Having established that the expression of BAF subunits ubiquitous in neurons and astrocytic RGCs tends to cease within AGPs, we investigated the impact of BAF complex depletion on astrocytic RGCs. We took this step to determine whether the loss of the BAF complex would cause that astrocytic RGCs detach from the VZ and translocate as proliferative AGPs.
We addressed this question in vivo by means of BAF complex deletion targeted to late RGCs. To this end, we generated a transgenic mutant mouse model with human glial fibrillary acidic protein (hGFAP) promoter-driven dcKO of BAF155 and BAF170 (hGFAP-Cre dcKO) (Narayanan et al., 2015;
Nguyen et al., 2018; Zhuo et al., 2001).
hGFAP promoter driven recombination
First, by means of ROSA-tdTomato system (Madisen et al., 2010) used in heterozygous (Het) mice (see Chapter 2. Materials and Methods. 2.1) we traced the activity of hGFAP promoter within astrocytic RGCs. As presented in Figure 12 the choice of hGFAP promoter as a driver of recombination allowed us to affect all known dorsal and ventral forebrain astrocytic germinal zones (Bayraktar et al., 2014; Minocha et al., 2015; Tsai et al., 2012). In agreement with the literature (Anthony and Heintz, 2008), we found that hGFAP-Cre driven recombination starts in hippocampal and medial cortical VZ (Figure 12 A), later propagating to all areas of DP as development proceeds
51 Figure 12 | hGFAP promoter drives recombination in all astrocytic germinal zones of murine forebrain.
(A-C) Coronal sections of brains of hGFAP-Cre Het tdTomato mice at: E13.5 (A), E15.5 (B) and E17.5 (C).
tdTomato (tdTOM) signal in red, DAPI in blue. A-C, middle: single channel tdTOM images. Arrowheads indicate astrocytic germinal zones with recombination detected at given stage in: Cx VZ (A), MGE VZ (B) and LGE VZ (C). A-C, right: are magnified pictures of tdTOM positive cells in given areas. (D-F) Schematics showing adult forebrain astrocytes born in embryonic astrocytic germinal zones affected by recombination: (D) CC and Cx astrocytes descend from Cx RGCs (E) these of VPI and Str from MGE RGCs and (F) Str and PCx astrocytes from LGE RGCs. Graphics prepared based on (Bayraktar et al., 2014; Tsai et al., 2012). CC, corpus callosum; Cx, cortex; MGE, medial ganglionic eminence; MCx, medial cortex; LGE, lateral ganglionic eminence; RGCs, radial glial cells; PCx, piriform cortex; Str, striatum; VPl, ventral pallidum.
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(Figure 12A, B). Interestingly the activity of hGFAP promoter within ventral astrocytic germinal zone of MGE started to emerge later than that of DP and was observed around E15.5 (Figure 12B). The astrocytic RGCs affected latest were these of LGE where the recombination occurred around E17.5 (Figure 12C).
Based on previously published tracing studies (Tsai et al., 2012), we could indicate forebrain astrocyte populations, progenitors (astrocytic RGCs) of which exhibited hGFAP-Cre driven recombination (thus will be affected by introduced BAF complex dcKO). Accordingly, it is known that astrocytes of cortex and corpus callosum originate from cortical astrocytic RGCs (recombination within cortical VZ starts as early as E13.5, Figure 12D). At the same time astrocytes populating striatum and ventral pallidum as well as striatum and piriform cortex were shown to be derivatives of astrocytic RGCs of MGE (affected at E15.5, Figure 12E) and LGE respectively (affected at E17.5, Figure 12F).
Overall performed tracing studies confirmed that hGFAP promoter allows for targeting of all known dorsal and ventral astrocytic RGCs populations, thereby allowing us to affect different forebrain astrocyte populations.
Expression of BAF complex subunits in hGFAP-Cre dcKO forebrain
It has been previously established that dcKO of BAF155 and BAF170 results in a complete deletion of the entire BAF complex from the affected cells (Narayanan et al., 2015).
