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First published online 3 January 2007
doi: 10.1242/dev.02763
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-cells and a small number of ß-cells in the pancreatic islet
1 Program in Molecular Biology, University of Colorado at Denver Health Sciences
Center, Aurora, CO 80045, USA.
2 Department of Biochemistry and Molecular Genetics, University of Colorado at
Denver Health Sciences Center, Aurora, CO 80045, USA.
* Author for correspondence (e-mail: lori.sussel{at}uchsc.edu)
Accepted 22 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cells, and, in its absence, these cell types are
converted to a ghrelin cell fate. To understand the molecular functions of
Nkx2.2 that regulate these early cell-fate decisions during pancreatic islet
development, we created Nkx2.2-dominant-derivative transgenic mice. In the
absence of endogenous Nkx2.2, the Nkx2.2-Engrailed-repressor derivative is
sufficient to fully rescue glucagon-producing -cells and to partially
rescue insulin-producing ß-cells. Interestingly, the insulin-positive
cells that do form in the rescued mice do not express the mature ß-cell
markers MafA or Glut2 (Slc2a2), suggesting that additional activator functions
of Nkx2.2 are required for ß-cell maturation. To explore the mechanism by
which Nkx2.2 functions as a repressor in the islet, we assessed the pancreatic
expression of the Groucho co-repressors, Grg1, Grg2, Grg3 and Grg4
(Tle1-Tle4), which have been shown to interact with and modulate Nkx2.2
function. We determined that Grg3 is highly expressed in the embryonic
pancreas in a pattern similar to Nkx2.2. Furthermore, we show that Grg3
physically interacts with Nkx2.2 through its TN domain. These studies suggest
that Nkx2.2 functions predominantly as a transcriptional repressor during
specification of endocrine cell types in the pancreas.
Key words: Nkx2.2, Transcriptional repression, Islet, ß-cells, -cells, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
ß, 
, PP and 
- each secreting a specific peptide hormone:
glucagon, insulin, somatostatin, PP and ghrelin, respectively. Considerable
progress has been made recently in understanding the transcription factor
network regulating the development of the pancreatic islet
(Habener et al., 2005
;
Servitja and Ferrer, 2004). In
particular, the hierarchy of transcription factors functioning during the
different stages of islet cell specification is being elucidated: Ptf1a and
Pdx1 are the earliest known factors in the pancreatic progenitors, and Ngn3
(also known as Neurog3 - Mouse Genome Informatics) is essential for further
specifying the endocrine progenitors
(Gradwohl et al., 2000;
Jonsson et al., 1994;
Kawaguchi et al., 2002;
Matsuoka et al., 2004;
Offield et al., 1996).
Subsequently, the upregulation of Pdx1 and Nkx6.1 (a ß-cell-specific
factor; also known as Nkx6-1 - Mouse Genome Informatics), and the activation
of MafA, directs the maturation of the insulin-producing ß-cells
(Henseleit et al., 2005;
Jonsson et al., 1994;
Matsuoka et al., 2004;
Sander et al., 2000). One of
the key regulators of the early cell-fate decisions in the islet is the
homeodomain transcription factor Nkx2.2 (also known as Nkx2-2 - Mouse Genome
Informatics). Nkx2.2 is expressed at the onset of pancreatic epithelium
formation, and becomes restricted to mature -, ß- and PP cells
(Sussel et al., 1998). Nkx2.2
has been identified as a crucial regulator for islet cell differentiation; in
Nkx2.2-homozygous-null embryos, all ß-cells and most -cells are
replaced by ghrelin-positive cells (Prado
et al., 2004).
Although Nkx2.2 is known to be essential for proper islet cell development,
little is known about its specific molecular functions or early direct targets
that specify cell-fate decisions. At the transcriptional level, Nkx2.2 has
been shown to have several molecular activities. In vitro studies have
implicated Nkx2.2 in the direct regulation of MafA, of the mouse
insulin genes and of Nkx2.2 itself
(Cissell et al., 2003;
Raum et al., 2006;
Watada et al., 2003). These
studies have suggested that Nkx2.2 may act as an activator through the
Nkx2.2 and MafA promoters, but as a repressor through the
Nkx2.2 consensus site in the insulin promoter. Watada et al.
(Watada et al., 2000) have
also identified a strong activation domain in the C-terminus of Nkx2.2;
however, full-length Nkx2.2 had weak repression activity in the ßTC3-cell
line. In the spinal cord, where Nkx2.2 is crucial for the specification of
ventral neural-progenitor cell fates, ectopic expression of a Nkx2.2-dominant
repressor fusion protein (Nkx2.2hd-EnR) mimics the ability of endogenous
Nkx2.2 to repress Pax6-positive neuronal progenitors. Furthermore, this
repression activity is dependent on the interaction with the Groucho
co-repressor, Grg4 (also known as Tle4 - Mouse Genome Informatics)
(Muhr et al., 2001). Finally,
it has been demonstrated that the Nkx2.2 Drosophila ortholog Vnd
differentially regulates its targets depending on the presence of different
cofactors (Yu et al., 2005).
