Fig 1: Blocking DCC signaling in rat embryos impairs TmesV axon projection and TH cell migration, and TmesV axons misproject in DCC mutant mouse embryos. Embryos cultured in presence of Ig-Fc as control (A,D) or with the DCC-Fc chimera to alter TmesV axon projection (B,E) followed by double immunostaining for TH and β-III tubulin; their approximate location is indicated in diagram in (C). Overall organization of TmesV neurons and their axon projection was dramatically altered by DCC-Fc (B,E) along with a drastic disorganization of TH neurons and their impaired caudal migration into the hindbrain. White arrows in (D,E) indicate TH+ cells apposed to β-III tubulin axons in single optical sections and orthogonal digital projections (D′,D″ and E′,E″ respectively); in (B) green arrows indicate TH+ cells that do not interact with β-III tubulin axons, white arrowhead indicates interruption of the longitudinal tract of the TmesV, and white arrow indicates lack of projection of the TmesV into the hindbrain. Panel (F) is a flat-mounted hemibrain of a wild-type mouse embryo stained for β-III tubulin and (G) is from a DCC−/− embryo. Arrow in (G) indicates misprojecting axons and arrowheads indicate dorsal displacement of the TmesV axon bundle. (H) The treatment with DCC-Fc reduced the density of TmesV axon bundles. The area covered by β-III tubulin axon bundles in projections of confocal Z-stacks was measured. Asterisk indicates statistical difference between controls and DCC-Fc treated embryos (Wilcoxon-Mann-Whitney test, p = 0.0143). (I) The TmesV axon bundle was dorsally displaced in DCC−/− embryos. The distance of the center of the axon bundle from the dorsal edge of the brain was measured at the isthmic region for wild-type and mutant embryos. Dorsal displacement is revealed by a significantly shorter distance of the bundle in mutant embryos. Asterisk indicates statistical difference between controls and DCC−/− embryos (Wilcoxon-Mann-Whitney test, p = 0.008). Scale bar: 100 μm. Bars in (A,D,F) also apply to (B,E,G), respectively.
Fig 2: TH migratory cells from the midbrain do not express the markers Phox2a and DCC present in LC cells. Double immunostaining for TH (red) and either Phox2a (A–C) or DCC (D,E) reveals that TH+ migratory cells from the midbrain do not express these markers found in NA neurons at this stage. In all panels, rostral is to the left and dashed lines indicate the MHB. Note that although expression of DCC was absent from TH migratory cells (D), it stained TH+ cells of the prospective LC (arrows in (E)) and longitudinal axons in the midbrain (arrow in (D)). Arrowhead in (D) indicates apposition of DCC axons and TH cells. Location of (B,C) is indicated in (A). Panels (D,E) correspond to locations similar to (B,C), respectively. LC, locus coeruleus. Scale bars: 100 μm. Bar in (E) also applies to (B–D).
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