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Human Molecular Genetics Advance Access originally published online on August 24, 2007
Human Molecular Genetics 2007 16(21):2640-2646; doi:10.1093/hmg/ddm221
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Increased expression of Grainyhead-like-3 rescues spina bifida in a folate-resistant mouse model

Peter Gustavsson{dagger}, Nicholas D.E. Greene*,{dagger}, Dina Lad, Erwin Pauws, Sandra C.P. de Castro, Philip Stanier and Andrew J. Copp

Neural Development Unit, Institute of Child Health, University College London, Guilford Street, London WC1N 1EH, UK

* To whom correspondence should be addressed. Tel: +44 2079052217; Fax: +44 2078314366; Email: n.greene{at}ich.ucl.ac.uk

Received May 21, 2007; Revised August 3, 2007; Accepted August 12, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Neural tube defects (NTDs), such as spina bifida, are common and severe birth defects in humans but the underlying causes are poorly understood. The pathogenesis and etiology of spina bifida in the curly tail mouse closely resemble defects in humans, providing a well-characterized model of NTDs. Grainyhead-like-3 (Grhl3), which encodes a transcription factor, was recently identified as a candidate gene for curly tail based on chromosomal location and the occurrence of spina bifida in Grhl3 null mice. However, the causative curly tail mutation has not been established, while the relationship between Grhl3 gene expression and the known cellular defect leading to NTDs in curly tail is unknown. Spina bifida in curly tail results from a cell type-specific proliferation defect in the hindgut endoderm, and we find that Grhl3 is expressed specifically in this tissue during the final stages of spinal neural tube closure in wild type embryos. Moreover, Grhl3 expression is diminished in the spinal region of neurulation-stage curly tail embryos. Curly tail mice do not carry a coding region mutation in Grhl3, however, we found a putative regulatory mutation upstream of the Grhl3 gene, which may be responsible for the expression deficit. In order to test the hypothesis that spina bifida in curly tail mice results from insufficient expression of Grhl3, we generated Grhl3-expressing curly tail mice by bacterial artificial chromosome-mediated transgenesis and demonstrated complete rescue of spina bifida. This study provides evidence for a critical role of diminished Grhl3 expression in causing spinal NTDs in the curly tail mouse model.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Neural tube defects (NTDs) are severe congenital malformations of the central nervous system that affect 0.5–2 per 1000 human pregnancies, and arise when the neural tube fails to form correctly during development (1). Mouse models provide an opportunity to investigate the genetic and environmental factors that determine susceptibility to NTDs, and the curly tail mouse is probably the most extensively characterized such model (1–3). Homozygous mutants (ct/ct) develop lumbo-sacral spina bifida and/or tail flexion defects in around 40% of fetuses, and infrequent anencephaly. NTDs in the curly tail mouse closely resemble the corresponding birth defects in humans, in terms of the location and pathogenesis of the defects, the association of hydrocephalus with spina bifida, higher frequency of anencephaly in females and elevated levels of {alpha}-fetoprotein in amniotic fluid (2). In particular, curly tail represents a model for those defects that are not preventable by folic acid, whereas inositol has a protective effect leading to the suggestion that inositol may provide a preventive therapy for folate-resistant NTDs in humans (4–7). The incidence of spina bifida among ct/ct embryos is strongly influenced both by genetic background and by environmental factors, including retinoic acid and hyperthermia (2). Thus, the curly tail model with its reduced penetrance, phenotypic heterogeneity and involvement of environmental factors resembles the complex etiology of human NTDs.

Although the ct mutant phenotype was first described in the 1950s (8), and the cellular basis of the spinal defects has been determined (9,10), the genetic defect has yet to be definitively identified. The ct locus was previously mapped to a region of mouse chromosome 4 (11,12) containing the Grainyhead-like-3 (Grhl3) gene (also known as Get-1), which encodes a transcription factor related to the Drosophila grainyhead protein (13,14). The occurrence of spina bifida in targeted Grhl3 null mice suggested Grhl3 as a candidate for ct (15,16), although no coding region mutation was identified. Genetic interaction between Grhl3 and ct was also observed (15) but cannot be taken as proof of allelism since genetic interactions between ct and NTD-causing mutations at other loci, for example Pax3 and Vangl2, have also been described (17,18). In addition to the major ct locus on chromosome 4, different modifier genes have been mapped (11,19), indicating a complex aetiology underlying curly tail NTDs.

