ADP-ribosylation Factor Like 4aa(arl4aa)is a member of ADP ribosylation factor(ARF)family,a group of GTPases controlling membrane and intracellular proteins trafficking.Previous study showed that Arl4aa expression was up-regulated in zebrafish chordin mutant,in which hematopoiesis was expanded,suggesting a hitherto undescribed role in hematopoiesis.Whole mount in-situ hybridization(WISH)showed that zebrafish arl4aa was preferentially expressed in the ventral wall of dorsal aorta(VDA)at 24 and 36 hpf and caudal hematopoietic tissue at 48 hpf.Morpholino(MO)knockdown(KD)and transcription activator-like effector nuclease(TALEN)knockout(KO)of arl4aa significantly reduced c-myb and rag1 expression in VDA and thymus.The integrity of Golgi complex at the VDA was disrupted as shown by both transmission electron microscopy(TEM)and immunostaining of Golgi membrane giantin.The Golgi complex at the developing neural tube,where arl4aa was not intensely expressed,was unaffected.Disruption of Golgi complex was also seen in HeLa cells when human ARL4A was KD by shRNA.Mechanistically,arl4aa KD down-regulated notch signaling in VDA in a double transgenic line(tp1:GFP;kdrl:mcherry).Expression of notch target genes and NICD protein were also reduced.Induction of NICD expression at 14-15 somite stage in a double transgenic line(hsp70l:gal4;UAS:NICD-myc)successfully restored c-myb+HSC population along the DA in arl4aa morphant at 36 hpf.This study identified arl4aa as an important factor regulating initiation of definitive hematopoiesis by maintaining the integrity of Golgi complex hence maturation of notch receptor.