Retinoic acid signaling plays a restrictive role in zebrafish primitive myelopoiesis.
ABSTRACT: 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:Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.
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.
Project description:Over the past few years it has become clear that over half of the mammalian heart derives from outside the heart field as originally defined. Such a second heart field, however, has not been described in zebrafish, which could explain its smaller, two-chambered heart. Instead, zebrafish have a population of haemangioblasts, which is absent in mammalian embryos, raising the possibility that these cells represent the evolutionary ancestor of the second heart field. Here, we show for the first time that the genetic programmes of these anterior haemangioblasts and the adjacent heart field are co-regulated, by transcription factors previously associated with heart but not blood or endothelial development. We demonstrate that gata4, gata5 and gata6 are essential for anterior haemangioblast specification, and for subsequent myelopoiesis, acting as early as cloche and upstream of scl. The requirement for gata4, gata5 and gata6 in myeloid, endothelial and cardiac specification is in the mesoderm, but these factors also control, from within the endoderm and the yolk syncytial layer, the migration of the cardiac precursors as they differentiate. This genetic link between the blood/endothelial and cardiac programmes supports the notion that this haemangioblast population in zebrafish is an evolutionary antecedent of the second heart field, and has implications for the differentiation of haemangioblasts and cardiomyocytes from pluripotent cells, and for the origins of stem cells in the adult heart.
Project description:Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1(+) erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4(-/-) mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.
Project description:SCL/Tal-1 is a transcription factor necessary for hematopoietic stem cell differentiation. Although SCL is also expressed in endothelial and neural progenitors, SCL function in these cells remains unknown. In the zebrafish mutant cloche (clo), SCL expression is nearly abolished in hematopoietic and vascular tissues. Correspondingly, it was shown previously that clo fails to differentiate blood and angioblasts. Genetic analysis demonstrates that the clo mutation is not linked to the SCL locus. Forced expression of SCL in clo embryos rescues the blood and vascular defects, suggesting that SCL acts downstream of clo to specify hematopoietic and vascular differentiation.
Project description:Normal heart development requires appropriate levels of retinoic acid (RA) signaling. RA levels in embryos are dampened by Cyp26 enzymes, which metabolize RA into easily degraded derivatives. Loss of Cyp26 function in humans is associated with numerous developmental syndromes that include cardiovascular defects. Although previous studies have shown that Cyp26-deficient vertebrate models also have cardiovascular defects, the mechanisms underlying these defects are not understood. Here, we found that in zebrafish, two Cyp26 enzymes, Cyp26a1 and Cyp26c1, are expressed in the anterior lateral plate mesoderm (ALPM) and predominantly overlap with vascular progenitors (VPs). Although singular knockdown of Cyp26a1 or Cyp26c1 does not overtly affect cardiovascular development, double Cyp26a1 and Cyp26c1 (referred to here as Cyp26)-deficient embryos have increased atrial cells and reduced cranial vasculature cells. Examining the ALPM using lineage tracing indicated that in Cyp26-deficient embryos the myocardial progenitor field contains excess atrial progenitors and is shifted anteriorly into a region that normally solely gives rise to VPs. Although Cyp26 expression partially overlaps with VPs in the ALPM, we found that Cyp26 enzymes largely act cell non-autonomously to promote appropriate cardiovascular development. Our results suggest that localized expression of Cyp26 enzymes cell non-autonomously defines the boundaries between the cardiac and VP fields within the ALPM through regulating RA levels, which ensures a proper balance of myocardial and endothelial lineages. Our study provides novel insight into the earliest consequences of Cyp26 deficiency that underlie cardiovascular malformations in vertebrate embryos.
Project description:Hematopoietic and endothelial cells develop from a common progenitor, the hemangioblast, or directly from mesodermal cells. The molecular pathway that regulates the specification of both cell lineages remains elusive. Here, we show that a lysocardiolipin acyltransferase, lycat, is critical for the establishment of both hematopoietic and endothelial lineages. We isolated lycat from the deletion interval of cloche, a zebrafish mutant that has dramatically reduced hematopoietic and endothelial cell lineages. Reduction of lycat mRNA levels in wild-type zebrafish embryos decreases both endothelial and hematopoietic lineages. Lycat mRNA rescues blood lineages in zebrafish cloche mutant embryos. E165R and G166L mutations in the highly conserved catalytic domain in lycat abolish its function in zebrafish hematopoiesis. Epistasis analysis supports that lycat acts upstream of scl and etsrp in zebrafish hemangioblast development. These data indicate that lycat is the earliest known player in the generation of both endothelial and hematopoietic lineages.