To assess the efficacy of this process within our hGFAP-Cre dcKO system (further referred to as dcKO for simplicity), we first compared the protein expression levels of BAF155 and BAF170 in WT and mutant E17.5 DP. The choice of this area allowed a proper evaluation of protein levels, as the cortical recombination in dcKO starts relatively early (around E13.5, Figure 12A), thus only a small fraction of tissue should contain KO unaffected cells.
Western blot (WB) analysis of E17.5 DP lysate proteomes (Figure 13) revealed a massive reduction in BAF155 and BAF170 protein abundance within dcKO cortex compared to WT (Figure 13A).
Quantitatively, the average levels of BAF155 and BAF170 expression in dcKO cortex amounted to 35-40% and 32-35% of these of WT (=100%), respectively (Figure 13F).
The observation of residual BAF155/BAF170 levels in dcKO prompted us to investigate the possible cellular sources of these proteins. Using immunochemistry, we evaluated BAF155 and BAF170 expression in E17.5 coronal sections of Het (used as control) and hGFAP-Cre dcKO tdTomato mice (Figure 13B). As expected, both BAF complex subunits were depleted in dcKO astrocytic germinal zones (Figure 13B and C) exhibiting hGFAP promoter activity (tdTOM tracing). A faint signal was observed in the LGE of dcKO (indicated by arrow head in Figure 13B). We conjectured that this was
53 Figure 13 | Loss of BAF complex subunits in astrocytic cortical RGCs affected by hGFAP-Cre dcKO. (A) BAF155 and BAF170 protein expression analysis in WT and dcKO murine E17.5 DP tissues. Left panel shows a schematic explanation of experimental principle. E17.5 WT/dcKO embryos were subjected to DP dissection, subsequent protein isolation and WB analysis. Right board shows representative blots indicating expression of BAF170 (MW~170kDa, upper blot), BAF155 (MW~155kDa, middle blot) and GAPDH (housekeeping gene, MW~36kDa, lower blot) in 3 independent WT (right panel) and 3 independent dcKO (left panel) E17.5 DPs after WB experiment. The molecular weight of proteins (in kDa) marked on the right side of each blot. Quantitative representation of WB analysis shown in F. (B-C) E17.5 Het and dcKO coronal sections stained with antibodies to BAF155/BAF170 (in green and grey).
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Endogenous tdTOM signal is red. (B) Overview pictures of Het (used as control) and dcKO medial area brain slices. For each down panels are single channel grey scale images. Regions exhibiting BAF155/BAF170 expression indicated with arrowheads. (C) Magnified insets of DP VZ (from the region of future M2 area, indicated in overview pictures by dashed line) showing downregulated expression of BAF155/BAF170 in the region of VZ in dcKO animals by comparison to WT. The surface of the VZ marked with dashed line. (D-E) Immunofluorescence analysis of mouse E17.5 WT and dcKO DP VZ coronal sections stained with antibody to astroglial progenitor marker – GLAST (in red) together with antibodies detecting BAF complex subunits (in grey): BRG1 (D) and BAF60a (E). Nuclear DAPI staining in blue.
Immunohistochemical analysis shown depletion of BRG1 and BAF60a in GLAST positive astrocytic RGCs of dcKO animals. For C, D and E full arrowheads indicate cells positive for given BAF complex subunit, empty arrowheads indicate cells lacking BAF subunit expression. (F) Box plot representing dcKO BAF170 and BAF155 PI shown as ratio of WT results measured from the blot for 3 independent animals. WB analysis indicated significantly reduced amount of BAF155 and BAF170 in E17.5 dcKO DP by comparison to WT.
(G) Percentages of given BAF subunit positive per total GLAST positive astrocytic RBCs in the DP of E17.5 WT and dcKO mice. Data are averages ± σ of n=3 animals. For F and G ***p≤0.001, *p≤0.05, in t student test. dcKO, double knockout; DP, dorsal pallium; Het, heterozygous; MW, molecular weight; PI, pixel intensity; VP, ventral pallium; VZ, ventricular zone; WB, Western blot WT, wild type.
due to the relatively late initiation of hGFAP promoter activity within this region, as described above.
In addition and importantly protoplasmic astrocytes found within ventral regions of postnatal dcKO brains were deprived of BAF complex subunits, indicating for the efficient dcKO within ventral astrocytic germinal zones (Appendix, Figure 43).
