1. Volume 37, Issue 1, Pages 29-40 (January 2003)
Colonic Epithelial Expression of ErbB2 Is Required for Postnatal Maintenance of the Enteric Nervous System 
Steven A. Crone, Alejandra Negro, Andreas Trumpp, Marco Giovannini, Kuo-Fen Lee 
Neuron 
Volume 37, Issue 1, Pages (January 2003)
DOI: /S (02)

2. Figure 1
ErbB2/Nestin-Cre Mutant Mice Display a Hirschsprung's Disease-like Phenotype
(A) A targeting vector was constructed by introducing a LoxP site into the first intron and a neor-cassette flanked by two LoxP sites 1.5 kb upstream of the first exon (gray box). This targeting vector was electroporated into ES cells. ES cell clones containing the targeted (T) allele were isolated and then transfected with plasmids expressing Cre recombinase under the control of the CMV promoter. ES cell clones in which the neor-cassette was removed but that maintained two LoxP sites were isolated. This is called the floxed (F) allele. In the recombined allele (R), the neor-cassette and first exon are removed, and only one LoxP site is retained.
(B) Southern blotting analysis of ES cell clones. Following digestion of ES cell DNA by BamHI and HindIII, probe A hybridizes to a 5.0 kb band for both F and T alleles and a 8.3 kb band for the wild-type allele (+). Following digestion with HindIII, probe B hybridizes to a 9.4 kb band for the T allele, a 8.3 kb band for the + and F alleles, and a 5.3 kb band for the R allele. ES clones containing the F allele were used to generate mutant mice.
(C) ErbB2 floxed animals were crossed with nestin-Cre transgenic mice. Brain DNA was isolated from newborn mice, digested with HindIII, and hybridized with probe B. Nearly complete recombination occurs in mice containing floxed alleles and the nestin-Cre transgene (F/R; Cre+). Note: germline recombination of one allele (R) occurs in mice in which a parent carries a floxed allele and the nestin-Cre transgene, even if the offspring does not carry the Cre transgene (F/R; Cre−) No recombination of the floxed allele occurs in the absence of Cre expression (F/+; Cre−).
(D) ErbB2/nestin-Cre mutant mice display distended proximal colons by 4 weeks of age (arrowheads in [C]). Control at left; erbB2/nestin-Cre mutant at right; St, stomach; Ce, cecum.
Neuron  , 29-40DOI: ( /S (02) )

3. Figure 2 Postnatal Enteric Neuron Loss in ErbB2/Nestin-Cre Mutants
(A) AChE staining of whole-mount intestines reveals fewer neurons in the colons of 2- and 5-week-old erbB2/nestin-Cre mice, but not in the colons of newborn (0 weeks of age) or small intestines of 5-week-old erbB2/nestin-Cre mice compared to controls.
(B) Myenteric neuron number in the colons of newborn mice. There is no statistically significant difference between the density of neurons in the colons of control and erbB2/nestin-Cre mutant mice at birth (control n = 3, erbB2/nestin-Cre n = 5; p = 0.121).
(C) Neuron number in the colons of P24 animals. There are 55% fewer neurons/mm2 in erbB2/nestin-Cre mutant compared to control colons (n = 3; p = 0.010). There is no statistically significant difference between the number of neurons/mm2 in the small intestine of controls and erbB2/nestin-Cre mutants (n = 3; p = 0.417).
Neuron  , 29-40DOI: ( /S (02) )

4. Figure 3
The Loss of Enteric Neurons and Glia in the Colon of ErbB2/Nestin-Cre Mutants Occurs Postnatally
Whole-mount immunohistochemistry with antibodies against TuJ1 (neurons) and S100β (glia) reveals a reduction of both neurons and glia in the colons of erbB2/nestin-Cre mutants compared to control littermates at 4 weeks of age. There is no apparent difference in TuJ1 or S100β immunoreactivity between erbB2/nestin-Cre mutants and controls at birth.
Neuron  , 29-40DOI: ( /S (02) )

