1 Max-Planck Institute for Biophysical Chemistry, Department of Molecular Cell
Biology, Am Fassberg, 37077 Göttingen, Germany
2 Hagedorn Research Institute, Department of Developmental Biology, Niels
Steensensvej 6, DK-2820 Gentofte, Denmark
3 DIBIT, San Raffaele Scientific Institute, Via Olgenittina 58, I-20132 Milano,
Italy
4 Centre de Biologie du Développement, UMR-5547 CNRS-Université P.
Sabatier, 118 Route de Narbonne, F-31062 Toulouse, Cedex 04, France
* Author for correspondence (e-mail: amansou{at}gwdg.de)
Accepted 19 April 2005
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SUMMARY |
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Key words: Endocrine pancreas development, Arx, Pax4, Mouse, Hyperglycaemia, Fate specification
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Introduction |
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In recent years, major advances have been made towards a better
understanding of the molecular mechanisms controlling endocrine cell genesis.
One of the first determinants controlling the endocrine specification program
was found to be the activation of the bHLH factor neurogenin 3 (Ngn3; Neurog3
- Mouse Genome Informatics) in the mouse E9 pancreatic epithelium
(Apelqvist et al., 1999;
Gu et al., 2002
;
Jensen et al., 2000
;
Schwitzgebel et al., 2000
;
Sommer et al., 1996
). Notably,
Ngn3-deficient mice fail to develop any hormone-producing endocrine
cells (Gradwohl et al., 2000
),
whereas the misexpression of Ngn3 in Pdx1 (Ipf1 -
Mouse Genome Informatics) expression domains results in the differentiation of
most of the pancreas into endocrine cells
(Apelqvist et al., 1999
;
Schwitzgebel et al., 2000
).
Following Ngn3 activation, several downstream factors participate in endocrine
subtype specification. These include the homeodomain-containing proteins
Nkx2.2, Nkx6.1, Pax4 and Pdx1 (Ahlgren et
al., 1998
; Mansouri et al.,
1999
; Sander et al.,
2000
; Smith et al.,
1999
; Sosa-Pineda et al.,
1997
; Sussel et al.,
1998
). Once this fate is established, additional transcription
factors such as Isl1, Pax6 and Pdx1 act to maintain specified islet cells
(Ahlgren et al., 1997
;
Guz et al., 1995
;
Jonsson et al., 1994
;
Offield et al., 1996
;
Sander et al., 1997
;
St-Onge et al., 1997
).
Recently, the involvement of an additional homeobox-containing gene
localized on the X chromosome, Arx, was demonstrated in the
-cell specification process
(Collombat et al., 2003
). Mice
deficient for Arx lack mature
-cells, whereas the numbers of
ß- and
-cells are proportionally increased so that the total islet
cell content is unaltered. Such phenotypic changes are opposite to those
observed in Pax4-deficient mice
(Sosa-Pineda et al., 1997
),
and it was suggested that, during the early stages of endocrine development, a
mutual inhibition operates between Arx and Pax4 to allocate endocrine fate.
These findings suggest that, early during islet cell specification, endocrine
progenitors are confronted with the choice of becoming precursors of either
ß-/
-cells or
-cells, the alternative cell fate being
promoted by Pax4 and Arx, respectively. To gain further insights into the
genetic program controlling the genesis of the different endocrine subtypes,
we generated mice deficient for both Arx and Pax4 genes. We
found that these animals died perinatally, after having developed a severe
hyperglycaemia. Immunohistochemical analysis of Arx/Pax4 mutant
pancreas revealed an absence both of
- and ß-cells. Strikingly, a
dramatic increase in the number of somatostatin-producing cells was observed,
whereas the total number of endocrine cells remained unchanged. Further
studies during embryogenesis suggested that the lack of both Arx and
Pax4 provokes an early-onset virtually exclusive generation of
somatostatin-expressing cells at the expense of the
- and ß-cell
lineages. Equally striking was the observation that, in Arx/Pax4
mutants, production of PP occurs in somatostatin-expressing cells only
following feeding onset, unravelling an epigenetic control. We provide
evidence that Arx and Pax4 inhibit transcription of one another by direct
interaction with their respective promoter regions in order to achieve proper
endocrine cell allocation. Finally, our study suggests an unrecognized
essential role for Pax4 in ß-cell fate specification.
