Specification of endocrine progenitors

Expression of Ngn3 in pancreatic precursors converts them into endocrine precursors.

However, expression of additional transcription factors is needed to trigger differentiation (specification) of these cells into the different endocrine lineages. This is illustrated by the importance of the timing of Ngn3 expression in pancreatic progenitors. Different subsets of endocrine lineages are specified at different stages of development, with glucagon⁺ cells induced first, followed by insulin⁺, PP⁺ and somatostatin⁺ cells [91]. In addition, pancreatic endocrine cell differentiation has shown to be plastic and it is possible to convert one endocrine cell type to another by the misexpression or knockout of one or more transcription factors (Fig. 4).

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FIG. 4: Schematic model representing the transcription factors implicated in the specification of the endocrine and exocrine pancreas [92].

3.3 Transcription factors directing pancreatic development and function of α- and β-cells

Several transcription factors are activated during early pancreatic development to ensure normal organogenesis and the subsequent differentiation of the different endocrine cell types.

Beta2/NeuroD1

Beta2/NeuroD1 is class B bHLH. It is a key transcription factor required for pancreatic development and endocrine cell differentiation. It is first expressed at E9, in a subset of epithelial cells most of which co-express glucagon. Later, Beta2/Neurod1 is expressed in all endocrine cells of the developing and adult pancreas indicating that, unlike Ngn3, Beta2/Neurod1 expression is maintained in differentiated endocrine cells [93].

The ubiquitous E2A family of proteins, including E12 and E47, function either as homo- or heterodimers with Beta2/NeuroD1 to bind and transactivate promoters via conserved

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sequence elements known as E-boxes [94-97]. Beta2/NeuroD1is, expressed in pancreatic endocrinecells, the intestine, and brain [93, 95, 98].

The heterodimer Beta2/NeuroD1-E47 represents an islet-specific transcription factor that controls both insulin [93, 99-102] and glucagon gene transcription [100]. ß2/E47 specifically has been reported to bind and activate the glucokinase promoter in the islet [103].

Beta2/NeuroD1binds and activates the promoter of Sur1 [104], which playsa major role in the activation of insulin secretion upon glucose stimulation [105], and may be controlled at the transcriptional and post-transcriptional level by an increase in intracellular Ca²⁺

concentration [104]. The principal role of Beta2/NeuroD1 may be in the maintenance of insulinexpression in mature ß-cells by active repressionof the somatostatin promoter [106].

Mutations in Beta2/NeuroD1are linked to MODY6 in humans and are associated withtype 2 diabetes in the heterozygous state [107]. Mice knockout for Beta2/Neurod1 die after or several months after birth of severe diabetes, depending on their genetic background [93, 108]. They have a 60% reduction of endocrine cells and fail to develop morphologically distinct islets [93].

Arx and Pax4

The transcription factor aristaless related homeobox (Arx), demonstrates plasticity in the specification of endocrine lineages [87, 109, 110]. Arx is detected in the pancreatic epithelium, in endocrine precursor cells along with Pax4 from E10.5 to E18.5, when Arx becomes α-cell specific.

The paired box 4 (Pax4) transcription factor, is first expressed in some cells of the ventral spinal cord and pancreas at E9.5. Like Ngn3, Pax4 is transiently expressed in pancreatic progenitors during development and is downregulated shortly after birth. In the developing pancreas, its expression is restricted to β-cells and some δ-cells but it is absent from α-cells [87].

The analysis of Pax4^-, Arx^- and Pax4^-Arx^- double knockout mice revealed that the choice between α- β- and δ- cell fate in the endocrine progenitors is regulated by the activity of these factors. Arx promotes the α-cell fate while Pax4 regulates specification of β-, and δ- cells. Both factors negatively regulate each other's expression, thereby regulating the

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proportion of these cells. Interestingly, over-expression of Arx in early pancreatic epithelium, embryonic endocrine cells or post-natal insulin⁺ cells resulted in conversion of β-cells into δ-cells under all conditions. This observation demonstrates a significant plasticity in endocrine lineages and opens new possibilities of converting the fate of other endocrine cells into insulin⁺ cells. Interestingly, insulin producing cells specified during development do undergo maturation steps before they become glucose-responsive β-cells [111-114].

