In Situ Hybridization

In situ hybridization was performed according to published procedures (22,49). Embryos were staged according to Hamburger and Hamilton (17), removed from the egg, washed in PBS, and the gut was removed on ice. The whole gut was divided into the anatomical regions as shown in Figure 1 and fixed in 4% paraformaldehyde prepared fresh in PBS, pH. 7.4, at 4°C for 4 h [embryonic day (ED) 4/5] to overnight (ED8–18). Following fixation, the tissue was extensively washed (16 × 15 min) in PBS and stored in 30% sucrose in PBS at 4°C. Frozen sections were cut at 10 μm from tissues embedded in O.C.T. (Miles, Inc., Elkhart, IN) mounting medium and thaw mounted onto SuperFrost Plus (Fisher, Pittsburgh, PA) slides; sections were stored at −20°C until used. In situ hybridization was performed using digoxigenin-labeled antisense cRNA probes corresponding to the full-length chicken cDNAs encoding HAND1 or HAND2 (22,23). There was no visible signal in sections hybridized with sense strand control probe (data not shown). For in situ hybridization, sections were dried at 50°C for 15 min, postfixed with 4% paraformaldehyde for 20 min, washed in PBS, and then treated with 50 μg/ml of proteinase K (Sigma, St. Louis, MO) for 8 min. Tissue sections were fixed again in 4% paraformaldehyde for 15 min and washed in PBS. Sections were prehybridized in buffer containing 50% formamide, 5 × SSC, 0.3 mg/ml yeast tRNA, 50 μg/ml heparin, 1× Denhardt’s solution, 0.1% Tween 20, 0.1% CHAPS, and 5 mM EDTA, for 3 h at 65°C in a humid chamber containing 50% formamide and 5 × SSC. The sections were then hybridized with cRNA probes (1 μg/ml in pre-hybridization buffer) overnight at 65°C in a humidified chamber. Following hybridization, excess probe was removed by washing at 65°C, in 5× SSC, 1 × 15 min, and in 0.2× SSC, 2 × 30 min. To detect sites of hybridization, sections were washed with PBT (1× PBS, 2 mg/ml BSA, 0.1% Triton X-100) and incubated in blocking solution containing PBT and 20% heat-inactivated horse serum for 30 min at room temperature. The sections were then incubated with anti-digoxygenin antibody conjugated to alkaline phosphatase (1:2000; Roche, Indianapolis, IN) in the blocking solution at 4°C overnight. Sites of antibody binding were detected using BCIP/NBT according to manufacturer’s directions (Roche). Color development was carried out in the dark from 20 min to 2 h and stopped by washing in PBS. For combined in situ hybridization and immunostaining with HNK-1 to detect neural crest-derived cells, in situ hybridization was followed by immunocytochemistry, as described below.