1 Department of Molecular and Cellular Biology, Harvard University, Cambridge,
MA 02138, USA
2 Department of Pathology, Massachusetts General Hospital, Charlestown,
Massachusetts 02129, USA
Author for correspondence (e-mail:
dmelton{at}biohp.harvard.edu)
Accepted 30 September 2003
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SUMMARY |
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Key words: Myt1, endoderm, Pancreas, Endocrine, Islets, Microarray
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Introduction |
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The pancreas derives from the endoderm germ layer
(Pictet et al., 1972;
Slack, 1995
), which in mouse
is a cup of cells enveloping the mesoderm and ectoderm at embryonic day 7.5
(E7.5). At this time, the endoderm receives signals from adjacent mesoderm and
ectoderm and becomes competent to respond to subsequent permissive signals
that establish organ domains along the anterior-posterior axis
(Wells and Melton, 1999
). By
E8.5, the endoderm begins to form a primitive gut tube, and the region
destined to become the pancreas receives signals from the notochord and dorsal
aorta, leading to the expression of essential pancreatic transcription factor
genes such as pancreatic-duodenal homeobox 1 [Pdx1, also known as
insulin-promoter factor 1 (Ipf1)] (Hebrok
et al., 1998
; Lammert et al.,
2001
). At E9.0, Pdx1 expression marks both the dorsal and
ventral domains of the developing pancreas, and defines where pancreatic buds
will appear around E10 (Guz et al.,
1995
). As pancreatic buds expand and branch, signals from adjacent
mesenchyme direct cells toward an endocrine or exocrine fate
(Guz et al., 1995
;
Miralles et al., 1998a
;
Miralles et al., 1998b
). Cells
that have adopted an endocrine cell fate express the bHLH transcription factor
neurogenin 3 (NGN3) (Gu et al.,
2002
).
Functional studies have identified several signaling pathways and
transcription factors important for pancreatic development. Initial pancreatic
specification of endoderm is mediated by the FGF, hedgehog, Notch and
TGFß/activin signaling pathways (Kim
and Hebrok, 2001). These signals result in the expression of genes
for several transcription factors in the developing pancreas including,
HNF1
(Tcf1
), HNF1ß
(Tcf1ß), HNF4
(Tcf4
), Pdx1,
NeuroD1, Ngn3, Pax4, Pax6 and others
(Edlund, 1998
). Mutations in
some of these genes are associated with maturity onset diabetes of the young
(MODY 1, 3, 4, 5 and 6), and genetic analyses in mice have begun to elucidate
how these transcription factors function during discrete stages of pancreas
development (Stride and Hattersley,
2002
). For example, loss of PDX1 results in defects of both early
pancreatic specification and budding
(Jonsson et al., 1994
;
Offield et al., 1996
), whereas
loss of NGN3 results in specific absence of endocrine cell development
(Gradwohl et al., 2000
).
Moreover, cell lineage analysis supports the idea that PDX1 functions to
establish the three basic lineages of the pancreas (ducts, acini, islets),
whereas NGN3 functions specifically to establish the endocrine lineages
(Gannon et al., 2000
;
Gu et al., 2002
;
Herrera, 2000
;
Herrera et al., 1998
;
Schwitzgebel et al.,
2000
).
Analyses of individual genes have begun to define some critical stages in
the development of the endocrine pancreas, yet the complex interactions of
extracellular signals and the responding genetic networks that control
endocrine cell growth and differentiation are largely unstudied. For example,
it is not known how Pdx1 is induced and restricted to a defined
region of the developing gut, nor is it known how Ngn3 expression is
temporally controlled resulting in the genesis of endocrine progenitor cells.
Recently, 3,400 genes expressed in the pancreas were used to generate an
endocrine pancreas microarray (PancChip), which is available through the
ß Cell Biology Consortium (Scearce et
al., 2002). The PancChip will probably be a valuable diagnostic
tool for the genetic analysis of pancreatic cell samples. However, the focus
of the Endocrine Pancreas Consortium was not to provide a complete and
quantitative analysis of the genes that are expressed during the formation of
the endocrine pancreas. A transcriptional profile of pancreatic and endocrine
progenitors would provide fundamental information about the processes
regulating normal development of the endocrine pancreas. Moreover, regulatory
factors identified in this screen might be used to promote regeneration of
endocrine cells in vivo, or used to direct the differentiation of embryonic
stem cells or adult stem/progenitor cells toward the ß cell lineage in
vitro.
We describe the fundamental gene expression profiles of several tissue or
cell samples that define distinct stages during pancreatic and endocrine islet
development. We used high-density microarrays from Affymetrix to
systematically analyze the genes that are expressed at four key stages of
pancreatic and endocrine development: E7.5 unspecified endoderm, E10.5
pancreatic cells that express Pdx1, E13.5 endocrine progenitor cells
that express Ngn3, and mature islets of Langerhans. This genetic
analysis is uniquely designed in several ways. First, we used a combination of
dissection and cell-sorting using an eGFP reporter that was under the control
of promoters of specific pancreatic genes to isolate highly purified cells
from these well-defined stages of pancreatic development. Second, we compared
both the temporal and spatial expression profile at each stage to more fully
define these cell types. Third, we validated our profiles by demonstrating the
cell-specific expression of several genes from each time point by RT-PCR and
in situ hybridization (ISH). Finally, we demonstrated that one gene we
identified, myelin transcription factor 1 (Myt1), might be a novel
regulator of , ß and
cell development in the pancreas.
