The Zebrafish Homologue of the Murine Ecotropic Viral Integration Site-1 (. Evi-1) gene Regulates Zebrafish Embryonic Blood Development.

Abstract Abstract 1461 Poster Board I-484 The ecotropic viral integration site-1 (Evi-1) locus was originally identified as a common site of retroviral integration in murine myeloid tumors and was later shown to be one of the most potent oncogenenes associated with murine and human myeloid leukemia. More recent data suggest involvement of Evi-1 in embryonic hematopoiesis (Goyama et al, Cell Stem Cell 2008; Yuasa et al, EMBO J, 2005), yet the precise role and molecular regulation of Evi-1 during blood development remains poorly understood. The zebrafish model offers powerful tools for genetic and embryonic studies. Here, we study zebrafish embryonic development and human pluripotent stem cells to understand how evi-1 modulates early hematopoietic development. Loss-of-function studies were performed in vivo by injecting Morpholino oligonucleotides in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. To control for off-target effects, two separate morpholinos were designed and injected. N=100 zebrafish were analysed pro experiment in each group. Inhibition of evi-1 was confirmed by quantitative PCR comparison in morpholino-injected and control embryos. Hematopoietic development was followed in both morphants and wild-type embryos by simple microscopy and in situ hybridizations using known hematopoeitic markers in order to investigate the developmental time-point in which evi-1 regulates blood development. evi-1 morpholino injected zebrafisch embryo showed severely reduced numbers of circulating blood cells, consistent with the phenotype observed in Evi-1−/− mice. Additionally, hemorrhages were observed, suggesting concomittant defects of the endothelial lineage in evi-1 deficient fish. In situ hybridization analysis on 11-12 somite stage embryos revealed strong reduction of myeloid embryonic hematopoiesis (measured by pu.1 expression in the anterior lateral plate mesoderm), while no change was observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Taken together, our studies demonstrate a strong impact of evi-1 on zebrafish blood development, confirming the results from Evi-1−/− mice. As gata1 expression and therefore erythroid precursor cells in the posterior blood islands are unaffected in evi-1 morphants, our results support the hypothesis that the reduction of primitive yolk-sac erythrocytes in mutant mice was caused from hemorrhages from pericardial effusions. Since erythroid and myeloid cells derive from a common precursor, but gata1 expression was unaffected in knock-down embryos, we anticipate that evi-1 plays a specific role in the myeloid lineage, as shown by abolished pu.1 expression in the anterior LPM. evi-1 therefore probably affects differentiation, survival or proliferation of myeloid cells. Previous reports in adult hematopoietic cells show that evi-1 can interact with both gata1 and pu.1. However, our data suggest that this is not the case during embryonic development, since gata1 expression remained unaltered in morpholino-injected embryos. Furthermore, data in mice suggest that Evi-1 may modulate embryonic hematopoiesis by affecting hematopoietic stem cell proliferation through regulation of Gata2. Currently ongoing experiments in our laboratories focus on characterization of genetic interactions between evi-1, gata2 and pu.1 during zebrafish blood development. Amongst other, gata2 and respectively pu.1 mRNA are co-injected in evi-1 morphants to analyse whether they can rescue the blood phenotype. Moreover, selected findings in zebrafish embryonic development will be verified in the human using using in vitro differentiating human induced pluripotent stem (iPS) cells. First expression data generated by real-time PCR analysis showed differential expression of EVI-1 in embryoid bodies generated from human iPS cells, confirming our hypothesis that EVI-1 has specific effects during human blood development. Disclosures No relevant conflicts of interest to declare.

