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First published online 13 September 2006
doi: 10.1242/dev.02537
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1 Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
2 Massachusetts General Hospital, Department of Medicine, 55 Fruit Street,
Boston, MA 02114, USA.
3 Department of Medicine, Harvard Medical School, 25 Shattuck Street, Boston, MA
02115, USA.
4 Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts
Avenue, Cambridge, MA 02139, USA.
5 Department of Neuroscience, Harvard Medical School, 25 Shattuck Street,
Boston, MA 02115, USA.
6 Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.
Author for correspondence (e-mail:
ramesh_shivdasani{at}dfci.harvard.edu)
Accepted 18 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Transcription factor, Organogenesis, Differentiation, Isx, Stomach, Intestine, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gilbert, 2000). Thus, an
essential step in studying the molecular basis of differentiation is to
identify TFs that are restricted in distribution and regulated during
development. Historically, this goal has relied on evolutionary conservation
of transcriptional regulatory pathways and enrichment of specific cis-elements
in batteries of tissue-selective gene loci. Coupled with gain- and
loss-of-function studies in model organisms, identification of crucial TFs has
improved understanding of differentiation in tissues ranging in complexity
from echinoderm and vertebrate endoderm to whole organs
(Brent and Tabin, 2002;
Cripps and Olson, 2002;
Davidson et al., 2002;
Inoue et al., 2005;
Orkin, 2000;
Shivdasani, 2002;
Wilson et al., 2003;
Zhu et al., 2005). We report a
novel approach to identify developmentally regulated tissue-restricted TF
genes, as applied in the mouse gastrointestinal (GI) tract.
The gut mucosa serves digestive, metabolic and barrier functions. It arises
and operates in intimate contact with mesenchyme and provides a useful model
to investigate tissue processes such as mesenchyme-epithelial interactions,
establishment and renewal of stem-cell compartments, and specification of
daughter lineages. Whereas gut development and homeostasis rely on many of the
same pathways that regulate other organs
(Radtke and Clevers, 2005),
the basis for cell-specific responses to the limited signal repertoire and the
range of tissue-restricted TFs remain unclear. Few such TFs are implicated
directly in regulating gut epithelial stem cells (Tcf4)
(Korinek et al., 1998) or
differentiated lineages: for example, Math1 and Hes1 in secretory cells
(Jensen et al., 2000;
Yang et al., 2001), KLF4 in
colonic goblet cells (Katz et al.,
2002), and Cdx2 in enterocytes
(Beck, 2004). Other
GI-restricted TFs, particularly homeodomain (HD) proteins, define boundaries
between discrete gut segments (Aubin et
al., 2002; Kim et al.,
2005; Offield et al.,
1996; Roberts et al.,
1998; Zakany and Duboule,
1999). However, current appreciation of TF activities and
hierarchies in the mammalian GI tract lags behind that in other tissues
(Cripps and Olson, 2002;
Lee and Pfaff, 2001;
Orkin, 2000;
Zhu et al., 2005), in part
because many key factors remain unknown.
We conducted an unbiased survey of the developing mouse gut for mRNA expression of all known and predicted DNA-binding proteins; transcript levels were evaluated over the period of greatest morphological change in fetal intestine and stomach. The ensuing analysis serves as a comprehensive assessment of TF gene expression in development of a mammalian organ. Some protein families are represented extensively and others sparingly, and we identified previously unknown gut expression of many known and novel TFs. Only a few dozen TF mRNAs reveal substantially different levels in the developing stomach and intestine or levels that increase with maturation of these organs. Such factors may be especially important for tissue differentiation, and we used various methods to isolate those expressed selectively in the gut, including a novel TF, intestine-specific homeobox (Isx). Targeted disruption of Isx in mice reveals its requirement in intestine-specific regulation of the high density lipoprotein (HDL) receptor and cholesterol transporter scavenger receptor class B, type I (Scarb1; previously SR-BI). Our approach thus uncovers promising candidates for transcriptional control of GI differentiation and outlines a general strategy to study a whole gene class in development or disorders of particular organs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-[32P] dCTP. For the expression survey, solutions containing all components required for PCR, except primers, were distributed into 96-well plates by a pipetting robot that subsequently dispensed PCR primers so that pairs specific for each TF were applied to a complete set of eight samples. PCR reactions were run to 31 cycles using Hi-Fidelity Taq polymerase (Invitrogen) on a Tetrad thermal cycler (MJ Research) at 62°C annealing temperature. PCR products were resolved on pre-cast 1% agarose E-gels (Invitrogen) containing ethidium bromide and imaged under identical conditions for all gels. Pixel intensities for each band were summated using Quantity One software (BioRad) (data not shown, but see http://genome.dfci.harvard.edu/~mhu/GIFT). Because E-gels permit multiple PCR products to be compressed into a single band, we resolved the PCR products from 33 TF mRNAs by conventional electrophoresis in 1% agarose. Only three out of the 33 reactions returned more than 1 band, and in every case the dominant product was of the predicted size and sequence.