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Materials and methods |
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An XbaI-SphI (partial digestion) fragment that contains
sufficient Ngn3 enhancers (Gu et
al., 2002) replaced the Pdx1 enhancer region in
p#48 to generate the Ngn3-eGFP construct (p#63,
Fig. 1). Insert was released by
SalI digestion to generate three transgenic lines. After verifying
that eGFP expression mimics that of NGN3 by double ISH
(Gu et al., 2002
), one line
P#63.1 was used to obtain embryos for cell sorting.
|
Tissue isolation and cell sorting
To obtain purified endoderm, mesoderm and ectoderm tissue, E7.5 embryos
(90) were isolated from timed pregnant female ICR mice (Taconic, Germantown,
New York) and the endoderm was manually dissected from the mesoderm and
ectoderm with a polished tungsten needle
(Wells and Melton, 2000).
Isolated germ layers were combined into two pools. Each pool of isolated
endoderm and mesoderm and ectoderm contained approximately 0.2-0.4 µg total
RNA, which was used for cRNA probe generation
(Baugh et al., 2001
).
To isolate Pdx1-eGFP+ cells, ICR or CD-1 mice were crossed with P#48 males, and the eGFP-expressing E10.5 embryos were identified under a fluorescence microscope. The pancreatic rudiments and the stomach and duodenum (Std) anlagen were separated by dissection. These tissues were trypsinized to single cells and sorted into eGFP+ and eGFP populations by FACS. From 350 eGFP+ embryos, 1.3 and 1.8 million eGFP+ cells were collected from the pancreatic or Std region, respectively. Meanwhile, five million eGFP cells were also collected from both dissected samples. From these cells, 6, 8 and 14 µg total RNA was isolated and used for cRNA probe generation (each of these RNA were maintained in several small pools respectively). Ngn3-eGFP-expressing cells were isolated by a similar approach except that only the pancreatic rudiment was isolated, and the stage used was E13.5 (from 1300 eGFP+ pancreata, 1.3 million Ngn3-eGFP+ cells were collected and 5 µg total RNA was made and maintained as two pools).
Mouse islets were isolated by perfusing the pancreas with a collagenase
solution (2 mg/ml), filtering the digested pancreas though a 300 µM wire
mesh, and centrifugation on a histopaque 1077 cushion
(Warnock et al., 1990). Islets
were hand-picked to minimize contamination with exocrine tissue. For our
analysis, pancreata from five adult animals were used to obtain 30 µg total
RNA.
cRNA probe generation and hybridization to Affymetrix microarray chips
Total RNA samples were used to generate cRNA probes by two rounds of
transcription (Baugh et al.,
2001). Basically, a poly(dT) primer (with its 5' end
carrying T7 promoter sequence) was used to synthesize cDNA from total RNA. The
cDNA were used to amplify cRNA using T7 polymerase. The cRNA product from this
first round amplification was used to generate more cDNA by random priming,
with the 3' end carrying a T7 promoter sequence. This cDNA was used to
transcribe biotinylated cRNA, which was used to hybridize to the Mu11K,
Mu74Av1 or MU74Av2 microarrays produced by Affymetrix, following the
manufacturer's protocol.
Data normalization and analysis
Two programs were used to analyze the data generated from the microarray
hybridization.
First, using MicroArray Suite 5.0 (Affymetrix) image files were examined for uniform image quality without significant scratches or smudged fluorescence patterns. The images were processed into intensity data that was scaled per chip to a target intensity of 1500. Chip reports were examined for evidence of high quality and uniform RNA, RNA labeling, hybridization and scanning using approaches similar to those described at (http://cardiogenomics.med.harvard.edu/groups/proj1/pages/Method_QC.html). In brief, control oligonucleotide signal corresponding to spiked and constitutive RNAs were strong, uniform, sensitive and properly interpreted by the Affymetrix software. Background values were uniformly less than 100 and the scaling factor SF that is used to normalize the signal across the entire chip to 1500 signal units was within a twofold range for all chips. GeneSpring 5.0.1 (Silicon Genetics, Inc., Redwood City, CA) was used to analyze the resulting data values obtained from MicroArray Suite 5.0. The values used for filtering and clustering were `Signal', `Signal Confidence', `Absolute Call' (Absent/Present). Data were normalized as follows: the 50th percentile of all measurements was used as a positive control for each array. Each measurement for each gene was divided by this synthetic positive control, assuming that this was at least 10. The bottom tenth percentile signal level was used as a test for correct background subtraction. The measurement for each gene in each sample was divided by the corresponding value in untreated samples, assuming that the value was at least 0.01. Throughout our analysis, only the genes that display more than threefold change between samples were listed and studied (P=0.01 in at least one statistical test).
Chick embryo electroporation
Chick embryo electroporations followed the reported protocol
(Grapin-Botton et al., 2001).
Briefly, electroporation was performed on embryos between the 18- and
25-somite stage (i.e., stage 13-15 HH). Eggs were windowed and DNA (2
µg/µl DNA in 1xPBS, 1 mM MgCl2, 3 mg/ml
carboxymethylcellulose, 50 µg/ml Nile Blue Sulfate) was injected in the
blastocoel. A negative electrode was inserted below the embryo, and a positive
electrode was held by a micromanipulator above the embryo and three square
pulses of 17 volts for 50 mseconds each were applied (BTX T-820). After
electroporation, eggs were incubated at 38°C for 48-60 hours, then
collected and fixed in 4% paraformaldehyde/PBS, and sectioned for
immunohistochemistry or in situ RNA analysis.
Immunohistochemistry
Electroporated embryos were sectioned and analyzed for hormone expression.
Transgenic mouse embryos with the Ngn3 promoter driving
dnMyt1 expression were analyzed by insulin and glucagon expression.