NK Family

NK-family members and homeodomain protein Nkx2.2 and Nkx6.1 are expressed in pancreatic endocrine cells and involved in their differentiation.

During pancreas development, Nkx2.2 is first expressed in the whole epithelium from E9.5 to E12.5. Then, as the pancreatic program resolves into endocrine and exocrine cells, Nkx2.2 expression persists only in the endocrine cells. In the adult pancreas, Nkx2.2 is expressed in α-, β- and PP-cells but not in δ-cells [115]. Nkx2.2 null-mice have a large number of endocrine cell precursors without hormone production. Nkx2.2 is necessary for the maturation of ß-cells [115], whereas its distant homologue Nkx6.1 controls their expansion [82].

The expression pattern of Nkx6.1 is similar to that of Nkx2.2. At E10, it is expressed in pancreatic progenitor cells in combination with Pdx1 and Ptf1a [116, 117]. Interestingly, Insulin+/Glucagon+ early hormone expressing cells do not express either Nkx6.1 or Pdx1 [118]. At the beginning of the second developmental transition, Nkx6.1 is expressed in ductal and periductal cells expressing Pdx1 or Ngn3 and in Insulin-expressing cells [82]. At E15.5, all Nkx6.1 expressing cells also express Nkx2.2, but many Nkx2.2 expressing cells do not express Nkx6.1. Finally, at later developmental stages and postnatally, Nkx6.1-expression is restricted to β-cells [82]. Interestingly the phenotype of Nkx2.2 and Nkx6.1 double mutant is similar to that of Nkx2.2, indicating that Nkx6.1 acts downstream of Nkx2.2 [82]. Nkx6.1 knockout mice have a selective reduction of β-cells with a normal amount of other endocrine cell types. Nkx2.2 is co-expressed with insulin [115] and Ngn3 [80, 115] during pancreatic development. Foxa2 and Ngn3 are proposed to lie upstream fromNkx2.2 in the hierarchy of

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ß-cell differentiation transcription factors [119]. Prado et al. suggest that high expression levels of both Nkx2.2 and Pax4 are independentlyrequired to specify or maintain ß-cell fate [120].The absence of either transcription factor resulted in ß-cellsfailing to form, with the ß-cells replaced by ghrelin-producingε-cells [120]. Pax6 expression depends on Nkx2.2 [121], thusit is probable that Nkx2.2 acts upstream of Pax6 in the samepathway to regulate ß- and ε-cell fates [120].

Pax6

The paired box 6 (Pax6) is a pairedomain-homeodomain transcription factor. In the pancreas, it is expressed in both ventral and dorsal buds from E9. In the endocrine differentiation cascade, Pax6 stands downstream of Ngn3 [18]. At E10.5, Pax6 is detected in glucagon-expressing cells and later at E15.5, Pax6-positive cells express either Insulin or Glucagon. Finally, in newborns, Pax6 is restricted to mature islet cells [122, 123].

Mutations in Pax6 are related to diabetes mellitus, aniridia, brain malformation, and other anomalies [124, 125]. Pax6 homozygous mutant mice fail to form islets; show a decreased number of β-, δ-, and pancreatic polypeptide-producing cells; a lack of α- cells; and an increase in ghrelin-positive ε-cells without any change in endocrine pancreatic mass [18, 126].

This thesis is aimed at better defining the role of Pax6 in α- and β- cells function. Therefore, the last sub-chapter of the introduction will further focus on the Pax family of transcription factors and in particular Pax6.

Sox4

Sox4 is a member of the Sry/hydroxymethylglutaryl (HMG) box family of transcription factors. Sox4 is expressed in all cells of the endocrine pancreas and the developing pancreas of mutant mice for Sox4 is similar to wild type up to E12.5 but die at die at E14 due to cardiac defects. However, when put in culture explants have 50% less α- and β- cells compared to normal mice [127, 128]. Furthermore, α-cell differentiation is drastically reduced in zebrafish mutants for Sox4 [129].

38 Large MAFs

The cloning of the insulin gene transcription factor RIPE3b1 as MafA [130] initiated systematic examination of the function of Maf factors in the endocrine pancreas. Maf proteins are subdivided into two classes, “large” (236-370 AA; c-Maf, MafB, NRL and MafA/L-Maf) and “small” (149-162 AA; MafF, MafG and MafK) [131-134]. The leucine zipper domain allows the Maf proteins to dimerize with themselves or other basic leucine zipper (bZIP) factors and thereby influence the DNA binding specificity of these factors [132, 135, 136].

