D alanine d ala

To decrease H₂O₂ levels, cells were treated with extracellular Catalase (CAT_ext; Sigma-Aldrich, #C1345, 400 U/mL). To increase H₂O₂ levels, cells expressing D-amino acid oxidase (DAO) were treated with 10 mM D-alanine (D-Ala; Sigma-Aldrich, #A7377) before the internalization or secretion assays were performed. To inhibit NOX activity, cells were pre-treated for 1 h with 10 μM VAS-2870 (NOX-i; #BML-El395-0010, Enzo Life Sciences, Inc.; Farmingdale, NY, USA) or an equivalent amount of DMSO as a control. To inhibit Rac1, cells were pre-treated for 6 h with 20 μM NSC23766, a Rac1-inhibitor (Rac1-i; No2161, Tocris). To inhibit Dock release from Elmo, cells were pre-treated for 6 h with 100 μM CPYPP (DOCK-i; No4568, Tocris). To inhibit Shh signaling, cells were pretreated for 24 h with 10 μM cyclopamine (Shh-i; #239803, Millipore).

D-alanine (D-ala) and L-alanine (L-ala) were purchased from Sigma-Aldrich. 55 mM D-ala or L-ala were used in the drinking water for the mice in the in vivo experiments. The drinking water was exchanged every second day.

Zebrafish were maintained and staged, as previously described [41 (link)]. Experiments were performed using the standard AB wild-type strain. The embryos were incubated at 28 °C. Developmental stages were determined and indicated as hours post fertilization (hpf). The animal facility obtained permission from the French Ministry of Agriculture for all the experiments described in this manuscript (agreement No. C 75-05-12). To decrease H₂O₂ levels, embryos were incubated in VAS-2870 (NADPH oxidase inhibitor; NOX-i) (100 nM) from Enzo Life Sciences (#BML-El395-0010, Enzo Life Sciences, Inc.; Farmingdale, NY, USA) or an equivalent amount of DMSO as a control for the duration of the time-lapse analysis. To enhance H₂O₂ levels, D-Alanine (D-Ala, Sigma-Aldrich, St. Louis, MO, USA #A7377) (10 mM) was injected in zebrafish ventricles one hour prior to H₂O₂ levels or filopodia analysis.