Little is known about the extracellular signals that drive beta cell specification from Ngn3 + progenitors. Animals lacking Nkx6.1 and Nkx2.2 have defects in beta cell formation. MafA has also been implicated in the terminal differentiation of beta cells, particularly in the beta cell-specific reactivation of Pdx1. However, several observations point to Pax4 as the main hallmark of beta cell differentiation: The knockout of this gene results in the total absence of beta cells, but not alpha cells; its expression peaks between e13.5 and e15.5, which coincides with the period of maximal differentiation of beta cell precursors ; and shortly after endocrine specification, Ngn3 colocalizes with Pax4 , which suggests that the latter may be one of the targets of the former.
Also, ES cells transfected with Pax4 were induced to express insulin at much higher levels than untransfected controls. Recent evidence indicates that Pax4 and Arx are mutually repressed, and that the balance between the two determines a ( Arx ) or beta ( Pax4 ) specification from Ngn3 + progenitors. Nkx2.2
The NK2 homeobox 2 (Nkx2.2) gene belongs to a family of genes involved in the differentiation of many tissues.Nkx2.2 expression is observed both in the ventral CNS and in mature alpha, beta, and PP cells. The gene is activated from very early on during pancreatic specification ( ~ e8), but – in a pattern similar to that of Pdx1 – becomes restricted to specific islet cell types later in development. Nkx2.2 mutant mice lack beta cells and have lower amounts of other islet endocrine cell types. Further analyses of these islets, however, show a sizeable population of hormone-negative undifferentiated cells. While Pdx1 expression remains largely unaffected in Nkx2.2 knockouts at the onset of pancreatic specification ( ~ e8.5), the relative strength of its signal is significantly reduced during the secondary transition ( ~ e13.5 onward). This led to the hypothesis that Nkx2.2 might be required for the terminal differentiation of beta cells.
Nkx6.1
Nkx6.1 is another member of the NK homeodomain family, which can be found both in the pancreas and the CNS. Nkx6.1 expression is first detected from e10.5 in the pancreatic epithelium. At e15.5, it colocalizes either with Pdx1 + cells (postmitotic beta cells, mostly) or with Ngn3 (immature progenitors). At later developmental stages, as well as in the adult organ, Nkx6.1 is exclusively restricted to beta cells. Knockouts display a marked deficiency of beta cells, which could be traced back to the initiation of the secondary transition, during which beta cell neogenesis was severely impaired. Nkx2.2 expression was not affected, suggesting that this gene is upstream of Nkx6.1. This was further confirmed by double Nkx2.2/Nkx6.1 knockout experiments, whose phenotype was identical to that of Nkx2.2 −/− .
Pax4
Paired box-containing gene 4 ( Pax4 ) belongs to a multigene family that shares a conserved motif, termed “paired box.” Both Pax4 and Pax6 have, in addition, a homeodomain. Pax4 expression is first detected in the pancreas at around e9.5, and is later restricted to beta cells. Knockout mice lack both beta and delta cells, but alpha cells appear to compensate for their absence with a much higher than normal representation in the islet, which is suggestive of a “default” alpha cell differentiation pathway. Several lines of evidence point at Pax4 as a direct downstream target of Ngn3. As discussed in the main text, the balance between Pax4 and Arx might be critical for the adoption of alpha or beta cell fates.
Pax6
Perhaps best known for its role in eye development, Pax6 is also expressed both in the developing pancreas (scattered throughout the fore/midgut cells at e9.5; colocalizing with arising alpha cells at e9.5; with alpha or beta cells at e15.5) and with alpha , beta, or delta cells in the adult pancreas – but not in acinar tissue. Knockout mice show both a very significant reduction in all hormone- producing cells (with alpha cells largely absent) and an abnormal distribution of the remaining ones within the islet.
Arx
The aristaless-related homeobox gene ( Arx ) contains both a C-terminal stretch of amino acids known as the OAR/aristaless domain and a prd-class homeobox domain. Like many other genes involved in pancreatic development, Arx was first identified in the mouse CNS. Arx expression is observed in the proliferating epithelium of the pancreatic anlage at e9.5, and then is progres sively restricted to endocrine cell types. Ngn3 knockouts do not express Arx, indicating that the former is upstream of the latter. Arx −/− mutants lack alpha cells and display a concomitant increase in delta cells (and, to a lesser extent, beta cells).This effect is exacerbated in double Pax4/Arx mutants, where both alpha and beta cell subsets are largely replaced by delta cells. Arx, however, does not seem to be required for early alpha cell differentiation, because glucagonpositive cells can be readily detected until e12.5 in mutant embryos, which suggests that the main role of Arx is during the secondary transition. Taken together, these observations support a model where Arx promotes alpha cell differentiation and antagonizes that of beta cells (from e12.5 onward) and delta cells (from e14.5 onward). Because Pax4 has an opposing activity, the relative levels of each protein will likely dictate cell fate after e12.5 .
A model for alpha , beta, and delta cell specification. Experimental evidence suggests that the first insulin-positive and glucagon-positive cells that appear prior to the secondary transition are a developmental dead end. Relative levels of the mutually repressing proteins Arx and Pax4 will define two populations of endocrine progenitors during the secondary transition. Both are characterized by the expression of Nkx2.2 and Pax6. However, Pax4 expression is only detected in those cells that will give rise to both beta and delta cells. The effect of knocking out either Pax6 or Pax4 is indicated in dotted lines .
MafA/MafB
Maf transcription factors (containing a basic leucine zipper) have been associated with the regulation of multiple differentiation processes. The best-characterized Maf factors expressed in the pancreas are MafA and MafB, which are preferentially expressed in beta and alpha cells, respectively. The role of these factors has been difficult to ascertain. MafA knockout mice, for instance, display glucose intolerance and develop diabetes; however, there are no developmental effects associated with the mutation. Recent evidence suggests that a switch from MafB to MafA might be critical for the embryonic maturation and prolonged survival/function of beta cells. However, MafB has also been recently shown to be essential for the appropriate regulation of Pdx1, Nkx6.1, and GLUT-2 in the final stages of maturation of beta cells.
Foxa2 (HNF3beta)
The winged-helix transcription factor Foxa2 (also known as hepatocyte nuclear factor 3beta [HNF3beta]) is one of the first true markers of definitive endoderm. Despite its expression throughout the development of the pancreas, not many studies have looked into its role in this process. These studies are chiefly based on conditional loss of function. Thus, selective abrogation of Foxa2 expression in beta cells results in insulin hypersecretion, leading to profound hypoglycemia. In fact, Foxa2 has been shown to control Pdx1 expression in mature beta cells. However, the onset of Pdx1 expression and pancreatic specification is not affected in mice where the Foxa2 has been selectively inactivated in the developing endoderm. These animals, however, are severely hypoglycemic and die within days of birth, due to a dramatic reduction (>90%) in the number of alpha cells. They show impaired maturation, but not initial specification of alpha cells.
Tags: alpha cells, aristaless related homeobox gene, C-terminal stretch of amino acids, delta cell, Foxa2 HNF3beta, hormone producing cells, Maf transcription factors, Ngn3 + progenitors, NK homeodomain family, Nkx6.1, Paired box-containing gene 4, Pax4, Pax6, winged-helix transcription factor Foxa2