Retinoic Acid Signaling Plays a Restrictive Role in Zebrafish Primitive Myelopoiesis

<div><p>Retinoic acid (RA) is known to regulate definitive myelopoiesis but its role in vertebrate primitive myelopoiesis remains unclear. Here we report that zebrafish primitive myelopoiesis is restricted by RA in a dose dependent manner mainly before 11 hpf (hours post fertilization) when anterior hemangioblasts are initiated to form. RA treatment significantly reduces expressions of anterior hemangioblast markers <em>scl</em>, <em>lmo2</em>, <em>gata2</em> and <em>etsrp</em> in the rostral end of ALPM (anterior lateral plate mesoderm) of the embryos. The result indicates that RA restricts primitive myelopoiesis by suppressing formation of anterior hemangioblasts. Analyses of ALPM formation suggest that the defective primitive myelopoiesis resulting from RA treatment before late gastrulation may be secondary to global loss of cells for ALPM fate whereas the developmental defect resulting from RA treatment during 10–11 hpf should be due to ALPM patterning shift. Overexpressions of <em>scl</em> and <em>lmo2</em> partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10–11 hpf, suggesting RA acts upstream of <em>scl</em> to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing <em>gata4/6</em> whereas the abolished primitive myelopoiesis in <em>gata4/5/6</em> depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down <em>aldh1a2</em>, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing <em>gata4/6</em> inhibits <em>aldh1a2</em> expression whereas depleting <em>gata4/5/6</em> increases <em>aldh1a2</em> expression. The results reveal that RA signaling acts downstream of <em>gata4/5/6</em> to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either <em>cloche</em> embryos or <em>lycat</em> morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of <em>gata4/5/6</em>, upstream of, or parallel to, <em>cloche</em>, and upstream of <em>scl</em>.</p>