Download Mice lacking the homeodomain transcription factor

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Development 125, 2213-2221 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV6347
Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due
to arrested differentiation of pancreatic β cells
L. Sussel1, J. Kalamaras2,*, D. J. Hartigan-O’Connor1,*,†, J. J. Meneses4, R. A. Pedersen4,
J. L. R. Rubenstein1 and M. S. German2,3,‡
1Nina
Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, 2Hormone Research Institute, 3Department of
Medicine, 4Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA 94143,
USA
*These two authors contributed equally to this work
†Present address: Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109-0618, USA
‡Author for correspondence at address 2 (e-mail: [email protected])
Accepted 2 April; published on WWW 19 May 1998
SUMMARY
The endocrine pancreas is organized into clusters of cells
called islets of Langerhans comprising four well-defined
cell types: α, β, δ and PP cells. While recent genetic
studies indicate that islet development depends on the
function of an integrated network of transcription factors,
the specific roles of these factors in early cell-type
specification and differentiation remain elusive. Nkx2.2 is
a member of the mammalian NK2 homeobox
transcription factor family that is expressed in the ventral
CNS and the pancreas. Within the pancreas, we
demonstrate that Nkx2.2 is expressed in α, β and PP cells,
but not in δ cells. In addition, we show that mice
homozygous for a null mutation of Nkx2.2 develop severe
hyperglycemia
and
die
shortly
after
birth.
Immunohistochemical analysis reveals that the mutant
embryos lack insulin-producing β cells and have fewer
glucagon-producing α cells and PP cells. Remarkably, in
the mutants there remains a large population of islet cells
that do not produce any of the four endocrine hormones.
These cells express some β cell markers, such as islet
amyloid polypeptide and Pdx1, but lack other definitive β
cell markers including glucose transporter 2 and Nkx6.1.
We propose that Nkx2.2 is required for the final
differentiation of pancreatic β cells, and in its absence, β
cells are trapped in an incompletely differentiated state.
INTRODUCTION
in development, the first differentiated cells of the pancreas,
the glucagon-producing α cells, appear (Herrera et al., 1991;
Teitelman et al., 1993; Upchurch et al., 1994). Insulinproducing β cells appear within the next 24 hours. As the dorsal
pancreatic bud grows and fuses with a ventral bud,
invaginations within the pancreatic epithelium develop into
ducts. Endocrine cells continue to differentiate from the ductal
epithelium throughout pancreatic development. At
approximately E14.0, several events occur simultaneously: the
termini of the ducts begin to form acini and differentiate into
the exocrine cells of the pancreas; the first somatostatinproducing δ cells appear; the endocrine cells start to form
clusters; and the number of endocrine cells and the levels of
insulin and glucagon production per cell increase dramatically
(Pictet and Rutter, 1972). Finally, at E18.0, shortly before birth
in the mouse, the pancreatic polypeptide (pp)-producing PP
cells appear, and the endocrine cells begin to form well
organized islets (Herrera et al., 1991).
Recently, mice with targeted disruptions of genes encoding
transcription factors that are expressed in the pancreas have
provided insights into the regulation of islet development. Pdx1,
a homeodomain protein, is normally expressed in all cells of the
The pancreas plays a central role in nutrient regulation through
the function of two distinct populations of cells: the exocrine
cells secrete digestive enzymes through the duct system into the
gut and the endocrine cells secrete hormones into the
bloodstream. The hormones produced by the endocrine pancreas
regulate nutrient metabolism in mammals; in particular, loss or
dysfunction of the insulin-producing β cells causes diabetes
mellitus. Development of the endocrine pancreas involves a
complex process of cell differentiation that ultimately gives rise
to four distinct hormone-producing cell types: α, β, δ and PP
cells, precisely organized within the islets of Langerhans (for
reviews on pancreas development, see Slack, 1995; Sander and
German, 1997). While all the cells of the endocrine pancreas are
thought to arise from a common endodermal precursor, the early
process of lineage determination and cell-type differentiation
within the pancreas remains unclear.
In the mouse, morphologic development of the pancreas first
appears at embryonic day (E) 9.5 as a small bud of epithelial
cells on the dorsal aspect of the gut at the junction of the
foregut and the midgut (Pictet and Rutter, 1972). At this point
Key words: Nkx2.2, Pancreas, Transcription factors, β cells, Diabetes
2214 L. Sussel and others
pancreatic bud (Guz et al., 1995); absence of Pdx1 arrests
pancreas development at the bud stage (Jonsson et al., 1994;
Offield et al., 1996). Islet-1 (Isl1) is a LIM homeodomain
protein that is first expressed in the dorsal pancreatic epithelium
and later is produced in all adult islet cells (Ahlgren et al.,
1997). Isl1 null mutants have an early block in the
differentiation of the endocrine pancreas. Two additional
transcription factors, Pax4 and Pax6, also appear to function in
determining endocrine cell fate. A null mutation in either gene,
individually, leads to a reduction in islet cell types (Sander et
al., 1997; Sosa-Pineda et al., 1997; St-Onge et al., 1997). Mice
lacking both Pax4 and Pax6 apparently fail to develop any
pancreatic endocrine cells (St-Onge et al., 1997), suggesting
that these genes may be required in concert to determine
endocrine cell fate in the pancreas. In contrast to the above
genes, a mutation in the helix-loop-helix gene BETA2/neuroD
arrests islet morphogenesis at approximately E15.5 indicating
that this transcription factor acts at a relatively late stage of islet
development and may be primarily involved in the maintenance
or proliferation of the islet cell types (Naya et al., 1997).