For conventional PCR, cycle numbers were adjusted to ensure linear amplification; products were resolved by agarose gel electrophoresis and detected by ethidium bromide staining. Real-time qPCR was carried out in an Applied Biosystems 7300 thermal cycler (94°C for 30 seconds, 62°C for 30 seconds, 72°C for 30 seconds) using SYBR-green and the results analyzed using software provided by the manufacturer. GAPDH mRNA controlled for equal sample loading in all these cases.
Bioinformatics
TFs were selected for survey as described previously
(Gray et al., 2004). Briefly,
public and private gene databases were screened for nonredundant predictions
of DNA-binding domains as defined in the Protein Families Database (Pfam;
http://www.sanger.ac.uk).
All annotations were confirmed manually, as reported
(Gray et al., 2004). Primers
were 20-22 bp in length, with 
40% GC content, and designed to amplify
700 bp products.
We determined pixel densities for each RT-PCR product and, based on background levels in blank and control lanes, selected 100 units as the threshold for presence of the transcript. Signal strength ranged from 100 to 3400 units, with a large and apparently linear dynamic range, but varied between PCR primers. Absolute transcript levels may therefore only be compared across time points and not between TFs, whereas comparison of changes in TF levels are always valid. To qualify for a change between two developmental stages, we required a greater than twofold difference in signal strength of the RT-PCR product; the algorithm gave greater weight for changes of higher magnitude and those that occur over more than two consecutive stages.
To identify GI-restricted factors, we examined an expression dataset that
profiled 42,000 mouse mRNAs across 55 tissues using customized 60-base
oligonucleotide probes (Zhang et al.,
2004) and referred to rigorous tissue isolation protocols. We
extracted genes for which the intestine (small or large intestine, colon)
showed normalized expression that was k-fold more than m of
51 adult tissues. Several combinations for k and m yielded
restrictive results on known digestive markers when k=2.5 and
m=40, thus identifying 1240 mRNAs with more than 2.5 fold levels in
gut compared with 78% of tissues. We compared this pool with genes expressed
in our TF survey using unique EntrezGene identifiers; 23 genes, representing
less than 2.5% of TFs expressed in the developing gut, show GI-enriched
expression postnatally. Manual review confirmed GI-enriched expression for 20
of these 23 genes in an independent profiling dataset
(Su et al., 2004).
Mice, tissue explants, and detection of RNA and proteins
Isx-deficient mice were generated by standard methods for targeted gene
disruption by homologous recombination in 129/Sv strain-derived TC1 embryonic
stem cells and maintained on a mixed 129/Sv-C57BL/6 background. Gene targeting
was monitored by Southern analysis using the probes and restriction enzymes
indicated in Fig. 6D. Organ
culture and electroporation of fetal CD-1 mouse stomachs were performed as
described previously for intestinal explants
(Tou et al., 2004). Membranes
blotted with adult mouse tissues or whole mouse embryos at sequential stages
(Seegene, Seoul, South Korea) were hybridized with a 700 bp probe
corresponding to the mouse Isx-coding sequence. Rabbit Scarb1
antiserum was raised against a keyhole limpet hemocyanin-conjugated peptide
corresponding to residues 495-509 (MSPAAKGTVLQEAKL) of murine Scarb1 and
reacts with a single 82 kDa band on liver immunoblots from wild-type but not
from Scarb1-/- mice. Northern blotting, immunochemistry
and microarray comparison of wild-type and Isx-/- terminal
ileum RNA were performed as described (Kim
et al., 2005).
