas (25).
Morphogen signaling in early pancreas development
It is well recognized that the differentiation of epithelial cells during development requires their interactions with growth factors, or morphogens, produced by the mesodermal mesenchyme and by vascular endothelium in a specific temporal and spatial fashion (26). Studies of early pancreatic development in the chick embryo have provided insight into mesenchymal-derived morphogenesis in the signaling pathways involved in differentiation of the prepatterned region of the foregut endoderm that becomes the pancreas. At early stages of the developing chick pancreas, the dorsal pancreatic bud is in close proximity to the notochord (15). Removal of the notochord in early embryogenesis, before the 13-somite stage, prevents the development of adjacent endoderm otherwise destined to become the dorsal pancreas (27). Candidate mediators by which the notochord signals to the dorsal pancreatic endoderm include the growth factors activin-? and fibroblast growth factor-2 (28). When applied to chick embryo cultures of foregut, these growth factors mimic the notochord in repression of the signaling factor Sonic Hedgehog in the dorsal pancreatic endoderm and permit expression of pancreatic genes such as glucagon and insulin. Repression of Sonic Hedgehog in the pancreatic endoderm in the presence of Pdx-1 expression appears to be a prerequisite for the pancreas to develop; targeted misexpression of Sonic Hedgehog directed by the Pdx-1 promoter to the developing pancreas in mice causes the development of a disorganized mixed phenotype of intestinal and pancreatic tissues and absence of the spleen (29). Inhibition of Sonic Hedgehog signaling by the steroid alkaloid cyclopamine promotes pancreatic development in Pdx-1-expressing segments of foregut in the chick embryo (30).
Signals from developing structures other than notochord must control the development of the ventral pancreatic bud, but have yet to be completely defined. A recent study demonstrated that the homeobox protein, Hex, controls the positioning of endoderm cells beyond the cardiogenic mesoderm and thereby dictates ventral pancreatic specifications (31). The LIM (Lin11, Isl-1, and Mec-3) homeoprotein Isl1 is required for the formation of dorsal, but not ventral, pancreatic mesenchyme. Mice in which Isl1 expression is disrupted by gene knockout fail to develop a dorsal pancreas, but partially execute the differentiation of ventral exocrine pancreas (32). The dorsal pancreatic endoderm obtained from Isl1-null mice and studied ex vivo responds to signals transmitted from cocultured 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 signaling pathways that are critical for development of the pancreas requires additional investigation.
Transcription Factors and Pancreas Development
Several transcription factors are now recognized to be critical regulators of pancreatic development in mouse models of gene expression in which specific genes have been disrupted (gene knockouts; Table 1 and Fig. 2) (for reviews, see Refs.1, 2, 3, 4, 5, 6, 7 and 33, 34, 35, 36, 37, 38). Expression patterns of transcription factors limit the boundaries of the developing pancreas and determine the differentiation programs of individual cell lineages. Transcription factors appear to serve dual functions in determining early cellular development and later in maintaining the phenotype of terminally differentiated cells. Increasing recognition of the importance of transcription factors as a cause of human disease highlights the significance of the identification of these factors involved in the regulation of pancreatic development. The transcription factor cascade depicted in Fig. 2 is likely to be a marked oversimplification. A recent study used chromatin immunoprecipitation combined with promoter microassays to identify target genes in hepatocytes and pancreatic islets for the transcription factors Hnf6, Hnf1, and Hnf4 (39). In islets, Hnf6, Hnf1, and Hnf4 bound to 189, 106, and 1400 target genes, respectively. A significant number of the target genes in islets encoded transcription factors, e.g. eight for Hnf1 and 14 for Hnf4.
TABLE 1. Endocrine pancreatic phenotypes of transcription factor knockouts
FIG. 2. A simplified hierarchy of transcription factor expression in the developing pancreas based on the temporal expression and phenotypic results of gene-specific knockouts of the factors in mice. The color scheme designates the type (class) of transcription factors involved in the proposed hierarchal cascade: pink, homeodomain proteins; purple, bHLH; green, Maf proteins; orange, Hnfs; and gray, unidentified factors. The model does not strictly represent direct signaling pathways, but, rather, a consensus of gene expression requirements that permits continued progression toward the terminal differentiated state.
Pdx-1
The pancreatic duodenal homeobox gene-1 (Pdx-1) is a master regulator of both pancreatic development and the differentiation of progenitor cells into the ?-cell phenotype (35, 36) (Fig. 2). The transcription factor Pdx-1 was identified by several laboratories and so has multiple names, including Ipf-1 (40), Stf-1 (41), and Idx-1 (42). Pdx-1 belongs to a ParaHox gene cluster that lies outside the major Hox cluster of homeodomain proteins (43). The ParaHox set of genes consists of Gsh, Pdx, and Cdx, expressed in anterior, middle, and posterior locations in the vertebrate axis. Because Pdx-1 is a pancreas-specific homeoprotein, it was cloned and identified as a ?- and -cell-specific regulatory factor for transcriptional expression of insulin and somatostatin genes and has subsequently been shown to regulate the expression of other islet-specific genes, including Glut-2 (44), islet amyloid polypeptide (45), and glucokinase (46). The stable introduction and expression of Pdx-1 in a glucagon-secreting -cell line are sufficient to induce expression of the ?-cell-specific genes insulin, glucokinase, and islet amyloid polypeptide (33, 47). In the differentiated ?-cell, Pdx-1 is a glucose-responsive regulator of insulin gene expression (48, 49, 50, 51). The function of Pdx-1 in response to glucose is regulated by both its phosphorylation (50, 52) and nuclear translocation (53).
The patterns of expression of Pdx-1 in the developing pancreas are maintained throughout development and provide both spatial and temporal contributions to the commitment of endoderm to a pancreatic phenotype (54). Pdx-1 expression is first detected in murine cells on e8.5 in a narrow band of foregut endoderm. On e9.5, Pdx-1 expression is seen in both ventral and dorsal pancreatic buds (54, 55). From e11.5 to e13.5, Pdx-1 is expressed throughout the developing ductal tree. As the exocrine pancreas appears and the islets begin to form into the hormone-producing cells (e14–e15), Pdx-1 expression shifts to the endocrine compartment. Colocalization of Pdx-1 and the exocrine cell-specific enzyme amylase is observed on e13.5, diminishes markedly by e16.5, and is nearly undetectable in the exocrine pancreas of adult mice. During the later stages of islet development, by e18.5, the expression of Pdx-1 becomes mostly restricted to the mature ?-cells of the endocrine pancreas (54, 55). In the adult pancreas, subpopulations of somatostatin-producing and pancreatic polypeptide-producing cells also express Pdx-1. Only a few glucagon-producing cells express Pdx-1 (40, 41, 42, 54).