It has recently been shown that members of the Nkx
homeobox gene family have restricted expression in the
pancreatic islet: Nkx2.2 mRNA is present in the adult
pancreatic islets and in pancreatic α and β cell lines; and
Nkx6.1 is expressed in adult β cells, and in α and β cell lines
(Rudnick et al., 1994; Jensen et al., 1996). In an attempt to
understand endocrine cell type differentiation, we have chosen
to analyze the role of Nkx2.2 in pancreatic development.
Nkx2.2 is the member of the vertebrate homeodomain
transcription factor gene family that is most homologous to the
Drosophila NK2/ventral nervous system defective (vnd) gene
(Kim and Nirenberg, 1989; Price, 1993; Jimenez et al., 1995).
Nkx2.2 was originally identified as a gene that is expressed in
ventral regions of the developing vertebrate CNS (Price et al.,
1992). In addition to Nkx2.2, five other family members have
been identified in mice: Nkx2.1, Nkx2.2, Nkx2.3 and Nkx2.4
are closely related (Price et al., 1992; Price, 1993), while
Nkx2.5 and Nkx2.6 represent more divergent members of the
family (Lints et al., 1993). NK2 family members have now
been shown to be key regulators of development and
differentiation in several tissues: Nkx2.1 is necessary for lung,
thyroid and ventral forebrain development and Nkx2.5 is
required for proper heart formation (Kimura et al., 1996; Lints
et al., 1995). Therefore, it is possible that Nkx2.2 may play a
similar role in the development of the pancreas.
In this study, we demonstrate that Nkx2.2 is expressed at the
onset of pancreatic bud evagination, and later becomes
restricted to specific endocrine cell types as islets develop. To
gain insight into the function of Nkx2.2 in the pancreas, we
generated mice carrying a null mutation of the gene. Our
analysis reveals that Nkx2.2 is involved in the differentiation
of three islet cell types. Furthermore, Nkx2.2 appears to play
a pivotal role in the final differentiation of insulin-producing β
cells, and loss of Nkx2.2 arrests these cells in a partially
differentiated state.
MATERIALS AND METHODS
Targeting construct
To generate Nkx2.2 mutant mice, three overlapping approx. 14 kb
genomic clones containing the entire Nkx2.2 gene were isolated from
a 129J mouse genomic library (provided by Anton Berns,
Amsterdam). The gene was mapped using a combination of Southern
analysis and restriction enzyme digests. A targeting construct was
generated using pBS KS- (Stratagene) as the cloning vector. The
organization of the targeting vector is shown in Fig. 2. Briefly, the 6.0
kb BglII-XhoI 5′ fragment was ligated 5′ to the PGK-neo cassette. The
4.0 kb BamHI 3′ fragment was placed downstream of PGK-neo.
Finally, the PGK-thymidine kinase (tk) cassette was inserted 3′ to the
4.0 kb Nkx2.2 fragment.
Generation of mutant animals
The generation of recombinant ES clones and Nkx2.2 mutant mice
was performed as described extensively in Qiu et al. (1995). Southern
analysis was used for all genotyping of ES cells and mice. The
southern probe is shown in Fig. 2.
Immunohistochemistry
Immunohistochemistry and immunofluorescence assays were
performed on paraffin sections as described previously (Sander et al.,
1997). Primary antibodies were: guinea pig anti-insulin (Linco) or
monoclonal anti-insulin (Sigma); guinea pig anti-glucagon (Linco);
rabbit anti-PP (Dako); rabbit anti-somatostatin (Dako); rabbit antiglucokinase (provided by C. Newgard); rabbit anti-glucose transporter
2 (Chemicon); rabbit anti-IAPP (Advanced Chemtech); rabbit antiprohormone convertase 1/3 (provided by D. Steiner); rabbit antiamylase (Sigma); monoclonal anti-synaptophysin (Biogenics);
monoclonal anti-Nkx2.2 (provided by T. Jessell); rabbit anti-Pdx1
(provided by H. Edlund); monoclonal anti-Isl1 (Developmental
Hybridoma Bank); rabbit anti-Pax6 (provided by S. Saule); rabbit
anti-Brain4 (provided by M. G. Rosenfeld) and rabbit anti-Nkx6.1
(antibodies were raised against a GST fusion protein containing the
carboxy-terminal 70 amino acids of hamster Nkx6.1). Secondary
antibodies were used as described previously (Sander et al., 1997).
