CULTIVOS DE ALGODÓN EN EL PERU

2.2.11.1. C. intestinalis

The eggs from 3 to 4 adults were dissected into separate petri-dishes filled with filtered sea water (FSW), whilst sperm was collected from each animal into separate microfuge tubes containing 1ml of FSW and kept on ice. Eggs and sperm were then mixed in a beaker for 10 minutes and then washed through a 100µm mesh filter to wash off excess sperm. Eggs were washed in FSW three times in order to clean excess sperm off, taking care to keep them covered in FSW at all times, and then transferred to a 15ml tube with no more than 2ml of FSW. Fertilised eggs were then

dechorionated using 2% sodium thioglycolate and 0.1% protease, prepared separately and then mixed prior to dechorionation. Dechorionation solution was added to the eggs and allowed to sit for 2 minutes in a 15ml tube, then a small sample taken and checked every 30 seconds in a petri dish under a benchtop microscope to check for dechorionation of 50% of fertilised eggs. Dechorionation times varied for animals from different locations, with zygotes from Croabh Haven requiring 3-3.5 minutes and zygotes from Arbroath 6-8mins. These were then washed with FSW several times in a 15ml falcon, gently spinning the zygotes down via a hand centrifuge for no more than 2 minutes in between washes, before transferring to pre-prepared 1% agarose-FSW coated petri-dishes

containing approximately 50ml of FSW. 5ml of 100µg/ml Gentamycin in FSW was then added to prevent bacterial growth and embryos allowed to develop at 16°C until the stage required. Embryos were then fixed with 4%-PFA:MOPS solution overnight at 4°C. These were then washed twice in PBT with rocking for 10 minutes, and once in 70% EtOH with rocking for 10 minutes before storing in a 1.5ml microfuge tube in 70% EtOH. All microfuge tubes, 15ml tubes and Pasteur pipettes used in this protocol were silicon-coated using Sigmacote-SL2 (Sigma) to avoid embryos sticking to equipment.

2.2.11.2. B.lanceolatum

Adult Branchiostoma lanceolatum were collected by Dr David Ferrier and Clara Coll Lladó at the facilities of Laboratoire Aragó in Banyuls-sur-mèr, France, in 2010. Adult animals were also

64 transferred straight to RNAlater for use in RNA extraction or preserved in absolute ethanol for use in gDNA extraction. Embryos were collected by spawning of ripe amphioxus. These were induced by heat stimulation according to (Fuentes et al., 2007) and embryonic stages (gastrula, early neurula, mid-neurula, late neurula and early larval stage) were collected at regular intervals and fixed in MOPS-PFA, 4% for 1 hour at room temperature or overnight at 4°C. After fixation, embryos for whole-mount in situ hybridization (WMISH) were washed three times in 70% ethanol and stored in 70% ethanol at -20°C. Larvae (first gill slit stage) and juvenile amphioxus from previous spawnings were kindly provided by Dr. Héctor Escrivà and Dr. Stéphanie Bertrand. Both developmental stages were fixed and stored in 70% ethanol following the same procedure described for the embryonic stages. Late neurula and early larval stages were kindly donated by Dr. Ildikó Somorjai to complete the amphioxus developmental series.

2.2.11.3. B. floridae

B.floridae adults were collected from Tampa Bay, Florida using a shovel and sieve by Tom Butts and Peter Osborne in 2006. Embryonic stages were collected according to Holland and Yu (2004) and fixed for 1 hour at room temperature or overnight at 4°C in MOPS-PFA, 4%. Embryos were then washed multiple times in 70% Ethanol and stored in 70% ethanol at -20°C. Adults were also preserved in absolute ethanol for use in gDNA extraction.

