Reelin controls position of autonomic neurons in the spinal cord

Abstract

Mutation of the reeler gene (Reln) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the Reln product, reelin, is thought to control cell-cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997) Dev. Dyn. 210, 157-172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997) Eur. J. Neurosci. 9, 1055-1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.

Figure 1

Anatomy of the mouse sympathetic nervous system.

Figure 2

Migratory pathways of sympathetic preganglionic neurons in upper thoracic spinal cord of wild-type and reeler mutant. Preganglionic neurons in embryos were retrogradely labeled with DiI crystals applied to the sympathetic ganglia; somatic motor neurons were retrogradely labeled with DiO crystals applied to the spinal nerves (a–d and f–i). Preganglionic neurons in postnatal mice were labeled by i.p. injection of Fluorogold (e and j). (a) E11 wild type. Transverse section shows that preganglionic neurons (Pn) have undergone their primary migration from the neuroepithelium to the ventrolateral spinal cord. (b) E12 wild type. Many preganglionic neurons (red) have migrated dorsally to separate from the somatic motor neurons (Mn, green). (c and d) E14 (c) and E16 (d) wild type. The majority of preganglionic neurons have completed their dorsal migration to arrive at their final location in the IML (Iml); a small number of neurons become localized to areas between the IML and the central canal (Cc). Note that in these micrographs, DiI labeling is found spanning the middle of the spinal cord as well as around the central canal. However, microscopic examination at higher magnification revealed that this is mostly fiber labeling. (e) Fluorogold labeling in a P30 mouse shows that the majority of sympathetic neurons are indeed located in the IML. (f) E11 reeler. Preganglionic neurons migrated, as in the wild-type mouse, to the ventrolateral spinal cord. (g) E12 reeler. Many preganglionic neurons stream toward the central canal, instead of migrating toward the IML as they normally do. The secondary migration of preganglionic neurons in the reeler mutant is, therefore, abnormal. (h and i) E14 (h) and E16 (i) reeler. The majority of preganglionic neurons have arrived at their final location adjacent to the central canal; a few neurons remain in the IML. Dh, dorsal horn. (j) Fluorogold labeling in a P30 reeler mutant confirms that the majority of preganglionic cell bodies are located adjacent to the central canal.

Figure 3

Blocking reelin function in slice culture of wild-type spinal cord disrupts preganglionic neuronal migration. E11.5 spinal cord slices were cultured for 2 days with and without reelin function-blocking antibody, CR-50. Preganglionic neurons were retrogradely labeled with FITC dextran amine. (a) Control culture without CR-50 shows that preganglionic neuronal migration to the IML is normal. (b) Embryo slice cultured in the presence of CR-50 shows abnormal migration of preganglionic neurons. As in the reeler mutant, the majority of preganglionic neurons (Pn) are located adjacent to the central canal, instead of at their normal location in the IML. Note that in this slice, the dorsal root ganglia (Drg) were inadvertently labeled because of their proximity to the sympathetic ganglia where dextran amine was injected. In the spinal cord, however, only preganglionic neurons are retrogradely labeled.

Figure 4

Relationship between reelin and migrating preganglionic neurons. (a) E11.5. CR-50 immunostaining shows that reelin is widely distributed in the newly formed ventral spinal cord. The only regions that are devoid of reelin are the neuroepithelium and the ventrolateral spinal cord, where preganglionic and somatic motor neurons are located (arrows). (b) E12. Retrograde dextran amine labeling of preganglionic neurons (green, arrows) combined with immunostaining of reelin (red) shows that reelin is present adjacent to migrating preganglionic neurons in the ventral spinal cord. (c) E12.5. Reelin is located more dorsally in the spinal cord. Again, the area devoid of reelin is where preganglionic neurons are located (arrows). (d) E14. Double immunostaining with nitric oxide synthase immunostaining for preganglionic neurons and CR-50 shows that reelin (red) is located more dorsally but is still adjacent to the preganglionic neurons (green, arrows) and separates them from regions of the central canal.