Evi-1 expression in Xenopus

doi:10.1016/j.modgep.2005.03.007

Gene Expression Patterns

Volume 5, Issue 5, June 2005, Pages 601-608

https://doi.org/10.1016/j.modgep.2005.03.007 Get rights and content

Abstract

The Evi-1 gene was first identified as a site for viral integration in murine myeloid leukemia. Evi-1 is a zinc finger transcription factor that has been implicated in the development of myeloid neoplasia. In humans, disruption of the Evi-1 locus, by chromosomal rearrangements, is associated with myeloid leukemia and myelodyplastic syndromes. Here, we report the cloning and developmental pattern of expression of Xenopus Evi-1. xEvi-1 is expressed during oogenesis and during embryonic development. In situ hydridization reveals that xEvi-1 has a dynamic expression profile during early embryonic development. Expression of Evi-1 is detected by in situ hybridization in the pronephric tissue, the brain and in neural crest derivatives of the head and neck.

Section snippets

Molecular cloning of Xenopus Evi1

A full-length cDNA was isolated from a Xenopus oocyte lambda phage library using a murine c-Evi-1 cDNA probe. The xEvi-1 cDNA was 4208 bp, with 235 bp of 5′-noncoding and 808 bp of 3′-noncoding sequence. The predicted amino acid sequence of the single open reading frame was 1051 amino acids in length and encodes a protein with a calculated molecular mass of 119 kD. Comparison of the human, mouse and Xenopus Evi-1 proteins indicates extensive amino acid identity (Fig. 1). Xenopus Evi-1 shares 76.6%

Oocytes and embryos

Ovaries were manually dissected and treated with collagenase type I (2 mg/ml) in modified Barth solution (MBS) for 15 h. Oocytes (from I to VI) were staged according to Dumont (Dumont, 1972). Stage VI oocytes were matured in vitro by treatment with 5 μg/ml progesterone for 17 h. Albino embryos for in situ hybridization were fertilized in vitro and staged according to Nieuwkoop and Faber (1967)).

cDNA cloning

A Xenopus oocyte cDNA library was screened using the first DNA binding domain of murine Evi-1 (700 bp Sph1

Acknowledgements

We thank Doug Melton for the gift of the Xenopus oocyte λ phage library. James Ihle is an Investigator with the HHMI. We thank Drs Joanne Doherty and Clair Kelley for careful review of this manuscript. This works was supported by the American Lebanese Syrian Associated Charities (ALSAC) and by grants from the NIH (5RO1 DK42932).

References (14)

L.C. Bradley et al.
The structure and expression of the Xenopus Krox-20 gene: conserved and divergent patterns of expression in rhombomeres and neural crest
Mech. Dev.
(1993)
T.J. Carroll et al.
Synergism between Pax-8 and lim-1 in embryonic kidney development
Dev. Biol.
(1999)
R.M. Harland
In situ hybridization: an improved whole-mount method for Xenopus embryos
Methods Cell Biol.
(1991)
P.R. Hoyt et al.
The Evi1 proto-oncogene is required at midgestation for neural, heart, and paraxial mesenchyme development
Mech. Dev.
(1997)
K. Morishita et al.
Retroviral activation of a novel gene encoding a zinc finger protein in IL-3-dependent myeloid leukemia cell lines
Cell
(1988)
B. Sadaghiani et al.
Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy
Dev. Biol.
(1987)
A.H. Brivanlou et al.
Expression of an engrailed-related protein is induced in the anterior neural ectoderm of early Xenopus embryos
Development
(1989)

There are more references available in the full text version of this article.

