A panel of monoclonal antibodies against reelin, the extracellular matrix protein defective in reeler mutant mice

Abstract

Reelin, the extracellular matrix protein defective in reeler mutant mice, plays a key role during brain development. We therefore raised antibodies directed against various reelin epitopes in order to facilitate biochemical and cell biological studies of this important molecule. Homozygous reeler mice with a large deletion of most of the reelin gene were immunized with fusion proteins and carrier-coupled peptides corresponding to parts of the reelin sequence. Monoclonal antibodies were produced using classical procedures, screened using ELISA and–or western blot prepared with the antigen, and tested by immunohistochemistry and immunoprecipitation assays to detect endogenous reelin. The labeling of Cajal–Retzius cells in the embryonic mouse telencephalon was selected as criterion for positivity in immunohistochemistry. A total of 11 monoclonal antibodies were obtained, providing useful additions to the widely used antibody CR-50. Five are directed against the N-terminal part of reelin, among which three recognize the region that has significant similarity with F-spondin, and two are specific for hinge region located downstream from the F-spondin similarity region and upstream from the reelin repeats. Six antibodies are directed against the C-terminal part of reelin, among which one anti-peptide antibody recognizes the highly basic C-terminal segment. Antibodies against the N-terminal region stain well in immunohistochemistry. By comparison, the labeling of embryonic Cajal–Retzius cells with antibodies directed against the C-terminal region is weaker, suggesting that this part of the molecule might be modified or not be as readily accessible in the tissue as the N-terminus.

Introduction

Reeler is an autosomal recessive mutation in the mouse that results in abnormal brain development (Caviness and Rakic, 1978, Goffinet, 1984, Goffinet, 1995). The product of the reeler gene has been mapped (Bar et al., 1985) and cloned (D'Arcangelo et al., 1995). It encodes a protein named reelin. The structure of reelin, deduced from the cDNA sequence, suggests that it is a secreted extracellular matrix protein, as was recently confirmed by cell biological analyses (D'Arcangelo et al., 1997). Reelin is 3461 amino acids long and displays the following organization (Fig. 1). A signal peptide is followed by a sequence similar to F-spondin (20–25%), separated from reelin repeats by a unique segment. This region is followed by eight similar repeats of 350–390 amino acids, I–VIII, each composed of two subrepeats named A and B separated by an EGF motif; subrepeats A and B display significant similarity. The reelin protein ends with a highly basic C-terminal sequence of 33 amino acids. Studies of reelin mRNA distribution using in situ hybridization revealed heavy expression in Cajal–Retzius cells in the marginal zone as well as in cerebellar granule cells and several other structures in the embryonic central nervous system such as mitral cells in the olfactory bulb, retina and spinal cord (Schiffmann et al., 1997). Studies of the reelin protein have so far relied upon the CR-50 antibody (Ogawa et al., 1995), which stains Cajal–Retzius cells and cerebellar granule cells (Miyata et al., 1996) in normal but not in reeler mice in which reelin is absent. This antibody was subsequently shown to recognize an epitope of reelin (D'Arcangelo et al., 1997) and to perturb the action of reelin in vivo and in vitro (Miyata et al., 1997, Nakajima et al., 1997). Polyclonal anti-peptides that recognize reelin have also been described (D'Arcangelo et al., 1997, de Bergeyck et al., 1997, Nakajima, personal communication). As reelin is such a large molecule, the understanding of its function would presumably benefit from the availability of additional antibodies directed against different portions of the molecule. In the present work, we report the production and characterization of monoclonal antibodies directed against the N-terminal and C-terminal regions of reelin.