The pancreata from five independently derived F1 transgenic E14.5
embryos were fixed, completely sectioned (6 µm sections), immunostained
with anti-insulin or glucagon antibodies, and the insulin+ and/or
glucagon+ cells were counted on alternate paraffin sections. As a
control, four littermate pancreata were counted in a similar fashion. Primary
antibodies used were guinea pig anti-insulin (Dako, Carpinteria, CA), guinea
pig anti-glucagon (Linco, St. Charles, MI) and rabbit anti-glucagon (Chemicon,
Temecula, CA). Secondary antibodies used were peroxidase-conjugated donkey
anti-guinea pig, FITC-conjugated donkey anti-guinea pig, and Cy3-conjugated
donkey anti-rabbit (Jackson Immunoresearch, West Grove, PA). In order to
obtain a significant number of insulin+ or glucagon+
cells, at least half of sections from each pancreas was counted.
RT-PCR and ISH
RT-PCR followed standard protocols. The primers used in our analyses were:
ApoAIV forward: 5'-aaggtgaagggcaacacggaag-3', reverse:
5'-cctcaagctggtacaagaagtgc-3'.
HPRT forward: 5'-gctggtgaaaggacctctc-3', reverse:
5'-cacaggactagaacacctgc-3'
(Johansson and Wiles, 1995).
Dkk1 forward: 5'-ggagatattccagcgctgtta-3', reverse:
5'-ggtaagtgccacactgaggat-3'.
Prss12 forward: 5'-agagagaggccacagaaaacag-3', reverse: 5'-ttgactccacatccataccccc-3'.
Eya2 forward: 5'-ttactcccattacccacgggtc3', reverse: 5'-gaagcctaaacaacgggcaaag-3'. Osteopontin forward: 5-gaagctttacagcctgcacccaga-3'; reverse: 5'-gcttttggttacaacggtgtttgc-3'; T7/osteopontin reverse: 5'-gtaatacgactcactatagggc aacagactaagctaagagccc-3'. Nkx2.2 forward: 5'-ccatgtcgctgaccaacacaaaga-3'; reverse; 5'-cgctcaccaagtccactgctgg-3'; T7/Nkx2.2 reverse: 5'-gtaatacgactcactatagggcggtgtgctgtcgggtactg-3'. Tm4sf3 (AF010499) forward: 5'-cagttccgctgtagcaatggctg-3'; reverse: 5'-cacacacactctaccactgagc-3'. T7/Tm4sf3 reverse: 5'-gtaatacgactcactatagggcagcacaaactacaaagaccca-3'. Spintz1 (AA57115): forward; 5'-gctgcaggcacacggatctctgc-3'; reverse: 5'-cagtgaatacctgtgaagatatc-3'. T7/Spintz1 reverse: 5'-gtaatacgactcactatagggcctcagtgagatacttcaataac-3'. Myt1 forward: 5'-gtctccggtggaagctcatggaca-3'; reverse: 5'-cttatggtgccctagtgtgtcatc-3'; T7/Myt1 reverse: 5'-gtaatacgactcactatagggccattaacataagagggtaa-3'. Rbp forward: 5'-ggctacatcataggtcccttttcg-3'; reverse: 5'-tactgcctctctaggcacagctc-3'; T7/Rbp reverse: 5'-gtaatacgactcactatagggctgtctctgggctcaggc-3'. Galphao forward: 5'-gcatgcacgagtctctcatgctc-3'; reverse: 5'-ctagacagactagcctgacatg-3'; T7/Galphao reverse: 5'-gtaatacgactcactatagggcgaggcgccaggcccag-3'. Foxa3 forward: 5'-ataaccatggctattcagcaggct-3'; reverse: 5'-cacaggtcaatcaagattgccaac-3'; T7/Foxa3 reverse: 5'-gtaatacgactcactatagggccatccaacatcacgaccatc-3'; actin control forward: 5'-atgccaacac agtgctgtctggtgg-3'; reverse: 5'-gcgaccatcctcctcttaggagtg-3'
Sectioned in situ analysis was performed as described previously
(Grapin-Botton et al., 2001).
Paraffin sections (6 µm) were collected on glass slides (Superfrost Plus),
dewaxed, treated with 1 µg/ml proteinase K for 7 minutes, and postfixed in
4% paraformaldehyde. Hybridization mix contained 1 µm/ml of probe, and
hybridization was done overnight at 70°C. Sections were washed in maleic
acid buffer and blocked with 20% lamb serum/2% Blocking Reagent (Boehringer
Mannheim, Indianapolis, IN) and incubated overnight with
anti-digoxigenin-alkaline phosphatase antibody (Boehringer Mannheim), 1:1000.
Slides were washed again and developed with NBT and BCIP.
Whole-mount ISH was performed as described previously
(Wilkinson and Nieto, 1993).
Briefly, E7.5 embryos were fixed, dehydrated in methanol, rehydrated, treated
with 6% hydrogen peroxide, proteinase K treated for 1.5 minutes, and postfixed
in 4% paraformaldehyde. Embryos were hybridized in buffer containing 1
µg/ml probe overnight at 70°C. Embryos were washed and incubated
overnight with an anti-digoxigenin antibody (1:1000). Embryos were developed
with BM purple (Boehringer Mannheim). Probe templates for ApoAV, Dkk1, Prss12
and Eya2 were generated by PCR amplification from an E7.5 endoderm library
(Harrison et al., 1995
) using
a gene-specific forward primer (mentioned above), and a vector specific
(pSPORT) reverse primer. The resulting amplified product contained the
3' end of the gene and an SP6 polymerase site from the pSPORT vector.
The amplified products were verified by sequencing and used in an in vitro
transcription reaction to generate antisense probes. To generate cRNA probes
for Foxa3, galphao, osteopontin, Myt1, Nkx2.2, Rbp, Spintz1,
and Tm4sf3, T7-reverse primers (has T7 promoter sequence at the
5' end, see above) were used to amplify cDNA fragments with
corresponding forward primers.