Targeted disruption of the pdx-1 gene in mice results in agenesis of the pancreas (55, 56). Nullizygous pdx-1 mice are viable, but die within days after birth (56). In these animals rudimentary pancreatic buds form, but then regress. Defects in development of the rostral duodenal epithelium and enteroendocrine cells also occur (55). The pancreatic mesenchyme is visibly normal in pdx-1-null mice, but the pancreatic epithelium is defective in its ability to respond to mesenchymal signals required to complete the differentiation program (57). A few insulin-expressing cells are detected in Pdx-1-null mice early in pancreas development, suggesting that a population of insulin-positive, Pdx-1-negative cells may occur distinct from the mature Pdx-1-expressing ?-cells of the developed pancreas (57). Notably, a child born without a pancreas (pancreatic agenesis) is homozygous for an inactivating mutation in pdx-1 (ipf-1) (58), underscoring the importance of the Pdx-1 transcription factor in the development of the human, as well as the mouse, pancreas.
Hlxb9
Hlxb9 is a bHLH transcriptional activator protein expressed early in pancreas development on e8. In mice homozygous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop (59, 60). The remnant Hlxb9-null pancreas has small islets with reduced numbers of insulin-producing ?-cells.
Isl1
The LIM homeodomain protein Isl1 is expressed in the developing pancreas and the central nervous system (61, 62). In adult animals, Isl1 is expressed in all of the hormone-producing cells of pancreatic islet cells, but its low level of expression in ?-cells argues against it having an important function to regulate insulin gene transcription. Targeted disruption of the isl-1 gene results in an early arrest of embryonic development on approximately e9.5 (63). Analysis of the pancreatic anlage reveals failure of development of the dorsal pancreatic mesenchyme and the complete absence of endocrine cells. Notably, development of the ventral pancreatic epithelium and associated mesenchyme in Isl-1-null mice is unaffected (32). The addition of mesenchyme derived from pancreas or lung to explants of dorsal pancreatic epithelium of isl-1-null animals results in the development of exocrine tissue, but no endocrine cells are generated. These findings suggest that Isl1 is required for development of the mesenchyme of the dorsal pancreatic bud and also for differentiation of the dorsal pancreatic epithelium to endocrine cells.
Hes1, Neurogenin-3 (Ngn3), and Notch Signaling
Notch signaling is an important factor in the periodicity of somatogenesis in the vertebrate embryo (64). Although its role as a key participant in the biological clock of embryonic development is slowly emerging, evidence of notch signaling in the developing pancreas suggests a primary role in governing cell fate. Signals exchanged between neighboring cells through the Notch receptor can amplify and consolidate differences in cellular phenotypes; thus, Notch signaling provides a developmental tool to influence morphogenesis and organ formation. Several recent studies suggest the involvement of Notch in the regulation of pancreatic exocrine and endocrine cell fate, reflecting the well established regulatory functions of Notch in neural development by a mechanism known as lateral inhibition (65). In the neural crest, expression of the bHLH transcription factor Ngn leads to the expression of extracellular ligands such as (dl), Serrate (Ser), and Jagged (Jag) in neural precursor cells that activate Notch receptors on adjacent cells. Intracellular recombination signal sequence binding protein mediates Notch receptor-dependent expression of Hes genes in the adjacent cell. Hes genes encode bHLH factors that function to repress the expression of Ngn and other target genes, thereby preventing neuronal differentiation in cells adjacent to developing neuroblasts. In essence, the lateral inhibition model provides a mechanism by which the cell that will differentiate into the endocrine lineage (ngn3 positive) inhibits its neighboring cells, forcing them to adopt a nonendocrine fate. In the generation of the pancreas, Ngn3 is required for the development of all endocrine cell lineages of the pancreas (9, 66) and has been designated as a marker of islet precursor cells (see below). Overexpression of Ngn3 in the developing pancreas results in accelerated differentiation of endocrine progenitor cells. Hes-1, although expressed in the pancreas, is absent from endocrine cells, in agreement with this model. Hes-1 knockout mice exhibit a rapid depletion of precursor cells and a resultant precocious development of endocrine cells (67). Furthermore, mice deficient for -like ligand-1 (dll1) display accelerated differentiation of pancreatic epithelial cells expressing Ngn3 (68).
As discussed above, the initiating step toward endocrine cell fate appears to depend on regulation of the bHLH transcription factor Ngn3. The role of Ngn3 as an early marker of cells differentiating toward a primary endocrine fate was clearly demonstrated by its ectopic expression under the regulatory control of the pdx-1 promoter in transgenic mice (68). Small and poorly branched pancreatic buds were evident on e12, followed by premature and precocious differentiation of pancreatic progenitor cells into endocrine cells, and the resultant depletion in precursor cells with the capacity to proliferate, branch, and differentiate into exocrine cells. Expression of Ngn3 starts on e9–e9.5, peaks on e15.5 during the major wave of endocrine cell genesis, and is greatly diminished at birth, with little or no detection of Ngn3 in the adult pancreas. Significantly, mice homozygous for an ngn3-null mutation failed to generate any endocrine cells or putative endocrine precursors during development (66). These observations led to the assumption that ngn3 expression is a functional marker of an islet cell precursor population in the developing pancreas.
Because Ngn3 is both necessary and sufficient to drive the differentiation program of islet cells during pancreatic development, it may be inferred that the activities of specific transcription factors that regulate cis-acting elements within the promoter region of the ngn3 gene, collectively dictate the endocrine fate of the cell. In the distal region of the ngn3 promoter, a cluster of binding sites for HNF1, HNF3?, and HNF6 was recently identified (69). Concurrent with the roles of these transcription factors in regulating ngn3 gene expression and the subsequent initiation of the endocrine program, disruption of their respective genes in the developing embryo results in corroborative phenotypes, affecting the progression of early pancreatic development and endocrine specification. Mice homozygous for a disrupted hnf1 gene have smaller islets and reduced insulin secretion (70). HNF3?-null mice show a lack of foregut formation (71, 72), and in mouse embryos lacking hnf6 expression, a marked reduction in endocrine cell differentiation is observed along with severely reduced levels of ngn3 gene expression (73). However, the independent expression of HNF factors appears not to be sufficient for ngn3 expression, but, rather, a cooperative mechanism exists between these accessory proteins in the cell type-restricted activation of the ngn3 gene promoter (69). Hes-1 represses the expression of the ngn3 gene promoter by binding to several silencer sites located close to the transcription initiation site and thus suppresses endocrine precursor patterning through the Notch signaling pathway.
Beta2/NeuroD
The bHLH transcription factor Beta2/NeuroD is a key regulator of both insulin gene transcription in pancreatic ?-cells and the terminal differentiation of neurons (74, 75). Beta2/NeuroD expression occurs in a subset of pancreatic epithelial cells as early as e9.5, colocalizing with glucagon expression (76). On e14.5, expression appears within and in proximity to ductal epithelium, and by e17.5, expression is restricted to islets (77). Beta2/NeuroD heterodimerizes with ubiquitous bHLH proteins of the E2A family to regulate transcription of the insulin gene and other ?-cell-specific genes. Expression of the Beta2/NeuroD gene is activated by Ngn3 (78). Mice homozygous for a targeted disruption of the ?2/neuroD gene survive to birth, but die within 3–5 d postpartum of severe hyperglycemia. The islets of these mice are dysmorphic and have markedly diminished numbers of endocrine cells arranged in streaks and irregular aggregates and reduced numbers of ?-cells (77).
