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RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons

¹ Departments of Pathology and Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Box 8118, St Louis, MO 63110, USA
² Department of Pediatrics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8116, St Louis, MO 63110, USA
³ Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8113, St Louis, MO 63110, USA

View larger version (68K): [in a new window]   Fig. 1. Generation of Ret-TGM mice. (A) Homologous recombination between Ret gene (top) and the targeting vector (second line) resulted in insertion of TGM and floxed-Tn5-neo (third line). Tn5-neo was successively removed in mice to generate Ret-TGM allele (bottom). Arrowheads indicate loxP sites. (B) Southern blot analysis for RetTGM mutation using genomic DNA from RW-4 ES cells (control), homologous recombinant ES clone (clone 108), and tails of wild-type (+/+), heterozygous (TGM/+) and homozygous (TGM/TGM) mice. The DNA samples were digested with NcoI and hybridized with radiolabeled probe, indicated in Fig. 1A. (C) Representative pictures of TGM expression in the nervous system. Signals obtained by RET immunohistochemistry (left) completely overlaps that of GFP fluorescent signal provided by TGM (middle) in trigeminal ganglion of newborn RetTGM/+ mice (merged image, right). (D) No RET protein expression is detected in spinal motoneurons (left) that are identified by GFP fluorescence (right) in RetTGM/TGM newborn mice. Abbreviations: K, KpnI; N, NcoI. Scale bar: 55 µm in C; 100 µm in D.

View larger version (115K): [in a new window]   Fig. 2. Aberrant position and impaired innervation of the SCG in newborn Ret-/- mice. (A,B) Parasagittal section of newborn mouse head stained with Hematoxylin and Eosin (rostral part is on the left). The SCG (white arrowheads) is located far caudal to its normal location near the bifurcation of the carotid artery (black arrows) in Ret-/- mouse. (C) Whole-mount TH immunohistochemistry showing connection of the SCG to the STG (arrowhead) by the cervical sympathetic trunk (white arrow). (D) Ganglion volume of the SCG. Closest vertebral level to the center of the ganglion is shown by x-axis. WT1,2, wild type; KO, Ret-/- mice; R, right SCG; L, left SCG. Note that the location and the volume vary between the right and left side of the same animal (KO1R versus KO1L, KO2R versus KO2L). (E,F) SCG neurons of Ret-/- mice are often small and display immature morphology. (G-J) The internal carotid nerve (white arrows) is abnormally branched and fails to travel rostrally in Ret-/- mice. Dotted circles depict the inner ear. (I,J) Dorsal view of the newborn head, showing total absence of the internal carotid nerve. The brain was removed in this preparation. (K,L) Absence of TH-positive fibers in the peripheral lobe of the submandibular gland of Ret-/- mice. Scale bar: 2 mm in A,B; 600 µm in C; 550 µm in E,F; 1 mm in G,H; 1.7 mm in I, J.

View larger version (91K): [in a new window]   Fig. 3. Newborn Ret-/- mice display deficits in the thoracic and lumbar sympathetic chain and prevertebral sympathetic ganglia. (A-J) Whole mount TH staining of the mouse body. (A,B) The STG (arrowheads) is small, and displays a defective axonal projection pattern in Ret-/- mice. Note the projection of the vertebral nerve (white arrows) is attenuated in Ret-/- mice. Black arrow indicates the SCG, which is located abnormally close to the STG. (C-F) Individual chain ganglia (black arrows) are small or missing (white arrow in D) at both thoracic (C,D) and lumbar (E,F) levels in Ret-/- mice. (G,H) Prevertebral sympathetic ganglion complex. The celiac superior mesenteric ganglion complex (yellow arrows) surrounding the exit of superior mesenteric artery (dotted circle) is dramatically reduced in size, whereas the aortico-renal ganglion (white arrowhead) is abnormally large in Ret-/- mice. (I,J) Sympathetic innervation along the branch of superior mesenteric artery is severely depleted in Ret-/- mice. Ad, adrenal gland. Scale bar: 900 µm in A,B; 600 µm in C,D; 550 µm in E-H; 100 µm in I,J.

View larger version (89K): [in a new window]   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.

View larger version (121K): [in a new window]   Fig. 5. Cell death of sympathetic neurons increases perinatally in Ret-/- mice, owing to lack of non-GFL neurotrophic factors. (A-F) Thionin (A,B), Phox2b and BrdU (C,D) and activated-caspase 3 (E,F) staining of E16.5 STG. The STG of Ret-/- embryos is mostly composed of neurons containing little cytoplasm (B), and with mixture of excessive dividing neuronal precursors (D) and dying cells (F). (G) Neuronal cell death is increased during the perinatal period in the SCG and STG of Ret-/- animals. Error bars indicate the standard deviation. (H-M) Ret gene expression monitored by GFP immunohistochemistry in the SCG of RetTGM/+ and RetTGM/TGM animals at E11.5 (H,I), E15.5 (J,K) and P0 (L,M). Ret expression is markedly downregulated in most developing neurons in the SCG by E15.5. The Ret-expressing population in the SCG is comparable in size between RetTGM/+ and RetTGM/TGM mice at P0. The broken line demarcates the SCG (L,M). (N-Q) GFP (green) and activated caspase 3 staining (red) of the SCG of newborn RetTGM/+ (N, P) and RetTGM/TGM (O,Q) mice. Arrows (N,O) depict cells that are doubly stained. Despite the apparent increase of caspase 3-positive cells in RetTGM/TGM mice, GFP and caspase 3-positive populations remain largely distinct (P,Q). Scale bar: 35 µm in A-F, 140 µm in H,I,L,M,P,Q; 80 µm in J,K; 50 µm in N,O; 100 µm in P,Q.

View larger version (83K): [in a new window]   Fig. 6. Expression analysis of Artn mRNA in developing rat embryos. Radioactive in situ hybridization for Artn mRNA on parasagittal sections of E12.5 (A), 14.5 (B,C) and 16.5 (D-F) rat embryos. High levels of Artn mRNA are detected in the dorsal aorta (A,B, white arrows), the superior mesenteric, vertebral and celiac arteries (B,D, arrowheads). At E14.5, the vertebral artery also expresses high levels of Artn mRNA (C). Note that Artn expression in the dorsal aorta is undetectable at E16.5 (D, arrow). By contrast, persistent high expression is detected in small branches of the mesenteric arteries (arrows in dark- (E) and bright- (F) field photographs) at E16.5. Scale bar: 500 µm in A,C,E,F; 250 µm in B; 1100 µm in D.

View larger version (51K): [in a new window]   Fig. 7. ARTN promotes neurite outgrowth and attracts growing axons. (A-F) Representative pictures of STG explants stained with anti-ß tubulin antibodies. Explants were dissected out from E13.5 mouse embryos and cultured in the presence of NT3 (100 ng/ml (B)), NRTN (20 ng/ml (C), 100 ng/ml (D)) and ARTN (20 ng/ml (E), 100 ng/ml (F)). (A) No factor. (E,F) Moderate and robust neurite outgrowth is induced by lower and higher concentrations of ARTN, respectively. (G) Axon-covering area was measured by use of NIH Image software (three explants for each condition; bars indicate mean standard errors). (H,I) ARTN-impregnated bead (dotted circle) induces robust neurite outgrowth of the sympathetic ganglion explant and directs growing axons toward the bead. Note that axons emanating from the distal side of the explant steer towards the source of ARTN (arrows in I). Scale bar: 350 µm for A-F; 250 µm for H,I.

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