Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 2015-2030 ◽  
Author(s):  
Maggie L. Kalev-Zylinska ◽  
Julia A. Horsfield ◽  
Maria Vega C. Flores ◽  
John H. Postlethwait ◽  
Maria R. Vitas ◽  
...  
Keyword(s):  
Transient Expression ◽  
Normal Function ◽  
Model System ◽  
Zebrafish Embryos ◽  
Lateral Plate ◽  
Vessel Development ◽  
Morpholino Oligonucleotides ◽  
Antisense Morpholino Oligonucleotides ◽  
Definitive Hematopoiesis

RUNX1/AML1/CBFA2 is essential for definitive hematopoiesis, and chromosomal translocations affecting RUNX1 are frequently involved in human leukemias. Consequently, the normal function of RUNX1 and its involvement in leukemogenesis remain subject to intensive research. To further elucidate the role of RUNX1 in hematopoiesis, we cloned the zebrafish ortholog (runx1) and analyzed its function using this model system. Zebrafish runx1 is expressed in hematopoietic and neuronal cells during early embryogenesis. runx1 expression in the lateral plate mesoderm co-localizes with the hematopoietic transcription factor scl, and expression of runx1 is markedly reduced in the zebrafish mutants spadetail and cloche. Transient expression of runx1 in cloche embryos resulted in partial rescue of the hematopoietic defect. Depletion of Runx1 with antisense morpholino oligonucleotides abrogated the development of both blood and vessels, as demonstrated by loss of circulation, incomplete development of vasculature and the accumulation of immature hematopoietic precursors. The block in definitive hematopoiesis is similar to that observed in Runx1 knockout mice, implying that zebrafish Runx1 has a function equivalent to that in mammals. Our data suggest that zebrafish Runx1 functions in both blood and vessel development at the hemangioblast level, and contributes to both primitive and definitive hematopoiesis. Depletion of Runx1 also caused aberrant axonogenesis and abnormal distribution of Rohon-Beard cells, providing the first functional evidence of a role for vertebrate Runx1 in neuropoiesis.To provide a base for examining the role of Runx1 in leukemogenesis, we investigated the effects of transient expression of a human RUNX1-CBF2T1 transgene [product of the t(8;21) translocation in acute myeloid leukemia] in zebrafish embryos. Expression of RUNX1-CBF2T1 caused disruption of normal hematopoiesis, aberrant circulation, internal hemorrhages and cellular dysplasia. These defects reproduce those observed in Runx1-depleted zebrafish embryos and RUNX1-CBF2T1 knock-in mice. The phenotype obtained with transient expression of RUNX1-CBF2T1 validates the zebrafish as a model system to study t(8;21)-mediated leukemogenesis.

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

Blood ◽  
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.

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

Blood ◽  
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.

scholarly journals Serotonin is required for pharyngeal arch morphogenesis in zebrafish

2014 ◽  
Author(s):  
Katarina Kotnik Halavaty ◽  
Michael Bader ◽  
Saleh Bashammakh ◽  
Salim Seyfried
Keyword(s):  
Neural Crest ◽  
Tryptophan Hydroxylase ◽  
Expression Patterns ◽  
Pharyngeal Arch ◽  
Pharyngeal Arches ◽  
Early Markers ◽  
Morpholino Oligonucleotides ◽  
Antisense Morpholino Oligonucleotides ◽  
Induced Defects

