Neural Tube Closure in Mouse Whole Embryo Culture

Department of Neurology/Neuroscience, Weill Cornell Medical College.
Journal of Visualized Experiments (Impact Factor: 1.33). 10/2011; DOI: 10.3791/3132
Source: PubMed


Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

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    • "The protocol for animal handling and treatment was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Hawaii. Culture of whole embryos collected from 8.5-dpc pregnant mice was performed according to the published protocols (Takahashi and Osumi, 2010; Gray and Ross, 2011; Behringer et al., 2014) with the following modifications. The medium used for whole-embryo culture was the same as the P19C5 culture medium, as described above. "
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