Fig. 4. Impaired migration and axon growth in sympathetic precursors of Ret^-/- embryos. (A-F) Transverse (A,D) and parasagittal (B,C,E,F) sections stained with anti-class III ß tubulin (TUJ1 antibodies; red) and anti-Phox2b (green) (A,D) or anti-TH antibodies (B,C,E,F). (A,D) While many sympathetic precursors with long axons are observed in wild-type embryos, few sympathetic precursors extend long axonal processes in Ret^-/- embryos at E10.5. (B,E) Tips of postganglionic fibers emanating from the SCG towards the internal carotid artery (B, white arrows) are absent in Ret^-/- embryos (E). (C,F) Sympathetic trunks exiting from the STG (C, white arrows) are absent in Ret^-/- embryos (F). (G-L) Pictures of whole-mount TH immunohistochemistry showing rostral and ventral region to the left and bottom, respectively. (G,J) Formation of primary sympathetic chain is comparable between Ret^-/- and wild-type littermates at E10.5. Black arrowheads depict the developing brachial plexus. (H,K) The SCG primordium is short and displays an exaggerated constriction (K, white arrows) in Ret^-/- embryos (K) at E11.5. Black arrows indicate C7 vertebral level. (I,L) While sympathetic precursors are evenly distributed in the lumbar chain and prevertebral regions of wild-type embryos (I), these precursors are found as clumps of cells in Ret^-/- embryos (L). (M,N) Ventral view of whole-mount TH staining of E15.5 embryos, showing rostral region on top. In this preparation, the lower jaw is removed for better visualization of the sympathetic trunks. Major sympathetic trunks of the SCG project rostrally in E15.5 wild-type embryos (M, arrow). In some Ret^-/- embryos, these trunks are thin and project caudally (N, arrows). Note that the vertebral nerve growing from the STG (M, arrowhead) is not yet formed in Ret^-/- embryos at this developmental stage. Abbreviations: Scg, the superior cervical ganglion; Stg, the stellate ganglion. Scale bar: 80 µm in A,D; 220 µm in B,C,E,F; 55 µm in G,H,J,K; 70 µm in I,L; 1 mm in M,N.