Adult (6- to 8-week-old) male and female C57BL/6 mice were used for this study. All animal studies were approved by the Chinese Association for Laboratory Animal Sciences. The age of mouse embryos was determined by the appearance of the vaginal plug, which was taken to be E0.5. The birth day of the pup was marked as P1 for these experiments. Generations of Isl1
mice have been reported previously
[30, 31]. In brief, we used a ‘floxed’ Isl1 allele (Isl1
) in which loxP sites were inserted into the introns flanking exon 4 of the Isl1 locus
, and a tamoxifen-inducible knockin Isl1 mER-Cre-mER allele
[31, 39]. Isl1
mice were mated with Isl1
ice to generate litters with equal numbers of Isl1
-inducible knockouts (Isl1
) and Isl1
ontrols. To induce excision in Isl1
embryos, pregnant females were administered an oral gavage of 300 μl of tamoxifen (T5648; Sigma, St. Louis, MO, USA) in sesame oil (10 mg/ml) at E11.5 for three consecutive days just before Isl1 expression sharply increased, and the embryos were harvested at E14.5 or E18.5.
Two patients with hypertrophic pyloric stenosis were selected from the 306th Hospital of People’s Liberation Army, Beijing. Pyloric tissue stored in the 4% Paraformaldehyde buffered in 0.01M PBS were selected from excess material collected from patients undergoing operations to retrieve surgical specimens. The study on human material was performed according to the instructions and guidelines of the 306th Hospital Ethics Committee. Approval of this study was granted by the Chinese Association for Laboratory Animal Sciences and the 306th Hospital Ethics Committee.
PCR, semi-quantitative PCR and real-time quantitative PCR
Genomic DNA was isolated from tail biopsies following the HotSHOT method
 and genotyping was performed using standard PCR methods with sequence-specific primers (Additional file
2: Table S1). Total RNA was extracted from the pyloric regions of stomachs at E14.5 and E18.5 using commercial reagents (12183–016; Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA was converted to cDNA using M-MLV reverse transcription reagents (M170A; Promega, Madison, WI, USA). RT-qPCR was performed using SYBR Green master mix (DRR420A; TaKaRa, Dalian, China) in the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and reactions were done in triplicate. RT-qPCR conditions were as follows: 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Relative RNA quantifications were normalized to endogenous control Gapdh. PCR and semi-quantitative PCR was performed in the PCR instrument (Bio-Rad Laboratories, Hercules, CA, USA) as follows: 94°C for 5 minutes (one cycle); 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds (32 cycles); 72°C for 10 minutes; and 4°C holding. PCR products were visualized on a 2% agarose gel with added ethidium bromide. Primers for detecting Isl1 knockdown efficiency and identifying gene expression change in Isl1
mouse embryos are listed in Additional file
2: Table S1.
Embryonic stomachs were lysed with RIPA buffer (9806; Cell Signaling, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (8553S; Cell Signaling). The protein concentration of each group was determined using a bicinchoninic acid assay reagent (Vigorous Biotechnology, Beijing, China) according to the manufacturer’s recommendations. Equal amount of proteins were electrophoresed on a 12% SDS-PAGE, and the bands were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). The membrane was blocked with 5% (w/v) non-fat dry milk for 3 hours and incubated with Isl1 antibody (40.2D6; Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and internal control Gapdh antibody (AM4300;= Ambion, Austin, TX, USA) overnight at 4°C. The PVDF membrane was then washed three times for 30 minutes in 0.1% Tween-20 in Tris-buffered saline (TBST) and incubated for 1 hour with horseradish peroxidase-conjugated goat anti-mouse IgG (Zhongshan, Beijing, China). After washing for 30 minutes with three changes of TBST, the membrane was treated with the pierce™ ECL 2 Western Blot Substrate (Thermo Scientific, Rockford, IL, USA).
Chromatin was prepared from the pyloric regions of C57BL/6 mouse embryos at E14.5 using the manufacturer’s instructions (17–371; Millipore, Darmstadt, Germany). Tissues were dissected in ice-cold PBS. Following a gentle digestion, cells were cross-linked with 1% formaldehyde (252549, Sigma) and chromatin was sheared by sonication to an average length of 500 bp. The antibody used for immunoprecipitation was the 39.4D5 Isl1 (Developmental Studies Hybridoma Bank). Reverse cross-linked immunoprecipitated chromatin was subjected to both RT-PCR and RT-qPCR using primers corresponding to the specific region (spanning the 10 putative Isl1 binding sites). Primers are listed in Additional file
2: Table S3. In all, we collected pylorus regions of more than 400 embryos and repeated ChIP reactions four times.
