Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Masatsugu Ema ◽  
Satoru Takahashi ◽  
Janet Rossant
Keyword(s):  
Growth Factor Receptor ◽  
Primitive Streak ◽  
Cell Types ◽  
Gene Encoding ◽  
Mesodermal Cell ◽  
Selection Cassette ◽  
Cis Acting ◽  
Endogenous Expression ◽  
Extraembryonic Mesoderm ◽  
Early Mouse

Abstract Flk1, the gene encoding the vascular endothelial growth factor receptor 2 (VEGFR-2), is a well-known marker for vascular and hematopoietic progenitors and is indispensable for normal hematopoiesis and vasculogenesis. Here we show that Flk1 expression in the early mouse embryo marks a broad spectrum of mesodermal progenitors exiting the primitive streak as well as later mesodermal cell types including some cardiomyocytes, portions of the somites, and all extraembryonic mesoderm cells. These findings made use of an Flk1-lacZ knock-in allele in which the neomycin selection cassette was removed, which resulted in full replication of the endogenous expression of Flk1. Targeted deletion of a region in intron 1 that has been proposed to direct endothelial expression produced no alteration in either endothelial or broader mesodermal expression of the Flk1-lacZ allele. Examination of lacZ expression in homozygotes for the Flk1lacZ neo-out allele revealed that lacZ-expressing mesodermal cells persisted in nonvascular regions. Thus, Flk1 expression marks progenitors with broad mesodermal potential but is not absolutely required for the development of all mesodermal lineages in which it is expressed.

Primordial germ cells in the mouse embryo during gastrulation

Development ◽  
1990 ◽  
Vol 110 (2) ◽  
pp. 521-528 ◽  
Author(s):  
M. Ginsburg ◽  
M.H. Snow ◽  
A. McLaren
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primitive Streak ◽  
Cell Types ◽  
Mesodermal Cell ◽  
Alp Activity ◽  
Cluster Area ◽  
Extraembryonic Mesoderm ◽  
Excised Tissue ◽  
Number Of Cells

With the aid of a whole-mount technique, we have detected a small cluster of alkaline phosphatase (ALP)-positive cells in whole mounts of mid-primitive-streak-stage embryos, 7–7 1/4 days post coitum (dpc). Within the cluster, about 8 cells contain a small cytoplasmic spot, intensely stained for ALP activity and possibly associated with an active Golgi complex. The cluster lies just posterior to the definitive primitive streak in the extraembryonic mesoderm, separated from the embryo by the amniotic fold. Towards the end of gastrulation, the number of cells containing the ALP-positive spot rises to between 50 and 80. Thereafter the number of cells in the extraembryonic cluster declines, and similar cells start to be seen in the mesoderm of the primitive streak and then in the endoderm. At 8 dpc, about 125 ALP-stained cells are found, mainly in the hindgut endoderm and also at the base of the allantois, their appearance and location at this stage agreeing closely with previous reports on primordial germ cells (PGCs). Embryos from which the cluster area has been removed at the 7-day stage are devoid of PGCs after culture for 48 h, whereas the excised tissue is rich in PGCs. We argue that the cells in the cluster are indeed primordial germ cells, at a stage significantly earlier than any reported previously. This would indicate that the PGC lineage in the mouse is set aside at least as early as 7 dpc, possibly as one of the first ‘mesodermal’ cell types to emerge, and that its differentiation, as expressed by ALP activity, is gradual.

Pattern of the insulin-like growth factor II gene expression during early mouse embryogenesis

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 151-159 ◽  
Author(s):  
J.E. Lee ◽  
J. Pintar ◽  
A. Efstratiadis
Keyword(s):  
Growth Factor ◽  
Immunohistochemical Analysis ◽  
Primitive Streak ◽  
Insulin Like Growth Factor ◽  
Surface Ectoderm ◽  
Factor Ii ◽  
Extraembryonic Mesoderm ◽  
Early Mouse ◽  
Lateral Mesoderm

