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First published online 30 May 2007
doi: 10.1242/dev.002691
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Department of Developmental and Cell Biology, University of California at Irvine, 4203 McGaugh Hall, Irvine, CA 92697-2300, USA.
* Author for correspondence (e-mail: msander{at}uci.edu)
Accepted 28 April 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Nkx6.1, Nkx6.2, Pdx1, Ngn3, Pancreas, Islet, Endocrine, Insulin, Glucagon, Development, Mouse, Transgenic
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The in
vitro generation of fully functional beta-cells for transplantation, however,
will require further improvement of existing differentiation protocols. Such
refinement will require detailed knowledge of the molecular mechanisms that
underlie beta-cell differentiation. Lineage-tracing studies in mice have shown
that all pancreatic lineages, which include the exocrine and endocrine
compartment, arise from a common, proliferative progenitor cell population
that is marked by the transcription factor Pdx1
(Gu et al., 2002). Expression
of the transcription factor Ngn3 (also known as Neurog3 - Mouse Genome
Informatics) further restricts progenitors to five distinct endocrine cell
fates: alpha-, beta-, delta-, PP- or epsilon-cells, which produce the hormones
glucagon, insulin, somatostatin, pancreatic polypeptide and ghrelin,
respectively (Gu et al., 2002;
Heller et al., 2005;
Prado et al., 2004).
Initially, scattered endocrine cells differentiate throughout the organ, but
these cells aggregate into so-called islets of Langerhans at the end of
gestation (Slack, 1995).
In recent years, major progress has been made in our understanding of the
molecular pathways that control endocrine differentiation
(Jensen, 2004). Much of this
knowledge stems from genetic gain- and loss-of-function experiments in mice.
Such studies have shown that Ngn3 activity is essential for the
differentiation of all endocrine cells and, conversely, that Ngn3 is
sufficient to restrict Pdx1-positive (Pdx1+) progenitors to an
endocrine fate (Apelqvist et al.,
1999; Gradwohl et al.,
2000; Schwitzgebel et al.,
2000). Based on the observation that only select endocrine
lineages are affected in null mutant mice for Nkx2.2
(Nkx2-2), NeuroD (Neurod1), Pax4, Arx, Hb9
(also known as Hlxb9 - Mouse Genome Informatics) or Nkx6.1
(Nkx6-1), these transcription factors have been proposed to be
downstream effectors of Ngn3 (Collombat et
al., 2003; Harrison et al.,
1999; Li et al.,
1999; Naya et al.,
1997; Sander et al.,
2000b; Schwitzgebel,
2001; Sosa-Pineda et al.,
1997; Sussel et al.,
1998; Wilson et al.,
2003). Although the loss of Pax4, Arx and NeuroD
expression in Ngn3 mutant mice
(Collombat et al., 2003;
Gradwohl et al., 2000) indeed
suggests a function of these genes downstream of Ngn3 in endocrine
differentiation, the evidence for Nkx6.1 being downstream of Ngn3 is less
clear.
Deficiency for Nkx6.1 results in a specific abrogation of beta-cell
neogenesis during embryogenesis without affecting cell survival or the
development of any other cell type in the pancreas
(Sander et al., 2000b). In
Nkx6.1 mutant mice, a marked reduction in beta-cell numbers is first
apparent at embryonic day (E)14, the time-point at which the first mature
beta-cells differentiate. The specific defect in the beta-cell lineage and the
maintenance of Ngn3 expression in Nkx6.1-deficient mice have led to the
suggestion that Nkx6.1 selectively functions in beta-cell differentiation
genetically downstream of Ngn3
(Schwitzgebel, 2001;
Wilson et al., 2003). However,
recent analysis of compound mutants for Nkx6.1 and its paralog
Nkx6.2 have suggested that the function of Nkx6.1 is not restricted
to beta-cell differentiation (Henseleit et
al., 2005). Although the pancreas develops normally in
Nkx6.2 single-mutant mice, Nkx6.1/Nkx6.2 double-mutant
embryos display a severe reduction in alpha-cell number. This demonstrates
redundant functions for the two Nkx6 factors and suggests a more general
requirement for Nkx6.1 in endocrine differentiation than just in the beta-cell
pathway. The expression domains of Nkx6.1 and Nkx6.2 diverge significantly
during development (Henseleit et al.,
2005). Co-expression of the two Nkx6 factors is only observed in
the early Pdx1+ progenitor cells at E10.5. Thereafter, Nkx6.2
expression becomes downregulated in Pdx1+ progenitor cells, by