Cited by (13)

MECOM-associated syndrome: A heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia
2018, Blood Advances
Citation Excerpt :
The MECOM locus encodes a number of differentially spliced transcripts yielding the MDS1, MDS1-EVI1, and EVI1 protein isoforms. From different animal models, it has become obvious that the physiological role of the gene lies in the regulation of embryonic development and regulation of hematopoietic stem-cell renewal.24-29 Spatiotemporal expression patterns were observed during mouse embryonic development, with expression in the developing lung, heart, urogenital system, and emerging limb buds.30
Heterozygous mutations in MECOM (MDS1 and EVI1 complex locus) have been reported to be causative of a rare association of congenital amegakaryocytic thrombocytopenia and radioulnar synostosis. Here we report on 12 patients with congenital hypomegakaryocytic thrombocytopenia caused by MECOM mutations (including 10 novel mutations). The mutations affected different functional domains of the EVI1 protein. The spectrum of phenotypes was much broader than initially reported for the first 3 patients; we found familial as well as sporadic cases, and the clinical spectrum ranged from isolated radioulnar synostosis with no or mild hematological involvement to severe bone marrow failure without obvious skeletal abnormality. The clinical picture included radioulnar synostosis, bone marrow failure, clinodactyly, cardiac and renal malformations, B-cell deficiency, and presenile hearing loss. No single clinical manifestation was detected in all patients affected by MECOM mutations. Radioulnar synostosis and B-cell deficiency were observed only in patients with mutations affecting a short region in the C-terminal zinc finger domain of EVI1. We propose the term MECOM-associated syndrome for this heterogeneous hereditary disease and inclusion of MECOM sequencing in the diagnostic workup of congenital bone marrow failure.
Zebrafish nephrogenesis is regulated by interactions between retinoic acid, mecom, and notch signaling
2014, Developmental Biology
Citation Excerpt :
More recent work has demonstrated that Mecom is required for long-term hematopoietic stem cell maintenance (Zhang, et al., 2011). With respect to vertebrate kidney development, transcripts encoding Mecom have been detected in the pronephros distal tubule and duct in Xenopus (Mead, et al., 2005) and zebrafish mecom has also been reported in the pronephros (Wingert, et al., 2007; Wingert and Davidson, 2011). In the zebrafish, mecom is initially expressed in the renal progenitor field, but its domain changes dynamically during nephrogenesis (Wingert et al., 2007; Wingert and Davidson 2011).
The zebrafish pronephros provides a conserved model to study kidney development, in particular to delineate the poorly understood processes of how nephron segment pattern and cell type choice are established. Zebrafish nephrons are divided into distinct epithelial regions that include a series of proximal and distal tubule segments, which are comprised of intercalated transporting epithelial cells and multiciliated cells (MCC). Previous studies have shown that retinoic acid (RA) regionalizes the renal progenitor field into proximal and distal domains and that Notch signaling later represses MCC differentiation, but further understanding of these pathways has remained unknown. The transcription factor mecom (mds1/evi1 complex) is broadly expressed in renal progenitors, and then subsequently marks the distal tubule. Here, we show that mecom is necessary to form the distal tubule and to restrict both proximal tubule formation and MCC fate choice. We found that mecom and RA have opposing roles in patterning discrete proximal and distal segments. Further, we discovered that RA is required for MCC formation, and that one mechanism by which RA promotes MCC fate choice is to inhibit mecom. Next, we determined the epistatic relationship between mecom and Notch signaling, which limits MCC fate choice by lateral inhibition. Abrogation of Notch signaling with the γ-secretase inhibitor DAPT revealed that Notch and mecom did not have additive effects in blocking MCC formation, suggesting that they function in the same pathway. Ectopic expression of the Notch signaling effector, Notch intracellular domain (NICD), rescued the expansion of MCCs in mecom morphants, indicating that mecom acts upstream to induce Notch signaling. These findings suggest a model in which mecom and RA arbitrate proximodistal segment domains, while MCC fate is modulated by a complex interplay in which RA inhibition of mecom, and mecom promotion of Notch, titrates MCC number. Taken together, our studies have revealed several essential and novel mechanisms that control pronephros development in the zebrafish.
