scholarly journals Single cells can sense their position in a morphogen gradient

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5309-5317 ◽  
Author(s):  
J.B. Gurdon ◽  
H. Standley ◽  
S. Dyson ◽  
K. Butler ◽  
T. Langon ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Single Cells ◽  
Morphogen Gradient ◽  
Rnase Protection ◽  
Lateral Contact ◽  
Related Response ◽  
Sense And Respond ◽  
Alternative Idea

Xenopus blastula cells show a morphogen-like response to activin by expressing different genes according to the concentration of activin to which they are exposed. To understand how cells recognize their position in a concentration gradient, it is essential to know whether each cell responds individually to activin concentration. An alternative idea, proposed by previous work, is that cells need to interact with their neighbours to generate a concentration-related response. To distinguish between these ideas, we have cultured blastula cells under conditions which provide different degrees of contact with other cells, allowing nil to maximum communication with their neighbours. The cultures include cells attached to fibronectin and cells resting unattached on an agarose base. The cultures also include cells that have no contact with any cell except their clonal progeny, cells that have lateral contact to neighbouring cells, and cells that are completely enveloped by other cells in a reaggregate. We have used RNase protection and in situ hybridization to assay the expression of the activin-responsive Xenopus genes Xbra, Xgsc, Xeomes, Xapod, Xchordin, Mix1, Xlim1 and Cerberus. We find no difference in gene expression between cells attached to fibronectin and those unattached on agarose. Most importantly, we find that cells respond to activin in a concentration-related way irrespective of their degree of contact with other cells. Therefore interaction among cells is not required for the interpretation of morphogen concentration, at least in the case of the early genes studied here. We conclude that isolated blastula cells can sense and respond individually to activin by expressing genes in a concentration-dependent way.

scholarly journals Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

1989 ◽  
Vol 37 (5) ◽  
pp. 697-701 ◽  
Author(s):  
F J Tang ◽  
P O Ts'o ◽  
S A Lesko
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
In Situ Hybridization ◽  
Single Cells ◽  
Specific Gene ◽  
Whole Cells ◽  
Beta Interferon ◽  
Ht1080 Cells ◽  
In Situ Detection

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

scholarly journals FISHing for chick genes: Triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos

2004 ◽  
Vol 229 (3) ◽  
pp. 651-657 ◽  
Author(s):  
Nathaniel Denkers ◽  
Pilar García-Villalba ◽  
Christopher K. Rodesch ◽  
Kandice R. Nielson ◽  
Teri Jo Mauch
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Avian Embryos ◽  
Overlapping Gene ◽  
Whole Mount

scholarly journals Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization.

1991 ◽  
Vol 88 (18) ◽  
pp. 8154-8158 ◽  
Author(s):  
A. M. Henney ◽  
P. R. Wakeley ◽  
M. J. Davies ◽  
K. Foster ◽  
R. Hembry ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Atherosclerotic Plaques

Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain

1992 ◽  
Vol 68 (3) ◽  
pp. 756-766 ◽  
Author(s):  
T. M. Perney ◽  
J. Marshall ◽  
K. A. Martin ◽  
S. Hockfield ◽  
L. K. Kaczmarek
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
K Channel ◽  
Rnase Protection ◽  
Hybridization Histochemistry ◽  
Adult Rat ◽  
In Situ Hybridization Histochemistry ◽  
Adult Rat Brain ◽  
Developing Rat

1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

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