Together, these data suggest that Nkx2.2 may have complex regulatory
activities during pancreatic development.
In this study, we test the ability of the Nkx2.2-dominant-activator or
-repressor derivatives characterized by Muhr et al.
(Muhr et al., 2001) to
substitute for endogenous Nkx2.2 in the developing mouse islet. The
Nkx2.2-repressor can partially rescue the Nkx2.2-null phenotype in the
pancreas. In neonatal islets, we see a full recovery of glucagon-expressing
cells and a corresponding decrease in ghrelin-positive cells. In addition, the
Nkx2.2-repressor is able to restore a small population of insulin-producing
cells; however, these cells appear to be incompletely differentiated and do
not express the mature ß-cell markers MafA and Slc2a2 (previously known
as Glut2, and hereafter referred to as Glut2). It appears that more complex
Nkx2.2 functions may be required for the full maturation and expansion of the
ß-cell population. Finally, we show that the Grg3 (also known as Tle3 -
Mouse Genome Informatics) co-repressor is specifically expressed in the
developing pancreatic epithelium and that it interacts with Nkx2.2. From these
studies, we conclude that Nkx2.2 primarily functions as a transcriptional
repressor during the specification of all -cells and a subset of
insulin-producing cells.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of Nkx2.2 derivative transgenic mice
A 1200 bp ClaI fragment encoding the Nkx2.2 homeodomain (hd) and
Drosophila Engrailed (En) repressor domain fusion protein
(Muhr et al., 2001) was
inserted into the pPdx1-EcoRI promoter vector, which
includes the 4.5 kb Pdx1 regulatory region and a ß-globin splice cassette
(Norgaard et al., 2003).
NheI sites were added, by PCR, to DNA fragments encoding the Nkx2.2
homeodomain and herpes simplex virus strong activator VP16 or the Nkx2.2-myc
tagged fusion proteins (Muhr et al.,
2001), and the fragments were each cloned into the
pPdx1-EcoRI promoter vector. The pPdx1:Nkx2.2hdEnR,
pPdx1:Nkx2.2hdVP16 and pPdx1:Nkx2.2myc plasmids were
linearized with SacII, NotI and DraIII,
respectively, for pronuclear injection (UCHSC UCCC Transgenic/KO Core
Facility).
Immunohistochemistry
Tissue was fixed for 3 hours or overnight in 4% paraformaldehyde, and were
then either cryopreserved or were fixed in 10% buffered formalin and paraffin
embedded. Immunofluorescence was performed on cryopreserved tissue or
paraffin-embedded tissue (Glut2). Primary antibodies include: rabbit
-amylase (Sigma; 1:1000), rabbit -glucagon (Phoenix
Pharmaceuticals, Belmont, CA; 1:200), guinea pig -glucagon (Linco
Research; 1:3000), guinea pig -insulin (Linco; 1:1000), rabbit
-ghrelin (Phoenix; 1:500), rabbit -Nkx6.1 (Beta cell biology
consortium; 1:800), rabbit -Pdx1 (Chemicon; 1:1000), rabbit
-Glut2 (a gift from B. Thorens, Lausanne, Switzerland; 1:100) and
rabbit -MafA (Bethyl Laboratory; 1:1000). Secondary antibodies include:
Alexa-fluor 488, 594 or 305 (Molecular Probes, Eugene, OR; 1:800). For
immunohistochemistry, biotinconjugated secondary antibodies were used (Jackson
ImmunoResearch; 1:400) with the Vectastain ABC and DAB kits (Vector
Laboratories). Images were obtained with a Leica DM5000 microscope, an
Evolution MP color camera and ImagePro software from Media Cybernetics.
Confocal images were obtained with a Zeiss LSM 510 Meta microscope.
In situ hybridization
RNA in situ hybridization was performed as previously described
(Prado et al., 2004) using
antisense riboprobes transcribed from linearized plasmids. Riboprobes were
generated for mouse Grg3 and Nkx2.2 from full-length cDNA. A
riboprobe-targeting mRNA encoding the Drosophila EnR repression
domain was generated from the pCS2:EnR plasmid and a riboprobe to
Nkx2.2hdVP16 was generated from TOPO:Nkx2.2hdVP16 cDNA.
