BACKGROUND
the pancreas is comprised of acinar and duct cells , producing and transporting digestive enzymes and bicarbonate, as well as endocrine cells, which secrete hormones to the blood stream.
the latter are aggregated into scattered clusters of cells, termed islets of langerhans.
these are typically composed of five cell subtypes, including alpha-, beta-, delta-, epsilon- and pp-cells that produce the hormones glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide , respectively.
insulin and glucagon are secreted co-ordinately to control glucose homeostasis, whereas somatostatin and pp regulate the secretion of other hormones and of exocrine enzymes  <cit> .
the hormone ghrelin has been shown to possess orexigenic properties and to play a role in glucose-stimulated insulin secretion  <cit> .
during pancreas development, the first wave of endocrine cell differentiation begins at embryonic day   <dig> and results in the emergence of glucagon-producing cells, followed by the appearance of few insulin-producing cells, often co-expressing glucagon  <cit> .
ghrelin-expressing cells can be detected from e <dig>  on  <cit> .
subsequently, the major wave of endocrine cell formation , initiates around e <dig> and results in the emergence of numerous insulin-producing beta-cells and glucagon-producing alpha-cells.
around e <dig>  the first somatostatin-producing delta-cells emerge  <cit>  while, shortly before birth, pp-expressing pp-cells differentiate.
concurrently, from e <dig> on, the endocrine cell mass continuously expands and self-organizes into well-shaped islets, a process lasting until approximately  <dig> weeks postpartum  <cit> .
a number of transcriptional regulators have been shown to have crucial roles in controlling the specification of pancreatic cells.
among these, pdx <dig> was found to exert a key function for the pancreatic cell lineage allocation.
later during pancreatic development, pdx <dig> activity is restricted to beta-cells and a subpopulation of delta-cells  <cit> .
the expression of the basic helix-loop-helix transcription factor ngn <dig> induces endocrine differentiation, as evidenced by gain- and loss-of-function studies as well as genetic lineage tracing  <cit> .
the specification towards the distinct endocrine cell types was found controlled by the concerted activities of specific transcription factors, including the homedomain-containing factors nkx <dig>   <cit> , nkx <dig>   <cit> , pax <dig>  <cit> , brn <dig>  <cit> , pax <dig>  <cit>  and arx  <cit> .
nkx <dig>  is broadly expressed in the early undifferentiated pancreatic epithelium and in endocrine progenitors.
along with the secondary transition, nkx <dig>  expression becomes restricted to mature beta-cells and a subset of alpha- and pp-cells, whereas it is not detected in delta-cells .
accordingly, mice depleted in nkx <dig>  display a complete beta-cell loss and reduced alpha- and pp-cell content, while the numbers of delta-cells remain unchanged.
notably, the ghrelin+ cell population appears considerably augmented  <cit> .
in this context, it has been shown that nkx <dig>  is decisive for the maintenance of normal beta-cell function and that the transcriptional repressor activity of nkx <dig>  is required for the proper specification of alpha-cells  <cit> .
nkx <dig>  has been found essential for the specification of multipotent progenitors towards the duct-/endocrine-fates in early pancreas development  <cit> .
post second transition, nkx <dig>  becomes exclusively restricted to developing beta-cells; at that stage it is essential for their expansion  <cit> .
the pax gene family member, pax <dig>  is produced in all endocrine cells throughout pancreas development with one exception that is a subpopulation of epsilon-cells  <cit> .
thus, in the absence of functional pax <dig>  alpha-, beta-, delta- and pp-cells are negatively affected with a near complete loss of glucagon-expression from alpha-cells  <cit> .
similar to nkx <dig>  mutants, the endocrine portion in pax6-/- pancreata mainly consists of cells expressing the ghrelin-hormone  <cit> .
pax <dig>  another pax family member, has been found essential for the formation of beta- and delta-cells  <cit> .
lineage tracing studies revealed that pax4+ precursor cells give rise to all four major endocrine cell types   <cit> .
nevertheless, in the absence of pax <dig>  beta- and delta-cell development is impaired and precursor cells acquire an alpha-cell-like fate, suggesting a predominant role of pax <dig> in the differentiation of beta-cells  <cit> .
this assumption is supported by recent work demonstrating that the in vivo misexpression of pax <dig> in pancreatic/endocrine progenitors results mainly in the formation of beta-cells.
moreover, the forced and ectopic expression of pax <dig> in mature alpha-cells was found sufficient to convert these into functional beta-like cells  <cit> .
mice lacking the aristaless homeobox gene arx display a phenotype opposite to the one observed in pax4-/- animals  <cit> .
depleted in alpha-cells, such mice exhibit proportionally enlarged beta- and delta-cell populations.
in fact, a direct and mutually antagonistic transcriptional repression between arx and pax <dig> was previously demonstrated and associated with the specification of the alpha- vs. beta-/delta-cell fates, respectively  <cit> .
this model was further supported by the forced expression of arx in pancreatic/endocrine progenitors, which led to the favouring of the alpha- but also pp-cell fate, at the expense of beta- and delta-cell lineages  <cit> .
interestingly, recent analyses of nkx <dig> -deficient pancreata outlined a 5-fold increase in the transcript content of the alpha-cell "specifier" arx despite an almost complete loss in glucagon-expressing cells  <cit> .
to address this apparent paradox, we undertook a thorough comparative analysis of the endocrine populations arising in pancreata of wild type, nkx <dig> - and pax6-deficient animals.
moreover, we further analysed the roles of nkx <dig>  and arx, by characterizing pancreatic endocrine cell fates in mice compound-deficient for nkx <dig>  and arx.
RESULTS
expression of arx and brn <dig> in e <dig>  pancreata of wild type, nkx <dig> -/- and pax6-/- mice
we previously investigated the antagonistic roles of arx and pax <dig> for proper endocrine cell allocation and thereby demonstrated the essential function of arx in promoting a subset of endocrine precursor cells specifically towards the alpha-cell fate  <cit> .
the apparent discrepancy between the severely reduced alpha-cell numbers and the 5-fold augmentation in arx transcripts upon loss of nkx <dig> , prompted us to examine the subcellular localization of arx in pancreata of such mice and thus gain further insights into the role of arx and nkx <dig>  during endocrine cell specification.
we initially focused our study at e <dig> , a developmental stage subsequent to a peak in mature endocrine cell generation.
in wild type pancreata, most arx-labelled cells co-expressed glucagon and were found scattered within the pancreatic tissue .
only few arx+/glucagon- cells were detectable, most likely corresponding to alpha-cell precursors that have not yet initiated hormone synthesis.
arx+ cells did not express ghrelin, except in rare instances .
interestingly, in nkx <dig> -deficient animals, an almost complete lack of alpha- and beta-cells accompanied by a five-fold increase in arx transcript levels were noted when compared to their control counterparts .
subsequent quantifications revealed a  <dig> -fold rise in the numbers of arx-immunoreactive cells in e <dig>  mutant pancreata compared to age-matched wild type embryos .
clearly, this number did not fully reflect the upregulation of arx transcripts, suggesting an increase in arx production at the single cell level.
these results led us to determine whether nkx <dig>  expression could be likewise affected in animals deficient for arx.
however, nkx <dig>  transcript levels in e <dig>  arx mutant pancreata were not significantly different from wild type controls, suggesting that nkx <dig>  transcription is not regulated by arx .
further analyses of the arx+ cell population in nkx <dig> -/- mice revealed that a vast majority expressed ghrelin.
approximately 90% of the ghrelin+ cells were found positive for arx .
besides this ghrelin+/arx+ population, only few scattered arx+/ghrelin- or ghrelin+/arx- cells were detectable in this genotype.
surprisingly, most of the few remaining glucagon-expressing cells present in nkx <dig> -deficient pancreata were negative for arx expression .
in order to further assess the identity of ghrelin+/arx+ cells, we analysed the expression of the alpha-cell-related marker gene brn <dig>  previous analyses of nkx <dig>  mutant pancreata had revealed that the numbers of brn4+ cells were reduced; correlating with the lower number of glucagon+ cells  <cit> .
consistent with these findings, the expression of brn <dig> was absent from the hyperplastic ghrelin+ cells noted in this genotype .
altogether, these results suggest that, in the absence of nkx <dig> , the beta- and alpha-cell lineages are disfavoured and replaced by a ghrelin+/arx+/brn4- cell fate.
