The Institutional Animal Care and Use Committee approved all protocols used in this study.
Sulf1 and Sulf2 targeting
Embryonic stem (ES) cells with retroviral gene trap vectors inserted into the Sulf1
(OST352220) and Sulf2
(OST311938) loci were generated in collaboration with Lexicon Genetics (The Woodlands, TX) using methods described previously 
was disrupted by insertion of the VICTR48 construct; this allele is therefore named Sulf1Gt(VICTR48)352220Lex
was disrupted by the VICTR37 retroviral construct, which included a promoterless β-galactosidase cassette in addition to the Neomycin resistance gene. The Sulf2
allele generated by the insertion mutation is named Sulf2Gt(VICTR37)311938Lex
. The insertion sites were determined by sequence analysis. The Sulf1
- or Sulf2
-targeted 129/SvEvBrd ES cells were microinjected into blastocysts and implanted into C57BL/6J females. Germline transmission was assessed in F1 pups by PCR analysis of genomic DNA. Heterozygous mice were either intercrossed to assess the phenotypes on a mixed strain background or backcrossed onto the C57BL/6N background. Unless otherwise specified, the results presented herein were performed using the N4 (Sulf2
), or N5 or N6 (Sulf1
) backcross generations. For simplicity's sake, we refer to the gene trap alleles of the genes as Sulf1−
and the wild-type alleles as Sulf1+
Generation of mice deficient for both Sulf1 and Sulf2
Sulf1−/− (N3, C57BL/6N) mice were crossed to Sulf2−/− (N2, C57BL/6N) mice to generate F1 Sulf1+/− Sulf2+/− mice. F1 mice were intercrossed to assess the viability of mice with the different possible genotypes. To generate sufficient Sulf1−/− Sulf2−/− mice to study, we routinely intercrossed F2 Sulf1+/− Sulf2−/− to Sulf1−/− Sulf2+/− mice to produce study cohorts with Sulf1+/− Sulf2+/− mice as controls.
Genotyping of Sulf1 and Sulf2 alleles
We utilized a PCR genotyping strategy to detect the wild type and disrupted alleles of Sulf1 and Sulf2. Tail DNA was prepared using the Extract-N-Amp extraction kit following the manufacturer's protocol (Sigma, St. Louis, MO). PCR conditions were as follows: 94°C for 4 min., 1 cycle; 94°C for 60 s, 60°C for 30 s, 72°C for 60 s (30–35 cycles); 72°C for 10 min., 1 cycle. For genotyping of tails from neonate and young adult mice, 30 cycles were sufficient. We increased the cycle number to 35 when detecting DNA from embryonic tissue.
The retroviral insertion in the Sulf1 locus was detected using primers Sulf1-R1 and LTR, which generated a 334-bp product. We detected the wild type Sulf1 allele using primers Sulf1-F and Sulf1-R1, which produced a 459-bp amplicon. Using a similar strategy, the insertion allele in the Sulf2 locus were detected using primers Sulf2-R and LTR2, producing a 440-bp band; the wild-type Sulf2 allele was amplified with primers Sulf2-F2 and Sulf2-R, making a 759-bp product. The appropriate three-primer sets were used simultaneously without evidence of interference when evaluating both modified genes. Sulf1; Sulf2 mice were genotyped as above, amplifying the Sulf1 and Sulf2 loci in separate reactions.
Sequences of genotyping primers were as follows, from 5′ to 3′: LTR, ATA AAC CCT CTT GCA GTT GCA TC; Sulf1-F, CCG CAA AGA CTT GGA ATT AAC TC; Sulf1-R1, CTT CAC ACA CTC CAC ACT CAG TTC T; LTR2, AAA TGG CGT TAC TTA AGC TAG CTT GC; Sulf2-F2, TTG ACT TTC TGG GGA GGG TGG ATG; and Sulf2-R, GAT GGG CCA CTC CTG AGA TAA CCT G.
RNA purification and quantitative RT-PCR
For quantitative real-time RT-PCR (Q-RT-PCR), total RNA was isolated using the RNeasy kit essentially according to the manufacturer's protocol, including the on-column DNase digestion (Qiagen, Valencia, CA). Immediately prior to column purification, frozen tissue was homogenized in RLT buffer containing β-mercaptoethanol using a stator homogenizer followed by a QiaShredder column (Qiagen). To remove all contaminating genomic DNA, we re-purified the eluted RNA over a fresh RNeasy column, including a second DNase treatment. This routinely reduced the amplification of a control reaction omitting reverse transcriptase to below the limit of detection, allowing us to conclude that signal was indeed due to RNA and not contaminating genomic DNA.
