Detailed staging of inbred C57BL/6 mice between Theiler's [1972] stages 18 and 21 (11-13 days of gestation) based on craniofacial development

A detailed staging table of inbred C57BL/6 embryonic mice was developed to facilitate a study of the stage-by-stage cellular and molecular mechanisms underlying cranial skeletal development and elucidation of the developmental mechanisms potentially involved in evolutionary changes in cranial skeletal morphology exhibited by different inbred strains of mice. Mice were mated for only 2 hr and embryos were recovered every 2 or 4 hr between 11 and 13 days of gestation. Theiler's [1972] stages 18 through 21 were divided into substages and divisions based on the development of five external structures-the frontonasal area, eyes, auditory meatus, mandibular and hyoid auricular hillocks/pinna, and vibrissae-and three internal histological structures-eyes, tongue, and vibrissae. Each substage and division was designated with a decimal point: e.g., substage 20.1 and division 20.11. Embryos were staged using the staging table and the relationship of the substages and divisions with days of gestation was examined. The results showed considerable intra- and inter-litter variation in stages of embryos, suggesting that days of gestation are not a good indicator for staging embryos. Our staging table offers a more reliable and precise method to standardize embryonic development. Regression analyses of substages on days of gestation showed that the duration of stages increased from stages 18 to 21. Estimated durations were 3.5 hr for stages 18 and 19, 8.8 hr for stage 20, and 38.8 hr for stage 21. Our staging table also provides baseline information on development of the frontonasal area (muzzle) and vibrissae and development and the transformation of the auricular hillocks into the pinna. The developmental sequence of mystacial and labial vibrissae indicated highly regulated differentiation and morphogenesis of vibrissal development at stages 20 and 21. Three hyoid auricular hillocks transiently became four hillocks at stage 19.1 before transforming to the pinna during stage 21. The second and third hyoid auricular hillocks were the major contributor to the pinna before stage 21.2, whereas mandibular auricular hillocks contributed to the pinna from stage 21.32 onward. The staging table has already served to demonstrate stage-specific skeletogenesis of the first arch cartilages in inbred C57BL/6 mice and to reveal differences in the onset of timing of skeletogenesis among inbred C57BL/6, C3H/He and CBA/J mice.