Mouse strains
The WdpcpCys40/Cys40 mouse and WdpcpFlox/Flox mouse have been previously described [17]. The Prx1-Cre mouse strain (Jackson Stock #005584) and Gli1-Lacz mouse strain (Jackson Stock #008211) were obtained from Jackson Laboratories. To avoid recombination in the female germline, male Prx1-Cre;WdpcpCys40/+ mice were crossed with the female WdpcpFlox/Flox mice to induce limb bud mesenchyme specific deletion of the WdpcpFloxallele [27]., resulting in litters with Prx1-cre;WdpcpFlox/+ (control) and Prx1-cre;WdpcpCys40/Flox(Wdpcp-KO) mice. Male Gli1-Lacz;Prx1-cre;WdpcpCys40/+ and female WdpcpFlox/Flox mice were crossed for limb bud assay. Plug date was identified as E0.5 for timed matings. All reported mice are from mixed background strains. All histological specimens are shown in comparison to littermates and representative of at least 3 biological samples.
Genotyping
For genotyping, DNA was extracted from livers of embryos and tail clips of newly weaned mice using the REDExtract-N-Amp Tissue PCR Kit (Sigma). Primers used for genotyping are included in Table 1. The WdpcpCys40 allele was genotyped using Sanger sequencing of a 150 base pair fragment to identify the relevant A>G base pair change (Supplemental Fig. 1A). The WdpcpFloxallele was genotyped using polymerase chain reaction (PCR) with primers to amplify a region containing an inserted flox site (424 base pair fragment) or lacking an inserted flox site (262 base pair fragment) that could be differentiated with agarose gel electrophoresis [17]. Presence of the Prx1-Cre allele was identified by PCR with primers specific to Cre that amplified a 102 base pair fragment (Supplemental Fig. 1B,C) [60].
Skeletal Preparations
Skeletal preparations were made by co-staining embryos with Alizarin Red S for ossified, calcium rich tissue and Alcian Blue for cartilage as described previously [61]. Briefly, embryos were scalded in hot tap water, skinned, and their abdominal and thoracic organs removed. Livers were saved for genotyping. Embryos were transferred into ethanol before staining 24 hours with 40% glacial acetic acid, 60% ethanol, 0.0001% Alcian Blue (Sigma) solution at room temperature. Embryos were destained for 24 hours in ethanol before being transferred to a solution containing 2% KOH (Sigma) in distilled water with 0.0015% Alizarin Red S (Sigma) for 5 hours. Embryos were subsequently destained in 2%KOH in distilled water overnight, followed by 1% KOH in 50% glycerol/50% distilled water. Embryos were transferred to 50% glycerol/50% distilled water for imaging. All images are representative of at least 3 specimens. For limb length quantification, the longest vector along each cartilage element was used. Six limbs from six separate animals were used for both mutants and controls.
Quantitative Real time RT-PCR (qRT-PCR)
Total RNA was extracted using TRIzol (Invitrogen) with RNeasy columns (Qiagen) for purification and removal of genomic DNA. RNA was quantified on nanodrop, and cDNA was synthesized from 200ng of total RNA using Superscript III (Invitrogen) with the Oligo(dT) primers per the manufacturer’s protocol. Quantitative real-time PCR was performed on Applied BioSystems StepOnePlus with Applied Biosystems SYBR Green PCR mastermix in 96-well plates. Primers were all used at a concentration of 200 nM. Cycling variables were as follows: 95 °C for 10 min, then 40 cycles of 15 second denaturation at 95 °C and 1 minute at 60 °C. Primer sequences are included in Table 2. Expression was reported normalized to housekeeping gene HPRT due to reported stability of expression of this gene during skeletal development [62, 63]. The mean and standard deviation represent 3-4 biological replicates each composed of RNA isolated from 3 experimental replicates. All statistics were performed on difference from HPRT cycle counts to avoid error propagation.
Western Blotting
The limb bud specific knockdown of Wdpcp was confirmed by individually genotyping embryos after forelimb buds had harvested for protein isolation. Forelimb bud protein content was isolated with Total Protein Extraction Kit (Millipore) supplemented with 5 mM EDTA and 1X Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Samples were sonicated to homogenize lysate and protein concentration was quantified using BCA assay kit (Pierce) and standardized to same concentration using isolation buffer dilution. Western blots were performed as previously described [64]. Protein samples were subjected to reducing SDS-PAGE and transferred to low-fluorescence background polyvinyl fluoride (PVDF) membranes (Millipore). Membranes were blocked in 3% milk in 0.25% Tween-20 in TBS (TBS-T) for 1 hour at room temperature and probed overnight at 4C with primary antibody in 1% milk/TBS-T. Primary antibodies used were goat anti-Wdpcp (1:1000) (Santa Cruz, Sc-245737 T-20) and mouse anti-β-actin (Santa Cruz sc-47778) (1:1000). After washing with TBS-T, membranes were incubated for 1 hour at room temperature with HRP conjugated anti-mouse (Abcam) (1:1000) or poly-HRP conjugated anti-goat antibody (Pierce) (1:2000) in 1% milk/TBS-T. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) on a Fotodyne imaging system. Each blot was repeated in duplicate, and representative blot is presented.