Quantitative real-time PCR
Total RNA was extracted from embryonic day (E)17.5 pancreatic tissue and
prepared using a Qiagen RNeasy kit. cDNAs were prepared with oligo(dT) primers
and Superscript III (Invitrogen). Real-time PCR was performed using Taqman
probes (ABI Assays on Demand) for insulin2, glucagon, ghrelin and 18S
ribosomal RNA (rRNA) or Gapdh on the ABI 7000. Taqman probes and
primers were designed for 2.2hdEnR (FAMCAGGGCCCCGGGCGA, forward:
5'-GGCCGAGAAAGGTATGAATTCGA-3'; reverse:
5'-GGGCTGCAGCGATCCT-3') and 2.2hdVP16
(FAM-ACGGCCGACTTCGAGTTTGAGC, forward: 5'-TACGGCGCTCTGG AT-3',
reverse: 5'-CGTACTGCTCAATTCCA-3') transgenes and Nkx2.2
(FAM-CCATTGACTCTGCCCCATCGCTTCT, forward: 5'-CCTCCCCGAGTGGCAGAT-3',
reverse: 5'-GAGTTCTATCCTCTCC AAAAGTTCAAA-3'). Each gene was
normalized to 18S rRNA or Gapdh. PCR products were quantified with
ABI prism software.
Co-immunoprecipitation assays
PANC1 cells (ATCC) were transfected with pcDNA3:Grg3 and pcDNA3:Nkx2.2 or
pcDNA3:Nkx2.2
TN using FuGene 6 (Roche) following the manufacturer's
instructions. Cells were harvested 48 hours post-transfection and lysates
collected. Lysate total protein (500 µg) was immunoprecipitated with
proteinG-sepharose beads (Sigma) overnight with 1 µg -Grg3 antibody
(rabbit polyclonal, Santa Cruz Biotechnology) or 1 µg -Nkx2.2
antibody (rabbit polyclonal, Santa Cruz Biotechnology). Proteins bound to the
beads were eluted by boiling, resolved by SDS-PAGE and western blotted
(n=3 experiments). The blots were probed with -Nkx2.2
antibody, 1:100 (mouse monoclonal, DSHB), followed by horseradish
peroxidase-conjugated -mouse secondary antibody and developed using
chemiluminescence (Pierce).
Luciferase-reporter assay
PANC1 and ßTC3-cells were transiently co-transfected with the
pFOxLuc1-7xNk2prl Nkx2.2-dependent luciferase reporter
(Watada et al., 2000) and the
pcDNA3 empty vector, pcDNA3:Nkx2.2-activator or pcDNA3:Nkx2.2-repressor
expression plasmids. Each experiment was performed in triplicate. The
transfected cells were harvested at 48 hours and assayed for luciferase
activity (Promega; n=3 experiments).
Morphometry and statistics
The total area of insulin- or glucagon-positive cells and the total
pancreatic area were measured using standard morphometric analysis with
ImagePro software on every tenth section (10 µm each) for wild-type
(n=2 for each time point) and Nkx2.2-/-;2.2hdEnR transgene
embryos (n=2 for each time point) at E15.5 and E18.5. The total area
of hormone-positive cells was normalized to the total pancreatic area. All
values are expressed as the mean±s.e.m. Statistical analysis was
carried out with a two-tailed Student's unpaired t-test. Results were
considered significant when P<0.05.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
addition, we used a control construct containing the Nkx2.2 homeodomain alone
(Nkx2.2hdCon) to ensure that the homeodomain does not function as a dominant
negative by competing with endogenous Nkx2.2
(Muhr et al., 2001). Muhr et
al. (Muhr et al., 2001)
previously demonstrated the ability of these fusion proteins to correctly
regulate transcription in Cos7 cells. To verify that the Nkx2.2 derivatives
function in the pancreas, we tested the transcriptional activity of
Nkx2.2hdVP16 and Nkx2.2hdEnR on a Nkx2.2-responsive luciferase reporter
plasmid, pFOxLuc1-7xNk2prl (Watada et al.,
2000), in ßTC3- and PANC1-cell lines. Similar to the report
by Muhr et al. (Muhr et al.,
2001), Nkx2.2hdVP16 activates and Nkx2.2hdEnR represses the
reporter gene, as expected (Fig.
1B), indicating that the Nkx2.2 derivatives are functional in
pancreatic cell lines.
To drive expression of these dominant derivatives in the embryonic
pancreas, the constructs were each placed under the control of the 4.5 kb
Pdx1-promoter fragment, which has been shown to faithfully
recapitulate the endogenous expression of Pdx1
(Gannon et al., 2001;
Stoffers et al., 1999). The
Pdx1 promoter was chosen because it has well-characterized regulatory
regions, is commonly used to drive transgene expression in the developing
pancreas and, unlike insulin and Nkx2.2 itself
(Cissell et al., 2003;
Watada et al., 2003), is not
regulated by Nkx2.2. Although Nkx2.2 has been shown to bind the Pdx1
promoter (Van Velkinburgh et al.,
2005), Nkx2.2 does not appear to regulate Pdx1 gene
transcription in vivo; Pdx1 mRNA levels are unaltered in Nkx2.2-null
mice. Furthermore, the expression pattern of Pdx1 is similar to Nkx2.2: it is
first expressed in the pancreatic epithelium at embryonic day (E)8.5 and
continues to be expressed throughout pancreatic development, predominantly in
ß-cells (Guz et al.,
1995). Nkx2.2 is also expressed throughout the pancreatic
epithelium, beginning at E9.5, and later becomes restricted to mature -
and ß-cells (Sussel et al.,
1998).
