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First published online 13 September 2006
doi: 10.1242/dev.02537

Development 133, 4119-4129 (2006)
Published by The Company of Biologists 2006
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A dynamic expression survey identifies transcription factors relevant in mouse digestive tract development

Michael Y. Choi1,2,3, Anthony I. Romer1, Michael Hu1, Maina Lepourcelet1,3, Ambili Mechoor1,3, Ayce Yesilaltay4, Monty Krieger4, Paul A. Gray1,5,* and Ramesh A. Shivdasani1,3,6,{dagger}

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.


Figure 1
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  		Fig. 1. Analysis and representative results from the GIfT expression survey. (A) Group breakdown of TFs according to temporal variation in the developing intestine; the fetal stomach showed virtually identical grouping. All RT-PCR data were quantified; classifications were derived computationally and verified manually. (B) Arbitrary examples from hundreds of TF mRNAs with constant levels during GI organogenesis. The data represent electrophoretic bands of RT-PCR products from our TF survey. (C) RT-PCR results of temporal modulation of TF transcripts during GI organogenesis, highlighting the similar trends observed for most TFs in the intestine and stomach. (D) Statistical analysis of differential gene expression. Most TF mRNAs are expressed either constitutively or with the same temporal variation in both stomach and intestine, and more than 18% of transcripts are not expressed in either site. Only ~8% (103 TFs) meet criteria for differential expression between intestine and stomach, including the examples shown in E and F. (E) Selected examples from ~25 TF mRNAs that are restricted to the developing stomach (grouped beside the red bar) or intestine (grouped by the blue bar). (F) Examples of TF transcripts that differ between stomach and intestine in pattern but not in absolute expression. The full set of original data can be analyzed at

 

Figure 2
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  		Fig. 2. Analysis of TF gene expression in the developing GI tract according to protein families. (A) TF gene families show divergent patterns of expression and temporal modulation during intestine development, with proportionally higher representation of basic-leucine zipper (bZip) and zinc-binding (ZnB) factors. Nuclear receptor (NR), homeodomain (HD) and high-mobility group (HMG) factors have the highest degree of temporal modulation. Except for HD and NR, most families are distributed similarly in intestine (In, shown here) and stomach (St, data are shown only for NR and HD). (B) Comparative gene expression for a single TF subfamily, Hox-cluster genes, commonly proposed as candidates for anteroposterior gut patterning. Expression in the developing gut is limited to genes at the 3' ends of collinear clusters and, with the few exceptions marked in red type, is very similar in extent and modulation in fetal stomach and small intestine. (C) As a group, NRs showed the greatest increase during intestine development, mostly from E11 to E13. One factor expressed with these dynamics is Nr2e3, previously regarded as a photoreceptor-specific product. (D) Nr2e3 mRNA, expressed in the developing gut, is abrogated in adult mice. (E) Fetal expression of Nr2e3 is restricted to the proximal small bowel. (F) In situ hybridization (E15 intestine) reveals Nr2e3 expression in the epithelial compartment (arrows).

 

Figure 3
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  		Fig. 3. Identification of novel GI-restricted TFs. (A) Schematic representation of relative expression levels, in light (lowest expression) to dark (highest) purple, of 161 sample transcripts out of 1240 that are enriched in the small and large intestine; colon was analyzed twice in the study from which the data are extracted (Zhang et al., 2004Go). The most common extra-intestinal sites of expression were stomach, liver and pancreas; most mRNAs are expressed sparingly in other tissues. (B) Intersection of these gene expression data (34 TFs) with the GIfT survey, revealing developmental representation of 23 TFs that are highly enriched in GI expression. (C) Detailed expression profile of these 23 TF genes in 55 adult and embryonic mouse tissues; again, data are taken from Zhang et al. (Zhang et al., 2004Go). In each row, the TF gene is listed towards the left, every square represents an organ and expression levels are represented by a color scale (red-orange, high; yellow-green, low; white, absent). Expression levels in adult small intestine and colon are marked with `I' and `C', respectively. Predominant phenotypes reported in knockout mice are indicated on the right. (D) Northern analysis of adult mouse tissues, showing significant intestine enrichment (and possibly exclusive expression) of the novel TF transcript corresponding to Gene ID 71597 (last row in panel C), for which we propose the name Isx.