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FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors

Stem Cell Reports ◽

10.1016/j.stemcr.2017.11.008 ◽

2018 ◽

Vol 10 (1) ◽

pp. 87-100 ◽

Cited By ~ 10

Author(s):

Charles Yoon ◽

Hannah Song ◽

Ting Yin ◽

Damaris Bausch-Fluck ◽

Andreas P. Frei ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Lateral Plate Mesoderm ◽

Lateral Plate ◽

Cardiac Progenitors

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Ecotropic viral integration site 1, stem cell self-renewal and leukemogenesis

Cancer Science ◽

10.1111/j.1349-7006.2012.02303.x ◽

2012 ◽

Vol 103 (8) ◽

pp. 1371-1377 ◽

Cited By ~ 29

Author(s):

Keisuke Kataoka ◽

Mineo Kurokawa

Keyword(s):

Stem Cell ◽

Integration Site ◽

Viral Integration ◽

Self Renewal

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Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v110.11.1243.1243 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1243-1243

Author(s):

Michal A. Levine ◽

Christopher Roxbury ◽

Elias T. Zambidis

Keyword(s):

Stem Cell ◽

Embryonic Development ◽

Transient Expression ◽

Embryoid Body ◽

Hematopoietic Progenitors ◽

Hematopoietic Development ◽

Rna Molecules ◽

Over Expression ◽

Definitive Hematopoiesis

Abstract Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd-6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

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Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v110.11.3745.5.3745.5 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3745.5-3745.5

Author(s):

Michal A. Levine ◽

Christopher Roxbury ◽

Elias T. Zambidis

Keyword(s):

Stem Cell ◽

Embryonic Development ◽

Transient Expression ◽

Embryoid Body ◽

Hematopoietic Progenitors ◽

Hematopoietic Development ◽

Rna Molecules ◽

Over Expression ◽

Definitive Hematopoiesis

Abstract Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd–6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

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Gdf3 is required for robust Nodal signaling during germ layer formation and left-right patterning

eLife ◽

10.7554/elife.28635 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 25

Author(s):

Jose L Pelliccia ◽

Granton A Jindal ◽

Rebecca D Burdine

Keyword(s):

Embryonic Development ◽

Nodal Signaling ◽

Lateral Plate Mesoderm ◽

Embryonic Patterning ◽

Lateral Plate ◽

Mesoderm Formation ◽

Germ Layer Formation ◽

Multiple Stages ◽

Tgfβ Superfamily

Vertebrate embryonic patterning depends on signaling from Nodal, a TGFβ superfamily member. There are three Nodal orthologs in zebrafish; southpaw directs left-right asymmetries, while squint and cyclops function earlier to pattern mesendoderm. TGFβ member Vg1 is implicated in mesoderm formation but the role of the zebrafish ortholog, Growth differentiation factor 3 (Gdf3), has not been fully explored. We show that zygotic expression of gdf3 is dispensable for embryonic development, while maternally deposited gdf3 is required for mesendoderm formation and dorsal-ventral patterning. We further show that Gdf3 can affect left-right patterning at multiple stages, including proper development of regional cell morphology in Kupffer’s vesicle and the establishment of southpaw expression in the lateral plate mesoderm. Collectively, our data indicate that gdf3 is critical for robust Nodal signaling at multiple stages in zebrafish embryonic development.

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Dermomyotome-derived endothelial cells migrate to the dorsal aorta to support hematopoietic stem cell emergence

10.1101/2020.05.14.096305 ◽

2020 ◽

Cited By ~ 1

Author(s):

Pankaj Sahai-Hernandez ◽

Claire Pouget ◽

Ondřej Svoboda ◽

David Traver

Keyword(s):

Endothelial Cells ◽

Stem Cell ◽

Paraxial Mesoderm ◽

Dorsal Aorta ◽

Similar Process ◽

Lateral Plate Mesoderm ◽

Lateral Plate ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Endothelial Precursors

AbstractDevelopment of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Earlier studies in the chick showed that paraxial mesoderm generates another subset of endothelial cells that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. Here we show that a similar process occurs in the zebrafish, where a population of endothelial precursors delaminates from the somitic dermomyotome to incorporate exclusively into the developing dorsal aorta. Whereas somite-derived endothelial cells (SDECs) lack hematopoietic potential, they act as local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of endothelial cells (ECs) contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

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Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis

Development ◽

10.1242/dev.122.5.1363 ◽

1996 ◽

Vol 122 (5) ◽

pp. 1363-1371 ◽

Cited By ~ 17

Author(s):

L. Pardanaud ◽

D. Luton ◽

M. Prigent ◽

L.M. Bourcheix ◽

M. Catala ◽

...

