Computational protocol: De novo mutations in PLXND1 and REV3L cause Möbius syndrome

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Protocol publication

[…] We counted the total number of motoneurons presents in the facial motor nucleus in paraffin sagittal sections processed with Nissl staining. We used five Plxnd1 wt, five Plxnd1 heterozygous and five Plxnd1 homozygous mice at E16.5 and five Rev3l wt and five Rev3l heterozygous P0 newborns, and we counted separately the number of motoneurons from the right and left hindbrain sides (around 12 sections each side) (20–25 slices total per animal). For the counting we used the open-source ImageJ Cell Counter software. Differences in the number of motoneurons between the heterozygous, homozygous and the wt mice were calculated by a comparative t-test, using Graphpad Software ( [...] We performed volume rendering of image stacks on serial light microscopic sections of 16 μm using the open-source ImageJ TrakEM2 (ref. ). For the volume rendering of the subarachnoid space around 100 sagittal slices of each of the five Rev3l wt and five Rev3l heterozygous mice at E16.5 were used. This embryonic stage was selected because the calcification of the cranial bones is not completed yet allowing the sectioning. For hindbrain volumes comparison, around 80 slices of each of the five Rev3l wt and five Rev3l heterozygous brains at P0 were used. After alignment of all images from each brain respectively, we highlighted the areas of interest in each section to measure their volumes. The boundaries of the hindbrain were delineated according to the Allen Developing Mouse Brain Atlas from rhombomere r1 until r11, including the cerebellum. […]

Pipeline specifications

Software tools ImageJ, TrakEM2
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Congenital Abnormalities, Nervous System Diseases