Figure 6. Loss of Id4 results in activation of quiescent RGLs in the adult hippocampus.

(A) Design of the experiment for acute and long-term deletion of Id4 from RGLs of the adult hippocampus using Id4^cKO mice. (B) Immunolabeling for GFAP, tdTomato, Ascl1 and DAPI staining in control and Id4^cKO mice after 5 days of tamoxifen administration. Yellow arrows indicate Ascl1-positive RGLs. Scale bar, 30 µm. (C–D) Quantification of Ascl1 protein in tdTomato+ RGLs in control, Id4^Het and Id4^cKO mice after 5 days of tamoxifen administration. Loss of both copies of Id4 results in increases in the number of Ascl1-expressing cells and in the levels of Ascl1 protein in RGLs (Control vs Het p=0.3276; Control vs cKO p=0.0067; Het vs cKO p=0.0381; protein levels p=2.01E-5). n = 4 for control, Id4^Het and Id4^cKO mice. (E) Quantification of Ascl1 protein in tdTomato+ RGLs control, Id4^Het and Id4^cKO mice 30 days after tamoxifen administration. The percentage of RGLs positive for Ascl1 is increased in Id4^cKO mice compared with control mice 30 days after Id4 deletion (Control vs Het p=0.996; Control vs cKO p=0.311; Het vs cKO p=0.315). n = 5 for control and Id4^Het mice, n = 6 for Id4^cKO mice. (F) Immunolabeling for GFAP, tdTomato, Ki67 and DAPI staining in control and Id4^cKO and control mice after 5 days of tamoxifen administration. Yellow arrows indicate Ki67-positive RGLs. Scale bar, 30 µm. (G–H) Quantification of the fraction of Ki67+ tdTomato+ RGLs in control, Id4^Het and Id4^cKO mice, 5 days (G) and 30 days (H) following tamoxifen administration. The percentage of Ki67+ tdTomato+ RGLs is strongly increased following acute deletion of both copies of the Id4 allele, and remained significantly increased, albeit to a lesser extent, following long-term deletion. (Control vs Het P65 p=0.7595, P90 p=0.980; Control vs cKO P65 p=0.0049, P90 p=0.0036; Het vs cKO p=0.0101, P90 p=0.0026). n = 4 for P65 control, Id4^Het and Id4^cKO mice at P65; n = 5 for P90 control, Id4^Het and Id4^cKO mice. (I) Immunolabeling for GFAP, tdTomato, Id1 and DAPI staining in control and Id4^cKO and control mice 30 days after tamoxifen administration. Yellow arrows indicate Id1-positive RGLs. Scale bar, 30 µm. (J) Quantification of the fraction of Id1+ tdTomato+ RGLs 30 days after tamoxifen administration in control and Id4^cKO mice. Loss of Id4 results in a 2-fold increase in the fraction of tdTomato+ RGLs positive for Id1 immunoreactivity, from 38.3 ± 4.5% to 74.1 ± 1.0% (p=0.0016). n = 3 for both control and Id4^cKO. (K) Immunolabeling for GFAP, tdTomato, Id3 and DAPI staining in control and Id4^cKO and control mice 30 days after tamoxifen administration. Yellow arrows indicate Id3-positive RGLs. Scale bar, 30 µm. (L) Quantification of the fraction of Id3+ tdTomato+ RGLs in (K). Id3 is increased by more than 8-fold in tdTomato+ RGLs following Id4 deletion, from 9.7 ± 1.0% to 75.3 ± 1.1% (p=1.87E-6). n = 4 for control mice and n = 3 for Id4^cKO mice. Error bars represent mean ± SEM. Significance values: ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. See also Figure 6—figure supplement 1.

Figure 6—figure supplement 1. Expression of Id4, Ascl1, Ki67, Id1 and Id3 in RGLs following loss of Id4.

(A) Structure of the Id4^flox allele. (B) Immunolabeling for GFAP, tdTomato, Id4 and DAPI staining in control and Id4^cKO mice demonstrating Id4 elimination in Id4^cKO mice after 5 days of tamoxifen administration. Yellow arrows indicate tdTom+Id4+ RGLs; white arrows indicate tdTom+Id4- RGLs. Scale bar, 30 µm. (C) Quantification of single molecule RNA in situ hybridization (RNAscope) for Ascl1 RNA in control and Id4^cKO mice 5 days of tamoxifen administration. Ascl1 transcripts are decreased following Id4 deletion, suggesting the increased protein level is a result of post-transcriptional regulation. n = 2 mice for both control and Id4^cKO. (D) Immunolabeling for tdTomato, Ascl1, Ki67 and DAPI staining in Id4^cKO mice after 5 days of tamoxifen administration. Ascl1 and Ki67 are frequently co-expressed in tdTomato+ RGLs, as indicated by the yellow arrows. Scale bar, 30 µm. (E) Quantification of the co-expression of Ascl1 and Ki67 in control and Id4^cKO mice 5 days of tamoxifen administration. The fraction of tdTomato+ RGLs expressing both Ascl1 and Ki67 is 2.8 ± 1.9% in control mice and 6.3 ± 0.8% in Id4^cKO mice. n = 2. (F) Quantification of the fraction of proliferating (Ki67+) tdTomato+ RGLs co-expressing Ascl1 in control and Id4^cKO mice 5 days of tamoxifen administration. 34.1 ± 14.1% of Ki67+tdTomato+ RGLs also express Ascl1 in control mice, which increases to 54.4 ± 8.9% in Id4^cKO mice, indicating that Ascl1 and Ki67 expression are highly correlated. n = 2. (G) Quantification of the number of tdTomato+ RGLs per mm³ in the DG of control, Id4^Het and Id4^cKO mice 5 days and 30 days after tamoxifen administration shows no change in overall stem cell number following Id4 deletion at either time-point. Significance determined using ordinary one-way ANOVA. For control mice, n = 4 at P65 and n = 5 at P90; n = 4 for P65 and n = 6 for P90 Id4^Het mice; n = 5 for Id4^cKO mice at P65 and P90. (H–I) Quantification of the fraction of Id1-positive and Id3-positive tdTomato+ RGLs in Id4^cKO mice compared with control mice 5 days after Id4 deletion shows both are strongly increased, from 47.4 ± 7.3% to 88.0 ± 3.1% for Id1 and 11.24 ± 2.9% to 74.4 ± 2.1% for Id3. n = 3 for control and Id4^cKO mice. (J–M) Single molecule RNA in situ hybridization (RNAscope) for Ascl1 RNA (magenta) along with immunostaining for GFAP and Id3 in P65 control mice shows that most Id3+ RGLS express Ascl1 (J, yellow arrow; quantified in L) and the vast majority of Id3+Ascl1+ cells in the SGZ co-express Id4 (K, yellow arrow; quantified in M). Scale bar, 30 µm. Panel three in (J) shows magnification of yellow box; scale bar 10 µm. n = pooled data from three mice. Error bars represent mean ± SEM. Significance values: ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.