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Maternal pak4 expression is required for primitive myelopoiesis in zebrafish.

ABSTRACT: Transcripts of pak4, the zebrafish ortholog of p21-activated kinase 4 (PAK4), are most abundant in the egg and fall to low levels by the end of gastrulation, after which expression is essentially ubiquitous. Translation of maternal mRNA into pak4 protein is first detectable at high stage (3.3hpf). Splice-blocking morpholino oligonucleotides (MOs) were used to prevent zygotic pak4 expression. This had no discernable effect on development through larval stages. In contrast, a translation-blocking MO, alone or in combination with the splice MOs, resulted in a complex lethal phenotype. In addition to disrupted somite development and other morphogenetic abnormalities, the knockdown of maternal pak4 expression led to alterations in regulatory gene expression in the primitive hematopoietic domains, leading to deficiencies in granulocyte and leukocyte lineages. At least some of the effects of pak4 knockdown on gene expression could be mimicked by treatment with actin depolymerization agents, suggesting a mechanistic link between regulation of microfilament dynamics by pak4 and regulation of gene expression in primitive myeloid cell differentiation.

SUBMITTER: Law SH

PROVIDER: S-EPMC4132851 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Maternal pak4 expression is required for primitive myelopoiesis in zebrafish.

Law Sheran H W SH Sargent Thomas D TD

Mechanisms of development 20120929 2-3

Transcripts of pak4, the zebrafish ortholog of p21-activated kinase 4 (PAK4), are most abundant in the egg and fall to low levels by the end of gastrulation, after which expression is essentially ubiquitous. Translation of maternal mRNA into pak4 protein is first detectable at high stage (3.3hpf). Splice-blocking morpholino oligonucleotides (MOs) were used to prevent zygotic pak4 expression. This had no discernable effect on development through larval stages. In contrast, a translation-blocking ...[more]

PMID: 23032194

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Project description: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 scl, lmo2, gata2 and etsrp 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 scl and lmo2 partially rescue the block of primitive myelopoiesis in the embryos treated with 250 nM RA during 10-11 hpf, suggesting RA acts upstream of scl to control primitive myelopoiesis. However, the RA treatment blocks the increased primitive myelopoiesis caused by overexpressing gata4/6 whereas the abolished primitive myelopoiesis in gata4/5/6 depleted embryos is well rescued by 4-diethylamino-benzaldehyde, a retinal dehydrogenase inhibitor, or partially rescued by knocking down aldh1a2, the major retinal dehydrogenase gene that is responsible for RA synthesis during early development. Consistently, overexpressing gata4/6 inhibits aldh1a2 expression whereas depleting gata4/5/6 increases aldh1a2 expression. The results reveal that RA signaling acts downstream of gata4/5/6 to control primitive myelopoiesis. But, 4-diethylamino-benzaldehyde fails to rescue the defective primitive myelopoiesis in either cloche embryos or lycat morphants. Taken together, our results demonstrate that RA signaling restricts zebrafish primitive myelopoiesis through acting downstream of gata4/5/6, upstream of, or parallel to, cloche, and upstream of scl.

Project description:MicroRNAs (miRNAs) play significant roles in both embryonic hematopoiesis and hematological malignancy. Zebrafish miR-462-731 cluster is orthologous of miR-191-425 in human which regulates proliferation and tumorigenesis. In our previous work, miR-462-731 was found highly and ubiquitously expressed during early embryogenesis. In this study, by loss-of-function analysis (morpholino knockdown combined with CRISRP/Cas9 knockout) and mRNA profiling, we suggest that miR-462-731 is required for normal embryonic development by regulating cell survival. We found that loss of miR-462/miR-731 caused a remarkable decrease in the number of erythroid cells as well as an ectopic myeloid cell expansion at 48 hpf, suggesting a skewing of myeloid-erythroid lineage differentiation. Mechanistically, miR-462-731 provides an instructive input for pu.1-dependent primitive myelopoiesis through regulating etsrp/scl signaling combined with a novel pu.1/miR-462-731 feedback loop. On the other hand, morpholino (MO) knockdown of miR-462/miR-731 resulted in an expansion of posterior blood islands at 24 hpf, which is a mild ventralization phenotype resulted from elevation of BMP signaling. Rescue experiments with both BMP type I receptor inhibitor dorsomorphin and alk8 MO indicate that miR-462-731 acts upstream of alk8 within the BMP/Smad signaling pathway and functions as a novel endogenous BMP antagonist. Besides, an impairment of angiogenesis was observed in miR-462/miR-731 morphants. The specification of arteries and veins was also perturbed, as characterized by the irregular patterning of efnb2a and flt4 expression. Our study unveils a previously unrecognized role of miR-462-731 in BMP/Smad signaling mediated hematopoietic specification of mesodermal progenitors and demonstrates a miR-462-731 mediated regulatory mechanism driving primitive myelopoiesis in the ALPM. We also show a requirement for miR-462-731 in regulating arterial-venous specification and definitive hematopoietic stem cell (HSC) production. The current findings might provide further insights into the molecular mechanistic basis of miRNA regulation of embryonic hematopoiesis and hematological malignancy.

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Maternal pak4 expression is required for primitive myelopoiesis in zebrafish.

Publications

Maternal pak4 expression is required for primitive myelopoiesis in zebrafish.

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