BACKGROUND
in vertebrates, the pancreas plays a fundamental role in nutritional homeostasis through the secretion of various enzymes and hormones by acinar and endocrine cells, respectively.
the endocrine cells form aggregates, called islets of langerhans, and are composed of five cell subtypes  each producing a distinct hormone .
all endocrine pancreatic cells differentiate during embryogenesis from progenitor cells specified in the dorsal and the ventral pancreatic buds, two protrusions that emerge from the endodermal embryonic gut  <cit> .
this differentiation process is controlled by a panoply of transcription factors  <cit> , such as pdx <dig> and ptf1a acting at early stages to specify the pancreatic progenitors  <cit> , then the hlh factors ngn <dig> and neurod inducing the endocrine precursors  <cit> , and the homeodomain factors nkx <dig> , nkx <dig> , pax <dig>  pax <dig> and arx controlling the formation of the five endocrine cell subtypes  <cit> .
in mouse embryos, pax <dig> gene expression first appears in endocrine precursors and then is detected transiently in numerous differentiating β-cells and occasional α-cells  <cit> .
pax <dig> expression seems to switch off upon terminal β-cell maturation  <cit> , although some studies have reported expression in adult β-cells  <cit> .
pax <dig> has at least two functions in the differentiation of murine pancreatic cells.
first, it favours the fate of the endocrine precursors toward the β- and δ-cell fate while repressing the α-cell lineage.
indeed, pax <dig> mutant mice display a lack of δ-cells, an almost complete loss of β-cells and an increase in α-cells  <cit> .
this first role is due, at least in part, to the repression by pax <dig> of the arx gene, which encodes for an aristaless homeodomain factor and is absolutely required for the differentiation of α-cells  <cit> .
inversely, arx is also able to repress pax <dig> gene expression and the arx mutant mice have no α-cells and an increase of β-and δ-cells.
so, the balance of α-cells versus β-/δ-cells in pancreatic islets is controlled in mouse by an antagonistic action of the two homeodomain factors arx and pax <dig>  while pax <dig> favours the δ- and β-cell fate, it has no role per se in δ-cell differentiation; indeed, the double arx-/-; pax4-/- mice have no α- and almost no β-cells but display a strong increase in δ-cells  <cit> .
furthermore, ectopic expression of pax <dig> in endocrine precursors or even in α-cells of transgenic mice is sufficient to force their conversion into β-cells, but not in δ-cells  <cit> .
these data demonstrate the essential role of pax <dig> in β-cell differentiation.
surprisingly, while birds and amphibians possess pancreatic β- and δ-cells, no pax <dig> gene has been reported in these organisms and examination of the chicken and xenopus tropicalis genomic sequences indicates a lack of pax <dig> ortholog in these two vertebrates.
a recent phylogenetic study strongly suggests that the pax <dig> gene is derived from a duplication pax6/eyeless gene which probably occurred at the so-called two-round  genome duplication in early vertebrates  <cit> .
this ancient pax <dig> gene could have been lost in birds and some amphibians.
in contrast, fish have the pax <dig> orthologous gene but its function in pancreatic cell differentiation is still unknown.
the lack of pax <dig> gene in chick and xenopus tropicalis is quite puzzling and raises the question about the pancreatic function of pax <dig> protein during early vertebrate evolution and notably in fish.
two hypotheses can be proposed: i) pax <dig> was important for β- and/or δ-cell differentiation in the first vertebrate organisms but the loss of pax <dig> gene in birds and amphibians has been compensated by another transcription factor or by others mechanisms, ii) the role of pax <dig> in β- and δ-cell differentiation appeared later in vertebrate evolution.
to tackle this question, we examined in the present study the expression and function of pax <dig> in zebrafish and investigated the regulatory links with the arx zebrafish orthologous gene.
we show that pax <dig> is dispensable in zebrafish for the differentiation of the β-cells deriving from the dorsal bud, but has a role in the reduction of α-cells.
this role is mediated by repression of arx gene whose essential function in α-cell formation appears to be maintained between zebrafish and mammals.
in addition, our data uncover a so far unappreciated autorepression of pax <dig> and arx genes.
we also analyzed the fate of endocrine cells after pax <dig> or arx knock-down.
in light of our data, we propose a model where the main role of pax <dig> during the initial phases of vertebrate evolution was to modulate the number of endocrine α-cells.
RESULTS
1) expression of zebrafish arx and pax <dig> genes during pancreatic development.
the zebrafish pax <dig> gene sequence was identified on chromosome  <dig> by using the tblast program and the corresponding pax <dig> cdna was amplified by rt-pcr and 3’-race.
sequence comparison of the deduced zebrafish pax <dig> protein with the human and mouse pax <dig> proteins shows that the homeodomain and the paired domain are well conserved while the c-terminal domain is highly divergent .
as the neighbouring genes located to the 5’ side from pax <dig> gene are the same in zebrafish, in mouse and in human , this indicates that the zebrafish pax <dig> gene is the actual ortholog of the mammalian pax <dig> genes .
the zebrafish proteins showing the closest similarity with the deduced pax <dig> zebrafish protein are pax6a and pax6b.
sequence comparison of vertebrates pax <dig> and pax <dig> proteins indicate that  <dig> amino acid positions are identical among the vertebrate pax <dig> proteins while distinct from the pax <dig> proteins .
all together, these data indicate that, while the sequence of pax <dig> genes were much less conserved during vertebrates evolution compared to the pax <dig> genes, these pax <dig> genes are actual pax <dig> orthologs.
the expression of zebrafish pax <dig> was analyzed by whole-mount in situ hybridization  at various stages of development.
as described for the murine pax <dig> gene, zebrafish pax <dig> transcripts were detected only in the pancreatic region, the first pax4+ cells appearing at the level of the dorsal pancreatic bud at around  <dig> hours post fertilization  .
pax <dig> pancreatic expression reaches its maximal level around 22- <dig> hpf, then progressively decreases and becomes hardly detectable after  <dig> hpf .
some pax4+ cells can occasionally be detected at larval stages  near the intra-pancreatic ducts .
while miura and collaborators have previously detected arx transcripts in the telencephalon, diencephalon, floor plate and somites of zebrafish embryos, they did not report arx expression in the pancreas  <cit> .
however, our wish using the arx probe revealed expression in the pancreatic dorsal bud from  <dig> hpf onward .