Expression, function and regulation of Evi-1 during embryonic avian development
2013, Gene Expression Patterns
Citation Excerpt :
The SET domain of chicken is 117 amino acids long and is expected to function in gene expression regulation during development, similar to its function in other vertebrate and invertebrate animals (Cui et al., 1998). Xenopus and mouse sequences have been published without the SET domain (Mead et al., 2005; Mucenski et al., 1988); however, they still show a high similarity to the chicken gene. The rest of the gene structure was found to be common for all the described species, with two domains of zinc fingers: fingers 1–7 in the N-terminal region and 8–10 in the C-terminal area.
Ecotropical viral integration site 1 (Evi-1) is a transcription factor essential for vascularisation and cell proliferation during embryonic development. The chimeric transcription factor AML1-EVI-1 is activated in leukaemia where it plays a role as a differentiation block and stimulator of proliferation. Here, we cloned chicken Evi-1 and analysed its expression during embryonic development. There was early expression in the pharyngeal arches, in the brain and intermediate mesoderm of chicken embryos at stage 15. Later at stage 20, Evi-1 mesenchymal expression was concentrated in the second pharyngeal arch, and weaker expression was found in the mandibular and maxillary prominences. Facial expression decreased in intensity during development. Evi-1 expression in the limb was also limited to the mesenchyme with the most prominent expression in the anterior margin. Evi-1 was not detectable in the posterior limb bud. At later stages, Evi-1 was expressed in the peripheral mesenchyme of the limb but not in the developing precartilage blastema. At stage 29, the expression became restricted to the perichondrium and interdigital areas; however, the cartilage condensations themselves were negative. To study the function of Evi-1 in chondrogenesis, we knocked down expression in limb micromass cultures using siRNA. Chondrogenesis was significantly reduced in both anterior and posterior cultures. Since Evi-1 was expressed adjacent to the apical ectodermal ridge and this area is a source of FGFs, we tested whether endogenous FGF receptor signalling was necessary to maintain its expression. Inhibitors of FGFRs (PD161570 and SU5402) were applied to wing mesenchyme, and downregulation of Evi-1 expression was observed after treatment with both inhibitors. Therefore, Evi-1 may be a transcription factor mediating the effects of FGF and may also be defining the size of cartilage elements in the limb.
Coordinating the timing of cardiac precursor development during gastrulation: A new role for Notch signaling
2009, Developmental Biology
Notch signaling has been shown to mediate a wide array of cell fate decisions during development. While previous work has demonstrated that Notch signaling plays an important role in regulating cardiac differentiation and morphogenesis, an earlier role during cardiac field formation has not yet been fully characterized. Previously, our lab demonstrated that perturbations in Notch signaling beginning at the onset of gastrulation affect the subdivision of germ layers. However due to the potential additive effects of misregulating Notch signaling over multiple stages of development, it was not possible to distinguish a specific role for this pathway during heart field specification. Here, we developed an innovative approach that takes advantage of temporally inducible constructs to isolate our manipulations to specific windows of development. In particular, we focused our studies on some of the earliest stages of cardiogenesis when heart field specification occurs. Our findings demonstrate a novel role for Notch signaling during the prepatterning of the cardiac mesoderm. Specifically, once relieved of aberrantly activated Notch signaling following gastrulation, cardiac precursors retain the ability to express markers of the cardiac field. Conversely, downregulating Notch signaling in cells fated to become heart tissue results in the induction of cardiac field genes in gastrula embryos. Finally, we provide evidence suggesting that this new role for Notch signaling is mediated at least in part via the Notch effector protein, Esr9 and the transcription factor GATA4. Taken together, these findings provide strong evidence for a novel role for Notch signaling in regulating the timing of heart field specification during early cardiogenesis.