The principal previously reported expression sites of Grhl3 are in the surface ectoderm and epidermis (15,20), whereas the site of the primary defect in ct/ct embryos is thought to be the hindgut endoderm (9,10). Prior to failure of closure of the posterior neuropore (PNP) in ct/ct embryos the hindgut endoderm exhibits a reduced cellular proliferation rate, which leads to excessive ventral curvature of the caudal region. This curvature mechanically opposes closure of the neural folds, and thereby causes spina bifida, or tail flexion defects in those embryos in which final closure is delayed but does eventually occur (2,9,10). In the present study, we investigated in detail the hypothesis that reduced expression of Grhl3 causes spina bifida in curly tail embryos.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
To determine whether NTDs in curly tail mice result from altered Grhl3 expression we first examined the expression pattern of Grhl3, in detail, throughout neural tube closure in ct/ct and genetically matched wild-type (+ct/+ct) embryos. During spinal neurulation, Grhl3 exhibits a dynamic expression pattern, being expressed in the tail bud, notochord and the open neural folds of the PNP at E9.0–9.5 (Fig. 1A, C and D). Grhl3 expression is not detected in the closed neural tube at this stage, with the exception of the ventral forebrain (Fig. 1A). Expression in the spinal neural folds is no longer detectable by E10–10.5 (Fig. 1B and E), whereas intense expression becomes apparent in the hindgut, in embryos with more than 25–26 somites (Fig. 1B, E). This expression correlates with the precise location and developmental stage at which the hindgut cell proliferation defect arises, which causes spina bifida in ct/ct embryos (2,9,10).


Figure 1
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  		Figure 1. Expression analysis of Grhl3 gene during spinal neural tube closure. In an E9.5+ct/+ct embryo (A, C, D) whole mount in situ hybridization reveals expression of Grhl3 mRNA in the ventral forebrain, and in the caudal part of the embryo where expression is localized in the caudal-most part of the notochord (n; C) and the open neural folds (nf) of the PNP (C, D). In the caudal region of an E10.5+ct/+ct embryo (B), Grhl3 transcripts are detected in the hindgut (hg) endoderm (E), and the allantois (arrow in B). Dashed lines in A and B indicate the axial level of sections in C–E. Dotted line in E shows outline of section. Scale bar represents 250 µm (A–B) or 30 µm (C–E).

 
Using in situ hybridization, no consistent differences were detected in intensity or location of Grhl3 mRNA between ct/ct and +ct/+ct embryos. We, therefore, performed quantitative RT–PCR to evaluate expression levels during neural tube closure. In order to study precisely the stage of neural tube closure, we compared Grhl3 expression in caudal regions isolated from ct/ct embryos and equivalent somite staged +ct/+ct embryos that are matched for genetic background. At the 28–29 and 30–31 somite stages, which correspond to the final stages of spinal neurulation, Grhl3 mRNA was reduced in abundance in ct/ct embryos compared with wild-type by 56 and 41%, respectively (Fig. 2), whereas no difference was apparent at the 25–27 somite stage. These findings support the idea that insufficient expression of Grhl3, in the hindgut endoderm located ventrally to the PNP, could be causally related to spina bifida in curly tail embryos.


Figure 2
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  		Figure 2. Relative quantification of Grhl3 expression in curly tail, wild-type and Grhl3-expressing curly tail transgenics. Quantitative real time RT–PCR reveals a significantly lower level of Grhl3 expression in the caudal region of ct/ct (grey boxes) compared with +ct/+ct (white circles) embryos at the 28–29 and 30–31 somite stages (during the latter stages of spinal neurulation at E10.5) and 1 day later, at E11.5 (*–significant difference compared with +ct/+ct at same stage; P < 0.01). In transgenic curly tail embryos (ct/ctTg(Grhl3)1ICH; black diamonds), which carry a BAC containing the intact Grhl3 gene, Grhl3 mRNA was more abundant than in ct/ct embryos at all stages examined (**–significant difference compared with ct/ct at same stage; P < 0.005).