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). We did not detect any exocrine phenotype in these
mice. Interestingly, both lines of Nkx2.2hdEnR mice expressed the transgene at
levels considerably lower than endogenous Nkx2.2 when quantified by real-time
PCR (Fig. 2C). Expression of
the transgene in line 7414 was approximately 50% of endogenous Nkx2.2 levels,
whereas line 7660 expressed lower levels of the transgene. Expression of the
transgene in line 7660 gradually decreased with each mouse generation,
suggesting that transgene loss or silencing was occurring. The Nkx2.2hdVP16
lines also did not express at supraphysiological levels, but did vary
significantly in their expression of the transgene: line 7318 expressed at
approximately 70% of the level of endogenous Nkx2.2, whereas line 7319
transgene expression was over twofold higher than endogenous Nkx2.2
(Fig. 2C). The relatively
normal or low expression of the transgenes somewhat alleviates concerns of
off-target effects that can arise from over-expressing a protein or protein
derivative. Consistent with this, transgenic mice from each of these lines and
the Nkx2.2hdCon lines were fertile and lived to a normal lifespan, suggesting
that these transgenes were not detrimental to islet development and did not
adversely affect endocrine hormone expression. In particular, the higher
expressing 7319 line of activator mice remained normoglycemic throughout its
lifespan.
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By contrast, the Nkx2.2hdVP16 transgene, as well as the Nkx2.2hdCon transgene did not rescue islet cell differentiation in the absence of endogenous Nkx2.2: insulin and glucagon were absent or reduced, respectively (compare Fig. 3G,I with 3C), and ghrelin expression remained high throughout the islet (compare Fig. 3H,J with 3D). We were unable to observe rescue with either Nkx2.2hdVP16 line, including line 7319, which expressed the Nkx2.2-activator transgene at twice the level of the endogenous Nkx2.2 protein (Fig. 2C). Because we were unable to measure Nkx2.2-activator-transgene-protein production in these mice with the available antibodies, it remains possible that Nkx2.2hdVP16 protein is not produced at adequate levels to achieve rescue. However, the Nkx2.2-repressor does appear sufficient to rescue the glucagon cells and a small subset of insulin-producing cells, suggesting that the functions of the Nkx2.2 activator may not be required at these early time points. Furthermore, the Nkx2.2hdVP16 fusion protein is competent to activate transcription in pancreatic cells lines (Fig. 1B), indicating that the Nkx2.2-activator fusion protein is functional.
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-cells and immature ß-cells-cell population by testing for
co-expression with other islet hormones. Glucagon and ghrelin double-positive
cells are normally found in a small percentage of islet cells throughout
development of the pancreas; however, this population is lost in Nkx2.2-null
mice (Heller et al., 2005;
Prado et al., 2004). In
Nkx2.2-/-;Nkx2.2hdEnR embryos, the glucagon and ghrelin
co-expressing population was also rescued; both the -cells and
glucagon/ghrelin double-positive population were expressed in numbers and
ratios similar to wild-type islets (Fig.
4A-D). We also observed no evidence that the rescued
glucagon-producing population abnormally co-expressed insulin, somatostatin or
PP (Fig. 3E and data not
shown), suggesting that the rescued cells are normal glucagon-producing
-cells. Furthermore, all glucagon-positive cells co-expressed Pax6, a
characteristic -cell transcription factor
(Fig. 4E,F).
Although the Nkx2.2-repressor fully rescued -cells in Nkx2.2-null
embryos, the number of rescued insulin-producing ß-cells was low and
these mice died shortly after birth. There are a number of possibilities for
this partial rescue of insulin-producing cells, including the timing and level
of transgene expression, or a requirement for additional activation functions
of Nkx2.2 in the ß-cell lineage. To begin to understand the incomplete
rescue of insulin-positive cells, we assessed the molecular features and
maturation status of the insulin-positive cells that were maintained in the
rescued animals. In the normal developing pancreas, insulin-producing cells
are present as early as E9.5 (Teitelman et
al., 1993). Several studies have proposed the existence of a
precursor population of cells at the earliest stages of development that
co-express insulin and glucagon (Pang et
al., 1994; Teitelman et al.,
1993; Upchurch et al.,
1994). It does not appear that the rescued insulin-positive cells
in Nkx2.2-/-; Nkx2.2hdEnR mice represent this
double-hormone-expressing population, because we were unable to detect any
glucagon and insulin double-positive cells at any stage of development
(Fig. 3E and data not shown).