For immunofluorescence assays, we used fluorescein coupled antirabbit, fluorescein coupled anti-mouse, rhodamine coupled antiguinea pig, rhodamine coupled anti-rabbit (Cappel) and marina blue
coupled anti-mouse (Molecular Probes). Biotinylated secondary
antibodies (Vector) were detected with the ABC Elite
immunoperoxidase system (Vector).
Glucose levels
Glucose levels were measured with the One Touch Glucose
Monitoring kit (Johnson and Johnson) using 20 µl of peripheral blood
from 2- to 3-day old mice.
Quantification of insulin and glucagon concentrations
Protein was extracted as described previously (Sander et al., 1997).
The concentrations of insulin and glucagon were determined by
radioimmunoassays (RIA) using commercially available kits (Linco).
RESULTS
To precisely determine the temporal and spatial domains of
Nkx2.2 expression in the embryonic pancreas, we used a
monoclonal antibody directed against chicken Nkx2.2 (Ericson
et al., 1997) to localize the protein (Fig. 1). Initial expression
of Nkx2.2 on day 9.5 of embryonic development (E9.5) is
coincident with the appearance of the dorsal pancreatic bud,
which forms near the junction of the foregut and midgut
endoderm (Fig. 1b). As the pancreatic epithelium grows and
develops into a branched structure at E12.5, Nkx2.2 expression
persists in most cells (Fig. 1c). However, by E15.5 when
distinct exocrine and endocrine compartments can be
identified, expression of Nkx2.2 becomes restricted to most of
Role of Nkx2.2 in pancreas development 2215
E9.0
E9.5
E12.5
adult
Fig. 1. Temporal and spatial expression of Nkx2.2 protein in the developing mouse pancreas. (a) A transverse section of an E9.0 (20-somite
stage) wild-type embryo; there is no detectable Nkx2.2 protein in the region of the midgut-foregut junction. In the same section, Nkx2.2 is
present in the spinal cord (data not shown). mg, midgut; fg, foregut. (b) By E9.5 (25-somite stage), a sagittal section shows Nkx2.2 is present in
the dorsal pancreatic bud as it develops from the primitive gut endoderm. (c) At E12.5, the pancreatic epithelium has grown and developed into
a branched stucture; at this stage, Nkx2.2 is expressed in most pancreatic epithelial cells. (d) In the adult pancreas (P30), Nkx2.2 protein is still
present in the islets. (e-h) Double immunofluorescence staining of E18.5 pancreas sections with anti-Nkx2.2 (fluorescein label, green) and
antibodies directed against each of the four pancreatic hormones (rhodamine label, orange), as indicated. Nkx2.2 is localized to the nucleus and
the hormones are detected in the cytoplasm of islet cells. (e) Nkx2.2 is co-expressed with all insulin positive cells. (f) Nkx2.2 is present in most
of the glucagon expressing cells. (g) Nkx2.2 can be detected in the nucleus of most cells expressing pp. (h) Nkx2.2 is not detectable in
somatostatin-expressing cells. Arrows indicate cells within the islet that are clearly representative of co-staining (f,g) or lack of co-staining (h).
Magnifications are indicated (a-d).
the endocrine cells (data not shown). Expression of Nkx2.2 is
maintained within the pancreatic islets into adulthood (Fig.
1d), and is not detectable in the exocrine tissue.
The mouse islet comprises four distinct endocrine cell
populations: α, β, δ, and PP cells, each of which produces one
of the four pancreatic hormones, glucagon, insulin,
somatostatin and pancreatic polypeptide (pp), respectively
(Slack, 1995; Sander and German, 1997). Glucagon expression
begins at E9.5 in the developing pancreatic bud and insulin
expression can be detected by E10.5. Somatostatin-producing
δ cells arise at E15.5 and PP cells are generated just before
birth, at E18.5. To determine which endocrine cell types
express
Nkx2.2
protein,
we
performed
double
immunohistochemistry to analyze the co-expression of Nkx2.2
protein and each of the four pancreatic hormones at E18.5. As
shown in Fig. 1, Nkx2.2 protein appears to be present in every
insulin-expressing
β
cell.
In
addition,
double
immunofluorescence reveals that Nkx2.2 co-localizes with
glucagon in approximately 80% of the α cells and with
pancreatic polypeptide in most, but not all, of the PP cells. In
contrast, Nkx2.2 is not expressed in somatostatin-producing δ
cells.
To address the role of Nkx2.2 in pancreatic development, we
generated mice in which the Nkx2.2 gene was deleted by
standard gene targeting techniques in ES cells (see Fig. 2 and
Materials and Methods). The deletion completely eliminates
Nkx2.2 activity since we are unable to detect any Nkx2.2
protein in the CNS and pancreas of homozygous mutant mice
by immunohistochemistry. Consistent with a null mutation in
Nkx2.2, there is complete penetrance of all phenotypes
observed in the homozygous mutant mice. Mice heterozygous
for the Nkx2.2 mutation survive to adulthood, are fertile and
are indistinguishable from wild-type animals.
