Figure 4.

CCK expression in olfactory system in vitro mimics in vivo expression pattern. A, Schematic of a nasal explant removed from an E11.5 mouse and maintained in serum-free media for 7 DIV. Ovals represent olfactory pit epithelium (OPE); in center is nasal midline cartilage (NMC) and surrounding mesenchyme. GnRH-1 neurons (dots) migrate from OPE and follow olfactory axons to the midline and off the explant into the periphery. Boxed region 1 within schematic is area shown in B, whereas boxed region 2 is area shown in C–E. B, Double immunocytochemistry was performed using antibodies to GnRH-1 (brown) and NCAM (blue). Many GnRH-1 neurons are detected that migrated off the explant into the periphery along NCAM-immunoreactive olfactory fibers (see insert, arrow; asterisk indicates GnRH-1 neuron; NCAM-positive axon bundles). It should be noted that ICC on nasal explants is performed on the entire tissue; thus, cells in the periphery of the explant and in the inner tissue mass undergo dimensional changes. GnRH-1 cells located in the periphery migrate on a monolayer of fibroblast and appear flatter than those on the explant. C, D, Double immunofluorescence was performed on 7 DIV explants using antibodies to NCAM (red) and CCK (green). C, Immunostaining for NCAM revealed robust staining in OPE structures as well as along olfactory axons emerging from OPE and directed toward midline area. The OPE structures are contained in the inner tissue mass of the explant where the thickness is ∼300 μm. Cells located inside these areas appear more round, and cell density is very high; thus molecule localization is not limited to a single dimension. D, CCK immunoreactivity displayed similar labeling pattern as NCAM. E, Simultaneous visualization of both fluorescent wavelengths indicated that both OPE cell soma as well as processes were positive for CCK and NCAM. Scale bars: B, 27 μm; inset in B, E, 10 μm; C, D, 40 μm.