2.2.12. In situ Hybridisation

2.2.12.1. C.intestinalis

In situ hybridisation was carried out as detailed in (Wada et al., 1995) with the following modifications. Embryos were rehydrated through an ethanol series into PBT and then digested for 10 minutes at room temperature in 2µg/ml proteinase K for gastrula to mid-tailbud embryos and 20 minutes for late-tailbud embryos. 4µl of 10% glycine was then added, swirled and the solution removed immediately and replaced with 10% glycine in PBT and washed for 5 minutes. This was then changed for 4% PFA in PBS and fixed for 1 hour at room temperature. After

triethanolamine/acetic anhydride washes, embryos were washed three times in PBT before being washed once in 50:50 Hybridisation buffer (HYB) to PBT, then once in Ciona-HYB. This was then changed to fresh Ciona-HYB and embryos were pre-hybridised at 60°C for 3 hours. Approximately 50-100ng of antisense RNA probe in fresh Ciona-HYB was denatured at 70°C for 10 minutes before being added to the embryos. Embryos were then incubated at 70°C for 2 minutes before being moved to an overnight hybridisation at 60°C, rocking gently. Hybridised embryos then underwent 3x

65 20 minute washes in Ciona Wash buffer 1 at hybridisation temperature. This was followed by 2x 20 minute washes at 37°C in Ciona wash buffer 2 and then 2x 5 minute washes at room temperature in Ciona Wash buffer 3. 3x 5 minute washes at 37°C were followed by 2x 20 minute washes at 50°C, all in Ciona Wash buffer 3. A single wash for 20 minutes at 50°C in Ciona wash buffer 4 was carried out before 3x 10 minute washes in PBT at room temperature. Embryos were then blocked for 3 hours in blocking solution before adding 1:3000 Anti-digoxigenin-AP Fab fragments in blocking solution and incubating O/N at 4°C. No modifications were made to the staining and post-staining procedures.

2.2.12.2. Amphioxus

In situ hybridisation on B.floridae and B.lanceolatum embryos was carried out according to (Holland et al., 1996) with the following modifications. Amphioxus embryos were rehydrated through an ethanol series into PBT and then digested for 5 minutes at room temperature in 2µg/ml proteinase K for mid-gastrula to early-neurula embryos, 10 minutes for mid neurula-late neurula embryos and then 15 minutes for premouth embryos and 2-day larvae. After triethanolamine/acetic anhydride washes, embryos were washed once in PBT for 1 minute rotating, then again in PBT for 5 minutes rotating. This was then changed for 100µl of HYB buffer, pre-warmed to 60°C, and rotated for 1 minute. This was then changed for fresh HYB and rocked in the hybridisation buffer for 2 hours. Antisense RNA probe was mixed in 1/200-1/50 dilutions in fresh warm HYB and then denatured at 70°C for 10 minutes, before being added to the embryos. These were then rocked overnight at either 60°C or 62°C in the hybridisation oven. No modifications were made to the day 2 until

blocking. RNase steps were carried out with 2µl 10mg/ml RNaseA and 1µl RNaseT1 (10,000U/ml) in 1 ml of Wash solution 3 and 250µl added per well. Wash solution 5 was then removed and 200µl of blocking solution was added to the embryos and rotated for 3 hours at room temperature. Blocking solution was then removed and 1:2000 Anti-digoxigenin-AP Fab fragments in blocking solution were added to the embryos and left incubating overnight at 4°C. On day 3, embryos were washed 4 times in NaPBT for 20 minutes each at room temperature, before 3 washes in AP- followed by 3 washes in AP+. AP+ was then exchanged for staining buffer and embryos were left in the dark at RT for the colour to develop. Signal typically came up overnight, but could take up to 4 days dependant on probe concentration. The final post-staining procedure consisted of 3 washes in AP- for 10 minutes each, rotating and kept dark, followed by 3 washes in NaPBT for 10 minutes each, rotating and kept dark. Embryos were cleaned during the NaPBT washes. They were then fixed in 4%PFA in NaPBS for 1 hour at RT. Finally embryos were washed twice in NaPBT for 10 minutes each before being transferred to 80% glycerol to clear.

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