Serotonin (5-HT) is not only a neurotransmitter but also a mediator of developmental processes in vertebrates. In this study, we analyzed the importance of 5-HT during zebrafish development. The expression patterns of three zebrafish tryptophan hydroxylase isoforms (Tph1A, Tph1B, Tph2), the rate-limiting enzymes in 5-HT synthesis, were analyzed and compared to the appearance and distribution of 5-HT. 5-HT was found in the raphe nuclei correlating with tph2 expression and in the pineal gland correlating with tph1a and tph2 expression. tph2 deficient fish generated with antisense morpholino oligonucleotides exhibited morphogenesis defects during pharyngeal arch development. The correct specification of neural crest cells was not affected in tph2 morphants as shown by the expression of early markers, but the survival and differentiation of pharyngeal arch progenitor cells were impaired. An organizing role of 5-HT in pharyngeal arch morphogenesis was suggested by a highly regular pattern of 5-HT positive cells in this tissue. Moreover, the 5-HT2B receptor was expressed in the pharyngeal arches and its pharmacological inhibition also induced defects in pharyngeal arch morphogenesis. These results support an important role of Tph2-derived serotonin as a morphogenetic factor in the development of neural crest derived tissues.

scholarly journals Dpysl2 (CRMP2) is required for the migration of facial branchiomotor neurons in the developing zebrafish embryo

2020 ◽  
Vol 64 (10-11-12) ◽  
pp. 479-484
Author(s):  
Carolina Fiallos-Oliveros ◽  
Toshio Ohshima
Keyword(s):  
Motor Neurons ◽  
System Development ◽  
Nervous System Development ◽  
Knock Out ◽  
Facial Branchiomotor Neurons ◽  
Morpholino Oligonucleotides ◽  
Mutant Phenotypes ◽  
Antisense Morpholino Oligonucleotides

Dihydropyrimidinase-like family proteins (Dpysls) are relevant in several processes during nervous system development; among others, they are involved in axonal growth and cell migration. Dpysl2 (CRMP2) is the most studied member of this family; however, its role in vivo is still being investigated. Our previous studies in zebrafish showed the requirement of Dpysl2 for the proper positioning of caudal primary motor neurons and Rohon-Beard neurons in the spinal cord.In the present study, we show that Dpysl2 is necessary for the proper migration of facial branchiomotor neurons during early development in zebrafish. We generated a dpysl2 knock-out (KO) zebrafish mutant line and used different types of antisense morpholino oligonucleotides (AMO) to analyze the role of Dpysl2 in this process. Both dpysl2 KO mutants and morphants exhibited abnormalities in the migration of these neurons from rhombomers (r) 4 and 5 to 6 and 7. The facial branchiomotor neurons that were expected to be at r6 were still located at r4 and r5 hours after the migration process should have been completed. In addition, mutant phenotypes were rescued by injecting dpysl2 mRNA into the KO embryos. These results indicate that Dpysl2 is involved in the proper migration of facial branchiomotor neurons in developing zebrafish embryos.

scholarly journals Knockdown of Laminin gamma-3 (Lamc3) impairs motoneuron guidance in the zebrafish embryo

2017 ◽  
Vol 2 ◽  
pp. 111
Author(s):  
Alexander M. J. Eve ◽  
James C. Smith
Keyword(s):  
Endothelial Cells ◽  
Zebrafish Embryo ◽  
Axonal Guidance ◽  
Loss Of Function ◽  
Cardinal Vein ◽  
Morpholino Oligonucleotides ◽  
Antisense Morpholino Oligonucleotides ◽  
Antisense Morpholino

Background: Previous work in the zebrafish embryo has shown that laminin γ-3 (lamc3) is enriched in endothelial cells marked by expression of fli1a, but the role of Lamc3 has been unknown. Methods: We use antisense morpholino oligonucleotides, and CRISPR/Cas9 mutagenesis of F0 embryos, to create zebrafish embryos in which lamc3 expression is compromised. Transgenic imaging, immunofluorescence, and in situ hybridisation reveal that Lamc3 loss-of-function affects the development of muscle pioneers, endothelial cells, and motoneurons. Results: Lamc3 is enriched in endothelial cells during zebrafish development, but it is also expressed by other tissues. Depletion of Lamc3 by use of antisense morpholino oligonucleotides perturbs formation of the parachordal chain and subsequently the thoracic duct, but Lamc3 is not required for sprouting of the cardinal vein. F0 embryos in which lamc3 expression is perturbed by a CRISPR/Cas9 approach also fail to form a parachordal chain, but we were unable to establish a stable lamc3 null line. Lamc3 is dispensable for muscle pioneer specification and for the expression of netrin-1a in these cells. Lamc3 knockdown causes netrin-1a up-regulation in the neural tube and there is increased Netrin-1 protein throughout the trunk of the embryo. Axonal guidance of rostral primary motoneurons is defective in Lamc3 knockdown embryos. Conclusions: We suggest that knockdown of Lamc3 perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance.

