Immunofluorescence.

The 4-dpf larvae were fixed with 4% PFA containing 0.1% TritonX-100 at 4 °C overnight. They were then washed sequentially with 1% PBT (PBS containing 1% TritonX-100) for 5 min and DWT (distilled water with 1% TritonX-100) for 5 min. After washing, 1 mL −20 °C cold acetone was used to treat embryos for 10 min at −20 °C, followed by three washes with PBST for 5 min. Then embryos were then blocked with 5% goat serum/PBT for 1 h at room temperature. The primary antibodies were diluted in the blocking buffer and incubated with the larvae overnight at 4 °C. On the second day, the larvae were washed three times with PBT for 30 min followed by a final wash for 1 h. Secondary antibodies were diluted in blocking buffer and incubated with the larvae overnight at 4 °C. On the third day, larvae were washed three times with PBT for 1 h. After staining, the larvae were ready for imaging. The primary antibodies used were anti-HuC/D (1:500; mouse; catalog no. A21271; ThermoFisher Scientific) and anti-DsRed (1:100; rabbit; catalog no. 632496; Clontech). The secondary antibodies used were Alex 647 donkey anti-mouse (1:500; catalog no. A31571; ThermoFisher Scientific) and Alex 555 donkey anti-rabbit (1:500; catalog no. A31572; ThermoFisher Scientific).