Zebrafish scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor) domain-containing protein 1) is involved in primitive hematopoiesis.
ABSTRACT: scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.
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:Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.
Project description:The hemangioblast is a multipotential progenitor, which is derived from the mesoderm and can further differentiate into hematopoietic and endothelial lineages. The molecular mechanism governing the specification of hemangioblasts is fundamental to regenerative medicine based on embryonic stem cells for the treatment of various hematologic and vascular diseases. Here we show that aggf1 acts at the top of the genetic regulatory hierarchy in the specification of hemangioblasts in zebrafish. Knockdown of aggf1 expression decreases expression of endothelial cell-specific markers (cdh5, admr) and disrupts primitive hematopoiesis as shown by a decreased number of erythroid cells and reduced expression of gata1 (marker for erythroid progenitors) and pu.1 (myeloid progenitors). Aggf1 knockdown also decreases expression of runx1 and c-myb, indicating that it is required for specification of hematopoietic stem cells (definitive hematopoiesis). Aggf1 knockdown led to dramatically reduced expression of hemangioblast markers fli1, etsrp, lmo2, and scl, and hematopoietic/endothelial defects in aggf1 morphants were rescued by messenger RNA for scl, fli-vp16, or etsrp. Taken together, these data indicate that aggf1 is involved in differentiation of both hematopoietic and endothelial lineages and that aggf1 acts upstream of scl, fli1, and etsrp in specification of hemangioblasts.
Project description:The bone morphogenetic protein (BMP) signaling pathway regulates multiple steps of hematopoiesis, mediated through receptor-regulated Smads, including Smad1 and Smad5. Here, we use loss-of-function approaches in zebrafish to compare the roles of Smad1 and Smad5 during embryonic hematopoiesis. We show that knockdown of Smad1 or Smad5 generates distinct and even opposite hematopoietic phenotypes. Embryos depleted for Smad1 have an increased number of primitive erythrocytes, but fail to produce mature embryonic macrophages. In contrast, Smad5-depleted embryos are defective in primitive erythropoiesis, yet have normal numbers of macrophages. Loss of either Smad1 or Smad5 causes a failure in the generation of definitive hematopoietic progenitors. To investigate the mechanism behind these phenotypes, we used rescue experiments and found that Smad5 is unable to rescue the Smad1 loss-of-function phenotype, indicating that the 2 highly related proteins have inherently distinct activities. Microarray experiments revealed that the 2 proteins redundantly regulate the key initiators of the hemato-vascular program, including scl, lmo2, and gfi1. However, each also regulates a remarkably distinct genetic program, with Smad5 uniquely regulating the BMP signaling pathway itself. Our results suggest that specificity of BMP signaling output, with respect to hematopoiesis, can be explained by differential functions of Smad1 and Smad5.
Project description:A genomic variant in the human ADTRP [androgen-dependent tissue factor (TF) pathway inhibitor (TFPI) regulating protein] gene increases the risk of coronary artery disease, the leading cause of death worldwide. TFPI is the TF pathway inhibitor that is involved in coagulation. Here, we report that adtrp and tfpi form a regulatory axis that specifies primitive myelopoiesis and definitive hematopoiesis, but not primitive erythropoiesis or vasculogenesis. In zebrafish, there are 2 paralogues for adtrp (i.e., adtrp1 and adtrp2). Knockdown of adtrp1 expression inhibits the specification of hemangioblasts, as shown by decreased expression of the hemangioblast markers, etsrp, fli1a, and scl; blocks primitive hematopoiesis, as shown by decreased expression of pu.1, mpo, and l-plastin; and disrupts the specification of hematopoietic stem cells (definitive hematopoiesis), as shown by decreased expression of runx1 and c-myb However, adtrp1 knockdown does not affect erythropoiesis during primitive hematopoiesis (no effect on gata1 or h-bae1) or vasculogenesis (no effect on kdrl, ephb2a, notch3, dab2, or flt4). Knockdown of adtrp2 expression does not have apparent effects on all markers tested. Knockdown of adtrp1 reduced the expression of tfpi, and hematopoietic defects in adtrp1 morphants were rescued by tfpi overexpression. These data suggest that the regulation of tfpi expression is one potential mechanism by which adtrp1 regulates primitive myelopoiesis and definitive hematopoiesis.-Wang, L., Wang, X., Wang, L., Yousaf, M., Li, J., Zuo, M., Yang, Z., Gou, D., Bao, B., Li, L., Xiang, N., Jia, H., Xu, C., Chen, Q., Wang, Q. K. Identification of a new adtrp1-tfpi regulatory axis for the specification of primitive myelopoiesis and definitive hematopoiesis.