Homozygous Nkx2.2 mutants survive through gestation and
are grossly indistinguishable from their wild-type and
heterozygous littermates at birth. The mutant neonates are as
active as their littermates and have milk in their stomachs,
indicating that they feed normally. By the second day after
birth, however, the homozygous mutant animals begin to
display severe growth retardation and are diabetic, with blood
glucose levels five-fold higher than the control littermates
(Table 1). Mutant animals do not survive longer than 6 days
postnatally. At the time of death, the gross morphology of the
pancreas appears normal. However, pancreas histology reveals
2216 L. Sussel and others
a
Fig. 2. Disruption of the
Nkx2.2 gene by homologous
recombination. (a) Schematic
drawings of the wild-type
Nkx2.2 genomic locus, the
targeting vector and the mutant
allele generated after
homologous recombination.
Exon 1 and 2 of the Nkx2.2
gene are indicated. The
homeodomain is represented
by a shaded box. The 5′ probe
for southern blotting is also
indicated. The cloning strategy
is described in Materials and
Methods. (b) Southern blot
analysis of ApaI-digested
genomic DNA from a litter
derived from an Nkx2.2+/−
intercross. The sizes of the
wild-type (8.6 kb) and targeted
(12.0 kb) alleles are indicated.
b
a general reduction in islet cell mass and the islets display an
unusual string-like morphology, whereas the exocrine cells
appear unaffected (data not shown).
To study the effect of Nkx2.2 deficiency on the endocrine
pancreas, we examined hormone expression from the four
endocrine cell types using immunohistochemistry.
Throughout all stages of embryonic development examined
(E10.5, E12.5 and E18.5), we are unable to detect any
insulin-positive cells in the mutant islets, even with increased
concentrations of the insulin antibody (Fig. 3e). In addition,
the number of glucagon-producing cells and the amount of
glucagon expressed per cell was significantly reduced (Fig.
3f). The decrease in glucagon-expressing cells can be
detected as early as E10.5, and by the end of embryogenesis
(E18.5) there are approximately 20% of the normal number
of α cells. There appears to be a modest reduction in both the
number of PP cells and the intensity of PP staining in the
mutant mice (Fig. 3g), while somatostatin expression appears
unaltered (Fig. 3h), which correlates with the absence of
Nkx2.2 expression in the δ cells (Fig. 1h).
Radioimmunoassays on pancreata from several litters of
E18.5 embryos revealed that insulin content in the mutant
pancreata is decreased by greater than 150 fold and glucagon
Table 1. Blood glucose levels in Nkx2.2 wild-type,
homozygous and heterozygous neonatal animals
Genotype
+/+
+/−
–/−
No. of
animals
Blood glucose levels
(mg/dl)
4
9
7
81.25±3.45
74.0±2.0
424.4±11.86
Blood glucose levels were determined from peripheral blood taken from
neonatal animals (see Materials and Methods). The values shown represent
the mean ± s.e.m. value from the number (No.) of samples assayed.
content is reduced by approximately 50 fold in comparison
to their wild-type and heterozygous littermates (Table 2).
Interestingly, a large proportion of the cells within the
mutant islet clusters do not express any of the four pancreatic
hormones (Figs 3 and 5d). To determine whether these cells
retain general endocrine characteristics, we analyzed them for
the expression of several known endocrine and exocrine
markers. Glucagon expression is used to delineate the
perimeter of the islet in serial sections (Fig. 4c,f). In the Nkx2.2
mutant at E18.5, expression of amylase, an exocrine enzyme,
is confined to the exocrine tissue and there is no ectopic
expression in the islets (Fig. 4d). At the same developmental
timepoint, synaptophysin, a representative endocrine marker
(Wiedenmann et al., 1986), is expressed in all cells in the
mutant islet clusters (Fig. 4e). Furthermore, all the mutant islet
cells at E18.5 express the neuroendocrine cell adhesion factor
N-CAM (Cirulli et al., 1994), despite the abnormal string-like
morphology of the mutant islets (data not shown).
The quantity and distribution of the atypical endocrine cells
in the mutant, combined with a complete absence of insulin
expression, suggested that the non-hormone producing cells in
Table 2. Quantification of hormone levels in pancreata
taken from E18.5 embryos carrying the Nkx2.2 null allele
and their wild-type and heterozygous littermates
Genotype
+/+
+/–
–/–
No. of
animals
4
6
5
µg insulin/
mg protein
14.25±1.58
14.38±1.29
0.09±0.0017
µg glucagon/
mg protein
1.58±0.58
1.02±0.13
0.03±0.009
Hormone concentrations were determined in protein extracts from
individual pancreata taken from wild-type, heterozygous and homozygous
E18.5 embryos (see Materials and Methods). The values shown represent the
mean ± s.e.m. value from the number (No.) of samples assayed.