The MDS1-EVI1 gene complex as a retrovirus integration site: Impact on behavior of hematopoietic cells and implications for gene therapy
2008, Molecular Therapy
Citation Excerpt :
By day 14.5 postcoitum, Evi1 expression in the heart disappears, only to reappear again in adult murine cardiac tissues. Similar patterns have been seen in Xenopus and zebrafish, suggesting that developmental regulation of this locus has been conserved throughout evolution, further supporting a central role in embryonic development.40,41 Thus far analysis of expression during development has not utilized probes that could discriminate Evi1 from Mds1–Evi1 expression.
Gene therapy trials have been performed with virus-based vectors that have the ability to integrate permanently into genomic DNA and thus allow prolonged expression of corrective genes after transduction of hematopoietic stem and progenitor cells. Adverse events observed during the X-linked severe combined immunodeficiency gene therapy trial revealed a significant risk of genotoxicity related to retrovirus vector integration and activation of adjacent proto-oncogenes, with several cases of T-cell leukemia linked to vector activation of the LMO2 gene. In patients with chronic granulomatous disease (CGD), rhesus macaques, and mice receiving hematopoietic stem and progenitor cells transduced with retrovirus vectors, a highly non-random pattern of vector integration has been reported. The most striking finding has been overrepresentation of integrations in one specific genomic locus, a complex containing the MDS1 and the EVI1 genes. Most evidence suggests that this overrepresentation is primarily due to a modification of primitive myeloid cell behavior by overexpression of EVI1 or MDS1–EVI1, as opposed to a specific predilection for integration at this site. Three different proteins can be produced from this complex locus: MDS1, MDS1–EVI1, and EVI1. This review will summarize current knowledge regarding this locus and its gene products, with specific focus on issues with relevance to gene therapy, leukemogenesis, and hematopoiesis. Insights into the mechanisms that result in altered hematopoiesis and leukemogenesis when this locus is dysregulated could improve the safety of gene therapy in the future.
The oncogene and developmental regulator EVI1: Expression, biochemical properties, and biological functions
2007, Gene
Citation Excerpt :
EVI1 orthologs also have specific functions in the neuronal development of invertebrates: the Drosophila EVI1 (or more precisely, MDS1/EVI1, see Section 4) ortholog hamlet specified single versus multiple dendrite morphology in the fly's peripheral nervous system (Moore et al., 2002); and the Caenorhabditis elegans EVI1 ortholog egl-43 was required for the migration of the hermaphrodite specific neurons (HSNs), and for proper morphological development of the PHA and PHB sensory neurons (Garriga et al., 1993). The recently reported expression patterns of Evi1 in Xenopus, chicken, and zebrafish demonstrated that many aspects of the developmental regulation of the Evi1 gene have been conserved throughout vertebrate evolution (Mead et al., 2005; Van Campenhout et al., 2006). As in the mouse, temporal and spatial specificity of Evi1 expression in these organisms argued for a role of Evi1 in development, which was experimentally confirmed for the formation of the Xenopus pronephros (Van Campenhout et al., 2006).
The EVI1 gene codes for a zinc finger transcription factor with important roles both in normal development and in leukemogenesis. Transcriptional activation of this gene through chromosome rearrangements or other, yet to be identified mechanisms leads to particularly aggressive forms of human myeloid leukemia. In vitro as well as in animal model systems, EVI1 affected cellular proliferation, differentiation, and apoptosis in cell type specific ways. Retroviral integrations into the EVI1 locus provided cells with increased abilities to engraft, survive, and proliferate in bone marrow transplantation experiments. Experimental overexpression of EVI1 by itself was insufficient to cause leukemia in animal model systems, but it cooperated with other genes in this process.
This review summarizes the currently available experimental evidence for the proposed biochemical and biological functions of this important oncogene.

View all citing articles on Scopus

View full text

Evi-1 expression in Xenopus

Abstract

Section snippets

Molecular cloning of Xenopus Evi1

Oocytes and embryos

cDNA cloning

Acknowledgements

Mech. Dev.

Dev. Biol.

Methods Cell Biol.

Mech. Dev.

Cell

Dev. Biol.

Expression of an engrailed-related protein is induced in the anterior neural ectoderm of early Xenopus embryos

Development