E15.5 is only detected in a small subset of glucagon+ and exocrine
cells, and it is no longer expressed during late gestation or in the adult
pancreas. By contrast, Nkx6.1 remains strongly expressed throughout the
Pdx1+ epithelium between E11 and E13, and is the only Nkx6 factor
expressed in committed endocrine progenitors or in newly differentiated or
adult beta-cells (Henseleit et al.,
2005; Sander et al.,
2000b). Therefore, questions arise as to whether beta-cell
differentiation requires sustained Nkx6 activity in Ngn3+
progenitors and whether the inability of Nkx6.2 to compensate for Nkx6.1 in
beta-cell differentiation results from differences in their spatiotemporal
expression or in their biochemical activities. To determine which progenitor
populations require Nkx6 activity for normal beta-cell formation, we used a
transgenic approach to restore Nkx6 expression in select progenitor pools in
Nkx6.1 mutants. Surprisingly, we found that expression of Nkx6.1 in
the Ngn3+ domain was not sufficient to rescue beta-cell
development, whereas the expression of either Nkx6.1 or Nkx6.2 in the
Pdx1+ domain resulted in a complete rescue of mature beta-cells.
Our findings suggest that beta-cell development requires Nkx6 activity in
Pdx1+ progenitors prior to the activation of Ngn3. This study shows
that the lack of redundancy between Nkx6.1 and Nkx6.2 in beta-cell development
is a result of their divergent expression patterns. Therefore, the Nkx6.1
expression domain rather than its biochemical activity determines the unique
requirement for Nkx6.1 in beta-cell specification.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). An
IRES-lacZ-polyA cassette was inserted 3' of the Nkx6.1
or Nkx6.2 coding sequence. The
Ngn3-Nkx6.1-IRES-eGFP transgene was generated by fusing the
rat Nkx6.1 coding sequence in frame to the start codon of
Ngn3 preceded by 6.7 kb of mouse Ngn3 regulatory sequence
(Jenny et al., 2002). An
IRES-NLS-GFP-polyA cassette was placed 3' of the
Nkx6.1 coding sequence. The transgenic constructs were linearized
prior to pronuclear injections into CB6F1 fertilized oocytes (performed by the
UC Irvine Transgenic Core Facility). Founders for the Pdx1-Nkx6.1,
Pdx1-Nkx6.2 and Ngn3-Nkx6.1 transgenic lines were identified by
PCR with lacZ or GFP internal primers, respectively.
Nkx6.1 mutant mice have been previously described and were
maintained on a CD1 background (Sander et
al., 2000b). Nkx6-transgene-carrying mice were crossed
with Nkx6.1+/- mice and
Nkx6.1+/-;transgene+ offspring were used in
timed matings with Nkx6.1+/- mice. Mid-day of the day on
which the vaginal plug was detected was considered as E0.5. In all
experiments, embryos of identical genetic background were compared and
littermates were used whenever possible. All genotyping was performed by
Southern blot analysis as described
(Sander et al., 2000b).
Immunohistochemistry and X-gal staining
Pancreata were fixed in 4% paraformaldehyde in phosphate-buffered saline
(PBS) for 4 hours and immunofluorescence was performed on 10 µm
cryopreserved sections. Immunohistochemical detection of proteins was
conducted as described previously (Sander
et al., 1997). The following primary antibodies were used in these
assays: mouse 
-glucagon diluted 1:5000 (Sigma); guinea pig
-insulin diluted 1:10,000 (Linco); rabbit -Nkx6.1 diluted
1:10,000 (Jensen et al.,
1996); rabbit -Pdx1 diluted 1:1000 (Chemicon); guinea pig
-Pdx1 diluted 1:10,000 (C. Wright, Vanderbilt University, Nashville,
TN); rabbit -ß-galactosidase diluted 1:500 (ICN, 55976); guinea
pig -Ngn3 diluted 1:1000 (Henseleit
et al., 2005); guinea pig -Nkx6.1 diluted 1:1000
(Henseleit et al., 2005);
rabbit -Glut2 (Slc2a2) diluted 1:200 (Alpha Diagnostics); rabbit
-MafA diluted 1:1500 (R. Stein, Vanderbilt University, Nashville, TN);
rabbit -Myt1 diluted 1:1000 (G. Gu, Vanderbilt University, Nashville,
TN); rabbit -Hb9 diluted 1:8000
(Harrison et al., 1999);
rabbit -Isl1 diluted 1:5000
(Tsuchida et al., 1994);
rabbit -IAPP diluted 1:2000 (Advanced Chemtech); rabbit -PC1/3
diluted 1:2000 (D. Steiner, University of Chicago, Chicago, IL); and rabbit
-GFP diluted 1:2000 (Molecular Probes, A-6455). Secondary -IgG
antibodies were as follows: Cy3-conjugated -guinea pig, -rabbit
and -mouse diluted 1:2000 (Jackson Laboratory); Alexa (488
nm)-conjugated -guinea pig and -rabbit diluted 1:2000 (Molecular
Probes); biotinylated -mouse diluted 1:200 (Vector Laboratories).
