Targeting Strategy and Mouse Strains
All breeding procedures, animal care and experimental protocols were approved by the Medical University of Ohio (renamed University of Toledo Health Sciences Campus) animal care and use committee prior to initiation of this work. Our strategy for targeting Hand2
is published elsewhere (Hendershot et al., 2007
). Our analysis of Hand2fl/fl
mice demonstrates that the mice are fertile, viable and phenotypically normal; introduction of lox
P sites in the 5′ UTR therefore does not negatively impact transcription at the targeted locus. Normal levels and patterns of Hand2
expression have been confirmed by in-situ
hybridization and qRT-PCR (Hendershot et al., 2007
; Hendershot et al., 2008
). Both systemic and targeted deletion of Hand2
is embryonic lethal (Srivastava et al., 1997
; Hendershot et al., 2008
). To generate embryos older than E11 (Hendershot et al., 2008
) pregnant dams are fed water containing a cocktail of catecholamines (100 μg/ml L-phenylephrine, 100 μg/ml isoproterenol, and 2 mg/ml ascorbic acid) beginning at embryonic day (E) 8. This pharmacological approach allows us to rescue Hand2flneo/flneo
embryos from pre-term death, a phenotype observed in other mouse models in which norepinephrine is absent or expression curtailed (Hendershot et al., 2007
; Lim et al., 2000
; Thomas et al., 1995
; Zhou and Palmiter, 1995
). Reporter mice were generated as previously described (Hendershot et al., 2007
). Homozygous R26RYFP females were mated to hemizygous Wnt1-Cre males and Hand2fl/+;Wnt1-Cre
males were mated to Hand2fl/fl;R262YFP
Immunocytochemistry and Histology
The antibodies used for the current studies are detailed in . Embryos were prepared and treated as previously described (Hendershot et al., 2007
; Hendershot et al., 2008
). Briefly, embryos or tissues are emersion fixed in 4% paraformaldehyde overnight at 4°C, extensively washed in PBS and stored in 30% sucrose unless otherwise stated. For analysis of heart development, hearts were removed in ice-cold PBS, fixed in 10% neutral buffered formalin, dehydrated through a graded series of ethanol and embedded in paraffin. Paraffin sections were prepared and stained with hematoxylin and eosin (University of Texas, Southwestern Medical center, Molecular Pathology Core Laboratory) using standard procedures. Frozen sections of Hand2wt/wt
E11 hearts were stained using a modified hematoxylin and eosin stain (Sanderson, 1994
). Briefly, tissue sections were post-fixed in 4% paraformaldehyde (10 minutes), rinsed in PBS (2 × 5 minutes) and stained in hematoxylin (1 minute), followed by washing in running water, differentiation in 1% HCl in 70% EtOH, bluing in Scott’s tap water (3.6 g sodium bicarbonate, 20g magnesium sulphate/litre) with counterstaining in Eosin Y for 10–20 seconds, followed by dehydration and mounting in Permount (Fisher Scientific, Fair Lawn, NJ) or EUKITT (O. Kindler, Germany). Immunostaining was done according to our established procedures (Howard et al., 1999
, Wu and Howard, 2002
, Liu et al., 2005
, Hendershot et al., 2007
). Tissue sections were blocked in a solution containing 0.1M Tris, pH. 7.5, 1.5% NaCl, 0.3% Triton-X 100 and 10% horse serum for 3 × 5 minutes. Primary antibody is applied in this same solution but containing 4% horse serum and incubated overnight at 4°C. Following washing in 0.1M Tris pH 7.5, 15% NaCl, secondary antibody was applied in this same solution with 4% horse serum for three hours at room temperature. Sections are then washed 3 × 5 minutes in 0.1M Tris, pH 7.5, 15% NaCl and mounted in Vectashield (Vector Laboratories, Burlingame, CA) or Fluoromount-G (Southern Biotech, Birmingham, Al). To view sites of antibody binding in whole embryos (Young et al., 1999
; Hendershot et al., 2007
; Hendershot et al., 2008
) samples were incubated overnight in PBS containing 0.3% Triton-X 100 with gentle shaking at 4°C. We used our standard blocking step and primary antibody was applied as described above and incubated for two days at 4°C with gentle shaking. Following extensive washing secondary antibody was applied and embryos incubated overnight at 4°C with shaking. Whole embryos were mounted on depression slides in Fluoromount-G (Southern Biotech, Birmingham, AL).
Antibodies used for immunostaining in this study are listed. The antigen, dilution, and secondary antibodies are listed. The source for each antibody is listed.
All confocal images were acquired using a Leica Microsystems multiphoton confocal microscope (TCS SP5). Confocal Z-stack images were captured using either a 10X objective (n.a.= 0.3) or 63X (oil immersion) objective (n.a.= 1.4) with 4X software magnification at 0.5 μm (tissue sections) or 1.0 μm (whole-mount) steps. To acquire images, fluorescent FITC–coupled, TRITC–coupled and AlexaFlour 647-coupled secondary antibodies were excited using argon, HeNE or 633 laser lines, respectively.
