Misexpression of the Drosophila argos gene, a secreted regulator of cell determination

Development ◽  
1994 ◽  
Vol 120 (8) ◽  
pp. 2297-2304 ◽  
Author(s):  
M. Freeman
Keyword(s):  
Cell Fate ◽  
Cell Types ◽  
Soluble Form ◽  
Loss Of Function ◽  
Wild Type ◽  
Over Expression ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Normal Expression ◽  
Transgenic Flies

I have examined the effects on cells in the developing eye of over-expressing the argos gene. Transgenic flies carrying argos expressed under hsp70 and sevenless control sequences were analysed. All cell types in the developing eye (except bristles) are sensitive to argos concentration: over-expression leads to too few cells forming, the opposite phenotype to that seen in argos loss-of-function mutants. This effect was only seen with HS-argos flies: sev-argos flies, which over-express the protein at a lower level are not affected, suggesting that a considerable over-expression is required to disrupt cell fate. However, sev-argos is able to rescue argos eye mutations completely, indicating that the normal expression pattern is not critical for wild-type eye development. By transfecting argos into tissue culture cells, I show that the protein is secreted in a soluble form.

Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster

1997 ◽  
Vol 110 (17) ◽  
pp. 1979-1988 ◽  
Author(s):  
H. Bousbaa ◽  
L. Correia ◽  
G.J. Gorbsky ◽  
C.E. Sunkel
Keyword(s):  
Drosophila Melanogaster ◽  
Metaphase Plate ◽  
Cell Types ◽  
Early Prophase ◽  
Mitotic Chromosomes ◽  
Microtubule Inhibitors ◽  
Phosphatase Inhibitor ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Late Telophase

The progression of cells from metaphase to anaphase is thought to be regulated by a checkpoint that delays entry into anaphase until all chromosomes reach a stable bi-polar attachment at the metaphase plate. Previous work has suggested that the 3F3/2 kinetochore phosphoepitopes are involved in this checkpoint system. We show that the 3F3/2 centromere phosphoepitopes are present in Kc cells, third instar larval neuroblasts and isolated chromosomes of Drosophila melanogaster. In tissue culture cells and neuroblasts isolated from third instar larvae, centromere labelling is detected from early prophase to the metaphase-anaphase transition but absent once cells center anaphase. During anaphase, the antibody stains the spindle mid zone and during telophase the midbody is labelled until cells separate. In both cell types, the 3F3/2 antibody stains the centrosome from prophase to late telophase. The 3F3/2 staining is retained in Kc cells and third instar larval neuroblasts arrested at the prometaphase state with microtubule inhibitors. Also, two mitotic mutants that show abnormal spindle morphology retain the centromere labelling in a metaphase-like configuration, suggesting that they activate the metaphase-anaphase checkpoint. Finally, mitotic chromosomes isolated in the presence of a phosphatase inhibitor show phosphoepitopes at the primary constriction on the surface of each chromatid, however, chromosomes isolated in the absence of a phosphatase inhibitor do not. Incubation of these chromosomes with ATP causes the rephosphorylation of the phosphoepitopes at the centromere.

scholarly journals Rap1A-Deficient T and B Cells Show Impaired Integrin-Mediated Cell Adhesion

2006 ◽  
Vol 26 (2) ◽  
pp. 643-653 ◽  
Author(s):  
Marlena Duchniewicz ◽  
Tomasz Zemojtel ◽  
Mateusz Kolanczyk ◽  
Steffen Grossmann ◽  
Jürgen S. Scheele ◽  
...  
Keyword(s):  
Functional Complementation ◽  
Wild Type ◽  
T And B Cells ◽  
Genetic Studies ◽  
Mild Phenotype ◽  
Cell Homing ◽  
Culture Cells ◽  
Cell Matrix ◽  
Tissue Culture Cells

ABSTRACT Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1−/− cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.

spiel ohne grenzen/pou2is required for zebrafish hindbrain segmentation

Development ◽  
2002 ◽  
Vol 129 (7) ◽  
pp. 1645-1655 ◽  
Author(s):  
Giselbert Hauptmann ◽  
Heinz-Georg Belting ◽  
Uta Wolke ◽  
Karen Lunde ◽  
Iris Söll ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Hox Genes ◽  
Ectopic Expression ◽  
Loss Of Function ◽  
Wild Type ◽  
Specific Expression ◽  
Size And Shape ◽  
Regulatory Cascade ◽  
Normal Expression ◽  
Segmental Identity

