3 MATERIALS AND METHODS
3.6 Embryological methods
Cysteine (Sigma): 2% LCysteine in 0.1X MBS (X. laevis) or in 1/9 MR (X. tropicalis);
pH 7.8 (X. laevis) or 7.5 (X. tropicalis); the solution is kept at RT.
Human Chorionic Gonadotropin (HCG): 2000 UI/ml HCG in milliQ water.
MEMFA: 0.1M 3-(N-Morpholino)-propanesulfonic acid (MOPS); 2mM EGTA; 1mM MgSo4; 3.7% formaldehyde (freshly set up for the use), pH 7.4.
Modified Barth’s saline (MBS, 1X): 880mM NaCl, 10mM KCl, 24mM NaHCO3, 8.2mM MgSO4, 3.3mM Ca(NO3)2, 4.1mM CaCl2, 100mM Hepes; pH 7.6. The solution is kept at RT.
Modified Barth’s saline (MBS) high salt (1X): 1X MBS supplemented with 50mM NaCl.
0.1X MBS/Gentamycin: 0.1X MBS supplemented with 10µg/ml Gentamycin.
0.8X MBS/CS: 0.8X MBS high salt with 20% chicken serum, 200U Penicillin/ml, 200 µg/ml streptomycin; the solution was stored at -20°C until use.
1X Modified Ringer Solution (MR): 0.1M NaCl, 1.8M KCl, 2mM CaCl2, 1mM MgCl2, 5mM Hepes-NaOH. 1/9 and 1/18 MR solutions were prepared with proper dilution of 1X MR. The solutions are kept at RT.
1X Ringer’s solution: 116mM NaCl, 2.9mM KCl, 1.8 CaCl2, 5mM Hepes. The solution was kept at RT.
10X Steinberg’s Solution (SS): 580mM NaCl, 6.7mM KCl, 3.4mM CaNO3, 8.3mM MgSO4, 50mM Tris, 0.1g Kanamycin; pH 7.4; the solution is filtered, autoclaved and kept at RT.
1X SS/Gentamycin: 1X SS supplemented with 10µg/ml Gentamycin.
1X SS/PIF: 1X SS supplemented with activin supernatant diluted 1:10 (Sokol et al., 1990).
3.6.2 Experimental animals
Adult wild type Xenopus laevis and Xenopus tropicalis frogs were purchased from commercial breeding farms (Xenopus Express, Nasco). Animal work has been conducted in accordance with Deutsches Tierschutzgesetz; experimental use of Xenopus embryos has been licensed by the Government of Oberbayern (Projekt/AK ROB: 55.2.1.54-2532.6-3-11). Animal husbandry and use for the work presented here complies to the Directive 2010/63/EU. Xenopus frogs were kept in 17-19°C (X. laevis) or 21°C (X. tropicalis) tap water. The animals were fed three times per week with Pondsticks Premium brittle (Interquell GmbH, Wehringen).
3.6.3 Superovulation of female frogs
Ovulation of Xenopus laevis females was stimulated by injection of 800 units of HCG into the dorsal lymph sac. In animals maintained at 18-20°C water temperature, egg lying started about 12-16h later.
Xenopus tropicalis females were stimulated to lay eggs following a two-step protocol: 12-20h before the main stimulation the females were primed with 10 units of HCG into the dorsal lymph sac. The second stimulation was performed by injecting 200 units of HCG in the dorsal lymph sac.
3.6.4 Testis preparation
A male frog was anaesthetized in 0.1% 3-Aminobenzoeacid-ethyl-ester in milliQ water for 30min, cooled down in ice-cold water and killed by decapitation. The two testes were taken from the abdominal cavity by pulling out the fat body through an incision of the skin, to which they are connected. Unless used, the testes from X. laevis were kept in MBS/CS at 4°C for a maximum of 6 days. For X. tropicalis, the testes were kept in 1X Ringer solution at 4°C for a maximum of 2 days.
