Fig. 1. Expression of transgenic and endogenous RNCAM isoforms in OSNs. (A) Schematic representation of OMP-GpiRNCAM and OMP-TmRNCAM transgene constructs. A 6.0 kb mouse genomic OMP sequence that spanned from an endogenous BamHI (B) site to the OMP initiation codon was cloned in frame with RNCAM cDNA coding sequence (white boxes). The cDNA corresponded to sequences between initiation codons and endogenous EcoRI (E) sites in the 3' UTRs of GpiRNCAM and TmRNCAM transcripts, respectively. Indicated are SV40 polyadenylation sites (SV40 pA, gray boxes), the transmembrane domain (TM) and two AU-rich boxes in the 3' UTR of the GpiRNCAM transcript. (B-H) In situ hybridization analyses of RNCAM expression in OE. Hybridization signals appear white after darkfield illumination. (B-D) Coronal sections of 2-week-old mice hybridized with a probe corresponding to the extracellular domain of RNCAM, common to both isoforms. Broken line indicates the Z1-Z2-border. RNCAM expression in OE of (B) control mouse, (C) GpiRNCAM transgenic mouse and (D) TmRNCAM transgenic mouse are shown. (E-H) Higher magnification of in situ hybridization analyses of RNCAM isoforms in transgenic mice. Sections were hybridized with a TmRNCAM-specific probe (E-F) or a GpiRNCAM-specific probe, (G-H). Signals corresponds to endogenous TmRNCAM expression in GpiRNCAM transgenic mice (E), endogenous and transgenic TmRNCAM expression in TmRNCAM transgenic mice (F), endogenous GpiRNCAM expression in TmRNCAM transgenic mice (G) and endogenous and transgenic GpiRNCAM expression in GpiRNCAM transgenic mice (H). The Z1-Z2 border (broken line) in transgenic mice was determined by hybridizing serial sections with probes corresponding to the isoform not overexpressed from the transgenic construct. E and H are serial OE sections of GpiRNCAM transgenic mice, while F and G are serial OE sections of TmRNCAM transgenic mice. In situ hybridization analyses shown in E-H were generated from sections that were processed, hybridized and exposed identically and in parallel. Results from analyses of two transgenic mouse lines for each RNCAM isoform were consistent. (I,J) Laminar distribution in OE of endogenous RNCAM mRNA isoforms. In situ hybridization analyses of serial OE sections of control mice. (I) In situ hybridization signal generated with a ³⁵S-labeled cRNA probe that recognized TmRNCAM transcripts throughout all cell layers of OSNs. (J) In situ hybridization with a ³⁵S-labeled cRNA probe that recognized GpiRNCAM transcripts preferentially located in the cell layer of OE containing immature OSNs close to the basal lamina (broken line).

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