Role of Nkx2.2 in pancreas development 2217
Fig. 3. Several endocrine hormones are absent or reduced in the pancreata of Nkx2.2 mutants. Immunohistochemistry of serial pancreas sections
from Nkx2.2+/+ (a-d) and Nkx2.2−/− (e-h) E18.5 embryos. The four principle islet cell hormones, insulin (a), glucagon (b), pp (c) and
somatostatin (d), are detected in characteristic patterns in Nkx2.2+/+ pancreas. In pancreata from Nkx2.2−/− embryos, there is a general
reduction in islet cell mass and insulin expression is undetectable (e). The arrow indicates the islet; ex, exocrine tissue. In addition, glucagon (f)
and pp (g) expression is clearly reduced in the mutant islet, whereas the proportion of islet cells expressing somatostatin, and the amount of
somatostatin expressed per cell, appears unchanged (h).
the mutant islets may represent immature or partially
differentiated β cells. To test the hypothesis, we examined the
expression of several β cell-specific proteins in the Nkx2.2
mutant islets. Islet amyloid polypeptide (IAPP) is a β cell
peptide hormone that is co-secreted with insulin (Cooper et al.,
1989), and prohormone convertase 1/3 is a proinsulin
processing enzyme that is restricted to β cells within the
pancreas (Marcinkiewicz et al., 1990). In the mutant islets,
both of these β cell markers are expressed at normal levels,
suggesting that the unidentified cells are incompletely
differentiated β cells (Fig. 5e,i). However, two additional β
cell-specific proteins that regulate glucose catabolism, glucose
transporter 2 and glucokinase (Matschinsky, 1990; German,
1993; Pang et al., 1994) are undetectable in the mutant islet
cells by immunohistochemistry (Fig. 5f,j). These
characteristics, coupled with the absence of insulin production,
suggest that the Nkx2.2 mutant islets contain the normal
proportion of β cells, but that these cells have failed to
differentiate completely and are defective in β cell function.
Although it is possible, we cannot assume that these cells
define a normal stage of β cell differentiation.
To elucidate the relationship between Nkx2.2 and other
transcription factors involved in pancreas development, the
expression of the homeodomain factors islet-1 (Isl1), Pax6,
Brain 4 (Brn4), Pdx1 and Nkx6.1 was examined in the Nkx2.2
mutants (Fig. 6). Isl1 is a LIM homeodomain protein involved
in the early formation of the pancreas (Ahlgren et al., 1997).
It is expressed in all post-mitotic islet cells and plays a key role
in endocrine development. Interestingly, expression of Isl1 is
unaffected in the Nkx2.2 mutant pancreas (Fig. 6g). Normal
Isl1 expression, in addition to normal levels of proliferation in
the mutant pancreas (data not shown), suggest that all the cells
of the islet, including the immature β cells are able to exit
mitosis normally. We next examined the expression of the
homeodomain protein Pdx1, which is required for the growth
and differentiation of the entire pancreas beyond the initial bud
stage (Jonsson et al., 1994; Offield et al., 1996). Normally,
Pdx1 is uniformly expressed in the early pancreatic bud and
becomes restricted to β cells later in development. In the
Nkx2.2 mutants, early expression of Pdx1 appears normal and
Pdx1 does become restricted to the defective β cells. However,
by E18.5, the level of Pdx1 expression within these cells
appears to be reduced (Fig. 6f).
Pax6 is a paired domain homeobox gene that is expressed
in all pancreatic endocrine cells. In Pax6 mutant mice, there
is a reduction in all four islet cell types, a decrease in
hormone expression, and abnormal islet morphology (Sander
et al., 1997; St.-Onge et al., 1997). Pax6 protein is detectable
at wild-type levels in the islet cells of the Nkx2.2 mutants
(Fig. 6h). We also looked at the expression of the POU
homeodomain protein, Brn4, which is localized to the α cells
(J. K. and M. G., unpublished observations; Hussain et al.,
1997). Consistent with the reduction of α cells in the mutant
islet, there are fewer cells expressing Brn4. However, the
remaining α cells appear to express Brn4 at levels comparable
to wild-type (Fig. 6j).
Finally, we looked at Nkx6.1, a homeodomain protein that
is β cell-specific in the mature islets (Jensen et al., 1996). Early
in the development of both mutant and wild-type littermates
(E10.5 to E12.5), Nkx6.1 is widely expressed in the pancreatic
epithelium (data not shown). However, at E18.5, by which time
Nkx6.1 expression is normally restricted to the β cells, Nkx6.1
protein is undetectable in the Nkx2.2 mutants (Fig. 6i).