The rescued insulin-producing cells also never abnormally co-expressed
somatostatin or PP (data not shown). We also assessed for ghrelin and insulin
co-expression to ensure that we were not merely rescuing insulin gene
transcription in the ghrelin cells; we did not observe the co-expression of
insulin and ghrelin in Nkx2.2-/-; Nkx2.2hdEnR mice
(Fig. 5C,D).
It has been postulated that, in wild-type mice, there are both early
insulin-positive, and glucagon and insulin double-positive populations that do
not give rise to the mature ß-cells of the islet
(Herrera, 2000;
Larsson, 1998). After E13.5,
during the secondary transition, a distinct lineage of endocrine cells forms
that give rise to the mature islet cell populations. The majority of
ß-cells are formed during this stage of islet development and proceed to
differentiate in a stepwise manner. In Nkx2.2-null mice, no insulin-producing
cell population could be detected during pancreatic islet development. In
Nkx2.2-/-; Nkx2.2hdEnR embryos, only approximately 18% of the
normal numbers of insulin-producing cells were rescued. This population of
rescued cells may correspond to the earliest terminal insulin-positive cells
that form prior to E13.5, to an immature ß-cell population that fails to
undergo complete differentiation and expansion, or they may represent reduced
numbers of fully differentiated ß-cells. The earliest (terminal) insulin
cells often co-express glucagon and are believed not to express Pdx1 and
Nkx6.1 (Kim and MacDonald,
2002). The majority of the precursor cells that give rise to
ß-cells express Pdx1 and Nkx6.1 (Kim
and MacDonald, 2002), and recent studies have indicated that MafA
and Glut2 become expressed later in the ß-cell differentiation process
(Kim and MacDonald, 2002;
Matsuoka et al., 2004). To
assess the differentiation state of the rescued ß-cell population, we
tested for the expression of Pdx1, Nkx6.1, MafA and Glut2. At E18.5, Pdx1 and
Nkx6.1 were expressed at normal levels in the insulin-producing cells that
were rescued by the Nkx2.2-repressor; however, the majority of these cells
(>90%) remained MafA negative (Fig.
6). Furthermore, the rescued ß-cells did not express Glut2,
which normally becomes activated in ß-cells after they have initiated
terminal differentiation (Boj et al.,
2001) (Fig. 6G-J).
This would suggest that the repressor activity of Nkx2.2 is sufficient to
specify the immature Pdx1- and Nkx6.1-expressing ß-cell population that
forms during the secondary transition, but is not adequate to promote the
ß-cell differentiation process; additional Nkx2.2 activities may be
required to complete the maturation and expansion process. Alternatively, a
higher threshold of Nkx2.2-repressor activity may be required to initiate
further steps of the ß-cell process.
|
).
Because Nkx2.2 appears to function as a repressor in the developing islet, we
wanted to determine whether Nkx2.2 also interacts with Groucho co-repressors
in the developing pancreas. We assessed the expression profiles of the four
full-length mouse Groucho co-repressors (Grg1, Grg2, Grg3 and Grg4; also
collectively known as Tle1-Tle4) in the developing pancreas. Although Grg4
interacts with Nkx2.2 in the spinal cord, we were unable to detect its
expression in the developing pancreatic epithelium (data not shown). By
contrast, Grg1 and Grg2 mRNAs are expressed at low levels
throughout the pancreatic epithelium, while Grg3 mRNA is expressed
robustly throughout the pancreatic epithelium at E12.5
(Fig. 7A; data not shown). As
pancreatic development proceeds, Grg3 mRNA becomes restricted to the
endocrine compartment (Fig. 7F)
and, throughout pancreas development, is expressed in a manner similar to
Nkx2.2, suggesting that it may be the interacting Groucho partner in the
pancreas that is needed to mediate the repressive activities of Nkx2.2.
Antibodies against Nkx2.2 and Grg3 that work well for immunolabeling of the
embryonic pancreas were not available to us; however, RNA in situ analysis of
Grg3 and Nkx2.2 expression on adjacent tissue sections shows
a high correlation between these two genes; at E12.5, both genes are expressed
throughout the pancreatic epithelium (Fig.
7A,B) and, by E16.5, both genes are expressed within the same cell
clusters (Fig. 7C,D). Using
combined RNA in situ hybridization and immunohistochemistry, we also
demonstrated that, similar to Nkx2.2, Grg3-positive cells co-express glucagon,
but do not overlap with amylase-expressing cells
(Fig. 7E,F). These expression
studies demonstrate that, although Grg4 appears to be the binding partner of
Nkx2.2 in the spinal cord, Grg3 is the more likely Groucho family member to
modulate Nkx2.2 function in the pancreatic islet.