Project description:Second heart field (SHF) progenitors perform essential functions during mammalian cardiogenesis. We recently identified a population of cardiac progenitor cells (CPCs) in zebrafish expressing latent TGF?-binding protein 3 (ltbp3) that exhibits several defining characteristics of the anterior SHF in mammals. However, ltbp3 transcripts are conspicuously absent in anterior lateral plate mesoderm (ALPM), where SHF progenitors are specified in higher vertebrates. Instead, ltbp3 expression initiates at the arterial pole of the developing heart tube. Because the mechanisms of cardiac development are conserved evolutionarily, we hypothesized that zebrafish SHF specification also occurs in the ALPM. To test this hypothesis, we Cre/loxP lineage traced gata4(+) and nkx2.5(+) ALPM populations predicted to contain SHF progenitors, based on evolutionary conservation of ALPM patterning. Traced cells were identified in SHF-derived distal ventricular myocardium and in three lineages in the outflow tract (OFT). We confirmed the extent of contributions made by ALPM nkx2.5(+) cells using Kaede photoconversion. Taken together, these data demonstrate that, as in higher vertebrates, zebrafish SHF progenitors are specified within the ALPM and express nkx2.5. Furthermore, we tested the hypothesis that Nkx2.5 plays a conserved and essential role during zebrafish SHF development. Embryos injected with an nkx2.5 morpholino exhibited SHF phenotypes caused by compromised progenitor cell proliferation. Co-injecting low doses of nkx2.5 and ltbp3 morpholinos revealed a genetic interaction between these factors. Taken together, our data highlight two conserved features of zebrafish SHF development, reveal a novel genetic relationship between nkx2.5 and ltbp3, and underscore the utility of this model organism for deciphering SHF biology.
Project description:A serine/threonine kinase, Melk, was initially cloned in mouse oocytes as a maternal gene, but whose function was unknown. In adult mice, Melk was strongly expressed in the thymus and bone marrow, suggesting a role for Melk in hematopoiesis. We cloned a Melk-like gene from zebra fish (zMelk). zMelk-like gene was expressed in the brain and lateral mesoderm at 12 hours postfertilization (hpf) and in several tissues of adult fish, including the kidney and spleen, both of which are known to be hematopoietic tissues in zebra fish. Abrogation of zMelk-like gene function by zMelk-like gene-specific Morpholino (MO) resulted in abnormal swelling around the tectum region. In addition, the start of blood circulation was severely delayed but, in contrast, the vessel formation seemed normal. Expression of scl, gata-1, and lmo-2 was down regulated at 12 to 14 hpf in the zMelk-like gene MO-injected embryos, and the coexpression of gata-1 rescued the anemic phenotype induced by zMelk-like gene MO. Expression of the zMelk-like gene in embryos enhanced gata-1 promoter-dependent enhanced green fluorescent protein expression, suggesting that the zMelk-like gene affects gata-1 expression at the transcriptional level. Taken together, our data suggest that the zMelk-like gene may play a role in primitive hematopoiesis by affecting the expression of genes critical for hematopoiesis.
Project description:The derivation of the primitive endoderm layer from the pluripotent cells of the inner cell mass is one of the earliest differentiation and morphogenic events in embryonic development. GATA4 and GATA6 are the key transcription factors in the formation of extraembryonic endoderms, but their specific contribution to the derivation of each endoderm lineage needs clarification. We further analyzed the dynamic expression and mutant phenotypes of GATA6 in early mouse embryos. GATA6 and GATA4 are both expressed in primitive endoderm cells initially. At embryonic day (E) 5.0, parietal endoderm cells continue to express both GATA4 and GATA6; however, visceral endoderm cells express GATA4 but exhibit a reduced expression of GATA6. By and after E5.5, visceral endoderm cells no longer express GATA6. We also found that GATA6 null embryos did not form a morphologically recognizable primitive endoderm layer, and subsequently failed to form visceral and parietal endoderms. Thus, the current study establishes that GATA6 is essential for the formation of primitive endoderm, at a much earlier stage then previously recognized, and expression of GATA6 discriminates parietal endoderm from visceral endoderm lineages.