DISCUSSION
Role of Nkx2.2 in islet cell differentiation
Development and differentiation of the pancreas requires the
coordinate activation of unique sets of genes. Key regulators
of these processes are transcription factors, and recent mouse
genetic studies have demonstrated that several transcription
2218 L. Sussel and others
Fig. 4. Normal exocrine and
endocrine compartments are
maintained in Nkx2.2 mutants.
The exocrine enzyme, amylase,
is present in exocrine cells (Ex)
and is absent from endocrine
tissue (En) in pancreata derived
from either Nkx2.2+/+ (a) or
Nkx2.2−/− (d) E18.5 embryos.
Similarly, there appears to be
no difference in the expression
of synaptophysin, a
representative endocrine
marker, between Nkx2.2+/+ (b)
or Nkx2.2−/− (e) embryos.
Serial sections stained with
anti-glucagon antibody to
delineate the perimeter of the
islet (c,f).
factors are required for normal pancreatic development
(Jonsson et al., 1994; Ahlgren et al., 1997; Offield et al., 1996;
Sander et al., 1997; Sosa-Pineda et al., 1997; St.-Onge et al.,
1997). Among these factors, Nkx2.2 plays a unique role in
endocrine pancreas development. Without Nkx2.2, there is a
complete arrest of β cell terminal differentiation leading to the
accumulation of a large group of cells that display endocrine
characteristics and express a subset of β cell markers, but lack
insulin. Furthermore, the phenotype of Nkx2.2 null mice
demonstrates that the specification of islet cell types and their
subsequent differentiation can be independent processes:
Nkx2.2 does not appear to be required for β cell specification,
but is essential for the differentiation of insulin-producing β
cells. In addition, Nkx2.2 is not essential for the maintenance
of these incompletely differentiated β cells, since there is a
large stable pool of these cells at E18.5.
Fig. 5. A large population of cells in the Nkx2.2−/− islet do not express any of the four principle endocrine hormones, and appear to be partially
differentiated, non-functional β cells. Immunofluorescence on serial sections of pancreas derived from Nkx2.2+/+ (a-c) and Nkx2.2−/− (d-e)
E18.5 embryos. (a,d) Triple immunofluorescent staining of insulin (marina blue label, blue), glucagon (rhodamine label, orange), and
somatostatin and pp (fluorescein label, green). In the Nkx2.2−/− pancreas, there is a large population of cells within the islet which does not
stain for the four principle endocrine hormones (d). (b,e) Double immunofluorescence on serial pancreas sections with glucagon (rhodamine
label, orange) delineating the perimeter of the islet, and the β cell-specific protein IAPP (amylin) (fluorescein label, green). The cells within the
mutant islet that are not expressing any of the four endocrine hormones clearly express IAPP (e). (c,f) Serial sections stained with glucagon
(rhodamine label, orange) and the cell surface β cell marker glut-2 (fluorescein label, green). Glut-2 is undetectable in the Nkx2.2−/− pancreas
(f). (g,i) Non-serial sections showing double immunoflourescence of glucagon (rhodamine label, orange) and the β cell-specific protein PC1/3
(fluorescein label, green). (h,j) Non-serial sections showing double immunoflourescence of glucagon (rhodamine label, orange) and the
membrane bound β cell protein, glucokinase (fluorescein label, green).
Role of Nkx2.2 in pancreas development 2219
Fig. 6. The expression of several transcription factors is affected in the pancreas of Nkx2.2−/− mice. Immunohistochemical analysis of the
expression of Pdx1 (a,f), Isl1 (b,g) Pax6 (c,h), Nkx6.1 (d,i) and Brn4 (e,j) in the pancreas of Nkx2.2+/+ (a-e) and Nkx2.2−/− (f-j) E18.5 mice. In
wild-type E18.5 mice, Pdx1 expression has become restricted to the pancreatic β cells (a), Isl1 and Pax6 can be detected in all islet cell types
(b,c) and Nkx6.1 is expressed specifically in the β cells (d). Brn4 is expressed in α cells (e). In the Nkx2.2 mutant, Pdx1 can be detected in the
non-functional β cells, but at reduced levels (f); Isl1 and Pax6 continue to be expressed in cells throughout the islet (g,h). Notably, in the Nkx2.2
mutant, there is no detectable expression of Nkx6.1 (i). The arrow in i indicates the islet cell mass. Brn4 is expressed in α cells at levels
comparable to wild type. However, because the total number of α cells are reduced in the mutant, there are fewer numbers of cells expressing
Brn4 (j).
Despite the presence of incompletely differentiated β cells,
insulin expression cannot be detected at any stage of
development in the Nkx2.2 null animals. This finding suggests
that Nkx2.2 may directly activate insulin gene transcription.
