Figure 1. Numbers of genes highly expressed in specific segments of the
adult mouse gastrointestinal (GI) tract. Graph shows the numbers of genes
highly expressed in each segment of the GI tract,including the number of
genes whose increased expression is limited to that particular segment
(unique to segment). Black bars indicate the numbers of genes expressed
in each tissue segment that are not highly expressed in non-GI tissues.
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Figure 2. Expression of selected genes in the adult mouse gastrointestinal
tract. Clones are grouped by functional category of the corresponding
protein and identified by protein identity and accession numbers. Clones that
are not expressed greater than 2-fold above reference in any normal adult
non-GI tissue in our mouse gene expression database are indicated. Expression
profiles are shown by colored squares, with two squares (one for each
microarray hybridization) for each tissue. Colors are graded with red indicating
increased expression and green low expression relative to reference. S,
stomach; D, duodenum; J, jejunum; I, ileum; Ce, cecum; PC, proximal colon;
DC, distal colon.
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Figure 3. Hierarchical clustering of genes highly expressed in adult mouse GI
tissues. A total of 571 cDNAs with 2-fold or greater increases in expression
relative to reference in replicate hybridizations for one or more of the seven adult
GI tissues were identified. Expression values were subjected to hierarchical
clustering using a minimum distance value of 0.001 and a separation ratio of 0.5.
Data for replicate hybridizations are shown. Colors are graded with red
indicating increased expression and green low expression relative to reference.
White tiles indicate microarray elements that did not pass quality control. (A)
Hierarchical clustering applied to log2-transformed ratios relative to reference,
using Pearson correlation. (B) Hierarchical clustering using log2-transformed
ratios for each gene normalized to its median level of expression among the GI
tissues (“each gene normalized”). The resulting hierarchy is identical to that in
panel A, but the colors more clearly depict expression differences between GI
tissues. (C) Hierarchical clustering with standard correlation applied to relative
expression ratio values for each gene normalized to its median level of expression
among the GI tissues (“each gene normalized”
[http://www.sigenetics.com/cgi/TNgen.cgi/GSnotes/Notes/what_options]). S,
stomach; D, duodenum; J, jejunum; I, ileum; Ce, cecum; PC, proximal colon;
DC, distal colon. PDF Figure 3.pdf HTML Figure 3.html

Figure 4. K-means classification of gene expression profiles in adult mouse GI
tissues. Analysis was performed with the “each gene normalized” expression
ratios, using GeneSpring 4.0 to specify a total of 30 different classifications. This
number of classifications was suggested by the types of A-P expression profiles
apparent in the hierarchical tree analyses (Figure 3). No gene profiles were left
unclassified. We found that the use of fewer classifications resulted in more
heterogeneous groupings of expression profiles (data not shown). K-means
classified profiles are grouped in the figure and colored according to shared
features of the profiles: A-D (pink), peak expression in stomach; E-H (blue), peak
expression in duodenum; I-K (blue), highest expression in jejunum; L-N (blue),
decreasing A-P gradient of expression across the small intestine; O-S (blue), peak
expression in ileum; T-Y (orange), peak expression in large intestine; Z-D' (green),
peaks of expression in more than one non-adjacent tissue. The number of cDNAs
in each classification were: A, 11; B, 1; C, 2; D, 12; E, 14; F, 18; G, 21; H, 49; I,
18; J, 9; K, 33; L, 22; M, 18; N, 46; O, 6; P, 17; Q, 16; R, 38; S, 58; T, 5; U,
14; V, 23; W, 20; X, 10; Y, 43; Z, 18; A', 7; B', 7; C', 9; D', 6. S, stomach; D,
duodenum; J, jejunum; I, ileum; Ce, cecum; PC, proximal colon; DC, distal colon.
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Figure 5. Expression patterns of genes with increased expression in adult
mouse GI tissues. (A) Levels of expression for highly expressed genes
(corresponding to 571 cDNAs with increased expression in at least one
GI tissue) were defined as log2 ratios (relative to reference). Expression
profiles for each cDNA are read horizontally, with a gray–black shade
corresponding to the log2 ratio for each tissue in which it was expressed
2-fold or more relative to reference. Expression profiles were sorted in
Microsoft Excel. (B) Gene expression patterns sorted by functional classification,
then by profile (as in A). Classifications (along vertical axis): C, cell-cell
communication; D, DNA/RNA processes; I, immune/defense; M, intermediary
metabolism; P, miscellaneous cell processes; S, structure/cytoskeleton; T,
transport; U, unknown/other. Tissues (along horizontal axis): S, stomach;
D, duodenum; J, jejunum; I, ileum; Ce, cecum; PC, proximal colon; DC, distal
colon. PDF Figure 5.pdf HTML Figure 5.html

