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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.
The appearance of embryos pre- and post- roller culture is illustrated in Figure 1. At the time of dissection, embryos should be in an unturned configuration (Fig. 1A,D) where the tail is behind the head folds. After 36 hrs in culture, embryos should have completed turning so that they are in the C-curved, fetal position, where the tail is in front of the head (Fig 1B,C,E,F). Pharmacological manipulation with RhoA kinase inhibitor (Y-27632), a known inhibitor of convergent extension during neurulation (Ybot-Gonzalez, 2007), results in a shortening of the embryos along their rostral-caudal axis (Fig. 1E,F) and inhibits cranial neural fold closure. Our data show that increasing doses of Y-27632 progressively impairs cranial fold closure (Figure 2A) and shortens the body axis (Fig. 2B), consistent with the role of downstream RhoA signaling in cranial neurulation and convergent extension.
Figure 1. Appearance of cultures embryos and manipulation with the pharmacological inhibitor Y-27632. (A,D) Dissected embryos at E8.5 prior to whole embryo culture (B,E) Embryos at E10 with the yolk sac still intact, subsequent to 36hrs of roller culture. (C,F) The yolk sac has been removed to illustrate successful turning and neural tube closure. Embryos that were treated with the Rho kinase inhibitor Y-27632 (E,F) failed to undergo proper convergent extension and illustrate a shortened body axis.
Figure 2. Effect of RhoA kinase inhibitor on cranial neural tube closure and axis elongation. (A) The percentage of embryos that were able to successfully close their cranial folds (%NTC= percentage neural tube closure) is compared to the dose of Y-27632 added to culture media. (B) The distance between the otic vesicle and forelimb was significantly reduced at increasing doses of Y-27632 (p<.05).
The ability to separate maternal, intrauterine factors from those that are intrinsic to the embryo during its development is an important tool for studying all stages of embryogenesis. Here we have analyzed the effects of a small molecule inhibitor of RhoA kinase on cranial neurulation ex utero, thereby removing the variable of maternal metabolism of the drug. This pharmacological manipulation has a profound effect on cranial neurulation and convergent extension. Sensitivity to this compound can be compared among different genetic mouse mutants. The method presented here can also be applied to studies of other molecular pathways in development, allowing the direct manipulation of cellular function in embryos using a variety of reagents.
No conflicts of interest declared.
We would like to thank the lab of A. Hadjantonakis (Sloan-Kettering Institute) and the lab of L. Niswander (U of Colorado-Denver) for helpful advice with dissection and culture techniques. This work has been supported by NRSA NS059562 to JDG and RO1NS05897 to MER in collaboration with L. Niswander and J. Nadeau (Institute for Systems Biology).