About the Project
One in every 40 pregnancies is affected by a birth defect, such as spina bifida or congenital heart anomaly. In Europe, 360,000 new-born infants have birth defects each year, and many experience a life-time of disability and surgical treatment. Birth defects arise early in pregnancy, 3-8 weeks after conception, when the human embryo is inaccessible inside the mother’s uterus. Since it is difficult to study early human embryos, scientists instead work with embryos of mice and rats to gain insight into the processes that cause or may prevent birth defects.
An alternative to studying birth defects in pregnant female mice or rats is to grow the embryos outside the mother. This whole embryo culture (WEC) procedure allows embryos to be grown in vitro through the early stages of organogenesis, overcoming the inaccessibility of in vivo embryos, within the uterus. WEC allows analysis of the direct effects of exogenous agents on the embryo, permits surgical/other interventions, and is used increasingly to study how gene dysfunction leads to birth defects.
In terms of the 3Rs, WEC is an efficient way of avoiding the manipulation of pregnant female rodents. Littermate embryos can be allocated to different treatment groups, enhancing comparability between groups, and counteracting inter-litter variability. WEC is already widely used: e.g. 82 publications in 2010-14. However, several factors currently limit its more general uptake, and this NC3Rs-funded PhD studentship aims to adapt WEC to overcome these limitations. The goals are to:
1. Validate and extend the scope of mouse whole embryo culture:
(i) Determine the effectiveness of serum-free embryo culture compared with the traditional method that uses lab-produced rat serum, to determine whether rat serum usage can be replaced by a defined medium in future;
(ii) Characterise cultured embryos at the transcriptomics level, using RNAseq, compared with in vivo developed embryos, in order to validate the use of cultured embryos in molecular developmental studies;
(iii) Optimise embryo culture for induction of conditional transgene expression by tamoxifen, in order to render embryo culture a suitable alternative to in vivo transgenic studies in developmental biology.
2. Use the optimised whole embryo culture method to examine a fundamental hypothesis about the genetic control of spina bifida development. Homozygous loss of function of Vangl2, a key gene in the non-canonical Wnt-dishevelled signalling pathway, leads to total failure of neural tube closure. It is unknown, however, whether loss of Vangl2 function later in development, after closure is initiated, may be a cause of spinal neurulation defects, with the generation of spina bifida. The student will use the tamoxifen-inducible embryo culture system to test this hypothesis.
This student project will first extend and validate WEC for contemporary birth defects research. Then, the student will use the optimised techniques, particularly induction of conditional transgene expression, to perform a hypothesis-driven analysis of the role of the Vangl2 gene in development of mouse spina bifida.
References
Greene N.D. and Copp A.J. (2014). Neural tube defects. Annu. Rev. Neurosci. 37: 221-242.
Pryor S.E., Massa V., Savery D., Greene N.D.E., and Copp A.J. (2012). Convergent extension analysis in mouse whole embryo culture. Methods Mol. Biol 839: 133-146.