at  <dig> hpf, pancreatic arx staining displays the shape of a ring which is reminiscent of the disposition of glucagon-expressing α-cells .
the levels of arx gene expression decreases at later developmental stages but its expression can still be detected in pancreas of larvae and adult zebrafish .
another gene related to arx was found in the zebrafish genome; this novel arx-like gene  is expressed in the cns but not in the pancreas .
in order to identify the pancreatic cell types expressing arx and pax <dig>  fluorescent in situ hybridizations were performed with various pancreatic markers between  <dig> hpf and  <dig> hpf .
arx transcripts were detected in all glucagon+ cells and in some ghrelin+ cells, but not in insulin+ cells and not in somatostatin+ cells .
at  <dig> hpf, all arx+ cells correspond to a subset of pancreatic post-mitotic isl1+ endocrine cells .
thus, the expression profile of arx is quite similar in zebrafish and mouse, being mainly restricted to α-cells.
in contrast, the pancreatic expression of pax <dig> seems different in mouse and zebrafish.
indeed, while pax <dig> was detected in numerous differentiating β-cells in mouse embryos, we never observed co-localization of pax <dig> and insulin transcripts in zebrafish embryos and larvae .
most α-cells do not express pax <dig> , although some cells coexpressing glucagon and pax <dig> could occasionally be found.
in contrast, pax <dig> was detected in many somatostatin+ cells  and ghrelin+ cells  .
in order to determine whether pax <dig> expression is restricted to hormone expressing cells, we performed a double staining of pax <dig> with a cocktail of the four hormone probes .
at  <dig> hpf, about half of the pax4+ cells were not labelled by the hormones.
isl <dig> being expressed in all post-mitotic endocrine cells, we also analyzed the expression of pax <dig> with this transcription factor.
while many pax4+ cells were isl1+, some cells were found to be labelled only by the pax <dig> probe .
to determine whether pax <dig> is expressed in endocrine precursors, we next compared its expression with sox4b, a marker of these precursors in zebrafish  <cit> .
many sox4+ cells were found to express pax <dig> .
finally, pax <dig> was not detected in pancreatic nkx <dig> + progenitors which are located ventrally to the developing islet   <cit> .
taken together, these data indicate that pax <dig> expression is restricted to pancreatic endocrine precursors and transiently in some differentiating endocrine cells such as δ- and ε-cells .
we next investigated whether pax <dig> and arx can be expressed in the same pancreatic cells.
at  <dig> hpf, a substantial number of cells were found to contains both transcripts ; however, arx and pax <dig> expression progressively segregates into distinct cells afterwards and only  <dig> or  <dig> cells per islet positive for the two transcripts were observed at  <dig> hpf .
2) function of arx and pax <dig> in zebrafish pancreatic cell differentiation.
to determine the function of the zebrafish pax <dig> and arx genes in pancreatic cell differentiation, we injected antisense morpholinos designed to target exon-intron junctions in both pre-mrna and to alter the splicing of these transcripts.
to determine the efficiency of these knock-down, mrna was extracted from these injected zebrafish embryos  and from control embryos, and we analyzed the pax <dig> and arx transcripts by rt-pcr .
the morpholino mo1pax <dig> targeting the exon2-intron <dig> boundary was highly efficient as injection of  <dig> ng was sufficient to block the removal of intron <dig> in almost all pax <dig> transcripts, while mo2pax <dig> targeting exon1-intron <dig> junction was less efficient and partially inactivate the splicing at that site.
thus, mo1pax <dig> was used for the subsequent functional studies.
similarly, 2ng of moarx morpholino was sufficient to block the splicing of intron <dig> for almost all arx transcripts .
analysis of arx morphants revealed a complete loss of α-cells while the number of the other endocrine cell types was not significantly modified .
injection of an unrelated morpholino  or of the arx morpholino containing  <dig> mismatches in the exon2-intron <dig> junction site did not cause any changes in the number of pancreatic cells.
in contrast to arx knock-down, injection of mo1pax <dig> did not decrease the number of any pancreatic endocrine cell types ; instead, a small but highly reproducible increase in glucagon+ cells number was observed  as well as a very slight increase in ghrelin+ cells .
consistent with the increased number of α-cells, an increase in arx+ cells was also detected  .
altogether, these data indicate that arx is required for α-cell formation, as described in mouse; however, pax <dig> is not required for the generation of the first β-cells of zebrafish embryos but appears to modulate α-cell number.
3) arx and pax <dig> negatively autoregulate their own gene transcription.
to determine whether transcription of the arx and pax <dig> genes was affected after the inactivation of these two transcription factors, we next analyzed arx and pax <dig> transcript levels by wish in arx and pax <dig> morphants, respectively .
surprisingly, while pax <dig> mrna was mainly cytoplasmic in control embryos as expected, the altered pax <dig> transcripts were located in cell nuclei in mo1pax <dig> morphants as revealed by colocalization with the nuclear marker topro <dig> .
the intensity of fluorescence in nuclei was also higher in pax <dig> morphants and the number of pax4-labelled cells was significantly increased.
similar data were obtained using the second mo2pax <dig> morpholino, albeit with slightly less pronounced effects .
these data suggested that splicing alteration disrupted the transport of pax <dig> transcripts from the nucleus to the cytoplasm and that a negative autoregulatory loop exists in the regulation of zebrafish pax <dig> gene.
interestingly, a similar phenomenon was observed for the arx gene.
indeed, while the level of arx staining was low and cytoplasmic in control embryos, it became nuclear and very strong in arx morphants .
this up-regulation was observed in all tissues expressing arx gene such as the floor plate, brain, spinal cord and somites, but not in the pancreas.
indeed, while about  <dig> pancreatic cells are labelled by arx probe in control embryos, very low staining was observed in a maximum of  <dig> pancreatic cells in arx morphants.
to determine if the retention of arx transcripts in nuclei and the stronger staining in most tissues were caused by splicing alteration induced by the morpholino, we injected another morpholino  targeting the translation start site of arx mrna.
this morpholino did not cause a nuclear retention of arx transcripts, but it produced a strong increase in the level of arx transcripts in all tissues , with the exception of pancreas where arx+ cells could hardly be detected .
all together, these data indicate that i) splicing disruption is the cause of nuclear localization of transcripts and ii) a negative feed-back loop exists for arx in all tissues, but is not detected in pancreas.
to verify that the up-regulation of arx transcripts in arx morphants is actually due to an auto-regulatory effect occurring at the level of arx gene transcription, we next used a zebrafish transgenic line in which gfp expression was driven by an enhancer of the human arx gene.
performing a systematic enhancer-screening assay  <cit> , a sequence was identified that regulated gfp in the central nervous system identical to the endogenous zebrafish arx gene .
when the arx morpholinos were injected in one of the transgenic lines, a significant up-regulation of gfp was observed compared to control embryos .
this confirms the existence of an inhibitory action of arx protein on the expression of its own gene, this auto-regulation being either direct or indirect.
