For all experiments, embryos were obtained by crossing adult Tg(sox10:tagRFP), Tg(col2a1a:EGFP), and AB wildtype fish. Embryos were maintained in E3 embryo medium at 28˚C. All zebrafish were maintained at The University of Texas El Paso according to the Institutional Animal Care and Use Committee (IACUC) guidelines protocol 811689-5. All adult larvae were obtained from the University of Colorado, Anschutz Medical Campus or the Zebrafish International Resource Center (ZIRC). Adult and larval zebrafish were euthanized and anesthetized according to guidelines from the American Veterinary Medical Association and approved IACUC protocols. For euthanasia, adults beyond the age of peak breeding age (>1.5 years old) were euthanized using a solution of 10g/L buffered solution of pharmaceutical grade MS 222. Fish were emerged in solution for 30 minutes at RT. All euthanized adults underwent secondary euthanasia with a cold ice bath (2-4 degrees C). Cessation of movement was indicative of euthanasia. Embryos (<7 days old) were euthanized using 1-10% sodium hypochlorite solution after being anesthetized in cold ice bath. For any genotyping and before fixation, all fish, adults and larvae, were anesthetized using MS 222 (150mg/L for adults and 300mg/L for embryos). The degree of anesthesia was monitored by operculum movement of adults and cessation of movement for larvae.
Antisense oligonucleotide morpholino design and microinjection
Two antisense oligonucleotide morpholino sequences were designed in conjugation with Gene Tool LLC. The first was a translation blocking morpholino (MO) with the sequence 5’-TATCCTCGCCCCCATTTCTGCCAA-3’, created to bind to the hspg2 translation start site and sterically knockdown perlecan translation in the developing larvae. The second was a random control morpholino with the sequence 5’-AAAAAAAAAAAAAAAAAAAAAAAA-3’, used to assure that the MO microinjections were not causing any form of cell death as described by previous literature (26,27). For all experiments, 3 experimental groups (morphants, random control, and wildtype) were injected (wildtype were non-injected) and compared. Both MOs were injected into embryos at the one cell stage with a stock concentration of 0.10 µM and at a volume of 0.5 nL per embryo.
Alcian Blue Staining and Imaging
Zebrafish larvae (aged 5 days post fertilization (dpf)) were fixed in 2% PFA in PBS, pH 7.5 for 1 hour at room temperature (RT). Samples were washed for 10 minutes with 100mM Tris pH 7.5/10mM MgCl2, stained with Alcian blue stain (pH 7.5: 0.4% Alcian blue (Anatech Ltd., MI) in 70% EtOH, Tris pH 7.5 (Fisher, MA), and 1 M MgCl2 (Fisher, MA)), and incubated overnight at RT. Samples were subsequently destained and rehydrated using an EtOH: Tris pH 7.5 gradient as previously described (28). Embryos were bleached (30% H2O2 (Sigma, St. Louis, MO), 20% KOH (Fisher, MA)) for 10 minutes at RT. Samples were washed twice for 10 minutes per wash in wash buffer (25% glycerol/0.1% KOH (Fisher, MA)) and stored at 4ºC in storage buffer (50% glycerol/0.1% KOH (Fisher, MA)) until imaged. The distance between the Meckel’s cartilage (an intermediate cartilaginous structure, which makes up the ventral component of the mandibular arch) (29) and the ceratohyal (a pharyngeal arch cartilage) (30) was measured for each embryo as previously described (17).
For imaging, a representative sample of each group (hspg2 morphants, random control, and non-injected wildtype) was dissected and the viscerocranium (the mandibular jaw region) was mounted on a glass slide with 100% glycerol. A Leica microscope was used to take high-resolution color images of each sample.
Whole mount in situ hybridization and quantitative real time polymerase chain reaction (PCR)
Whole mount in situ hybridization was performed as described by Thisse and Thisse (31). Larvae were harvested and dechorionated at the indicated time point and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, PA) overnight at 4ºC. Larvae were then dehydrated using a methanol: PBS gradient and stored in 100% methanol overnight at -20°C. Embryos were rehydrated using a PBS:Methanol gradient, washed in PBS with 0.1% Tween 20 and permeabilized with proteinase K (10ug/ml) for the time indicated by Thisse and Thisse (31). Permeabilized larvae were prehybridized for 2 hours in hybridization buffer (HB) (50% deionized formamide (Fisher), 5X SSC (Fisher), 0.1% Tween 20 (Fisher), 50 µg/m heparin (Sigma), 500 µg/mL of RNase-free tRNA (Sigma), and 1M citric acid (Fisher). Larvae were then incubated overnight in fresh HB with probe (dlx2a and nkx3.2 at 127 ng) at 70°C. Samples were washed according to protocol, blocked in 2% sheep serum (Sigma) and 2 mg/ml bovine albumin serum (Sigma) for 2 hours at room temperature. Samples were then incubated with anti-DIG Fab fragments (1:10,000) (Sigma) overnight at 4°C. Samples were developed with BM purple AP substrate (Sigma) and imaged with a Zeiss Discovery Stereo Microscope fitted with Zen Software. Statistical analysis was performed using a Fisher’s exact test. For quantitative polymerase chain reaction (qPCR), RNA was isolated from embryos at the indicated time point using Trizol (Fisher) according to manufacturer’s protocol. Reverse transcription was performed using a Verso cDNA Synthesis Kit (Fisher) and total RNA was normalized across all samples. PCR was performed using an Applied Biosystem’s StepOne Plus machine with Applied Biosystem’s software. Sybr green (Fisher) based primer pairs for each gene analyzed are as follows: dlx2a fwd CCTCACGCAAACACAGGTTA, dlx2a rev TGTTCATTCTCTGGCTGTGC, nkx3.2 fwd GCAGATTTAGCGGACGAGAC, nkx3.2 rev GCTTCAACCACCAGCGTTAT, sox10 fwd ACGCTACAGGTCAGAGTCAC, sox10 rev ATGTTGGCCATCACGTCATG, rpl13a fwd TCCCAGCTGCTCTCAAGATT, and rpl13a rev TTCTTGGAATAGCGCAGCTT. Analysis performed using 2ΔΔct. Statistical analysis of messenger RNA (mRNA) expression was performed using a Student t test on biological replicates. All qPCR was performed in biological duplicates.
Confocal Imaging and Transgenic Cell Counts
Transgenic larvae (Tg(sox10:tagRFP) and Tg(col2a1a:EGFP)) were fixed at given time points using 4% paraformaldehyde. Fixed larvae were mounted in 0.6% low-melt agar in a glass bottom dish (Fisher). Imaging was performed on a Zeiss LSM 700 at 20X and 40X Oil magnification. Images were restricted to the larval craniofacial region. For each fish, a minimum of 20 to 30 z-stacks were collected. Maximum intensity projection images were processed and saved (32). For cell counts, the number of cells per z-stack (20-30/fish) at each jaw joint region (3 rows of chondrocytes on the left side of the joint and 5 rows on the right) were counted using ImageJ. Statistical significance was obtained using a Student t test (32).