Nkx2.2 could function by binding directly to the insulin
promoter, by physically associating with an insulin geneactivating complex, or by inducing the expression of other
proteins that regulate insulin gene transcription. The consensus
DNA binding site for Nkx2.2 is not known, however the
consensus binding site for the closely related Nkx2.1 protein
has been well characterized (Guazzi et al., 1990). Since Nkx2.1
and Nkx2.2 are nearly identical in their DNA-binding
homeodomain regions and Nkx2.2 can bind to the Nkx2.1
consensus site (H. Ee and M. German, unpublished results), we
scanned the insulin promoter sequence for close sequence
matches to the Nkx2.1 consensus and identified a putative
Nkx2.1/Nkx2.2 binding site at the C1 regulatory element
(Hwung et al., 1990). However, preliminary experiments
suggest that Nkx2.2 is not a binding factor for the C1 element
and, in addition, does not bind other homeobox sites present
within the insulin gene regulatory region (H. Ee and M.
German, unpublished results). Therefore, we cannot yet
determine whether Nkx2.2 is directly involved in insulin
transcription or whether the lack of insulin production in the
mutant islets is secondary to the defect in β cell differentiation.
Whereas Nkx2.2 is involved in the terminal differentiation
of β cells, it appears to play a different role in the development
of the other islet cell types. In the Nkx2.2 null animals,
glucagon- and PP-expressing cells are easily detectable,
although at reduced numbers. This suggests there is a defect in
the specification or maintenance of these cell types, but not a
block in differentiation, as is the case for the β cells. The
reduction in α cell number is first evident at E9.5, when
glucagon can initially be detected, providing additional
evidence for a defect in α cell formation. Furthermore, these
cells do not appear to be present in an immature state, since
most, if not all, of the incompletely differentiated cells in
mutant islets express the β cell markers IAPP, PC1/3, and
Pdx1, but do not express the non-β cell marker Brn4. However,
since there are fewer markers for the α and PP cell types, we
cannot rule out the possibility that a small subset of these cells
are also trapped in a partially differentiated state.
From our studies, it is clear that the functional development
of the majority of α and PP cells require Nkx-2.2. However,
the fact that some α and PP-expressing cells remain in the
mutant islet clusters, although at reduced numbers, suggests
that there may be another regulatory factor that can partially
compensate for the function of Nkx2.2 in these cells.
Alternatively, since Nkx2.2 is not expressed in all α and PP
cells in wild-type animals, there may exist a small, but distinct
class of glucagon- or PP-expressing cells which form
independently of Nkx2.2. With our current limited
understanding of islet cell determination and lineage, and the
lack of additional α and PP cell molecular markers, it is
difficult to distinguish between these two possibilities.
It is not surprising that somatostatin expression is normal in
the Nkx2.2 null animals considering that Nkx2.2 is not
expressed in δ cells. Yet, since δ cells arise at E14.0 when
Nkx2.2 expression usually becomes restricted within the
pancreas, it is possible that exclusion of Nkx2.2 from δ cells
is a necessary step for their differentiation. However, in the
mutant embyros, the loss of Nkx2.2 does not appear to result
in a shift to the δ cell lineage, therefore the absence of Nkx2.2
alone cannot commit a cell to the δ cell lineage. It is likely that
the formation of a δ cell may require other, presumably
positive, signals in addition to the absence of Nkx2.2.
Endocrine cell lineage
There is now reasonably good evidence that exocrine and
endocrine cells arise from a common endodermal precursor
that expresses Pdx1 (Guz et al., 1995). Subsequently, these two
cell groups diverge and progress through distinct pathways of
2220 L. Sussel and others
differentiation. It is generally assumed that all islet cell types
develop from a common endocrine precursor and then pass
through intermediate precursors that give rise to subsets of
endocrine cell types, and several lineage models have been
proposed (Alpert et al., 1988; Guz et al., 1995; St.-Onge et al.,
1997). However, to date, no direct lineage tracing has been
performed in the pancreas, and the identification of precursor
cells and their descendants has been inferred through
expression studies of hormones and transcription factors. As
more expression patterns are analyzed, no unifying hypothesis
emerges, suggesting there is a greater degree of complexity in
lineage determination than expected. For example, our studies
of Nkx2.2 could be interpreted as suggesting that the δ cell and
β cell lineages are not closely linked, in direct contrast to the
conclusions drawn from studies of Pax4 (Sosa-Pineda et al.,
1997) and Pdx1 (Guz et al., 1995).
It seems likely that a more complicated mechanism is
responsible for the determination and differentiation of each
islet cell type. Each cell type population is specified by a
transcription factor code, where the presence of a particular set
of transcription factors determines cellular fate and the final
cellular phenotype. In this regard, it is interesting that Nkx2.2,
Pdx1 and Nkx6.1 are all initially expressed globally in the
pancreatic bud, but later become restricted to specific
endocrine cell types. The inactivation of the genes encoding
these factors clearly plays as important a role in pancreatic cell
type differentiation as their activation. The sequential
activation and inactivation of transcription factors may occur
in the different cell types as they progress through a common
lineage pathway, or as they differentiate along distinct, but
parallel, pathways. Furthermore, the cell type restriction of
these regulatory factors between E12.5 and E15.5 may be
analogous to the mechanism of lateral inhibition that restricts
neural cell differentiation during development of the nervous
system. Determining the lineage relationships among the
endocrine cell types will require conventional lineage tracing
studies, while careful analysis of transcription factor
expression patterns and function will reveal the mechanisms
that control these lineage patterns.