5. Figure 4
A Loss of Neuropeptide-Expressing Neurons in Both the Myenteric and Mucosal Plexuses of the ErbB2/Nestin-Cre Mutant Mice
Transverse sections of colons of 4-week-old control and erbB2/nestin-Cre mutant mice were stained with antibodies to the following neuropeptides: vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), calcitonin gene related peptide (CGRP), and substance P (SP). Mutant mice display a loss of immunoreactivity for all of these neuropeptides in both the myenteric (My) and mucosal (Muc) layers of the colon.
Neuron  , 29-40DOI: ( /S (02) )

6. Figure 5
Loss of ErbB2 Expression in the Colonic Epithelia of ErbB2/Nestin-Cre Mutants
(A and B) Immunohistochemical detection of β-galactosidase was used to analyze where Cre-mediated recombination occurs in transverse sections of the colons of ROSA26-R/nestin-Cre mice at 3 weeks of age. Recombination occurs in the myenteric and mucosal plexuses of the enteric nervous system (arrows) in the colon and small intestine, as well as the mucosa of the colon (arrowheads), but not of the small intestine. My, myenteric layer; Muc, mucosal layer.
(C) Western blot analysis demonstrates decreased erbB2 protein levels in the colons of erbB2/nestin-Cre mutants (CM) compared to control littermates (CNT). ErbB3 protein levels are the same. Actin protein levels were measured to control for protein loading.
(D) Immunoreactivity for neuregulin is detectable in transverse sections of wild-type colons in the colonic epithelia (arrowheads), as well as the enteric ganglia (arrows). My, myenteric layer; Muc, mucosal layer.
(E, F, I, and J) In situ hybridization of colon sections from 2-week-old mice demonstrates decreased expression of erbB2 mRNA (E and F), but not erbB3 mRNA (I and J), in the mucosal layer of the colons of erbB2/nestin-Cre mutants. No erbB2 or erbB3 mRNA is detected in the myenteric layer or the enteric ganglia.
(G, H, K, and L) Bright-field images of hematoxylin-eosin stained sections shown in (E), (F), (I), and (J). My, myenteric layer; Muc, mucosal layer.
Neuron  , 29-40DOI: ( /S (02) )

7. Figure 6
The Requirement for ErbB2 for Enteric Neuron Survival Is Not Cell Autonomous
(A and B) Immunohistochemical detection of β-galactosidase (blue) was used to detect Cre-mediated recombination in enteric ganglia in whole mount preparations of colon from ROSA26-R/nestin-Cre (A) and ROSA26-R/P0-Cre (B) mice. TuJ1 (green) immunoreactivity was used to visualize enteric ganglia.
(C) Immunohistochemical detection of β-galactosidase was used to detect Cre-mediated recombination in transverse sections of colons from ROSA26-R/P0-Cre mice. Recombination occurs in enteric ganglia (arrows) but not in the mucosa. My, myenteric layer; Muc, mucosa.
(D) Quantitation of recombined cells in the enteric ganglia demonstrates no significant difference between ROSA26-R/nestin-Cre and ROSA26-R/P0-Cre mice (n = 4; p = 0.746).
(E and F) AChE staining of whole-mount colons demonstrates intact enteric ganglia in the myenteric plexus of erbB2/P0-Cre mutants (F) compared to controls (E).
Neuron  , 29-40DOI: ( /S (02) )

8. Figure 7
Nonautonomous Role for ErbB2 in Maintaining the Enteric Nervous System
Neuregulin (NRG) produced by the enteric nervous system or mucosal epithelia induces cells in the mucosa of the colon (via erbB2 and erbB3 receptors) to produce survival factors, which in turn are required for the postnatal maintenance of the enteric nervous system.
Neuron  , 29-40DOI: ( /S (02) )