Images were collected on a Zeiss Axioplan2 microscope with a Zeiss AxioCam
driven by Zeiss AxioVision v. 3.1 software.
Using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a
substrate, whole-mount X-gal staining was performed on either whole embryos or
on isolated abdominal organs as described previously
(Mombaerts et al., 1996)
Quantitative morphometry and cell counting
Cell counting and/or morphometry was performed on every section through the
pancreas at E14.5 or E15.5 and on every tenth section at E18.5 from a minimum
of three embryos per genotype. Morphometry was conducted using Image-Pro Plus
v. 5.0.1 (Media Cybernetics).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
our findings suggest that, during beta-cell differentiation, progenitors
undergo a progression from
Pdx1high/Ngn3-/insulin- 
Pdx1high/Ngn3+/insulin-
Pdx1low/Ngn3+/insulin-
Pdx1high/Ngn3-/insulin+. Next, we characterized the expression of Nkx6.1 with respect to Pdx1 and Ngn3. Nkx6.1 and Pdx1 showed largely overlapping domains of expression during development. Both proteins were co-expressed in the undifferentiated pancreatic epithelium at E10.5 and E12.5 (Fig. 1D and data not shown) and in differentiated beta-cells at E18.5 (Fig. 1F). At E14.5, co-localization of Nkx6.1 and Pdx1 was observed in the majority of cells in the central part of the epithelium, whereas the peripherally located exocrine acinar cells expressed only Pdx1 (Fig. 1E). In contrast to Pdx1, high levels of Nkx6.1 expression were maintained in the majority of Ngn3+ endocrine progenitors until E14.5 (Fig. 1G-I). After E14.5, however, the expression domains of Ngn3 and Nkx6.1 diverged and only 26% of Ngn3+ cells also expressed Nkx6.1 at E16.5 (Fig. 2C). Because the restriction of Nkx6.1 to a subset of Ngn3+ cells coincides with the major wave of beta-cell differentiation, we hypothesized that Nkx6.1 activity may commit Ngn3+ endocrine progenitors to a beta-cell fate.
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). Because
the Ngn3-Nkx6.1 transgene did not target all Ngn3+ cells,
we selectively studied the fate of transgene-expressing cells by counting
their relative contribution to the insulin and glucagon lineages. Although
Ngn3 is normally not detected in hormone+ cells
(Apelqvist et al., 1999;
Gradwohl et al., 2000;
Schwitzgebel et al., 2000),
Ngn3-promoter-directed eGFP can be traced to insulin+ and
glucagon+ cells due to the protein stability of eGFP
(Li et al., 1998). To study
whether expression of Nkx6.1 directs Ngn3+ progenitors to the
beta-cell lineage, we compared the ratios of
eGFP+;insulin+ and eGFP+;glucagon+
cells to the total number of eGFP+ cells in Ngn3-Nkx6.1
and in Ngn3-IRES-eGFP control mice. We did not detect a
difference in the percentages of eGFP+;insulin+ and
eGFP+;glucagon+ cells or in the total area of
insulin+ cells between transgenic and control mice at E18.5 (data
not shown), and therefore conclude that Nkx6.1 is not sufficient to confer
beta-cell identity to Ngn3+ progenitors.