Segmentation of the vertebrate hindbrain leads to the formation of a series of rhombomeres with distinct identities. In mouse, Krox20 and kreisler play important roles in specifying distinct rhombomeres and in controlling segmental identity by directly regulating rhombomere-specific expression of Hox genes. We show that spiel ohne grenzen (spg) zebrafish mutants develop rhombomeric territories that are abnormal in both size and shape. Rhombomere boundaries are malpositioned or absent and the segmental pattern of neuronal differentiation is perturbed. Segment-specific expression of hoxa2, hoxb2 and hoxb3 is severely affected during initial stages of hindbrain development in spg mutants and the establishment of krx20 (Krox20 ortholog) and valentino (val; kreisler ortholog) expression is impaired. spg mutants carry loss-of-function mutations in the pou2 gene. pou2 is expressed at high levels in the hindbrain primordium of wild-type embryos prior to activation of krx20 and val. Widespread overexpression of Pou2 can rescue the segmental krx20 and val domains in spg mutants, but does not induce ectopic expression of these genes. This suggests that spg/pou2 acts in a permissive manner and is essential for normal expression of krx20 and val. We propose that spg/pou2 is an essential component of the regulatory cascade controlling hindbrain segmentation and acts before krx20 and val in the establishment of rhombomere precursor territories.

Sectors expressing the homeobox gene liguleless3 implicate a time-dependent mechanism for cell fate acquisition along the proximal-distal axis of the maize leaf

Development ◽  
1997 ◽  
Vol 124 (24) ◽  
pp. 5097-5106 ◽  
Author(s):  
G.J. Muehlbauer ◽  
J.E. Fowler ◽  
M. Freeling
Keyword(s):  
Cell Fate ◽  
Homeobox Gene ◽  
Longitudinal Axis ◽  
Cell Types ◽  
Transverse Dimension ◽  
Leaf Sheath ◽  
Wild Type ◽  
Cell Fates ◽  
Maize Leaf ◽  
Gradual Process

The longitudinal axis of the maize leaf is composed of, in proximal to distal order, sheath, ligule, auricle and blade. The semidominant Liguleless3-O (Lg3-O) mutation disrupts leaf development at the ligular region of the leaf midrib by transforming blade to sheath. In a previous study, we showed that leaf sectors of Lg3 mutant activity are cell nonautonomous in the transverse dimension and can confer several alternative developmental fates (Fowler, Muehlbauer and Freeling (1996) Genetics 143, 489–503). In our present study we identify five Lg3 sector types in the leaf: sheath-like with displaced ligule (sheath-like), sheath-like with ectopic ligule (ectopic ligule), auricle-like, macro-hairless blade and wild-type blade. The acquisition of a specific sector fate depends on the timing of Lg3 expression. Early Lg3 expression results in adoption of the sheath-like phenotype at the ligule position (a proximal cell fate), whereas later Lg3 expression at the same position results in one of the more distal cell fates. Furthermore, sheath-like Lg3 sectors exhibit a graded continuum of phenotypes in the transformed blade region from the most proximal (sheath) to the most distal (wild-type blade), suggesting that cell fate acquisition is a gradual process. We propose a model for leaf cell fate acquisition based on a timing mechanism whereby cells of the leaf primordium progress through a maturation schedule of competency stages which eventually specify the cell types along the proximal to distal axis of the leaf. In addition, the lateral borders between Lg3 ‘on’ sectors and wild-type leaf sometimes provide evidence of no spreading of the transformed phenotype. In these cases, competency stages are inherited somatically.

scholarly journals EPEN-05. MUTATIONAL ANALYSIS OF THE C11ORF95 DOMAIN AND SINGLE-CELL RNA-SEQ PROFILE OF A MOUSE MODEL OF SUPRATENTORIAL EPENDYMOMA

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i14-i14
Author(s):  
Kevin Truong ◽  
James He ◽  
Gavin Birdsall ◽  
Ericka Randazzo ◽  
Jesse Dunnack ◽  
...  
Keyword(s):  
Mouse Model ◽  
Single Cell ◽  
Tumor Cells ◽  
Cell Fate ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Cell Types ◽  
Marker Genes ◽  
Rna Seq ◽  
Loss Of Function