4) antagonistic actions of zebrafish arx and pax <dig> in pancreatic cell differentiation.
the increase of arx transcripts in the somites and the central nervous system of arx morphants contrasts with the clear down-regulation of arx transcripts in pancreatic cells.
this suggests the involvement of a pancreas-specific factor thwarting or impeding the negative feed-back loop.
as pax <dig> is described as a repressor of the arx gene in mouse and is selectively expressed in pancreas, this prompted us to verify whether the down-regulation of arx transcripts in pancreatic cells of arx morphants was due to pax <dig> action.
to that end, we compared the expression of arx transcripts in the arx morphants and in the double arx;pax <dig> morphants.
we found that indeed, that the levels of the pancreatic arx transcripts were much higher in the double morphants .
these data demonstrate that zebrafish pax <dig> represses arx gene expression in zebrafish and is the factor thwarting the arx negative feed-back loop in the pancreas.
to further analyse the antagonistic actions of arx and pax <dig>  we determined the pancreatic expression of these two genes by fluorescent wish in the single and double arx;pax <dig> morphants at  <dig> hpf .
in control embryos, arx and pax <dig> transcripts were mostly detected in distinct cells at this stage.
following pax <dig> knock-down, we detected an up-regulation of both arx and pax <dig> transcripts and the nuclei of many arx+ cells were also co-labeled by the altered pax <dig> transcripts .
in arx morphants, besides downregulation of arx gene transcription, increased number of pax4+ cells was noticed, with some of them expressing low amount of altered arx transcripts .
this co-expression was more obvious in the embryos injected with both arx and pax <dig> morpholinos, as the two altered transcripts were strongly detected in many nuclei and around 50% of arx+ or pax4+ cells were co-labeled by the two probes .
in conclusion, as arx and pax <dig> are expressed in distinct cells in control embryos while the two genes are co-transcribed in many cells in the double morphants, this confirms the mutual repression of these two factors on the each gene.
5) characterization of arx- and pax4-deficient cells in zebrafish morphants.
our observation of the altered arx and pax <dig> transcripts in the nucleus of pancreatic cells after their knock-down allows us to characterize these arx+ and pax4+ cells and thereby we determined whether their identity is modified after their respective knock-down.
as already mentioned above, in wild-type embryos at  <dig> hpf, pax <dig> is expressed in sox4b+ endocrine precursors, in some δ- and ε-cells and is excluded from α cells .
in the pax <dig> morphants, the expression profile of the altered pax <dig> transcripts remained essentially the same as in wild-type except that they were also detected in the nuclei of α-cells .
these data suggest that, in the absence of functional pax <dig> activity, the endocrine progenitors that should have expressed pax <dig> can give rise to α-cells as well as to δ- and ε-cells.
as arx gene transcription is strongly inhibited following arx knock-down, it was difficult to evaluate the identity of arx+ cells in arx morphants using the same strategy.
to circumvent this problem, we analyzed instead the identity of arx+ cells in the double arx;pax <dig> morphants where arx transcripts are easily detected.
like in the control embryos, arx transcripts were not detected in β- or δ-cells in the double morphants .
no glucagon expression could be detected confirming the efficiency of arx knock-down.
only a minority of arx+ cells expressed ghrelin, while the majority was not stained by any other of the hormone probes .
these data indicate that the fate of arx+ cells has not been switched to another cell type after arx;pax <dig> knock-down and that these arx+ cells are probably blocked in their differentiation process toward α cells.
discussion
previous studies in mouse have shown that the balance of α-cells versus β/δ-cells is controlled by the antagonistic action of arx and pax <dig> homeodomain factors, arx being required for α-cell identity and, inversely, pax <dig> favoring β- and δ-cell fate at the expense of α-cells   <cit> .
pax <dig> is also sufficient in mice to convert α-cells to β-cells  <cit> .
however, the lack of pax <dig> gene in birds and in some amphibians, which possess β-cells, raises the question of its original function in lower vertebrates such as fish.
in this study, we show that pax <dig> is not required for β- and δ-cell differentiation in zebrafish embryos but modulates the number of α-cells through repression of the arx gene .
zebrafish arx is required for α-cell differentiation and has a repressive action on pax <dig> gene expression, like in mouse.
thus, we can reasonably conclude that the mutual repression between pax <dig> and arx was established early in vertebrate evolution and that pax <dig> was not required for β-cell differentiation in the first vertebrate species such as fish; the crucial role of pax <dig> in β-cell differentiation could have appeared much later, possibly during mammals’ evolution.
comparison of the peptidic sequence of zebrafish pax <dig> with its human and murine orthologs reveals strong divergence beside the homeodomain and the paired domain.
in contrast, the sequence of the closely related pax <dig> proteins has been extremely conserved.
nevertheless, several observations indicate that the zebrafish pax <dig> protein is the actual ortholog of the mammalian pax4; indeed, i)  <dig> amino acid residues of the homeodomain and paired domain are common between the pax <dig> proteins of different species while divergent in pax <dig> proteins, ii) synteny is clearly identified between the vertebrate pax <dig> genes, iii) the zebrafish pax <dig> gene, like mouse pax <dig>  is specifically expressed in pancreatic endocrine precursors and not in other tissues, and iv) we observe an antagonism between the zebrafish pax <dig> and arx genes as reported in mouse.
surprisingly, we could not detect any expression of zebrafish pax <dig> in differentiating β-cells in the embryos as well as in the  <dig> dpf larvae .
as a second pax <dig> paralog could not be identified in the zebrafish genome and as the efficiency of our pax <dig> knock-down experiment was high, we can conclude that pax <dig> is clearly not essential for the generation of β-cells in zebrafish embryos.
in contrast to pax <dig>  the pancreatic expression and function of arx gene seems to have been well maintained throughout vertebrate evolution.
indeed, this gene is expressed in all α-cells and is required for their differentiation as in mouse.