Pax4
Pax4 is a paired-box homeoprotein whose expression is restricted to the central nervous system and the developing pancreas. By e9.5, Pax4 is expressed in both ventral and dorsal buds of the developing pancreas, and by the time of birth, expression is restricted to ?-cells. Targeted disruption of Pax4 in mice results in a striking pancreatic phenotype (79). The pax4-null mice survive birth, but die within 3 d of hyperglycemia and dehydration. There is a virtual absence of ?- and -cells, but -cells are increased. PDX-1 expression in pax4-null pancreas is absent, a finding consistent with the absence of differentiated ?-cells. Thus, Pax4 functions early in the development of islet cells to promote the differentiation of ?- and -cells.
Pax6
Another paired-box homeoprotein, Pax6, is important for islet cell development. The expression of Pax6 is restricted to the eye, the central nervous system, the nose, and the endocrine pancreas (80, 81). Pax6 is expressed early in the epithelium of the developing pancreas on e9.0 in both dorsal and ventral pancreatic buds and is expressed in differentiated -, ?-, -, and PP cells. A spontaneous mutation of the pax6 gene in mice, known as Small eye (Sey–/–), has defined the importance of Pax6 in the development of the lens placode of the eye and portions of the forebrain (82). Small eyeNeu mice have abnormal organization of islets, with decreased -, ?-, -, and PP cells (83). The production of glucagon and insulin is substantially reduced in these animals. Pax6 regulates the promoters of the glucagon, insulin, and somatostatin genes, suggesting a mechanism to explain the decreased hormone production observed in Small eye mice (83). Knockout pax6 nullizygous mice die within minutes of birth. These animals fail to form islets (84). The disorganization of islets is proposed to relate to a possible function of Pax6 in the regulation of cell adhesion molecules (84, 85). These mouse models of gene knockouts implicate both Pax4 and Pax6 as key regulators of the terminal steps in cellular differentiation of the endocrine pancreas.
Nkx2.2 and Nkx6.1
Members of the NK2 family of homeoprotein transcription factors, Nkx2.2 and Nkx6.1, are regulators of the differentiation of pancreatic endocrine cells (86). Nkx2.2 is expressed early in developing pancreatic buds and is later restricted to -, ?-, and PP cells of islets. Nkx6.1 is expressed primarily in ?-cells of adult islets (87, 88). Disruption of either the Nkx2.2 or Nkx6.1 gene in mice results in death soon after birth due to severe diabetes (86). The pancreata of Nkx2.2 and Nkx6.1 knockout mice have no insulin-producing cells, and glucagon-producing cells are diminished, although the exocrine pancreas is histologically normal. Nkx2.2-null mice have a large number of hormone-negative, arrested precursor cells that express endocrine pancreas-specific proteins, such as synaptophysin, neuroendocrine cell adhesion molecule, amyloid polypeptide, and prohormone convertase 1/3, but no hormones. Nkx6.1-null mice have a selective reduction of ?-cells with a normal complement of other endocrine cell types (89). Notably, it appears that in both Nkx2.2- and Pax4-null mice that fail to develop insulin-producing cells, the arrested progenitor ?-cell expresses the hormone ghrelin (90).
MafA and MafB
MafA and MafB are transcription factors previously recognized to be important in development of the lens of the eye. During pancreatic development, MafA is expressed in early ?-cells during the second developmental transition on e14 (91, 92). MafB appears to be expressed in glucagon-producing -cells on e15. The phenotypic consequences of MafA and MafB gene knockouts in mice have not yet been reported.
Mist1
Mist1 is a bHLH transcription factor expressed in pancreatic acinar cells (93). Mist1-null mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation, and acinar tissue acquires a ductal phenotype. It has been proposed that Mist1-null mice represent a genetic model for chronic pancreatic injury and that Mist1 serves as a key regulator of acinar cell function and stability.
Relevance of pancreatic development to pathogenesis of diabetes mellitus
Diabetes mellitus results from a combination of insulin resistance and deficient or dysregulated insulin secretion by pancreatic ?-cells. Human genetic studies indicate that dysfunction of ?-cell transcriptional regulators causes some forms of type 2 diabetes. Maturity-onset diabetes of the young (MODY) is a type of autosomal dominant monogenic diabetes with early onset (age of 30 yr or less). The prevalence of MODY-type diabetes is higher than initially proposed (94), now accounting for 5–10% of type 2 diabetes. The autosomal dominant inheritance pattern of this type of diabetes has facilitated genetic analysis of familial type 2 diabetes. The first genetic mutations linked with MODY were in the glucokinase gene (MODY2), an enzyme essential for glucose-sensing in ?-cells (95, 96). The five additional types of MODY identified to date are linked to mutations in transcription factors relevant to pancreatic ?-cell function (Table 2), although additional MODY genes remain to be defined (97). These transcription factors function in both pancreatic development and the differentiated ?-cell. MODY3 results from mutations in the Hnf1 gene. Hnf1 is a homeodomain and POU-like DNA-binding protein that transactivates insulin and IGF-I gene transcription (98, 99). Disruption of the Hnf1 gene in mice results in Laron dwarfism, decreased insulin gene expression, and diabetes. Humans with Hnf1 (MODY3) mutations have defective glucose utilization, insulin secretion, and glucose disposal (100). MODY1 is a result of mutations in the transcription factor Hnf4. Hnf4 activates the HNF1 promoter (101) (Fig. 3), which is relevant in light of the linkage of Hnf1 mutations to MODY3 (102). Both Hnf4 and Hnf1 are essential for normal pancreatic ?-cell function, and human subjects with mutations in either gene develop impaired glucose tolerance and/or diabetes (103, 104). MODY5 results from mutations in Hnf1?, a POU (Pit-1, Oct-1, and Unc-86)-type homeoprotein, structurally related to Hnf1. Selective deletion of the Hnf1? gene in mice manifests in impaired glucose tolerance and impaired glucose-dependent insulin release (105). In addition, the pancreata of Hnf1? mice have increased Pdx1 and Hnf1 mRNA levels and decreased levels of Hnf4 mRNA.
TABLE 2. Transcription factors involved in pancreas development associated with diabetes
FIG. 3. Transcription factor cascade involved in the regulation of the rat insulin-1 gene promoter. The enhancers E2-A4/A3 (and E1/A1, not shown) bind and are activated by bHLH proteins and the homeodomain protein Ipf-1 (Pdx-1). HNF1 also binds to the E2-A4/A3 complex. Hnf4, in turn, is regulated by Hnf1? and possibly Hnf3? (activated by Hnf6). Mutations in Hnf4, Hnf1, Ipf-1, Hnf1?, and Beta2 are responsible for MODY1, -3, -4, -5, and -6, respectively. CREB, cAMP response element-binding protein; Basal, basal transcription factors; Trx, transcriptional activation of rat insulin-1 gene promoter (rInsulin-1 gene).