P1 and P6 regions of Gata3 and α-SMA promoter gene were amplified from mouse genomic DNA by RT-PCR method using specific primers. Primers are listed in Additional file
2: Table S2. The forward primer contained a restriction enzyme site of SacI and the reverse primer contained a restriction enzyme site of HindIII. The PCR product was purified from agarose gel, digested, and cloned into SacI and HindIII sites of pGL3.0-basic luciferase reporter vector (E1910; Promega). Mutated Gate3-P1 and Gata3-P6 promoter regions were done using over-lap PCR, and ATTA/TAAT were mutated to CGGC/GCCG in each putative Isl1 binding site sequence. All of the constructs were verified by sequencing.
Transient transfection and luciferase assays
Human embryonic cells (293FT) were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Invitrogen) supplemented with 100 IU/ml penicillin and 100 IU/ml streptomycin. 293FT cells were plated at a density 5 × 104 cells per well in 24-well plates. After 24 hours in culture, cells were transfected with the Isl1 expression vector (Institute of Molecular and Cell Biology, Singapore) or pXJ40-Myc control vector, Gata3 or α-SMA luciferase reporter vectors, and pTK-Ranilla vector (E2241; Promega) at a ratio of 10:4:1 using the VigoFect transfection reagent (Vigorous Biotechnology). Cells were harvested 24 hours after transfection. Using the same method, the pcDNA-Gata3 expression vector (plasmid 1332; Addgene, Cambridge, MA, USA) and α-SMA luciferase reporter vector were co-transfected into 293FT cells. Luciferase activity was measured using a dual-luc assay kit (E1960; Promega) on a Modulus™ Microplate Luminometer (Turner Biosystems, Sunnyvale, CA, USA). Values shown by the fluc to rluc ratio were normalized to an empty luciferase reporter control. All transfection experiments were performed at least three times.
Hematoxylin and eosin staining
Hematoxylin and eosin staining was performed as previously described
. Briefly, sections were dewaxed, rehydrated, stained with hematoxylin, incubated in bluing solution, counterstained with eosin, dehydrated, and equilibrated with xylene. Glass coverslips were mounted with Permount Mounting Media (SP15-100; Fisher Scientific, Pittsburgh, PA, USA). Sections were photographed under bright-field microscope photograph system (Leica Microsystems, Buffalo Grove, IL, USA).
Stomach samples or embryos were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. Serial sections were dewaxed and rehydrated, and antigen retrieval was performed by microwaving the sections in 0.01 M sodium citrate buffer (pH 6.0). Sections were then blocked using 10% normal animal serum in PBS for 1 hour at room temperature, and incubated with primary antibodies overnight at 4°C. Subsequently, sections were washed and incubated with appropriate secondary antibodies for 2 hours at room temperature. For signal amplification, slides were washed and incubated with appropriate tertiary antibodies for 2 hours. Sections were counterstained with DAPI (10236276001; Roche Applied Science, Basel, Switzerland) for 10 minutes and mounted on plus-coated slides that were cover-slipped using Vectashield (H-1000; Vector Laboratories, Burlingame, CA, USA). Finally, sections were photographed under a fluorescence microscope photograph system (Leica Microsystems).
Primary antibodies used were goat polyclonal to Isl1 (AF1837; R&D, Minneapolis, MN, USA); mouse monoclonal to α-SMA (A2547; Sigma); mouse monoclonal to Gata3 (sc-268; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal to Pdx1 (ab47267; Abcam, Cambridge, UK); rabbit polyclonal to PGP9.5 (AB1761; Millipore); rabbit polyclonal to Sox9 (AB5535; Millipore); rabbit monoclonal to cleaved Caspase 3 (9664S; Cell Signaling), and mouse polyclonal to BrdU (G3G4; Developmental Studies Hybridoma Bank). Secondary antibodies used were biotinylated conjugated donkey anti-goat IgG (sc-2042; Santa Cruz Biotechnology), CY2-conjugated goat anti-mouse IgG (115-225-146; Jackson ImmunoResearch, West Grove, PA, USA), and 488 donkey anti-rabbit IgG (A21206; Life Technologies, Carlsbad, CA, USA). Tertiary antibodies used were TRITC-conjugated streptavidin (7100–03; SouthernBiotech, Birmingham, AL, USA). See Additional file
2: Table S4 for details of specific immunofluorescence protocols.
For BrdU immunofluorescence, DNA was denatured in 2 N HCl at 37°C for 30 minutes and BrdU-incorporated sites were exposed by 0.01% trypsin at 37°C for 12 minutes. After incubation with animal serum, other-step process described above.
Paraffin sections were processed as described above (see Immunofluorescence). Mouse monoclonal antibody to Cxd2 (AM392; BioGenex, San Ramon, CA, USA) and Isl1 antibody (40.2D6; Developmental Studies Hybridoma Bank, Iowa City, IA,USA) were incubated on sections overnight at 4°C. Sections were washed and incubated with a biotinylated goat anti-mouse IgG (115-065-146; Jackson ImmunoResearch) for 2 hours at room temperature. Slides were then washed and incubated for horseradish peroxidase-conjugated streptavidin (123-065-021; Jackson ImmunoResearch) for 2 hours at room temperature. Peroxidase activity was detected with the addition of diaminobenzidine (D4293; Sigma) and 0.1% H2O2. Sections were counterstained with hematoxylin, dehydrated, and covered with coverslips. Sections were photographed as described above (see Hematoxylin and eosin staining). See Additional file
2: Table S4 for details of specific immunohistochemistry protocols.