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Materials and methods |
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Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde overnight at 4°C, embedded in
paraffin wax and 6-µm sections were applied to slides. These sections were
assayed as described previously (Collombat
et al., 2003). The primary antibodies used were: mouse monoclonal
anti-insulin, anti-glucagon (1/1000, Sigma), anti-somatostatin (1/100,
Promega), anti-Ghrelin (1/1000, kindly provided by C. Tomasetto), anti-CA812
(undiluted), anti-Ngn3 (1/500); guinea pig anti-insulin, anti-glucagon
(1/1000, Sigma), rabbit anti-somatostatin (1/600, Dako), anti-PP (1/200,
Dako), anti-Nkx6.1 (1/3000), anti-Nkx2.2 (1/1000, kindly
provided by T. Jessell), anti-Pax6 (1/500, kindly provided by S.
Saule), anti-Arx (1/1000), and anti-CART (1/1000). The secondary antibodies
(1/1000, Molecular Probes) used for immunofluorescence were: 594-Alexa
anti-mouse, 488-Alexa anti-mouse, 594-Alexa anti-rabbit, 488-Alexa
anti-rabbit, 594-Alexa anti-guinea pig; and 488-Alexa anti-guinea pig.
Pictures were processed using confocal microscopy.
Glucose levels
Glucose levels (mg/dl) were determined with the One Touch Glucose
monitoring kit (Johnson & Johnson) using 15 µl of peripheral blood.
Blood glucose levels are represented as an average±s.e.m.
ß-galactosidase staining
Whole-embryos were isolated at E10.5 and the yolk sac saved for DNA
preparation and genotyping. After fixing in 4% paraformaldehyde, embryos were
washed in PBS and stained in 4 mM K3[Fe(CN)6], 4 mM
K4[Fe(CN)6], 0.02% NP-40, 0.01% Na-deoxycholate, 5 mM EGTA, 2 mM
MgCl2 and 0.4 mg/ml
5-bromo-4-chloro-3-inodolyl-D-galactopyranoside.
Sequence processing
Sequence comparisons were performed online using the Vista program
(http://genome.lbl.gov/vista/index.shtml).
The search for P4BS was performed using the consensus sequence from
Fujitani et al. (Fujitani et al.,
1999) with a program of our own conception (available upon
request). Twenty-seven potential candidate sites were thereby obtained.
Plasmid construction
The full-length cDNA clones for the mouse Arx and Pax4
genes were subcloned into the pBluescript KSII vector (Stratagen) for in vitro
translation, and, in the case of Pax4, into a modified pCDNA vector
(Invitrogen) containing an intron and a HA epitope (kindly provided by R.
Lührman). The Pax4- or Arx-responsive luciferase (Luc) reporter
constructs were created by cloning five copies of P4BS or
ArBS, respectively, into the XhoI site present in the
T81-Luc vector (kindly provided by S. Nordeen).
Cell culture and transfection
The ß-cell-derived ßTC 13 T cells, COS cells, and the
-cell-derived
TC 1.9 cells were grown in DMEM medium
supplemented with 10% heat-inactivated foetal calf serum (FCS), penicillin and
streptomycin. Twenty-four hours before transfection experiments, the cells
were replated in 100-mm-diameter plates (approximately 3x106
cells/plate). Transfection experiments were performed using the Fugene 6
transfection reagent (Roche), according to the manufacturer's
instructions.
Electrophoretic mobility shift assay
Complementary single-stranded oligonucleotides (IBA-Göttingen) were
incubated in a medium containing 10 mM Tris-HCl, 5 mM MgCl2 and 100
mM NaCl, and then denatured at 80°C for 5 minutes in a waterbath.
Annealing was performed after switching off the waterbath and leaving the
mixture cool down overnight. The resulting double-stranded oligonucleotides
were end-labelled with the T4 polynucleotide kinase and
[-32P]ATP. The Pax4 and Arx proteins, generated by in vitro
translation using the TNT reticulocyte lysate transcription/translation kit
(Promega) according to the manufacturer's instructions, were incubated with
the labelled probe and processed as described in Fujitani et al.
(Fujitani et al., 1999
). For
competition studies, a 10-, 100-, or 200-fold molar excess of unlabeled
oligonucleotide competitor was added together with the probe.