Mutations in the Beta2/NeuroD gene appear to have defined a MODY6. At least two distinct mutations in the Beta2/NeuroD gene appear to be responsible for the onset of type 2 diabetes (MODY6) (106). The linkage of MODY with three members of the Hnf regulatory cascade of transcription factors suggests that additional genes in the Hnf signaling cascade may be involved in causing diabetes. For example, Hnf6 is expressed in the developing exocrine pancreatic cells and in islets and may be important in stimulating the transcriptional expression of Hnf4 and Hnf3?. Hnf3? is a transcription factor expressed in embryonic pancreatic buds and islets and functions as an activator of the expression of the Hnf4, Hnf1, and Pdx-1 genes.
An inactivating mutation in transcription factor Pdx-1 (Ipf1) causes MODY4. Pdx-1 is required for pancreatic development. Individuals heterozygous for the inactivating mutation in the Pdx-1 gene develop diabetes. Evaluation of this inactivating Pdx-1 mutation in vitro suggests that diabetes results from both diminished gene dosage (haploinsufficiency) and dominant negative inhibition of transcriptional activation of the intact PDX-1 allele. In the mouse model of Pdx-1 inactivation, Pdx-1 hemizygous animals are glucose intolerant, and conditional inactivation of Pdx-1 after the pancreas has developed results in diabetes. Several missense and insertional mutations in the Pdx-1 gene are now shown to be associated with the development of late-onset type 2 diabetes in the French and United Kingdom populations (107, 108). ?-Cell dysfunction due to altered cellular levels of Pdx-1 may occur in the absence of mutations. Suppression of Pdx-1 expression in transgenic mice expressing a conditional Pdx-1 antisense ribozyme targeted by the rat insulin promoter develop age-dependent diabetes (109). In ?-cell models of glucotoxicity, sustained exposure to hyperglycemia down-regulates Pdx-1 expression concordant with decreased insulin gene transcription.
Several additional transcription factors essential for pancreatic development or for differentiated ?-cell function, including the regulation of insulin gene transcription, are candidate diabetes genes. These transcription factors include Isl-1, Pax4, Nkx2.2, Nkx6.1, and Ngn3, based on the findings that targeted disruptions of the genes encoding these factors in mice result in impaired development of the pancreas and/or pancreatic islets (?-cells). A subset of these transcription factors, such as Pdx-1, may be critical in mediating both ?-cell development and differentiated ?-cell function. Investigations of pancreatic development, islet gene regulation, and the genetics of diabetes mellitus are rapidly converging, with the potential to generate novel and potent therapies for pancreatic disease.
Acknowledgments
We thank the members of the Laboratory of Molecular Endocrinology for their helpful suggestions, and Melissa Fannon and Kimberly MacDonald for help with the preparation of the manuscript.
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Address all correspondence and requests for reprints to: Dr. Joel F. Habener, Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, WEL320, Boston, Massachusetts 02114. E-mail: jhabener@partners.org.
Abstract
Considerable progress has been made in the understanding of the sequential activation of signal transduction pathways and the expression of transcription factors during pancreas development. Much of this understanding has been obtained by analyses of the phenotypes of mice in which the expression of key genes has been disrupted (knockout mice). Knockout of the genes for Pdx1, Hlxb9, Isl1, or Hex results in an arrest of pancreas development at a very early stage (embryonic d 8–9). Disruption of genes encoding components of the Notch signaling pathway, e.g. Hes1 or neurogenin-3, abrogates development of the endocrine pancreas (islets of Langerhans). Disruption of transcription factor genes expressed more downstream in the developmental cascade (Beta2/NeuroD, Pax4, NKx2.2, and Nkx6.1) curtails the formation of insulin-producing ?-cells. An understanding of the importance of transcription factor genes during pancreas development has provided insights into the pathogenesis of diabetes, in which the mass of insulin-producing ?-cells is reduced.
RECENT STUDIES OF the biology of pancreas development have shed new light on the cause of type 2 diabetes. The identification of transcription factors involved in regulation of the expression of key genes required in the developing and adult endocrine and exocrine pancreas has been provided by observing the phenotypic consequences of targeted disruptions of the genes (gene knockouts) encoding these factors in mice. Remarkably, almost without exception, disruption of these genes has resulted in phenotypes of impaired development of the pancreas and consequent diabetes. Furthermore, lessons learned from the gene knockouts in mice have been used to successfully identify mutations in several of the corresponding orthologous genes in individuals with familial monogenic type 2 diabetes.
In this minireview we attempt to encapsulate a rapidly growing body of knowledge that is providing insight into the genetic contributions to the development of diabetes. The thematic emphasis is on the impact that genetic polymorphisms or mutations in genes encoding transcription factors essential for pancreas development have on predisposition for diabetes. For more comprehensive, in-depth information on this topic, the reader is referred to several recent excellent reviews (1, 2, 3, 4, 5, 6, 7, 8, 9).
Anatomical and Morphological Development of the Pancreas
The pancreas consists of three main tissue cell types (in addition to vascular and stromal supporting tissues): the exocrine acinar tissue that produces digestive enzymes; the endocrine cells (islets of Langerhans) that produce the hormones involved in nutrient homeostasis, such as insulin and glucagon; and the elaborately branched ductal tree (10). The pancreas originates early in development [embryonic d 8.5 (e8.5) to e9.5 in the mouse] by the outcropping of two buds (ventral and dorsal) of cells from a specialized prepatterned endodermal epithelium located in the region of the foregut that is to become the duodenum (Fig. 1). By e10.5, the partially differentiated epithelium of the two buds undergoes branching morphogenesis into a ductal tree that by e12.5 results in the formation of two primordial pancreas organs consisting predominantly of an undifferentiated ductal epithelium (first developmental transition).
FIG. 1. Schematic diagram of pancreatic development in the mouse. On e8, prepatterned endodermal epithelium of the foregut forms dorsal (DP) and ventral (VP) buds by e9.5, which then develop into branching ducts and undifferentiated epithelium (e12.5; first development transition). Single endocrine cells are interspersed among the undifferentiated epithelium. The buds begin to differentiate into endocrine and exocrine cellular lineages by e14 and proliferate and expand extensively (second development transition). By e15, the dorsal and ventral pancreases rotate, fuse, and form a nearly fully developed pancreas by e19, containing the endocrine cells organized into isolated clusters that condense into the islets of Langerhans (third developmental transition). The third transition, consisting of maturation of endocrine cells and their acquisition of full nutrient responsiveness, continues for 2–3 wk after birth. The representative transcription factors expressed during the program of development are indicated in blue and are diagrammed in a simplified developmental cascade in Fig. 2. The approximate embryonic age (in days) is designated for each stage of development.
Between e13 and e14, the dorsal and ventral pancreata rotate and fuse into a single organ. During e14.5 and e15.5, the exocrine pancreas differentiates from the ductal epithelium; on e15.5, acini are clearly discernible from ducts. Endocrine cells are present from the very beginning of development (e9.5), but up until e14 they are arrayed as single cells within the ductal epithelium, after which they undergo extensive proliferation (second developmental transition). On e16, the endocrine cells begin to organize into islet-like clusters. The islets are not fully formed until shortly before birth on e18–e19 and undergo additional remodeling and maturation for 2–3 wk after birth (third developmental transition).