as the analyses of arx and pax <dig> morphants were performed in this study at  <dig> hpf, the function of arx and pax <dig> genes was tested only for the pancreatic endocrine cells deriving from the dorsal pancreatic bud.
do the arx and pax <dig> factors have the same role in the second wave of endocrine cells appearing in the zebrafish larvae after  <dig> dpf and which derive from the ventral pancreatic bud?
while our unpublished data show the absence of glucagon in a minority of arx morphants and the presence of secondary insulin cells in some pax <dig> morphants at  <dig> dp, it is difficult to draw clear-cut conclusions as the knock-down efficiency decreases in the zebrafish larvae  <dig> days after morpholinos injection.
late function of arx and pax <dig> genes will require the generation and analysis of arx and pax <dig> mutants.
an interesting point raised by the present study is the observation that altering the splicing of a particular rna causes its retention in the nucleus.
retention of incorrectly spliced transcript has been previously described in yeast and seems to be part of a quality control mechanism set up in eukaryotes to prevent the synthesis of truncated and potentially deleterious proteins  <cit> .
the nuclear location of altered pax <dig> and arx transcripts allowed us to characterize the pax4- and arx-deficient cells in morphants.
using this strategy, we could highlight that the cells expressing the altered pax <dig> transcripts correspond to endocrine precursors, ε- and δ-cells like in control embryos, but also to α-cells in pax <dig> morphants.
pax <dig> transcripts could not be detected in β-cells either in control embryos or in pax <dig> morphants.
this is in sharp contrast with data obtained in mouse and reinforces our conclusion that, in zebrafish, pax <dig> is not involved in β-cell differentiation.
arx was expressed in all α-cells and in some ε-cells in control embryos.
analysis of the double arx/pax <dig> morphants revealed no change in the fate of the arx+ cells and rather indicated a blockage in their differentiation process towards α-cells.
such data are also contrasting with those reported in mouse where the pancreatic cells switch from α-cell toward β- or δ-cell fate in the arx ko mice  <cit> .
the reason for such a difference is unclear but a likely explanation could be again the lack of pax <dig> function in β/δ fate decision in zebrafish.
indeed, if pax <dig> was sufficient to specify β-cell fate in zebrafish like in mouse, the increased number of pax4-expressing cells in arx morphants should have resulted in additional β-cells.
the nuclear retention of unspliced transcript is an useful phenomenon allowing to i) to easily verify the efficiency of the knock-down induced by morpholinos in a single zebrafish morphant, and ii) to identify and characterize the cells that should normally have expressed the studied factor.
the blockage of mrna nuclear export seems to occur only if an entire intron remains within the mrna.
indeed, in other cases, morpholinos targeting exon-intron junctions can also lead to the use of neighbouring cryptic splicing sites or to the removal of whole exon, as previously shown for pax6b or bmp2a <cit> .
in these latter cases, we did not observe a nuclear retention of the altered transcripts .
our study also demonstrates a negative auto-regulation of arx and pax <dig> on their own gene expression.
indeed, the knock-down of pax <dig> leads to an increase in the number of pancreatic cells expressing high level of altered pax <dig> transcripts.
similarly, arx knock-down causes a striking increase in the level of arx transcripts in all tissues expressing this gene, except in the pancreas.
direct negative auto-regulation is a common feature found in many organisms allowing repressors to rapidly reach a steady-state in their expression level and preventing an excess of their expression.
however in the pancreas, the levels of arx transcripts were dramatically reduced following arx knock-down, indicating that the negative feed-back was thwarted or blocked by another regulatory circuit.
we demonstrated that this is due to the repressive action of pax <dig> whose expression level is up-regulated in arx morphants.
the feed-back loop of arx on its own gene was further verified on a transgene driving gfp expression under the control of arx regulatory sequence.
these data demonstrate that the regulation occurs at the gene transcription level; if this effect is direct or mediated by  factor regulated by arx protein, is still unknown.
it is noteworthy to mention that, while both pax <dig> and arx are negative regulators of each other and of their own gene, the transcription of these  <dig> genes is regulated in opposite ways after their respective knock-down: indeed, loss of pax <dig> activity leads to an up-regulation of pax <dig> transcripts while loss of arx activity causes a down-regulation of arx transcripts in pancreatic cells.
how to explain such opposite behaviors as the actions of both factors seems identical on each gene ?
one explanation could be the different timing of their onset.
indeed, as pax <dig> expression starts at  <dig> hpf long before arx which appears around  <dig> hpf, the knock-down of pax <dig> leads to an increase of pax <dig> transcription that cannot be thwarted by arx between  <dig> and  <dig> hpf.
in contrast, the knock-down of arx activity will directly enhance pax <dig> expression which will then represses arx gene expression.
interestingly, a negative autoregulation of human pax <dig> gene has been previously reported through in vitro experiments  <cit> .
thus, it is likely that the auto-regulation we describe here for zebrafish arx and pax <dig> genes exists in many vertebrates such as humans.
CONCLUSIONS
our study demonstrates that, on one hand, the role of arx in α-cell differentiation has been maintained from fish to mammals, but on the other hand, pax <dig> has no apparent function in the formation of β-cell in zebrafish embryos.
thus, we propose that pax <dig> acquired its essential role in β-cells differentiation quite late in vertebrates’ evolution.
this can explain why chicken or xenopus generate β-cells although they do not possess any pax <dig> gene.
transcription factors shown to be crucial for pancreatic cell differentiation in mammals generally display conserved pancreatic expression in lower vertebrates such as zebrafish, suggesting that the regulatory network controlling pancreatic cell differentiation is similar across vertebrate species.
the present study shows that there are nevertheless some differences in the action of particular transcription factors, like pax <dig>  another zebrafish transcription factor could play the same role as the mammalian pax <dig>  one possible candidate is the mnx1/hb <dig> homeodomain protein which is required for β-cell differentiation in zebrafish  <cit>  and has been recently shown to repress α-cell fate  <cit> , as described for pax <dig> in mouse.
further experiments will be required to test this hypothesis.

