Multi-modal Developmental Gene Expression

Digoxigenin-UTP-labeled antisense RNA probes were transcribed using MEGAscript Kit (Ambion) according to the manufacturer's instructions. Whole-mount in situ hybridizations were performed according to previously published methods (Ning et al., 2013 (link)). In particular, fluorescent in situ hybridization in immunostained embryos was conducted as previously described (Mao et al., 2021 (link)). Briefly, for the detection of cxcr4a and tie1 expression, Tg(nkx2.5:ZsYellow) embryos were first immunostained using affinity-purified anti-ZsYellow antibody (1:800; TA180004, Origene), and then subjected to in situ hybridization with digoxigenin-labeled cxcr4a or tie1 probe. Anti-digoxigenin-HRP (1:400; Roche, 11633716001) was used as primary antibody to detect the probes and the hybridization signals were visualized by incubating embryos with fluorescein tyramide (1:50; PerkinElmer, NEL701A001KT). For the detection of cxcl12b expression, Tg(sox17:GFP) embryos were fluorescently stained with anti-GFP (1:1000; A11120, Invitrogen) antibody. The resulting hybridization signals were similarly generated with cyanine 3 tyramide (1:50; PerkinElmer, NEL701A001KT).