considering the crucial role exerted by arx in promoting the alpha-cell lineage, it is conceivable that such cells correspond to alpha-cell precursors that failed to acquire the full complement of alpha-cell characteristics.
however, the observation that the proportion of ghrelin+/arx+/brn4- cells present in nkx <dig> -/- pancreata is similar to the proportion of beta-cells normally detected in wild-type mice led us to favour an alternative explanation where cells normally fated towards the beta-cell lineage fail to properly differentiate and ectopically express arx and ghrelin.
the dramatic increase in arx transcripts, combined with the profound loss in pax <dig> expression in nkx <dig> -deficient pancreata , prompted us to get more insights into putative interplay between these three factors.
in pax <dig> knockout mice  <cit> , the early glucagon+ population emerging around embryonic day  <dig>  is present and properly express the associated markers brn <dig> and isl <dig>  <cit> .
however, post secondary transition, glucagon+ cell numbers are significantly reduced .
consistent with these findings, it was shown that pax <dig>  both indirectly and directly, potentiates the expression of the glucagon gene  <cit> .
interestingly, upon pax <dig> deficiency, the beta-cell population was found reduced concomitantly with a  <dig> -fold increase in the numbers of ghrelin+ cells  <cit> .
notably, both nkx <dig>  transcript content and expression pattern were found almost normal in pax6-/- pancreata as compared to their wild-type counterparts .
surprisingly, in e <dig>  pax <dig> mutant pancreata, arx transcripts were found  <dig>  times increased as compared to age-matched wild-type pancreata.
however, the numbers of arx+ cells were not significantly different from those observed in wild type, indicating that the deficiency in nkx <dig>  or pax <dig> leads to a similar increase in arx transcripts at the single cell level .
interestingly, and similar to what we observed in nkx <dig> -deficient mice, most of the arx+ cells present in pax6-deficient pancreata, express ghrelin .
approximately 80% of the ghrelin+ cells were found to express arx, although scattered arx+/ghrelin- and ghrelin+/arx- cells were also detectable .
the expression of brn <dig> appeared unchanged in pax6-deficient mice at e <dig>  and e <dig>  .
brn <dig> expression was clearly noted in approximately 45% of the ghrelin-expressing cells, suggesting at least a partially overlapping expression with arx .
notably, beside the ghrelin+/brn4+ cell population, brn4+ cells were also found outside of the ghrelin-stained areas .
together, our data indicate that the reduction of alpha- and beta-cell numbers in nkx <dig> -/- or pax6-/- pancreata is accompanied by an increase in the numbers of cells co-expressing arx and ghrelin.
interestingly, the loss of arx does not seem to impact on nkx <dig>  or pax <dig> expression.
comparative analysis of ghrelin+ cells in pancreata of wild type, nkx <dig> -/- and pax6-/- mice
to further assess the identity of the ghrelin+/arx+ cell population arising in pancreata of nkx <dig> -/- and pax6-/- mice, we assayed the expression of a number of beta-cell-related markers, such as insulin, iapp, pc1/ <dig> and pdx <dig> at e <dig>  .
most of these proteins have previously been found expressed in both mutant pancreata, however their expression relative to ghrelin has not been fully resolved  <cit> .
previous detailed analyses of wild type ghrelin-expressing cells had led to the conclusion that these could be subdivided into glucagon+ghrelin+ alpha-cells and glucagon-ghrelin+ epsilon-cells.
it should be noted that only a subset of alpha-cells were actually found positive for ghrelin, such cells retaining glucagon, arx and brn <dig> expression .
for technical reasons, we did not distinguish between these two populations in our analysis of wild type pancreata.
notably, none of the wild type ghrelin+ cells did express insulin, pc1/ <dig>  or pdx <dig> .
however, approximately 20% of ghrelin+ cells expressed iapp at e <dig>  .
next, we analysed the distribution of these markers within the hyperplastic ghrelin+ population found in nkx <dig> -deficient pancreata.
notably, pdx <dig> expression had been detected in these cells, although at markedly lower levels compared to wild type pancreata  <cit> .
our analysis revealed, that approximately 80% of ghrelin-expressing cells expressed low levels of pdx <dig>  and iapp in this genotype .
moreover, we frequently detected pdx1+/ghrelin- cells close to hormone-producing cells in the pancreatic epithelium of such animals .
the prohormone convertase 1/ <dig> was found localized in approximately 54% of ghrelin+ cells .
thus, the majority of the ghrelin+/arx+ cells that emerge upon nkx <dig>  deficiency significantly differ from wild type ghrelin+ cells.
the direct association of these beta-cell markers with ghrelin+, and hence with arx+ cells, provides a strong evidence that at least a subpopulation of the ghrelin+/arx+ cells exhibit committed beta-cell characteristics, and do not consist of putative alpha-cell precursors.
interestingly, similar analyses of the ghrelin+/arx+ population in pax6-deficient pancreata outlined different alterations.
notably, a few apparently normal beta-cells develop in the absence of pax <dig>  these expressing insulin, pdx <dig>  hb <dig>  nkx <dig>  and mafa  <cit> .
however, we did not detect co-expression of ghrelin and insulin, except in very rare instances .
at comparably low frequencies were detected iapp+/ghrelin+ co-expressing cells, however, the bulk of ghrelin+ cells appeared negative for iapp .
moreover, and in contrast to the ghrelin+ population noted in nkx <dig> -deficient pancreata, we did not observe any co-expression of ghrelin and pdx <dig> in pancreata of such mice .
surprisingly, almost all ghrelin+ cells were immunoreactive for pc1/ <dig> .
consequently, our data reveal significant differences in the expression profile of ghrelin+ cells arising in pancreata of nkx <dig> -/- and pax6-/- mice.
while most ghrelin+ cells from nkx <dig> -deficient embryos are positive for iapp and pdx1low, the majority of the pax6-/- ghrelin-cells do not express these markers.
importantly, the expression of iapp and pdx <dig> is not per se impeded upon pax <dig> deficiency as it can clearly be detected outside of the ghrelin-stained areas, probably labelling the remaining beta-cells.
hence, our data supports the notion that, upon the loss of nkx <dig>  or pax <dig> functional alleles, endocrine precursor cells are still committed towards the beta-cell lineage, do initiate the expression of some beta-cell-specific determinants, but fail to reach a mature beta-cell state and rather misexpress arx and ghrelin.
next we assayed the expression of pdx <dig>  iapp and pc1/ <dig> in relation with arx in nkx <dig> -/- pancreata.
arx+ cells do not express pdx <dig> or pc1/ <dig> in wild type pancreata at e <dig>  .
scattered arx+ cells co-express iapp at this age; however the bulk does not .
in contrast, the majority of the arx+ cells arising in nkx <dig> -/- pancreata do co-express pdx1low, iapp or pc1/ <dig> .
nkx <dig> -/-arx- mice pancreata lack alpha- and mature beta-cells and exhibit a hyperplastic ghrelin/somatostatin co-positive cell population
our results pointing towards a putative failure of beta-cell development upon nkx <dig>  deficiency, prompted us to investigate the role of arx in this context.
specifically, we asked  whether the loss of nkx <dig>  is directly responsible for this failure or  whether the subsequent increase in arx expression, resulting from nkx <dig>  deficiency, could lead to such phenotypic alteration.
to address this issue and gain further insight into the mutual roles of nkx <dig>  and arx in the processes underlying the specification towards the alpha- and beta-cell lineages, we generated animals doubly mutant for nkx <dig>  and arx.
double mutant mice were born following normal mendelian inheritance, indicating no embryonic lethality.
in contrast to their nkx <dig> - and arx-single mutant littermates surviving until postnatal day   <dig> and  <dig>  respectively, compound animals did not suckle and died within few hours postpartum.
analyses of their blood glucose levels indicated a hypoglycaemic condition with blood glucose levels lower than  <dig> mg/dl.
using immunofluorescence, we examined the different endocrine cell populations at p <dig> .
although arx-single mutants were found to exhibit a slight increase in the number of beta-cells, we could not detect any insulin-immunoreactive cells in compound mutant pancreata, further demonstrating that functional nkx <dig>  is essential for the formation of beta-cells .
as reported in arx-deficient pancreata, glucagon-immunoreactive cells were lost in double-mutant mice , concurring with previous findings outlining arx as an alpha-cell lineage-defining factor  <cit> .