Gene Array Analysis
For this array (Affymetrix 430 2.0, mouse array) total cellular RNA was isolated from Hand2wtwt
E10.5 hearts using RNeasy (Qiagen, Valencia, CA) according to manufacturer’s directions. The RNA samples were submitted to the NIH Neurosciences Microarray Consortium (Phoenix, AZ, USA; http://arrayconsortium.tgen.org/np2/home.do
;) for array preparation and analysis. The screen included three biological replicates for each genotype (3 chips/genotype). Expression and statistical analyses were compiled using GeneSpring (GX V 7.3.1 (Agilent Technologies, Santa Clara CA). The signal intensities from each probe set were normalized at the 50th percentile for each chip; the expression level for each gene was normalized to the median level across the arrays. The linear regression for all replicates was greater than 0.9 indicating that replicates correlated very well. A list of significantly expressed genes was generated based on a comparison of 3 arrays derived from control and three arrays derived form mutant hearts. The list was filtered first for the absent genes, secondly for a fold change cutoff of 2, and thirdly for p value of ≤ 0.05 using Welch’s t-test. Genes whose expression was significantly altered by Hand2
deletion in the neural crest were subjected to network analysis using GeneGo (St. Joseph, MI), MetaCore and amiGO to group genes based on function and/or pathway analysis. We chose genes for further analysis based on their fold change, relationship to Hand2
and cardiac development.
Expression of transcripts encoding proteins identified in the gene array analysis were verified using quantitative reverse transcription-real time PCR as previously described (Zhou et al., 2004
; Liu et al., 2005b
; Hendershot et al., 2008
). Briefly, we calculated the levels of transcript from the Ct values and normalized them based on expression of GAPDH. A detailed description of the protocol and equations can be found in Zhou et al., (2004)
or Liu et al., (2005b)
. Each triplicate sample was analyzed (in triplicate) using cDNA corresponding to 10 ng of input RNA isolated from either Hand2wt/fl
E10.5 hearts. cDNA was mixed with Taqman Gene Expression master mix (Applied Biosystems, Foster City, CA, USA) and premixed (20X) Taqman probes (5 μM) and primers (18 μm). Forward and reverse primers (1.1 μM final concentration) with 6-FAM (carboxyfluorescein reporter dye) and a non-fluorescent quencher dye incorporated at 5′ and 3′ ends, respectively were used for all assays. Primers and probes were purchased from Applied Biosystems (Foster City, CA, USA) from their inventory of pre-developed Taqman® Gene Expression Assays. Triplicate PCR reactions were run and analyzed using a 7500 Fast Real-Time PCR sequence detection system (Applied Biosystems); the increase in product is monitored directly and reflects the threshold number of cycles (C
) required for a detectable change in fluorescence based on release of probe. The efficiency of each primer and probe set was determined in separate reactions and based on the slope of C
versus input cDNA dilution. Efficiency values were ≥ 1.875 for all transcripts.
Chromatin Immunoprecipitation (ChIP) Assay
To determine whether Cx40 is a direct Hand2 target, we employed Chromatin Immunoprecipitation (ChIP) on genomic DNA cross-linked, immunoprecipitated and purified from H92c cells, using a protocol modified from manufacturer’s guidelines (Millipore Corp., Billerica, MA). Briefly, ~1 × 106 H9c2 cells (~75% confluent, 10 cm dish) were cross-linked in 1% formaldehyde for 10 minutes at RT. Cross linking was quenched in 1 ml of 1.25 M glycine in PBS with shaking for 5 minutes at RT. Cells were rinsed twice with ice-cold PBS, collected to 1.5 ml Eppendorf tubes, and pelleted at 2,000× g for 4 minutes at 4°C. Cell pellets were washed with 1 ml of PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml pepstatin, Roche Inc., Madison WI) then resuspended in 200 μl of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-Cl, pH 8.1, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml pepstatin). The cell suspensions were incubated on ice for 10 minutes and sonicated on ice 4 times, 30 seconds each, at 1 minute intervals using a setting of 3 (Fisher Scientific Model: 550 Sonic Dismembrator). The appropriate sonication protocol was determined empirically. Lysates were centrifuged (1000 rpm, 7 minutes) and supernatants were added to 1.8 ml of dilution buffer (1.1% Triton X-100, 0.01% SDS, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl, pH 8.1, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml pepstatin), then pre-cleared with 50 μl of Protein A/G PLUSAgarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 hour with end-over-end rotation at 4°C. Protein A/G PLUSAgarose was removed by brief centrifugation and immunoprecipitation was performed overnight at 4°C with 5 μg of Hand2 antibody.