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)
- a ten amino acid domain similar to the Engrailed eh1 domain, which binds to
Drosophila Groucho (Jimenez et
al., 1997; Tolkunova et al.,
1998). When we deleted the TN domain from Nkx2.2 in these assays,
the interaction between Nkx2.2 and Grg3 was abolished
(Fig. 7G, lane 6). These
results suggest that Nkx2.2 interacts with unique Groucho co-repressors in
different cell types to influence its transcriptional activity during
development. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cells and a small population of
immature insulin-producing ß-cells. For this analysis, we expressed the
previously characterized Nkx2.2hd derivatives
(Muhr et al., 2001) in the
pancreas using Pdx1-promoter regulatory elements. We acknowledge that
there are caveats associated with this experimental system, including the use
of the Pdx1 promoter, which is expressed in similar, but not
identical, expression domains as Nkx2.2. In particular, Pdx1 is expressed at
an earlier developmental time point in the pancreatic epithelium and, unlike
Nkx2.2, becomes downregulated in the -cell population. However, the
fact that we were able to partially restore islet cell differentiation with
these Pdx1:Nkx2.2hdEnR derivatives suggests that they have sufficient
normal functions to replace some endogenous Nkx2.2 activities. The full rescue
of the -cell population is particularly intriguing, precisely because
Pdx1 expression is normally downregulated in -cells, where endogenous
Nkx2.2 expression would normally be maintained. This would suggest that the
repressor activity of Nkx2.2 is required early in the Pdx1-positive progenitor
population to initiate the complete specification and differentiation program
of -cells; maintenance of Nkx2.2-repressor function in the
glucagon-producing -cells does not appear to be necessary for the full
differentiation of these cells. The concomitant reduction in ghrelinexpressing
cell numbers also indicates that expression of the Nkx2.2-repressor in
Pdx1-positive precursors is sufficient to regulate these particular cell-fate
decisions.
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-cell population, only a small
number of insulin-producing cells are rescued by the Nkx2.2-repressor
transgene. Interestingly, the rescued insulin-producing cells persist through
birth, however, they fail to activate MafA, the only transcription factor
known to be expressed exclusively in the differentiated ß-cell population
that forms after E13.5 (Matsuoka et al.,
2004). Furthermore, Glut2 is not expressed on the surface of these
cells, indicating that the rescued ß-cells are non-functional. Based on
marker analysis, we favor the hypothesis that the Nkx2.2-repressor rescues an
immature ß-cell that is unable to complete the differentiation process;
however, it is difficult to rule out that the rescued insulin-positive cells
do not represent the earliest `terminal' insulin-expressing population
(Herrera, 2000). Currently,
there are no markers that exclusively localize to the postulated first wave of
insulin-producing cells and, therefore, we cannot fully resolve this issue. In
support of our hypothesis, in the Nkx2.2-repressor-rescued mice,
double-positive cells expressing both glucagon and insulin were not observed.
These cells are thought to be characteristic of this early population
(Herrera, 2000). Furthermore,
insulin-positive cells that are specified express Pdx1 and Nkx6.1, markers,
which are thought to be absent in the early hormone-positive population
(Kim and MacDonald, 2002).
The inability to express MafA in the rescued insulin-producing cells
suggests that there may be a requirement for additional Nkx2.2-activator
functions in the ß-cell-maturation process
(Fig. 8). This would be
consistent with the recent finding that Nkx2.2 binds to the promoter of
MafA and is important for the activation of MafA in
ß-cells, both in vitro and in vivo
(Raum et al., 2006).
Alternatively, the partial rescue of the ß-cell phenotype may reflect the
low level of transgene expression in these cells or the inability of the
truncated Nkx2.2 protein to regulate all of its downstream targets. We
attempted to address this issue by assessing the degree of rescue that could
be achieved by a Pdx1:Nkx2.2 full-length transgene; however, the
introduction of this transgene into a wild-type background caused perinatal
lethality and we were unable to establish stable transgenic lines (Grasch and
L.S., unpublished data). These results are consistent with our inability to
establish more highly expressed Nkx2.2-derivative lines and suggest that
tightly regulated levels of Nkx2.2 protein are crucial for appropriate islet
cell development. We are pursuing these studies using the Tet-On system to
establish inducible Nkx2.2 full-length and derivative transgenic lines.
Interestingly, we did not observe rescue with the Nkx2.2-activator
transgenic lines. Although we detected Nkx2.2hdVP16 mRNA throughout
the pancreatic epithelium, it is possible that the lack of rescue is due to
insufficient amounts of the Nkx2.2-activator fusion protein in these mice.