pp-expressing cells were detected in wild type, single- and double-mutant mice although relatively rare in pancreata of nkx <dig> -/- and nkx <dig> -/-arx- newborns .
next, we assayed somatostatin-expressing delta-cells.
wild type delta-cells classically do not express nkx <dig> .
accordingly, they appeared in normal numbers in nkx <dig> -deficient mice .
as previously reported, the delta-cell population was found significantly increased in arx-mutants as compared to age-matched controls  .
hence, we expected an unopposed delta-cell differentiation in the absence of both factors.
importantly, we noticed a dramatic increase in the number of somatostatin-producing cells at all stages tested  as compared to wild type, arx- or nkx <dig> -deficent pancreata .
in p <dig> wild type pancreata, a small population of single-hormone ghrelin+ epsilon-cells is detectable beside glucagon+/ghrelin+ alpha-cells  <cit> .
the loss of functional arx specifically depletes the population of glucagon+/ghrelin+ alpha-cells but does not significantly impact on the numbers of single-hormone ghrelin+ epsilon-cells  <cit> .
similarly, glucagon+/ghrelin+ alpha-cells were found lacking in pancreata of nkx <dig> -/- animals  <cit> .
however, in the absence of both nkx <dig>  and arx, a drastic augmentation in ghrelin+ cells was observed, such increase being similar to that of nkx <dig> -/- pancreata .
surprisingly, somatostatin and ghrelin co-detection revealed a great number of cells co-expressing both hormones .
this later result was confirmed using two different sets of anti-ghrelin and anti-somatostatin antibodies .
notably, such bi-hormonal cells were not detectable in p <dig> wild type, nkx <dig> - or arx single mutant mice .
moreover, pancreata of double heterozygous mice as well as of nkx <dig> -/-arx+/- or nkx <dig> +/-arx- mice did not display such bi-hormonal cells .
we further quantified the different endocrine cell subtypes present at p <dig> .
in nkx <dig> -/- pancreata, the numbers of ghrelin+ cells was found approximately  <dig> times increased compared to ghrelin+ cells in wild type pancreata.
interestingly, a similar augmentation in the total amount of ghrelin+ cells was noted comparing pancreata of nkx <dig>  single and nkx <dig> /arx double mutants; however, approximately 80% of such ghrelin+ cells also expressed the somatostatin hormone in the latter genotype.
this suggests that arx suppresses somatostatin expression in nkx <dig> -/- ghrelin+ cells.
it should be noted that the numbers of single-hormone positive somatostatin-producing cells observed in nkx <dig> -/-arx- were comparable to those of nkx <dig> -/- single knockout and wild type pancreata.
equally important is the observation that the content of ghrelin+somatostatin+ cells found in nkx <dig> -/-arx- or of ghrelin+ noted in nkx <dig> -/- were similar to the beta-cell numbers found in their wild type counterparts.
this finding further supports the notion that beta-cells are correctly specified but fail to properly differentiate in these genotypes.
altogether, our findings demonstrate that the combined loss of nkx <dig>  and arx prevents the formation of both insulin- and glucagon-expressing endocrine cells, but has no effect on the number of single-hormone positive somatostatin-producing cells.
however, an alternative cell subtype develops, such cells expressing both ghrelin and somatostatin hormones.
nkx <dig> /arx-deficient endocrine cells express iapp as well as low levels of pdx <dig> and pax6
pdx <dig> expression persists in developing and mature wild type beta-cells and can be found in scattered delta-cells, these two endocrine cell subtypes also expressing islet amyloid polypeptide  .
our observations prompted us to further characterize the identity of the endocrine cells present in the pancreata of nkx <dig> -/-arx- mice by investigating the expression of pdx <dig> and iapp in association with somatostatin or ghrelin.
moreover, we also assessed the expression of the endocrine marker pax <dig> that had been suggested to be nkx <dig> -dependent  <cit> .
in contrast to the scattered expression of pdx <dig> classically observed in delta-cells of wild type, nkx <dig> -/- or arx- embryos, pdx <dig> could be detected in almost all somatostatin+ cells present in nkx <dig> -/-arx- double mutant pancreata.
notably, pdx <dig> expression levels were markedly lower as compared to wild type pancreata but comparable to the low pdx <dig> levels found in nkx <dig> -/- pancreata .
furthermore, all somatostatin+ cells were found to express iapp  and low levels of pax <dig> .
subsequent analyses of ghrelin-secreting cells in double mutant pancreata expectedly revealed that a vast majority likewise expressed these factors .
the expression of these beta-cell associated genes can also be observed in double-mutant pancreata at embryonic day  <dig> .
hence, the alterations of pdx <dig>  iapp and pax <dig> expression appear similar in mice lacking nkx <dig>  and animals deficient for both nkx <dig>  and arx, outlining the importance of nkx <dig>  in sustaining normal expression levels of these genes.
consequently, the main endocrine cell type found in pancreata of double mutant mice is characterized by the expression of ghrelin, somatostatin, pdx1low, pax6low and iapp.
therefore, such cells display the same beta-cell-like features as the ghrelin+ cells found in nkx <dig>  single mutants and differ only in somatostatin expression.
taken into account that, in nkx <dig>  single mutants, the population of ghrelin+/pdx1low/iapp+ cells is also expressing high levels of arx, it is conceivable that the loss of arx promotes ectopic somatostatin expression in such cells.
expression of pax <dig> and nkx <dig>  in pancreata of normal, single- and double-mutant animals
to further determine the developmental stage at which beta-cell differentiation is impaired in our mutant animals, we subsequently assayed the expression of the endocrine precursor marker pax <dig> .
broadly expressed in the embryonic pancreas around e <dig> , its expression successively declines and is rarely detectable in newborns  <cit> .
however, low expression levels have been noted in adult beta-cells  <cit> .
moreover, pax <dig> has been shown to be essential for the formation of beta- and delta-cells, and for the expression of a normal set of genes associated with beta-cell characteristics.
in accordance with previous studies detecting pax <dig> transcripts in arx- or nkx <dig> -deficient mice, we observed numerous pax4+ cells at embryonic day  <dig>  in the pancreatic epithelium of such animals using immunohistochemical detection .
we have previously reported that the arx factor binds the pax <dig> promoter and negatively regulates pax <dig> expression, thereby specifying alpha vs. beta-cell fates  <cit> .
however, despite markedly elevated arx transcript levels upon the loss of functional nkx <dig> , we did detect normal pax4+ cell numbers in pancreata of e <dig>  nkx <dig> -/- mice, suggesting that, in this genotype and at this age, pax <dig> is not downregulated by arx.
interestingly, the combined loss of nkx <dig>  and arx did not impact in the number of pax4+ cells as compared to nkx <dig>  mutant pancreata, thus indicating that their formation does not depend on either factor .
notably, and in agreement with previous findings, pax <dig> expression was not detectable in ghrelin+ cells of wild type or mutant pancreata.
next, we analysed the temporal expression of nkx <dig>  in pancreata of all four genotypes .
importantly, previous studies had shown that both pax <dig> and nkx <dig>  are essential to maintain the expression of nkx <dig>  in developing beta-cells  <cit> ; in the absence of either factor, a decrease in nkx <dig> + cell numbers was seen from e <dig>  on, progressing to a complete loss of nkx <dig> + cells in newborns.
the loss of arx had no impact on nkx <dig>  expression as it was found maintained and properly located in emerging beta-cells .
in nkx <dig> -/-arx- compound mutant mice, we observed a temporal expression pattern of nkx <dig>  comparable to that of nkx <dig> -deficient pancreata; broadly expressed in the undifferentiated pancreatic epithelium at e <dig> , the number of nkx <dig> + cells was found drastically reduced at e <dig> , while no nkx <dig> + cells could be detected at p <dig> .
recent studies have demonstrated that nkx <dig> + cells arising during embryonic pancreatogenesis can be subdivided into sox9+ and sox9- cells, representing ductal/endocrine progenitors and committed beta-cells, respectively  <cit> .
in order to characterize the nkx <dig> + population within the different genotypes, we co-detected nkx <dig>  and sox <dig> using immunofluorescence.
in e <dig>  wild type and arx-mutant pancreata, numerous nkx <dig> +sox9- beta-cells are detectable besides nkx <dig> +sox9+ progenitor cells .
interestingly, in nkx <dig> -deficient genotypes, nkx <dig> + cells were found associated to the uncommitted progenitor population .
