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