Whole mount in situ hybridization (WMISH)

To visualize the spatial and temporal pattern of endogenous transcripts, a whole mount in situ hybridization (WMISH) was performed. The performance essentially based on (Harland, 1991; Hollemann and Pieler, 1999; Nieber et al., 2009). For the detection, a Digoxygenin-11-UTP (Dig) labeled anti-sense RNA probe was used.

The visualization was done by the use of an alkaline phosphatase-coupled anti-Dig antibody. All steps were performed at ambient temperature with mild shaking.

2.2.4.1 Fixation and X-Gal staining

10x MEM: 1 M Mops; 20 mM EGTA; 10 mM MgSO4; pH 7.4 (stored light

protected)

10x PBS: 1.75 M NaCl; 1 M KCl; 65 mM Na2HPO4; 18 mM KH2PO4; pH 7.4

K₃FE(CN)₆: 0.5 M in H₂O (stored light protected) K₄FE(CN)₆: 0.5 M in H₂O (stored light protected) MEMFA: 4% (v/v) formaldehyde (37%) in 1x MEM

X-Gal: 40 mg/ml 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside in formamide (stored -20 °C light protected)

X-Gal staining solution: 1 mg/ml X-Gal; 5 mM K3FE(CN)6; 5 mM K₄FE(CN)₆; 2 mM MgCl₂ in 1x PBS

Embryos were fixated with MEMFA in glas vials for 1 hr at RT following three washing steps with 100 % ethanol for 5 min. β-gal injected embryos were fixated in MEMFA for only 25 min following three washing steps with 1x PBS for 10 min. The co-injection of RNA encoding for β-galactosidase (glb1) was used as a control for the purpose of knockdown and over-expression studies. To visualize the β-gal presence, X-Gal staining was performed (Bourguignon et al., 1998). Therefore, the embryos were incubated in X-Gal staining solution under light protection until the desired staining level was achieved. To stop the staining reaction, the embryos were washed three times for 10 min in 1x PBS and refixated for 25 min in MEMFA.

Finally, the embryos were washed three times for 5 min in 100 % ethanol and stored at -20 °C.

55 2.2.4.2 Rehydration

PTw: 0.1 % Tween-20 in 1x PBS

Embryos were rehydrated with an ethanol series (75 %, 50 %, 25 %) to PTw and washed three times in PTw for 10 min each at RT.

2.2.4.3 Proteinase-K treatment Proteinase-K: 10 µg/ml in PTw

To allow a better penetration of the anti-sense RNA probe, embryos were treated with Proteinase-K. The embryos were incubated in 2 ml of PTw/proteinase-K solution for a defined time period and temperature depending on the developmental stage.

stage time [min] temperature

explants 1 RT

10 to12 4 RT

32 to 35 18 RT

39 to 41 15 37°C

2.2.4.4 Acetylation and refixation

Triethanolamine: 0.1 M (0.93 g in H₂O); pH 7.5 (SIGMA) Acetic anhydride: (SIGMA)

PTw-FA: 4 % formaldehyde (v/v) in PTw

The proteinase-K treatment was stopped by two washing steps with triethanolamine for 5 min. To prevent an unspecific reaction of the Dig-labeled anti-sense RNA probe, free amino-acid ends were blocked by the treatment with 25 µl acetic anhydride in triethanolamine. Next, the embryos were washed two times with PTw for 5 min and refixated in PTw-FA for 20 min. Afterwards the embryos were washed 5 times with PTw for 5 min.

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2.2.4.5 Hybridization

Hybridization Mix: 50 % (v/v) formamide; 1 mg/ml Torula RNA (SIGMA);

100 µg/ml Heparin, 1x Denhardt´s; 0.1 % (v/v) Tween- 20; 0.1 % (w/v) CHAPS; 10 mM EDTA in 5x SSC 100x Denhardt´s: 2 % BSA; 2 % Polyvinylpyrollidone (PVP); 2 % Ficoll

400 in H₂O (w/v)

20x SSC: 3 M NaCl; 0.3 M NaCitrate; pH 7.2-7.4

The Hybridization with the anti-sense RNA probe was preceded by a pre-hybridization with 1 ml pre-hybridization mix for 5 min followed by incubation with fresh hybridization mix for at least 5 h at 65 °C. Afterwards the embryos were incubated in 1 ml Dig-labeled anti-sense RNA containing Hybridization mix over night at 65 °C.

2.2.4.6 Washing

20x SSC: 3 M NaCl; 0.3 M NaCitrate; pH 7.2-7.4 MAB: 100 mM maleic acid; 150 mM NaCl; pH 7.5

RNase solution: 20 µg/ml RNase A and 10 U/ml RNase T1 (Fermentas) in 2x SSC

The next day, the anti-sense RNA probe was collected and stored at -20°C for reuse. The embryos were washed in hybridization mix for 10 min at 65 °C and three times with 2x SSC for 15 min at 65 °C. To remove the non-hybridized RNA molecules, the embryos were incubated in RNase solution for 1 h at 37 °C.

Afterwards the embryos were washed once in 2x SSC at 37 °C and twice in 0.2x SSC at 65 °C. Next, the buffer was exchanged to MAB.

2.2.4.7 Blocking and antibody incubation

MAB: 100 mM maleic acid; 150 mM NaCl; pH 7.5

MAB/BMB: 2 % BMB (Bohringer Mannheim blocking reagent) in 1x MAB

MAB/BMB/HS: 20 % heat-treated horse serum (HS) in MAB/BMB MAB/BMB/HS/AK: 1:5000 sheep-anti-Dig antibody linked to alkaline

phosphatase (AP) in MAB/BMB/HS (SIGMA)

57 To minimize unspecific binding of the AP-linked anti-Dig antibody, embryos were blocked in MAB/BMB for 20 min and in MAB/BMB/HS for 40 min. For the detection, the embryos were incubated with anti-Dig antibody containing MAB/BMB/HS/AK for 4h at RT. Next, the embryos were extensively washed with MAB, first three times for 10 min and then overnight at 4 °C.

2.2.4.8 Staining reaction

APB: 100 mM Tris-HCL; 50 mM MgCl2; 100 mM NaCl; 0.1 % Tween-20; pH 9.0

NBT: 100 mg/ml in 70 % Dimethylformamide (light sensitive) BCIP: 50 mg/ml in 100 % Dimthylformamide (light sensitive) Staining solution: 0.8 µl NBT and 3.5 µl BCIP in 1 ml APB

MEMFA: 4% (v/v) formaldehyde (37%) in 1x MEM

The washing with MAB was continued by three times for 10 min with MAB. Then the caps of the glas vials were exchanged and three additional washing steps with MAB followed. Next, the embryos were two times washed in APB for 5 min at 4 °C and then the staining solution was added. This solution contained NBT and BCIP, substrates for the alkaline phosphatase that were converted to a colored product.

The staining reaction was performed in the dark at 4 °C until the staining was sufficient. To stop the colour reaction and to remove background staining, the embryos were transferred to 100 % methanol. Then the embryos were rehydrated with a methanol series (75 %, 50 %, 25 %) to MEMFA and incubated for 30 min.

2.2.4.9 Bleaching

20x SSC: 3 M NaCl; 0.3 M NaCitrate; pH 7.2-7.4 MEMFA: 4% (v/v) formaldehyde (37%) in 1x MEM Bleaching solution: 50 % formamide and 1 – 2 % H₂O₂ in 5x SSC

To remove the pigments for an easier documentation of the staining, the embryos were first washed twice in 5x SSC and then incubated with the bleaching solution.

Afterwards, the embryos were washed again twice in 5x SSC and re-fixated with MEMFA.

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