Keyword(s):

Endothelial Cells ◽

Body Wall ◽

The Body ◽

Paraxial Mesoderm ◽

Lateral Plate Mesoderm ◽

Hemopoietic Precursors ◽

Lateral Plate ◽

Visceral Organs ◽

Endothelial Precursors

We have shown previously by means of quail/chick transplantations that external and visceral organs, i.e., somatopleural and splanchnopleural derivatives, acquire their endothelial network through different mechanisms, namely immigration (termed angiogenesis) versus in situ emergence of precursors (or vasculogenesis). We have traced the distribution of QH1-positive cells in chick hosts after replacement of the last somites by quail somites (orthotopic grafts) or lateral plate mesoderm (heterotopic grafts). The results lead to the conclusion that the embryo becomes vascularized by endothelial precursors from two distinct regions, splanchnopleural mesoderm and paraxial mesoderm. The territories respectively vascularized are complementary, precursors from the paraxial mesoderm occupy the body wall and kidney, i.e., they settle along with the other paraxial mesoderm derivatives and colonize the somatopleure. The precursors from the two origins have distinct recognition and potentialities properties: endothelial precursors of paraxial origin are barred from vascularizing visceral organs and from integrating into the floor of the aorta, and are never associated with hemopoiesis; splanchnopleural mesoderm grafted in the place of somites, gives off endothelial cells to body wall and kidney but also visceral organs. It gives rise to hemopoietic precursors in addition to endothelial cells.

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EVI1 Is Activated During the Generation of Induced Pluripotent Stem (iPS) Cells.

Blood ◽

10.1182/blood.v114.22.5048.5048 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5048-5048

Author(s):

Leopoldo Laricchia-Robbio ◽

Nuria Montserrat ◽

Alessandra Giorgetti ◽

Juan Carlos Izpisúa Belmonte

Keyword(s):

Stem Cell ◽

Conflicts Of Interest ◽

Integration Site ◽

Ips Cells ◽

The Self ◽

Genomic Locus ◽

Self Renewal ◽

Hematopoietic Stem ◽

Retroviral Integration ◽

Induced Pluripotent

Abstract Abstract 5048 EVI1 gene was first identified as a common site of retroviral integration in murine leukemia models. This gene is part of a complex genomic locus, MDS1-EVI1, that has been described as a target for retroviral integration that may lead to the emergence of a non-malignant dominant hematopoietic stem cell (HSC) clone in mice, in primates, and in humans. These studies suggested that one of the genes encoded by this locus could affect the self-renewal potential of HSC. Recent studies in mice revealed that indeed EVI1 plays an essential role in cell proliferation and it also enhances the self-renewal ability of HSC. The intense attention focused on the MDS1-EVI1 locus as retrovirus integration site prompted us to investigate whether EVI1 might have a role in somatic cell re-programming generated with retroviruses. Recent developments in stem cell research have enabled the re-programming of somatic cells to a pluripotent state using exogenous factors. Induced pluripotent stem (iPS) cells have the potential to differentiate into any cells types and that might be used in the future for clinical therapy. In order to elucidate the molecular events allowing the conversion of adult somatic into pluripotent stem cell, we evaluated EVI1 expression during this process. We found that EVI1 is activated in the early stages of re-programming and then it is silenced once the cells has been fully re-programmed. EVI1 seems to facilitate the initiation of cell re-programming by up-regulating a subset of genes previously described as potent stimulators of stem cells expansion. Disclosures No relevant conflicts of interest to declare.

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