EXPERIMENTAL PROCEDURES
1. Embryos handling
In order to obtain Xenopus laevis embryos, each adult female used were injected with 800-1000 units of human chorionic gonadotropin (Gonasi HP 5000 U.I., Amsa) 12-14 hours prior to eggs collection. Ovulated eggs were fertilized with testis homogenate and let to develop in O.1X MMR. Jelly coats were removed by using the dejelling solution.
2. Embryos microinjection and manipulation
For knock-down experiments, morpholino antisense oligonucleotides (Mo, Gene Tools, LLC), were injected together with nuclear-β-galactosidase (n-β-gal) and Green Fluorescent Protein (GFP) in vitro transcribed capped mRNAs (see Results).
For overexpression experiments, XHas2 in vitro transcribed capped mRNA was injected together with nuclear-β-galactosidase (β-gal) capped mRNA (see Results).
Morpholinos or capped mRNAs were injected into one blastomere of two-cells stage embryos. Microinjections were performed using a Drummond "Nanoject" apparatus. Embryos were injected in 0.1X MMR and 3% Ficoll-400 and cultured over night (O./N.) at 14° C in the same solution. Embryos were subsequently transferred in 0,1X MMR and incubated at Room Temperature (R.T.) until the desired developmental stage. Embryos were staged according to Nieuwkoop and Faber (1967).
When n-β-gal mRNA was used as a lineage tracer in microinjection experiments, β-gal staining was detected by fixing embryos in 1X MEMFA for 30 min. at R.T., washing in 1X PBS pH 7.3 and than incubating them in β-gal staining solution for 20-30 min. at 37°C. Embryos were than fixed again in 1X MEMFA for 30 min. at R.T. and then gradually dehydrated with ethanol to be
processed by whole mount in situ hybridization. The embryos treatment before in situ hybridization on sectioned embryos, Tunel assay or bHABC staining will be detailed afterward.
2.1. Morpholino antisense oligos
For each down regulation experimental assay, two non-overlapping morpholino antisense oligos have been used: one targeted to the 5’ untranslated region (Mo-5’UTR) and the other one targeted to the first 25 nucleotides of the coding region of the specific gene (Mo-ATG).
Morpholino antisense oligonucleotides (Gene Tools, LLC) sequences:
XHas1-Mo-5’UTR a: 5´-CACACACATGGGATATGCCAAGTTT-3´ XHas1-Mo-5’UTR b: 5’-GTTGCCGAATGAAGAGGCCCCAAGA-3’ XHas2-Mo-5’UTR: 5´-CTTGGTTATTGCCTTGGTCCTGTGG-3´ XHas2-Mo-ATG: 5´-TGCATATAAACCGTTCACAGTGCAT-3’ XCD44-Mo-5’UTR: 5’-TGTGCTCCGCAGACAAGAGGCTCCT-3´ XCD44-Mo-ATG: 5’-TAACAATCCACAGCATTGAGGCCAT-3’
For each gene, a control morpholino was synthesized as a random sequence of the same length (Gene Tools, Philomath, OR).
2.2. CAPPED mRNAs
Capped mRNAs were synthesized in vitro from the template cDNAs using the SP6 mMESSAGE mMACHINE Kit (Ambion). After DNase digestion to remove template cDNA, transcripts were purified with 0.1 volume of Ammonium Acetate to avoid embryos gastrulation defects, extracted with 1 volume of phenol/chloroform, precipitated with 2 volume of 100% ethanol, resuspended in water at the 500 ng/µl. concentration and stored at -20°C. Concentration and length of transcripts were estimated by agarose gel electrophoresis.
2.2.1. Constructs used for overexpression analysis: linearization and capped mRNA transcription
- n-β-gal (NotI/Sp6) (Chitnis et al,. 1995) that contains the nuclear-β-galactosidase reporter inserted into the pCS2+ vector.
- pCMTEGFP (NotI/Sp6) (a gift from D. Gilmour) that contains the Enhanced Green Fluorescent Protein reporter gene inserted into the pCS2+ vector.