RT-PCR products were cloned into Topo II (Invitrogen), confirmed by DNA sequencing and amplified by PCR. The amplified fragments were transcribed to produce digoxigenin-labeled antisense and sense riboprobes. CD1 mouse embryos at E13, E15 and E17 were fixed in 4% paraformaldehyde at 4°C for 2, 3 and 12 hours, respectively; soaked overnight in 0.5 M sucrose at 4°C; and embedded in OCT compound (Sakura Finetech, Torrance, CA). Sections (12 µm) were fixed further in 4% paraformaldehyde for 20 minutes and then treated as described previously to detect mRNA localization, with hybridization temperatures of 58-62°C and washing stringency of 0.2x SSC. Images were captured using an Olympus BX41 compound microscope, CCD camera and Photoshop 7.0 software (Adobe).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). About 6%
of all genes encode potential DNA-binding factors, and recent studies estimate
nearly 1500 human TFs, including over 700 ZnB proteins
(Messina et al., 2004). Our
survey of 1381 genes covers the vast majority of known or predicted mammalian
TFs, and Table 1 compares
sub-groups of human TFs (Messina et al.,
2004) with the mouse genes we examined.
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100 bp amplicon length that is optimal for
monitoring by qPCR is difficult to verify and limits other applications. We
used conventional PCR to amplify 700 bp fragments, so that products could
be resolved by gel electrophoresis, isolated for sequencing and used to
prepare probes for in situ hybridization. When possible, TF-specific PCR
primers corresponded to coding sequences separated by introns. Non-reverse
transcribed samples did not result in amplification. PCR products were
quantified by the relative intensity of electrophoretic bands.
To optimize quantitation, we assessed 16 genes known to be present at
levels typical for TF transcripts in the developing gut, fewer than five
copies per 105 mRNA molecules
(Lepourcelet et al., 2005).
Fifteen of these 16 products could be detected in the linear range of
amplification after 31 cycles of PCR (data not shown). In an interim analysis
of the first 72 TFs, 21 primer pairs had failed to generate a PCR product;
increasing the reaction to 36 cycles yielded weak signals for only two
additional transcripts, whereas saturated PCR signals masked temporal changes
in the levels of five factors. Conversely, among 31 genes that initially gave
a saturated signal, reducing the reaction to 27 or 24 cycles uncovered subtle
temporal change in just three transcripts. Another test midway through the
analysis yielded similar results, and we completed the survey at 31 PCR
cycles.
To judge the quality of the results, we interrogated a set of 213 TFs using
two to five independent primer pairs. In small intestine, 42 (19.7%) of these
transcripts never yielded a PCR product, whereas 29 (13.6%) gave weak signals
with one primer pair and none with the others. These results agree roughly
with our assessment that about 25% of all TF mRNAs are absent from the
developing stomach or small bowel (Fig.
1A). One-hundred and thirty-nine of the remaining 142 mRNAs showed
concordant patterns with multiple primer pairs. Second, using independent RNA
isolates to examine 63 TFs that show developmental regulation of mRNA levels,
we reproduced the results with high accuracy for 58; all but one of the
failures were associated with weak and unreliable signals. The results are
thus highly reproducible and strongly validated through redundancy among PCR
primers. Third, 62% of reactions that failed to amplify a product from gut
cDNA generated the correct product, verified by DNA sequence, when mouse brain
served as the template (Gray et al.,
2004), thus excluding primer design as the basis for PCR failure.
Although failure to detect a PCR product may reflect low expression levels,
some factors may be absent from both fetal brain and gut, as our method to
detect TF transcripts appears to be highly sensitive. Fourth, DNA sequences of
all 63 tested PCR products confirmed amplification of the expected product.
