Figure 1.
Contactin1 as a candidate gene for this mutation.
A) The genetic interval of Chromosome 15 between D15Mit242 and D15Mit192 (2.5 Mb) is shown. Genomic features including protein-coding genes are indicated. The image is from the Ensembl genome browser (http://ensembl.org/index.html). B) The structure of CNTN1 is schematized; the protein has an N-terminal signal peptide for secretion, 6 immunoglobulin domains (Ig), 4 fibronectin repeats (FN), and a GPI-linked carboxy-terminal modification for attachment to membranes. C) Immunoblot of CNTN1 in spinal cord and brain from two unaffected littermate control mice and two affected mice reveals an absence of CNTN1 protein in the affected mice. β-actin was used as a loading control.
Figure 2.
Neuromuscular analysis of Cntn1 mutant mice.
A) Neuromuscular junctions in wild type P11 mice have a plaque-like field of postsynaptic acetylcholine receptors (red) that is beginning to become convoluted. This is completely overlapped by the presynaptic motor nerve terminal (green). B) A similar NMJ morphology is seen in Cntn1 mutant mice. Histological examination of longitudinal and cross sections of control (C,D), and mutant (E,F) hind limb muscles at P13 did not reveal hallmarks of myopathy. G,H) Transmission electron microscopy was used to evaluate sarcomere anatomy in the tibialis anterior muscle. The structure was not adversely affected by the mutation. I) The absolute maximal contractile force of the extensor digitorum longus muscle was reduced in mutant mice. J) When normalized for muscle weight, contractile force of mutant muscle was not significantly different than control. The scale bar in H represents 14 µm in A, B, 72 µm in C–F, and 3 µm in G, H.
Table 1.
Additional in vitro contractile properties of directly stimulated EDL muscles.
Figure 3.
Dystrobrevin and Syntrophin localization in Cntn1 mutant muscle.
Cross sections of triceps surae from three mutant and three littermate control mice (ages P14, P15, and P16) were labeled with antibodies recognizing dystrobrevins and syntrophins (green). NMJs were counterstained with α-bungarotoxin (red). Merged images as well as the green channel with NMJs denoted by arrowheads are shown. Immunolabels are noted at left, genotypes are noted at top (control left, mutant right). The scale bar in the lower right is 25 µm.
Figure 4.
Expression and localization of Cntn1 in the retina.
A–C In situ hybridization for Cntn1 expression in the wild type mouse retina at P5 (A), P10 (B), and P21 (C). A subset of cells in the retinal ganglion cell layer (bottom) and inner nuclear layer are positive for Cntn1 expression. Photoreceptors in the outer nuclear layer (top) do not have signals above background. D) Double label in situ hybridization with Cntn1 and syntaxin1a at P21 demonstrates that some amacrine cells in the inner nuclear layer are positive for Cntn1 expression (arrowheads). Other Cntn1-positive cells are likely to be bipolar cells based on their position (arrows). E) In the retinal ganglion cell layer, a majority of cells expressing Cntn1 at P21 also express Thy1, a marker of ganglion cells. F) Immunolabeling of retinas with anti-CNTN1 antibodies at P14 revealed strong labeling of the synaptic plexiform layers, as well as immunoreactivity in the cellular layers, particularly the inner nuclear layer. G) Immunolabeling retinas from Cntn1 mutant mice revealed a marked reduction, but not an elimination of signal intensity in images collected with equivalent parameters.
Figure 5.
Normal retina development in Cntn1 mutant mice.
A,B) The retina and optic nerve head of wild type and Cntn1 mutant mice stained with H&E did not reveal any defects. C,D) More peripheral areas of the retina were also normal in the mutant. E,F) Staining for dopaminergic amacrine cells (red, anti-tyrosine hydroxylase) and horizontal cells and calbindin-positive amacrine cells (anti-calbindin, green), showed that cell bodies and dendritic arbors of these cells were in the appropriate anatomical location. G,H) Staining for intrinsically photoresponsive ganglion cells (anti-melanopsin, green) and starburst amacrine cells (anti-choline acetyltransferase, red) showed that these cells were also unaffected by the loss of Cntn1. The scale bar in H represents 145 µm in A–D and 72 µm in E–H.