3.6.5 In vitro fertilization of eggs and embryos culture
For in vitro fertilization of X. laevis eggs a small piece of testis was minced in 1X MBS and subsequently mixed with freshly laid eggs. After 3-4min the eggs were incubated in 0.1X MBS at 16-23°C in 110 mm Petri dish. For in vitro fertilization of X.
tropicalis eggs half a testis was minced in 300µl 1X Ringer solution, and subsequently mixed with freshly laid eggs. After 3-4min eggs were covered with 0.1X MBS and put at 23°C in 110mm Petri dish.
3.6.6 Removal of egg jelly coat
Amphibian eggs are encapsulated in a multi-layered protein networks, the jelly coat. Due to its elastic properties, it needs to be removed prior to microinjection.
For X. laevis embryos, the jelly coat was routinely removed approx. 60min post-fertilization in order not to interfere with fertilization-associated developmental events, such as cortical rotation. The egg jelly coat was removed by incubating the embryos in 0.1X MBS plus 2% Cysteine solution, pH 7.8 for about 5min with gentle agitation in a glass flask. Embryos were then washed three times with 0.1X MBS and finally cultured in 0.1X MBS/Gentamycin at 16-23°C.
For X. tropicalis the egg jelly coat was removed, after 20min post fertilization, by incubating the embryos in 1/9 MR plus 2% Cysteine solution, pH 7.5 for about 10- 15min. While in Cysteine solution, embryos were gently mixed. Embryos were then washed 3 times with 0.1X Barth solution and twice with 1/9 MR and subsequently incubated in 1/9 MR at 23°C.
3.6.7 Injection of embryos
Injection needles were created from capillaries with the Microneedle Puller (settings: heat: 800; pull: 35; vel: 140; time: 139; Sutter Instrument, model P-87). The needles were placed into the needle holder of the injection equipment (Medical System, model Pi-100). The tip of the needle was broken back carefully with a forceps in order to create an appropriate opening. The injection volume was adjusted by choosing the proper injection pressure (15-30psi) and/or the injection duration (30ms -1s). 2.5 or 5nl drops were injected during the experiments. Embryos were injected at two to eight cell stages into specific animal or vegetal blastomeres. After injection, not more than 50 embryos per dish were incubated in 0.1X MBS/Gentamycin at 16-23°C until the desired developmental stages in a 60mm Petri dish, covered with 1% agarose in 0.1X MBS (for X. laevis injections) or in 1/9 MR (for
Ficoll in 1/9 MR solution for 1h, then transferred in 1/9 MR solution for 30min; finally embryos were put in 1/18 MR solution and incubated at 23°C. The saline was exchanged every day to increase the survival rates of the embryos. For each experiment uninjected/untreated embryos were cultured in parallel with the injected/treated ones.
3.6.8 Animal cap explants preparation and culturing
Animal cap explants were manually dissected from embryos in 60mm Petri dish covered with 1% agarose in 1X SS and containing 1X SS/Gentamycin. Animal caps were explanted with a pair of forceps and singly transferred into wells of a 96- well plate covered with 90µl of 1% agarose in 1X SS and filled with 150µl of 1X SS/Gentamycin. For neural induction, embryos were injected into the animal pole with Noggin mRNA (60pg per embryo) alone or together with xSuv4-20h1 and h2 morpholinos (40ng each per embryo) at two- to four-cell stage. For mesoderm induction, embryos were injected animally 4 times with 2.5nl of control morpholino (80ng per embryo) or a mix of xSuv4-20h1 and h2 morpholinos (40ng each) at two or four cell stage. Subsequently, animal cap explants were incubated in 1X SS/Gentamycin buffer containing the P388D1-derived inducing factor (PIF) from the mouse macrophage cell line P388D1, previously described as a strong inducers of mesodermal tissues (Sokol, Wong et al. 1990). For Oct-25-VP16 and –EnR overexpression experiments, embryos were injected animally 4 times with 2.5nl of each mRNAs (100pg per embryo). For epistasis experiments on animal caps, embryos were injected 4 times with 2.5nl of xSuv4-20h1 and h2 Morpholinos (40ng each per embryo) and Oct-25 Morpholino (30ng per embryo) at two or four cell stage.