Finally, where possible, we compared spatial and temporal variation revealed
in our analysis with the results of published studies; expression of
well-known TFs of the Hox, Cdx, Gata and Hnf groups were reproduced
accurately. (The full dataset is not discussed here, but is available at
http://genome.dfci.harvard.edu/~mhu/GIFT.)
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). Although we cannot judge how this fraction compares with TF
gene expression in other organs, expression of such a large number of TFs in
the GI tract implies a significant overlap in TF use among tissues.
Furthermore, temporal modulation of TF genes during a period of substantial
morphogenesis is modest (Fig.
1A). Among expressed transcripts, most (48% in small intestine,
50% in stomach) show minimal modulation over time (e.g.
Fig. 1B) or similar variation
(5% of transcripts) in the two organs (e.g.
Fig. 1C); the extent of
temporal variation for 7% (intestine) to 10% (stomach) of transcripts fell
within margins of error. Gut development and maturation thus occur with
limited specific changes in TF gene expression, which emphasizes the likely
importance of those TFs that are temporally regulated: according to `relaxed'
criteria, up to 253 in the intestine and 191 in the stomach (e.g.
Fig. 1C). Slightly fewer TF
transcripts increase in expression over the E11-E17 period than decrease in
that interval. In small bowel, for example, 114 transcripts decrease in
abundance compared with 90 that increase steadily, whereas 49 TF genes show
peak expression at E13 or E15. Such TFs probably serve stage-specific
functions.
The stomach and intestine, derivatives of a common primordium, develop
vastly different features. Over the embryonic stages we examined, 103 TFs
(8%) are expressed differentially between the two organs
(Fig. 1D). Although our survey
readily identified known intestine-specific genes such as Cdx2 (data
not shown), fewer than 25 TF mRNAs appear to be expressed exclusively in the
fetal stomach or intestine (e.g. Fig.
1E). Most spatial differences occur at the level of signal
strength or of temporal variation in mRNA levels (e.g.
Fig. 1F). Based solely on
differential gene expression, we recently proposed and demonstrated a vital
role for the homeobox TF Barx1 in stomach epithelial specification
(Kim et al., 2005). The
limited extent to which TF genes are expressed only in one organ or the other
implies that such examples are rare. Nevertheless, spatially restricted TF
expression (Fig. 1D) is
probably a good predictor of tissue-specific functions.
Expression of TF families in gut development
There is wide variation in the extent to which the members of
well-characterized TF families are expressed during gut development
(Fig. 2A). About 90% of all ZnB
factors and basic-leucine zipper (bZip) proteins are expressed, whereas the
smallest fractions are detected among forkhead (32%) and HD (45%) proteins
(Fig. 2A). The two TF families
with highest proportional representation (ZnB and bZip) also have the largest
fraction of genes expressed constitutively from E11 to E17. By contrast, the
HD family shows an especially high degree of regulated expression, with over
60% of genes showing differential expression in space or time
(Fig. 2A,
Table 2; data not shown). Two
families, HD and nuclear receptors (NR), account for the bulk of differential
TF gene expression between the stomach and intestine
(Fig. 2A).
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, Nr2a1 (HNF-4), Nr1f3
(ROR-) and Nr5a2 (LRH-1) are known to regulate region- and
stage-specific intestinal genes or crypt functions
(Drori et al., 2005;
Kan et al., 2004;
Ladias et al., 1992;
Raspe et al., 2001;
Sauvaget et al., 2002;
Botrugno et al., 2004;
Schoonjans et al., 2005).
Biological functions correlate precisely with spatiotemporally restricted
expression in each of these cases, and our survey has the potential to
identify other factors with limited expression. Indeed, late in intestine
development we observed high levels of Nr2e3
(Fig. 2C,D), a transcript
previously believed to be present only in the retina
(Chen et al., 2005;
Kobayashi et al., 1999). We
confirmed presence of Nr2e3 mRNA in fetal gut by northern analysis
and cloning of the PCR product (data not shown); in situ hybridization
localized the transcript in epithelial cells
(Fig. 2F). Nr2e3
expression is confined to the period between E15 and 3 weeks after birth and
to the proximal bowel (Fig.
2E). These findings suggest the possibility of regional or
stage-specific functions, and highlight the potential for gene discovery
through the GIfT resource.