This is not likely to be the case in the -cell population because
Nkx2.2-repressor activity is sufficient to fully rescue -cells;
however, it does remain possible that Nkx2.2-activator activities are required
in ß-cell formation and maturation. In addition, because we are unable to
regulate the timing of Nkx2.2-activator expression in these studies, we cannot
rule out the possibility that Nkx2.2-activator activity will be involved in
the continued maturation of the ß-cell population once the ß-cells
have been initially specified. Because many transcription factors are known to
have dual activities and because evidence exists for Nkx2.2 regulating the
transcriptional activation of several important ß-cell genes, including
MafA, it is likely that Nkx2.2 will be found to have complex
regulatory activities in the different islet cell types and at different
stages of development. Importantly, this study reveals that the repressor
activity of Nkx2.2 is sufficient for the correct specification of
-cells, and it is likely that repressor and activator activities will
be required to promote ß-cell differentiation.
This study also resulted in the unexpected finding that Grg3, and not Grg4,
is the Groucho-family member expressed in the pancreatic islet. Previous
studies have demonstrated that Nkx2.2 interacts with Grg4 to mediate its
repressor activity in ventral neuron specification
(Muhr et al., 2001). Because
of the conservation of the Grg proteins, it is not surprising that we also
demonstrated an interaction between Nkx2.2 and Grg3 in the pancreas. However,
the different Grg factors are not identical at the protein level, and the
expression of distinct family members in the different cell types may
contribute to the differential gene regulation that must occur to specify
cell-type specific gene expression. Further exploration of these unique
protein interactions and the resulting downstream regulatory consequences in
different tissues will help to elucidate the complex regulatory pathways that
allow overlapping sets of transcription factors to specify unique cell
types.
In summary, this study highlights the importance of understanding the
distinct molecular functions of a transcriptional regulator, such as Nkx2.2,
and how its different biological activities influence cell-fate lineages.
Furthermore, it emphasizes the need to elucidate the protein modifications and
protein-protein interactions that modulate the molecular activities of a
transcription factor at different stages of development. We have discovered
that the repressor activities of Nkx2.2 are sufficient for the differentiation
of one islet cell type, the -cells; however, more-complex activities
are required to generate a fully functional ß-cell. These results have
serious implications for the manipulation of cell-fate choices, both in vivo
and in vitro, and for advancing our ability to engineer pancreatic islet cells
from alternative sources for therapeutic purposes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Boj, S. F., Parrizas, M., Maestro, M. A. and Ferrer, J.
(2001). A transcription factor regulatory circuit in
differentiated pancreatic cells. Proc. Natl. Acad. Sci.
USA 98,14481
-14486.
Cissell, M. A., Zhao, L., Sussel, L., Henderson, E. and Stein,
R. (2003). Transcription factor occupancy of the insulin gene
in vivo. Evidence for direct regulation by Nkx2.2. J. Biol.
Chem. 278,751
-756.
Gannon, M., Gamer, L. W. and Wright, C. V. (2001). Regulatory regions driving developmental and tissue-specific expression of the essential pancreatic gene pdx1. Dev. Biol. 238,185 -201.[CrossRef][Medline]
Gradwohl, G., Dierich, A., LeMeur, M. and Guillemot, F.
(2000). neurogenin3 is required for the development of the four
endocrine cell lineages of the pancreas. Proc. Natl. Acad. Sci.
USA 97,1607
-1611.
Guz, Y., Montminy, M. R., Stein, R., Leonard, J., Gamer, L. W., Wright, C. V. and Teitelman, G. (1995). Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. Development 121, 11-18.[Abstract]
Habener, J. F., Kemp, D. M. and Thomas, M. K.
(2005). Minireview: transcriptional regulation in pancreatic
development. Endocrinology
146,1025
-1034.
Heller, R. S., Jenny, M., Collombat, P., Mansouri, A., Tomasetto, C., Madsen, O. D., Mellitzer, G., Gradwohl, G. and Serup, P. (2005). Genetic determinants of pancreatic epsilon-cell development. Dev. Biol. 286,217 -224.[CrossRef][Medline]
Henseleit, K. D., Nelson, S. B., Kuhlbrodt, K., Hennings, J. C.,
Ericson, J. and Sander, M. (2005). NKX6 transcription factor
activity is required for alpha- and beta-cell development in the pancreas.
Development 132,3139
-3149.
Herrera, P. L. (2000). Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development 127,2317 -2322.[Abstract]
Jimenez, G., Paroush, Z. and Ish-Horowicz, D.
(1997). Groucho acts as a corepressor for a subset of negative
regulators, including Hairy and Engrailed. Genes Dev.
11,3072
-3082.