 in vertebrates, the pancreas plays a fundamental role in nutritional homeostasis through the secretion of various enzymes and hormones by acinar and endocrine cells, respectively. the endocrine cells form aggregates, called islets of langerhans, and are composed of five cell subtypes each producing a distinct hormone. all endocrine pancreatic cells differentiate during embryogenesis from progenitor cells specified in the dorsal and the ventral pancreatic buds, two protrusions that emerge from the endodermal embryonic gut. this differentiation process is controlled by a panoply of transcription factors, such as pdx and ptf1a acting at early stages to specify the pancreatic progenitors, then the hlh factors ngn and neurod inducing the endocrine precursors, and the homeodomain factors nkx, nkx, pax pax and arx controlling the formation of the five endocrine cell subtypes. in mouse embryos, pax gene expression first appears in endocrine precursors and then is detected transiently in numerous differentiating β-cells and occasional α-cells. pax expression seems to switch off upon terminal β-cell maturation, although some studies have reported expression in adult β-cells. pax has at least two functions in the differentiation of murine pancreatic cells. first, it favours the fate of the endocrine precursors toward the β- and δ-cell fate while repressing the α-cell lineage. indeed, pax mutant mice display a lack of δ-cells, an almost complete loss of β-cells and an increase in α-cells. this first role is due, at least in part, to the repression by pax of the arx gene, which encodes for an aristaless homeodomain factor and is absolutely required for the differentiation of α-cells. inversely, arx is also able to repress pax gene expression and the arx mutant mice have no α-cells and an increase of β-and δ-cells. so, the balance of α-cells versus β-/δ-cells in pancreatic islets is controlled in mouse by an antagonistic action of the two homeodomain factors arx and pax while pax favours the δ- and β-cell fate, it has no role per se in δ-cell differentiation; indeed, the double arx-/-; pax4-/- mice have no α- and almost no β-cells but display a strong increase in δ-cells. furthermore, ectopic expression of pax in endocrine precursors or even in α-cells of transgenic mice is sufficient to force their conversion into β-cells, but not in δ-cells. these data demonstrate the essential role of pax in β-cell differentiation. surprisingly, while birds and amphibians possess pancreatic β- and δ-cells, no pax gene has been reported in these organisms and examination of the chicken and xenopus tropicalis genomic sequences indicates a lack of pax ortholog in these two vertebrates. a recent phylogenetic study strongly suggests that the pax gene is derived from a duplication pax6/eyeless gene which probably occurred at the so-called two-round genome duplication in early vertebrates. this ancient pax gene could have been lost in birds and some amphibians. in contrast, fish have the pax orthologous gene but its function in pancreatic cell differentiation is still unknown. the lack of pax gene in chick and xenopus tropicalis is quite puzzling and raises the question about the pancreatic function of pax protein during early vertebrate evolution and notably in fish. two hypotheses can be proposed: i) pax was important for β- and/or δ-cell differentiation in the first vertebrate organisms but the loss of pax gene in birds and amphibians has been compensated by another transcription factor or by others mechanisms, ii) the role of pax in β- and δ-cell differentiation appeared later in vertebrate evolution. to tackle this question, we examined in the present study the expression and function of pax in zebrafish and investigated the regulatory links with the arx zebrafish orthologous gene. we show that pax is dispensable in zebrafish for the differentiation of the β-cells deriving from the dorsal bud, but has a role in the reduction of α-cells. this role is mediated by repression of arx gene whose essential function in α-cell formation appears to be maintained between zebrafish and mammals. in addition, our data uncover a so far unappreciated autorepression of pax and arx genes. we also analyzed the fate of endocrine cells after pax or arx knock-down. in light of our data, we propose a model where the main role of pax during the initial phases of vertebrate evolution was to modulate the number of endocrine α-cells.  1) expression of zebrafish arx and pax genes during pancreatic development. the zebrafish pax gene sequence was identified on chromosome by using the tblast program and the corresponding pax cdna was amplified by rt-pcr and 3’-race. sequence comparison of the deduced zebrafish pax protein with the human and mouse pax proteins shows that the homeodomain and the paired domain are well conserved while the c-terminal domain is highly divergent. as the neighbouring genes located to the 5’ side from pax gene are the same in zebrafish, in mouse and in human, this indicates that the zebrafish pax gene is the actual ortholog of the mammalian pax genes. the zebrafish proteins showing the closest similarity with the deduced pax zebrafish protein are pax6a and pax6b. sequence comparison of vertebrates pax and pax proteins indicate that amino acid positions are identical among the vertebrate pax proteins while distinct from the pax proteins. all together, these data indicate that, while the sequence of pax genes were much less conserved during vertebrates evolution compared to the pax genes, these pax genes are actual pax orthologs. the expression of zebrafish pax was analyzed by whole-mount in situ hybridization at various stages of development. as described for the murine pax gene, zebrafish pax transcripts were detected only in the pancreatic region, the first pax4+ cells appearing at the level of the dorsal pancreatic bud at around hours post fertilization. pax pancreatic expression reaches its maximal level around 22- hpf, then progressively decreases and becomes hardly detectable after hpf. some pax4+ cells can occasionally be detected at larval stages near the intra-pancreatic ducts. while miura and collaborators have previously detected arx transcripts in the telencephalon, diencephalon, floor plate and somites of zebrafish embryos, they did not report arx expression in the pancreas. however, our wish using the arx probe revealed expression in the pancreatic dorsal bud from hpf onward. at hpf, pancreatic arx staining displays the shape of a ring which is reminiscent of the disposition of glucagon-expressing α-cells. the levels of arx gene expression decreases at later developmental stages but its expression can still be detected in pancreas of larvae and adult zebrafish. another gene related to arx was found in the zebrafish genome; this novel arx-like gene is expressed in the cns but not in the pancreas. in order to identify the pancreatic cell types expressing arx and pax fluorescent in situ hybridizations were performed with various pancreatic markers between hpf and hpf. arx transcripts were detected in all glucagon+ cells and in some ghrelin+ cells, but not in insulin+ cells and not in somatostatin+ cells. at hpf, all arx+ cells correspond to a subset of pancreatic post-mitotic isl1+ endocrine cells. thus, the expression profile of arx is quite similar in zebrafish and mouse, being mainly restricted to α-cells. in contrast, the pancreatic expression of pax seems different in mouse and zebrafish. indeed, while pax was detected in numerous differentiating β-cells in mouse embryos, we never observed co-localization of pax and insulin transcripts in zebrafish embryos and larvae. most α-cells do not express pax, although some cells coexpressing glucagon and pax could occasionally be found. in contrast, pax was detected in many somatostatin+ cells and ghrelin+ cells. in order to determine whether pax expression is restricted