We next determined whether reconstitution of Nkx6.1 expression in
Ngn3+ progenitors is sufficient to rescue beta-cell development in
Nkx6.1-/- null mutants. The intercrossing of
Nkx6.1+/-;Ngn3-Nkx6.1 transgenic mice with
Nkx6.1 heterozygous-mutant mice produced
Nkx6.1-/-;Ngn3-Nkx6.1 mice, in which all Nkx6.1
protein is derived from the Ngn3-promoter-driven transgene. Because
Ngn3 expression is not affected in Nkx6.1-deficient embryos
(Henseleit et al., 2005;
Sander et al., 2000b), the
Ngn3-Nkx6.1 transgene should be similarly expressed in wild-type and
in Nkx6.1-/- mutant backgrounds. Indeed, 60% of
Ngn3+ cells co-expressed Nkx6.1 in
Nkx6.1-/-;Ngn3-Nkx6.1 embryos
(Fig. 3C), which is similar to
the fraction of Ngn3+ cells expressing GFP in Ngn3-Nkx6.1
mice in a wild-type background (Fig.
2B). Consistent with the absence or low abundance of Pdx1 in the
Ngn3+ domain in wild-type embryos
(Fig. 1C), the cells targeted
by the Ngn3-Nkx6.1 transgene were either Pdx1-negative or expressed
low levels of Pdx1 (Fig. 3G).
Notably, we observed that the Ngn3-Nkx6.1 transgene also directed
Nkx6.1 expression in a subset of beta-cells
(Fig. 3K). This was unexpected,
because Ngn3 expression is normally lost before insulin becomes detectable
(Apelqvist et al., 1999;
Gradwohl et al., 2000;
Schwitzgebel et al., 2000).
The ectopic expression in insulin+ cells could be explained by
differences in protein stability of Nkx6.1 and Ngn3, or alternatively by
ectopic expression from the Ngn3 promoter fragment.
Because our analysis demonstrated mainly correct expression of Nkx6.1 from the Ngn3-Nkx6.1 transgene in Nkx6.1-deficient mice, we next studied whether reconstitution of Nkx6.1 expression in Ngn3+ progenitors is sufficient to rescue beta-cell formation in Nkx6.1-/- embryos. Embryos from both Ngn3-Nkx6.1 lines were analyzed, but each failed to display an obvious rescue of beta-cell number at E14.5 and E18.5 (see Fig. S1A-C in the supplementary material; data not shown). This was corroborated further by quantitative morphometric area measurements for insulin comparing Nkx6.1-/-;Ngn3-Nkx6.1 embryos to Nkx6.1-/- embryos at E18.5 (see Fig. S1D in the supplementary material). Although it is formally possible that the Ngn3 promoter specifically fails to target Ngn3+ beta-cell progenitors, the most likely conclusion from these results is that development of the beta-cell lineage requires Nkx6.1 activity outside the Ngn3+ domain.
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) (Fig. 2F-I).
This was confirmed by direct co-staining for ß-galactosidase (ß-gal)
and Pdx1 (Fig. 2J-L), which
showed ß-gal expression in the vast majority of Pdx1+ cells.
The pancreas of Pdx1-Nkx6.1-IRES-lacZ embryos (hereafter
abbreviated to Pdx1-Nkx6.1) appeared morphologically and
histologically normal. Additional morphometric area measurements for insulin
and glucagon at E14.5 as well as measurements for pancreatic insulin and
glucagon content at birth also failed to show a difference between wild-type
and Pdx1-Nkx6.1 mice (data not shown). We conclude that
overexpression of Nkx6.1 in Pdx1+ cells does not perturb
pancreatic morphogenesis and cell differentiation.
To test whether expression of Nkx6.1 in the Pdx1+
domain is sufficient to restore beta-cell formation in
Nkx6.1-/--null mice, we crossed four Pdx1-Nkx6.1
transgenic lines that had confirmed transgene expression at E10.5 into the
Nkx6.1-/- background. Consistent with the lack of
significant Pdx1/Ngn3 co-expression at E14.5
(Fig. 1C), the
Pdx1-Nkx6.1 transgene resulted in a mosaic pattern of very weak or
absent Nkx6.1 expression in the Ngn3+ domain
(Fig. 3D). Notably, Nkx6.1
protein was absent from the Pdx1+ exocrine acini
(Fig. 3H, arrows), although
co-staining for ß-gal and Pdx1 demonstrated expression of the transgene
in the entire Pdx1+ domain (Fig.