these data clearly demonstrate that the expression of nkx <dig>  is not dependent on nkx <dig>  activities within sox9+ progenitor cells.
however, as soon as a cell enters the endocrine lineage, nkx <dig>  expression is found highly dependent on nkx <dig> .
these results also imply that the steady decline in nkx <dig> + cell numbers observed in nkx <dig> -/- pancreata, most likely reflects the normal decrease of progenitor cells classically noted in the last days of embryonic pancreas development  <cit> .
taken together, our results demonstrate that nkx <dig>  and arx are dispensable for the generation of pax4+ cells.
moreover, the alterations in the expression of pdx <dig>  nkx <dig>  and pax <dig>  appear similar when comparing pancreata of nkx <dig> -single and nkx <dig> /arx compound-mutants, suggesting that the failure of beta-cell differentiation and the associated ghrelin misexpression are solely caused by the loss of nkx <dig> .
similarly, the dramatic alterations in alpha-cell development in nkx <dig> -/- pancreata, where arx expression is even more pronounced, sustain the notion that nkx <dig>  is also necessary for the acquisition of mature alpha-cell fate.
discussion
in this study we have  examined the consequences of the loss-of-function of pax <dig>  arx or nkx <dig> , as well as of the combined loss of nkx <dig>  and arx, on the development of pancreatic endocrine hormone-producing cells.
previous studies had shown that the loss of nkx <dig>  or pax <dig> results in the formation of numerous ghrelin-expressing cells at the expense of the beta- and alpha-cell populations  <cit> .
herein, we demonstrate that the majority of such ghrelin+ cells express the alpha-cell specifier arx.
however, further expression analyses focusing on ghrelin and alpha- or beta-cell-related genes suggest a partial association of the mutant ghrelin+/arx+ population to the beta-cell lineage.
the combined loss of nkx <dig> - and arx likewise results in the formation of numerous ghrelin+ cells at the expense of alpha- and beta- cells, whereas delta-cell formation appears unaffected.
notably, the majority of the ghrelin+ cells were, yet again, found to display some of the features classically associated to beta-cells, but also to ectopically express somatostatin, suggesting that the additional loss of arx promotes the ectopic expression of somatostatin.
ghrelin+ cells arising in nkx <dig> -deficient pancreata display different characteristics as compared to ghrelin+ cells arising in wild type or pax6-deficient mice
previous reports have revealed that the loss of pax <dig>  nkx <dig>  or pax <dig> is accompanied by the emergence of numerous ghrelin+ cells at the expense of the beta-cell population  <cit> .
notably, in either mutant pancreas, the expression of the two other genes is detectable, rendering it difficult to attribute putative ghrelin-suppressing activities to one of the three candidates.
at least for pax <dig>  such ghrelin-suppressing function has been demonstrated in vitro, whereas nkx <dig>  has been shown to exhibit ghrelin-activating functions in cell culture experiments  <cit> .
however, pax <dig> expression remains detectable in nkx <dig> - or pax6-mutant pancreata, suggesting that ghrelin-silencing requires multiple factors.
interestingly, in all three mutants, arx transcripts levels are found increased and arx is often associated with ghrelin-expressing cell populations .
the possibility that arx acts as a putative ghrelin-activator, however, appears unlikely as ghrelin-expression is also broadly maintained in cells arising in nkx <dig> /arx compound mutants.
in an effort to better characterize such mutant ghrelin-expressing cells, we analysed co-expression of ghrelin and alpha-or beta-cell related markers in pancreata of wild type, pax6-/- and nkx <dig> -/- mice.
the bulk of the nkx <dig> -deficient ghrelin+ cells expresses arx, iapp, pc1/ <dig> and pdx <dig> and thus strongly differs from wild type epsilon-cells, excluding a specific augmentation of epsilon-cell numbers upon nkx <dig>  depletion.
as previously suggested, our data strengthens the notion, that the majority of ghrelin+ cells arising in nkx <dig> -deficient mice represent committed beta-cells that fail to properly differentiate  <cit> .
wang et al.
suggested that nkx <dig>  acts in synergy with pax <dig> during the early steps of beta-cell differentiation  <cit> .
interestingly, the population of ghrelin+/glucagon+ cells emerging in pancreata of pax4-deficient mice share many characteristics with the one observed in nkx <dig> -deficient mice, including the expression of arx, iapp, pdx1low and pc1/ <dig> .
it is therefore conceivable, that such cells likewise represent committed beta-cell precursors and that the presence of nkx <dig>  and arx allows their maturation towards a glucagon-expressing state.
wang et al.
also outlined that synergistic activities of nkx <dig>  and pax <dig> are essential in the early steps of beta-cell differentiation in order to activate crucial factors, such as hb <dig>  nkx <dig>  and pdx1high  <cit> .
furthermore, we previously demonstrated that pax <dig> strongly suppresses arx expression in early beta-cells  <cit> .
together with these findings, our current data provides strong evidence for the essential synergistic activity of nkx <dig>  and pax <dig> to suppress arx expression in early committed beta-cells.
a strict control of arx production appears essential as arx is sufficient to convert mature beta-cells into pp-like or alpha-like cells  <cit> .
the facts that nkx <dig>  is also expressed in alpha-cells and that alpha-cell development is impaired upon nkx <dig>  deficiency, lead to the assumption that the lineage determining activities of both arx and pax <dig> require nkx <dig>  activity.
we also demonstrate that the lack of functional pax <dig> results in the formation of ghrelin+/arx+ cells.
pax <dig> was thought to exert a more general role in the maintenance of the beta-cell identity.
the presence of a few but apparently normal beta-cells in pancreata of pax6-deficient mice even led to the suggestion of an alternate pax6-independent pathway leading to mature beta-cells  <cit> .
interestingly, we frequently detected scattered insulin+/ghrelin+ cells in such mutant pancreata.
such cells might represent a transitional state of beta-cells converting into ghrelin+ cells.
tracing the lineage of insulin-expressing cells in the pax6-deficient background would doubtless shed more light on this interesting issue.
however, pax6-deficient ghrelin+ cells express neither iapp nor pdx <dig>  thus differing from the ghrelin+ populations arising in nkx <dig> - or pax4-deficient pancreata.
the presence of iapp and pdx <dig> in the remaining pax6-deficient beta-cells indicates that pax <dig> is not necessary for the activation of these genes.
however, it is conceivable that pax <dig> function is essential for maintaining their expression as soon as a beta-cell reaches a certain stage of differentiation.
the almost complete loss of glucagon+ cells in pax6-deficient mice indicates a more essential role of pax <dig> in alpha-cells.
in this context, it has been demonstrated that pax <dig> binds and activates the glucagon gene  <cit> , however it remains unknown whether pax <dig> is also involved in the determination of the alpha-cell lineage.
the continued expression of three alpha-cell related genes  in pax6-deficient pancreata might be an indication for a predominant involvement of pax <dig> in later stages of alpha-cell differentiation.
ghrelin+/somatostatin+ cells arising in nkx <dig> /arx compound mutants represent a non-delta-cell type
our analysis shows that the combined loss of nkx <dig>  and arx prevents the formation of mature alpha- and beta-cells, thus confirming the requirement of arx and nkx <dig>  activities for the formation of these cell types.
however, the presence of pax4+ cells indicates that an endocrine precursor state is attained without nkx <dig>  and/or arx.
this, and previous studies, suggest that nkx <dig>  is essential for the maintenance of high levels of pdx <dig> and pax <dig> as well as the activation of nkx <dig>  in developing beta-cells  <cit> .
consistent with these assumptions, the alterations found in the expression of pdx <dig>  pax <dig> and nkx <dig>  appear similar, comparing pancreata of nkx <dig>  single- and nkx <dig> /arx compound-mutant mice.
these data once more imply that arx is dispensable for the beta-cell fate and that the proper expression of these genes can be attributed to nkx <dig>  alone.
however, the additional loss of arx results in the formation of numerous ghrelin+/somatostatin+ cells beside populations of single-hormone positive somatostatin+ or ghrelin+ cells.
we did not detect ghrelin expression in delta-cells arising in wild type or either single-knockout pancreata.
together with the observation that neither nkx <dig>  nor arx is expressed in wild type delta-cells, we conclude that such double positive cells do not represent delta-cells ectopically expressing ghrelin but rather a population comparable to the ghrelin+ cells arising in nkx <dig> -deficient mice.
this is further supported by the fact that ghrelin+/somatostatin+ cells display the same broad expression of pdx1low as observed in nkx <dig> -deficient ghrelin+ cells.
hence, we conclude that 80% of the ghrelin+ cells initiate somatostatin expression upon the additional loss of arx.
not all ghrelin-labelled cells co-express somatostatin in double-mutant pancreata.
this might be attributed to their distinct predeterminations and partially reflect the presence of normal epsilon-cells.
nevertheless, our data also imply that arx or a not yet identified downstream mediator exerts a suppressive effect on somatostatin expression.
in this context, previous studies demonstrated that arx activities strongly antagonize delta-cell formation  <cit> .
however, whether arx directly antagonizes somatostatin expression would require further investigations.