South-western blot
The in vitro-translated Arx protein was manually spotted onto nylon
membranes that were incubated in 5% non-fat dry milk in 10 mM Hepes (pH 8.0)
for 1 hour at room temperature. The membranes then were incubated overnight in
binding buffer [10 mM Hepes (pH 8.0), 50 mM NaCl, 10 mM MgCl2, 0.1
mM EDTA, 1 mM dithiothreitol, 0.25% non-fat dry milk] containing
1x105 cpm of 32P-labeled DNA per ml. After
extensive washes in binding buffer containing 0.3 M NaCl, the membranes were
exposed to X-ray film.
Chromatin immunoprecipitation (ChIP) assay
Embryonic tissues or cells transfected with a vector encoding HA-tagged
Pax4 were sonificated and treated as described
(Spieler et al., 2004). The
following primers were used:
5'-TGAGCACCACGTTTTAAAATATACACATAGTC-3' with
5'-GTAGTGGGTACTGACCTAGACAGACATGTCATC-3' for the amplification of
the P4BS-containing 200-bp fragment; and
5'-GACAAAGTCTCACTAGGTAACGCAGCCCAGCC-3' with
5'-TAAAGATATATCATAGGGGCGGGGCTGGTCGA-3' as negative control (2.2 kb
downstream of the 9.7 kb NheI-EcoRV fragment
(Fig. 5A). The
5'-AGTCTGGCGCGTTGGGCAGAGGGCTGAGTGACTGA-3' and
5'-ATAGACAGCTGCCTGAGGGTGTACGTAGGGGTGTT-3' were used for the
amplification of the ArBS-containing 256-bp fragment and
5'-TTGAACTTGGTATAACAATAATCCTTAGGTAGCGA-3' with
5'-ATTTACGTGATTGTTTTGGAATTGCACACGTTAGG-3' was used as a negative
control (3.5 kb upstream of the Pax4 enhancer, see
Fig. 5F).
Reporter assay
The luciferase constructions (2 µg each) were co-transfected together
with over-expression vectors into appropriate cells. A co-transfected
lacZ reporter (1 µg) was used to normalize transfection
efficiency.
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Results |
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Arx/Pax4 double-mutant mice appeared to be indistinguishable from their littermates at birth. However, phenotypic differences arose within the first day postpartum: despite normal feeding, as evidenced by the presence of milk in the stomach; double-deficient animals rapidly developed growth retardation and died around postnatal day 2 (P2). To determine whether the observed lethality was related to an endocrine pancreatic dysfunction, blood glucose levels were measured for all the different genotypes of the offspring of Arx+/- Pax4+/-::Pax4+/- crosses. Twenty-four hours after birth, blood glucose levels were normal in all littermates (Table 1). Differences first became apparent at P2 and were amplified shortly before death: Arx- and Pax4-single-deficient mice displayed severe and lethal hypo- and hyperglycaemia, respectively (Table 1). Strikingly, unlike their Arx single mutant counterparts, Arx- Pax4+/- mice did not die at P2, but survived until P8-P12, with an initially mild hypoglycaemia that progressively became more severe (Table 1 and data not shown). Importantly, the animals lacking both Arx and Pax4 genes died around P1-P2 exhibiting an acute hyperglycaemia that contrasted with glucose levels of age-matched wild-type and single or double heterozygous Arx/Pax4 animals.
|
|
As development proceeds, a peak of endocrine cell genesis occurs, at about
E14.5, leading to the formation of cells contributing to the definitive islet
of Langerhans. When quantitatively assayed at E15, Arx- and
Pax4-deficient pancreata were found to be already entirely lacking
glucagon-expressing cells (Fig.
3C,D), whereas only ten percent of the normal number of
insulin-labelled cells were present (Fig.
3E-J). The number of cells positive for the pro-endocrine marker
Ngn3 was unchanged (Fig.