2J-L). Because Pdx1 expression is relatively low in acinar cells,
this could reflect a difference in the sensitivity of the anti-ß-gal and
anti-Nkx6.1 antibodies. Consistent with the expression of Pdx1 in beta-cells
(Guz et al., 1995), the
Pdx1-Nkx6.1 transgene targeted Nkx6.1 to insulin+ cells
(Fig. 3L,
Fig. 8K). Thus, with the
exception of the early acinar cells, the transgene restored Nkx6.1 expression
in the entire Pdx1+ domain.
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). Together, our results demonstrate that beta-cell
development requires Nkx6.1 activity in the Pdx1+ domain, whereas
expression in Ngn3+ progenitors is insufficient to induce beta-cell
formation. Notably, because the mice died postnatally due to a defect in motor
neuron development (Sander et al.,
2000a), we could not conduct studies of beta-cell function in
Nkx6.1-/-;Pdx1-Nkx6.1 mice.
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). The
lack of compensation in beta-cell formation could be explained either by
differences in the biochemical properties of the two proteins or by their
distinct spatial and temporal expression domains in the developing pancreas.
To distinguish between these possibilities, we genetically compared the
ability of Nkx6.1 and Nkx6.2 to restore beta-cell formation in
Nkx6.1-deficient mice. Analogous to the approach described for
Pdx1-Nkx6.1 transgenic mice, we generated
Pdx1-Nkx6.2-IRES-lacZ (Pdx1-Nkx6.2) mice
(Fig. 5A) and identified four
mouse lines that expressed the lacZ reporter exclusively in the
Pdx1+ domain (Fig.
5B). The transgene was expressed in virtually all Pdx1+
cells at E14.5 (Fig. 5C-E).
Similar to the Pdx1-Nkx6.1 line, mosaic transgene expression in the
Pdx1+ domain was observed at E18.5
(Fig. 5F-H). We crossed the four Pdx1-Nkx6.2 mouse lines into the Nkx6.1-/--null mutant background and compared the ability of the Pdx1-Nkx6.2 and Pdx1-Nkx6.1 transgenes to rescue beta-cell formation. Similar to the Pdx1-Nkx6.1 transgene, expression of the Pdx1-Nkx6.2 transgene resulted in a significant rescue of insulin+ cells as early as E14.5 (Fig. 6C). At E18.5, Nkx6.1-/-;Pdx1-Nkx6.2 embryos displayed large islet clusters that were indistinguishable from Nkx6.1-/-;Pdx1-Nkx6.1 or wild-type embryos (Fig. 6F). As expected, the Pdx1-Nkx6.2 transgene correctly targeted ß-gal expression to the insulin+ cells (Fig. 8L). These results demonstrate that Nkx6.1 and Nkx6.2 have a similar ability to induce beta-cell formation and islet morphogenesis. Again mirroring our observation in Pdx1-Nkx6.1 rescues, morphometric quantification of five independent embryos from one transgenic founder revealed a cohort with a partially and a cohort with a fully restored insulin cell area in Nkx6.1-/-;Pdx1-Nkx6.2 embryos (Fig. 6G). Overall, we found that only one out of the four transgenic lines significantly rescued beta-cell development. In lines that failed to rescue, we detected little or no ß-gal expression at E18.5 (data not shown).
Nkx6.1 and Nkx6.2 both restore expression of Nkx6.1 targets and beta-cell maturation
To study whether the Pdx1-Nkx6 transgenes fully restore a normal
program of beta-cell differentiation, we analyzed rescued embryos for
expression of the endocrine differentiation factor Myt1 (myelin transcription
factor 1). Myt1 is a zinc-finger transcription factor that has recently been
shown to function as an obligatory cofactor of Ngn3 in endocrine
differentiation (Gu et al.,
2004). Because pancreatic Myt1 expression is reduced in
Nkx6.1 mutants (Henseleit et al.,
2005), we tested whether the Pdx1-Nkx6 transgenes restore
the expression of Myt1. Confirming the findings from our in situ
hybridization experiments (Henseleit et
al., 2005), immunofluorescence staining with an anti-Myt1 antibody
showed a marked reduction in the number of Myt1+ cells in
Nkx6.1 mutant embryos (Fig.
