Figure 1.
Figure 1. Defective MF axon pruning in Lnx1 null mice. (A) The schematic indicates the strategy for generating Lnx1 knockout by substituting Lnx1 with LacZ gene. 5′ untranslated sequences and exon coding sequences are shown as gray and dark boxes, respectively, and introns and 5′ and 3′ nontranscribed regions are shown as lines. EcoRI (E) and NcoI (N) restriction sites are indicated. The Lnx1-LacZ targeting vector, including a PGK-Neo cassette, was designed to replace Lnx1 exons including the p80 and p70 promoters after homologous recombination. The 5′ and 3′ external probes used to confirm the results by Southern blot are shown with the expected sizes indicated (see Fig. S1 B). (B) Immunofluorescence of β-gal in Lnx1 mutant mice indicated specific expression of Lnx1 in hippocampal CA3 pyramidal cell layer. Bars: 200 µm (left); 50 µm (right). (C) NeuroTrace dye–labeled CA3 pyramidal cells in 3-wk-old Lnx1−/− mice are loosely packed compared with WT littermates. Calbindin staining showed that the IPB axon–penetrated CA3 pyramidal neurons layer are much longer in Lnx1−/− mice compared with WT littermates. White brackets delineate IPB length, and distance between arrowheads delineates the IPB length in CA3 pyramidal neurons. Bars: 200 µm (left); 100 µm (right). (D) Quantification of the length ratio of IPB to CA3 area (from the hilus to the curvature) in Lnx1−/− mice and WT littermates during postnatal development. n = 5–7 mice. (E) Comparison of IPB length within CA3 pyramidal neurons in Lnx1−/− mice and WT littermates during postnatal development. n = 5–7 mice. (F) Diagram of coculture assay. (G) TdT+ primary hippocampal axons (arrowheads) cocultured with Lnx1−/− neurons showed fewer pruned axons than with WT neurons. Bar, 100 µm. (H) Comparison for neurite lengths in neuronal coculture with WT or Lnx1−/− neurons. (I) Comparison of primary neurite numbers in neuronal coculture with WT or Lnx1−/− hippocampal neurons. n = 72–403 neurons per group in H and I. (J) Comparison of longest neurite length (arrowheads) of calbindin-negative neurons and calbindin-positive neurons dissected from TdT+ hippocampi cocultured with WT or Lnx1−/− hippocampal neurons. n = 16–19 neurons per group. Bar, 100 µm. Means ± SEM; two-way ANOVA with Tukey’s post hoc test (J) or Student’s t test (D, E, and I); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Defective MF axon pruning in Lnx1 null mice. (A) The schematic indicates the strategy for generating Lnx1 knockout by substituting Lnx1 with LacZ gene. 5′ untranslated sequences and exon coding sequences are shown as gray and dark boxes, respectively, and introns and 5′ and 3′ nontranscribed regions are shown as lines. EcoRI (E) and NcoI (N) restriction sites are indicated. The Lnx1-LacZ targeting vector, including a PGK-Neo cassette, was designed to replace Lnx1 exons including the p80 and p70 promoters after homologous recombination. The 5′ and 3′ external probes used to confirm the results by Southern blot are shown with the expected sizes indicated (see Fig. S1 B). (B) Immunofluorescence of β-gal in Lnx1 mutant mice indicated specific expression of Lnx1 in hippocampal CA3 pyramidal cell layer. Bars: 200 µm (left); 50 µm (right). (C) NeuroTrace dye–labeled CA3 pyramidal cells in 3-wk-old Lnx1−/− mice are loosely packed compared with WT littermates. Calbindin staining showed that the IPB axon–penetrated CA3 pyramidal neurons layer are much longer in Lnx1−/− mice compared with WT littermates. White brackets delineate IPB length, and distance between arrowheads delineates the IPB length in CA3 pyramidal neurons. Bars: 200 µm (left); 100 µm (right). (D) Quantification of the length ratio of IPB to CA3 area (from the hilus to the curvature) in Lnx1−/− mice and WT littermates during postnatal development. n = 5–7 mice. (E) Comparison of IPB length within CA3 pyramidal neurons in Lnx1−/− mice and WT littermates during postnatal development. n = 5–7 mice. (F) Diagram of coculture assay. (G) TdT+ primary hippocampal axons (arrowheads) cocultured with Lnx1−/− neurons showed fewer pruned axons than with WT neurons. Bar, 100 µm. (H) Comparison for neurite lengths in neuronal coculture with WT or Lnx1−/− neurons. (I) Comparison of primary neurite numbers in neuronal coculture with WT or Lnx1−/− hippocampal neurons. n = 72–403 neurons per group in H and I. (J) Comparison of longest neurite length (arrowheads) of calbindin-negative neurons and calbindin-positive neurons dissected from TdT+ hippocampi cocultured with WT or Lnx1−/− hippocampal neurons. n = 16–19 neurons per group. Bar, 100 µm. Means ± SEM; two-way ANOVA with Tukey’s post hoc test (J) or Student’s t test (D, E, and I); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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