In the pancreas, MafA, MafB, and cMaf are expressed in pancreatic endocrine cells [111].

MafA, which is -cell specific, is a glucose-responsive insulin gene transcription factor that binds to the insulin Maf Responsive Element (MARE) and activates insulin gene expression [130, 137-140]. MafA expression is restricted to Insulin-expressing cells from E13.5 to adults.

Its absence in Nkx6.1 mutant mice suggests that it lies downstream in the β-cell differentiation cascade [141]. Interestingly, mice deficient in MafA have normal pancreatic islets at birth, but the ratio of  to  cells is gradually reduced, resulting in glucose intolerance by 8-12 weeks, and development of diabetes with age [142]. Identification of downstream targets of MafA as critical regulators of insulin synthesis and secretion [143, 144] (such as, insulin, Pdx1, Nkx6.1, GLUT2, GK, GLP1 receptor, prohormone convertase 1/3 (PC1/3), pyruvate carboxylase and granuphilin [145]) suggests a mechanism by which MafA may regulate the maturation process.

MafB is restricted to α-cells in adults. During pancreatic development, at E10.5, MafB is expressed in glucagon+ cells and at E15.5, although all glucagon+ cells express MafB, a significant proportion of cells express only MafB. These early MafB+/Glucagon^- cells express the panendocrine marker Synaptophysin and some even express Insulin [111, 140], suggesting that MafB could play a role in the differentiation of β-cells. In addition, MafB was also shown to regulate the expression of genes specific for β-cell differentiation such as Pdx1, Nkx6.1 and GLUT2 [146, 147].

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cMaf is expressed in both α- and β- cells during embryonic development and in adults. cMaf knock out mice die shortly after birth and they have a decreased amount of insulin- and glucagon- producing cells.

Pax family of transcription factors and Pax6 4.1 Pax Family

Paired box (Pax) genes are a family of tissue specific transcription factors containing a paired domain and usually a partial or complete homeodomain. Pax proteins are important in early animal development and formation of several tissues from all germ layers in the mammalian embryo. This family is composed of nine genes, Pax1-9.

The original finding that the paired domain conferred a sequence-specific DNA-binding function, together with a Proline–Serine–Threonine-rich (PST) carboxy terminus that is characteristic of other transcriptional activators, led to the supposition that Pax proteins would be involved in transcriptional regulation [148, 149] (Fig. 5).

FIG. 5: Functional domains of Pax6. Structure of the human full length protein. PD, paired domain; G, Gap; HD, homeodomain; PST, Proline-serine-threonine (transactivating domain).

4.2 Pax6 in detail

Pax6 is a Paired homeodomain transcription factor expressed in the eye, nose, pancreas, and central nervous system from the early stages of embryonic development [150].

Pax6 appears to be necessary for the correct execution of islets’ endocrine cell differentiation [151, 152]. It is expressed early in the developing pancreas (E 9.0) in cells destined to an endocrine cell fate and does not seem to be required for cell type specification [122, 126]. However, Pax6 is essential for the normal expression of final differentiation markers such as insulin, glucagon and somatostatin [153]. Pax6 regulates the C2 element of the insulin gene [122], the G1 and G3 elements of the glucagon gene [154, 155] and somatostatin gene expression [156].

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Pax6 Heterozygous mutations in humans cause congenital eye anomalies such as aniridia.

Heterozygous mutations in Pax6 might also lead to glucose intolerance characterized by impaired insulin secretion [157]. Therefore, haploinsufficiency of this gene is sufficient to obtain a phenotype, illustrating that regulation of transcription factor levels are of great importance.

Previous studies have shown that various forms of Pax6 with different molecular weights exist, and at least four variants of Pax6 (p46, p48, p43, and p32) were detected in cellular extracts (Fig. 6) [158, 159]. The various forms are the result of alternative splicing [158]. All forms of Pax6 bear a conserved C-terminal transactivation domain, which contains relatively rich proline, PST residues. Several phosphorylation sites have been identified in this region of the human and zebrafish Pax6. It has been shown that phosphorylation of these sites in Pax6 is carried out by p38, ERK [160], and homeodomain-interacting protein kinase 2 [150]. On the other hand, dephosphorylation of Pax6 remains largely unknown.