7A,B,E). In embryos, in which insulin expression was rescued by
the Pdx1-Nkx6.1 and Pdx1-Nkx6.2 transgenes, the number of
Myt1+ cells was also restored to almost wild-type values
(Fig. 7A,C-E). These findings
confirm that Myt1 is a target of Nkx6 factors, and demonstrate that Nkx6.1 and
Nkx6.2 have similar activity in restoring Myt1 expression in
Nkx6.1-deficient pancreata.
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Interestingly, although absent from beta-cells, `strings' of
Glut2+ cells were found in the vicinity of the developing islets in
Nkx6.1-/- mutants (Fig.
8B). These cells did not express endocrine hormones, the
islet-marker glucagon, chromogranin A, IAPP, PC1/3 or islet transcriptions
factors, such as Pdx1, Hb9, Isl1 or Pax6 (data not shown). In wild-type
embryos, similar `strings' of hormone-negative Glut2+ cells were
detected in the ductal network of the embryonic pancreas at E14
(Pang et al., 1994) (data not
shown), but were no longer present at birth
(Fig. 8A). Because beta-cells
have been suggested to develop from the Glut2+ ductal network
(Pang et al., 1994), it is
possible that the Glut2+ cells in Nkx6.1-/-
mutant pancreata represent arrested precursor cells.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Nkx6.1 and Nkx6.2 possess fully equivalent functions in pancreatic endocrine development
Interestingly, despite the inability of endogenous Nkx6.2 to compensate for
Nkx6.1 in beta-cell development, we found that Nkx6.2 restores beta-cell
formation and maturation in Nkx6.1 mutants when expressed under the
Pdx1 promoter. Moreover, Nkx6.2 was able to induce the expression of
Nkx6.1 downstream effectors and beta-cell-specific markers. This demonstrates
that both Nkx6 factors possess equivalent biochemical activities in endocrine
differentiation. Similarly, although only Nkx6.1 is required for motor neuron
development in vivo, Nkx6.1 and Nkx6.2 are both able to induce motor neurons
when misexpressed in the neural tube
(Vallstedt et al., 2001).
Hence, the functional differences of Nkx6.1 and Nkx6.2 seem to be largely
determined by the divergent expression pattern of the two genes during
embryogenesis. Whereas Nkx6.2 is strongly expressed in Pdx1+
pancreatic progenitors between E8.5 and E10.5, its expression rapidly declines
thereafter, and Nkx6.1 becomes the predominantly expressed Nkx6 factor between
E11.5 and E13 (Henseleit et al.,
2005; Pedersen et al.,
2005). It is therefore possible that the inability of Nkx6.2 to
compensate for Nkx6.1 in beta-cell development is a result of insufficient
Nkx6.2 levels in undifferentiated progenitors between E11.5 and E13. This
model fits remarkably well with the results of a recent study, which indicates
that specification of the different endocrine subtypes is controlled in a
temporal manner (Johansson et al.,
2007). Via rescue experiments with an inducible Ngn3
transgene in Ngn3 mutants, Johansson et al. have shown that induction
of the Ngn3 transgene between E8.5 and E10.5 specifically restores
alpha-cell development, whereas beta-cells are restored after E11.5
(Johansson et al., 2007).
Thus, the time-window of competence for beta-cell formation correlates with
the peak of Nkx6.1 activity and with the downregulation of Nkx6.2 in
Pdx1+ pancreatic progenitors. Our observation that Nkx6.1 and
Nkx6.2 are functionally equivalent has important implications for
understanding the role of Nkx6 factors in endocrine development. Together with
our previous finding that alpha-cell development is redundantly controlled by
Nkx6.1 and Nkx6.2 (Henseleit et al.,
2005), it lends further support to the idea that Nkx6 factors
specify the endocrine compartment as a whole, rather than determining the fate
choice towards a specific endocrine subtype. This implies that subtype
identity is conferred by additional factors that may function in concert with
Nkx6.1 or Nkx6.2.
Both with the Pdx1-Nkx6.1 and the Pdx1-Nkx6.2 transgenes, we observed incomplete rescue of beta-cell mass in a subset of embryos. This could be explained by the variant degrees of mosaicism in transgene expression that we observed or by differences in endogenous Nkx6.1 protein levels. An alternative possibility is that partial restoration of beta-cell mass in Nkx6.1 mutants could reflect spatial differences in the expression domains of endogenous Nkx6.1 and the Pdx1-promoter-driven Nkx6.1 transgene. Indeed, a key difference between the expression of Pdx1 and Nkx6.1 is that Nkx6.1 is expressed at high levels in Ngn3+ endocrine progenitors, whereas Pdx1 expression is markedly downregulated in these cells (see Fig. 1C and Fig. 3D). We consider it unlikely, however, that the lack of Nkx6.1 in Ngn3+ cells accounts for the incomplete rescue with the Pdx1-Nkx6.1 transgene, because we did not observe a consistent increase in beta-cell mass when an additional Ngn3-Nkx6.1 transgene was crossed into the Nkx6.1-/-;Pdx1-Nkx6.1 background (data not shown).