Jonsson, J., Carlsson, L., Edlund, T. and Edlund, H. (1994). Insulin-promoterfactor 1 is required for pancreas development in mice. Nature 371,606 -609.[CrossRef][Medline]
Kawaguchi, Y., Cooper, B., Gannon, M., Ray, M., MacDonald, R. J. and Wright, C. V. (2002). The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat. Genet. 32,128 -134.[CrossRef][Medline]
Kim, S. K. and MacDonald, R. J. (2002). Signaling and transcriptional control of pancreatic organogenesis. Curr. Opin. Genet. Dev. 12,540 -547.[CrossRef][Medline]
Larsson, L. I. (1998). On the development of the islets of Langerhans. Microsc. Res. Tech. 43,284 -291.[CrossRef][Medline]
Matsuoka, T. A., Artner, I., Henderson, E., Means, A., Sander,
M. and Stein, R. (2004). The MafA transcription factor
appears to be responsible for tissue-specific expression of insulin.
Proc. Natl. Acad. Sci. USA
101,2930
-2933.
Muhr, J., Andersson, E., Persson, M., Jessell, T. M. and Ericson, J. (2001). Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104,861 -873.[CrossRef][Medline]
Norgaard, G. A., Jensen, J. N. and Jensen, J. (2003). FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development. Dev. Biol. 264,323 -338.[CrossRef][Medline]
Offield, M. F., Jetton, T. L., Labosky, P. A., Ray, M., Stein, R. W., Magnuson, M. A., Hogan, B. L. and Wright, C. V. (1996). PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122,983 -995.[Abstract]
Pang, K., Mukonoweshuro, C. and Wong, G. G. (1994). Beta cells arise from glucose transporter type 2 (Glut2)-expressing epithelial cells of the developing rat pancreas. Proc. Natl. Acad. Sci. USA 9,9559 -9563.
Prado, C. L., Pugh-Bernard, A. E., Elghazi, L., Sosa-Pineda, B.
and Sussel, L. (2004). Ghrelin cells replace
insulin-producing beta cells in two mouse models of pancreas development.
Proc. Natl. Acad. Sci. USA
101,2924
-2929.
Raum, J. C., Gerrish, K., Artner, I., Henderson, E., Guo, M.,
Sussel, L., Schisler, J. C., Newgard, C. B. and Stein, R.
(2006). FoxA2, Nkx2.2, and PDX-1 regulate islet
beta-cell-specific mafA expression through conserved sequences located between
base pairs -8118 and -7750 upstream from the transcription start site.
Mol. Cell. Biol. 26,5735
-5743.
Sander, M., Sussel, L., Conners, J., Scheel, D., Kalamaras, J., Dela Cruz, F., Schwitzgebel, V., Hayes-Jordan, A. and German, M. (2000). Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development 127,5533 -5540.[Abstract]
Servitja, J. M. and Ferrer, J. (2004). Transcriptional networks controlling pancreatic development and beta cell function. Diabetologia 47,597 -613.[CrossRef][Medline]
Stoffers, D. A., Heller, R. S., Miller, C. P. and Habener, J.
F. (1999). Developmental expression of the homeodomain
protein IDX-1 in mice transgenic for an IDX-1 promoter/lacZ transcriptional
reporter. Endocrinology
140,5374
-5381.
Sussel, L., Kalamaras, J., Hartigan-O'Connor, D. J., Meneses, J. J., Pedersen, R. A., Rubenstein, J. L. and German, M. S. (1998). Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. Development 125,2213 -2221.[Abstract]
Teitelman, G., Alpert, S., Polak, J. M., Martinez, A. and Hanahan, D. (1993). Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development 118,1031 -1039.[Abstract]
Tolkunova, E. N., Fujioka, M., Kobayashi, M., Deka, D. and
Jaynes, J. B. (1998). Two distinct types of repression domain
in engrailed: one interacts with the groucho corepressor and is preferentially
active on integrated target genes. Mol. Cell. Biol.
18,2804
-2814.
Upchurch, B. H., Aponte, G. W. and Leiter, A. B. (1994). Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor. Development 120,245 -252.[Abstract]
Van Velkinburgh, J. C., Samaras, S. E., Gerrish, K., Artner, I.
and Stein, R. (2005). Interactions between areas I and II
direct pdx-1 expression specifically to islet cell types of the mature and
developing pancreas. J. Biol. Chem.
280,38438
-38444.
Watada, H., Mirmira, R. G., Kalamaras, J. and German, M. S.
(2000). Intramolecular control of transcriptional activity by the
NK2-specific domain in NK-2 homeodomain proteins. Proc. Natl. Acad.
Sci. USA 97,9443
-9448.
Watada, H., Scheel, D. W., Leung, J. and German, M. S.
(2003). Distinct gene expression programs function in progenitor
and mature islet cells. J. Biol. Chem.
278,17130
-17140.
Yu, Z., Syu, L. J. and Mellerick, D. M. (2005).
Contextual interactions determine whether the Drosophila homeodomain protein,
Vnd, acts as a repressor or activator. Nucleic Acids
Res. 33,1
-12.
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