Project description:Recent lineage studies suggest that hematopoietic stem cells (HSCs) may be derived from endothelial cells. However, the genetic hierarchy governing the emergence of HSCs remains elusive. We report here that zebrafish ets1-related protein (etsrp), which is essential for vascular endothelial development, also plays a critical role in the initiation of definitive hematopoiesis by controlling the expression of 2 stem cell leukemia (scl) isoforms (scl-alpha and scl-beta) in angioblasts. In etsrp morphants, which are deficient in endothelial and HSC development, scl-alpha alone partially rescues angioblast specification, arterial-venous differentiation, and the expression of HSC markers, runx1 and c-myb, whereas scl-beta requires angioblast rescue by fli1a to restore runx1 expression. Interestingly, when vascular endothelial growth factor (Vegf) signaling is inhibited, HSC marker expression can still be restored by scl-alpha in etsrp morphants, whereas the rescue of arterial ephrinb2a expression is blocked. Furthermore, both scl isoforms partially rescue runx1 but not ephrinb2a expression in embryos deficient in Vegf signaling. Our data suggest that downstream of etsrp, scl-alpha and fli1a specify the angioblasts, whereas scl-beta further initiates HSC specification from this angioblast population, and that Vegf signaling acts upstream of scl-beta during definitive hematopoiesis.
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:CUL4A and CUL4B are closely related members in Cullin family and can each assemble a Cullin-RING E3 ligase complex (Cullin-RING Ligase 4A or 4B, CRL4A, or CRL4B) and participate in a variety of biological processes. Previously we showed that zebrafish cul4a, but not cul4b, is essential for cardiac and pectoral fin development. Here, we have identified cul4a as a crucial regulator of primitive erythropoiesis in zebrafish embryonic development. Depletion of cul4a resulted in a striking reduction of erythroid cells due to the inhibition of erythroid differentiation. Transcript levels for early hematopoietic regulatory genes including scl, lmo2, and gata1 are significantly reduced in cul4a-deficient embryos. Mechanistically, we demonstrated that scl and gata1, the central regulators of primitive hematopoiesis for erythroid determination, are transcriptionally upregulated by cul4a. These findings demonstrate an important role for cul4a in primitive erythropoiesis and may bear implications in regeneration medicine of anemia and related diseases.
Project description:The stem-cell leukemia (SCL, also known as TAL1) gene encodes a basic helix-loop-helix transcription factor that is essential for the initiation of primitive and definitive hematopoiesis, erythrocyte and megakarocyte differentiation, angiogenesis, and astrocyte development. Here we report that the zebrafish produces, through an alternative promoter site, a novel truncated scl (tal1) isoform, scl-beta, which manifests a temporal and spatial expression distinct from the previously described full-length scl-alpha. Functional analysis reveals that while scl-alpha and -beta are redundant for the initiation of primitive hematopoiesis, these two isoforms exert distinct functions in the regulation of primitive erythroid differentiation and definitive hematopoietic stem cell specification. We further demonstrate that differences in the protein expression levels of scl-alpha and -beta, by regulating their protein stability, are likely to give rise to their distinct functions. Our findings suggest that hematopoietic cells at different levels of hierarchy are likely governed by a gradient of the Scl protein established through temporal and spatial patterns of expression of the different isoforms.
Project description:Hematopoiesis involves the production of stem cells, followed by the orchestrated differentiation of the blood lineages. Genetic screens in zebrafish have identified mutants with defects that disrupt specific stages of hematopoiesis and vasculogenesis, including the cloche, spadetail (tbx16), moonshine (tif1g), bloodless, and vlad tepes (gata1) mutants. To better characterize the blood program, gene expression profiling was carried out in these mutants and in scl-morphants (scl(mo)). Distinct gene clusters were demarcated by stage-specific and mutant-specific gene regulation. These were found to correlate with the transcriptional program of hematopoietic progenitor cells, as well as of the erythroid, myeloid, and vascular lineages. Among these, several novel hematopoietic and vascular genes were detected, for instance, the erythroid transcription factors znfl2 and ncoa4. A specific regulation was found for myeloid genes, as they were more strongly expressed in vlt mutants compared with other erythroid mutants. A unique gene expression pattern of up-regulated isoprenoid synthesis genes was found in cloche and scl(mo), possibly in migrating cells. In conjunction with the high conservation of vertebrate hematopoiesis, the comparison of transcriptional profiles in zebrafish blood mutants represents a versatile and powerful tool to elucidate the genetic regulation of blood and blood vessel development.