The mouse insulin-like growth factor II (IGF-II) gene encodes a polypeptide that plays a role in embryonic growth. We have examined the temporal and spatial pattern of expression of this gene in sections of the mouse conceptus between embryonic days 4.0 and 8.5 by in situ hybridization. Abundant IGF-II transcripts were detected in all the trophectodermal derivatives, after implantation. Labeling was then observed in primitive endoderm, but was transient and disappeared after formation of the yolk sac. Expression was next detected in extraembryonic mesoderm at the early primitive streak stage. Labeling in the embryo proper appeared first at the late primitive streak/neural plate stage in lateral mesoderm and in anterior-proximal cells located between the visceral endoderm and the most cranial region of the embryonic ectoderm. The position of the latter cells suggests that their descendants are likely to participate in the formation of the heart and the epithelium of the ventral and lateral walls of the foregut, where intense labeling was observed at the neural fold stage. Hybridization was also detected in cranial mesenchyme, including neural crest cells. The intensity of hybridization signal increased progressively in paraxial (presomitic and somitic) mesoderm, while declining in the ectoplacental cone. The neuroectoderm and surface ectoderm did not exhibit hybridization at any stage. Immunohistochemical analysis indicated co-localization of IGF-II transcripts, translated pre-pro-IGF-II, and the cognate IGF-II/mannose-6-phosphate receptor. These correlations are consistent with the hypothesis that IGF-II has an autocrine function.

scholarly journals Identification of highly conserved regulatory domains and protein-binding sites in the promoters of the rat and human genes encoding the stress-inducible 78-kilodalton glucose-regulated protein.

1988 ◽  
Vol 8 (10) ◽  
pp. 4579-4584 ◽  
Author(s):  
E Resendez ◽  
S K Wooden ◽  
A S Lee
Keyword(s):  
Regulatory Element ◽  
Calcium Ionophore ◽  
Cell Types ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Gene Encoding ◽  
Cis Acting ◽  
Regulatory Domains ◽  
Nuclear Extracts ◽  
Basal Level Expression

The gene encoding GRP78 has been shown to be constitutively expressed in many cell types and is inducible by the calcium ionophore A23187. To understand the regulation of GRP78 transcription, we analyzed the components that control its basal-level expression. By transfecting deletions into cells, we have identified a 54-nucleotide cis-acting regulatory element important for high basal-level expression and a contiguous 50-nucleotide element for both basal-level expression and A23187 induction. Using DNase footprinting assays with both rat and human GRP78 promoters, we demonstrated that the protein factors present in the HeLa cell nuclear extracts bind to the regulatory regions identified by the deletion studies. This domain contains a palindromic sequence and is highly conserved among GRP genes in Caenorhabditis elegans, chicks, rats, and humans.

The eed mutation disrupts anterior mesoderm production in mice

Development ◽  
1995 ◽  
Vol 121 (2) ◽  
pp. 273-285 ◽  
Author(s):  
C. Faust ◽  
A. Schumacher ◽  
B. Holdener ◽  
T. Magnuson
Keyword(s):  
Ectopic Expression ◽  
Primitive Streak ◽  
Cell Types ◽  
Growth Defect ◽  
Mrna In Situ Hybridization ◽  
Embryonic Ectoderm Development ◽  
Distinct Node ◽  
Extraembryonic Mesoderm ◽  
Or Organization

Mouse embryos homozygous for the mutation embryonic ectoderm development (eed) exhibit a growth defect and fail to gastrulate normally. While extraembryonic mesoderm is produced extensively, very little embryonic mesoderm is detected in eed mutant embryos, and there is no subsequent organization of mesoderm into node, notochord, or somites. The phenotype is consistent with a defect in the distal primitive streak. Here we report additional phenotypic analyses that include mRNA in situ hybridization of genes whose expression reflects the function of different regions of the primitive streak and their derivatives. These studies have confirmed that mesoderm derived from the proximal primitive streak is specified appropriately. Despite the absence of a morphologically distinct node, sparse axial mesoderm cells in eed mutant embryos are specified, as reflected by expression of Brachyury (T), Sonic hedgehog, and Tcf3b/HNF-3 beta, and definitive endoderm is produced. Specification of these cell types is also independent of correct expression of nodal, Fgf4, and gsc. Finally, T and Evx1 display ectopic expression in cells not normally fated to ingress through the primitive streak. The data presented are discussed in terms of mechanisms for establishment of the eed phenotype, and are consistent with the eed gene product playing an early role in primitive streak formation and/or organization.