Authors frequently comment on a special role for HD proteins, including the
products of Hox-cluster genes, in gut development
(Beck et al., 2000;
Grapin-Botton and Melton,
2000). Although HD subfamilies are unequally represented in the
fetal gut, ranging from five out of 28 Paired-class proteins to nine out of 13
factors from the Msx-Dlx group (Table
2), every sub-family is developmentally regulated, either in
changes over time or in differences between stomach and intestine. Levels of
most HD TFs, including the products of Hox clusters, decline late in fetal gut
development, which suggests that their functions may concentrate at early
stages. Our data extend previous work that investigated a limited number of
Hox-cluster genes and developmental stages or relied on methods less sensitive
than RT-PCR (Kawazoe et al.,
2002; Pitera et al.,
1999), and we detect several Hox mRNAs previously reported to be
absent from the gut. All Hox gene transcription in developing gut occurs from
genes located near the 3' end of Hox clusters, with strongest expression
from paralogous sub-groups 5, 6 and 7 (Fig.
2B); mRNAs derived from subgroups 9-13 are absent. With the
exception of HoxC4, HoxB6, HoxA7 and HoxC8, expression dynamics are remarkably
concordant in the developing stomach and intestine. Thus, whereas genes from
paralogous subgroups 6, 7 and 8 may serve in regional specification, Hox gene
expression is unlikely to act alone to distinguish the two organs from one
another.
Identification of TFs expressed in a restricted distribution
TFs that are confined to the gut or differentially expressed within the GI
tract are especially likely to regulate tissue-specific genes, and we adopted
three methods to reveal such TFs. The first approach sought to identify TFs
that are highly enriched in mature intestine and reinforced the idea of
tissue-restricted functions for selectively expressed genes. We mined an
expression database of 51 adult mouse tissues
(Zhang et al., 2004) and found
all mRNAs expressed at more than 2.5-fold higher levels in intestine compared
with at least 40 other sites. For these 1240 transcripts (partially
represented in Fig. 3A), the
commonest sites of additional expression are the stomach, liver and pancreas,
organs that share embryonic origin and metabolic or digestive functions with
intestine. Thirty-four intestine-enriched genes encode TFs
(http://www.geneontology.org).
The GIfT survey identified 23 of them in the developing gut
(Fig. 3B) and we confirmed
restricted expression for 20 TFs in another database of 61 organ profiles
(http://symatlas.gnf.org).
Detailed review of published information on knockout mice
(Fig. 3C; see Table S1 in the
supplementary material) revealed that the principal abnormalities uniformly
lie in restricted or dominant sites of gene expression. Whereas mice deficient
in these TFs occasionally show anatomic GI defects, a common factor is
defective metabolism of bile acids, lipids, glucose or xenobiotics, which
reflects the metabolic function of endoderm-derived tissues. These
observations support the idea of organ-specific functions for GI-restricted
TFs and drew attention to an uncharacterized gene within the group. Northern
analysis confirmed that this mRNA (Gene ID 71597) is highly restricted to the
intestine (Fig. 3C, bottom;
Fig. 3D). It encodes a novel HD
protein and entered our survey based on ESTs derived from a cecum library. As
its mRNA expression is highly restricted, we propose the name Isx
(intestine-specific homeobox).
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Finally, Cdx2, the best characterized TF in intestinal epithelium, is
implicated in control of intestine-specific genes
(Beck, 2004). Cdx2
haploinsufficiency promotes limited stomach-type differentiation in the colon
(Beck et al., 1999;
Chawengsaksophak et al., 1997)
and transgenic mice with forced Cdx2 expression in the stomach mucosa display
intestinal features in that tissue (Mutoh
et al., 2002; Silberg et al.,
2002). Intestinal TFs that are normally excluded from the stomach
but are present within Cdx2-induced intestinal metaplasia may be especially
important in intestinal gene regulation. We therefore examined stomachs
derived from FoxA3-Cdx2 transgenic mice
(Silberg et al., 2002) for
presence of 15 TF mRNAs that met stringent criteria for selective expression
late in normal intestine development. Consistent with reports that Cdx2
induces partial intestine differentiation in this animal model
(Silberg et al., 2002), only
one TF, Isx, was induced by the transgene to appreciable levels
(Fig. 5A). Thus, three
independent approaches converged on Isx as a novel intestine-restricted TF,
and its unique appearance in the Foxa3-Cdx2 transgenic stomach may
particularly reflect a role in intestinal gene regulation.