Q-RT-PCR was done according to the Taqman
protocol (Applied Biosystems, Foster City, CA). Using 100 ng total RNA per 50 µl reaction, we detected and quantitated the amplification products using an ABI 7500 system (Applied Biosystems), normalizing to either the GAPDH or RPL19 housekeeping genes. Relative changes in gene expression were quantitated using the 2−ΔΔCt
. Primer/probe sets used were as follows, with forward primers denoted by F, reverse primers by R, and FAM/TAMRA-labeled probes as T: Sulf1-4535F, CCT CGA CGT GCT AAA CTT GA; Sulf1-4611R, TAT TCC CGC AGG ATT TAT TTC; Sulf1-4556T, TAG CAG AAA GGC ATG GCT CAC AAT G; Sulf2-3364F, CCC TTG AGC TTT CAG ACA TTT; Sulf2-3428R, CAG TTC TGG GAT GGA TAA CAA A; Sulf2-3386T, TTC CTG CCC GGG ATT CGT TC; RPL19-F2, AGA AGG TGA CCT GGA TGA GAA; RPL19-R2, TGA TAC ATA TGG CGG TCA ATC T; RPL19-T2, CTT CTC AGG AGA TAC CGG GAA TCC AAG; GAPDH-F, ATG TTC CAG TAT GAC TCC ACT CAC G; GAPDH-R, GAA GAC ACC AGT AGA CTC CAC GAC A; and GAPDH-T, AAG CCC ATC ACC ATC TTC CAG GAG CGA GA.
Whole-mount embryo RNA in situ hybridization
Embryos were harvested at E8.5-E12.5 (with noon of the day the vaginal plug was observed designated as E0.5) and fixed in freshly prepared 4% paraformaldehyde (PFA) in PBS (pH 7.4) overnight at 4°C. Primers used to amplify the Sulf1 transcript from a mouse embryo cDNA library to TOPO-clone into pCR-II-TOPO (Invitrogen, Carlsbad, CA) were Sulf1-wmISH-S, ACC AGT CAG CCA GAG CGT and Sulf1-wmISH-AS, CTT CCC ATC CAT CCC ATA ACT. Following sequencing to ensure fidelity of the amplification and orientation of insertion, we generated antisense, DIG-labeled probe from linearized plasmid template using the mMESSAGE mMACHINE kit (Ambion, Austin, TX), following the manufacturer's instructions. Hybridization and detection of DIG-labeled probe were done according to standard protocols.
Section RNA in situ hybridization
Following fixation in freshly prepared 4% PFA in PBS (pH 7.4) overnight at 4°C, we equilibrated embryos (E9.5–E12.5) overnight in 30% sucrose in PBS, followed by 1 h in Tissue-Tek O.C.T. (Sakura). Specimens then were embedded in fresh O.C.T. and frozen in an ethanol/dry ice bath. In situ hybridization was carried out on 20-µm transverse sections of wild type embryos. To generate 35S-radiolabeled RNA probe for in situ hybridization, we used the mRNAlocator kit (Ambion) according to the included instructions. The template for the probe synthesis was a PCR product with a T7 promoter included in the primer, using the following primers: Sulf1-sISH-S, TAT CCG GTG CAA GCA ACA T; Sulf1-sISH-T7-AS, TAA TAC GAC TCA CTA TAG GGA GGC TGT TCA GAT GCA GGG TTT G; Sulf2-sISH-S, CCC ACA ACT TCC TCT TCT GC; and Sulf2-sISH-T7-AS, TAA TAC GAC TCA CTA TAG GGA GGC CCA TAG CTG TCC CAG TGA T.
hybridization using DIG-labeled probes specific to Collagen Type X
and Collagen Type II
was performed on paraformaldehyde-fixed, paraffin-embedded tissue, as previously described 
β-Galactosidase expression pattern analysis
All solutions were made in 0.1 M phosphate buffer (pH 7.3). Embryos (E10.5–E12.5) were washed in unadulterated buffer and then fixed for 30–45 min in buffer containing 0.2% glutaraldehyde, 5 mM EGTA, and 2 mM MgCl2. Embryos were then washed 3×15 min in buffer containing 2 mM MgCl2, 0.01% deoxycholate, 0.02% Nonidet-P40 and stained in the dark for 1–12 hours at room temperature in buffer containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.02% NP-40, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, IBI Shelton Scientific, Peosta, IA). Embryos were then washed in PBS (pH 7.4) and post-fixed in neutral buffered 10% formalin (NBF). Stained specimens were evaluated as whole mounts and then, in selected instances, in 12-µm-thick transverse cryosections of O.C.T.-embedded material.
Near-term (E18.5) fetuses or neonates were killed, flayed, eviscerated, and fixed in 100% ethanol for 1–2 days, followed by an overnight acetone treatment. Specimens were stained in 0.015% alcian blue, 0.005% alizarin red in 5% acetic acid, 60% ethanol for 2–3 days at room temperature; washed briefly in tap water; then cleared in 1% KOH for several days. Cleared specimens were passed through a series of glycerol solutions of increasing concentration prior to visualization on a Zeiss dissecting microscope.