Identification of highly conserved regulatory domains and protein-binding sites in the promoters of the rat and human genes encoding the stress-inducible 78-kilodalton glucose-regulated protein

1988 ◽  
Vol 8 (10) ◽  
pp. 4579-4584
Author(s):  
E Resendez ◽  
S K Wooden ◽  
A S Lee
Keyword(s):  
Regulatory Element ◽  
Calcium Ionophore ◽  
Cell Types ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Gene Encoding ◽  
Cis Acting ◽  
Regulatory Domains ◽  
Nuclear Extracts ◽  
Basal Level Expression

The gene encoding GRP78 has been shown to be constitutively expressed in many cell types and is inducible by the calcium ionophore A23187. To understand the regulation of GRP78 transcription, we analyzed the components that control its basal-level expression. By transfecting deletions into cells, we have identified a 54-nucleotide cis-acting regulatory element important for high basal-level expression and a contiguous 50-nucleotide element for both basal-level expression and A23187 induction. Using DNase footprinting assays with both rat and human GRP78 promoters, we demonstrated that the protein factors present in the HeLa cell nuclear extracts bind to the regulatory regions identified by the deletion studies. This domain contains a palindromic sequence and is highly conserved among GRP genes in Caenorhabditis elegans, chicks, rats, and humans.

scholarly journals Identification of Jade1, a Gene Encoding a PHD Zinc Finger Protein, in a Gene Trap Mutagenesis Screen for Genes Involved in Anteroposterior Axis Development

2003 ◽  
Vol 23 (23) ◽  
pp. 8553-8552 ◽  
Author(s):  
Elena Tzouanacou ◽  
Susan Tweedie ◽  
Valerie Wilson
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Ventricular Zone ◽  
Primitive Streak ◽  
Cell Types ◽  
Gene Trap ◽  
Protein Isoforms ◽  
Tail Bud ◽  
Gene Encoding ◽  
Finger Protein

ABSTRACT In a gene trap screen for genes expressed in the primitive streak and tail bud during mouse embryogenesis, we isolated a mutation in Jade1, a gene encoding a PHD zinc finger protein previously shown to interact with the tumor suppressor pVHL. Expressed sequence tag analysis indicates that Jade1 is subject to posttranscriptional regulation, resulting in multiple transcripts and at least two protein isoforms. The fusion Jade1-β-galactosidase reporter produced by the gene trap allele exhibits a regulated expression during embryogenesis and localizes to the nucleus and/or cytoplasm of different cell types. In addition to the primitive streak and tail bud, β-galactosidase activity was found in other embryonic regions where pluripotent or tissue-specific progenitors are known to reside, including the early gastrulation epiblast and the ventricular zone of the cerebral cortex. Prominent reporter expression was also seen in the extraembryonic tissues as well as other differentiated cell types in the embryo, in particular the developing musculature. We show that the gene trap mutation produces a null allele. However, homozygotes for the gene trap integration are viable and fertile. Database searches identified a family of Jade proteins conserved through vertebrates. This raises the possibility that the absence of phenotype is due to a functional compensation by other family members.

scholarly journals DRES-13. DUAL KINASE INHIBITION TO COMBAT EGFR-INHIBITOR RESISTANCE IN GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi74-vi74
Author(s):  
Erin Smithberger ◽  
Abigail Shelton ◽  
Madison Butler ◽  
Alex Flores ◽  
Ryan Bash ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Alternative Pathway ◽  
Growth Factor Receptor ◽  
Cell Types ◽  
Genetically Engineered ◽  
Differentially Expressed ◽  
Egfr Inhibitor ◽  
Tumor Resistance ◽  
Pten Deletion ◽  
In Cells