The endocrine cells of the pancreas arise from stem/progenitor cells located within the early (e9.5) gut endoderm (Fig. 1). Previous ideas that pancreatic endocrine cells are derived from the neural crest have been disproved by decisive quail-chick chimera experiments, and it is now established that they are of endodermal origin. It has been shown that isolated pancreatic endodermal-derived duct cells from embryonic rat pancreas can directly differentiate into hormone-expressing cells when cultured in the presence of gut mesenchyme (11). The earliest endocrine cells detected in the pancreatic anlage in the foregut (e9.5) express glucagon and members of the PP-fold family of hormones, peptide YY, pancreatic polypeptide, and neuropeptide Y.
Soon thereafter (e10–e10.5), the cells coexpress glucagon and insulin and then divide into distinct lineages that express glucagon (-cells) and insulin (?-cells). On about e14, somatostatin-expressing cells (-cells) arise. The precise temporal sequence of the expression of the hormones during pancreatic development remains somewhat controversial; the above description represents a consensus based on immunocytochemical examination of the mouse pancreas during embryonic development. Specific ablations of the -, ?-, and PP cell lineages have been accomplished by creating transgenic mice in which the cell-cidal diphtheria A toxin was targeted to the developing pancreas using the glucagon, insulin, and PP promoters (12). The targeted diphtheria A toxin driven by these respective promoters effectively ablated the -, ?-, and PP cell lineages, but only PP ablation alone impaired the formation of - and ?-cells, suggesting that PP-expressing cells, and not glucagon- or insulin-expressing cells, are precursors of the other endocrine cell types.
The signaling factors involved in specifying the segregation of the endocrine and exocrine compartments are of great interest. Studies of rat pancreatic rudiments grown in culture indicate that signals derived from the mesenchyme are either instructive or permissive for the determination of endocrine or exocrine cell lineages. Pancreas buds obtained from e12.5 rat embryos cultured in the presence of pancreatic mesenchyme develop into exocrine pancreatic tissue along with only a few immature insulin-producing endocrine cells. Buds cultured in the absence of pancreatic mesenchyme develop into endocrine tissue, and the development of exocrine tissue is retarded. Follistatin, present in e12.5 mesenchyme, mimics the effects of pancreatic mesenchyme to accelerate exocrine and limit endocrine differentiation (13).
Low levels of expression of endocrine and exocrine-specific genes are detectable for 2–3 d before development of the acinar cells characteristic of the differentiated pancreas (14, 15, 16) (first developmental transition). Segregation of insulin-expressing cells from amylase-expressing cells occurs on e15 (17). Levels of mRNAs encoding endocrine hormones and exocrine proteins increase markedly from e14 to e20 (16) by several thousand-fold (18) (second developmental transition) (Fig. 1).
The observation of a coordinate regulation of several genes expressed in the exocrine pancreas led to the identification of a complex of cell-specific DNA-binding proteins (PTF1) that activate the promoters of the several enzyme genes. PTF1 is detectable in acinar cells from e15 throughout adulthood and consists of a heterooligomer comprised of three subunits (19, 20). One of the three proteins, Ptf1a/P48, is an exocrine pancreatic-specific transcription factor of the basic helix-loop-helix (bHLH) type. Ptf1a RNA is detectable in the developing pancreas on e12 and was believed to be produced exclusively in the exocrine, and not the endocrine, pancreas (21). Mice nullizygous for the Ptfla/P48 gene die within hours of birth and have no detectable exocrine pancreatic tissue. Endocrine cells develop and migrate from the intestinal mesentery to colonize the spleen (22). A subsequent study showed that Ptfla is expressed in ductal, endocrine, and exocrine progenitors (23). Inactivation of Ptf1a switches pancreatic progenitors to intestinal epithelial progenitors. Transgenic expression of Pdx1 targeted by the Ptf1a promoter in Pdx1-null mice restores the development of pancreatic tissue. The Forkhead group of proteins, hepatic nuclear factor-3? (HNF3?; Foxa2) and Hnf3, binds to regulatory DNA sequences in the amylase promoter and contributes to exocrine-specific gene expression (24). Transcription of the amylase gene is stimulated by insulin, providing a possible mechanism for cross-talk between genes specific to the endocrine and exocrine compartments of the pancreas (25).
Morphogen signaling in early pancreas development
It is well recognized that the differentiation of epithelial cells during development requires their interactions with growth factors, or morphogens, produced by the mesodermal mesenchyme and by vascular endothelium in a specific temporal and spatial fashion (26). Studies of early pancreatic development in the chick embryo have provided insight into mesenchymal-derived morphogenesis in the signaling pathways involved in differentiation of the prepatterned region of the foregut endoderm that becomes the pancreas. At early stages of the developing chick pancreas, the dorsal pancreatic bud is in close proximity to the notochord (15). Removal of the notochord in early embryogenesis, before the 13-somite stage, prevents the development of adjacent endoderm otherwise destined to become the dorsal pancreas (27). Candidate mediators by which the notochord signals to the dorsal pancreatic endoderm include the growth factors activin-? and fibroblast growth factor-2 (28). When applied to chick embryo cultures of foregut, these growth factors mimic the notochord in repression of the signaling factor Sonic Hedgehog in the dorsal pancreatic endoderm and permit expression of pancreatic genes such as glucagon and insulin. Repression of Sonic Hedgehog in the pancreatic endoderm in the presence of Pdx-1 expression appears to be a prerequisite for the pancreas to develop; targeted misexpression of Sonic Hedgehog directed by the Pdx-1 promoter to the developing pancreas in mice causes the development of a disorganized mixed phenotype of intestinal and pancreatic tissues and absence of the spleen (29). Inhibition of Sonic Hedgehog signaling by the steroid alkaloid cyclopamine promotes pancreatic development in Pdx-1-expressing segments of foregut in the chick embryo (30).
Signals from developing structures other than notochord must control the development of the ventral pancreatic bud, but have yet to be completely defined. A recent study demonstrated that the homeobox protein, Hex, controls the positioning of endoderm cells beyond the cardiogenic mesoderm and thereby dictates ventral pancreatic specifications (31). The LIM (Lin11, Isl-1, and Mec-3) homeoprotein Isl1 is required for the formation of dorsal, but not ventral, pancreatic mesenchyme. Mice in which Isl1 expression is disrupted by gene knockout fail to develop a dorsal pancreas, but partially execute the differentiation of ventral exocrine pancreas (32). The dorsal pancreatic endoderm obtained from Isl1-null mice and studied ex vivo responds to signals transmitted from cocultured 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 signaling pathways that are critical for development of the pancreas requires additional investigation.
