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doi: 10.1242/10.1242/dev.00317

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Differential function of RNCAM isoforms in precise target selection of olfactory sensory neurons

Department of Molecular Biology, Umeå University, Umeå, S-901 87, Sweden

View larger version (115K): [in a new window]   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).

View larger version (84K): [in a new window]   Fig. 2. Distinct distribution of RNCAM isoforms on axons of OSNs and Z1-specific NADPHd-activity. Images were processed to show RNCAM immunoreactivity in red, RNCAM in situ hybridization signal in white, NADPHd-activity in yellow and nuclear counterstain in blue. (A-C) Anti-RNCAM immunohistochemistry analyses of nerve and glomeruli layers in a region of the OB innervated by Z1 axons lacking endogenous RNCAM expression in (A) control mice lacking RNCAM immunoreactivity, (B) GpiRNCAM transgenic mice with RNCAM immunoreactivity predominantly localized to the in nerve layer, and (C) TmRNCAM transgenic mice with RNCAM immunoreactivity localized to both nerve and glomerular layers. The broken line indicates the border between the two layers. (D) In situ hybridization analyses showing RNCAM expression in Z2-4; (E) NADPHd histochemistry analyzes showing a signal selectively confined to Z1 OSNs (Z1). Note the complementary signals on either side of the Z1/Z2 border (broken line). (F) NADPHd histochemistry analysis showing signal in the nerve and the glomerular layers of the OB. (G) Anti-RNCAM immunohistochemistry analysis showing RNCAM immunoreactivity in the nerve and the glomerular layers of the OB. Note the complementary signals on either side of the broken line. Experiments were carried out using serial coronal sections of OE (D,E) and OB (F,G).

View larger version (156K): [in a new window]   Fig. 3. Unaltered distribution of NADHPd-positive glomeruli in RNCAM transgenic mice. (A-F) Histochemistry analyzes of NADPHd activity (black) in the OB. (A-C) Dorsal views of whole-mount OB preparations; control mouse (A), GpiRNCAM transgenic mouse (B) and TmRNCAM mice (C). (D-F) Coronal sections of the OB at a rostrocaudal position indicated by a broken line in A-C from control mouse (D), GpiRNCAM transgenic mouse (E) and TmRNCAM transgenic mice (F). Although the intensity and exact position of NADPHd-positive glomeruli varied between individual mice in an RNCAM-independent manner, both control and RNCAM-transgenic mice showed an identical general topography of NADPHd-positive and NADPHd-negative OB regions.

View larger version (59K): [in a new window]   Fig. 4. Glomerular morphology and trajectory of misrouted P2 axons in GpiRNCAM transgenic mice. (A-D) High power images of P2 innervated glomeruli. Major P2 glomeruli showed a similar morphology in control mouse (A) and GpiRNCAM transgenic mouse (B). An increased number of a distinct type of P2 glomeruli, in which P2 axons contributed only fractionally to the total number of innervated axons, were detected in transgenic mice. These semi-innervated P2 glomeruli showed a similar morphology in mice heterozygous (C) and homozygous (D) for the targeted P2 allele. (E) Location (vertical lines) of serial coronal sections shown in E1-9. The semi-innervated P2 glomeruli analyzed was located 360 µm caudal to the major lateral P2 glomeruli. The drawing depicts a side view, with the major lateral P2 glomerulus (L), axon trajectory of P2 axons (red line) and semi-innervated P2 glomeruli (circle) located on the lateral side, whereas the major medial P2 glomerulus (M) is located on the opposite (medial) side of the olfactory bulb. (E1-9) High power images of serial coronal olfactory bulb sections from a GpiRNCAM transgenic mouse. Misguided P2 axons segregated from a lateral division of P2 axons close to the major lateral P2 glomerulus (E1), bypassed their correct target and coursed in a caudal and ventral direction (E2-7) to a glomerulus primarily innervated by axons of another OR specificity (E8-9). Photographs were processed to show ß-galactosidase staining in red, while UV visualized the nuclear counterstain in blue.

