Disruption of hippocampal development in vivo by CR-50 mAb against reelin - PubMed (original) (raw)

Disruption of hippocampal development in vivo by CR-50 mAb against reelin

K Nakajima et al. Proc Natl Acad Sci U S A. 1997.

Abstract

We previously generated a monoclonal alloantibody, CR-50, by immunizing reeler mutant mice with homogenates of normal embryonic brains. This antibody recently was shown to recognize a Reelin protein, which is coded by the recently identified candidate gene for the reeler mutation. However, it is still unclear whether Reelin, especially the CR-50 epitope region, is indeed responsible for the reeler phenotype in vivo. Here we show that Reelin is localized on Cajal-Retzius neurons in the hippocampus and that intraventricular injection of CR-50 at the embryonic stage disrupts the organized development of the hippocampus in vivo, converting it to a reeler pattern. Labeling experiments with 5-bromodeoxyuridine demonstrated that the labeled cells in the stratum pyramidale of the CR-50-treated mice were distributed in a pattern similar to that of reeler. Thus, Cajal-Retzius neurons play a crucial function in hippocampus development, and the CR-50 epitope on Reelin plays a central role in this function.

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Figures

Figure 1

Figure 1

Immunolocalization of the CR-50 epitope in the developing hippocampus. (A) Toluidine blue staining of wild-type mouse hippocampus at E17. (B and C) Immunohistochemical staining of E17 hippocampus with CR-50, showing localization of its epitope in the outer marginal zone of the hippocampus proper and in the dentate marginal zone. AD, area dentata; dmz, dentate marginal zone; hp, hippocampal plate; imz, inner marginal zone; omz, outer marginal zone; RI, regio inferior; RS, regio superior. (Scale bars, 100 μm.)

Figure 2

Figure 2

Characterization of the CR-50-positive cells. (A_–_C) Double-staining with CR-50 and anti-MAP2 on wild-type mouse hippocampus at E17. CR-50-positive cells (A) are also positive for anti-MAP2 (B) (arrowheads). A double-exposed photomicrograph of A and B is shown in C. (Scale bar in C, 50 μm.) (D) BrdUrd injected at E9.5 was detected with anti-BrdUrd antibody (red) at E17, showing that the CR-50-positive cells (green) arose at this stage (arrowheads). Some other CR-50-positive cells were labeled at E10 or E11 (not shown). (Scale bar, 10 μm.) (E_–_G) Double-staining with CR-50 (green) (E) and anti-calretinin (red) (F) on E17 mouse hippocampus revealed the existence of both calretinin-positive cells (arrowheads) and calretinin-negative cells (arrows) among the CR-50-positive population in the marginal zones. A double-exposed photomicrograph is shown in G. Early granule neurons in the area dentata are also calretinin-positive, but negative for CR-50. (Scale bar in G, 50 μm.) (H_–_J) Embryonic hippocampus was cultured at E17 and fixed the next day. The cells were double-stained with CR-50 (green) (H) and anti-calretinin (red) (I). A double-exposed photomicrograph is shown in J. There were both calretinin-positive cells (arrowheads) and calretinin-negative cells (arrows) among the CR-50-positive population. (Scale bar in J, 20 μm.)

Figure 3

Figure 3

Disruption of the organized cell layering in the stratum pyramidale of the regio superior and conversion to the reeler pattern with CR-50. (A) Binding of CR-50 to its epitope in vivo visualized by FITC-conjugated secondary antibody alone. (Scale bar, 50 μm.) (B) Cresyl violet staining of the regio superior of a P1 reeler mutant. Cell-poor rifts are shown with an arrowhead. (Scale bar, 50 μm.) (C) Wild-type P1 mouse that was injected with control mouse IgG at E12 and E14. (D_–_G) Wild-type P1 mice that were injected with CR-50 at E12 and E14 (D) or at E13 and E15 (E_–_G). Note the presence of cell-poor rifts in the stratum pyramidale and that the cells are rather loosely packed. (H_–_K) Dividing cells were labeled with BrdUrd at E13 and immunostained with anti-BrdUrd at P1. (H) Noninjected reeler, (I) noninjected wild-type mouse, (J) CR-50-injected wild-type mouse, and (K) control mouse IgG-injected wild-type mouse. Intensely labeled cells are shown with arrowheads. (Scale bar in H, 20 μm.) so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum.

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