Our previous study indicated that dcKO of BAF155 and BAF170 in ESCs, cultured neurons and early cortical development results in a complete deletion of the entire BAF complex from the affected cells (Narayanan et al., 2015). We therefore determined whether astrocytic RGCs of dcKO mice express BAF subunits (Figure 13C-E). Accordingly, we immunohistochemically assessed GLAST positive astrocytic RGCs of cortex VZ of E17.5 dcKO (that presumably did not stain for BAF155/BAF170, Figure 13C) and Het cortical for the expression of 2 BAF complex subunits: BRG1 (Figure 13D) and BAF60a (Figure 13E). Strikingly, cortical VZ of dcKO (shown to be deprived of BAF155 and BAF170) exhibited staining for both, BRG1 and BAF60a. However, as indicated in Figure 13D, E and G numbers of GLAST positive E17.5 dcKO cortical astrocytic RGCs expressing BRG1 or BAF60a were highly reduced comparing to control animals (Figure 13G), indicating BAF complex degradation within these cells.
Thus we confirm the full elimination of BAF complex from astrocytic RGCs achieved by means of hGFAP-Cre driven dcKO of BAF155 and BAF170.
BAF complex depleted mice - initial severity assessment
The post-partum mortality of generated dcKO animals was sorely high, limiting their average lifespan to P3-4. The external appearance of P3-4 mutant mouse body did not differ from WT or Het
55 littermates, apart from their slightly smaller size. However a striking anatomical difference was observed between the brains of WT and dcKO mice (Figure 14A-D).
Figure 14 | Reduced cortical size of BAF complex depleted postnatal mouse brains. (A) Bright field images of WT and dcKO brains of P3 mice. (B) Box plot representing differences between R-C and M-L extent in WT and dcKO brains. The cartoon below indicates the dimensions measured. (C) DAPI staining of WT and dcKO coronal P3 murine brain slices showing visible anatomical differences. (D) Cortical thickness differences between M1 and S1BF areas of P3 WT and dcKO mouse brains. Horizontal lines within each bar indicate average value for (A) n=10-12 experimental replicates (WT RC/ML extent: n=10, dcKO RC/ML extent: n=12), (D) n=11-13 experimental replicates (WT M1 area: n=11, WT S1BF area: n=12, dcKO M1 area: n=13, dcKO S1BF area: n=12); for (B) R-C extent ***p≤0.001 in t-student test, for (B) M-L extent ***p≤0.001 in Mann-Whitney Rank Sum test, for (D) M1 area ***p≤0.001 in Mann-Whitney Rank Sum test and for (D) S1BF area *p≤0.05 in t-student test. dcKO, double knockout; M1, primary motor cortex; M-L, medio-lateral; R-C, rostro-caudal; S1BF, primary somatosensory barrel cortex; WT, wild type.
The undeveloped P3 dcKO brain displayed an atrophied cortex and cerebellum (Figure 14A).
Noticeable changes to the cortex were in its rostro-caudal and medio-lateral extents, which were measurably smaller (Figure 14B). Notably, as shown in Figure 14C, dcKO brains did not develop a hippocampal structure and featured a significantly reduced cortical thickness, particularly in medial
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areas. Quantitatively, measurements of primary motor cortex (M1) thickness indicated an almost 2.5 fold reduction compared to WT (Figure 14D). Strikingly, in more lateral areas, the thickness of cortex tended to normalize, with only minimal discrepancies between genotypes in S1BF area (Figure 14D).
Foreword to astroglial phenotype assessment
The purpose of this study was to elucidate the role of BAF complex in astrocytes development. As explained above, having generated transgenic mutant mouse model with specific hGFAP-Cre driven dcKO of BAF155 and BAF170, we were capable of targeting all 3 astrocytic germinal zones and eliminating BAF complex from astrocytic RGCs. Because our model featured high juvenile mortality, we could not assess astrogliogenesis beyond P3. As a result, the analysis presented henceforth will mostly focus on this developmental postnatal stage. We also evaluated the initial steps of cortical astroglia formation on E17.5 dcKO and WT tissues.