Figure 1.
Necl-4 expression in embryonic and postnatal spinal cords.
A–H: Spinal cord sections from E16.5, E18.5, P0, P4, P7, P15, P30 and P64 were subjected to ISH with Necl-4 riboprobe. Necl4 expression was detected in spinal cord white matter after E18.5, and persisted till adulthood. Scale bar, 100 µm.
Figure 2.
Co-expression of Necl-4 with neuronal and oligodendroglial markers.
Spinal cord sections from P15 were subjected to Necl-4 ISH (in blue), followed by immunohistochemical staining for NeuN and APC, respectively. The arrows and arrowheads indicate representative double stained neurons and mature oligodendrocyte in the ventral white matter, respectively. Scale bar, 50 µm.
Figure 3.
Reduced Necl-4 expression in Olig1 and Nkx2.2 null mutant spinal cords.
Spinal cord sections from wild-type, Olig1−/− and Nkx2.2 −/− mice at P3 were subjected to Necl-4 ISH. As indicated by the arrow, Necl-4 expression was significantly reduced in the white matter of mutant spinal cords. Scale bar, 100 µm. E. The counts of the Necl-4+ cells in the spinal cord white matter of wild type, Olig1 null mutant and Nkx2.2 null mutant mice. Student's t-test, n = 3, **p<0.01. Error bar, standard deviation.
Figure 4.
Expression of Necl-4 in brain oligodendrocytes.
A–B, Necl-4 expression in corpus callosum as detected by ISH. C–D, The forebrain coronal sections (P15) and cerebellum sagittal sections (P7) were subjected to ISH with Necl-4 probe (in blue) followed by immunohistochemical staining with anti-Olig2 antibody (in brown). The insets are the higher magnification of double positive cells. Scale bar, 50 µm.
Figure 5.
Generation of Necl-4 knockout mice.
A. Schematic representation of Necl-4 gene-targeting vector. In the targeting vector, the first exon of Necl-4 was replaced with the Cre-ERT2 and Neo cassette. B. Genotyping of F2 pups by Southern hybridization with 3′-probe. C. RT-PCR was performed to confirm the deletion of Necl-4 transcript. Primers were designed to detect transcription of the targeted 5′ end cDNA (exon 1 and exon2) and the full-length cDNA, respectively. GAPDH was the control. D. Spinal cord sections from WT and homozygous mice at P7 were subjected to in situ hybridization with Necl-4 riboprobe. Scale bar, 50 µm. E. Western immunoblotting of spinal cord tissues (from P7 wild-type, Necl-4 heterozygous and homozygous pups) with anti-Necl-4 monoclonal antibody. β-actin was used as the internal control.
Figure 6.
Oligodendrocyte differentiation and axonal myelination in Necl4 null mutant mice.
A-D. Immunofluorescent staining of wildtype and Necl-4−/− spinal cord tissues at P15 with mature markers MBP and APC. Scale bar, 100 µm. E. Statistical analysis of the number of APC+ cells at P15 (n = 4). Student's t-test, p = 0.11. Error bar, standard deviation. F–K. Myelination in spinal cord and sciatic nerves at P7 from the wild type and Necl-4 null mutants was examined under electron microscope. Scale bars, 5 µm. High magnification images of individual axons were shown as the inserts. H. Statistical analysis of the percentage of myelinated axons (n = 3). Student's t-test, p = 0.34. Error bar, standard deviation. K. Statistical analysis of the G-ratio of sciatic nerves (n = 3). Student's t-test, p = 0.39. Error bar, standard deviation. SC, spinal cord. SN, sciatic nerve.
Figure 7.
The expression level of Necl1, Necl2 and Necl3 proteins was not changed in the CNS of Necl4 mutants.
A. Western immuneblotting of brain tissues (from P10 wild-type and Necl-4 homozygous mice) with anti-Necl-1, anti-Necl-2 and anti-Necl-3 antibodies. β-actin was the internal control. B. Statistical analysis on the relative expression level of Necl1, Necl2 and Necl3 with Student's t-test (n = 3. Necl1, p = 0.70. Necl2, p = 0.72. Necl3, p = 0.69). Error bar, standard deviation.