Abstract Glioblastoma (GBM) is an aggressive primary brain tumor with a poor survival rate. One of the most common molecular alterations seen in GBM is amplification and/or mutation of the Epidermal Growth Factor Receptor (EGFR), which has made it an attractive therapeutic target. However, several EGFR tyrosine kinase inhibitors have been tested clinically in GBM with minimal success. One reason for this lack of efficacy could be due to acute, adaptive resistance via alternative pathway activation. To investigate this mechanism of tumor resistance, we used RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze the transcriptomes and kinomes of genetically engineered murine astrocytes with common GBM genotypes. We have previously shown that 38% of the expressed kinome varied among a panel of diverse nGEM astrocytes harboring Cdkn2a deletion (C) plus Pten deletion (CP), wild-type human EGFR (CE) or EGFRvIII (CEv3) overexpression or both EGFRvIII overexpression and Pten deletion (CEv3P). Although CE have a similar transcriptional profile to C cells at baseline, when treated with the EGFR inhibitor afatinib, CE respond more similarly to CEv3 cells. When cells containing endogenous murine EGFR (C and CP) are treated with afatinib, fewer than 0.5% of kinases showed differential expression. In cells with EGFR overexpression alone, more than 6% of kinases were differentially expressed upon afatinib treatment, including Ntrk3, Fgfr2 and 3, Lyn, Bmx, Epha2 and 5, Fn3k, a kinase involved in fructosamine processing, and Nrbp2, a kinase involved in regulation of apoptosis. This effect was blunted in cells lacking Pten in addition to having EGFRvIII (CEv3P), resulting in less than 2% of kinases being differentially expressed. The only kinase upregulated in all three EGFR-overexpressing cell types was Coq8a, which is involved in electron transport and response to DNA damage. Given this overlap in response, Coq8a could be a potential dual treatment target for GBM.

scholarly journals Multiple cis-acting elements are required for RNA polymerase III transcription of the gene encoding H1 RNA, the RNA component of human RNase P.

1991 ◽  
Vol 266 (34) ◽  
pp. 22796-22799
Author(s):  
G.J. Hannon ◽  
A. Chubb ◽  
P.A. Maroney ◽  
G. Hannon ◽  
S. Altman ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Rnase P ◽  
Gene Encoding ◽  
Cis Acting ◽  
Polymerase Iii ◽  
Human Rnase

scholarly journals PET and SPECT Imaging of the EGFR Family (RTK Class I) in Oncology

2021 ◽  
Vol 22 (7) ◽  
pp. 3663
Author(s):  
Sara S. Rinne ◽  
Anna Orlova ◽  
Vladimir Tolmachev
Keyword(s):  
Single Photon ◽  
Photon Emission ◽  
Growth Factor Receptor ◽  
Spect Imaging ◽  
Emission Tomography ◽  
Class I ◽  
Whole Body ◽  
Her Family ◽  
Endogenous Expression ◽  
Egfr Family

The human epidermal growth factor receptor family (EGFR-family, other designations: HER family, RTK Class I) is strongly linked to oncogenic transformation. Its members are frequently overexpressed in cancer and have become attractive targets for cancer therapy. To ensure effective patient care, potential responders to HER-targeted therapy need to be identified. Radionuclide molecular imaging can be a key asset for the detection of overexpression of EGFR-family members. It meets the need for repeatable whole-body assessment of the molecular disease profile, solving problems of heterogeneity and expression alterations over time. Tracer development is a multifactorial process. The optimal tracer design depends on the application and the particular challenges of the molecular target (target expression in tumors, endogenous expression in healthy tissue, accessibility). We have herein summarized the recent preclinical and clinical data on agents for Positron Emission Tomography (PET) and Single Photon Emission Tomography (SPECT) imaging of EGFR-family receptors in oncology. Antibody-based tracers are still extensively investigated. However, their dominance starts to be challenged by a number of tracers based on different classes of targeting proteins. Among these, engineered scaffold proteins (ESP) and single domain antibodies (sdAb) show highly encouraging results in clinical studies marking a noticeable trend towards the use of smaller sized agents for HER imaging.

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