Abstract We used a recently developed mouse model to better understand the cellular and molecular determinants of tumors driven by the oncogenic fusion protein C11orf95-RELA. Our approach makes use of in utero electroporation and a binary transposase system to introduce human C11orf95-RELA sequence, wild type and mutant forms, into neural progenitors. We used single cell RNA-seq to profile the cellular constituents within the resulting tumors in mice. We find that approximately 70% of the cells in the tumors do not express the oncogene C11orf95-RELA and these non-oncogene expressing cells are a combination of different non-tumor cell cell-types, including significant numbers of T-cells, and macrophages. The C11orf95-RELA expressing tumor cells have a unique transcriptomic profile that includes both astrocytic and neural progenitor marker genes, and is distinct from glioblastoma transcriptomic profiles. Since C11orf95-RELA is believed to function through a combination of both activation of NF-κB response genes by constitutive activation of RELA, and genes not activated by NF-κB, we assessed the expression of NF-κB response genes across the populations of cells in the tumor. Interestingly, when tumor cells highly expressing C11orf95-RELA were analyzed further, the subclusters identified were distinguished by upregulation of non-NF-kB pathways involved in cell proliferation, cell fate determination, and immune activation. We hypothesized that the C11orf95 domain may function to bring RELA transcriptional activation to inappropriate non-NF-κB targets, and we therefore performed a point mutation analysis of the C11orf95 domain. We found that mutations in either of the cysteines or histidines that make up a possible zinc finger domain in C11orf95 eliminate the ability of the fusion to induce tumors. In cell lines, these loss-of-function point mutants still trafficked to nuclei, and activated NF-κB pathways. We are currently using RNAseq and CRISPR loss-of function to identify genes downstream of C11orf95-RELA that are required for tumorigenesis.

Delta-Notch signaling induces hypochord development in zebrafish

Development ◽  
2002 ◽  
Vol 129 (11) ◽  
pp. 2555-2563 ◽  
Author(s):  
Andrew J. Latimer ◽  
Xinhong Dong ◽  
Youlia Markov ◽  
Bruce Appel
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Cell Types ◽  
Paraxial Mesoderm ◽  
Loss Of Function ◽  
Cell Type ◽  
No Tail ◽  
Vertebrate Embryos ◽  
Different Cell Types ◽  
Her4 Expression

Different cell types that occupy the midline of vertebrate embryos originate within the Spemann-Mangold or gastrula organizer. One such cell type is hypochord, which lies ventral to notochord in anamniote embryos. We show that hypochord precursors arise from the lateral edges of the organizer in zebrafish. During gastrulation, hypochord precursors are closely associated with no tail-expressing midline precursors and paraxial mesoderm, which expresses deltaC and deltaD. Loss-of-function experiments revealed that deltaC and deltaD were required for her4 expression in presumptive hypochord precursors and for hypochord development. Conversely, ectopic, unregulated Notch activity blocked no tail expression and promoted her4 expression. We propose that Delta signaling from paraxial mesoderm diversifies midline cell fate by inducing a subset of neighboring midline precursors to develop as hypochord, rather than as notochord.

Cell-specific targeting of caveolin-1 to caveolae, secretory vesicles, cytoplasm or mitochondria

2001 ◽  
Vol 114 (7) ◽  
pp. 1397-1408 ◽  
Author(s):  
W.P. Li ◽  
P. Liu ◽  
B.K. Pilcher ◽  
R.G. Anderson
Keyword(s):  
Endocrine Cells ◽  
Secretory Pathway ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Lipid Complex ◽  
Intracellular Traffic ◽  
Caveolin 1 ◽  
Culture Cells ◽  
Tissue Culture Cells

In commonly used tissue culture cells, caveolin-1 is embedded in caveolae membranes. It appears to reach this location after being cotranslationally inserted into ER membranes, processed in the Golgi and shipped to the cell surface. We now report that caveolae are not the preferred location for caveolin-1 in all cell types. Skeletal muscle cells and keratinocytes target caveolin-1 to the cytosol while in exocrine and endocrine cells it accumulates in the secretory pathway. We also found that airway epithelial cells accumulate caveolin-1 in modified mitochondria. The cytosolic and the secreted forms appear to be incorporated into a soluble, lipid complex. We conclude that caveolin-1 can be targeted to a variety of intracellular destinations, which suggests a novel mechanism for the intracellular traffic of this protein.

