Article Figures & Data
Figures
- Figure 1.
Nrg4 and ErbB4 mRNA expression in single cells of the adult mouse cortex obtained from RNA-sequencing public repository data. A, Fraction in percentage of single cells expressing Nrg4 (purple) and ErbB4 (violet) in each class of cells: glutamatergic, GABAergic, non-neuronal, and endothelial. B, Fraction and levels of expression of Nrg4 and ErbB4 mRNA in seven subclasses of glutamatergic cortical neurons. C, Nrg4 and ErbB4 mRNA levels of expression in single cells in all the clusters in the glutamatergic neurons class from the ALM L5 IT (L5 IT ALM) subclass. P10 (D–I) and P30 (L–Q) IHC co-labeling of NRG4 and Fezf2, NRG4 and Gpr88, ErbB4 and Fezf2, and ErbB4 and Gpr88. NRG4 and ErbB4 co-expression at P10 (J, K) and P30 (R, S). Arrows indicate co-expression of both proteins. Dashed boxes indicate where higher magnifications were taken from. Scale bars: 100 μm (high magnification) and 50 μm (lower magnification).
- Figure 2.
Neuronal soma size is decreased in motor cortex L2/3 and L5 neocortical pyramidal neurons of Nrg4−/− mice. Representative micrographs of Golgi-impregnated pyramidal neurons in L2/3 (A) and L5 (D) of the motor cortex at P10, P30, and P60 Nrg4+/+ and Nrg4−/− mice. Scale bar: 25 μm. B, E, Quantification of the area and perimeter. Data are mean ± SEM. C, F, One-way ANOVA with Fisher’s post hoc test for multiple comparison, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
- Figure 3.
Unaffected neuronal morphology and soma size of multipolar interneurons from Nrg4−/− mice. A, H, Representative micrographs of Golgi-stained multipolar interneurons from Nrg4+/+ and Nrg4−/− mice at P10 and P30, respectively. Scale bar: 25 μm. Total area (B, I), perimeter (C, J), total dendritic length (D, K), number of branch points (E, L), number of dendrites (F, M), and Sholl analysis (G, N). Data are mean ± SEM.
- Figure 4.
Neuronal soma size is reduced in vitro in Nrg4−/− pyramidal neurons and it is rescued by NRG4 treatment. A, Representative micrographs of cortical pyramidal neurons from Nrg4+/+ and Nrg4−/− embryos at 3 and 9 DIV stained with calcein-AM (upper panel) and transfected with GFP (lower panel), respectively. Nrg4−/− neurons were treated with recombinant NRG4 (100 ng/ml). Scale bar: 20 μm. Quantification of the area (B, D) and perimeter (C, E) of 90 neurons per condition. Data are mean ± SEM. One-way ANOVA with Tukey’s post hoc test for multiple comparison, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significant.
- Figure 5.
The loss of NRG4 impairs motor skills. A, Nrg4+/+ P60 male mice spent significantly more time on the accelerating Rotarod after trial 6 compared with Nrg4−/− mice. Data are mean ± SEM. Repeated measurements ANOVA with Bonferroni’s multiple comparison test versus T1; **p < 0.01 B, Comparison of slope values from each animal tested obtained after logarithmic regression of each individual performance. Horizontal lines represent mean ± SEM. Unpaired t test *p < 0.05. C, Traces of individual performance (T1–T7).
- Figure 6.
The loss of NRG4 does not affect anxiety or response to novelty. A, Time spent in the open arms of the elevated plus maze (EPM) was no different among P60 Nrg4+/+ and Nrg4−/− mice. B, Total distance traveled and time spent in the center (C) of the open-field was similar between Nrg4+/+ and Nrg4−/− mice. Novel object recognition (NOR) test. D, P60 Nrg4−/− mice spent a similar amount of time exploring a familiar object and a novel object. Data are mean ± SEM. E, The object discrimination percentage was similar between genotypes (calculated as the total amount of time spent exploring the novel object divided by the total time spent exploring multiple by 100).
- Figure 7.
NRG4 and ErbB4 co-expression with Ctip2, a major FEZF2 effector, in the motor cortex of young postnatal brain. CTIP2 and FEZF2 (A, B), CTIP2 and NRG4 (C, D), and CTIP2 and ERBB4 (E, F) co-labeling in neurons from the motor cortex at P10. Scale bar: 50 μm.
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