A novel role of ADP-ribosylation factor like 4aa (Arl4aa) in zebrafish hematopoiesis

Abstract

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 based on gene expression profiling showed that the expression of arl4ab, the isoform arl4aain zebrafish was up-regulated in a chordin mutant in which the hematopoiesis was expanded during embryonic development, suggesting a previously undescribed role of arl4ain hematopoiesis. Whole mount in-situ hybridization (WISH) showed that zebrafish arl4aawas preferentially expressed in thedorsal aorta (DA) at 24 and 36 hpf, especially the ventral wall of dorsal aorta (VDA) where the hematopoietic stem cell (HSC) arises. At 48hpf, arl4aawas expressed inthe caudal hematopoietic tissue (CHT) in which HSC expands. Morpholino (MO) knockdown and transcription activator-like effector nuclease (TALEN) knockout of arl4aasignificantly reduced expression of c-myb(definitive HSC marker) in VDA and CHT as well that of rag1 (a marker of T-lymphoid development that arise from definitive HSC)in thymus. Endothelial-to-hematopoietic transition (EHT) was significantly impaired, as shown by reduced number of cells co-expressing c-myband kdrl in a double transgenic line (c-myb:GFP; kdrl:mCherry). TALENed F2 embryos also showed significant reduction in c-myb and rag1expression. Golgi complex integrity was disrupted in the VDA of both arl4aaknockout F0 and morphant embryos as shown by transmission electron microscopy(TEM) and immunostaining of Golgi membrane giantin. Similar observations were identified in HeLa cells where human ARL4Awas knocked down by shRNA. Mechanistically, arl4aaknockdown significantly reduced expression of Notch target genes as well as protein expression of NICD. Decrease in Notch signaling in VDA of arl4aamorphant was shown by a Notch reporter transgenic line. Effects of arl4aaknockdown on definitive hematopoiesis could be restored by inducible expression of NICD, providing a mechanistic link between arl4aa, Golgi complex integrity, Notch receptor processing and signaling during developmental hematopoiesis in zebrafish embryos. Furthermore, knockdown and knockout of arl4aainduced significant increase in gene expression associated with primitive macrophages (mpeg1and l-plastin) but not other myeloid lineage markers (pu1and mpo), erythropoiesis (gata1andɑ-hemoglobin), and primitive progenitors (scland lmo2) in MO-injected morphant and TALEN F0 mutants. The mechanistic link between arl4aaand macrophage development would have to be further examined.

This study identified arl4aaas an important factor in the initiation of definitive hematopoiesis by maintaining Golgi complex integrity and Notch receptor signaling.