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Results |
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The stages shown in Fig. 1
were chosen for the following reasons. (1) E7.5 endoderm. At E7.5, the
endoderm is a sheet of cells on the outside of the embryo. At this stage,
endoderm cells are plastic and are not yet determined to form the pancreas
(Wells and Melton, 2000).
Analysis of undifferentiated endoderm should provide a genetic baseline and
highlight genes involved in endoderm plasticity and pancreas differentiation.
(2) Pdx1-expressing cells of the E10.5 pancreatic rudiment.
PDX1+ cells will yield both the exocrine and endocrine components
of the adult pancreas and are therefore considered pancreatic progenitor cells
(Gannon et al., 2000
;
Gu et al., 2002
). At this
stage, PDX1+ cells are also found in the stomach and duodenum
(Offield et al., 1996
). A
transcriptional analysis of PDX1+ cells from the pancreas versus
PDX1+ cells from the stomach and duodenum, or from
PDX1 cells, should highlight genes that specify
pre-pancreatic cells from their gastrointestinal neighbors. (3) Endocrine
progenitor cells (NGN3+) of the E13.5 pancreas. The cells that
express Ngn3 at this stage will form only endocrine tissue
(Gu et al., 2002
). A
comparison of the transcriptional profile of NGN3+ cells with
NGN3 cells was aimed at distinguishing the endocrine and
exocrine compartments of the embryonic pancreas. (4) Adult islets. Adult
islets represent mature, differentiated endocrine cells and will highlight the
genes that need to be up-regulated, as well as down-regulated, in order to
form the endocrine pancreas. This experimental approach was designed to
quantitatively identify genes that are temporally and spatially regulated
during endocrine development.
The following methods were used to obtain tissue samples for
transcriptional analysis. (1) The endoderm from 90 E7.5 embryos was manually
separated from mesoderm/ectoderm. (2) The mouse Pdx1 promoter, which
recapitulates the endogenous Pdx1 expression
(Gu et al., 2002;
Wu et al., 1997
), was used to
drive expression of eGFP, and eGFP expression was used to FACS sort
PDX1+ from PDX1 from dissected pancreas, stomach
and duodenum. A total of 1.3x106 or 1.8x106
Pdx1-eGFP+ cells (from pancreatic or stomach/duodenumal regions,
respectively) were isolated from 350 E10.5 embryos. The trypsinization of
tissue before cell sorting did not alter the ability of these cells to
differentiate into insulin-producing cells in vitro (G.G. and D.A.M.,
unpublished data), nor did it dramatically alter the presence or absence of
predicted gene expression in this analysis. However, we cannot rule out the
possibility that the expression levels of some genes were altered by this
isolation method. (3) The Ngn3 promoter, which recapitulates
endogenous Ngn3 expression (Gu et
al., 2002
), was used to drive eGFP expression in endocrine
progenitor cells. 1.3x106 Ngn3-eGFP+ cells were
collected from 1,300 E13.5 embryos. (4) Islets were isolated from 10 adult
mice. All tissue or cell samples were separated into duplicates and used to
generate labeled cRNA samples using an in vitro transcription-based linear
amplification protocol (Baugh et al.,
2001
). Amplified RNA samples were hybridized to the Affymetrix
microarrays (Materials and methods), and the data were analyzed using
GeneSpring and Resolver clustering analysis software. Genes expressed at each
stage of development were grouped according to biological function
(Fig. 2B and tables), and
separated into classes that are temporally or spatially regulated during
endocrine development. Genes that were expressed in the pancreas, but were not
temporally or spatially regulated were generally not listed in the tables (see
supplemental data for a complete listing of genes expressed in these samples:
http://dev.biologists.org/supplemental).
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Endoderm cells express many transcripts involved in pattern-formation
E7.5 endoderm expresses 193 genes or ESTs (out of the 12,000 on the
microarray) at greater than threefold higher levels than cells at later stages
of pancreas development. These include 25 growth factors or other
signaling-related molecules and 44 transcription factors or other nuclear
proteins (Fig. 2). Many of
these factors were previously implicated in embryonic pattern formation. For
example, endoderm expresses molecules involved in TGFß signaling,
including Nodal, cerberus 1, and follistatin and the Wnt antagonist
dickkopf (Bouwmeester et al.,
1996
; Conlon et al.,
1994
; Mukhopadhyay et al.,
2001
). Endoderm-expressed transcription factors including
Cdx1, Hesx1, Irx3, Gata3, MespI and Sox17 (see Table S1,
http://dev.biologists.org/supplemental).
In addition, we have implicated several new signaling pathways in endoderm and
pancreatic development by virtue of their abundant expression. Some examples
include the cKit ligand, Edg2 (G-protein coupled receptor)
and Epha2 (Eph receptor A2). The cKit pathway is known to
function during hematopoiesis and germ cell migration and development
(Ueda et al., 2002
) and both
of these processes involve interactions with endoderm. Thus, the role of
endodermally expressed cKit may be restricted to hematopoietic and
germ cell development.
Gene expression complexity decreases as cells become restricted to the pancreatic lineage
The PDX1+ cells of the E10.5 pancreas (precursors to all
components of the developing pancreas) expressed 60 genes at enriched levels
(Fig. 2), a smaller number than
the endoderm-specific genes. This is consistent with the PDX1+
cells being a fate-restricted population while the endoderm cells contain
progenitors for all endoderm-derived organs
(Wells and Melton, 1999).