Figure 6. Expression profiles of genes encoding proteins of assigned
functional categories "gene normalized" profiles were sorted by functional
category of the encoded protein.The color used for each individual profile
is the same as in Figure 4 (pink, peak expression in stomach; blue, peak
expression in small intestine; orange, peak expression in colon; green,
peaks of expression in more than one non-adjacent tissue). S, stomach;
D, duodenum; J, jejunum; I, ileum; Ce, cecum; PC, proximal colon; DC, distal colon.
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Figure 7. Locations of large changes in gene expression along the adult
mouse GI A-P axis. Duplicate expression levels (log2 ratio) for each cDNA
represented on the mouse GEM1 microarray in each tissue were averaged,
and differences between adjacent tissues were calculated. The number of
cDNAs with increases or decreases of indicated magnitude between adjacent
tissues are shown. The expression of 273 cDNAs changed 4-fold or more
between adjacent GI segments, including 65 not identified as highly expressed
in adult GI tissues, i.e., not on the list of 571 highly expressed adult genes.
Because some of these genes had more than one 4-fold change in expression
in adjacent tissues, there were a total of 363 transitions identified. Stom,
stomach; Duod, duodenum; Jej, jejunum; Prox Col, proximal colon; Dist Col,
distal colon. PDF Figure 7.pdf HTML Figure 7.html

Figure 8. Consensus cis-regulatory/transcription factor binding elements
present in the 5' regions of coordinately regulated intestinal genes. (A)
Expression profiles of 6 genes with a sharp boundary of expression between
the ileum and cecum. cDNAs with highest expression in the ileum (SI group)
are shown in blue: AA245078, intestinal fatty acid binding protein (IFABP);
AA471960, cubilin; and AA239282, T-cell death associated gene (TDAG).
cDNAs with highest expression in the cecum (LI group) are shown in orange:
AA498312, methionine adenosyltransferase 2A subunit (Mat2A), AA498457,
guanylin, and W18397, Reg IV (a member of the calcium dependent lectin
superfamily; Hartupee et al., Biochim Biophys Acta 1518:287-29, 2001.
(B) Shared 5' cis-regulatory/transcription factor binding elements in the
5' regions. Elements present only in the SI group (n = 5) or LI group (n = 4)
are shown in blue and orange, respectively; those shared between the groups
(n = 4) are shown in black. Abbreviations: C/EBP, CCAAT/enhancer binding protein;
CDP, CCAAT- displacement protein; CRBP, CReb binding protein family; Fkhd,
forkhead/winged helix domain factors; ETSF, ETS family; HNF-1, hepatocyte nuclear
factor-1. (C-D) Locations of putative cis-regulatory/transcription factor binding
elements in 500-600 bp of the 5' upstream regions of the selected genes. SI
genes are shown in (C), LI genes are shown in (D). Numbers designate elements
as in (B). Below the schematic 5' region for four of the genes is a gray box with
lines designating regions of high sequence identity (50-100%) over a window
of at least 200 bp between the corresponding regions of the mouse and human
gene orthologues. Boxes under the IFABP gene show regions previously shown to
be important for regulation of gene expression along the A-P axis (Cohn et al.,
J Cell Biol 119:27-44, 1992 ): A, –1178 to –277 bp; B, –277 to –185 bp; C,
–103 to +28 bp. PDF Figure 8.pdf HTML Figure 8.html

Figure 9. Gene discovery using unknown/uncharacterized genes that are
highly expressed in the adult mouse GI tract.

(A) A total of 20 unknown clones that could not be characterized using
UniGene and BLASTsearches were selected based on interesting GI and
non-GI expression patterns. The list isordered based on hierarchical clustering
of the expression profiles of these clones in the GI tract, using log2-transformed
ratios relative to reference. GI expression profiles are shown by colored squares,
with two squares (one for each microarray hybridization) for each tissue. Colors
are graded with red indicating increased expression and green low expression
relative to reference. Sequences of the clones were compared to the mouse
genome database of Celera Genomics [http://www.celera.com/]. The location of
expression (greater than 2-fold above reference) in normal adult non-GI tissues
in our mouse gene expression database, location of the clone sequence relative
to the gene, and the identified or putative gene or gene family are indicated.
S, stomach; D, duodenum; J, jejunum; I, ileum; Ce, cecum; PC, proximal colon;
DC, distal colon; Chr, chromosome. PDF Figure 9A.pdf
HTML Figure 9A.html

Figure 9. Gene discovery using unknown/uncharacterized genes that are
highly expressed in the adult mouse GI tract.

(B) RT-PCR analysis of Amnionless gene expression in adult mouse ileum.
cDNA was prepared from poly A+ RNA obtained from adult ileum as described
and used for PCR to detect expression of Amnionless and GAPDH (control).
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