1. Volume 134, Issue 4, Pages 1104-1115 (April 2008)
Perturbation of Hoxb5 Signaling in Vagal Neural Crests Down-Regulates Ret Leading to Intestinal Hypoganglionosis in Mice 
Vincent C.H. Lui, William W.C. Cheng, Thomas Y.Y. Leon, Danny K.C. Lau, Maria–Mercedes Garcia–Bareclo, Xiao P. Miao, Mandy K.M. Kam, Man T. So, Yan Chen, Nancy A. Wall, Mai H. Sham, Paul K.H. Tam 
Gastroenterology 
Volume 134, Issue 4, Pages (April 2008)
DOI: /j.gastro
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2. Figure 1
NCC express Hoxb5 in embryonic intestine. Adjacent sections of mouse embryos were immunostained for p75NTR (A and C) and Hoxb5 (B and D). By E11.5, NCC colonized the stomach and the proximal midgut and arrived at the cecum (A). NCC (arrowheads) at the stomach (a), at the proximal midgut (b), and at the migration front at the cecum (c) were immunoreactive for Hoxb5 (arrowheads; d–f). Hoxb5 immunoreactivity was also localized at the mesenchyme of the stomach and duodenum (arrows; B). By E12.5, NCC arrived at the colon (C) and expressed Hoxb5 (D). Regions highlighted with dotted lines were enlarged as shown. Abbreviations: st, stomach; du, duodenum; ce, cecum; pmg, proximal midgut; co, colon; h, heart, lb, limb bud.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Generation and genotyping of enb5 mice. (A) Transgenic scheme to express enb5 in vagal NCC in mouse using Cre-loxP. Transcription from chicken β-actin gene promoter (CAG) stops at the first polyadenylation signal (pA) 3′ of the chloramphenicol acetyl transferase (CAT) gene in the enb5 transgenic mouse. Enb5 mouse was crossed to b3-IIIa-Cre mouse to obtain a mouse transgenic for both enb5 and b3-IIIa-Cre (enb5/Cre). Cre expression in vagal NCC resulted in the excision of CAT gene and the first polyadenylation signal in NCC in enb5/Cre mice. Transcription from the recombined transgenic allele extends to the cDNA encoding the enb5 chimeric protein. (B) PCR genotyping of enb5 and b3-IIIa-Cre transgenic loci. Amplification from the enb5 locus and b3-IIIa-Cre locus generated PCR fragments of 555 bp (enb5) and 374 bp (Cre), respectively. Primer locations are indicated with arrows.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Element b3-IIIa directs vagal NCC expression of Cre in embryos. (A) NCC (p75NTR+ brown; solid arrowheads) were found migrating from the neural tube into the foregut at E10.5. (B) Immunohistochemical analysis on adjacent sections indicated that Cre immunoreactivity (brown) overlapped with p75NTR (compare A and B). Immunofluorescence staining for Cre (green) and p75NTR (red) revealed Cre expression in a subset of NCC (C; arrowheads). Cre-expressing NCC have colonized the stomach and the proximal midgut at E10.5 (D, D‵, D″). By E11.5, NCC have arrived at the midgut (E), and Cre immunoreactivity was not detected in NCC (F). Whole-mount alkaline phosphatase activity staining of E13.5 b3-IIIa-Cre/Z/AP embryonic gut localized AP+ cells (marker for Cre-expressing NCC and their descendants) along the entire gut (G). AP+ cells (arrows) were localized in the mesenchyme of embryonic gut (H). Boxed regions were enlarged as shown on the right. Abbreviations: nt, neural tube; da, dorsal aorta; cv, cardinal vein; es, esophagus; st, stomach; pmg, proximal midgut; mg, midgut.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Transgenic mice expressing enb5 displayed ENS abnormalities. One enb5/Cre mouse developed a distended abdomen at P21, with enlarged duodenum and jejunum, and constricted ileum (B). Transgenic littermate (enb5) was included as control (Ctrl) (A). Sections of respective gut regions of this enb5/Cre mouse (E and F) and control (C and D) were immunostained for neurons (Tuj1). Neurons (arrowheads; brown) were localized at the jejunum of enb5/Cre mouse (E) and at the jejunum (C) and ileum (D) of control mouse. In contrast, neurons were absent from the ileum (F) of enb5/Cre mouse. Phenotypically indistinguishable enb5/Cre mice displayed hypoganglionosis and defective peristalsis. Neurons in the proximal and distal small intestine of enb5/Cre mice and single transgenic control (Ctrl) littermates (enb5 or Cre) were stained with Cuprolinic Blue (arrows, G and H). Representative photographs of stained distal small intestines of double (H) and single (G) transgenic mice are shown. The neurons in each field were counted with ×200 magnification, and neurons/field was calculated as shown (I and J). BaSO4 transit assay was performed to determine the time needed for the stomach content to travel the entire length of the intestine. Double