Developmental Screen
General Outline (details are available on request)
Embryos derived from matings of G1 males with G2 females are thoroughly examined on E18.5 (day of plug = E 0.5) to detect gross and microscopic abnormalities.
First, they are examined for overt external and internal (organ) gross abnormalities: specifically, mice are examined for palate and facial clefts, number of digits, posture and movement, edema, and general morphology. The mice are then dissected, the internal organs examined and the head, diaphragm, and gut are removed for additional processing.
For that purpose, the head is immersion fixed in Bouin's fixative and subsequently sectioned in a 1mm matrix using a teflon razor blade. The slices are stained in Hematoxylin and examined under a stereo dissecting microscope. The diaphragm is fixed in 2% buffered paraformaldehyde and rhodamine-labelled alpha-bungarotoxin (1:1000; Molecular Probes, Eugene, Oregon) is used to visualize the acetylcholine receptors on the muscle fibers. This way the motor innervation pattern and postsynaptic differentiation can be seen in a whole-mount preparation.
The gut is removed, fixed in paraformaldehyde and stained histochemically for acetylcholine esterase to visualize the enteric nervous system. This is done by Dr. Robert Heuckeroth at Washington University, St. Louis.
Once deviants are established as heritable, they are analyzed with more detailed histological procedures as appropriate.
