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. 2020 May 26;9:e54903. doi: 10.7554/eLife.54903

Figure 3. Multiplex RNA in situ hybridization validation of proposed immature PV IN markers.

(A) Heatmap of MGE IN marker genes for cluster 1 Sst- and Sst+ INs. (B–D’’) Lower magnification fluorescent images of multiplex RNA in situ hybridization for Mef2c (B–B’’), Sp9 (C–C’’) and Igfbp4 (D–D’’). (E–G’’’) Higher magnification fluorescent images of multiplex RNA in situ hybridization for Mef2c (E–E’’’), Sp9 (F–F’’’) and Igfbp4 (G–G’’’). White arrows point to INs that are marker (Mef2c, Sp9 or Igfbp4) and tdTomato positive but Sst negative. Violin plots showing the normalized expression value (Y-axis) of each cell analyzed in each group for Mef2c (H), Sp9 (J) and Igfbp4 (L). Quantification of the percentage of tdTomato+; Sst- and tdTomato+; Sst+ INs in the neocortex and in the hippocampus that are either Mef2c+(I), Sp9+(K) or Igfbp4+(M). Scale bar in (B) = 100 um, (B’’) = 200 um and (G) = 25 um. 2 WTs and multiple brain sections per animal were used for quantification. For statistical analysis, multiple independent t-tests without same standard deviation assumption were conducted to compare the expression of each gene in each brain region. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3.

Figure 3—figure supplement 1. Schema depicting the hypothesis behind candidate PV IN marker discovery In Pai et al., 2019, we fate-mapped the Sst-IRES-Cres lineage and discovered that > 90% of Sst-IRES-Cre labeled CINs remain to be SST-expressing in adulthood.

Figure 3—figure supplement 1.

Very few Sst-IRES-Cre CINs become PV-expressing. We hypothesize that the MGE-derived immature CINs that are SST-negative have a high likelihood to become PV CINs.
Figure 3—figure supplement 2. Multiplex RNA in situ hybridization validation of proposed immature PV IN markers.

Figure 3—figure supplement 2.

(A–C’’) Lower magnification fluorescent images of multiplex RNA in situ hybridization for Tcf12(A-A’’), Elmo1(B-B’’) and Arl4d(C-C’’). (D–F’’’) Higher magnification fluorescent images of multiplex RNA in situ hybridization for Tcf12(D-D’’’), Elmo1 (E–E’’’) and Arl4d (F–F’’’). White arrows point to INs that are marker (Tcf12, Elmo1 or Arl4d) and tdTomato positive but Sst negative. Violin plots showing the normalized expression value (Y-axis) of each cell analyzed in each group for Tcf12(G), Elmo1(I) and Arl4d(K). Quantification of the percentage of tdTomato+; Sst- and tdTomato+; Sst+ INs in the neocortex and in the hippocampus that are either Tcf12+(H), Elmo1+(J) or Arl4d+(L). Scale bar in (A) = 100 um, (A’’) = 200 um and (D) = 25 um. 2 WTs and multiple brain sections per animal were used for quantification. For statistical analysis, multiple independent t-tests without same standard deviation assumption were conducted to compare the expression of each gene in each brain region. *p<0.05, **p<0.01, ***p<0.001.
Figure 3—figure supplement 3. Higher magnification view of HINs from multiplex fluorescent in situ hybridization.

Figure 3—figure supplement 3.

Higher magnification of HINs that are marker positive [Igfbp4(A-A’’’), Sp9(B-B’’’), Mef2c(C-C’’’), Tcf12(D-D’’’), Elmo1(E-E’’’) and Arl4d(F-F’’’)] that are either in the Sst-lineage or in the proposed immature PV-lineage. Circle: representative cell that is Sst+ In but has the proposed PV marker; Triangle: representative cell that is proposed to be PV candidate IN; Square: representative cell that is Sst+ IN. Scale bar in (A) = 25 um.