 
Sequencing of the cDNA confirmed that curly tail mice do not carry a mutation in the Grhl3 coding sequence, nor were we able to identify any additional exons by 5' or 3' RACE or RT–PCR (data not shown). We hypothesized that reduced abundance of Grhl3 mRNA in ct/ct embryos could result from a regulatory mutation in a promoter or enhancer element and we, therefore, sequenced 30 kb of genomic DNA upstream of the Grhl3 start codon. In this entire stretch of non-coding DNA, sequence analysis revealed only six changes, including one known SNP and variation in length of four different nucleotide repeats (Supplementary Material, Table S1). In addition to these differences, a novel C to T nucleotide substitution was identified at position –21350 in curly tail DNA, compared with the partially congenic wild-type strain which carries a region of SWR encompassing the ct locus (Fig. 3A). The C to T nucleotide substitution is not a known polymorphism according to dbSNP. The wild-type sequence, corresponding to nucleotide C, was also present in C57/BL6 and four other strains. A genomic region of 180 bp encompassing the site of the substitution is 88% identical to a region on rat chromosome 5 and the C nucleotide is conserved (NCBI BLAST). Homology was not detected to human, chimpanzee or chick genomic sequence.


Figure 3
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  		Figure 3. A nucleotide substitution in curly tail lies within a putative enhancer region upstream of the Grhl3 coding sequence. (A) Sequencing of genomic DNA, reveals a C to T nucleotide substitution between +ct/+ct and ct/ct, 5' to the coding region of Grhl3 at nucleotide position –21350. Transgenic ct/ctTg(Grhl3)1ICH mice contain both T (ct DNA) and C (wild-type sequence of the BAC) sequences at nucleotide –21350. (B) A 577 bp sequence, encompassing the site of the putative ct mutation, in either sense (En S) or anti-sense (En AS) orientation relative to the SV40 promoter, increases promoter activity in a luciferase activity. Representative data for the wild-type sequence is shown, from three separate assays in 293T cells. *–indicates significant difference compared with promoter activity of SV40 alone (P < 0.01).

 
In order to investigate a possible functional role of this region of genomic DNA we cloned a 577 bp fragment, encompassing the site of the nucleotide change, upstream of the SV40 promoter in a luciferase reporter plasmid. In both human (293T) and mouse (3T3) cell lines this region consistently showed enhancer activity (P < 0.05, Fig. 3B). The putative enhancer-containing region was tested in both sense and anti-sense orientations relative to the promoter. In each case, the anti-sense construct exhibited higher enhancer activity than the sense construct, presumably due to favourable orientation of the insert to the SV40 promoter.

In order to experimentally test the hypothesis that reduced expression of Grhl3 causes spina bifida, we reasoned that reinstatement of Grhl3 expression in ct/ct embryos should prevent the occurrence of spina bifida. Therefore, we generated a transgenic ct/ct line in which Grhl3 is over-expressed from a bacterial artificial chromosome (BAC), containing the complete Grhl3 gene together with 120 kb of upstream sequence that encompasses the site of the putative ct mutation (Fig. 4A). BAC-positive transgenic mice were viable and fertile and displayed no obvious abnormalities up to 9 months of age.


Figure 4
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  		Figure 4. Generation of transgenic curly tail mice that express Grhl3. (A) BAC RP24-327D13 contains 176 kb genomic DNA, encompassing the intact Grhl3 gene plus 120 kb upstream DNA, including the site of the C to T substitution at nucleotide position –21350, identified in ct DNA. Fragments of one other gene, IL28ra, and one predicted gene, I493055I21Rik, are also present in the BAC, but neither gene is complete. The majority of the pTARBAC1 vector was removed by NotI digestion, prior to micro-injection. (B–C) At E10.5, Grhl3 is expressed in the hindgut (hg) of ct/ctTg(Grhl3)1ICH embryos, as in ct/ct embryos (compare with Fig. 1E). In addition, expression is also detected in the ventral neural tube (nt). Dotted line in B indicates level of section in C. (D) At E10.5, the mitotic index in the hindgut endoderm is significantly higher in ct/ctTg(Grhl3)1ICH embryos than in ct/ct embryos with a large PNP (*P < 0.02; Mann Whitney Rank Sum Test). Scale bar represents 250 µm (B) and 30 µm (C).

