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. 2019 Jun 13;8:e45545. doi: 10.7554/eLife.45545

Figure 2. Loss of Shh signaling in astrocytes results in increased spine density on layer V neurons.

(A-C) GFP immunostaining of representative apical dendritic segments from (A) layer V neurons, (B) layer II/III neurons, and (C) CA1 neurons in WT and Gfap Smo CKO mice. Scale bar, 5 μm. (D) Spine density of apical dendrites from layer V neurons across various postnatal ages and in adult mice. Data points represent individual cells, bars represent mean ± SEM. Statistical analysis performed by two-way ANOVA with Tukey’s post-hoc test for multiple comparisons. *, p<0.05; **, p<0.01; ****, p<0.0001. Significance is stated as Gfap Smo CKO compared to WT at a given age. (E) Spine density of apical dendrites from layer V neurons in adult CamKIIa Smo CKO mice. (F–G) Spine density of apical dendrites from layer II/III (F) and CA1 pyramidal neurons (G) in WT and Gfap Smo CKO mice. Data points represent individual cells, bars represent mean ± SEM. Statistical significance assessed by Student’s t-test. n ≥ 3 animals per genotype for all ages except P14, where n = 2 animals per genotype.

Figure 2—source data 1. Analysis of spine density.
elife-45545-fig2-data1.xlsx (22.8KB, xlsx)
DOI: 10.7554/eLife.45545.010

Figure 2.

Figure 2—figure supplement 1. Gfap-Cre recombination occurs primarily in astrocytes.

Figure 2—figure supplement 1.

(A) Representative image of tdTomato expression in the cortex of Gfap-Cre;Ai14 mice. w.m., white matter. Scale bar, 125 μm. (B–D) Expression of the astrocyte marker S100β and tdTomato in somatosensory cortex. Scale bar, 25 μm. (E) Fraction of recombined cells identified as astrocytes in the cortex of adult Gfap-Cre;Ai14 mice. (F–H) Expression of the neuronal marker NeuN and tdTomato in somatosensory cortex. (I) Fraction of recombined cells identified as neurons. (J– L) Expression of the oligodendrocyte marker CAII and tdTomato in somatosensory cortex. (M) Fraction of recombined cells identified as oligodendrocytes. White arrows show double-labeled cells, blue arrows show cells that express the indicated marker but are tomato-negative, and yellow arrows show single-labeled tomato-positive cells. Data points represent individual brain sections analyzed. Bar graphs represent mean ± SEM. n = 2 animals, four sections per animal.
Figure 2—figure supplement 2. Gfap Smo CKO mice have reduced levels of Smo.

Figure 2—figure supplement 2.

Quantitative PCR of cortical RNA shows a significant reduction in Smo expression. Data points represent individual animals, bars represent mean ± SEM. Statistical analysis assessed by unpaired Student’s t-test.
Figure 2—figure supplement 2—source data 1. Quantification of Smo mRNA.
elife-45545-fig2-figsupp2-data1.xlsx (13KB, xlsx)
DOI: 10.7554/eLife.45545.008
Figure 2—figure supplement 3. Spine density on layer V basal dendrites are not affected in adult Gfap Smo CKO mice.

Figure 2—figure supplement 3.

(A–B) Representative images of basal dendrites analyzed from layer V neurons expressing Thy1-GFPm in both WT (Smofl/fl) and Gfap Smo CKO (Gfap Smofl/fl) animals. Scale bar, 5 μm. (C) Graph comparing the density of spines on basal dendrites from WT and Gfap Smo CKO animals. Data points represent individual cells, bars represent mean ± SEM. Statistical significance was assessed using unpaired Student’s t-test. n = 3 animals per genotype.