3C-F). At E18, a massive increase of the somatostatin-marked cell
population (+720%) was clearly apparent
(Fig. 3G,H,K,L), although these
cells arose at the proper developmental stages (data not shown). Strikingly,
the number of PP-cells was found to be normal at this stage
(Fig. 3I-L). The results of
co-immunodetection experiments showed that somatostatin-producing cells did
not express the PP hormone, and vice versa
(Fig. 3K-L). Taken together,
these data provide evidence that Pax4 and Arx are not
required for the specification and differentiation of any of the islet
endocrine cell types at early developmental stages. However, from E15 onwards,
in the absence of both genes, there is an early-onset loss of mature -
and ß-cells, and a proportionate increase of the somatostatin-producing
cell population, while the number of PP-cells remains unchanged.
|
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To validate these data, expression constructs were generated for
Arx and Pax4. Each construct was introduced, together with a
luciferase reporter gene, into the Arx+ - or
Pax4+ ß-derived cell lines (TC1.9- and HC 13 T-cells,
respectively), the luciferase reporter constructs containing five copies of
the respective binding sites characterized above. Our results demonstrate that
both Pax4 and Arx efficiently repress basal reporter activity, 2.7-fold and
3.8-fold, respectively (Fig.
5E,I). Taken together, these results suggest that Arx directly
interacts with the Pax4 enhancer domain thereby antagonizing
Pax4 transcription and, as a consequence, promoting the
-cell
fate. Similarly, Pax4 appears to act early during endocrine cell genesis to
favour the ß-/
-cell fate, at the expense of the
-cell
destiny, through a direct inhibition of Arx transcription.
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Discussion |
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Arx and Pax4 are required for the proper differential genesis of endocrine cells
Endocrine pancreas morphogenesis is associated with the early emergence of
cells that often co-express glucagon and insulin. Despite an early expression
of both Pax4 and Arx genes in the pancreatic primordium
(Collombat et al., 2003;
Sosa-Pineda et al., 1997
), it
has been shown that neither gene is necessary for the formation of these early
cells. The phenotypic defects observed in Arx/Pax4 double-knockout
mice corroborate these findings, and rule out the possibility of redundant
activity between Arx and Pax4 in this respect. Specifically, we demonstrate
that, during endocrine development, two distinct populations of insulin- and
glucagon-producing cells arise successively; the early population is
unaffected by the deficiency of Arx and/or Pax4, whereas the
latter one corresponds to mature ß- and
-cells whose correct
development depends on the concerted activities of both factors. Accordingly,
in Arx/Pax4 double mutants, an early-onset loss of mature
-
and ß-cells is observed, concomitantly with an increase in
somatostatin-expressing cell numbers. Along the same line, all the
developmental markers associated with ß- and
-cell lineages,
including Nkx6.1, Glut2, Pdx1, Nkx2.2 and ghrelin, are missing or are found
dramatically reduced. Interestingly, the supernumerary somatostatin-producing
cells do not ectopically express any of these genes but are positive for the
-cell specific markers CART and CA812. These data suggest that these
cells do not share any characteristics with normal
- or ß-cells,
but rather express a
-cell-specific complement of transcription
factors. However, further analysis and lineage tracing experiments would be
required to fully characterize the identity of such somatostatin-expressing
cells, and to prove whether they correspond to
-cells or not.
|
Another interesting finding was that new-born
Arx-/Pax4+/- animals survived until
P8-P12, with a mild hypoglycaemia that became more severe with time. This
result was unexpected because Arx mutant mice die at P2 and
Pax4 heterozygous mice do not exhibit any obvious endocrine
alteration. However, the significant increase in -cell number and the
proportional reduction in ß-cell content observed in
Arx-/Pax4+/- animals when compared
with Arx mutants reveals a dose-dependent requirement of Pax4 for
ß-cell fate specification at the expense of
-cell destiny. In
addition, the extended life expectancy suggests that the decrease in
insulin-expressing cells, and/or the increase in somatostatin-producing cells,
might attenuate the hypoglycaemia observed in Arx mutants, possibly
due to an increased secretion of somatostatin, a known inhibitor of insulin
secretion (Strowski et al.,
2000
). It is also important to notice that the loss of a single
Arx allele does not affect the content in the different endocrine
cell subtypes, as compared to wild-type animals, suggesting that Arx
might escape the X-inactivation processes. However, further work would be
required to validate these statements.
Our present data, together with results obtained previously
(Collombat et al., 2003), lead
us to conclude that Arx is required for the acquisition of
-cell fate,
whereas Pax4 is necessary for ß-cell destiny; the simultaneous loss of
these factors results in an alternative outcome in which cells presenting most
of the characteristics of
-cells develop. Finally, a Pax4
haploinsufficiency phenotype can be recognized in an Arx-deficient
background.