scholarly journals LET-711, the Caenorhabditis elegans NOT1 Ortholog, Is Required for Spindle Positioning and Regulation of Microtubule Length in Embryos

2006 ◽  
Vol 17 (11) ◽  
pp. 4911-4924 ◽  
Author(s):  
Leah R. DeBella ◽  
Adam Hayashi ◽  
Lesilee S. Rose
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Regulation Of Gene Expression ◽  
Early Embryo ◽  
Cell Cortex ◽  
Loss Of Function ◽  
Wild Type ◽  
Spindle Positioning ◽  
C Elegans ◽  
Associated Proteins

Spindle positioning is essential for the segregation of cell fate determinants during asymmetric division, as well as for proper cellular arrangements during development. In Caenorhabditis elegans embryos, spindle positioning depends on interactions between the astral microtubules and the cell cortex. Here we show that let-711 is required for spindle positioning in the early embryo. Strong loss of let-711 function leads to sterility, whereas partial loss of function results in embryos with defects in the centration and rotation movements that position the first mitotic spindle. let-711 mutant embryos have longer microtubules that are more cold-stable than in wild type, a phenotype opposite to the short microtubule phenotype caused by mutations in the C. elegans XMAP215 homolog ZYG-9. Simultaneous reduction of both ZYG-9 and LET-711 can rescue the centration and rotation defects of both single mutants. let-711 mutant embryos also have larger than wild-type centrosomes at which higher levels of ZYG-9 accumulate compared with wild type. Molecular identification of LET-711 shows it to be an ortholog of NOT1, the core component of the CCR4/NOT complex, which plays roles in the negative regulation of gene expression at transcriptional and post-transcriptional levels in yeast, flies, and mammals. We therefore propose that LET-711 inhibits the expression of ZYG-9 and potentially other centrosome-associated proteins, in order to maintain normal centrosome size and microtubule dynamics during early embryonic divisions.

scholarly journals Improved localization of intracellular sites of phosphatases using cerium and cell permeabilization.

1985 ◽  
Vol 33 (8) ◽  
pp. 749-754 ◽  
Author(s):  
J M Robinson
Keyword(s):  
Cell Surface ◽  
Surface Reaction ◽  
Intracellular Localization ◽  
Cell Types ◽  
Cell Type ◽  
Cell Permeabilization ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Cytochemical Reaction ◽  
Triton X

A simple permeabilization method has been developed that allows for intracellular localization of acid phosphatase in neutrophils and several types of tissue culture cells with cerium. This permeabilization procedure also facilitates intracellular alkaline phosphatase localization in neutrophils without the loss of cell surface reaction in this cell type. Only the cell surface reaction was detected in the absence of permeabilization. Glutaraldehyde-fixed cells were permeabilized with detergent during the cytochemical reaction. Triton X-100 at 0.0001-0.0002% gave the best results for the enzymes and cell types tested.

scholarly journals PDZD7-MYO7A complex identified in enriched stereocilia membranes

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Clive P Morgan ◽  
Jocelyn F Krey ◽  
M'hamed Grati ◽  
Bo Zhao ◽  
Shannon Fallen ◽  
...  
Keyword(s):  
Hair Cells ◽  
Protein Complexes ◽  
Molecular Pathways ◽  
Wild Type ◽  
New Paradigm ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Myosin Viia ◽  
Mechanosensory Hair Cells ◽  
And Function

While more than 70 genes have been linked to deafness, most of which are expressed in mechanosensory hair cells of the inner ear, a challenge has been to link these genes into molecular pathways. One example is Myo7a (myosin VIIA), in which deafness mutations affect the development and function of the mechanically sensitive stereocilia of hair cells. We describe here a procedure for the isolation of low-abundance protein complexes from stereocilia membrane fractions. Using this procedure, combined with identification and quantitation of proteins with mass spectrometry, we demonstrate that MYO7A forms a complex with PDZD7, a paralog of USH1C and DFNB31. MYO7A and PDZD7 interact in tissue-culture cells, and co-localize to the ankle-link region of stereocilia in wild-type but not Myo7a mutant mice. Our data thus describe a new paradigm for the interrogation of low-abundance protein complexes in hair cell stereocilia and establish an unanticipated link between MYO7A and PDZD7.

Sign in / Sign up

Export Citation Format

Share Document