Neural tube (NT) formation in the spinal region of the mammalian embryo involves a wave of “zippering” that passes down the elongating spinal axis, uniting the neural fold tips in the dorsal midline. ...Failure of this closure process leads to open spina bifida, a common cause of severe neurologic disability in humans. Here, we combined a tissue-level strain-mapping workflow with laser ablation of live-imaged mouse embryos to investigate the biomechanics of mammalian spinal closure. Ablation of the zippering point at the embryonic dorsal midline causes far-reaching, rapid separation of the elevating neural folds. Strain analysis revealed tissue expansion around the zippering point after ablation, but predominant tissue constriction in the caudal and ventral neural plate zone. This zone is biomechanically coupled to the zippering point by a supracellular F-actin network, which includes an actin cable running along the neural fold tips. Pharmacologic inhibition of F-actin or laser ablation of the cable causes neural fold separation. At the most advanced somite stages, when completion of spinal closure is imminent, the cable forms a continuous ring around the neuropore, and simultaneously, a new caudal-to-rostral zippering point arises. Laser ablation of this new closure initiation point causes neural fold separation, demonstrating its biomechanical activity. Failure of spinal closure in pre-spina bifida Zic2Ku
mutant embryos is associated with altered tissue biomechanics, as indicated by greater neuropore widening after ablation. Thus, this study identifies biomechanical coupling of the entire region of active spinal neurulation in the mouse embryo as a prerequisite for successful NT closure.
Background
Whole embryo culture is a valuable research method in mammalian developmental biology and birth defects research, enabling longitudinal studies of explanted organogenesis‐stage rodent ...embryos. Rat serum is the primary culture medium, and can sustain growth and development over limited periods as in utero. However, the cost, labor, and time to produce culture serum are factors limiting the uptake of the methodology. The goal of replacing or at least reducing rat serum usage in culture would be in accordance with the principles of “replacement, reduction, and refinement” of animals in research (the 3Rs).
Methods
We performed cultures of mouse embryos for 24 hr from embryonic day 8.5 in serum‐free media or in rat serum diluted with defined media, compared with 100% rat serum. Developmental parameters scored after culture included yolk sac circulation, dorsal axial length, somite number, protein content, and completion of cranial neural tube closure.
Results
A literature review revealed use of both serum‐free and diluted rat serum‐based media in whole embryo culture studies, but with almost no formal comparisons of culture success against 100% rat serum. Two serum‐free media were tested, but neither could sustain development as in 100% rat serum. Dilution of rat serum 1:1 with Glasgow Minimum Essential Medium plus defined supplements supported growth and development as well as whole rat serum, whereas other diluent media yielded substandard outcomes.
Conclusion
Rat serum usage cannot be avoided, to achieve high quality mouse embryo cultures, but rat usage can be reduced using medium containing diluted serum.
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