Arx and Pax4 interact through direct mutual transcriptional inhibition
The previous findings that both Arx and Pax4 transcription factors are
initially co-expressed and that one predominates to promote a particular islet
subtype fate (Collombat et al.,
2003) prompted us to investigate the detailed mechanisms involved
in this phenomenon. Thus, through a study combining transgenic, EMSA, ChIP and
reporter assay approaches, we have characterized Arx and Pax4 binding sites
and have provided evidence that Arx inhibits Pax4 transcription by
interacting with the Pax4 enhancer domain, whereas Pax4 antagonizes
Arx transcription by binding to a 3' Arx enhancer
region. The finding that both Arx and Pax4 can behave as transcriptional
repressors is supported by in vitro studies or analyses performed in C.
elegans (Fujitani et al.,
1999
; Seufert et al.,
2004
; Smith et al.,
1999
). However, the detailed mechanisms regulating the prevalence
of one factor over the other remain to be elucidated.
Together with previous data obtained from Arx and Pax4
single mutant phenotypes, our results support the model depicted in
Fig. 6A. During early pancreas
morphogenesis, endocrine precursor cells express both Arx and Pax4. As each
protein directly inhibits transcription of the gene of the other, the initial
co-expression may reflect a production of inactive precursors. Next, the
selective activation of Arx or Pax4 will promote an -cell specification
or a ß-/
-cell fate, respectively
(Fig. 6A, 1). The mechanisms
involved are unclear but it is likely that an as yet undiscovered molecule
selectively induces, in a concentration-dependent manner, the expression of
Arx or Pax4. The factor thus induced will directly inhibit
the other at the transcriptional level, thereby further reinforcing its
dominance: if it is Arx, Pax4 transcription is repressed and the
-cell fate is favoured, whereas Pax4 activation induces a
ß-/
-cell fate through the inhibition of Arx expression
(Fig. 6A, 1). All our data
point to an early requirement for Arx in
-cell genesis, as, in
Arx and Arx/Pax4 mutant pancreas, the ß-
and/or
-cell lineages are promoted (Fig.
6B and
6D, respectively). Importantly,
in contrast to what was previously assumed
(Collombat et al., 2003
;
Sosa-Pineda et al., 1997
),
Pax4 does not appear to be necessary for
-cell genesis; rather, it
seems that it acts only early during pancreatic islet specification to inhibit
Arx, thereby specifying a ß-/
-cell fate. Indeed, the
depletion in Pax4 results in a failure of ß-/
-cell specification,
as Arx activity promotes an
-cell fate
(Fig. 6C). However, in
Arx/Pax4 double mutants, single-hormone
somatostatin-producing
-cells develop normally concomitantly with an
excess of cells presenting most of the characteristics of
-cells
(Fig. 6D). From these data, it
seems that, in addition to its repressive action on Arx, Pax4 induces
a ß-cell fate at the expense of the
-cell lineage. The loss of a
single Pax4 allele in Arx-deficient pancreas, resulting in a
significant decrease in the number of ß-cells and a simultaneous increase
in the
-cell content, is in agreement with this notion. It seems likely
therefore that a third player ('factor X') is required to specify the
-cell fate at the expense of ß-cell formation
(Fig. 6A, 2,3). At later
developmental stages, an additional transcription factor, Pax6, is believed to
act in the terminal differentiation of the endocrine cell subtypes
(Ashery-Padan et al., 2004
).
Despite these advances towards an understanding of the differential genesis of
endocrine cells, numerous questions remain. (1) Which factors determine
whether Arx or Pax4 will prevail during the early steps of islet cell
specification? (2) What is the identity of `factor X'? (3) What mechanisms
underlie the transition from endocrine progenitors to ß-/
-cell
precursors? The study of the proteins interacting with Arx and Pax4 should
provide a greater insight into the processes leading to the selection of a
particular factor at the expense of the other (question 1). Likewise, a
scrutiny of the transcriptome of the mutant analyzed in this study should
enable the
-cell genetic determinants to be characterized (question 2),
thus shedding light on the mechanisms and molecules implicated in the
selection of a particular endocrine cell fate (question 3).
In summary, our analysis establishes the requirement of Arx and Pax4 at
multiple stages of -, ß- and
-cell specification. Our
findings uncover an essential role for Pax4 in ß-cell specification at
the expense of the
-cell lineage, and are consistent with a model in
which fate allocation occurs through repressive interactions between
transcription factors.
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ACKNOWLEDGMENTS |
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