Examination of these genes suggested that Notch activity and Wnt signaling
might play roles in promoting endoderm to adopt a pancreatic fate, since the
genes for the Notch ligand Dlk1 and Wnt signaling antagonist
Sfrp1 were highly expressed in these PDX1+ cells
(Table 1). In addition, genes
for several transcription factors, including Barx1, Nkx6.2, Onecut1,
Sox11 and a few other zinc finger proteins were highly expressed in the
PDX1+ cells. Several ECM proteins, including collagens I1,
I
2, V
2, tenascin and vinin 1 were also highly enriched in the
PDX1+ cells, suggesting that these molecules could be involved in
the budding process of the early pancreatic epithelium (reviewed by
Kim and Hebrok, 2001
).
|
Genes expressed in adult islets
The islet preparation contained the four major endocrine cell types,
endothelial cells, some exocrine cells, and other cells that contaminated the
islet preparations. We found that the expression of 217 genes
(Fig. 2; Table S1,
http://dev.biologists.org/supplemental)
were enriched at this stage, and most of these are associated with the
function of the adult organ. Among these, the transcripts for four endocrine
hormones, hormone processing enzymes, secretory apparatus, prolactin receptor,
REG1 and REG3, were found at very high levels. In addition, we identified the
novel expression of numerous regulatory molecules in adult islets (Table S1,
http://dev.biologists.org/supplemental).
Genes for the transcription factors that were expressed include activating
transcription factor 5 (Atf5), myelin transcription factor 1-like
(Myt1l), putative homeodomain transcription factor (Phtf),
and short stature homeobox 2 (Shox2 also Prx3). Although the
role of these transcription factors in islet function or maintenance is not
known, Atf5, Mytl1 and Shox2 are all expressed in the CNS,
implicating them in neuroendocrine as well as pancreatic endocrine development
and function (Angelastro et al.,
2003; Kim et al.,
1997a
; van Schaick et al.,
1997
). There were also components of several signaling pathways
expressed, including Notch 4, inhibin
, Wnt4, leukemia inhibitory
factor receptor, and epidermal growth factor, to name a few. These molecules
and pathways are possibly involved in regulation of islet size, function and
perhaps maintenance.
The adult islets also expressed many of the same transcription factors that
function in embryonic pancreatic development. One example is Pdx1,
which is expressed in entire embryonic pancreas, but is restricted to ß
cells in the islets. PDX1 was shown to regulate expression of several genes in
islets including insulin, glucagon, somatostatin, islet amyloid polypeptide
(Iapp), glucokinase and Glut2
(Brissova et al., 2002;
Perfetti et al., 2001
). PDX1
is also implicated in ß cell maintenance in the adult
(Sharma et al., 1999
;
Wells and Melton, 1999
),
suggesting that one additional role of some embryonic transcription factors
might be maintain progenitor cells in the adult.
Gene expression levels as an indicator of differentiation, plasticity and transformation
As endocrine progenitor cells differentiate and form islets, the number of
transcriptional and growth factor molecules expressed in endocrine cells
decreased. These data suggest that maintenance of progenitor cell plasticity
may depend on low-level expression of multiple regulatory genes.
Alternatively, the fact that progenitor cells expressed numerous regulatory
genes at low levels could reflect the heterogeneity of the progenitor pools.
Analyses of genes expressed in single cells of the E10.5 pancreas suggested
that Pdx1-expressing cells are a relatively heterogeneous population
(Chiang and Melton, 2003).
Another interpretation of that data is that only a subset of PDX1+
cells are specified toward pancreatic lineages and the remainder are still
plastic. This idea is supported by cell lineage studies which demonstrated
that many of the cells of the embryonic pancreas, once thought to be
pancreatic progenitor cells, never actually contribute to the adult organ
(Herrera, 2000
).
To identify additional genes that might regulate early cell plasticity, we performed a clustering analysis to identify genes that were down-regulated as a function of differentiation. This cluster of genes contains many known regulators of differentiation, proliferation and plasticity during development (Fig. S1; Table S2, http://dev.biologists.org/supplemental). Included in this cluster of `down-regulated genes' were numerous tumor-associated genes such as Tera (teratocarcinoma expressed, serine rich), Tacc3 (transforming acidic coiled coil containing protein 3), Ptov1 (prostate tumor over expressed 1), Tacstd2 (tumor-associated calcium signal transducer 2), Trap1a (tumor rejection antigen 1), Frat1 (frequently arranged in advanced T-cell lymphomas), and Lag (leukemia associated gene). Although the function of these factors in pancreas development is unknown, they were all identified by their abundant expression in different types of tumors and are thus implicated in cellular transformation.
Analysis of genes that are spatially restricted during islet cell development
Our temporal analysis of gene expression identified genes that were known
to regulate temporal cell differentiation during endocrine cell development.
However, it is equally important to identify the genes that define developing
pancreatic cells from their neighbors. For example, how are PDX1+
cells of the pancreas different from the PDX1+ cells of stomach or
duodenum, and how do the NGN3+ cells differ from
NGN3 cells? To catalog the genes that control these cell
fate decisions, we have generated a transcriptional profile from developing
endocrine progenitor cells and from adjacent cells at each stage of
development (Fig. 1, green
boxes).
Genes differentially expressed in the early endoderm as compared to mesoderm and ectoderm
In order to identify the genes whose expression is spatially restricted to
endoderm at E7.5, we compared gene expression profiles between E7.5 endoderm
and mesoderm plus ectoderm (Fig.
1, green box 1). We identified 203 transcripts that are greater
than threefold enriched in endoderm, while 262 were enriched in the mesoderm
plus ectoderm (Fig. 3,
Table 2; Table S3,
http://dev.biologists.org/supplemental).
We have verified endodermal expression of 25 genes by RT-PCR, and 17 of these
were further analyzed by ISH analysis (Fig.
3; Table S6,
http://dev.biologists.org/supplemental).
The gene expression patterns shown in Fig.