Micro-computed tomography (µCT) image acquisition
The mouse embryos were imaged with a µCT40 (SCANCO Medical, Basserdorf, Switzerland) x-ray micro-computed tomography system. A sagittal scout image, comparable with a conventional planar x-ray, was obtained to define the start and end point for the axial acquisition of a series of µCT image slices. The location and number of axial images were chosen to provide complete coverage of the embryo. The embryos were imaged with air as the background media. The µCT images were generated by operating the x-ray tube at an energy level of 45 kV, a current of 177 µA and an integration time of 300 milliseconds. Axial images were obtained at an isotropic resolution of 16 µm.
Analysis of µCT images
The relationship between the image intensity values and bone mineral density was assumed to be linear and was obtained by scanning a 97% pure hydroxyapatite (HA) sample (2.91 gHA/cm3). Three-dimensional (3D) surface renderings were created from the µCT data with the use of Analyze (AnalyzeDirect Inc., Lenexa, KS), an image analysis software package. The bone surface renderings were generated by applying a bone mineral density of threshold of 0.54 gHA/cm3. The bones of interest (femur, humerus, supraoccipital, interparietal, parietal, frontal, basisphenoid and basioccipital) were extracted for quantitative analysis by the applying a series of image analysis steps (thresholding, morphological filtering and region growing operations) to the volumetric image data. For the long bones, the distance between the ends of the bone was measured as a straight line and an average was computed using the contralateral bone measurements. The volume (mm3) and mean bone density (gHA/cm3) of the defined bones was measured and an average was obtained for the ipsilateral and contralateral bones for each embryo.
Necropsy and histological preparation of conceptuses and neonates
Fetuses (E15.5 and E18.5) were removed from the uteri of anesthetized dams. The tip of each fetus's tail was harvested for genotyping by PCR, after which the fetus and placenta were fixed by immersion in either 4% PFA, 10% NBF, or Bouin's solution. Some fetuses were bisected longitudinally just to the left of the midline, while others were decapitated so that the heads could be cut coronally. A few neonates (including placentas) were handled in a similar manner. Fixed tissues were processed into paraffin using a conventional protocol. Serial 5-µm-thick coronal sections of each head block were acquired at several steps, each separated by a distance of 250 µm. Serial 5-µm-thick sections of each torso block were acquired at several steps, each separated by a distance of 500 µm. Serial sections at each step were stained with hematoxylin and eosin (H&E) or cresyl violet (to better delineate subcellular characteristics of neural cells). Lesions were scored (with foreknowledge of the fetus's genotype) using a tiered, semi-quantitative grading scale: absent, minimal, mild, moderate, marked. Subsequently, a coded (“blinded”) histopathological assessment for a given organ was performed by inverting all slides and then sorting them into groups using criteria established in the uncoded examination.
Mouse embryonic fibroblast isolation
E12.5 embryos were dissected from the extraembryonic tissues in PBS, then the liver and head were removed. After mincing with scissors, the remaining tissue was digested in 0.1% trypsin/EDTA in PBS for 20 min at 37°C, with mixing every 5 min. After washing twice with culture medium (DMEM with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin/streptomycin), the cells from a single embryo were plated in a 25-cm2 flask. One day later, cells were trypsinized, counted, and either frozen in aliquots or used immediately for signaling experiments.
FGF signaling in embryonic fibroblasts
First- or second-passage fibroblasts were serum-starved overnight in assay medium containing 0.5% FBS at a density of 105
cells per well of a 6-well plate. The following day, prewarmed FGF1- or FGF2-containing assay medium (or assay medium lacking FGF) was added to the cells, which were then returned to the 37°C incubator for 10 minutes. Cells were lysed for 15 minutes at 4°C, with rocking, in 1×RIPA buffer (Upstate) supplemented with Complete protease inhibitors (Roche), 2 mM sodium orthovanadate, and 5 mM sodium fluoride. Lysates were cleared by centrifugation (10 minutes at 11,000 g
), then prepared for electrophoresis using LDS buffer and DTT (Invitrogen). Equal amounts of protein were loaded per lane of a 4–12% Bis-Tris NuPAGE gel and then separated according to the manufacturer's recommended protocol (Invitrogen). After transferring the protein to nitrocellulose membranes, non-specific binding was blocked using 5% milk in Tris-buffered saline solution containing 0.2% Tween-20 (TBST). Primary antibodies were incubated overnight at 4°C in 1% BSA in TBST (Pierce). Peroxidase-conjugated secondary antibodies were used for 1 hour at room temperature. Peroxidase activity was detected using ECL Plus (GE Healthcare). Antibodies and dilutions used: Phospho-FRS2, 1
1000 (Upstate); Total FRS2, 1
1000 (Upstate); anti-rabbit-HRP, 1
10,000 (Amersham). Bands were quantitated using the integrated density function of ImageJ. Data shown are representative of three independent experiments.
Histopathologic (ordinal) data from the blinded microscopic evaluation were assessed using the non-parametric chi-square test. Nominal data were analyzed by two-tailed Student's t-test. All calculations were done using commercial JMP statistical software (v. 5.0; SAS Institute, Cary, NC), and significance assigned to p-values ≤0.05.