T he autosomal recessive mouse mutant reeler, which exhibits ataxia of gait, dystonic posture, and tremor (1), has provided a genetic model for neural development for half a century (2-4). Characteristic of the reeler mutant are abnormal lamination of the cerebral, cerebellar, and hippocampal cortices and neuronal ectopia in several brainstem nuclei (5-13). The gene Reln, the mutation of which is responsible for the reeler phenotype, has recently been cloned (14). Its protein product, reelin, has been identified as an extracellular matrix molecule (15). Despite such progress, the role of reelin in neuronal migration remains unknown. The migration of sympathetic preganglionic neurons reported here provides a simple model system that could facilitate studies of reelin function.Sympathetic preganglionic neurons undergo extensive migration during the development of the spinal cord. In the rat (16-18), it has been shown that postmitotic preganglionic neurons first migrate from the neuroepithelium along radial glial fibers to the ventrolateral spinal cord. There, along with somatic motor neurons, they form a primitive motor column. Preganglionic neurons next segregate from the somatic motor neurons and undergo a secondary dorsolateral migration toward the intermediolateral column (IML) region. This secondary migration is perpendicular to radial fibers and is independent of radial glial fibers. Upon terminal migration, the majority of preganglionic neurons become localized to the IML. A small number of preganglionic neurons settle in areas adjacent to the central canal.Results from our present study show that the migration of preganglionic neurons in the reeler mutant is disrupted. Moreover, reelin expression and in vitro function blocking studies suggest that reelin acts as a barrier to migrating preganglionic neurons. Materials and MethodsAnimals. The reeler mouse colony was originally derived from heterozygous B6C3Fe-a͞a-rl adults (The Jackson Laboratory). Homozygous and heterozygous mice were obtained by mating homozygous males with heterozygous females. The day on which a vaginal plug was detected was designated as embryonic day 0.5 (E0.5). Embryonic staging was verified by using the criteria of Rugh (19). Embryos were genotyped by using PCR (20).Sympathetic Nervous System of the Mouse. The anatomy of the sympathetic nervous system is shown in Fig. 1. Sympathetic preganglionic neurons are located primarily in the IML region of the thoracic spinal cord. Their axons exit the spinal cord in the ventral roots to enter into the paravertebral ganglia. Most preganglionic axons terminate in the paravertebral ganglia. Some axons pass through the paravertebral ganglia to innervate the prevertebral ganglia. Postganglionic neurons innervate smooth muscle, cardiac muscle, and glands. Identification of Preganglionic Neurons in the Mouse Embryo.Preganglionic neurons in embryos were retrogradely labeled with 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine (DiI) or fluorescent dextran amines (Molecular Probes). For dextran ...

Our previous study showed that the migration of sympathetic preganglionic neurons (SPN) in the spinal cord is affected in the reeler mutant. The present study, using morphometric analysis to describe and compare the location of SPN at progressive developmental stages, provides detailed information on how SPN migrate in the presence or absence of the reelin gene. We found that the initial migration (prior to E11.5) of SPN from the neuroepithelium to the ventrolateral spinal cord is similar in both control (wild-type and heterozygous) and reeler mice. However, as development progressed (E12.5-E15.5), SPN in control mice migrated dorsally toward the intermediate lateral spinal cord region, where 80% settled to form the intermediolateral column (IML); the rest migrated medially to locations between the IML and the central canal. In reeler, 80% of SPN migrated dorsomedially to cluster around the central canal, with the rest distributed between the central canal and the intermediate lateral spinal cord region. The present study also examined the relationship among SPN, Reelin, and radial glial fibers in control and reeler mice. Confocal microscopic studies showed that during their initial migration, SPN in both control and reeler mice were closely apposed to radial glial fibers in the ventrolateral spinal cord. The majority of SPN in control mice then migrated dorsolaterally, in a direction perpendicular to radial glial fibers, to form the IML. In contrast, the majority of SPN in reeler migrated in the same orientation as radial glial fibers back toward the central canal, instead of migrating dorsolaterally to form the IML. A possible explanation for these results is that Reelin acts to prevent SPN from back-migration on radial glial fibers toward the central canal.

The Reelin signaling pathway in the brain involves the binding of Reelin to very-low-density lipoprotein receptors (VLDLR) and apolipoprotein E receptor 2 (ApoER2). After Reelin binds the lipoprotein receptors on migrating neurons, the intracellular adaptor protein Disabled-1 (Dab1) becomes phosphorylated, ultimately resulting in the proper positioning of cortical neurons. Previous work showed that Reelin also affects the positioning of sympathetic preganglionic neurons (SPN) in the spinal cord (Yip et al. [2000] Proc Natl Acad Sci USA 97:8612-8616). We asked in the present study whether components of the Reelin signaling pathway in the brain also function to control SPN migration in developing spinal cord. Results showed that Reelin and reelin mRNA are found adjacent to migrating SPN. In addition, dab1 mRNA and protein are expressed by migrating SPN, and dab1-null mice show abnormal SPN migration similar to that seen in reeler. Finally, vldlr and apoER2 are also expressed in migrating SPN, and mice lacking both vldlr and apoER2 show aberrant SPN location that is identical to that of reeler and dab1-null mice. Because molecules known to be involved in Reelin signaling in the brain are present in the developing spinal cord, it is likely that the Reelin signaling pathways in the brain and spinal cord function similarly. The relative simplicity of the organization of the spinal cord makes it a potentially useful model system with which to study the molecular and cellular function of the Reelin signaling pathway in control of neuronal migration.

No abstract