- XHas2-pCS2+ (Asp718/Sp6) that was available in laboratory
3. In situ hybridization
3.1. Purification of plasmidic DNA
Plasmidic DNA were prepared according to the alkaline lysis procedure (Sambrok et al,. 1989), followed by chromatography over QIAGEN columns. After purification, circular plasmids were digested with appropriate restriction enzymes, in order to linearize templates for in vitro transcriptions.
3.1.1. Constructs used for gene expression analysis: linearization and RNA transcription to generate antisense mRNA probes
XCD44-pGEM-T: (NotI/T7) (Ori et al., 2006)
XDll4-pGEM-T: (NotI/T7) (Papalopulu and Kintner, 1993) XFoxD3: (NotI/T7) (Pohl and Knochel, 2001)
XHas1-pGEM-Teasy: (NcoI/Sp6) (Nardini et al., 2004) XHas2-pGEM-Teasy: (NcoI/Sp6) (Nardini et al., 2004) XRHAMM: (SpeI/T7) (see Results).
XSlug-sp72: (EcoRV/SP6) (Mayor et al., 1995)
XSox9-pGEM-Teasy: (NcoI/Sp6) (Spokony et al., 2002) XVersican: (NcoI/SP6) (see Results)
3.2. Synthesis of digoxigenin (DIG) labeled probes
Linearized plasmids were used as template to synthesize digoxigenin-11-UTP-labeled (Boehringer Mannheim) antisense mRNA probes employing the RNA polymerases indicated above. In particular, transcription reactions were incubatedin the presence of 10 mM each of rATP, rCTP, rGTP, rUTP/DIG-11-rUTP (3/2) 2 hours at 37°C. After DNase digestion to remove template DNA, the amount and length of transcripts were estimated by agarose gel electrophoresis. Probes were than diluted in hybridization mix at a final concentration of 10 µg/ml and stored at -20°C for several months.
3.3. Whole mount in situ hybridization
Whole-mount in situ hybridization was performed by using standard procedures as described in Harland (1991), except that BM purple (Roche) was used as a substrate for the alkaline phosphatase. After colour development embryos were post-fixed on 1X MEMFA and bleached over a fluorescent light for about 1 hour to remove the pigment. Control experiments were performed with sense probes.
For histological examination, whole-mount in situ hybridization processed embryos were embedded in a gelatin albumin solution according to the protocol described in Levin (2004) whit minor modification, and then sectioned at 50 µm thickness using a Leica VT1000S vibratome.
3.4. In situ hybridization on frozen tissue sections
Embryos collected for in situ hybridization on cryostat cut tissue sections were fixed 2 hours in 4% paraphormaldeyde in 1X PBS, cryo-protected with 20% sucrose in 1X PBS and subsequently embedded in the optimal cutting temperature (O.C.T.) compound (Sakura Finetek) and stored at -80°C.
4. bHABC staining
The biotinylated hyaluronan binding complex (bHABC) was used to histologically localize hyaluronan (Tammi et al., 1994).
Embryos collected for bHABC staining on cryostat tissue sections were fixed 2 hours in 4% paraphormaldeyde in 1X PBS, cryo-protected with 20% sucrose in 1X PBS and subsequently embedded in the O.C.T. compound.
12 µm cryostat sections were incubated with 0.5% H202 in 1X PBS for 15
min to block endogenous peroxidases, washed with 1X PBS and incubated in 1% bovine serum albumin (BSA) in the same buffer for 30 min. at 37°C to block aspecific binding sites. The slides were incubated with the bHABC (1µg/ml, diluted in 1% BSA and 1X PBS) overnight at 4°C. The following day, slides were washed three times for 5 min with 1X PBS and treated with avidin-biotin-peroxidase (1:200, Vector Laboratories; Imine. CA) for 1 hr at R.T. The sections were then washed with 1X PBS and incubated in 0.05% DAB (3,3'-diaminobenzidine; SIGMA) and 0.03% H202 in 1X PBS at R.T. for 5 min.