Transcription Factors and Pancreas Development
Several transcription factors are now recognized to be critical regulators of pancreatic development in mouse models of gene expression in which specific genes have been disrupted (gene knockouts; Table 1 and Fig. 2) (for reviews, see Refs.1, 2, 3, 4, 5, 6, 7 and 33, 34, 35, 36, 37, 38). Expression patterns of transcription factors limit the boundaries of the developing pancreas and determine the differentiation programs of individual cell lineages. Transcription factors appear to serve dual functions in determining early cellular development and later in maintaining the phenotype of terminally differentiated cells. Increasing recognition of the importance of transcription factors as a cause of human disease highlights the significance of the identification of these factors involved in the regulation of pancreatic development. The transcription factor cascade depicted in Fig. 2 is likely to be a marked oversimplification. A recent study used chromatin immunoprecipitation combined with promoter microassays to identify target genes in hepatocytes and pancreatic islets for the transcription factors Hnf6, Hnf1, and Hnf4 (39). In islets, Hnf6, Hnf1, and Hnf4 bound to 189, 106, and 1400 target genes, respectively. A significant number of the target genes in islets encoded transcription factors, e.g. eight for Hnf1 and 14 for Hnf4.
TABLE 1. Endocrine pancreatic phenotypes of transcription factor knockouts
FIG. 2. A simplified hierarchy of transcription factor expression in the developing pancreas based on the temporal expression and phenotypic results of gene-specific knockouts of the factors in mice. The color scheme designates the type (class) of transcription factors involved in the proposed hierarchal cascade: pink, homeodomain proteins; purple, bHLH; green, Maf proteins; orange, Hnfs; and gray, unidentified factors. The model does not strictly represent direct signaling pathways, but, rather, a consensus of gene expression requirements that permits continued progression toward the terminal differentiated state.
Pdx-1
The pancreatic duodenal homeobox gene-1 (Pdx-1) is a master regulator of both pancreatic development and the differentiation of progenitor cells into the ?-cell phenotype (35, 36) (Fig. 2). The transcription factor Pdx-1 was identified by several laboratories and so has multiple names, including Ipf-1 (40), Stf-1 (41), and Idx-1 (42). Pdx-1 belongs to a ParaHox gene cluster that lies outside the major Hox cluster of homeodomain proteins (43). The ParaHox set of genes consists of Gsh, Pdx, and Cdx, expressed in anterior, middle, and posterior locations in the vertebrate axis. Because Pdx-1 is a pancreas-specific homeoprotein, it was cloned and identified as a ?- and -cell-specific regulatory factor for transcriptional expression of insulin and somatostatin genes and has subsequently been shown to regulate the expression of other islet-specific genes, including Glut-2 (44), islet amyloid polypeptide (45), and glucokinase (46). The stable introduction and expression of Pdx-1 in a glucagon-secreting -cell line are sufficient to induce expression of the ?-cell-specific genes insulin, glucokinase, and islet amyloid polypeptide (33, 47). In the differentiated ?-cell, Pdx-1 is a glucose-responsive regulator of insulin gene expression (48, 49, 50, 51). The function of Pdx-1 in response to glucose is regulated by both its phosphorylation (50, 52) and nuclear translocation (53).
The patterns of expression of Pdx-1 in the developing pancreas are maintained throughout development and provide both spatial and temporal contributions to the commitment of endoderm to a pancreatic phenotype (54). Pdx-1 expression is first detected in murine cells on e8.5 in a narrow band of foregut endoderm. On e9.5, Pdx-1 expression is seen in both ventral and dorsal pancreatic buds (54, 55). From e11.5 to e13.5, Pdx-1 is expressed throughout the developing ductal tree. As the exocrine pancreas appears and the islets begin to form into the hormone-producing cells (e14–e15), Pdx-1 expression shifts to the endocrine compartment. Colocalization of Pdx-1 and the exocrine cell-specific enzyme amylase is observed on e13.5, diminishes markedly by e16.5, and is nearly undetectable in the exocrine pancreas of adult mice. During the later stages of islet development, by e18.5, the expression of Pdx-1 becomes mostly restricted to the mature ?-cells of the endocrine pancreas (54, 55). In the adult pancreas, subpopulations of somatostatin-producing and pancreatic polypeptide-producing cells also express Pdx-1. Only a few glucagon-producing cells express Pdx-1 (40, 41, 42, 54).
Targeted disruption of the pdx-1 gene in mice results in agenesis of the pancreas (55, 56). Nullizygous pdx-1 mice are viable, but die within days after birth (56). In these animals rudimentary pancreatic buds form, but then regress. Defects in development of the rostral duodenal epithelium and enteroendocrine cells also occur (55). The pancreatic mesenchyme is visibly normal in pdx-1-null mice, but the pancreatic epithelium is defective in its ability to respond to mesenchymal signals required to complete the differentiation program (57). A few insulin-expressing cells are detected in Pdx-1-null mice early in pancreas development, suggesting that a population of insulin-positive, Pdx-1-negative cells may occur distinct from the mature Pdx-1-expressing ?-cells of the developed pancreas (57). Notably, a child born without a pancreas (pancreatic agenesis) is homozygous for an inactivating mutation in pdx-1 (ipf-1) (58), underscoring the importance of the Pdx-1 transcription factor in the development of the human, as well as the mouse, pancreas.
Hlxb9
Hlxb9 is a bHLH transcriptional activator protein expressed early in pancreas development on e8. In mice homozygous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop (59, 60). The remnant Hlxb9-null pancreas has small islets with reduced numbers of insulin-producing ?-cells.
Isl1
The LIM homeodomain protein Isl1 is expressed in the developing pancreas and the central nervous system (61, 62). In adult animals, Isl1 is expressed in all of the hormone-producing cells of pancreatic islet cells, but its low level of expression in ?-cells argues against it having an important function to regulate insulin gene transcription. Targeted disruption of the isl-1 gene results in an early arrest of embryonic development on approximately e9.5 (63). Analysis of the pancreatic anlage reveals failure of development of the dorsal pancreatic mesenchyme and the complete absence of endocrine cells. Notably, development of the ventral pancreatic epithelium and associated mesenchyme in Isl-1-null mice is unaffected (32). The addition of mesenchyme derived from pancreas or lung to explants of dorsal pancreatic epithelium of isl-1-null animals results in the development of exocrine tissue, but no endocrine cells are generated. These findings suggest that Isl1 is required for development of the mesenchyme of the dorsal pancreatic bud and also for differentiation of the dorsal pancreatic epithelium to endocrine cells.