to hormone expressing cells, we performed a double staining of pax with a cocktail of the four hormone probes. at hpf, about half of the pax4+ cells were not labelled by the hormones. isl being expressed in all post-mitotic endocrine cells, we also analyzed the expression of pax with this transcription factor. while many pax4+ cells were isl1+, some cells were found to be labelled only by the pax probe. to determine whether pax is expressed in endocrine precursors, we next compared its expression with sox4b, a marker of these precursors in zebrafish. many sox4+ cells were found to express pax. finally, pax was not detected in pancreatic nkx + progenitors which are located ventrally to the developing islet. taken together, these data indicate that pax expression is restricted to pancreatic endocrine precursors and transiently in some differentiating endocrine cells such as δ- and ε-cells. we next investigated whether pax and arx can be expressed in the same pancreatic cells. at hpf, a substantial number of cells were found to contains both transcripts; however, arx and pax expression progressively segregates into distinct cells afterwards and only or cells per islet positive for the two transcripts were observed at hpf. 2) function of arx and pax in zebrafish pancreatic cell differentiation. to determine the function of the zebrafish pax and arx genes in pancreatic cell differentiation, we injected antisense morpholinos designed to target exon-intron junctions in both pre-mrna and to alter the splicing of these transcripts. to determine the efficiency of these knock-down, mrna was extracted from these injected zebrafish embryos and from control embryos, and we analyzed the pax and arx transcripts by rt-pcr. the morpholino mo1pax targeting the exon2-intron boundary was highly efficient as injection of ng was sufficient to block the removal of intron in almost all pax transcripts, while mo2pax targeting exon1-intron junction was less efficient and partially inactivate the splicing at that site. thus, mo1pax was used for the subsequent functional studies. similarly, 2ng of moarx morpholino was sufficient to block the splicing of intron for almost all arx transcripts. analysis of arx morphants revealed a complete loss of α-cells while the number of the other endocrine cell types was not significantly modified. injection of an unrelated morpholino or of the arx morpholino containing mismatches in the exon2-intron junction site did not cause any changes in the number of pancreatic cells. in contrast to arx knock-down, injection of mo1pax did not decrease the number of any pancreatic endocrine cell types; instead, a small but highly reproducible increase in glucagon+ cells number was observed as well as a very slight increase in ghrelin+ cells. consistent with the increased number of α-cells, an increase in arx+ cells was also detected. altogether, these data indicate that arx is required for α-cell formation, as described in mouse; however, pax is not required for the generation of the first β-cells of zebrafish embryos but appears to modulate α-cell number. 3) arx and pax negatively autoregulate their own gene transcription. to determine whether transcription of the arx and pax genes was affected after the inactivation of these two transcription factors, we next analyzed arx and pax transcript levels by wish in arx and pax morphants, respectively. surprisingly, while pax mrna was mainly cytoplasmic in control embryos as expected, the altered pax transcripts were located in cell nuclei in mo1pax morphants as revealed by colocalization with the nuclear marker topro. the intensity of fluorescence in nuclei was also higher in pax morphants and the number of pax4-labelled cells was significantly increased. similar data were obtained using the second mo2pax morpholino, albeit with slightly less pronounced effects. these data suggested that splicing alteration disrupted the transport of pax transcripts from the nucleus to the cytoplasm and that a negative autoregulatory loop exists in the regulation of zebrafish pax gene. interestingly, a similar phenomenon was observed for the arx gene. indeed, while the level of arx staining was low and cytoplasmic in control embryos, it became nuclear and very strong in arx morphants. this up-regulation was observed in all tissues expressing arx gene such as the floor plate, brain, spinal cord and somites, but not in the pancreas. indeed, while about pancreatic cells are labelled by arx probe in control embryos, very low staining was observed in a maximum of pancreatic cells in arx morphants. to determine if the retention of arx transcripts in nuclei and the stronger staining in most tissues were caused by splicing alteration induced by the morpholino, we injected another morpholino targeting the translation start site of arx mrna. this morpholino did not cause a nuclear retention of arx transcripts, but it produced a strong increase in the level of arx transcripts in all tissues, with the exception of pancreas where arx+ cells could hardly be detected. all together, these data indicate that i) splicing disruption is the cause of nuclear localization of transcripts and ii) a negative feed-back loop exists for arx in all tissues, but is not detected in pancreas. to verify that the up-regulation of arx transcripts in arx morphants is actually due to an auto-regulatory effect occurring at the level of arx gene transcription, we next used a zebrafish transgenic line in which gfp expression was driven by an enhancer of the human arx gene. performing a systematic enhancer-screening assay, a sequence was identified that regulated gfp in the central nervous system identical to the endogenous zebrafish arx gene. when the arx morpholinos were injected in one of the transgenic lines, a significant up-regulation of gfp was observed compared to control embryos. this confirms the existence of an inhibitory action of arx protein on the expression of its own gene, this auto-regulation being either direct or indirect. 4) antagonistic actions of zebrafish arx and pax in pancreatic cell differentiation. the increase of arx transcripts in the somites and the central nervous system of arx morphants contrasts with the clear down-regulation of arx transcripts in pancreatic cells. this suggests the involvement of a pancreas-specific factor thwarting or impeding the negative feed-back loop. as pax is described as a repressor of the arx gene in mouse and is selectively expressed in pancreas, this prompted us to verify whether the down-regulation of arx transcripts in pancreatic cells of arx morphants was due to pax action. to that end, we compared the expression of arx transcripts in the arx morphants and in the double arx;pax morphants. we found that indeed, that the levels of the pancreatic arx transcripts were much higher in the double morphants. these data demonstrate that zebrafish pax represses arx gene expression in zebrafish and is the factor thwarting the arx negative feed-back loop in the pancreas. to further analyse the antagonistic actions of arx and pax we determined the pancreatic expression of these two genes by fluorescent wish in the single and double arx;pax morphants at hpf. in control embryos, arx and pax transcripts were mostly detected in distinct cells at this stage. following pax knock-down, we detected an up-regulation of both arx and pax transcripts and the nuclei of many arx+ cells were also co-labeled by the altered pax transcripts. in arx morphants, besides downregulation of arx gene transcription, increased number of pax4+ cells was noticed, with some of them expressing low amount of altered arx transcripts. this co-expression was more obvious in the embryos injected with both arx and pax morpholinos, as the two altered transcripts were strongly detected in many nuclei and around 50% of arx+ or pax4+ cells were co-labeled by the two probes. in conclusion, as arx and