XMeis3 protein activity is required for proper hindbrain patterning in Xenopus laevis embryos

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3415-3426 ◽  
Author(s):  
Charna Dibner ◽  
Sarah Elias ◽  
Dale Frank
Keyword(s):  
Spinal Cord ◽  
Fusion Protein ◽  
Cell Fate ◽  
Ectopic Expression ◽  
Neural Patterning ◽  
Protein Activity ◽  
Neural Markers ◽  
Morpholino Oligonucleotides ◽  
Antisense Morpholino Oligonucleotides

Meis-family homeobox proteins have been shown to regulate cell fate specification in vertebrate and invertebrate embryos. Ectopic expression of RNA encoding the Xenopus Meis3 (XMeis3) protein caused anterior neural truncations with a concomitant expansion of hindbrain and spinal cord markers in Xenopus embryos. In naïve animal cap explants, XMeis3 activated expression of posterior neural markers in the absence of pan-neural markers. Supporting its role as a neural caudalizer, XMeis3 is expressed in the hindbrain and spinal cord. We show that XMeis3 acts like a transcriptional activator, and its caudalizing effects can be mimicked by injecting RNA encoding a VP16-XMeis3 fusion protein. To address the role of endogenous XMeis3 protein in neural patterning, XMeis3 activity was antagonized by injecting RNA encoding an Engrailed-XMeis3 antimorph fusion protein or XMeis3 antisense morpholino oligonucleotides. In these embryos, anterior neural structures were expanded and posterior neural tissues from the midbrain-hindbrain junction through the hindbrain were perturbed. In neuralized animal cap explants, XMeis3-antimorph protein modified caudalization by basic fibroblast growth factor and Wnt3a. XMeis3-antimorph protein did not inhibit caudalization per se, but re-directed posterior neural marker expression to more anterior levels; it reduced expression of spinal cord and hindbrain markers, yet increased expression of the more rostral En2 marker. These results provide evidence that XMeis3 protein in the hindbrain is required to modify anterior neural-inducing activity, thus, enabling the transformation of these cells to posterior fates.

scholarly journals Pathology as a phenomenological tool

2021 ◽  
Author(s):  
Havi Carel
Keyword(s):  
Normal Function ◽  
Phenomenological Method ◽  
Experience Of Illness ◽  
Passive Object

AbstractThe phenomenological method (or rather, methods) has been fruitfully used to study the experience of illness in recent years. However, the role of illness is not merely that of a passive object for phenomenological scrutiny. I propose that illness, and pathology more generally, can be developed into a phenomenological method in their own right. I claim that studying cases of pathology, breakdown, and illness offer illumination not only of these experiences, but also of normal function and the tacit background that underpins it. In particular, I claim that the study of embodiment can be greatly enhanced, and indeed would be incomplete, without attending to bodily breakdown and what I term bodily doubt. I offer an analogy between illness and Husserl’s epoché, suggesting that both are a source of distancing, and therefore motivate a reflective stance.

scholarly journals Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

1998 ◽  
Vol 9 (7) ◽  
pp. 1803-1816 ◽  
Author(s):  
Michael C. Brown ◽  
Joseph A. Perrotta ◽  
Christopher E. Turner
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Binding Sites ◽  
Protein Localization ◽  
Model System ◽  
Lim Domain ◽  
Amino Terminal ◽  
Lim Domains ◽  
Inside Out

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.

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