 
Litters containing transgenic offspring were generated by intercrossing of Grhl3 BAC-positive ct/ct males with ct/ct females. Pups were phenotyped for spinal defects presenting as tail flexion defects and E11.5 embryos were phenotypically scored for spina bifida and/or curled tail. Among ct/ct pups there was a 38% frequency of tail flexion defects which correlates with the frequency in the curly tail colony, whereas no defects were observed among Grhl3-expressing transgenics (P < 0.001; Table 1). Analysis of non-transgenic ct/ct fetuses at E11.5 revealed a 58% incidence of spina bifida and/or tail flexion defects (Table 1), the increased incidence compared with pups being due to perinatal lethality of fetuses that exhibit spina bifida. In contrast, among 41 ct/ct fetuses that carried the Grhl3 BAC, spinal defects were never observed (Table 1), demonstrating complete rescue of the ct defect (P < 0.001).


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  		Table 1. Spinal neural tube closure defects in curly tail mutants are rescued by expression of Grhl3

 
Quantitative RT–PCR confirmed that rescue of spina bifida correlated with a significant up-regulation of Grhl3 expression in transgenic ct/ct embryos, at all stages examined (Fig. 2). We also performed whole mount in situ hybridization to assess the localization of Grhl3 expression in transgenic embryos at E10–10.5. Expression was detected in the same sites as in stage-matched ct/ct and +ct/+ct embryos, including the hindgut (Fig. 4B and C), and presumably represents the combined expression from the BAC and the endogenous gene. Grhl3 expression was not detected in novel locations, suggesting that appropriate regulatory elements, that determine expression pattern, are present within the BAC. Interestingly however, expression in the ventral neural tube persisted until E10.5 (30–31 somite stage) in ct/ctTg(Grhl3)1ICH embryos, such that the ventral neural tube and hindgut were positive at the same developmental stage (Fig. 4C). In contrast, neural tube expression is not detected after the 20–22 somite stage in ct/ct and +ct/+ctembryos, prior to onset of expression in the hindgut (Fig. 1B and E).

Finally, we tested the hypothesis that enhanced expression of Grhl3 would correct the cell proliferation defect in the hindgut that is the known cause of spina bifida in ct/ct embryos (2). Phospho-histone H3 immunostaining of transverse sections through the caudal region was used to determine the mitotic index in the hindgut of ct/ctTg(Grhl3)1ICH and ct/ct embryos, sub-divided according to the presence of a large (affected) or small (unaffected) PNP, as categorized previously (21). In Grhl3-transgenic ct/ct embryos the mitotic index was significantly higher than in the hindgut of pre-spina bifida ct/ct embryos with a enlarge PNP (Fig. 4D). Thus, an increase in proliferation rate in the hindgut correlates with normalization of spinal neural tube closure in ct/ct embryos.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The shared features of curly tail and human NTDs, such as multifactorial etiology involving genetic and environmental factors, has stimulated a number of studies aimed at understanding the mechanisms underlying the development and possible prevention of curly tail NTDs. These studies identified a proliferation defect in the hindgut as the primary cellular defect (2), and showed that inositol supplementation can prevent the spinal NTDs, in contrast to folic acid which is ineffective (4). This latter finding has led to inositol supplementation of a human pregnancy at high risk of NTD (22), and to an ongoing clinical trial of inositol as an adjunct therapy to folic acid for prevention of NTDs (http://www.pontistudy.ich.ucl.ac.uk). Despite these previous advances, the genetic causation of NTDs in curly tail has not been identified.

The chromosomal location and severe NTD phenotype of null mutant embryos identified Grhl3 as a candidate gene for curly tail (15), and the finding of apparent stage- and tissue-specific expression of Grhl3 in the present study is, therefore, particularly interesting. Prior to E10, Grhl3 transcripts are found in the neural folds but not in the hindgut of wild-type embryos. However, precisely at E10–10.5 (the 25–30 somite stage), when the hindgut cell proliferation defect is observed (10), we detect strong Grhl3 expression in hindgut epithelial cells. Moreover, when comparing the caudal region of ct/ct embryos with stage-matched +ct/+ct controls, we detect a quantitative reduction in Grhl3 expression solely at the 28–31 somite stage, in close agreement with the timing of Grhl3 expression in the hindgut. Finally, increase of Grhl3 expression above wild-type level via BAC transgenesis completely rescues the spinal NTDs in ct/ct mice. The complete prevention of spina bifida is particularly notable as, by generation of a BAC-transgenic on the curly tail genetic background, all the deleterious modifier genes (11,19) are still present. Taken together, these data strongly support the idea that reduced expression of Grhl3 is the cause of spina bifida in curly tail embryos.