FIG. 6: Summary of multiple forms of Pax6 and its conserved PST domain. A, diagram of four different variants of Pax6 (p33/32, p43, p46, and p48). PST, proline-, serine- and threonine-enriched activation domain; PD, paired domain; HD, homeodomain. B, phosphorylation sites identified in the PST domain from zebrafish or human Pax6. Adapted from [161].

4.2.1 Pax6 knockout models

There are several Pax6 knock out models, including cortex- and eye- specific knockout [162, 163]. The general Pax6 knockout mice as well as the endocrine specific knockout mice develop and die from diabetes few days after birth.

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Three general knockout mice are available- the Sey ,Sey^Neu and Pax6-LacZ mice [164]. Sey and Sey^Neu mice carry semi-dominant Pax6 alleles which are due to point mutations in the Pax6 locus resulting in truncated Pax6 proteins [122, 165]. In Sey mutants the protein is truncated directly after the paired domain while in Sey^Neu mice the protein is truncated after the homeodomain, thus leaving both protein domains intact. To generate complete knockout mice, the Pax6 start codon along with the entire paired box was replaced with the β-galactosidase gene (Pax6-LacZ) resulting in mutant mice in which no protein is detectable [126]. In the homozygous state, Sey^Neu and Pax6 knockout mutants lack eyes, show severe brain defects, and die shortly after birth. However, differences in pancreatic defects are observed in the mutant animals. Whereas Pax6-LacZ knockout mice do not form glucagon-producing α-cells throughout all developmental stages, Sey^Neu mice express normal levels of glucagon during the early stages of pancreas development. A decrease in the number of glucagon-positive cells becomes evident around E10.5, and at E12.5 glucagon- and also insulin-positive cells are reduced significantly in Sey^Neu mice. Although glucagon-producing α-cells are most strongly affected in Sey^Neu mice, all four endocrine cell types are decreased significantly in number by E18.5. In contrast, Pax6-LacZ knockout mice only lack cells of the α-cell lineage. Furthermore, islet morphology is disrupted in Pax6-LacZ knockout mice.

Insulin expression is detectable from E13.5 onwards, and expression of Pdx1 appears to be normal [164]. The exocrine tissue on the other hand seems unaffected, synthesizing α-amylase. In Sey^Neu mutant mice, islet morphology is also altered although in a more subtle manner. The initial aggregation of endocrine cells seems unaffected and the number of developing islets appears normal. However, Sey^Neu/Sey^Neu islets do not form properly with a β-cell core and a mantle of α-, δ- and PP-cells but the cell populations appear mixed within a given islet. Furthermore, hormone production is markedly reduced in Sey^Neu mutants, pointing to a decrease in Pax6-regulated glucagon and insulin gene transcription.

Thus, although the phenotypes of Sey^Neu and Pax6 knockout largely overlap, there are some important differences. First, Pax6 knockout mice do not form α-cells whereas in Sey^Neu mutant mice the number of α-cells is reduced. Secondly, in Sey^Neu mutant mice all endocrine cell types seem to be affected whereas in Pax6-LacZ knockout mice only α-cells are missing.

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A study in the Sey/Sey mice shows that E19 Pax6^sey/sey mutant embryos displayed an excess of ghrelin-expressing cells [18].

Another Pax6 knockout available is the Cre/loxP to inactivate Pax6 in the endocrine pancreas only. To obtain Pax6^flox/flox mice, Cre is activated in order to inactivate Pax6, specifically from the developing pancreas, thus avoiding perinatal death [153]. In these mice, very few α cells are present. GLUT2 (glucose transporter 2) protein was not detected in the Pax6-deficient pancreas suggesting that Pax6 plays a role in GLUT2 regulation [153]. Pax6 has been proposed to regulate Pdx1 expression asPax6-binding sites have been detected in the Pdx1 promoter [166]. Pax6-deficient islets maintained expression of PC1/3,a proinsulin processing enzyme, suggesting that the reductionin insulin in Pax6-deficient mice may be due to direct changes in insulin expression and/or the inability of these cells to respond to elevated glucose levels because of reduced GLUT2expression [153].