3 (ApoAIV, Dkk1, Prss12, and Eya2) are
representative examples of genes that were expressed in endoderm. The
expression of these genes in endoderm validates that our approach was
successful in identifying endodermally expressed genes.
|
|
Genes differentially expressed in PDX1+ cells of the pancreas, stomach and duodenum
Pdx1 expression marks all pancreatic progenitors of the E8.5-10.5
pancreas (Gannon et al., 2000;
Gu et al., 2002
). Yet,
Pdx1 is also expressed in cells in rostral stomach, and the mucosal
cells of the duodenum (Offield et al.,
1996
), demonstrating that additional factors are necessary to
specify pancreatic fate. We isolated PDX1+ cells of the pancreas,
stomach and duodenum to identify the genes that are specifically expressed in
pancreatic progenitor cells (Fig.
1, green box 2). Cell lineage analyses have demonstrated that
PDX1+ cells in the pancreatic buds at E10.5 give rise to all
pancreatic tissues whereas the PDX1+ cells in the stomach/duodenum
rudiment do not give rise to pancreatic tissues
(Gu et al., 2002
). We also
analyzed PDX1 cells from the mesoderm surrounding the
pancreas, stomach and duodenum. These include the mesenchymal cells
surrounding the endoderm and PDX1 epithelial cells.
We identified the transcripts that are enriched in the pancreatic PDX1+ cells by comparing the expression profile of these cells with that of the combined expression profile of the PDX1+ cells of the stomach and duodenum, as well as that of the PDX1 cells. This clustering analysis identified 158 genes that are enriched in the PDX+ pancreatic buds. 208 transcripts were enriched in the stomach, duodenum, and the PDX1 cells (Fig. 4A and Table 3; Table S4, http://dev.biologists.org/supplemental). We verified the expression pattern of 25 candidate genes whose transcripts were enriched in the PDX1+ pancreatic cells by RT-PCR (25-30 cycles) and 12 by ISH. We determined that the transcripts of 21 of the 25 genes were highly enriched in the pancreatic epithelium, compared to that of the duodenum or stomach and surrounding mesenchymes. Expression of the remaining four candidates was not detectable in any tissue (Fig. 4; Table S6, http://dev.biologists.org/supplemental). Similarly, 15 of the 18 candidate genes whose transcripts were enriched in the nonpancreatic cells were found by RT-PCR and/or ISH to be enriched only in the mesenchyme, stomach or duodenum (Table S6, http://dev.biologists.org/supplemental). The remaining three transcripts were not detected in any tissues (Fig. 4B-E and data not shown). We increased the number of PCR cycles in our analysis to 45 and found that we could detect the seven low-abundance transcripts. Data from our Affymetrix analysis predicted these seven genes to be expressed at low levels.
|
|
Identification of new pathways or factors that are expressed in pancreatic PDX+ cells
Several genes that were not known to be involved in pancreatic development
were found to be expressed by the pancreatic PDX1+ cells. Examples
include a G protein (RhoB), a related signaling member [diaphonos
homolog 1 (Dab1)] and calmodulin (Cldn,
Table 3). Because Rho plays an
essential role in focal adhesion formation, another molecule, FAK (focal
adhesion kinase), also detected in these cells, (data not shown) may interact
with the four above-mentioned molecules to control the morphogenesis of the
pancreatic rudiment.
Genes differentially expressed in early endocrine (NGN3+) progenitors
Pancreata from E13.5 embryos were dissected from animals expressing eGFP
from the Ngn3 promoter, and cells were dissociated and separated into
NGN3+ and NGN3 cells based on their eGFP
expression [The Ngn3 promoter used in these experiments recapitulates
endogenous Ngn3 expression (Gu et
al., 2002)]. We determined that 204 genes were enriched in
endocrine progenitors, as compared to 256 genes that were enriched in
non-Ngn3-expressing cells (Fig.
5A, Table 4; Table
S5,
http://dev.biologists.org/supplemental).
All genes known to be important for islet development were detected at high
levels only in the NGN3+ cell samples (Table S5a). In addition,
transcripts of many genes not previously identified as playing roles in
endocrine development were also enriched in the Ngn3-eGFP+ cells.
In the Ngn3-eGFP cells, Ngn3 transcripts were not
detected, demonstrating that our sorted Ngn3-eGFP pool was
devoid of Ngn3-expressing cells. We used ISH to analyze the
expression pattern of 18 candidate genes whose transcripts were only present
in endocrine progenitor (NGN3+) cells. 12/18 candidates analyzed
were expressed in a scattered cell population in the E10.5, E12.5 and E15.5
pancreat ic rudiments (Fig.
5B-E; Table S6,
http://dev.biologists.org/supplemental),
an expression pattern that is highly similar to that of Ngn3
(Gradwohl et al., 2000
). Six
of the 18 candidates cannot be detected by ISH, possibly because they are
expressed at low levels, which would be consistent with their low
hybridization intensity on the microarray (data not shown).
|
|
Several G-protein signaling components were enriched in endocrine progenitors
Transcripts encoding several G protein-coupled receptors (GPR27
and GPR56) and multiple guanine nucleotide-binding proteins,
including G0, Rab3D, Rab7 and a GDP
dissociation inhibitor (Table
4), were highly enriched in the NGN3+ cells.
Transcripts for several calcium signaling-related molecules, a calcium-binding
protein (ALG2), a calcium-dependent activator (Cadps),
calcium-dependent kinase II (Camk2b), and a calcium-independent
phospholipase A II (Pla2g6) were also highly enriched in endocrine
progenitors. The presence of these molecules suggests that G-protein-mediated
signaling, through receptor GPR27 or GPR56, and calcium mediated signaling
might participate in endocrine development or function.
