Localization of messenger RNA in the cortex of Chaetopterus eggs and early embryos

Development ◽  
1983 ◽  
Vol 75 (1) ◽  
pp. 225-239
Author(s):  
William R. Jeffery ◽  
Linda J. Wilson
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Polar Lobe ◽  
Early Embryos ◽  
Cell Embryo ◽  
Ooplasmic Segregation ◽  
Actin Mrna

The distribution of mRNA in Chaetopterus pergamentaceus eggs was examined by in situ hybridization with poly(U) and specific cloned DNA probes. Eggs contain three distinct regions; the cortical ectoplasm, endoplasm, and a plasm released from the germinal vesicle (GV) during maturation. The ectoplasm of the mature egg showed a 15-fold enrichment in poly(A) and in histone and actin mRNAs relative to the endoplasm and the GV plasm after in situ hybridization. More than 90% of the total mass of egg poly (A) + RNA and histone and actin messages was estimated to be present in the ectoplasm. The mRNA molecules codistributed with ectoplasmic inclusion granules during ooplasmic segregation. During the extensive cytoplasmic rearrangements which occur at the time of the first cleavage the ectoplasm was divided into animal and vegetal portions. The animal portion was segregated evenly between the AB and CD blastomeres, whereas the vegetal portion entered the polar lobe and was preferentially segregated to the CD blastomere. Histone and actin mRNA entered both the AB and CD blastomeres of the 2-cell embryo. The results demonstrate that mRNA is quantitatively localized in the cortex of the Chaetopterus egg and early embryo.

Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1239-1249 ◽  
Author(s):  
C.A. Whittaker ◽  
D.W. DeSimone
Keyword(s):  
In Situ Hybridization ◽  
Rnase Protection Assay ◽  
Early Embryo ◽  
Mrna Levels ◽  
Rnase Protection ◽  
Dorsal Midline ◽  
Transmembrane Receptors ◽  
Early Embryos ◽  
Whole Mount

Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289–298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.

scholarly journals Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

1989 ◽  
Vol 108 (6) ◽  
pp. 2343-2353 ◽  
Author(s):  
R H Singer ◽  
G L Langevin ◽  
J B Lawrence
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Colloidal Gold ◽  
Messenger Rna ◽  
Signal To Noise Ratio ◽  
Simultaneous Detection ◽  
Gold Particles ◽  
Rna Molecules ◽  
Nucleic Acid Molecule

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

In Situ Hybridization of Messenger RNA in Sleep Research

2019 ◽  
pp. 167-179 ◽  
Author(s):  
Tarja Porkka-Heiskanen ◽  
Jussi Toppila ◽  
Dag Stenberg
Keyword(s):  
In Situ Hybridization ◽  
Messenger Rna ◽  
Sleep Research

scholarly journals Synthesis of transforming growth factor-beta 1 by megakaryocytes and its localization to megakaryocyte and platelet alpha-granules

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 1946-1955 ◽  
Author(s):  
RA Fava ◽  
TT Casey ◽  
J Wilcox ◽  
RW Pelton ◽  
HL Moses ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Messenger Rna ◽  
Transforming Growth Factor ◽  
Immunoperoxidase Technique ◽  
Storage Site ◽  
Tgf Beta ◽  
Growth Factor Beta

We have directly demonstrated that megakaryocytes are a major site of synthesis and storage of transforming growth factor-beta 1 (TGF/beta 1) by combined immunohistochemical, immunocytochemical, and in situ hybridization methods. The presence of TGF/beta 1 messenger RNA (mRNA) in mature megakaryocytes in adult rat spleen and bone marrow (BM) was established by in situ hybridization. Localization of TGF/beta 1 protein to intact alpha-granules of megakaryocytes, its putative storage site, was accomplished in glycol-methacrylate embedded porcine BM with an immunoperoxidase technique and light microscopy. The TGF/beta 1 was sequestered in intracytoplasmic granules in a pattern virtually identical to that of another alpha-granule marker protein, fibrinogen. This observation strongly suggests packaging of TGF/beta 1 into this organelle within megakaryocytes. That TGF/beta 1 mRNA was localized to megakaryocytes suggests that the TGF/beta 1 found in the alpha-granules in platelets originates with megakaryocyte synthesis. The alpha-granule localization of TGF/beta 1, as well as fibrinogen, was also demonstrated in isolated platelets at the ultrastructural level by electronmicroscopy (EM) and postembedding colloidal-gold immunocytochemistry, thus directly demonstrating that alpha-granules are the final storage site for TGF/beta 1 in mature platelets.

Sign in / Sign up

Export Citation Format

Share Document