The following day, an additional 50 μl of Protein A/G PLUSAgarose was added and the incubation continued for 2 hours at 4°C. Immuno-complexes were collected by centrifugation at 1000 rpm for 1 minute at 4°C and subjected to sequential 5 minute washes in 1 ml of each of the following buffers: low salt immune complex wash buffer (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt immune complex wash buffer (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and LiCl immune complex wash buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Precipitates were washed twice with TE buffer, then eluted twice in 250 μl of elution buffer (1% SDS, 0.1M NaHCO3) for 15 min at RT. The eluates were pooled and 20 μl of 5M NaCl was added; the eluates were subsequently heated at 65°C for 4 hours. Protein was removed from the samples by addition of 10 μl 0.5 M EDTA, 20 μl 1M Tris-HCl, pH 6.5 and 1 μl of 20 mg/ml Proteinase K for 1 hour at 45°C. DNA fragments were recovered by phenol/chloroform extraction and ethanol precipitation; 10 μg of yeast tRNA was added to each eluate to aid in recovery of DNA pellets. Following centrifugation (12,000 × g, 20 minutes at 4°C), the pellets were resuspended in 50 μl of TE; 1 μl was used per PCR reaction. PCR primers were as follows: Cx40(F)ChIP[−1144]: 5′-ACTGTCCCTCAGTTTCCCTG-3′, Cx40(R)ChIP[−814]: 5′-ACAGAGGGTCAAGGACATGG-3′, Cx40(F)ChIP[−655]: 5′-GAACACTCTGATTGGTGGGG-3′, Cx40(R)ChIP[−283]: 5′-CCAGTGGCTGTCCTTGTTTT-3′, GAPDH: 5′-TCCCACTCTTCCACCTTC-3′, GAPDH: 5′-CTGTAGCCGTATTCATTGTC-3′. PCR was conducted using GoTaq polymerase (Promega Corp., Madison, WI) with a 58°C, 1 min annealing step over 40 cycles of amplification.
Neural crest-derived cells were FACsorted from 20 E10.5 hearts isolated from wild-type Wnt1-reporter embryos and samples were prepared according to the manufacturer’s ChIP-chip user’s guide (Roche NimbleGen, Inc. Madison, WI). Briefly, hearts were extensively washed in ice-cold PBS, incubated in 0.5% trypsin in CMF-PBS for 15 minutes and dissociated into single cells using a fire-polished Pasteur pipette. The cells were washed one time in PBS, pelleted, and cross linked in 1% formaldehyde for 8 minutes at RT. The cross linking was quenched in 125 mM glycine. The washed cells were filtered through a 70 μm nylon cell strainer (Falcon #352350, Bedford, MA) and subjected to FACsorting (BDFacs Aria, BD Biosciences, San Jose, CA); the non-neural crest cells from the non-sorted sample were used for the controls. The sorted neural crest cells were lysed and sonicated (Fisher Scientific Model: 550 Sonic Dismembrator) for 4 rounds for 15 seconds on a setting of 3 with a one minute incubation on ice in between each round. The appropriate settings for sonication were determined on genomic DNA and the fragment size verified in a 1% agarose gel. For IP, we used our Hand2 antibody (raised in rabbit) coupled to Dynabeads (M-20, Invitrogen, Carlsbad, CA). Complexes were allowed to form for 2 hours at 4° C with rotation and isolated on a magnetic table. Sample DNA was amplified according to manufacturer’s directions (WGA3, Sigma-Aldrich, St. Louis, MO) and sent to Roach for processing (2.1×106 Deluxe Promoter Array).
Transfection and Luciferase Reporter Assay
HEK293a cells were grown in DMEM plus 10% FBS supplemented with l-glutamine, Na-pyruvate and 100 mg/ml Pen/Stryp (Invitrogen, Carlsbad, CA); cells were transfected when 50% confluent using CaPO4
according to our published procedures (Firulli et al., 1994). In each well, 2.5 μg of pcDNA-Hand2 and/or pCS2-MT+E12 (plus empty pcDNA vector to make a total of 5 μg of plasmid DNA) was co-transfected with 5 μg of Cx40
luciferase reporter (Cx40+pXP2) and 150 ng of Renilla reporter (pRL-CMV) as a transfection control. Cultures were harvested 48 hours after transfection and diluted ~10 fold in lysis buffer; 50 μl of this lysate was assayed. Luciferase assays were performed as previously reported (Xu et al., 2003
) using the dual luciferase assay kit (Promega Corp., Madison, WI) according to the manufacturer’s protocol. Luciferase activities were read using a 96-well microtiter plate luminometer (Thermo Labsystems, Franklin, MA). Assays were done in triplicate.
Data are presented as the mean ± S.E.M unless otherwise stated. Statistical significance was determined using Student’s unpaired t test or ANOVA and Bonferroni post hoc test, unless otherwise stated. At least four embryos were examined for each condition from at least three different matings unless otherwise stated. For microarray analysis, three biological replicates were run and each in triplicate.