Components of the notch-signaling pathway are expressed by endocrine progenitor cells
Our screen not only revealed the presence of the transcripts for Notch
signaling members, but we also discovered that of a Notch modifier, manic
fringe (Mfng) and a transcription factors, Myt1
(Bellefroid et al., 1996) that
participate in Notch signaling (Table
4). This finding suggests that Mfng and Myt1
could be involved in endocrine cell development.
Signaling molecules expressed by NGN3 cells
The NGN3 cells included several tissue types, such as
progenitor cells that had not been specified toward the endocrine cell fate
(by virtue of its Ngn3 expression), precursors that give rise to the
exocrine pancreas, and mesodermally derived tissues within the pancreas.
Consequently, diverse signaling pathways were found to be expressed by the
NGN3 cells. Transcripts enriched in
Ngn3-eGFP cells included the endothelin receptor, PDGFR,
thrombin receptor, which are known for hematopoietic development. However it
is still possible that these genes are important for endocrine
development.
Analysis of Myt1 function during endocrine cell development
One goal of our gene expression analysis was to identify new genes that are
functionally involved in endocrine islet development. Of the genes whose
transcripts are enriched in the endocrine progenitors, one gene,
Myt1, is a promising candidate regulator of endocrine development. In
Xenopus laevis, xMyt1 has been shown to cooperate with xNgn1 to
induce neurogenesis (Bellefroid et al.,
1996). Because islet development has many similarities with that
of neuronal development (Gu et al.,
2003
), we wanted to determine whether Myt1 is involved in
endocrine differentiation.
The Myt1 locus produces two isoforms by utilizing alternative
transcriptional starts, Myt1 (noted as Myt1a) and
Nzf2b, both containing C2HC zinc fingers and a
transcriptional activation domain. These two isoforms differ in their
N-terminal 100 amino acid residues
(Matsushita et al., 2002),
such that NZF2b has an extra zinc finger (MYT1a has 6 zinc fingers and NZF2b
has 7 zinc fingers). For simplicity, we refer to both RNA isoforms from the
Myt1 locus as Myt1, and we refer to the 6-zinc-finger
Myt1 cDNA as Myt1a (Kim
et al., 1997a
; Matsushita et
al., 2002
). Our semi-quatitative RT-PCR results showed that
Myt1a and Nzf2b are both expressed in the developing
pancreas, with Nzf2b being expressed at much higher levels (data not
shown). In situ analysis using probes common to both isoforms demonstrated
that Myt1 is expressed in a few cells of the developing gut (E8.5)
where the pancreatic buds will form [between the seventh and ninth somites,
adjacent to the dorsal aorta (Fig.
6B and data not shown)], as well as in the nervous system
(Fig. 6B). As embryogenesis
proceeds, Myt1 is expressed in the pancreas in a similar fashion to
that of Ngn3, i.e. in a scattered subset of epithelial cells that are
adjacent to or within characteristic duct-like structures
(Fig. 6C). After E15.5,
Myt1 transcripts were considerably reduced
(Fig. 6D), yet not abolished
(longer exposure of these tissue sections yields positive Myt1 mRNA
hybridization signals, data not shown). The expression pattern of
Myt1 suggests that it functions, like Ngn3, during the early
stages of endocrine cell specification. We utilized gain-of-function and
loss-of-function approaches to determine if Myt1 was involved in
development of the endocrine pancreas, using both mouse and chicken embryos as
model systems.
|
|
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Discussion |
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New signaling molecules in endocrine development
In addition to those genetic pathways known to play a role in pancreatic
development, our results have newly implicated several additional pathways
(Table 6). In endoderm, we
detected the expression of a genetic network that has been well studied in eye
development. This network includes the genes Eyes absent 2 (Eya2) and
Sine oculis-related homeobox 1 (Six1) that genetically interact
during eye development in flies and mice
(Heanue et al., 1999;
Pignoni et al., 1997
;
Ridgeway and Skerjanc, 2001
).
In addition, we have identified multiple components of the Wnt
pathway, including Wnt ligands, Wnt receptors
(Fzds), Wnt receptor antagonists [secreted frizzled-related
1 and 3 (SfrPs)], and its downstream targets (Dvls) in early
endoderm, general pancreatic progenitors and endocrine progenitors. In
PDX1+ cells in the pancreatic region (E10.5), RhoB, Dab1,
Cldn and FAK are all expressed at enriched level. These genes
have been shown to function in modifying cell cytoskeleton and they might be
involved in pancreatic epithelia morphogenesis. In the endocrine progenitors,
we found the specific expression of G-protein cascade, GPR14, GPR27,
GPR56, Ga0, Rab3d, Rab7 and a GDP dissociation factor genes.
These factors might interact with each other and participate in endocrine
lineage differentiation. In addition, members of the calcium-activated
signaling cascade may also participate in islet formation or function.
|
The same signaling molecules regulate different cell fate decisions
We detected components of all common signaling pathways in each cell
population representing different stages of islet generation (data not shown).
Several specific growth factor receptors are expressed at each stage of
development, yet probably direct the expression of different target genes,
depending on the cell in which it is expressed. For example, cells at all
stages analyzed expressed the activin receptor 2b. Activin/TGFß signaling
can be regulated by extracellular modifiers like Cer1, and receptors can
transduce a signal via several different downstream Smads. It is
therefore easy to speculate that the response of any given cell to activin
signaling depends on many other cell-intrinsic and extrinsic factors according
to the levels of signal strength and/or the competence factors present in the
cells. This highlights the belief that a relatively small number of regulatory
molecules can be used to determine the eventual cell type.