Hierarchy of transcription factors
Genetic studies are beginning to outline the hierarchy of
transcription factors involved in β cell development. For
example, in Nkx2.2 mutant embryos, early expression of the β
cell specific transcription factor Nkx6.1 is unaffected. However,
between E12.5 and E18.5, when there are major changes taking
place within the pancreas (differentiation of exocrine tissue; β
cell proliferation; δ and PP cell formation) Nkx6.1 expression
disappears. The data are consistent with a model where Nkx2.2
is required for the maintenance of Nkx6.1 expression as β cells
differentiate, and that continued expression of Nkx6.1 is
necessary for complete β cell differentiation.
The genetic relationship between Nkx2.2 and Pdx1 is more
complicated. Early in development, Pdx1 is expressed in all
cells of the pancreatic bud, and is required for expansion of the
bud (Guz et al., 1995; Jonsson et al., 1994; Offield et al., 1996).
However, later in embryonic development, Pdx1 becomes
progressively restricted to β cells (and some δ cells); and at
approximately E14.5, Pdx1 becomes upregulated in β cells
(Ohlsson et al., 1993) suggesting it plays a role in β cell
differentiation. In the Nkx2.2 mutant, early expression of Pdx1
is not affected. The later β cell restriction of Pdx1 expression
also occurs, but is quantitatively reduced in comparison to
wild-type β cells (see Fig. 6). Therefore, Nkx2.2 may be
required for inducing high level expression of Pdx1 in β cells,
and the up-regulation of Pdx1 may be a necessary step in the
final differentiation of the β cell.
In contrast to Nkx6.1 or Pdx1 expression, the expression of
Isl1, Pax6 and Brn4 during embryogenesis does not appear to
require Nkx2.2. These genes may therefore either lie upstream
or in different pathways relative to the Nkx genes. Furthermore,
since Isl1 is expressed in islet cells soon after they exit the cell
cycle (Ahlgren et al., 1997), normal expression of Isl1 in the
Nkx2.2 mutant suggests that all the islet cells are able to
normally exit a proliferating state and proceed with a program
of differentiation. This result supports the hypothesis that the
immature β cells are able to initiate β cell development and it
is subsequent steps of terminal differentiation that are blocked.
Additionally, immunohistochemistry using antibody directed
against proliferating cell nuclear antigen (PCNA) reveals a
normal number of proliferating cells in the mutant islet (data
not shown). Therefore, the incompletely differentiated β cells
do not result from the accumulation of an undifferentiated,
dividing precursor.
Given the importance of the islet transcription factor genes
in the development and maintenance of functional β cells, it is
not surprising that mutations in these genes have been
associated with the development of human diabetes. Maturityonset diabetes of the young (MODY) 1 and 3 are types of noninsulin-dependent diabetes mellitus (NIDDM) caused by
mutations in hepatocyte nuclear factor-4α (HNF-4α) and
HNF-1α, respectively (Yamagata et al., 1996a,b). MODY 4 is
linked to the human IPF1 (Pdx1) gene (Stoffers et al., 1997).
Analogously, partial defects in Nkx2.2, or in other islet
transcription factor genes could contribute to the etiology of
the more common late onset form of NIDDM (Type 2
diabetes). In contrast to human MODY and Type 2 diabetes,
Nkx2.2 null animals develop diabetes immediately after birth
due to the lack of insulin production, and homozygous
mutations of the islet transcription factors neuroD/BETA2 and
Pdx1 cause similar syndromes in mice (Naya et al., 1997;
Jonsson et al., 1994; Offield et al., 1996). Therefore these genes
and others involved in β cell differentiation should also be
considered candidate genes for the analogous human
syndrome, neonatal diabetes, a rare but devastating disease of
newborns (von Muhlendahl and Herkenhoff, 1995).
We thank members of the German lab and J. Long for critical
readings of the manuscript. We also thank C. Newgard (antiglucokinase), D. Steiner (anti-prohomone convertase 1/3), T. Jessell
(anti-Nkx2.2), H. Edlund (anti-Pdx1), M. G. Rosenfeld (anti-Brn4)
and S. Saule (anti-Pax6) for generously providing antibodies. The
islet1 monoclonal antibody developed by T. M. Jessell was obtained
from the Developmental Studies Hybridoma Bank maintained by the
University of Iowa under contract NO1-HD-7-3263 from the NICHD.
This work was supported by research grants from the NIH (M. S. G.).;
Nina Ireland, NARSAD and NIMH (J. L. R. R.); NIH (R. A. P.); and
Bank of America-Giannini Foundation, Scottish Rite Foundation and
NIH (L. S.).
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