In this study, we created NeuroD1 and Nkx2.2 DKO mice to understand the relative roles of these two essential transcription factors in islet development. Surprisingly, we demonstrated that there is a unique genetic interaction between Nkx2.2 and NeuroD1 during α, PP and ε cell differentiation that is only revealed when Nkx2.2 and NeuroD1 are simultaneously deleted from the developing pancreas. These observations provide the first evidence that these two essential regulatory factors can form cell type-dependent functional interactions to differentially regulate the individual islet cell fates. Interestingly, it also appears that Nkx2.2 and NeuroD1 functionally cooperate to specify the fate decision between L cells and ghrelin cells in the intestine. In addition, these studies have uncovered a novel role for NeuroD1 in directing early α, PP and ε cell fate decisions that is distinct from its role in later stages of α cell development and β cell differentiation and survival.
The majority of recent pancreatic islet research has focused on β cell development due to the importance of β cells in islet function, as well as their relative abundance compared to the other islet cell types. As a result of this extensive body of research, a more defined picture of β cell differentiation is beginning to emerge. Specifically, many β cell regulators and markers have been identified and distinct stages of β cell differentiation have been characterized (Cerf et al., 2005
; Doyle et al., 2007
; Holland et al., 2005
; Lantz and Kaestner, 2005
; Nishimura et al., 2006
; Sosa-Pineda, 2004
). The Nkx2.2 and NeuroD1 single knockout β cell phenotypes suggest that Nkx2.2 and NeuroD1 function at different stages of β cell differentiation; Nkx2.2 is required for the initial specification of all β cells, while NeuroD1 appears to be required for late stage differentiation and maintenance. Furthermore, NeuroD1 expression is reduced in the Nkx2.2 knockout mice, whereas Nkx2.2 expression is unchanged in the NeuroD1 mutant, supporting the idea that Nkx2.2 functions upstream of NeuroD1 in β cells. Consistent with this, we do observe a significant reduction of NeuroD1-expressing cells as early as e12.5 (prior to the formation of many of the individual islet cell types) and there appear to be fewer cells expressing high levels of NeuroD1. At this point, however, we cannot rule out that the down regulation of NeuroD1 is merely secondary to the loss of specific islet cell types, especially late in gestation. The fact that the Nkx2.2−/−;NeuroD1−/− DKO mice display an Nkx2.2 KO phenotype also suggests that Nkx2.2 is epistatic to NeuroD1, the caveat being that the complete absence of β cells may preclude observing phenotypes associated with the NeuroD1 deletion. A conditional mutation of Nkx2.2 is being developed to resolve these issues and to further explore the relationship between NeuroD1 and Nkx2.2 specifically in the β cell.
In contrast to the β cells, much less is understood about the regulation of the non-β cell lineages. Although glucagon-producing α cells are also essential for proper islet function, little is known about the regulation of α cell formation and the stages of α cell differentiation. The restoration of the glucagon-producing α cell population in the Nkx2.2−/−;NeuroD1−/− DKO mice, compared to the Nkx2.2−/− mice implicates NeuroD1 as an important regulator of α cell determination. While these studies are beginning to clarify the role of NeuroD1 in α cell fate determination, the molecular mechanism by which NeuroD1 operates remains unclear. NeuroD1 is present in glucagon-producing α cells throughout embryogenesis; however, the NeuroD1−/− mice do not display an α cell phenotype until late in gestation (Chu et al., 2001
; Naya et al., 1997
). In contrast, NeuroD1 activity appears to be required to maintain repression of the α cell fate in the absence of Nkx2.2. Furthermore, incremental reductions in NeuroD1 activity, as seen in the NeuroD1 heterozygous mice, can restore to an intermediate level the α cell defects associated with loss of Nkx2.2. We propose that Nkx2.2 may be responsible for the normal repression of NeuroD1 function in early α cells, possibly through activation of a negative regulator of NeuroD1, such as an Id protein (Norton, 2000
). Alternatively, Nkx2.2 and NeuroD1 may function in parallel to modulate the formation of α cells during islet development.