pax are expressed in distinct cells in control embryos while the two genes are co-transcribed in many cells in the double morphants, this confirms the mutual repression of these two factors on the each gene. 5) characterization of arx- and pax4-deficient cells in zebrafish morphants. our observation of the altered arx and pax transcripts in the nucleus of pancreatic cells after their knock-down allows us to characterize these arx+ and pax4+ cells and thereby we determined whether their identity is modified after their respective knock-down. as already mentioned above, in wild-type embryos at hpf, pax is expressed in sox4b+ endocrine precursors, in some δ- and ε-cells and is excluded from α cells. in the pax morphants, the expression profile of the altered pax transcripts remained essentially the same as in wild-type except that they were also detected in the nuclei of α-cells. these data suggest that, in the absence of functional pax activity, the endocrine progenitors that should have expressed pax can give rise to α-cells as well as to δ- and ε-cells. as arx gene transcription is strongly inhibited following arx knock-down, it was difficult to evaluate the identity of arx+ cells in arx morphants using the same strategy. to circumvent this problem, we analyzed instead the identity of arx+ cells in the double arx;pax morphants where arx transcripts are easily detected. like in the control embryos, arx transcripts were not detected in β- or δ-cells in the double morphants. no glucagon expression could be detected confirming the efficiency of arx knock-down. only a minority of arx+ cells expressed ghrelin, while the majority was not stained by any other of the hormone probes. these data indicate that the fate of arx+ cells has not been switched to another cell type after arx;pax knock-down and that these arx+ cells are probably blocked in their differentiation process toward α cells. discussion previous studies in mouse have shown that the balance of α-cells versus β/δ-cells is controlled by the antagonistic action of arx and pax homeodomain factors, arx being required for α-cell identity and, inversely, pax favoring β- and δ-cell fate at the expense of α-cells. pax is also sufficient in mice to convert α-cells to β-cells. however, the lack of pax gene in birds and in some amphibians, which possess β-cells, raises the question of its original function in lower vertebrates such as fish. in this study, we show that pax is not required for β- and δ-cell differentiation in zebrafish embryos but modulates the number of α-cells through repression of the arx gene. zebrafish arx is required for α-cell differentiation and has a repressive action on pax gene expression, like in mouse. thus, we can reasonably conclude that the mutual repression between pax and arx was established early in vertebrate evolution and that pax was not required for β-cell differentiation in the first vertebrate species such as fish; the crucial role of pax in β-cell differentiation could have appeared much later, possibly during mammals’ evolution. comparison of the peptidic sequence of zebrafish pax with its human and murine orthologs reveals strong divergence beside the homeodomain and the paired domain. in contrast, the sequence of the closely related pax proteins has been extremely conserved. nevertheless, several observations indicate that the zebrafish pax protein is the actual ortholog of the mammalian pax4; indeed, i) amino acid residues of the homeodomain and paired domain are common between the pax proteins of different species while divergent in pax proteins, ii) synteny is clearly identified between the vertebrate pax genes, iii) the zebrafish pax gene, like mouse pax is specifically expressed in pancreatic endocrine precursors and not in other tissues, and iv) we observe an antagonism between the zebrafish pax and arx genes as reported in mouse. surprisingly, we could not detect any expression of zebrafish pax in differentiating β-cells in the embryos as well as in the dpf larvae. as a second pax paralog could not be identified in the zebrafish genome and as the efficiency of our pax knock-down experiment was high, we can conclude that pax is clearly not essential for the generation of β-cells in zebrafish embryos. in contrast to pax the pancreatic expression and function of arx gene seems to have been well maintained throughout vertebrate evolution. indeed, this gene is expressed in all α-cells and is required for their differentiation as in mouse. as the analyses of arx and pax morphants were performed in this study at hpf, the function of arx and pax genes was tested only for the pancreatic endocrine cells deriving from the dorsal pancreatic bud. do the arx and pax factors have the same role in the second wave of endocrine cells appearing in the zebrafish larvae after dpf and which derive from the ventral pancreatic bud? while our unpublished data show the absence of glucagon in a minority of arx morphants and the presence of secondary insulin cells in some pax morphants at dp, it is difficult to draw clear-cut conclusions as the knock-down efficiency decreases in the zebrafish larvae days after morpholinos injection. late function of arx and pax genes will require the generation and analysis of arx and pax mutants. an interesting point raised by the present study is the observation that altering the splicing of a particular rna causes its retention in the nucleus. retention of incorrectly spliced transcript has been previously described in yeast and seems to be part of a quality control mechanism set up in eukaryotes to prevent the synthesis of truncated and potentially deleterious proteins. the nuclear location of altered pax and arx transcripts allowed us to characterize the pax4- and arx-deficient cells in morphants. using this strategy, we could highlight that the cells expressing the altered pax transcripts correspond to endocrine precursors, ε- and δ-cells like in control embryos, but also to α-cells in pax morphants. pax transcripts could not be detected in β-cells either in control embryos or in pax morphants. this is in sharp contrast with data obtained in mouse and reinforces our conclusion that, in zebrafish, pax is not involved in β-cell differentiation. arx was expressed in all α-cells and in some ε-cells in control embryos. analysis of the double arx/pax morphants revealed no change in the fate of the arx+ cells and rather indicated a blockage in their differentiation process towards α-cells. such data are also contrasting with those reported in mouse where the pancreatic cells switch from α-cell toward β- or δ-cell fate in the arx ko mice. the reason for such a difference is unclear but a likely explanation could be again the lack of pax function in β/δ fate decision in zebrafish. indeed, if pax was sufficient to specify β-cell fate in zebrafish like in mouse, the increased number of pax4-expressing cells in arx morphants should have resulted in additional β-cells. the nuclear retention of unspliced transcript is an useful phenomenon allowing to i) to easily verify the efficiency of the knock-down induced by morpholinos in a single zebrafish morphant, and ii) to identify and characterize the cells that should normally have expressed the studied factor. the blockage of mrna nuclear export seems to occur only if an entire intron remains within the mrna. indeed, in other cases, morpholinos targeting exon-intron junctions can also lead to the use of neighbouring cryptic splicing sites or to the removal of whole exon, as previously shown for pax6b or bmp2a. in these latter cases, we did not observe a nuclear retention of the altered transcripts. our study also demonstrates a negative auto-regulation of arx and pax on their own gene expression. indeed, the knock-down of pax leads to an increase in the number of pancreatic cells expressing high level of altered pax transcripts. similarly, arx knock-down