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|
), Isx mRNA appears late in development
(Fig. 6A), just before the
villus transition in intestine morphogenesis. Isx transcripts are
restricted to the epithelial compartment in both adult
(Fig. 6B) and fetal
(Fig. 4B) intestine.
Phylogenetic comparison of the Isx HD with those of nearly 200 other HD
proteins places Isx within the Paired family; homology is closest to Pax3,
Pax7 and Prrx1 (Fig. 6C), which
serve organ-specific functions. To test Isx functions in vivo, we generated
mutant mice. Our strategy for targeted gene disruption replaced the first exon
with a neomycin-resistance cassette (Fig.
6D) and resulted in elimination of Isx transcripts, as judged by
northern analysis (Fig. 6F).
Isx-null mice are born in the expected numbers and appear healthy for at least
1 year. Under standard housing conditions, they gain weight at the same rate
as their littermates and display normal histological features in the gut (data
not shown).
Intestinal gene expression defect in Isx-null mice
Intestinal genes tend to express in anteroposterior (AP) gradients, with
few transcripts exclusive to a given segment
(Bates et al., 2002). To
determine if Isx might control regionally restricted gene expression, we used
qPCR to measure mRNA levels of Upa, Adh1, Ephx2, Slc2a2 (all enriched
in the duodenum), Cdx1, Pap, cubilin and guanylin (ileum-enriched
transcripts) (Bates et al.,
2002). No consistent differences were evident between control and
mutant samples (data not shown). Thus, Isx appears to be dispensable for gross
intestinal development and function or to establish the AP axis.
We hypothesized that, like other gut-restricted TFs
(Fig. 3C), Isx may control some
activities relevant to metabolism. To identify such a role, we performed RNA
microarray analysis of adult terminal ileum, the bowel segment with highest
Isx expression (Fig. 5B).
Although levels of very few mRNAs differed significantly between
Isx+/- and Isx-/- samples, transcripts for the scavenger
receptor, class B, type I (Scarb1), an HDL receptor and lipid
transporter (Rigotti et al.,
2003), are increased 10-fold in Isx-/-
gut. We detected this anomaly with three independent probe sets (e.g.
Fig. 7A) and verified the
results by qPCR analysis on multiple independent samples
(Fig. 7B). Scarb1 is
expressed more strongly in the liver and in adrenal steroidogenic cells than
in the gut (Acton et al., 1996;
Rigotti et al., 2003). In
Isx-/- mice, however, changes in Scarb1 expression are not
observed in the liver or adrenal glands but are confined to the gut
(Fig. 7B,C).
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;
Hauser et al., 1998;
Voshol et al., 2001), opposite
to the increasing duodenum-ileum gradient for Isx
(Fig. 5B). Scarb1 mRNA
levels in Isx-/- mice are higher in both duodenum and
ileum (Fig. 7B), and immunoblot
analysis confirms that ileal protein levels are substantially (more than
fivefold) elevated (Fig. 7C).