An unexpected finding emerged from our characterization of the glucagon-positive cell population in Nkx2.2−/−;NeuroD1−/− DKO mice with regards to α cell marker expression in the Nkx2.2−/− ghrelin cell population. While Brn4 expression levels corresponded to the presence and absence of glucagon-producing α cells in each of the genotypes analyzed, Arx and Irx2 expression levels corresponded more closely with the Nkx2.2−/− ghrelin cell population. In our previous analyses of the Nkx2.2−/− ghrelin cells, these two transcription factors had not yet been identified as α cell markers and were not assessed. It is possible, that the ghrelin-producing cells in the Nkx2.2−/− mice are α cells that merely misexpress ghrelin, however none of the ghrelin cells in the Nkx2.2−/− islets co-express glucagon or Brn4. It is also possible that Arx and Irx2 are also expressed in wild type ghrelin cells, a hypothesis we could not test due to the lack of antibodies (Irx2) or incompatibility of antibodies (Arx); however, this explanation is not supported by the phenotype of the Arx null mice in which there is a loss of glucagon cells and the glucagon+ ghrelin+ population, but no effect on the ghrelin ε cells (Collombat et al., 2003
; Heller et al., 2005
). An intriguing possibility is that the ghrelin-producing cells that form in the Nkx2.2−/− mice represent a novel bipotential intermediate cell that expresses Arx, but in the absence of Nkx2.2 cannot inactivate ghrelin and initate the expression of Brn4 and glucagon to allow mature α cell formation. If this is the case, then the simultaneous loss of NeuroD1 in the Nkx2.2−/− mice may function to partially suppress this block and allow normal differentiation of α cells to proceed. While we have yet to resolve this issue and the mechanism underlying the phenotypes we observe, it does appear that the loss of NeuroD1 in the Nkx2.2−/− mice allows the restoration of apparently normal α cells that express Brn4 and Arx.
Previous studies of the NeuroD1−/− mice have suggested that NeuroD1 is required for the terminal differentiation of α and β cells; at birth both these cell populations are significantly depleted compared to wild type mice (Chu 2001
, Naya 1997
). While the absence of normal numbers of β cells appears to be caused by increased levels of apoptosis, the mechanism underlying the decrease in α cell numbers was not determined, possibly due to the relatively small number of α cells available for analysis (Naya et al., 1997
). We have demonstrated that the elimination of NeuroD1 in the Nkx2.2−/− mice restores α cell formation during the secondary transition; however, α cell numbers never return to wild type levels. Based on the previously characterized role for NeuroD1 in the terminal differentiation of α cells (Naya et al., 1997
), we hypothesize that NeuroD1 also plays a later role in α cell survival that is independent of Nkx2.2. Alternatively, an additional complex relationship may exist between NeuroD1 and Nkx2.2 in maintaining the α cell population, and the loss of Nkx2.2 may partially rescue the survival of NeuroD1-deficient α cells. The generation of temporal and cell type specific knockout alleles of Nkx2.2 and NeuroD1 will allow us to explore these questions further.
In summary, a complex and overlapping regulatory network that requires Nkx2.2 and NeuroD1 determines the cell fate decisions in the islet and the intestine. A large number of mutational studies from many labs have uncovered many of the regulatory factors that contribute to these processes, however it is likely that additional regulatory factors and/or novel roles for known factors have yet to be identified. Using genetic epistasis analysis, we have demonstrated that Nkx2.2 and NeuroD1 functionally cooperate during islet cell differentiation and we have uncovered a novel function for NeuroD1 in the regulation of α, PP and ε cell fate determination suggesting there exists a unique interaction between Nkx2.2 and NeuroD1 in these early cell fate decisions. The corresponding decrease in ε cell numbers that occurs with the increase in α cells in the Nkx2.2−/−;NeuroD1−/− DKO mice provides further evidence that Nkx2.2 regulates the cell fate choice between these two islet lineages and implicates NeuroD1 as mediator of these important developmental fate decisions. NeuroD1 and Nkx2.2 also appear to interact in their regulation of PP cell fate determination. It is clear from this analysis that simple single gene mutations may not uncover the full functional repertoire of known transcription factors or the full complexities of these regulatory pathways. Additional studies will be required to determine epistatic relationships between known and novel islet factors to gain a complete understanding of their relative functions in directing pancreatic islet cell fates.