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. 2018 May 23;4:109–126. doi: 10.1016/j.isci.2018.05.014

Chromatin Remodeling BAF155 Subunit Regulates the Genesis of Basal Progenitors in Developing Cortex

Ramanathan Narayanan 1,7, Linh Pham 1, Cemil Kerimoglu 2, Takashi Watanabe 3, Ricardo Castro Hernandez 1, Godwin Sokpor 1, Pauline Antonie Ulmke 1, Kamila A Kiszka 1,5, Anton B Tonchev 3,6, Joachim Rosenbusch 1, Rho H Seong 4, Ulrike Teichmann 3, Jens Frahm 3, Andre Fischer 2,5, Stefan Bonn 2, Anastassia Stoykova 3,5,∗, Jochen F Staiger 1,5, Tran Tuoc 1,5,8,∗∗
PMCID: PMC6147019  PMID: 30240734

Summary

The abundance of basal progenitors (BPs), basal radial glia progenitors (bRGs) and basal intermediate progenitors (bIPs), in primate brain has been correlated to the high degree of cortical folding. Here we examined the role of BAF155, a subunit of the chromatin remodeling BAF complex, in generation of cortical progenitor heterogeneity. The conditional deletion of BAF155 led to diminished bIP pool and increased number of bRGs, due to delamination of apical RGs. We found that BAF155 is required for normal activity of neurogenic transcription factor PAX6, thus controlling the expression of genes that are involved in bIP specification, cell-cell interaction, and establishment of adherens junction. In a PAX6-dependent manner, BAF155 regulates the expression of the CDC42 effector protein CEP4, thereby controlling progenitor delamination. Furthermore, BAF155-dependent chromatin remodeling seems to exert a specific role in the genesis of BPs through the regulation of human RG-specific genes (such as Foxn4) that possibly acquired evolutionary significance.

Subject Areas: Neuroscience, Molecular Neuroscience, Developmental Neuroscience

Graphical Abstract

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Highlights

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    BAF155 potentiates PAX6 transcriptional activity

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    BAF155 and PAX6 co-regulate bIP genesis in developing cerebral cortex

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    BAF155 and PAX6 control bRG genesis through aRG delamination non-cell-autonomously

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    BAF155 suppresses the expression of human RG-specific genes in developing mouse cortex

Neuroscience; Molecular Neuroscience; Developmental Neuroscience

Introduction

The mammalian cerebral cortex is a sheet of cells covering the cerebrum and responsible for complex brain functions, such as sensory perception, motor response, and cognition, especially in higher primates. Cortical development is a tightly regulated process that involves waves of differentiative divisions of multiple progenitor types in the forebrain germinative zones, the ventricular (VZ) and subventricular zone (SVZ), to generate specific neuronal subtypes that make up the six cortical layers (Borrell and Gotz, 2014, Gotz and Huttner, 2005, Kriegstein et al., 2006, Leone et al., 2008, Molyneaux et al., 2007, O'Leary et al., 2007, Tuoc et al., 2014). Based on morphology, lineage commitment, molecular characteristics, and the site of cell division, the progenitor cells of the developing cortex are grouped into two major categories: apical (APs) and basal progenitors (BPs). The APs are cells that divide at the apical surface of the VZ and include neuroepithelial cells (NEs) and apical radial glial cells (aRGs). The BPs are derivatives of APs that divide in the SVZ and include two subtypes, the basal radial glial cells (bRGs) and basal intermediate progenitors (bIPs) (Lui et al., 2011, Florio and Huttner, 2014, Taverna et al., 2014, Tuoc et al., 2014, Dehay et al., 2015, Fernandez et al., 2016). In the course of mammalian evolution, the cerebral cortex has diversified from a smooth (lissencephalic) cortex in rodents to a folded (gyrencephalic) cortex in primates, including human. A specific trait of the primate and human cortices is the abundant presence of bRGs and bIPs, which together form a distinct zone, the outer sub-ventricular zone (oSVZ), located basal to the inner SVZ (iSVZ; identical to SVZ in rodents) (Betizeau et al., 2013, Fietz et al., 2010, Hansen et al., 2010, Kelava et al., 2012, Lukaszewicz et al., 2005, Wang et al., 2011). The remarkable enlargement of the oSVZ during evolution is strongly associated with an increased neuronal output, cortical expansion, and folding of the primate/human cortex (Borrell and Gotz, 2014, Dehay et al., 2015, Fernandez et al., 2016, Florio and Huttner, 2014, Lui et al., 2011, Sun and Hevner, 2014, Taverna et al., 2014, Tuoc et al., 2014). Despite the importance of the molecular machinery that regulates the genesis of BPs in cerebral cortex evolution, it is only beginning to be elucidated.