 

Figure 4
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  		Fig. 4. Tissue-enriched and compartmentalized expression of TF mRNAs. (A) Partial results from comparative expression of 66 TF mRNAs in multiple fetal mouse tissues at E13 (left) and E17 (right), determined by RT-PCR. Twelve factors are considerably enriched in the developing stomach and/or intestine, with persistent expression of some TFs in the adult GI tract. GAPDH provides a mRNA loading control, and three examples of TFs with broad tissue distribution are included. (B) Illustrative examples of two TFs, an uncharacterized ZnB protein (left) and Isx (right) revealed by in situ hybridization to be expressed in the E13 mesentery (left) and E15 mucosa (right, arrows), respectively. (C) Pie-chart representation of mRNA localization of the 66 tested TFs, which either increase during intestine development or differ notably between stomach and intestine.

 

Figure 5
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  		Fig. 5. Relation between Isx and the gut homeotic regulator Cdx2. (A) Representative RT-PCR analysis of 15 late-activated, intestine-restricted TFs in the stomach of FoxA3-Cdx2 transgenic mice. Isx is the only tested transcript present in the metaplastic stomach. (B) Isx mRNA shadows that of the intestine regulator Cdx2 in time and space. Both TFs are virtually absent from stomach and are activated simultaneously between E11 and E13 in developing mouse intestine (top, RT-PCR analysis); concentration of both mRNAs is highest in adult ileum and cecum (bottom, northern analysis). GAPDH or 28S RNA serve as loading controls. (C) Forced expression of GFP-tagged Cdx2 in fetal mouse stomach explants (left, organ margin outlined in white) consistently induces ectopic expression of Isx mRNA (right, RT-PCR analysis).

 

Figure 6
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  		Fig. 6. Characterization of the novel, intestine-restricted homeobox gene Isx. (A) Northern analysis of whole mouse embryos at the indicated post-fertilization (E) days shows absence of Isx mRNA expression before E14. This feature distinguishes Isx from Cdx2, which is also expressed at peri-implantation stages. (B) In situ hybridization reveals epithelium-restricted expression of Isx transcripts in adult intestine; right panel shows results with a sense probe. (C) The Isx HD is most closely related to that found in the Paired family. Unrooted phylogenetic analysis using the CLUSTAL_W algorithm (20 of 28 Paired proteins are shown) reveals closest homology to Pax3, Pax7 and Prrx1. (D) Isx gene targeting strategy. 3.5 kb EcoRI (E) and 2.5 kb XbaI (X) genomic fragments were isolated from a 129/Sv BAC clone and used to flank a PGK-NeoR cassette, positioned in reverse orientation, in the targeting construct. Positions of KpnI (K) sites enabled confirmation of gene targeting. (E) Correct targeting, with deletion of exon 1, was determined by Southern analysis of KpnI-digested DNA probed with the genomic fragments indicated in D. Homologous recombination produced the expected 12 kb and 4.5 kb bands with the 5' and 3' probes, respectively. (F) Northern blots confirmed loss of Isx expression after targeted gene disruption. RNA isolated from the cecum (Cec) and stomach (Sto) of nullizygous mutant (KO) mice and littermate controls (WT) was probed with Isx cDNA. Ethidium bromide staining of 28S RNA shows equal or excess loading of mutant samples.

 

Figure 7
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  		Fig. 7. Altered intestinal gene expression in mice lacking Isx function. (A) Microarray data for increased Scarb1 mRNA levels in Isx-/- (KO) ileum compared with control (WT) littermates. Dark signals represent absence and yellow the presence of hybridization; in each set, the top row shows probes that perfectly match the target transcript and the bottom row shows probes with single-base mismatches. Similar results were obtained for two independent Scarb1-specific probe sets. (B) qPCR confirmation of significant elevations in Scarb1 mRNA in Isx-/- ileum and duodenum (Duod) but not in the other sites of Scarb1 expression, adrenal gland and liver, where Isx is absent. All mutant (KO) values are expressed in relation to the control (WT, assigned a value of 1.0) for that tissue. Scarb1 mRNA levels in liver and adrenal glands are higher than in intestine, but increases in Isx-/- mice are confined to the gut. (C) Immunoblot confirmation of elevated Scarb1 protein levels in Isx-/- intestine but not liver (data not shown) or adrenal glands. (D,E) Immunohistochemistry with Scarb1 antiserum. Wild-type ileum (D) reveals no specific signal as Scarb1 is primarily a duodenal product, whereas Scarb1 localizes (arrowheads) in the mutant (KO) ileal apical brush border (E).

 

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© The Company of Biologists Ltd 2006