CONCLUSIONS
our data indicate that although proper endocrine differentiation/hormone expression is impaired in pax6- or nkx <dig> -deficient pancreata, committed cells retain some of the characteristics normally associated with mature beta-cells .
our analysis of nkx <dig> /arx single- and double-mutant pancreata allow us, together with the data from others, to conclude that both pax <dig> and arx require the activity of nkx <dig>  in order to maintain the transcriptional cascades initiated in early committed beta- and alpha-cell precursors, respectively.
our findings also suggest that one of the coupled functions of nkx <dig>  and pax <dig> is the control of arx gene activity in committed beta-cell precursors.

 the pancreas is comprised of acinar and duct cells, producing and transporting digestive enzymes and bicarbonate, as well as endocrine cells, which secrete hormones to the blood stream. the latter are aggregated into scattered clusters of cells, termed islets of langerhans. these are typically composed of five cell subtypes, including alpha-, beta-, delta-, epsilon- and pp-cells that produce the hormones glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide, respectively. insulin and glucagon are secreted co-ordinately to control glucose homeostasis, whereas somatostatin and pp regulate the secretion of other hormones and of exocrine enzymes. the hormone ghrelin has been shown to possess orexigenic properties and to play a role in glucose-stimulated insulin secretion. during pancreas development, the first wave of endocrine cell differentiation begins at embryonic day and results in the emergence of glucagon-producing cells, followed by the appearance of few insulin-producing cells, often co-expressing glucagon. ghrelin-expressing cells can be detected from e on. subsequently, the major wave of endocrine cell formation, initiates around e and results in the emergence of numerous insulin-producing beta-cells and glucagon-producing alpha-cells. around e the first somatostatin-producing delta-cells emerge while, shortly before birth, pp-expressing pp-cells differentiate. concurrently, from e on, the endocrine cell mass continuously expands and self-organizes into well-shaped islets, a process lasting until approximately weeks postpartum. a number of transcriptional regulators have been shown to have crucial roles in controlling the specification of pancreatic cells. among these, pdx was found to exert a key function for the pancreatic cell lineage allocation. later during pancreatic development, pdx activity is restricted to beta-cells and a subpopulation of delta-cells. the expression of the basic helix-loop-helix transcription factor ngn induces endocrine differentiation, as evidenced by gain- and loss-of-function studies as well as genetic lineage tracing. the specification towards the distinct endocrine cell types was found controlled by the concerted activities of specific transcription factors, including the homedomain-containing factors nkx, nkx, pax, brn, pax and arx. nkx is broadly expressed in the early undifferentiated pancreatic epithelium and in endocrine progenitors. along with the secondary transition, nkx expression becomes restricted to mature beta-cells and a subset of alpha- and pp-cells, whereas it is not detected in delta-cells. accordingly, mice depleted in nkx display a complete beta-cell loss and reduced alpha- and pp-cell content, while the numbers of delta-cells remain unchanged. notably, the ghrelin+ cell population appears considerably augmented. in this context, it has been shown that nkx is decisive for the maintenance of normal beta-cell function and that the transcriptional repressor activity of nkx is required for the proper specification of alpha-cells. nkx has been found essential for the specification of multipotent progenitors towards the duct-/endocrine-fates in early pancreas development. post second transition, nkx becomes exclusively restricted to developing beta-cells; at that stage it is essential for their expansion. the pax gene family member, pax is produced in all endocrine cells throughout pancreas development with one exception that is a subpopulation of epsilon-cells. thus, in the absence of functional pax alpha-, beta-, delta- and pp-cells are negatively affected with a near complete loss of glucagon-expression from alpha-cells. similar to nkx mutants, the endocrine portion in pax6-/- pancreata mainly consists of cells expressing the ghrelin-hormone. pax another pax family member, has been found essential for the formation of beta- and delta-cells. lineage tracing studies revealed that pax4+ precursor cells give rise to all four major endocrine cell types. nevertheless, in the absence of pax beta- and delta-cell development is impaired and precursor cells acquire an alpha-cell-like fate, suggesting a predominant role of pax in the differentiation of beta-cells. this assumption is supported by recent work demonstrating that the in vivo misexpression of pax in pancreatic/endocrine progenitors results mainly in the formation of beta-cells. moreover, the forced and ectopic expression of pax in mature alpha-cells was found sufficient to convert these into functional beta-like cells. mice lacking the aristaless homeobox gene arx display a phenotype opposite to the one observed in pax4-/- animals. depleted in alpha-cells, such mice exhibit proportionally enlarged beta- and delta-cell populations. in fact, a direct and mutually antagonistic transcriptional repression between arx and pax was previously demonstrated and associated with the specification of the alpha- vs. beta-/delta-cell fates, respectively. this model was further supported by the forced expression of arx in pancreatic/endocrine progenitors, which led to the favouring of the alpha- but also pp-cell fate, at the expense of beta- and delta-cell lineages. interestingly, recent analyses of nkx -deficient pancreata outlined a 5-fold increase in the transcript content of the alpha-cell "specifier" arx despite an almost complete loss in glucagon-expressing cells. to address this apparent paradox, we undertook a thorough comparative analysis of the endocrine populations arising in pancreata of wild type, nkx - and pax6-deficient animals. moreover, we further analysed the roles of nkx and arx, by characterizing pancreatic endocrine cell fates in mice compound-deficient for nkx and arx.  expression of arx and brn in e pancreata of wild type, nkx -/- and pax6-/- mice we previously investigated the antagonistic roles of arx and pax for proper endocrine cell allocation and thereby demonstrated the essential function of arx in promoting a subset of endocrine precursor cells specifically towards the alpha-cell fate. the apparent discrepancy between the severely reduced alpha-cell numbers and the 5-fold augmentation in arx transcripts upon loss of nkx, prompted us to examine the subcellular localization of arx in pancreata of such mice and thus gain further insights into the role of arx and nkx during endocrine cell specification. we initially focused our study at e, a developmental stage subsequent to a peak in mature endocrine cell generation. in wild type pancreata, most arx-labelled cells co-expressed glucagon and were found scattered within the pancreatic tissue. only few arx+/glucagon- cells were detectable, most likely corresponding to alpha-cell precursors that have not yet initiated hormone synthesis. arx+ cells did not express ghrelin, except in rare instances. interestingly, in nkx -deficient animals, an almost complete lack of alpha- and beta-cells accompanied by a five-fold increase in arx transcript levels were noted when compared to their control counterparts. subsequent quantifications revealed a -fold rise in the numbers of arx-immunoreactive cells in e mutant pancreata compared to age-matched wild type embryos. clearly, this number did not fully reflect the upregulation of arx transcripts, suggesting an increase in arx production at the single cell level. these results led us to determine whether nkx expression could be likewise affected in animals deficient for arx. however, nkx transcript levels in e arx mutant pancreata were not significantly different from wild type controls, suggesting that nkx transcription is not regulated by arx. further analyses of the arx+ cell population in nkx -/- mice revealed that a vast majority expressed ghrelin. approximately 90% of the ghrelin+ cells were found positive for arx. besides this ghrelin+/arx+ population, only few scattered arx+/ghrelin- or ghrelin+/arx- cells were detectable in this genotype. surprisingly, most of the few remaining glucagon-expressing cells present in nkx -deficient pancreata were negative for arx expression. in order to further assess the identity of ghrelin+/arx+ cells, we analysed the expression of the alpha-cell-related marker gene brn previous analyses of nkx mutant pancreata had revealed that the numbers of brn4+ cells were reduced; correlating with the lower number of glucagon+ cells. consistent with these findings, the expression of brn was absent from the hyperplastic ghrelin+ cells noted in this genotype. altogether, these results suggest that, in the absence of nkx, the beta- and alpha-cell lineages are disfavoured and replaced by a ghrelin+/arx+/brn4- cell fate. considering the crucial role exerted by arx in promoting the alpha-cell lineage, it is conceivable that such cells correspond to alpha-cell precursors that failed to acquire the full complement of alpha-cell characteristics. however, the observation that the proportion of ghrelin+/arx+/brn4- cells present in nkx -/- pancreata is similar to the proportion of beta-cells normally detected in wild-type mice led us to favour an alternative explanation where cells normally fated towards the beta-cell lineage fail to properly differentiate and ectopically express arx and ghrelin. the dramatic increase in arx transcripts, combined with the profound loss in pax expression in nkx -deficient pancreata, prompted us to get more insights into putative interplay between these three factors. in pax knockout mice, the early glucagon+ population emerging around embryonic day is present and properly express the associated markers brn and isl. however, post secondary transition, glucagon+ cell numbers are significantly reduced. consistent with these findings, it was shown that pax both indirectly and directly, potentiates the expression of the glucagon gene. interestingly, upon pax deficiency, the beta-cell population was found reduced concomitantly with a -fold increase in the numbers of ghrelin+ cells. notably, both nkx transcript content and expression pattern were found almost normal in pax6-/- pancreata as compared to their wild-type counterparts. surprisingly, in e pax mutant pancreata, arx transcripts were found times increased as compared to age-matched wild-type pancreata. however, the numbers of arx+ cells were not significantly different from those observed in wild type, indicating that the deficiency in nkx or pax leads to a similar increase in arx transcripts at the single cell level. interestingly, and similar to what we observed in nkx -deficient mice, most of the arx+ cells present in pax6-deficient pancreata, express ghrelin. approximately 80% of the ghrelin+ cells were found to express arx, although scattered arx+/ghrelin- and ghrelin+/arx- cells were also detectable. the expression of brn appeared unchanged in pax6-deficient mice at e and e. brn expression was clearly noted in approximately 45% of the ghrelin-expressing cells, suggesting at least a partially overlapping expression with arx. notably, beside the ghrelin+/brn4+ cell population, brn4+ cells were also found outside of the ghrelin-stained areas. together, our data indicate that the reduction of alpha- and beta-cell numbers in nkx -/- or pax6-/- pancreata is accompanied by an increase in the numbers of cells co-expressing arx and ghrelin. interestingly, the loss of arx does not seem to impact on nkx or pax expression. comparative analysis of ghrelin+ cells in pancreata of wild type, nkx -/- and pax6-/- mice to further assess the identity of the ghrelin+/arx+ cell population arising in pancreata of nkx -/- and pax6-/- mice, we assayed the expression of a number of beta-cell-related markers, such as insulin, iapp, pc1/ and pdx at e. most of these proteins have previously been found expressed in both mutant pancreata, however their expression relative to ghrelin has not been fully resolved. previous detailed analyses of wild type ghrelin-expressing cells had led to the conclusion that these could be subdivided into glucagon+ghrelin+ alpha-cells and glucagon-ghrelin+ epsilon-cells. it should be noted that only a subset of alpha-cells were actually found positive for ghrelin, such cells retaining glucagon, arx and brn expression. for technical reasons, we did not distinguish between these two populations in our analysis of wild type pancreata. notably, none of the wild type ghrelin+ cells did express insulin, pc1/ or pdx. however, approximately 20% of ghrelin+ cells expressed iapp at e. next, we analysed the distribution of these markers within the hyperplastic ghrelin+ population found in nkx -deficient pancreata. notably, pdx expression had been detected in these cells, although at markedly lower levels compared to wild type pancreata. our analysis revealed, that approximately 80% of ghrelin-expressing cells expressed low levels of pdx and iapp in this genotype. moreover, we frequently detected pdx1+/ghrelin- cells close to hormone-producing cells in the pancreatic epithelium of such animals. the prohormone convertase 1/ was found localized in approximately 54% of ghrelin+ cells. thus, the majority of the ghrelin+/arx+ cells that emerge upon nkx deficiency significantly differ from wild type ghrelin+ cells. the direct association of these beta-cell markers with ghrelin+, and hence with arx+ cells, provides a strong evidence that at least a subpopulation of the ghrelin+/arx+ cells exhibit committed beta-cell characteristics, and do not consist of putative alpha-cell precursors. interestingly, similar analyses of the ghrelin+/arx+ population in pax6-deficient pancreata outlined different alterations. notably, a few apparently normal beta-cells develop in the absence of pax these expressing insulin, pdx hb nkx and mafa. however, we did not detect co-expression of ghrelin and insulin, except in very rare instances. at comparably low frequencies were detected iapp+/ghrelin+ co-expressing cells, however, the bulk of ghrelin+ cells appeared negative for iapp. moreover, and in contrast to the ghrelin+ population noted in nkx -deficient pancreata, we did not observe any co-expression of ghrelin and pdx in pancreata of such mice. surprisingly, almost all ghrelin+ cells were immunoreactive for pc1/. consequently, our data reveal significant differences in the expression profile of ghrelin+ cells arising in pancreata of nkx -/- and pax6-/- mice. while most ghrelin+ cells from nkx -deficient embryos are positive for iapp and pdx1low, the majority of the pax6-/- ghrelin-cells do not express these markers. importantly, the expression of iapp and pdx is not per se impeded upon pax deficiency as it can clearly be detected outside of the ghrelin-stained areas, probably labelling the remaining beta-cells. hence, our data supports the notion that, upon the loss of nkx or pax functional alleles, endocrine precursor cells are still committed towards the beta-cell lineage, do initiate the expression of some beta-cell-specific determinants, but fail to reach a mature beta-cell state and rather misexpress arx and ghrelin. next we assayed the expression of pdx iapp and pc1/ in relation with arx in nkx -/- pancreata. arx+ cells do not express pdx or pc1/ in wild type pancreata at e. scattered arx+ cells co-express iapp at this age; however the bulk does not. in contrast, the majority of the arx+ cells arising in nkx -/- pancreata do co-express pdx1low, iapp or pc1/. nkx -/-arx- mice pancreata lack alpha- and mature beta-cells and exhibit a hyperplastic ghrelin/somatostatin co-positive cell population our results pointing towards a putative failure of beta-cell development upon nkx deficiency, prompted us to investigate the role of arx in this context. specifically, we asked whether the loss of nkx is directly responsible for this failure or whether the subsequent increase in arx expression, resulting from nkx deficiency, could lead to such phenotypic alteration. to address this issue and gain further insight into the mutual roles of nkx and arx in the processes underlying the specification towards the alpha- and beta-cell lineages, we generated animals doubly mutant for nkx and arx. double mutant mice were born following normal mendelian inheritance, indicating no embryonic lethality. in contrast to their nkx - and arx-single mutant littermates surviving until postnatal day and respectively, compound animals did not suckle and died within few hours postpartum. analyses of their blood glucose levels indicated a hypoglycaemic condition with blood glucose levels lower than mg/dl. using immunofluorescence, we examined the different endocrine cell populations at p. although arx-single mutants were found to exhibit a slight increase in the number of beta-cells, we could not detect any insulin-immunoreactive cells in compound mutant pancreata, further demonstrating that functional nkx is essential for the formation of beta-cells. as reported in arx-deficient pancreata, glucagon-immunoreactive cells were lost in double-mutant mice, concurring with previous findings outlining arx as an alpha-cell lineage-defining factor. pp-expressing cells were detected in wild type, single- and double-mutant mice although relatively rare in pancreata of nkx -/- and nkx -/-arx- newborns. next, we assayed somatostatin-expressing delta-cells. wild type delta-cells classically do not express nkx. accordingly, they appeared in normal numbers in nkx -deficient mice. as previously reported, the delta-cell population was found significantly increased in arx-mutants as compared to age-matched controls. hence, we expected an unopposed delta-cell differentiation in the absence of both factors. importantly, we noticed a dramatic increase in the number of somatostatin-producing cells at all stages tested as compared to wild type, arx- or nkx -deficent pancreata. in p wild type pancreata, a small population of single-hormone ghrelin+ epsilon-cells is detectable beside glucagon+/ghrelin+ alpha-cells. the loss of functional arx specifically depletes the population of glucagon+/ghrelin+ alpha-cells but does not significantly impact on the numbers of single-hormone ghrelin+ epsilon-cells. similarly, glucagon+/ghrelin+ alpha-cells were found lacking in pancreata of nkx -/- animals. however, in the absence of both nkx and arx, a drastic augmentation in ghrelin+ cells was observed, such increase being similar to that of nkx -/- pancreata. surprisingly, somatostatin and ghrelin co-detection revealed a great number of cells co-expressing both hormones. this later result was confirmed using two different sets of anti-ghrelin and anti-somatostatin antibodies. notably, such bi-hormonal cells were not