causes a striking increase in the level of arx transcripts in all tissues expressing this gene, except in the pancreas. direct negative auto-regulation is a common feature found in many organisms allowing repressors to rapidly reach a steady-state in their expression level and preventing an excess of their expression. however in the pancreas, the levels of arx transcripts were dramatically reduced following arx knock-down, indicating that the negative feed-back was thwarted or blocked by another regulatory circuit. we demonstrated that this is due to the repressive action of pax whose expression level is up-regulated in arx morphants. the feed-back loop of arx on its own gene was further verified on a transgene driving gfp expression under the control of arx regulatory sequence. these data demonstrate that the regulation occurs at the gene transcription level; if this effect is direct or mediated by factor regulated by arx protein, is still unknown. it is noteworthy to mention that, while both pax and arx are negative regulators of each other and of their own gene, the transcription of these genes is regulated in opposite ways after their respective knock-down: indeed, loss of pax activity leads to an up-regulation of pax transcripts while loss of arx activity causes a down-regulation of arx transcripts in pancreatic cells. how to explain such opposite behaviors as the actions of both factors seems identical on each gene? one explanation could be the different timing of their onset. indeed, as pax expression starts at hpf long before arx which appears around hpf, the knock-down of pax leads to an increase of pax transcription that cannot be thwarted by arx between and hpf. in contrast, the knock-down of arx activity will directly enhance pax expression which will then represses arx gene expression. interestingly, a negative autoregulation of human pax gene has been previously reported through in vitro experiments. thus, it is likely that the auto-regulation we describe here for zebrafish arx and pax genes exists in many vertebrates such as humans.  our study demonstrates that, on one hand, the role of arx in α-cell differentiation has been maintained from fish to mammals, but on the other hand, pax has no apparent function in the formation of β-cell in zebrafish embryos. thus, we propose that pax acquired its essential role in β-cells differentiation quite late in vertebrates’ evolution. this can explain why chicken or xenopus generate β-cells although they do not possess any pax gene. transcription factors shown to be crucial for pancreatic cell differentiation in mammals generally display conserved pancreatic expression in lower vertebrates such as zebrafish, suggesting that the regulatory network controlling pancreatic cell differentiation is similar across vertebrate species. the present study shows that there are nevertheless some differences in the action of particular transcription factors, like pax another zebrafish transcription factor could play the same role as the mammalian pax one possible candidate is the mnx1/hb homeodomain protein which is required for β-cell differentiation in zebrafish and has been recently shown to repress α-cell fate, as described for pax in mouse. further experiments will be required to test this hypothesis. 
this transcription factor specifies β- and δ-cell fate at the expense of α-cell identity by repressing arx gene expression and ectopic expression of pax in α-cells is sufficient to convert them into β-cells. such analyses demonstrated that arx knock-down in zebrafish does not lead to a switch of cell fate, as reported in mouse, but rather blocks the cells in their differentiation process towards α-cells. genetic studies in mouse have demonstrated the crucial function of pax in pancreatic cell differentiation. in addition to the mutual repression between arx and pax these two transcription factors negatively regulate the transcription of their own gene. surprisingly, no pax orthologous gene can be found in chicken and xenopus tropicalis raising the question of the function of pax gene in lower vertebrates such as in fish. interestingly, disruption of pax rna splicing or of arx rna splicing by morpholinos targeting exon-intron junction sites caused a blockage of the altered transcripts in cell nuclei allowing an easy characterization of the arx- and pax4-deficient cells. pax gene is transiently expressed in the pancreas of zebrafish embryos and is mostly restricted to endocrine precursors as well as to some differentiating δ- and ε-cells but was not detected in differentiating β-cells. in zebrafish, pax is not required for the generation of the first β- and δ-cells deriving from the dorsal pancreatic bud, unlike its crucial role in the differentiation of these cell types in mouse. conversely, knock-down of arx caused to a complete loss of α-cells and a concomitant increase of pax expression but had no effect on the number of β- and δ-cells. these data suggests that the main original function of pax during vertebrate evolution was to modulate the number of pancreatic α-cells and its role in β-cells differentiation appeared later in vertebrate evolution. pax knock-down in zebrafish embryos caused a significant increase in α-cells number while having no apparent effect on β- and δ-cell differentiation. on the other hand, the mutual repression between arx and pax is observed in both mouse and zebrafish. in the present study, we have analyzed the expression and the function of the orthologous pax gene in zebrafish.
 genetic studies in mouse have demonstrated the crucial function of pax in pancreatic cell differentiation. this transcription factor specifies β- and δ-cell fate at the expense of α-cell identity by repressing arx gene expression and ectopic expression of pax in α-cells is sufficient to convert them into β-cells. surprisingly, no pax orthologous gene can be found in chicken and xenopus tropicalis raising the question of the function of pax gene in lower vertebrates such as in fish. in the present study, we have analyzed the expression and the function of the orthologous pax gene in zebrafish.  pax gene is transiently expressed in the pancreas of zebrafish embryos and is mostly restricted to endocrine precursors as well as to some differentiating δ- and ε-cells but was not detected in differentiating β-cells. pax knock-down in zebrafish embryos caused a significant increase in α-cells number while having no apparent effect on β- and δ-cell differentiation. this rise of α-cells is due to an up-regulation of the arx transcription factor. conversely, knock-down of arx caused to a complete loss of α-cells and a concomitant increase of pax expression but had no effect on the number of β- and δ-cells. in addition to the mutual repression between arx and pax these two transcription factors negatively regulate the transcription of their own gene. interestingly, disruption of pax rna splicing or of arx rna splicing by morpholinos targeting exon-intron junction sites caused a blockage of the altered transcripts in cell nuclei allowing an easy characterization of the arx- and pax4-deficient cells. such analyses demonstrated that arx knock-down in zebrafish does not lead to a switch of cell fate, as reported in mouse, but rather blocks the cells in their differentiation process towards α-cells.  in zebrafish, pax is not required for the generation of the first β- and δ-cells deriving from the dorsal pancreatic bud, unlike its crucial role in the differentiation of these cell types in mouse. on the other hand, the mutual repression between arx and pax is observed in both mouse and zebrafish. these data suggests that the main original function of pax during vertebrate evolution was to modulate the number of pancreatic α-cells and its role in β-cells differentiation appeared later in vertebrate evolution. insulinglucagonpancreaspax4arxmrna exportdevelopmentzebrafish 