1. Volume 125, Issue 5, Pages 1428-1440 (November 2003)
Genetic background modifies intestinal pseudo-obstruction and the expression of a reporter gene in Hox11L1−/− mice1  
Melissa A Parisi, Audrey E Baldessari, Malissa H.K Iida, Christine M Clarke, Barbara Doggett, Senji Shirasawa, Raj P Kapur 
Gastroenterology 
Volume 125, Issue 5, Pages (November 2003)
DOI: /j.gastro

2. Figure 1
Construction of the Hox11L1 gene disruption and Hox11L1-nlacZ transgene. (A) Structure of the wild-type Hox11L1 gene. (B) The targeting vector used to create the disrupted Hox11L1 allele replaced a SmaI fragment containing a portion of the promoter and first exon with a PGK-neomycin resistance cassette in inverse orientation. (C) In the Hox11L1-nlacZ transgene, the Hox11L1 gene is replaced at the starting methionine codon within the first exon (NcoI site) with the β-galactosidase gene and a polyadenine cassette (pA) derived from the mouse protamine gene. The approximate locations of the tissue-specific enhancer and a Retinoic Acid Response Element (RARE) are indicated in the 5′ flanking region. Exons are indicated by solid boxes and Roman numerals. Selected restriction enzyme restriction sites are mapped. Arrows indicate the primer pairs used to genotype mice by PCR amplification (see Materials and Methods section).
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
Developmental pattern of Hox11L1-nlacZ expression. At E10.5 (A and D), β-gal activity is detected in cranial ganglia (white arrows), paravertebral sympathetic chain ganglia (thick black arrow), and dorsal root ganglia (thin black arrow), but no expression is detected in the gut (D). At E11.5 (B and E), the staining pattern is virtually identical, except that rare cells in the wall of proximal small intestine (arrow in E) are now detected. By E18.5 (C, F, and I), many X-gal-positive cells are detected throughout most of the gastrointestinal tract, including the distal ileum (di), cecum (ce), proximal large intestine (li), and stomach (F). In addition, β-gal activity is evident in the adrenal medulla (ad) and prevertebral autonomic ganglia (g) but not the kidneys (k in I), distal large intestine (li in C), or esophagus (e in F) until after birth. Sites of expression in adult mice (G, H, J, and K) include neurons in the distal esophagus (e) and proximal stomach, with only rare positive cells in the pylorus (p), abundant neurons in the distal ileum (di) and proximal colon (pc), and many in the cecum (ce). In addition, expression is evident in adrenal medulla (J, arrowheads) and brain (K, parasagittal section). co, adrenal cortex; ts, triangular septal nucleus; ∗, subcommisural organ; sc, superior colliculus.
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Immunohistochemical colocalization of neurochemical markers with neurons expressing the Hox11L1-nlacZ transgene (X-gal) in paraffin sections of gut (unless otherwise indicated as whole-mount sections). (A) Whole-mount section with cuprolinic blue (dark blue-gray) colocalization with X-gal (bright light blue) staining. (B) GFAP (glial fibrillary acidic protein; red-brown) staining does not colocalize with X-gal neurons. (C) Antibody to smooth muscle actin stains circular smooth muscle brown and does not colocalize with X-gal neurons. (D) Whole-mount section stained with NOS (NADPH diaphorase; purple) showing absence of colocalization with X-gal. (E) Substance P (brown) colocalizes with some X-gal neurons. (F) Calbindin (brown) colocalizes with some X-gal neurons. (G) Calretinin (brown) colocalizes with some X-gal neurons. (H) VIP (vasoactive intestinal peptide; brown) colocalizes with an occasional X-gal-positive neuron. (I) BrdU (bromo-deoxy-uridine; purple) never colocalizes with X-gal neurons. Arrows identify examples of colocalization of both markers.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
RT-PCR of endogenous Hox11L1 expression in adult murine tissues from null (Hox11L1−/−) and wild-type (Hox11L1+/+) animals: mixed B6/SVJ/DBA background. (A) The Hox11L1 transcript gives a PCR product of 570 bp. (B) The housekeeping gene DHFR gives a PCR product of 455 bp, which serves as a control for equal amounts of mRNA amplified from each tissue source.
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Phenotypes of Hox11L1−/− murine guts in the near-congenic B6 background strain. (A) Wild-type mouse gut at P60. (B) Hox11L1-null mouse gut showing proximal colonic distention (arrowhead), killed at P60 without obvious symptoms. (C) Symptomatic Hox11L1-null mouse gut showing megacecum (killed at P34) (bar = 1 cm). ce, cecum; di, distal ileum; re, rectum.
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Hox11L1 RNA is detected by RT-PCR analysis of tissues from N strain congenic Hox11L1+/+, but not Hox11L1−/−, mice. Upper panels in each column show the position of RT-PCR products for Hox11L1 mRNA. Bottom panels show RT-PCR products for dihydrofolate reductase mRNA (DHFR) from the same samples, which was used as a positive control for mRNA integrity. SCG, superior cervical ganglion; Ad, adrenal; stom, stomach; jej, jejunum; cec, cecum.
Gastroenterology  , DOI: ( /j.gastro )

8. Figure 7
Transgene expression in N10-congenic B6 (A, C, and E) vs. N10-congenic 129 genetic backgrounds. Representative whole-mount preparations demonstrate a relatively dense distribution of X-gal-positive myenteric neurons (blue dots) in the ileocecal junction (A) and midcolon (C) of a N10-congenic B6 mouse, in contrast to completely markedly reduced ileocecal (B) and completely absent (D) midcolonic X-gal staining in a N10-congenic 129 animal. Expression in the superior colliculi and triangular septal nuclei (arrows) persist in B6 (E) and 129 (F) mice. The intensity of CNS staining appears less in F only because the plane of section is slightly different from B and intersects small portions of the nuclei. TI, terminal ileum; PC; proximal colon.
Gastroenterology  , DOI: ( /j.gastro )