Global trends in gene expression to study complex biological processes
Our transcriptional profile of the developing endocrine pancreas has
generated a quantitative gene expression database that can be used to analyze
complex gene expression networks that would be impossible to study by other
strategies. For example, our analysis suggests that the most plastic cell
type, E7.5 endoderm, expressed many genes involved in cell fate specification,
and the number of these genes becomes progressively fewer as endocrine cells
begin to differentiate. Adult endocrine cells expressed the fewest number of
cell fate regulatory genes but abundantly expressed genes associated with the
adult function of the islets. The progressive decrease in the number of
cell-fate regulators during endocrine development is consistent with the
hypothesis that differentiation is a function of cells becoming progressively
restricted toward one lineage. We identified another group of genes that were
down regulated as a function of differentiation (Fig. S1 and Table S2,
http://dev.biologists.org/supplemental).
There is a significant number of tumor associated genes associated with this
gene cluster, suggesting that the genetic machinery underlying cell plasticity
in the embryo might overlap with the genes involved in the
`de-differentiation' that occurs during oncogenesis.
Expression data from these experiments will be available at www.genet.cchcc.org and can be directly compared to the expression profiles generated from other studies to look for informative biological trends between cell types and across organ systems. For example, a comparison of expression profiles between the developing and regenerating pancreas, or between two branching organs such as the pancreas and kidney could potentially uncover molecular trends associated with these processes.
Gene discovery: markers and regulators of developing pancreatic progenitor cells
Given the current research emphasis on deriving functional islets from stem
or other cell types, the identification of new endocrine regulatory genes and
markers is timely. There is ample evidence suggesting that many of the genes
involved in endocrine pancreatic development also function in the homeostasis
of the adult islet (Wells,
2003). It was our intention that a transcriptional profile of the
developing endocrine pancreas would be an important resource for the diabetes
research community. The genes identified in this study should facilitate
analysis of the putative stem cells identified in the pancreatic ducts
(Abraham et al., 2002
;
Cornelius et al., 1997
;
Ramiya et al., 2000
;
Zulewski et al., 2001
). In
addition, this catalog of signaling molecules and transcription factors
expressed during endocrine development will expedite attempts to promote stem
cell, embryonic or adult, differentiation into the islet cell lineage
(Hori et al., 2002
;
Lumelsky et al., 2001
).
Our temporal and spatial analysis of genes expressed in embryonic endocrine cells has generated a database of potential progenitor cell markers. For example, we have cross referenced our spatial and temporal analysis of genes expressed in E7.5 endoderm and identified 60 genes that were both spatially and temporally restricted to E7.5 endoderm (Table S1, http://dev.biologists.org/supplemental). These include genes of known endodermally expressed factors (Sox17, Foxa2, Dkk1, Cer1), and novel markers of endoderm (Eya2, cKit ligand, Prss12). Similar analyses revealed temporally and spatially restricted expression of genes in pancreatic and endocrine progenitor cells. We identified 16 genes that are highly enriched or only expressed in E10.5 PDX1+ cells of the pancreatic rudiment (Table 4; Table S1, http://dev.biologists.org/supplemental), and 36 genes whose expression was temporally and spatially enriched in NGN3+ endocrine precursors (Table 4; Table S1, http://dev.biologists.org/supplemental).
Thus far, we have not identified any genes that are exclusively restricted
to developing endocrine cells. For example, Eya2 and Kit
ligand are expressed in E7.5 endoderm and in other tissues at later stages of
development (Godin et al.,
1991; Motro et al.,
1991
; Xu et al.,
1997
). Ngn3 and Myt1 are both expressed in
endocrine progenitor cells, as well as a set of neural progenitors in the
developing nervous system (Apelqvist et
al., 1999
; Gradwohl et al.,
2000
; Kim et al.,
1997b
). It is possible that some of the ESTs in our database are
truly expressed in a cell-specific manner. Alternatively, our results suggest
that embryonic precursor cells seem to express many genes as a function of
maintaining plasticity, where as adult islets expressed cell-type-specific
genes. Nonetheless, the expression of a combination of several genes within
each group may provide us with a diagnostic standard to determine whether
cells are of endoderm, general pancreatic progenitor, endocrine precursors or
mature islets.
Myt1 function might be necessary for endocrine islet development
Other than revealing general gene expression trends during islet
development, our analysis also uncovered many candidate genes whose function
could be required for islet development. One such example is Myt1.
Our results suggested that the NZF2b isoform of Myt1
promotes the formation of glucagon and somatostatin-expressing cells when
ectopically expressed in chicken embryonic gut endoderm when it is expressed
in developing endoderm (before endogenous pancreatic cells appear). MYT1a and
NZF2b seemingly have different activity with regards to regulating the
formation of hormone-expressing cells (insulin, glucagon and somatostatin).
Differences in their activity could be due to differential stability of the
proteins, or differences in post-translational modification or nuclear
localization, or their different DNA binding activity.
We have also demonstrated that a dominant negative MYT1 partially inhibits
endocrine cell differentiation in transgenic mouse embryos and efficiently
inhibits NGN3 activity in chicken gut endoderm. Combine with the ectopic gene
expression analysis, these results suggest that Myt1 is involved in
endocrine islet differentiation, and may function along the same pathway as
NGN3. Although the ß- and -differentiation-inhibitory effect of
dnMYT1 could result from the functional inhibition of other MYT1-like
molecules, the other two Myt1 homologues, Myt1l
(Kim et al., 1997a
) and
Myt3 (GeneBank acc. no.: BC032273) are not expressed in the
developing pancreas (data not shown).
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Program of Developmental Biology, Department of Cell and
Developmental Biology, Vanderbilt Medical Center, Nashville, TN 37232, USA
These two authors contributed equally to this work and are listed
alphabetically
Present address: Division of Developmental Biology, Cincinnati Children's
Research Foundation, Cincinnati, OH 45229-3039, USA
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