detectable in p wild type, nkx - or arx single mutant mice. moreover, pancreata of double heterozygous mice as well as of nkx -/-arx+/- or nkx +/-arx- mice did not display such bi-hormonal cells. we further quantified the different endocrine cell subtypes present at p. in nkx -/- pancreata, the numbers of ghrelin+ cells was found approximately times increased compared to ghrelin+ cells in wild type pancreata. interestingly, a similar augmentation in the total amount of ghrelin+ cells was noted comparing pancreata of nkx single and nkx /arx double mutants; however, approximately 80% of such ghrelin+ cells also expressed the somatostatin hormone in the latter genotype. this suggests that arx suppresses somatostatin expression in nkx -/- ghrelin+ cells. it should be noted that the numbers of single-hormone positive somatostatin-producing cells observed in nkx -/-arx- were comparable to those of nkx -/- single knockout and wild type pancreata. equally important is the observation that the content of ghrelin+somatostatin+ cells found in nkx -/-arx- or of ghrelin+ noted in nkx -/- were similar to the beta-cell numbers found in their wild type counterparts. this finding further supports the notion that beta-cells are correctly specified but fail to properly differentiate in these genotypes. altogether, our findings demonstrate that the combined loss of nkx and arx prevents the formation of both insulin- and glucagon-expressing endocrine cells, but has no effect on the number of single-hormone positive somatostatin-producing cells. however, an alternative cell subtype develops, such cells expressing both ghrelin and somatostatin hormones. nkx /arx-deficient endocrine cells express iapp as well as low levels of pdx and pax6 pdx expression persists in developing and mature wild type beta-cells and can be found in scattered delta-cells, these two endocrine cell subtypes also expressing islet amyloid polypeptide. our observations prompted us to further characterize the identity of the endocrine cells present in the pancreata of nkx -/-arx- mice by investigating the expression of pdx and iapp in association with somatostatin or ghrelin. moreover, we also assessed the expression of the endocrine marker pax that had been suggested to be nkx -dependent. in contrast to the scattered expression of pdx classically observed in delta-cells of wild type, nkx -/- or arx- embryos, pdx could be detected in almost all somatostatin+ cells present in nkx -/-arx- double mutant pancreata. notably, pdx expression levels were markedly lower as compared to wild type pancreata but comparable to the low pdx levels found in nkx -/- pancreata. furthermore, all somatostatin+ cells were found to express iapp and low levels of pax. subsequent analyses of ghrelin-secreting cells in double mutant pancreata expectedly revealed that a vast majority likewise expressed these factors. the expression of these beta-cell associated genes can also be observed in double-mutant pancreata at embryonic day. hence, the alterations of pdx iapp and pax expression appear similar in mice lacking nkx and animals deficient for both nkx and arx, outlining the importance of nkx in sustaining normal expression levels of these genes. consequently, the main endocrine cell type found in pancreata of double mutant mice is characterized by the expression of ghrelin, somatostatin, pdx1low, pax6low and iapp. therefore, such cells display the same beta-cell-like features as the ghrelin+ cells found in nkx single mutants and differ only in somatostatin expression. taken into account that, in nkx single mutants, the population of ghrelin+/pdx1low/iapp+ cells is also expressing high levels of arx, it is conceivable that the loss of arx promotes ectopic somatostatin expression in such cells. expression of pax and nkx in pancreata of normal, single- and double-mutant animals to further determine the developmental stage at which beta-cell differentiation is impaired in our mutant animals, we subsequently assayed the expression of the endocrine precursor marker pax. broadly expressed in the embryonic pancreas around e, its expression successively declines and is rarely detectable in newborns. however, low expression levels have been noted in adult beta-cells. moreover, pax has been shown to be essential for the formation of beta- and delta-cells, and for the expression of a normal set of genes associated with beta-cell characteristics. in accordance with previous studies detecting pax transcripts in arx- or nkx -deficient mice, we observed numerous pax4+ cells at embryonic day in the pancreatic epithelium of such animals using immunohistochemical detection. we have previously reported that the arx factor binds the pax promoter and negatively regulates pax expression, thereby specifying alpha vs. beta-cell fates. however, despite markedly elevated arx transcript levels upon the loss of functional nkx, we did detect normal pax4+ cell numbers in pancreata of e nkx -/- mice, suggesting that, in this genotype and at this age, pax is not downregulated by arx. interestingly, the combined loss of nkx and arx did not impact in the number of pax4+ cells as compared to nkx mutant pancreata, thus indicating that their formation does not depend on either factor. notably, and in agreement with previous findings, pax expression was not detectable in ghrelin+ cells of wild type or mutant pancreata. next, we analysed the temporal expression of nkx in pancreata of all four genotypes. importantly, previous studies had shown that both pax and nkx are essential to maintain the expression of nkx in developing beta-cells; in the absence of either factor, a decrease in nkx + cell numbers was seen from e on, progressing to a complete loss of nkx + cells in newborns. the loss of arx had no impact on nkx expression as it was found maintained and properly located in emerging beta-cells. in nkx -/-arx- compound mutant mice, we observed a temporal expression pattern of nkx comparable to that of nkx -deficient pancreata; broadly expressed in the undifferentiated pancreatic epithelium at e, the number of nkx + cells was found drastically reduced at e, while no nkx + cells could be detected at p. recent studies have demonstrated that nkx + cells arising during embryonic pancreatogenesis can be subdivided into sox9+ and sox9- cells, representing ductal/endocrine progenitors and committed beta-cells, respectively. in order to characterize the nkx + population within the different genotypes, we co-detected nkx and sox using immunofluorescence. in e wild type and arx-mutant pancreata, numerous nkx +sox9- beta-cells are detectable besides nkx +sox9+ progenitor cells. interestingly, in nkx -deficient genotypes, nkx + cells were found associated to the uncommitted progenitor population. these data clearly demonstrate that the expression of nkx is not dependent on nkx activities within sox9+ progenitor cells. however, as soon as a cell enters the endocrine lineage, nkx expression is found highly dependent on nkx. these results also imply that the steady decline in nkx + cell numbers observed in nkx -/- pancreata, most likely reflects the normal decrease of progenitor cells classically noted in the last days of embryonic pancreas development. taken together, our results demonstrate that nkx and arx are dispensable for the generation of pax4+ cells. moreover, the alterations in the expression of pdx nkx and pax appear similar when comparing pancreata of nkx -single and nkx /arx compound-mutants, suggesting that the failure of beta-cell differentiation and the associated ghrelin misexpression are solely caused by the loss of nkx. similarly, the dramatic alterations in alpha-cell development in nkx -/- pancreata, where arx expression is even more pronounced, sustain the notion that nkx is also necessary for the acquisition of mature alpha-cell fate. discussion in this study we have examined the consequences of the loss-of-function of pax arx or nkx, as well as of the combined loss of nkx and arx, on the development of pancreatic endocrine hormone-producing cells. previous studies had shown that the loss of nkx or pax results in the formation of numerous ghrelin-expressing cells at the expense of the beta- and alpha-cell populations. herein, we demonstrate that the majority of such ghrelin+ cells express the alpha-cell specifier arx. however, further expression analyses focusing on ghrelin and alpha- or beta-cell-related genes suggest a partial association of the mutant ghrelin+/arx+ population to the beta-cell lineage. the combined loss of nkx - and arx likewise results in the formation of numerous ghrelin+ cells at the expense of alpha- and beta- cells, whereas delta-cell formation appears unaffected. notably, the majority of the ghrelin+ cells were, yet again, found to display some of the features classically associated to beta-cells, but also to ectopically express somatostatin, suggesting that the additional loss of arx promotes the ectopic expression of somatostatin. ghrelin+ cells arising in nkx -deficient pancreata display different characteristics as compared to ghrelin+ cells arising in wild type or pax6-deficient mice previous reports have revealed that the loss of pax nkx or pax is accompanied by the emergence of numerous ghrelin+ cells at the expense of the beta-cell population. notably, in either mutant pancreas, the expression of the two other genes is detectable, rendering it difficult to attribute putative ghrelin-suppressing activities to one of the three candidates. at least for pax such ghrelin-suppressing function has been demonstrated in vitro, whereas nkx has been shown to