Both Drosophila grainyhead and mouse Grhl3 are required for the barrier function of the surface epidermal barrier, playing a role in development of the fly cuticle, and in differentiation of the mouse epidermis (14,16,23). Expression analysis reveals that Grhl3 regulates a number of genes that mediate the differentiation, adhesive properties and barrier function of the epidermis (16). The occurrence of spina bifida in 100% of null mutant mice clearly demonstrates that Grhl3 is also required earlier in development (15,16), but the role in spinal neural tube closure has not been defined. We propose that expression of Grhl3 is required for enhanced hindgut cell proliferation that is observed in normal embryos, but not in affected ct/ct embryos (10). Our data suggest that prevention of spina bifida by BAC-transgenic expression of Grhl3 acts via correction of the cell proliferation imbalance, as previously seen in inositol-treated embryos (6). We suggest, therefore, that the enhancer function of the upstream genomic region in which we have detected a C to T nucleotide substitution is specific for Grhl3 expression in the hindgut at E10–10.5, and that the increase in expression from the 27 somite stage in wild-type embryos is required for adequate cell proliferation. This hypothesis suggests that the putative Grhl3 enhancer should drive reporter gene expression specifically to the hindgut epithelium at E10–10.5 in transgenic mice, a prediction that will be tested in future studies.

An intriguing feature of NTDs in curly tail, in common with a number of mouse mutants, is the partial penetrance with variable expressivity in which some homozygous mice are unaffected, while others exhibit spina bifida or tail flexion defects alone. Given the hypothesis that curly tail represents a hypomorphic allele of Grhl3, it is plausible that the extent of the expression deficit determines penetrance. However, although we detected variability in Grhl3 expression level between individual ct/ct embryos, there was no apparent correlation between expression level and severity of the defect in spinal neurulation, as indicated by the length of the PNP. Therefore, it is possible that penetrance is influenced by variability in expression or function of key genes that act downstream of, or parallel to, Grhl3. This could include Grhl3-regulated genes or independent modifier genes whose effect influences the defective pathway.

In summary, the stage-specific onset and reduced abundance of Grhl3 expression in the hindgut correlates precisely with the development of spina bifida in ct/ct embryos, and potentially results from mutation of an upstream enhancer element. Rescue of spina bifida by Grhl3 demonstrates that this expression deficit plays a causative role. It is interesting to speculate that GRHL3 could also be involved in the etiology of human NTDs, for which the genetic causation remains poorly understood (24).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Mice
Animal studies were carried out under regulations of the Animals (Scientific Procedures) Act 1986 of the UK Government, and according to guidance issued by the Medical Research Council, UK, in Responsibility in the Use of Animals for Medical Research (July 1993). Curly tail mice were maintained as a homozygous, closed random-bred colony (2). The wild-type, partially congenic curly tail strain (+ct/+ct) was generated previously (2), and was maintained as a random-bred closed colony. This strain is at N5 (five generations of backcrossing) such that 96% of background genes are expected to be homozygous and shared with the curly tail strain. Experimental litters were generated by timed matings in which mice were paired overnight and females checked for copulation plugs the following morning, designated embryonic day (E) 0.5. Pregnant females were killed by cervical dislocation and embryos were dissected from the uterus in Dulbecco's Modified Eagle's Medium (Invitrogen) containing 10% fetal calf serum (Sigma). Embryos were washed in phosphate buffered saline (PBS) and either immediately frozen prior to RNA isolation or fixed in 4% paraformaldehyde (PFA) in PBS at 4°C overnight for in situ hybridization.

Whole mount in situ hybridization
Whole mount in situ hybridization, followed by preparation of 8 µm paraffin sections, was performed as described previously (4,25), using a digoxygenin-labelled cRNA probe which was complementary to a 494 bp fragment of Grhl3 corresponding to nucleotides 130–624 (GenBank accession number NM001013756).