As in the duodenum of wild-type animals
(Hauser et al., 1998;
Voshol et al., 2001), Scarb1
protein in the mutant ileum resides in the epithelial brush border
(Fig. 7E), where low endogenous
levels are undetectable in the normal ileum
(Fig. 7D). Mice with forced
Scarb1 expression in the gut absorb dietary cholesterol and triglycerides more
efficiently (Bietrix et al.,
2006); although Scarb1-null mice are not deficient in these
functions (Mardones et al.,
2001), they show reduced absorption of other lipids such as
carotenoids, lutein and vitamin E (Reboul
et al., 2006; van Bennekum et
al., 2005). Thus, regulation of the location and extent of
intestinal Scarb1 expression by Isx probably reflects an underlying
function in lipid absorption or homeostasis in this organ, similar to the
essential metabolic roles of other gut-restricted TFs
(Fig. 3C). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our study reveals some surprising features in the TF landscape during gut
morphogenesis. A remarkably high fraction (78%) of all TF mRNAs is expressed,
similar to the high representation of TF transcripts in the developing mouse
nervous system (Gray et al.,
2004). These data highlight the likely redundant manner in which
tissues deploy a finite TF repertoire. Second, despite many functional and
morphological differences between the stomach and intestine, at the resolution
of our analysis, expression of only a few dozen TFs differs appreciably
between these tissues. Thus, to the extent that differential TF expression is
important, as strongly implied in the literature, a few TFs might suffice to
help achieve complex developmental differences. Of course, differences other
than total mRNA expression are also important, including the cellular context
and post-transcriptional modulation of TF activities, localization and protein
interactions.
Nevertheless, TFs that are restricted in distribution are especially likely to regulate tissue-specific genes, and our analysis of the reported requirements for gut-restricted TFs (Fig. 3) underscores the high frequency at which tissue distribution predicts gene functions. Together, these results point to the limited pool of spatiotemporally regulated TFs as an enriched source of factors that are especially pertinent in tissue differentiation. Thus, a particular value of GIfT lies in its examination of all TFs on a common methodological platform, whereas the experimental approach outlined in this report highlights the ability to generate cogent functional hypotheses based initially on differential mRNA expression. Indeed, intestinal expression of TFs such as Nr2e3 and Isx may not have been revealed as readily by methods other than a global survey.
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-defensin-related genes (M.Y.C. and R.A.S., unpublished) normally
expressed in intestinal Paneth cells, our comparison of
Isx+/- and Isx-/- ileum emphasizes
deregulation of the epithelial HDL receptor and lipid transporter Scarb1.
Consistent with GI-restricted expression of Isx, Scarb1 levels in
Isx-/- mice are altered only in the gut, to comparable
degrees in proximal (duodenum) and distal (ileum) segments. Scarb1
transcription is activated by C/EBP family TFs and sterol regulatory element
binding protein 1 (Malerod et al.,
2002) and suppressed post-transcriptionally by treatments that
reduce intestinal bile delivery, such as bile duct ligation or surgical
diversion (Voshol et al.,
2001). Our data suggest that Isx serves specifically to refine the
extent and domain of intestinal Scarb1, which is normally confined to the
duodenum, the principal site for uptake of dietary lipids and most
nutrients.
Absorption of dietary lipids is an essential intestinal function mediated
by brush border membrane receptors, including the class B scavenger receptors
Scarb1 and CD36. Scarb1 is required to absorb ß-carotene and vitamin E in
mice (Reboul et al., 2006;
van Bennekum et al., 2005),
and displays high affinity for cholesterol in vitro
(Acton et al., 1996). Although
forced Scarb1 expression in heterologous cells confers lipid uptake properties
that resemble those of enterocytes (van
Bennekum et al., 2005), cholesterol absorption is intact in
Scarb1-/- mice
(Mardones et al., 2001),
possibly because CD36 or other factors compensate for its absence. As a
multiligand receptor with diverse functions, it is probably important that
Scarb1 expression is restricted. Indeed, Villin promoter-driven
Scarb1 overexpression, mainly in the intestine but also to a lesser degree in
the liver, accelerates absorption of dietary cholesterol and triglycerides
(Bietrix et al., 2006). Ileal
expression, which is normally absent, may be especially detrimental as this
distal intestinal region has important functions in absorption of bile salts
and integrity of enterohepatic bile acid circulation. Thus, Isx, among other
possible activities, tailors gene expression to meet physiological needs in
the intestine. It is one of a few dozen TFs our study identified, by virtue of
regional and restricted expression, as a candidate for specific gut functions.
The approach we have taken with TFs can readily be extended to other protein
classes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/20/4119/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H. and Krieger, M. (1996). Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 271,518 -520.[Abstract]
Aubin, J., Dery, U., Lemieux, M., Chailler, P. and Jeannotte,
L. (2002). Stomach regional specification requires
Hoxa5-driven mesenchymal-epithelial signaling.
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