Measurement of pyloric sphincter constriction
A single section from at least six independent Isl1
embryos was examined by immunofluorescence for α-SMA, as described above (see Immunofluorescence). The shortest distance between the smooth muscle layers on opposite sides of the pyloric lumen was measured with Image J (United States National Institutes of Health, Bethesda, MA, USA)
BrdU was conducted by intraperitoneal injection of BrdU (50 mg/kg) into the pregnant female 2 hours before euthanasia by cervical dislocation. The embryos were removed and analyzed as described above.
Whole mount in situ hybridization
WISH was performed as previously described
. Tissues were fixed in 4% paraformaldehyde for 4 hours, dehydrated in methanol, and stored in 100% MeOH at -20°C until use. Samples were rehydrated, pretreated with proteinase K, and hybridized with DIG-labeled cRNA probes after washing with 2× SSC/50% formamide three times at 70°C. The signal was detected using an alkaline phosphatase-conjugated anti-DIG antibody (11093274910; Roche). Tissues were incubated in the BM Purple alkaline phosphatase substrate (11442074001; Roche) at 4°C for several hours until the signal developed to the desired extent. Probes for Gata3 564 nucleotide (1028 to 1591 bp), Nkx2.5 825 nucleotide (628 to 1452 bp), Gremlin 550 nucleotide (758 to 1307 bp), and Isl1 780 nucleotide (524 to 303 bp) were generated using DIG RNA Labeling Kit (11 175 025 910; Roche). Primers are provided in Additional file
2: Table S2.
Electrophoretic mobility shift assays
pcDNA3.1-Isl1 plasmid was used as a template for in vitro transcription and translation of Isl1 using the TNT Coupled Reticulocyte Lysate System (Promega; L4611) and pcDNA3.1 was used as control. 5′-biotin-labeled oligonucleotides were obtained from Sangon Biotech (Shanghai, China). Double-stranded DNA probes were generated by incubating complementary oligonucleotides at 90°C for 5 minutes, room temperature for 15 minutes, and 4°C for 5 minutes in a buffer containing 10 mM Tris, 1 mM EDTA and 100 mM NaCl (pH 8.0). pcDNA3.1-Isl1 was generated by cloning a fragment encoding C-terminal 216 amino acids of Isl1 into the pcDNA3.1/Hygro (+) vector. N-terminal 133 amino acids including Isl1 LIM domains have been shown previously to inhibit DNA binding in vitro. Recombinant Isl1 protein was prepared by pcDNA3.1-Isl1 in vitro transcription and translation using the TNT Coupled Reticulocyte Lysate System (L4611; Promega) and pcDNA3.1 was used as control. DNA binding reactions (20 μl final volume) were proceeded at room temperature for 20 minutes in 1 × binding buffer (40 mM KCl, 15 mM HEPES (pH 7.9), 1 mM EDTA, 0.5 mM DTT, 5% glycerol and 50 ng/μl poly (dI•dC)) containing 2 μl of in vitro translated recombinant Isl1 or control reticulocyte lysate and 2 nM of 5′-biotin-labeled oligo probe. Oligonucleotide sequences were as follows: number 1 wild type: GTCCTCTTTCCCAATTACCCACTGTCAGTC, mutant: GTCCTCTTTCCCACGGCCCCACTGTCAGTC; number 2 wild type: GGACCGGCTGGGAATTACATGTTAAATACC, mutant: GGACCGGCTGGGACGGCCATGTTAAATACC; number 3 wild type: CCTGGAGGGGCCTATTAGATATTTTGTTTT, mutant: CCTGGAGGGGCCTCGGCGATATTTTGTTTT. Competition experiments were performed using 100-fold excess of unlabeled wild-type or mutant oligonucleotides pre-incubated with the Isl1 protein at room temperature for 10 minutes before adding the DNA probes. Antibody super-shift assays were performed using 1 μl of Isl1 antibody (40.2D6, 400 μg/mL) pre-incubated with Isl1 protein at room temperature for 20 minutes before adding the DNA probes. All DNA binding samples were electrophoresed on 6% non-denaturing polyacrylamide gels at 100 V for 45 minutes in 0.5 × tris-borate-EDTA buffer. Gels were transferred to a nylon membrane at 380 mA for 45 minutes in 0.5 × tris-borate-EDTA buffer. The biotin-labeled DNA was detected with a LightShift chemiluminescent EMSA kit (20148; Thermo Scientific).
Data are expressed as means ± SEM. Statistical analysis was performed with GraphPad Prism 6.0. Comparisons between two groups were analyzed by Student’s t-test. More than two groups were compared using a one-way factorial analysis of variance, followed by Student’s t-test. A value of P <0.05 was considered to be statistically significant.