Quantitative real time RT–PCR
Total mRNA was extracted from the spinal region of E10.5 embryos (caudal to the 20th somite), isolated by micro-dissection using a needle. First strand cDNA was synthesized from 700 ng template RNA by reverse transcription using Superscript II (Invitrogen) with random hexamers. Grhl3 gene expression was determined by quantitative real time PCR using the SYBR GreenER reagent kit (Invitrogen) according to the manufacturer's instructions on a 7500 Fast Real Time PCR system (Applied Biosystems), with each sample analysed in triplicate. Specific primers were designed to amplify exons 10–13 of the mouse Grhl3 cDNA. Primers were 5'-CCAGACTCCAGTAACAATG-3' and 5'-AAGGGTGAGCAGGTTCGCTT-3', generating a 218 bp PCR fragment. All results were normalized with respect to Gapdh expression which was analysed for each sample in all runs. Gapdh primers were '-ATGACATCAAGAAGGTGGTG-' and 5'-CATACCAGGAAATGAGCTTG-', generating a 177 bp PCR fragment. PCR conditions are available on request.

Sequence analysis
Sixty seven overlapping genomic DNA fragments spanning a total of 30 kb of DNA upstream of the Grhl3 gene were amplified by PCR (primers and PCR conditions available on request). Purified PCR products were sequenced using big dye terminator chemistry (Applied Biosystems) and analysed on a MegaBACE 1000 (Amersham). Sequence reads derived from both strands were assembled, aligned and analysed for nucleotide differences using Sequencher (GeneCodes).

Luciferase assay
A potential enhancer effect of the region containing the C-21350T nucleotide substitution was analysed by luciferase assay using the SV40 promoter-containing pGL3 vector (Promega, UK). Primers with additional KpnI sites were used to amplify a 577 bp genomic DNA fragment from wild-type (+ct/+ct) genomic DNA, using Pfx polymerase (Invitrogen, UK), which was cloned into the pGL3 promoter vector. Primer sequences were 5'-CGGGGTACCTGTATTTTCTTGCTTGAAACG-3' and 5'-CGGGGTACCTCAGCGTAAGA AAGCTGTG-3'. 293T and 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 2 mM L-glutamine, non-essential amino acids and antibiotics. Transfections were performed in 96-well plates using 50 ng of pGL3 plasmid DNA together with 0.15 µl FuGene (Roche) reagent per well. Firefly and Renilla luciferase activities were measured using a Luciferase Reporter Gene Assay System (Luclite, Perkin Elmer) on a Fluostar Optima plate reader (BMG Labtech).

Generation of BAC Grhl3 transgenic curly tail mice
BAC RP24-327D13 was identified (in UCSC, NCBI and ENSEMBL) as containing the intact Grhl3 gene and no other intact known genes. Linear BAC DNA was purified (see Supplementary Methods for details) and used for pronuclear micro-injection of fertilized curly tail oocytes which were then transferred to CD1 surrogate mothers. Transgenic litters were screened for the presence of the BAC by PCR. The presence of a tail flexion defect was recorded for pups at P2, and confirmed at weaning.

Immunohistochemistry and calculation of mitotic index
Embryos were fixed in 4% PFA, dehydrated through an ethanol series and embedded in paraffin wax. Transverse 7 µm sections through the caudal region were de-waxed and treated for antigen retrieval by steaming in Declere (Firefly Scientific) for 40 min. All washes were performed in TBST (500 mM Tris, 9% NaCl, pH7.6, 0.1% Triton X-100). Blocking (30 min) and incubation with primary and secondary antibodies were performed with 5% heat-inactivated goat serum (HIGS, SIGMA) in TBST containing 0.15% glycine and 2 mg/ml BSA. Slides were incubated with rabbit polyclonal IgG anti-phospho-Histone H3 antibody (1:500 dilution, Upstate Cell Signaling Solutions), followed by polyclonal goat anti-rabbit IgG biotinylated secondary antibody (1:250 dilution, DAKO). Detection was performed using streptavidin-Alexa Fluor 555 conjugate in TBS (1:300 dilution, Molecular Probes). Slides were washed with TBS and mounted in vectashield with DAPI (Vector Laboratories). Images were captured on a Leica DC500 microscope using Fire Cam Software. The mitotic index (number of phospho-histone H3 positive cells/total cell number x 100) in the hindgut was obtained from alternate transverse sections at matched levels of the caudal region (n = 27 sections from three embryos for each experimental group).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was funded by the Wellcome Trust, Child Health Appeal Trust and the Swedish Research Council.


   ACKNOWLEDGEMENTS
 
We are grateful to Ulla Dennehy, Matthew Grist, Nicoletta Kessaris, Dawn Savery and Mona Al-Qatari for valuable assistance and to Henny van Straaten and Madeleine Brouns for stimulating discussion.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first 2 authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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