View larger version (12K): [in a new window]   Fig. 5. Increased number of semi-innervated P2 glomeruli in transgenic mice. Quantification of glomeruli in control and transgenic mice. P2 axons were visualized by ß-galactosidase staining and innervated glomeruli were counted manually. (A) Graph represents total number of major P2 glomeruli/olfactory bulb in control (cont) (n=36), GpiRNCAM (Gpi) (n=42) and TmRNCAM (Tm) (n=26) transgenic animals. Control and transgenic mice had the same number of major glomeruli. (B) Graph represents total number of semi-innervated P2 glomeruli/olfactory bulb in control mice (cont) (n=36), GpiRNCAM (Gpi) (n=42) and TmRNCAM (Tm) (n=26) transgenic animals. Both GpiRNCAM and TmRNCAM transgenic mice showed a significant increased number of semi-innervated P2 glomeruli. Results are mean±s.e.m. (***P<0,0001, Student's t-test). The data were obtained from progeny of two transgenic founder lines transgenic for each RNCAM isoform.

View larger version (24K): [in a new window]   Fig. 6. Two distinct RNCAM isoform-dependent activities influence segregation of P2 axons. Frontal (left) and side view (right) of olfactory bulb. The location of P2 semi-innervated glomeruli (white circles) relative to major lateral (L) and medial (M) P2 glomeruli (black circles) are shown in ten mice of each genotype. (A) Control mice (cont), (B) TmRNCAM transgenic mice (Tm), (C) GpiRNCAM transgenic mice (Gpi) and (D) double GpiRNCAM/TmRNCAM transgenic mice (Gpi/Tm). Three different domains (broken circles) of the OB, with an approximately diameter of 200 µm, contained semi-innervated P2 glomeruli. The trajectories of P2 axons innervating incorrect glomeruli within the domains are indicated with arrows. Note that overexpression of TmRNCAM results in targeting errors in close proximity to both the medial and the lateral P2 glomeruli within a domain in which spontaneous targeting errors can be found at a low frequency in control mice. Overexpression of GpiRNCAM results in targeting errors located caudally, distant from the main lateral P2 glomeruli. The phenotype of mice overexpressing both RNCAM isoforms is similar to that of TmRNCAM transgenic mice. These results indicate that two independent RNCAM isoform-specific mechanisms influence OR-specific axon segregation. The data were obtained from progeny of two transgenic founder lines transgenic for each RNCAM isoform.

View larger version (14K): [in a new window]   Fig. 7. RNCAM isoform-dependent variations in the number of laterally and medially located semi-innervated P2 glomeruli. Number of semi-innervated P2 glomeruli was quantified on serial sections of olfactory bulb stained for ß-galactosidase activity. Graphs represent total number of semi-innervated P2 glomeruli located in the medial (M) and lateral (L) hemisphere of the olfactory bulb in control mice (cont) (n=36), TmRNCAM transgenic mice (Tm) (n=26), GpiRNCAM transgenic mice (Gpi) (n=42) and TmRNCAM/GpiRNCAM (Gpi/Tm) double transgenic mice (n=16). Results are mean±s.e.m. ***P<0.0001, **P<0.005, *P<0.05; Student's t-test. The fractions of semi-innervated P2 glomeruli located proximal (37%) and caudal (63%) to the main lateral P2 glomerulus in GpiRNCAM transgenic mice were calculated from analyses (shown in Fig. 6) that were carried out to determine both rostrocaudal and mediolateral distances. The data were obtained from progeny of two transgenic founder lines transgenic for each RNCAM isoform.

View larger version (85K): [in a new window]   Fig. 8. Incorrectly targeted glomeruli are located in the NADPHd-negative region of OB. Shown are double NADPHd and ß-galactosidase histochemistry analyses of coronal OB sections of a double GpiRNCAM/P2-IRES-tau-lacZ transgenic mouse. (A) Image of a rostral section with semi-innervated P2 glomeruli on the lateral side of the OB. One lateral semi-innervated P2 glomerulus (boxed) is shown at high magnification. (B) Schematic representation of NADPHd (gray) and ß-galactosidase histochemistry (black) of P2 glomeruli on serial coronal sections throughout the rostrocaudal extension of the OB. Indicated are semi-innervated P2 glomeruli (white circles), major lateral and lateral P2 glomeruli (black circles). (C) Image of a rostral section with a semi-innervated P2 glomerulus located on the medial side of the OB. Semi-innervated P2 glomerulus and the major medial P2 glomerulus are boxed.