For the fluorescent labelling, after the bHABC incubation, slides were incubated with 5 µg/ml streptavidin Alexa Fluor 532 conjugate (INVITROGEN) for 1 hour at R.T.
After washes, the sections were counterstained with Hoechst to visualize nuclei and cover-slips were mounted with Aqua-Polymount (Polysciences, Inc.).
5. Tunel assay
Tunel staining was performed on whole-mount embryos as described in Hensey and Gautier (1998). For histological examination, processed embryos were vibratome sectioned as previously described.
6. Alcian Blue staining
For Alcian blue staining, embryos were raised over 10–11 days at 20°C until they reached stage 49, selected for asymmetrical distribution of the lineage tracer GFP and fixed O./N. in 4% paraphormaldehyde. The following day
embryos were gradually dehydrated on 80% Ethanol and 20% Acetic Acid and stained for 6 hours in 0.05% Alcian blue in 80% Ethanol and 20% Acetic Acid. Specimens were destained O./N. in 80% Ethanol and 20% Acetic Acid and transferred into a solution of 1% KOH e 3% H2O2 in 1X PBT (1X PBS and 0,1%
Tween 20) for about 3 hours. Subsequently, the tissue was cleared in 0.05% Trypsin (SIGMA) dissolved in saturated Sodium Tetraborate for 1-2 hours. Cleared embryos were rinsed in 1X PBT and then stored in 0.1% NaN3 in 1X PBS.
Specimens were dissected by removing the skin and by cutting the ethmoidal plate on the midline and flat-mounted on slides in order to make visible the visceral skeleton.
Specimens were visualized with a Nikon SMZ1500 stereo dissecting microscope equipped with epifluorescence optics, and digital images were generated using a Photometrics COOLSNAP CCD camera
7- RNA extraction and RT-PCR
Total RNA was extracted from staged embryos using the TRIZOL reagent (Gibco BRL). The corresponding cDNAs were synthesized using Superscript Reverse Transcriptase (Gibco BRL) and further PCR analysis was carried out using primers specific to XRHAMM, Xversican and Xenopus Ornithine decarboxylase (ODC) genes. ODC gene expression represents an normalizing control, as it is a housekeeping gene whose expression does not change between the stages analyzed.
The following primers and thermocycling parameters were used:
XRHAMM
For.: 5'- CAAACAAGTGGCGCACCC -3' Rev.: 5'- AGTCATTGCCCAGTCAGC -3’
XVersican
For.: 5'-CAAGTCAAGACCCATGCAAGG-3’ Rev.: 5'-GAGTCTTGCCAAGTCCTGCTC-3'
Thermocycling: 94°C 25sec. / 58° C 30sec. / 72° C 60sec. (30 cycles)
XODC
For.: 5'-AATGGATTTCAGAGACCA-3’ Rev.: 5'-CCAAGGCTAAAGTTGCAG-3'
Thermocycling: 94°C 25sec. / 55° C 30sec. / 72° C 40sec. (30 cycles)
8. Solutions BLEACHING SOLUTION 2% H2O2 5% Formamide 0.5X SSC 1X PBS DEJELLING SOLUTION DTT (dithiothreitol) 3.2 mM Tris pH 8.8, 0.2 M
GELATIN ALBUMIN SOLUTION 0.5% gelatine
30% albumin 1X PBS
fix with 0.6% glutaraldehyde
MEMFA salts 10X
MOPS (pH 7.4) 100 mM EGTA 2 mM
MgSO4 1 mM
MMR (Marc’s Modified Ringer’s) 10X NaCl 0.1M KCl 2mM MgSO4 1mM CaCl2 2mM HEPES (pH 7.8) 5mM EDTA 0.1mM
PBS (Phosphate Buffer Saline) 10X NaCl 137mM
KCl 2.07mM Na2HPO4 10mM
KH2PO4 2mM
pH 7.3
β-gal STAINING SOLUTION
K3Fe(CN)6 (Potassium Ferricyanide) 5mM
K4Fe(CN)6 (Potassium Ferrocyanide) 5mM
MgCl2 2mM
X-gal or red-gal substrate 1 mg/ml 1X PBS