Hedgehog responsiveness experiments
Mesenchymal progenitor cells used in these experiments were isolated and expanded from E13.5 forelimbs. For all drug treatment experiments, cells were grown to confluence, trypsinized, and re-plated at full confluence (150,000 cells/cm2) in appropriate size culture well in growth medium for 1 day before changing to starvation medium (DMEM supplemented with 0.25% MSC qualified FBS). Previous studies had demonstrated that cell cycle arrest as induced by serum starvation and confluence is necessary to induce optimal ciliation and hedgehog responsiveness [65]. Following 2 days of culture with starvation medium, medium was changed to starvation medium supplemented with pharmacological agent dissolved in DMSO (Sigma) for 24 hours. RNA for RT-PCR and/or protein for Western Blots was isolated from cells at this time point. For expression studies, results presented are from 3 independent biological replicates. All drugs were diluted in dimethyl sulfoxide (DMSO) and added 1:1000 to culture medium. DMSO was added 1:1000 for carrier control. The Click-iT Plus Edu 594 assay was used to quantify cell proliferation over final 24 hours of cell culture. Following serum starvation period, 10 mM EdU dissolved in PBS was added to culture medium at 1:1000 for a final concentration of 10 uM. Following fixation with 4% paraformaldehyde for 15 min at room temperature, EdU staining was performed per the manufacturer’s protocol (Lifetechnologies). Hoechst staining was performed to identify all nuclei. Images were acquired on Zeiss inverted epifluorescent microscope with 10X objective. Three fields were captured for each treatment condition and presented images are representative. Three biologic replicates were performed per experimental condition.
Micromass Culture
Forelimbs were obtained from 11.5 dpc Prx1-cre;WdpcpCys40/Floxand control embryos. Micromass cultures were prepared as previously described [66-68]. Dissected limbs were dissociated in 1 unit/mL dispase (Stem Cell Technologies) for 1.5 h at 37 °C. Digested limbs were pipetted up and down with 200 uL pipette to break up cells before being diluted in 10 mL 2:3 DMEM/F-12 medium containing 10% MSC qualified FBS (Invitrogen) to neutralize dispase. This suspension was passed through a 40 um filter and spun down to a pellet at 1200g. Cells were resuspended in 2:3 DMEM/F-12 medium and counted with hematocytometer. Cells were then divided into experimental groups, spun down and resuspended in 2:3 DMEM/F-12 medium supplemented with either DMSO or appropriate pharmacological agent dissolved in DMSO. Cells were diluted to 10 million cell/mL and spotted in 10 uL droplets on Nunc 24–well culture dishes. Cells were allowed to adhere for 1.5h in a cell incubator with humidified atmosphere containing 5% CO2. After two hours, wells were flood with growth medium containing appropriate pharmacological agent. Cultures were harvested after 3 days. For Alcian Blue staining, cultures were fixed for 15 minutes at room temperature in 4% paraformaldehyde in 1X phosphate buffered saline before staining overnight at 4 °C with 1% Alcian Blue stain solution pH 1.0 (EK Industries). Stained cultures were washed 3x with phosphate buffered saline before imaging. Alcian Blue staining shows a representative image of 3 to 4 independent biological replicates [60].
Histology and Immunohistochemistry
Specimens were fixed in 4% paraformaldehyde at indicated time point for 24h overnight at 4C before being dehydrated and paraffin embedded. Forelimbs were sectioned at 7 micron thickness. For Safranin O/Fast Green staining, sections were deparaffinized and rehydrated before being stained in Weigert's Iron Hematoxylin (Sigma) and 0.02% aqueous Fast Green (Sigma) followed by a rinse in 1% acetic acid and 0.1% aqueous Safranin-O (Sigma). For immunohistochemistry, ImmPRESS Excel amplified polymer staining kit (Vector Labs) was used [60]. Primary antibodies used for immunohistochemistry were rabbit anti-Osx/Sp7 (1:500) (Abcam, Ab22552), rabbit anti-Runx2 (1:500) (Santa Cruz, Sc-10758 M-70), and rabbit anti-Gli1 (Santa Cruz, Sc-20687 H-300) (1:200).
Mesenchymal progenitor isolation
Mesenchymal progenitor cells were isolated from the limbs of E13.5 embryos that were digested in 0.5% Trypsin EDTA and mechanically disrupted. Trypsin was neutralized after 5 minutes at 37C with DMEM supplemented with 10% MSC qualified FBS (Gibco) with 1X Penicillin/Streptomycin/Fungizone (Gibco) growth medium. Cells were pipetted up and down to break up remaining tissue before spinning at 1000g to pellet in 15 mL Vulcan tube. Cells were then resuspended in growth medium and plated on T75 at what was considered passage 0. Cells were expanded two days with growth medium changed each day. Cells were passaged on day 2 and split (1:4). Cells were grown to 90% confluence, passaged, and frozen in cryoprotective freezing medium (Lonza) for storage in liquid nitrogen. All experiments using were conducted at passage 3 or 4 [60].
Osteogenic differentiation assays
Following expansion, mesenchymal progenitor cells were seeded at 70% confluence (100,000 cells/cm2) in growth medium for two days before changing to standard osteogenic medium containing 1 mM β-glycerolphosphate, 100 nM ascorbic acid, and 20 ng/mL rBMP2 (Peprotech) supplemented with 10 nM smooth agonist (SAG, Calbiochem) similar to prior studies [10, 53]. Osteogenic medium was changed every 4 days. Alkaline phosphatase activity was measured with paranitrophenyl phosphate assay (Sigma) at day 4 of osteogenic differentiation. Eight independent biological replicates were used for quantification. Alizarin red staining was used to assay calcium matrix deposition of osteogenic cultures. Cells were fixed in 70% ethanol before being stained for 15 minutes with 2% alizarin red solution at pH 4.2 (Rowley Biochemical Institute). For quantification, 1 mL of cetylpyridinium chloride was used per 6 well plate well to solubilize dye. DNA was isolated from an analogous 6 well plate well with 1 mL of 0.5% Triton X-100 in distilled water and quantified using Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). Soluble alizarin red was quantified spectroscopically (A580) [60]. Six independent biologic replicates were used for quantification.