Hes1, Neurogenin-3 (Ngn3), and Notch Signaling
Notch signaling is an important factor in the periodicity of somatogenesis in the vertebrate embryo (64). Although its role as a key participant in the biological clock of embryonic development is slowly emerging, evidence of notch signaling in the developing pancreas suggests a primary role in governing cell fate. Signals exchanged between neighboring cells through the Notch receptor can amplify and consolidate differences in cellular phenotypes; thus, Notch signaling provides a developmental tool to influence morphogenesis and organ formation. Several recent studies suggest the involvement of Notch in the regulation of pancreatic exocrine and endocrine cell fate, reflecting the well established regulatory functions of Notch in neural development by a mechanism known as lateral inhibition (65). In the neural crest, expression of the bHLH transcription factor Ngn leads to the expression of extracellular ligands such as (dl), Serrate (Ser), and Jagged (Jag) in neural precursor cells that activate Notch receptors on adjacent cells. Intracellular recombination signal sequence binding protein mediates Notch receptor-dependent expression of Hes genes in the adjacent cell. Hes genes encode bHLH factors that function to repress the expression of Ngn and other target genes, thereby preventing neuronal differentiation in cells adjacent to developing neuroblasts. In essence, the lateral inhibition model provides a mechanism by which the cell that will differentiate into the endocrine lineage (ngn3 positive) inhibits its neighboring cells, forcing them to adopt a nonendocrine fate. In the generation of the pancreas, Ngn3 is required for the development of all endocrine cell lineages of the pancreas (9, 66) and has been designated as a marker of islet precursor cells (see below). Overexpression of Ngn3 in the developing pancreas results in accelerated differentiation of endocrine progenitor cells. Hes-1, although expressed in the pancreas, is absent from endocrine cells, in agreement with this model. Hes-1 knockout mice exhibit a rapid depletion of precursor cells and a resultant precocious development of endocrine cells (67). Furthermore, mice deficient for -like ligand-1 (dll1) display accelerated differentiation of pancreatic epithelial cells expressing Ngn3 (68).
As discussed above, the initiating step toward endocrine cell fate appears to depend on regulation of the bHLH transcription factor Ngn3. The role of Ngn3 as an early marker of cells differentiating toward a primary endocrine fate was clearly demonstrated by its ectopic expression under the regulatory control of the pdx-1 promoter in transgenic mice (68). Small and poorly branched pancreatic buds were evident on e12, followed by premature and precocious differentiation of pancreatic progenitor cells into endocrine cells, and the resultant depletion in precursor cells with the capacity to proliferate, branch, and differentiate into exocrine cells. Expression of Ngn3 starts on e9–e9.5, peaks on e15.5 during the major wave of endocrine cell genesis, and is greatly diminished at birth, with little or no detection of Ngn3 in the adult pancreas. Significantly, mice homozygous for an ngn3-null mutation failed to generate any endocrine cells or putative endocrine precursors during development (66). These observations led to the assumption that ngn3 expression is a functional marker of an islet cell precursor population in the developing pancreas.
Because Ngn3 is both necessary and sufficient to drive the differentiation program of islet cells during pancreatic development, it may be inferred that the activities of specific transcription factors that regulate cis-acting elements within the promoter region of the ngn3 gene, collectively dictate the endocrine fate of the cell. In the distal region of the ngn3 promoter, a cluster of binding sites for HNF1, HNF3?, and HNF6 was recently identified (69). Concurrent with the roles of these transcription factors in regulating ngn3 gene expression and the subsequent initiation of the endocrine program, disruption of their respective genes in the developing embryo results in corroborative phenotypes, affecting the progression of early pancreatic development and endocrine specification. Mice homozygous for a disrupted hnf1 gene have smaller islets and reduced insulin secretion (70). HNF3?-null mice show a lack of foregut formation (71, 72), and in mouse embryos lacking hnf6 expression, a marked reduction in endocrine cell differentiation is observed along with severely reduced levels of ngn3 gene expression (73). However, the independent expression of HNF factors appears not to be sufficient for ngn3 expression, but, rather, a cooperative mechanism exists between these accessory proteins in the cell type-restricted activation of the ngn3 gene promoter (69). Hes-1 represses the expression of the ngn3 gene promoter by binding to several silencer sites located close to the transcription initiation site and thus suppresses endocrine precursor patterning through the Notch signaling pathway.
Beta2/NeuroD
The bHLH transcription factor Beta2/NeuroD is a key regulator of both insulin gene transcription in pancreatic ?-cells and the terminal differentiation of neurons (74, 75). Beta2/NeuroD expression occurs in a subset of pancreatic epithelial cells as early as e9.5, colocalizing with glucagon expression (76). On e14.5, expression appears within and in proximity to ductal epithelium, and by e17.5, expression is restricted to islets (77). Beta2/NeuroD heterodimerizes with ubiquitous bHLH proteins of the E2A family to regulate transcription of the insulin gene and other ?-cell-specific genes. Expression of the Beta2/NeuroD gene is activated by Ngn3 (78). Mice homozygous for a targeted disruption of the ?2/neuroD gene survive to birth, but die within 3–5 d postpartum of severe hyperglycemia. The islets of these mice are dysmorphic and have markedly diminished numbers of endocrine cells arranged in streaks and irregular aggregates and reduced numbers of ?-cells (77).
Pax4
Pax4 is a paired-box homeoprotein whose expression is restricted to the central nervous system and the developing pancreas. By e9.5, Pax4 is expressed in both ventral and dorsal buds of the developing pancreas, and by the time of birth, expression is restricted to ?-cells. Targeted disruption of Pax4 in mice results in a striking pancreatic phenotype (79). The pax4-null mice survive birth, but die within 3 d of hyperglycemia and dehydration. There is a virtual absence of ?- and -cells, but -cells are increased. PDX-1 expression in pax4-null pancreas is absent, a finding consistent with the absence of differentiated ?-cells. Thus, Pax4 functions early in the development of islet cells to promote the differentiation of ?- and -cells.
Pax6
Another paired-box homeoprotein, Pax6, is important for islet cell development. The expression of Pax6 is restricted to the eye, the central nervous system, the nose, and the endocrine pancreas (80, 81). Pax6 is expressed early in the epithelium of the developing pancreas on e9.0 in both dorsal and ventral pancreatic buds and is expressed in differentiated -, ?-, -, and PP cells. A spontaneous mutation of the pax6 gene in mice, known as Small eye (Sey–/–), has defined the importance of Pax6 in the development of the lens placode of the eye and portions of the forebrain (82). Small eyeNeu mice have abnormal organization of islets, with decreased -, ?-, -, and PP cells (83). The production of glucagon and insulin is substantially reduced in these animals. Pax6 regulates the promoters of the glucagon, insulin, and somatostatin genes, suggesting a mechanism to explain the decreased hormone production observed in Small eye mice (83). Knockout pax6 nullizygous mice die within minutes of birth. These animals fail to form islets (84). The disorganization of islets is proposed to relate to a possible function of Pax6 in the regulation of cell adhesion molecules (84, 85). These mouse models of gene knockouts implicate both Pax4 and Pax6 as key regulators of the terminal steps in cellular differentiation of the endocrine pancreas.
Nkx2.2 and Nkx6.1
Members of the NK2 family of homeoprotein transcription factors, Nkx2.2 and Nkx6.1, are regulators of the differentiation of pancreatic endocrine cells (86). Nkx2.2 is expressed early in developing pancreatic buds and is later restricted to -, ?-, and PP cells of islets. Nkx6.1 is expressed primarily in ?-cells of adult islets (87, 88). Disruption of either the Nkx2.2 or Nkx6.1 gene in mice results in death soon after birth due to severe diabetes (86). The pancreata of Nkx2.2 and Nkx6.1 knockout mice have no insulin-producing cells, and glucagon-producing cells are diminished, although the exocrine pancreas is histologically normal. Nkx2.2-null mice have a large number of hormone-negative, arrested precursor cells that express endocrine pancreas-specific proteins, such as synaptophysin, neuroendocrine cell adhesion molecule, amyloid polypeptide, and prohormone convertase 1/3, but no hormones. Nkx6.1-null mice have a selective reduction of ?-cells with a normal complement of other endocrine cell types (89). Notably, it appears that in both Nkx2.2- and Pax4-null mice that fail to develop insulin-producing cells, the arrested progenitor ?-cell expresses the hormone ghrelin (90).