The inactivation of Pax6 exclusively in the endocrine cell types prolonged the life of the mutants by only a few days, as they died suffering from an overt diabetic phenotype, including hyperglycemia, hypoinsulinemia, weight loss, and ketosis, indicating an essential role for Pax6 in β-cell functions. As mentioned, GLUT2 expression was downregulated, but expression of several transcription factors essential for endocrine development (Nkx2.2, Nkx6.1, Isl1, and Pdx1) was maintained in the Pax6-deficient pancreas. This suggests that postnatal neogenesis does not seem to compensate for the early developmental defects in the endocrine pancreas of the Pax6-deficient mice. Lineage tracing of the Pax6-deficient cells using the Z/AP reporter line revealed that Pax6 is not required for the specification, formation, or survival of β-cells. However, it is essential for the normal expression of final differentiation markers such as insulin and GLUT2 in these cells. Furthermore, the findings suggest that Pax6 function is in parallel to Nkx2.2, Nkx6.1, and Pdx1 in some of the β-cells during the late stages of pancreas development [153].

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Objectives

The aim of this study is to understand the role of Pax6 in α- and β-cells of the endocrine-pancreas, producing glucagon and insulin, respectively. The study provides new insights into the intricate regulation of the endocrine cell function and differentiation. It further identifies new direct and indirect targets of Pax6.

In the first result section and the annexed study, we identify direct and indirect targets of Pax6 in α-cells implicated in glucagon transcription, glucagon processing and α-cell development.

In the last two results sections we identify direct and indirect targets of Pax6 in β-cells implicated in insulin transcription, glucose sensing, insulin secretion in response to glucose, incretins and free fatty acids as well as genes implicated in β-cell development.

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Chapter 1- Pax6 Regulates the Proglucagon Processing Enzyme PC2 and Its Chaperone 7B2

PC2 is expressed as a pro-convertase. In the secretory vesicles, it binds the 7B2 molecular chaperon, as the vesicle matures both PC2 and 7B2 are cleaved. The processed PC2 can now cleave the proglucagon precursor to obtain the α-cell hormone, glucagon. This process is tightly regulated in a spatial and temporal manner.

We previously showed that Pax6 controls the expression of glucagon through:

1. Binding to the G1 and G3 sites on its gene promoter.

2. Regulating the transcription of the cMaf, Beta2/NeuroD1 and Isl1 genes (described in the annex), all of them known to regulate glucagon gene transcription.

This paper describes a transcriptional network where Pax6 controls glucagon biosynthesis by:

1. Directly binding the promoter of the gene encoding 7B2, the molecular chaperon of PC2, important for the spatial and temporal activation of PC2.

2. Indirectly through cMaf and Beta2/NeuroD1 which bind both PC2 and 7B2 promoters and activate them.

This chapter was published in Mol Cell Biol. 2009 April; 29(8): 2322–2334

45 Winner of the Servier research award, 2009.

Mol Cell Biol. 2009 April; 29(8): 2322–2334.

Published online 2009 February 17. doi: 10.1128/MCB.01543-08.

PMCID: PMC2663301

Copyright © 2009, American Society for Microbiology

Pax6 Regulates the Proglucagon Processing Enzyme PC2 and Its Chaperone 7B2

Liora S. Katz,^* Yvan Gosmain, Eric Marthinet, and Jacques Philippe

Diabetes Unit, Division of Endocrinology, Diabetes and Nutrition, University Hospital, University of Geneva Medical School, 1211 Geneva, Switzerland

*Corresponding author. Mailing address: Diabetes Unit, University Hospital, 1211 Geneva, Switzerland. Phone: 41 22 3724237. Fax: 41 22 3729326. E-mail: Liora.Katz@unige.ch

Received October 3, 2008; Revised November 30, 2008; Accepted January 31, 2009.

Abstract

Pax6 is important in the development of the pancreas and was previously shown to regulate pancreatic endocrine differentiation, as well as the insulin, glucagon, and somatostatin

Pax6 is important in the development of the pancreas and was previously shown to regulate pancreatic endocrine differentiation, as well as the insulin, glucagon, and somatostatin