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Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Soares, Miguel L. and Haraguchi, Seiki and Torres-Padilla, Maria-Elena and Kalmar, Tibor and Carpenter, Lee and Bell, Graham and Morrison, Alastair and Ring, Christopher J. A. and Clarke, Neil J. and Glover, David M. and Zernicka-Goetz, Magdalena (2005) Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi. BMC Developmental Biology, 5 . Art. No. 28. ISSN 1471-213X. PMCID PMC1363358. doi:10.1186/1471-213x-5-28. https://resolver.caltech.edu/CaltechAUTHORS:20190419-105737131

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Abstract

Background: Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. Results: We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Conclusion: Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1186/1471-213x-5-28DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1363358/PubMed CentralArticle
ORCID:
AuthorORCID
Glover, David M.0000-0003-0956-0103
Zernicka-Goetz, Magdalena0000-0002-7004-2471
Additional Information:© Soares et al; licensee BioMed Central Ltd. 2005. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 30 July 2005. Accepted: 28 December 2005. Published: 28 December 2005. We thank Richard Behringer for the Lhx1 probe, Hiroshi Hamada for the Lefty-1 probe and Stephen Frankenberg for the Cer-l and Eomes probes. We are indebted to Berenika Plusa and Joanna Grabarek for invaluable technical assistance, and to Phil Elstob for logistical assistance. This work was supported by grants from Cancer Research UK (CRUK, grant number C3/A2943), Cyclacel Ltd, and BBSRC (for M.Z.-G). M.Z.-G. is a Wellcome Trust Senior Research Fellow; M-E.T-P. is recipient of an EMBO long-term fellowship. Authors' contributions: MLS designed and carried out most of the experimental work, and drafted the manuscript; SH assisted in experimental design and participated significantly in most experiments; METP performed part of post-implantation embryo recovery and assisted in confocal microscopy; TK and LC assisted in molecular analyses, experimental design and interpretation of data; GB, AM, and CJAR assisted in analysis and interpretation of results; NJC, DMG and MZG coordinated the project; MZG conceived and supervised the project; MLS prepared the manuscript with MZG and DMG. All authors read and approved the final manuscript. The author(s) declare that they have no competing interests.
Funders:
Funding AgencyGrant Number
Cancer Research UKC3/A2943
Cyclacel Ltd.UNSPECIFIED
Biotechnology and Biological Sciences Research Council (BBSRC)UNSPECIFIED
Wellcome TrustUNSPECIFIED
European Molecular Biology Organization (EMBO)UNSPECIFIED
Subject Keywords:Blastocyst Stage; Bmp4 Expression; Hepes Buffer Saline; Visceral Endoderm; Pseudopregnant Female
PubMed Central ID:PMC1363358
DOI:10.1186/1471-213x-5-28
Record Number:CaltechAUTHORS:20190419-105737131
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190419-105737131
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:94814
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:23 Apr 2019 17:12
Last Modified:16 Nov 2021 17:08

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