transgenic mice (enb5/Cre) showed a significantly longer transit time than controls (Ctrl) (K). Values represent the mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Absence of enb5-expressing ganglia in the distal intestine of enb5/Cre mice. Using an antibody specific for the repressor domain of en protein, enb5 expression in the stomach and small intestine of enb5/Cre mice was analyzed. (B) Neurons (Tuj1+, green) expressing enb5 (red, arrowheads) were localized in the stomach of adult enb5/Cre mouse; enb5− neurons (arrows) were also detected. No immunoreactivity for enb5 was seen in single transgenic control littermates (Ctrl) (A). Neurons (Tuj1+, brown, C) expressing enb5 (brown, arrowheads, D) and not expressing enb5 (arrows, D) were found in the stomach. Region highlighted with dotted line was enlarged as shown in inset. enb5+ Neurons (arrowheads) were scarce in the proximal small intestine (F), and none were detected in the distal small intestine (H) of enb5/Cre mice. Adjacent sections were stained with Tuj1 to show the development of ganglia in the enb5/Cre stomach (C) and proximal (E) and distal small intestine (F). Abbreviations: lm, longitudinal muscle; cm, circular muscle.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Retarded migration of enb5-expressing NCC in embryonic gut. Adjacent sections of E11.5 enb5/Cre embryo were immunostained for p75NTR and enb5 to localize enb5-expressing NCC. NCC (p75NTR+ arrows) have migrated to the small intestine (A). In contrast, only a few enb5-expressing NCC have entered the small intestine, although they were abundant in the stomach (B; solid arrowheads). (C) NCC (stained blue; arrowhead) have migrated to the cecum (C′) in control E11.5 embryos. Differential NCC (arrowhead) migration was observed in enb5/Cre embryos, either only halfway down the small intestine (D and D′) or just into the proximal small intestine (E and E′). Representative photographs are shown with the number (n) of embryos showing that degree of migration. Regions highlighted by dotted lines were enlarged and shown underneath. Abbreviations: st, stomach, si, small intestine; c, cecum.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Down-regulation of Ret in enb5/Cre mice. Using Phox2b as an NCC marker (green), Ret expression (red) in control (enb5) and enb5/Cre embryos was analyzed by coimmunofluorescence. Most NCC in the stomach of E11.5 (A, A′, and A″) and E13.5 (C and C′) enb5/Cre embryos expressed no Ret signal (arrowheads), whereas NCC at the small intestine of E13.5 enb5/Cre embryos expressed strong Ret signal (arrows; C″). NCC in the E11.5 (B and B′) and E13.5 (D and E) control embryos expressed a high level of Ret (arrows), whereas intense Ret immunoreactivity was localized to a few NCC in the stomach of enb5/Cre embryos (arrows; A′, A″, C′). Regions highlighted by dotted lines were enlarged and shown on the right. (C) Immunohistochemical staining revealed that Ret was highly expressed by the enteric ganglia (brown) in control stomach (enb5) (F and F′). In contrast, Ret was barely detectable in the myenteric region in enb5/Cre stomach (H and H′). Regions highlighted by dotted lines were enlarged and shown. Tuj1 staining of the myenteric regions of the control (G) and enb5/Cre (I) stomach are shown. (J) A reporter plasmid was constructed with the Ret promoter linked to the luciferase gene and transfected into HTB11 cell with hoxb5- and enb5-expressing vectors either alone or in combination. Transfection with an empty vector was the control. Fold induction of luciferase activity in different combinations was calculated with respect to the luciferase activity in the control, which was arbitrarily regarded as 1. Values reported in the Figure represent the mean ± SEM. Abbreviations: st, stomach; si, small intestine.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Analysis of mutations/variations of HOXB5 gene in HSCR patients. (A) Homology profile with conservation criteria of 75% and resolution of 100 bp of the comparison between human HOXB5 sequence and mouse Hoxb5 sequence. Four amplicons (I–IV) covering the exons, UTR, and CNS of the HOXB5 gene were generated for sequencing analysis. Locations of the 3 SNPs (DS1–DS3) are indicated. (B) Genotype and allele frequencies of SNPs in patients and controls. SNPs were analyzed using Haploview. DS1, DS2, and DS3 are in Hardy–Weinberg equilibrium. Abbreviations: UTR, untranslated region; CNS, conserved noncoding sequence, DS, downstream.
Gastroenterology  , DOI: ( /j.gastro )
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