Mechanisms of beta-cell specification
Our finding that the Pdx1-Nkx6.1, but not the
Ngn3-Nkx6.1, transgene restored beta-cell number in Nkx6.1
mutants implies that beta-cell development requires Nkx6.1 expression
in Pdx1+ progenitors. Because Pdx1 displays a dynamic pattern of
expression during beta-cell development, with high levels in undifferentiated,
multipotential pancreatic progenitors, absence or low levels in
Ngn3+ endocrine progenitors and again high levels in differentiated
beta-cells (see Fig. 1)
(Jensen et al., 2000;
Maestro et al., 2003), this
raises the question of which cell population requires Nkx6.1 activity to
restore beta-cell formation in Nkx6.1 mutants? It could reflect a
requirement for Nkx6.1 activity either in early multipotential progenitors
before Ngn3 expression is initiated or in late beta-cell progenitors
that have reactivated Pdx1 expression. Several lines of evidence
support the notion that the earliest requirement for Nkx6.1 activity is at the
level of multipotential Pdx1+ progenitors prior to the activation
of Ngn3. First, if Nkx6.1 controlled the development of both alpha-
and beta-cells exclusively in late progenitors after the downregulation of
Ngn3 expression, one would expect co-expression of Nkx6.1 and Nkx6.2
in early glucagon+ cells. However, whereas Nkx6.1 and Nkx6.2 are
co-expressed in early Pdx1+ progenitors, Nkx6.1 expression is
absent from the glucagon+ domain
(Henseleit et al., 2005;
Sander et al., 2000b). Second,
if beta-cell specification required Nkx6.1 solely in late beta-cell
progenitors, we would expect to have seen some degree of beta-cell rescue with
the Ngn3-Nkx6.1 transgene, because this transgene was ectopically
expressed in beta-cells (see Fig.
3K) and therefore targeted at least a subpopulation of late
beta-cell progenitors. Therefore, our findings imply that the initiation of
beta-cell development requires Nkx6.1 activity in mitotically active,
multipotential Pdx1+ pancreatic progenitors. In this respect, the
mechanism by which Nkx6.1 controls beta-cell development appears to be
distinct from other transcription factors with solely lineage-restricted
functions in endocrine differentiation, such as Pax4 and Arx. Unlike Pax4 and
Arx, which are both expressed in a Ngn3-dependent manner
(Collombat et al., 2003;
Gradwohl et al., 2000),
expression of Nkx6.1 is maintained in Ngn3-deficient embryos (S.B.N.,
unpublished observation). Moreover, loss of Pax4 or Arx
expression is not associated with a reduction in overall endocrine mass, but
with an endocrine-fate switch from beta-cells to ghrelin+ cells in
Pax4 mutants and from alpha-cells to beta- and delta-cells in
Arx mutants (Collombat et al.,
2005; Collombat et al.,
2003; Prado et al.,
2004).
Importantly, a requirement for Nkx6.1 in beta-cell specification before the initiation of Ngn3 expression does not preclude an additional function for Nkx6.1 in beta-cell maturation. Such an additional role for Nkx6.1 in beta-cells is consistent with our observation that some of the rescued insulin+ clusters in Nkx6.1-/-;Pdx1-Nkx6.1 embryos failed to restore MafA expression (Fig. 8G). Because expression of the Pdx1-Nkx6.1 transgene in mature beta-cells is partially mosaic, this could reflect a role for Nkx6.1 in activating MafA expression in beta-cells. Moreover, it is possible that the Glut2+/hormone-negative cells that we observed adjacent to the endocrine clusters in Nkx6.1 mutant embryos represent arrested beta-cell precursors. To substantiate this hypothesis, it needs to be tested whether the Glut2+ cells in Nkx6.1 mutants arise from Ngn3+ progenitors.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2491/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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