Evolutionarily Conserved, Alternative Splicing of Reelin during Brain Development Catherine Lambert de Rouvroit, Be ´atrice Bernier, Ine `s Royaux, Vinciane de Bergeyck, and Andre ´ M. Goffinet Neurobiology Unit, University of Namur School of Medicine, B-5000 Namur, Belgium Received August 10, 1998; accepted September 28, 1998 Reelin is the protein defective in reeler mutant mice and plays a pivotal role in brain development. How- ever, some uncertainties remain about the relation- ship between reelin and the reeler phenotype. It is generally believed that reelin, secreted by specific neuronal types such as Cajal–Retzius cells, acts at short distance via the extracellular matrix on target neurons, the response of which requires the Dab1 gene product. However, the pattern of reelin expression in some structures such as olfactory bulb, retina, and spinal cord suggests that the protein might be en- dowed with different functions. In the present study, we identify two uncommon, evolutionarily conserved splicing events in the 3' part of the transcript that result in different forms of the protein. First, a 6-nucle- otide, brain-specific microexon is skipped in about 10% of reelin RNA. In addition, an alternative polyadenyla- tion event involving 10–25% of reelin mRNA results in secretion of a truncated protein lacking the terminal, highly basic stretch. This alternative reelin is gener- ally expressed in the same cells as the major form, but is almost undetectable in retina and spinal cord. Both alternative splicing events are present in mouse, rat, and man, suggesting that the corresponding reelin forms are functionally important.  1999 Academic Press Key Words: brain development; cerebral cortex; reeler; reelin; disabled-1; alternative splicing; polyadenyla- tion; microexon. INTRODUCTION Reelin is the product of the gene affected in reeler mutant mice (4) and thus plays a key role in neural development, particularly in the organization of cell patterns at the end of radial neuronal migration (12, 21, 22, 33). In addition, a possible involvement for reelin in behavioral disorders was recently proposed (20). Reelin is a secreted extracellular matrix glycopro- tein (5). A phenotype indistinguishable from reeler is generated by null mutations of the disabled-1 (Dab1) gene (16, 17, 39, 43). Dab1 encodes a cytoplasmic protein with features of a putative tyrosine kinase adapter (16). Studies of reelin expression using in situ hybridization (4, 19, 37) and anti-reelin antibodies (6, 7, 29) showed that reelin is expressed by specific neuronal types that are mostly unaffected by the reeler trait. By contrast, Dab1 expression appears more closely correlated with the mutant phenotype (17,39), suggest- ing that reelin acts locally, in a juxtacrine fashion, on target neurons which use Dab1 as a key element of their response to reelin (13). However, there is high reelin expression in the olfactory bulb, retina, spinal cord, and some other structures that are barely affected in reelin or Dab1-deficient mice. Furthermore, expres- sion of reelin is these structures is high even in nonmammals (21, 22). Such observations suggest that various reelin isoforms may serve specific functions, evocative in this respect of the isoforms of N-CAM or immunoglobulins (reviewed, for example, in 8). In the present work, we examined whether different forms of the reelin message occur and report the characteriza- tion of two evolutionarily conserved alternative splic- ing events in the 3' end of the reelin gene, namely the brain-specific inclusion of a 6-nucleotides bona fide microexon and the presence of alternative polyadenyla- tion. MATERIALS AND METHODS Animals Normal BALB/c mouse and Wistar rat embryos were obtained by checking for a vaginal plug, the day of the plug being day zero (E0); the day of birth is postnatal day zero (P0). Fetal brains were removed after killing the mother by cervical dislocation under deep ether anesthesia. Cold and ether anesthesia were used respec- tively for neonatal–early postnatal animals and for mice age P10 and more. 3' RACE 3' RACE was performed as described (11) with the following modifications. Poly(A) RNA was prepared from E15 mouse brain total RNA, using the PolyA Tract kit (Promega). First-strand cDNA synthesis was primed Experimental Neurology 156, 229–238 (1999) Article ID exnr.1999.7019, available online at http://www.idealibrary.com on 229 0014-4886/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.