Refinement of inducible gene deletion in embryos of pregnant mice.

CreERT2-mediated gene recombination is widely applied in developmental biology research. Activation of CreERT2 is typically achieved by injection of tamoxifen in an oily vehicle into the peritoneal cavity of mid-gestation pregnant mice. This can be technically challenging and adversely impacts welfare. Here we characterize three refinements to this technique: Pipette feeding (not gavage) of tamoxifen, ex vivo CreERT2 activation in whole embryo culture and injection of cell-permeable TAT-Cre into Cre-negative cultured embryos. We demonstrate that pipette feeding of tamoxifen solution to the mother on various days of gestation reliably activates embryonic CreERT2, illustrated here using ß-Actin CreERT2 , Sox2 CreERT2 , T CreERT2 , and Nkx1.2 CreERT2 . Pipette feeding of tamoxifen induces dose-dependent recombination of Rosa26 mTmG reporters when administered at E8.5. Activation of two neuromesodermal progenitor-targeting Cre drivers, T CreERT2 , and Nkx1.2 CreERT2 , produces comparable neuroepithelial lineage tracing. Dose-dependent CreERT2 activation can also be achieved by brief exposure to 4OH-tamoxifen in whole embryo culture, allowing temporal control of gene deletion and eliminating the need to treat pregnant mice. Rosa26 mTmG reporter recombination can also be achieved regionally by injecting TAT-Cre into embryonic tissues at the start of culture. This allows greater spatial control over Cre activation than can typically be achieved with endogenous CreERT2, for example by injecting TAT-Cre on one side of the midline. We hope that our description and application of these techniques will stimulate refinement of experimental methods involving CreERT2 activation for gene deletion and lineage tracing studies. Improved temporal (ex vivo treatment) and spatial (TAT-Cre injection) control of recombination will also allow previously intractable questions to be addressed.

Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium.

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.

Biomechanical coupling facilitates spinal neural tube closure in mouse embryos.

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.