MafA and MafB
MafA and MafB are transcription factors previously recognized to be important in development of the lens of the eye. During pancreatic development, MafA is expressed in early ?-cells during the second developmental transition on e14 (91, 92). MafB appears to be expressed in glucagon-producing -cells on e15. The phenotypic consequences of MafA and MafB gene knockouts in mice have not yet been reported.
Mist1
Mist1 is a bHLH transcription factor expressed in pancreatic acinar cells (93). Mist1-null mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation, and acinar tissue acquires a ductal phenotype. It has been proposed that Mist1-null mice represent a genetic model for chronic pancreatic injury and that Mist1 serves as a key regulator of acinar cell function and stability.
Relevance of pancreatic development to pathogenesis of diabetes mellitus
Diabetes mellitus results from a combination of insulin resistance and deficient or dysregulated insulin secretion by pancreatic ?-cells. Human genetic studies indicate that dysfunction of ?-cell transcriptional regulators causes some forms of type 2 diabetes. Maturity-onset diabetes of the young (MODY) is a type of autosomal dominant monogenic diabetes with early onset (age of 30 yr or less). The prevalence of MODY-type diabetes is higher than initially proposed (94), now accounting for 5–10% of type 2 diabetes. The autosomal dominant inheritance pattern of this type of diabetes has facilitated genetic analysis of familial type 2 diabetes. The first genetic mutations linked with MODY were in the glucokinase gene (MODY2), an enzyme essential for glucose-sensing in ?-cells (95, 96). The five additional types of MODY identified to date are linked to mutations in transcription factors relevant to pancreatic ?-cell function (Table 2), although additional MODY genes remain to be defined (97). These transcription factors function in both pancreatic development and the differentiated ?-cell. MODY3 results from mutations in the Hnf1 gene. Hnf1 is a homeodomain and POU-like DNA-binding protein that transactivates insulin and IGF-I gene transcription (98, 99). Disruption of the Hnf1 gene in mice results in Laron dwarfism, decreased insulin gene expression, and diabetes. Humans with Hnf1 (MODY3) mutations have defective glucose utilization, insulin secretion, and glucose disposal (100). MODY1 is a result of mutations in the transcription factor Hnf4. Hnf4 activates the HNF1 promoter (101) (Fig. 3), which is relevant in light of the linkage of Hnf1 mutations to MODY3 (102). Both Hnf4 and Hnf1 are essential for normal pancreatic ?-cell function, and human subjects with mutations in either gene develop impaired glucose tolerance and/or diabetes (103, 104). MODY5 results from mutations in Hnf1?, a POU (Pit-1, Oct-1, and Unc-86)-type homeoprotein, structurally related to Hnf1. Selective deletion of the Hnf1? gene in mice manifests in impaired glucose tolerance and impaired glucose-dependent insulin release (105). In addition, the pancreata of Hnf1? mice have increased Pdx1 and Hnf1 mRNA levels and decreased levels of Hnf4 mRNA.
TABLE 2. Transcription factors involved in pancreas development associated with diabetes
FIG. 3. Transcription factor cascade involved in the regulation of the rat insulin-1 gene promoter. The enhancers E2-A4/A3 (and E1/A1, not shown) bind and are activated by bHLH proteins and the homeodomain protein Ipf-1 (Pdx-1). HNF1 also binds to the E2-A4/A3 complex. Hnf4, in turn, is regulated by Hnf1? and possibly Hnf3? (activated by Hnf6). Mutations in Hnf4, Hnf1, Ipf-1, Hnf1?, and Beta2 are responsible for MODY1, -3, -4, -5, and -6, respectively. CREB, cAMP response element-binding protein; Basal, basal transcription factors; Trx, transcriptional activation of rat insulin-1 gene promoter (rInsulin-1 gene).
Mutations in the Beta2/NeuroD gene appear to have defined a MODY6. At least two distinct mutations in the Beta2/NeuroD gene appear to be responsible for the onset of type 2 diabetes (MODY6) (106). The linkage of MODY with three members of the Hnf regulatory cascade of transcription factors suggests that additional genes in the Hnf signaling cascade may be involved in causing diabetes. For example, Hnf6 is expressed in the developing exocrine pancreatic cells and in islets and may be important in stimulating the transcriptional expression of Hnf4 and Hnf3?. Hnf3? is a transcription factor expressed in embryonic pancreatic buds and islets and functions as an activator of the expression of the Hnf4, Hnf1, and Pdx-1 genes.
An inactivating mutation in transcription factor Pdx-1 (Ipf1) causes MODY4. Pdx-1 is required for pancreatic development. Individuals heterozygous for the inactivating mutation in the Pdx-1 gene develop diabetes. Evaluation of this inactivating Pdx-1 mutation in vitro suggests that diabetes results from both diminished gene dosage (haploinsufficiency) and dominant negative inhibition of transcriptional activation of the intact PDX-1 allele. In the mouse model of Pdx-1 inactivation, Pdx-1 hemizygous animals are glucose intolerant, and conditional inactivation of Pdx-1 after the pancreas has developed results in diabetes. Several missense and insertional mutations in the Pdx-1 gene are now shown to be associated with the development of late-onset type 2 diabetes in the French and United Kingdom populations (107, 108). ?-Cell dysfunction due to altered cellular levels of Pdx-1 may occur in the absence of mutations. Suppression of Pdx-1 expression in transgenic mice expressing a conditional Pdx-1 antisense ribozyme targeted by the rat insulin promoter develop age-dependent diabetes (109). In ?-cell models of glucotoxicity, sustained exposure to hyperglycemia down-regulates Pdx-1 expression concordant with decreased insulin gene transcription.
Several additional transcription factors essential for pancreatic development or for differentiated ?-cell function, including the regulation of insulin gene transcription, are candidate diabetes genes. These transcription factors include Isl-1, Pax4, Nkx2.2, Nkx6.1, and Ngn3, based on the findings that targeted disruptions of the genes encoding these factors in mice result in impaired development of the pancreas and/or pancreatic islets (?-cells). A subset of these transcription factors, such as Pdx-1, may be critical in mediating both ?-cell development and differentiated ?-cell function. Investigations of pancreatic development, islet gene regulation, and the genetics of diabetes mellitus are rapidly converging, with the potential to generate novel and potent therapies for pancreatic disease.
Acknowledgments
We thank the members of the Laboratory of Molecular Endocrinology for their helpful suggestions, and Melissa Fannon and Kimberly MacDonald for help with the preparation of the manuscript.
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