exhibit ghrelin-activating functions in cell culture experiments. however, pax expression remains detectable in nkx - or pax6-mutant pancreata, suggesting that ghrelin-silencing requires multiple factors. interestingly, in all three mutants, arx transcripts levels are found increased and arx is often associated with ghrelin-expressing cell populations. the possibility that arx acts as a putative ghrelin-activator, however, appears unlikely as ghrelin-expression is also broadly maintained in cells arising in nkx /arx compound mutants. in an effort to better characterize such mutant ghrelin-expressing cells, we analysed co-expression of ghrelin and alpha-or beta-cell related markers in pancreata of wild type, pax6-/- and nkx -/- mice. the bulk of the nkx -deficient ghrelin+ cells expresses arx, iapp, pc1/ and pdx and thus strongly differs from wild type epsilon-cells, excluding a specific augmentation of epsilon-cell numbers upon nkx depletion. as previously suggested, our data strengthens the notion, that the majority of ghrelin+ cells arising in nkx -deficient mice represent committed beta-cells that fail to properly differentiate. wang et al. suggested that nkx acts in synergy with pax during the early steps of beta-cell differentiation. interestingly, the population of ghrelin+/glucagon+ cells emerging in pancreata of pax4-deficient mice share many characteristics with the one observed in nkx -deficient mice, including the expression of arx, iapp, pdx1low and pc1/. it is therefore conceivable, that such cells likewise represent committed beta-cell precursors and that the presence of nkx and arx allows their maturation towards a glucagon-expressing state. wang et al. also outlined that synergistic activities of nkx and pax are essential in the early steps of beta-cell differentiation in order to activate crucial factors, such as hb nkx and pdx1high. furthermore, we previously demonstrated that pax strongly suppresses arx expression in early beta-cells. together with these findings, our current data provides strong evidence for the essential synergistic activity of nkx and pax to suppress arx expression in early committed beta-cells. a strict control of arx production appears essential as arx is sufficient to convert mature beta-cells into pp-like or alpha-like cells. the facts that nkx is also expressed in alpha-cells and that alpha-cell development is impaired upon nkx deficiency, lead to the assumption that the lineage determining activities of both arx and pax require nkx activity. we also demonstrate that the lack of functional pax results in the formation of ghrelin+/arx+ cells. pax was thought to exert a more general role in the maintenance of the beta-cell identity. the presence of a few but apparently normal beta-cells in pancreata of pax6-deficient mice even led to the suggestion of an alternate pax6-independent pathway leading to mature beta-cells. interestingly, we frequently detected scattered insulin+/ghrelin+ cells in such mutant pancreata. such cells might represent a transitional state of beta-cells converting into ghrelin+ cells. tracing the lineage of insulin-expressing cells in the pax6-deficient background would doubtless shed more light on this interesting issue. however, pax6-deficient ghrelin+ cells express neither iapp nor pdx thus differing from the ghrelin+ populations arising in nkx - or pax4-deficient pancreata. the presence of iapp and pdx in the remaining pax6-deficient beta-cells indicates that pax is not necessary for the activation of these genes. however, it is conceivable that pax function is essential for maintaining their expression as soon as a beta-cell reaches a certain stage of differentiation. the almost complete loss of glucagon+ cells in pax6-deficient mice indicates a more essential role of pax in alpha-cells. in this context, it has been demonstrated that pax binds and activates the glucagon gene, however it remains unknown whether pax is also involved in the determination of the alpha-cell lineage. the continued expression of three alpha-cell related genes in pax6-deficient pancreata might be an indication for a predominant involvement of pax in later stages of alpha-cell differentiation. ghrelin+/somatostatin+ cells arising in nkx /arx compound mutants represent a non-delta-cell type our analysis shows that the combined loss of nkx and arx prevents the formation of mature alpha- and beta-cells, thus confirming the requirement of arx and nkx activities for the formation of these cell types. however, the presence of pax4+ cells indicates that an endocrine precursor state is attained without nkx and/or arx. this, and previous studies, suggest that nkx is essential for the maintenance of high levels of pdx and pax as well as the activation of nkx in developing beta-cells. consistent with these assumptions, the alterations found in the expression of pdx pax and nkx appear similar, comparing pancreata of nkx single- and nkx /arx compound-mutant mice. these data once more imply that arx is dispensable for the beta-cell fate and that the proper expression of these genes can be attributed to nkx alone. however, the additional loss of arx results in the formation of numerous ghrelin+/somatostatin+ cells beside populations of single-hormone positive somatostatin+ or ghrelin+ cells. we did not detect ghrelin expression in delta-cells arising in wild type or either single-knockout pancreata. together with the observation that neither nkx nor arx is expressed in wild type delta-cells, we conclude that such double positive cells do not represent delta-cells ectopically expressing ghrelin but rather a population comparable to the ghrelin+ cells arising in nkx -deficient mice. this is further supported by the fact that ghrelin+/somatostatin+ cells display the same broad expression of pdx1low as observed in nkx -deficient ghrelin+ cells. hence, we conclude that 80% of the ghrelin+ cells initiate somatostatin expression upon the additional loss of arx. not all ghrelin-labelled cells co-express somatostatin in double-mutant pancreata. this might be attributed to their distinct predeterminations and partially reflect the presence of normal epsilon-cells. nevertheless, our data also imply that arx or a not yet identified downstream mediator exerts a suppressive effect on somatostatin expression. in this context, previous studies demonstrated that arx activities strongly antagonize delta-cell formation. however, whether arx directly antagonizes somatostatin expression would require further investigations.  our data indicate that although proper endocrine differentiation/hormone expression is impaired in pax6- or nkx -deficient pancreata, committed cells retain some of the characteristics normally associated with mature beta-cells. our analysis of nkx /arx single- and double-mutant pancreata allow us, together with the data from others, to conclude that both pax and arx require the activity of nkx in order to maintain the transcriptional cascades initiated in early committed beta- and alpha-cell precursors, respectively. our findings also suggest that one of the coupled functions of nkx and pax is the control of arx gene activity in committed beta-cell precursors. 
nkx and arx represent key transcription factors implicated in the specification of islet cell subtypes during pancreas development. the combined loss of nkx and arx likewise results in the formation of a hyperplastic ghrelin+ cell population at the expense of mature alpha- and beta-cells. hence in order to precise the functional link between both factors we performed a comparative analysis of nkx /arx single- and double-mutants but also of pax6-deficient animals. in nkx mutant animals, alpha- and beta-cell development is severely impaired whereas a ghrelin-expressing cell population is found augmented. surprisingly, such nkx -/-arx- ghrelin+ cells also express the somatostatin hormone. we show that most of the ghrelin+ cells emerging in pancreata of nkx - and pax6-deficient mice, express the alpha-cell specifier arx, but also additional beta-cell related genes.
 nkx and arx represent key transcription factors implicated in the specification of islet cell subtypes during pancreas development. mice deficient for arx do not develop any alpha-cells whereas beta- and delta-cells are found in considerably higher numbers. in nkx mutant animals, alpha- and beta-cell development is severely impaired whereas a ghrelin-expressing cell population is found augmented. notably, arx transcription is clearly enhanced in nkx -deficient pancreata. hence in order to precise the functional link between both factors we performed a comparative analysis of nkx /arx single- and double-mutants but also of pax6-deficient animals.  we show that most of the ghrelin+ cells emerging in pancreata of nkx - and pax6-deficient mice, express the alpha-cell specifier arx, but also additional beta-cell related genes. in nkx -deficient mice, arx directly co-localizes with iapp, pc1/ and pdx suggesting an nkx -dependent control of arx in committed beta-cells. the combined loss of nkx and arx likewise results in the formation of a hyperplastic ghrelin+ cell population at the expense of mature alpha- and beta-cells. surprisingly, such nkx -/-arx- ghrelin+ cells also express the somatostatin hormone.  our data indicate that nkx acts by reinforcing the transcriptional networks initiated by pax and arx in early committed beta- and alpha-cell, respectively. our analysis also suggests that one of the coupled functions of nkx and pax is to counteract arx gene activity in early committed beta-cells. arxnkx somatostatinghrelinpax6pax4 