The transcription factor (TF) PAX6 is involved in many processes of mammalian corticogenesis, including generation of bRGs (Asami et al., 2011, Wong et al., 2015) and bIP specification (Georgala et al., 2011, Gotz et al., 1998, Quinn et al., 2006, Sansom et al., 2009, Tuoc et al., 2009). Recently, we and other groups reported that TF PAX6 interacts with multiple subunits of the chromatin-remodeling mSWI/SNF (or BRG1/BRM-associated factor, BAF) complex (Bachmann et al., 2016, Ninkovic et al., 2013, Tuoc et al., 2013a, Tuoc et al., 2013b). More specifically, we found that temporal interaction between TF PAX6 and BAF170 during early corticogenesis (in mouse, E10.5-E14.5) leads to down-regulation of bIP-specific gene expression by recruiting a transcriptional co-repressor complex to PAX6 downstream target genes, thus prohibiting bIP genesis (Tuoc et al., 2013a). In contrast, in the absence of BAF170, as seen in E14.5-E16.5 developing mouse cortex, the BAF complex includes an additional BAF155 subunit, which causes augmentation of the pool of bIPs with formation of a thicker cortex.

In this study, we examined the role of BAF155 in cortical development. We found that, BAF155 potentiates PAX6-dependent transcriptional activity in the developing mouse cortex such that the synergistic interaction between BAF155 and PAX6 controls the fate choice of aRG, hence promoting bIP genesis but suppressing the genesis of bRGs. We show results suggesting a cell-autonomous role for BAF155 in regulating aRG division and non-cell-autonomous mechanisms (PAX6-dependent and PAX6-independent) that appear to underlie the delamination of aRGs from the VZ to generate bRGs.

Results

Expression of BAF155 in Cortical Progenitors and Conditional Inactivation of Baf155 in Developing Mouse Cortex

Our previous study revealed a dynamic competition between BAF170 and BAF155 subunits within the BAF complex during progression of cortical neurogenesis and that they display complementary expression patterns (Tuoc et al., 2013a). Following a high expression during early corticogenesis (E10.5-E13.5), the expression of BAF170 in progenitors progressively decreases, becoming nearly absent during E15.5. In contrast, following a low expression during earlier stages, the expression of BAF155 peaks at E15.5-E16.5 (Tuoc et al., 2013a). To define which of the progenitor populations (in VZ or SVZ) expresses BAF155, we performed dual-IHC on cortical sections from E15.5 wild-type (WT) embryos with antibodies for BAF155 and markers for cortical progenitor subtype, including PAX6 for RG (Gotz et al., 1998) and TBR2 for bIPs (Hevner et al., 2006), followed by quantification of the double-positive cell populations: BAF155+PAX6+ and BAF155+TBR2+. We found around 72 ± 4% of BAF155+ cells to be PAX6+, whereas only 26 ± 1.89% were TBR2+ cells, implying that BAF155 co-localized predominantly with the RG marker PAX6 in the VZ (Figure 1A) as compared with the bIP marker TBR2 in the SVZ (Figure 1B).

Figure 1.

Figure 1

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Expression of BAF155 in Developing Cerebral Cortex

(A and B) Confocal imaging of coronal wild-type brain sections at E15.5 after immunostaining for BAF155 and RG marker PAX6 (A) or BAF155 and bIP marker TBR2 (B). Higher magnification of the VZ (white box) is presented in the inner panel.

(C) Statistical analysis revealed that most of BAF155+ cells are PAX6+ aRGs (72 ± 4%), whereas BAF155+/TBR2+ bIPs (26 ± 1.89%) were less than one-third of the BAF155+ cells. Abbreviations: VZ, ventricular zone; RG, radial glia; bIP, basal intermediate progenitor. Scale bars, 100 μm (A), 25 μm (B). See also Figure S1.

To investigate the functions of BAF155 in vivo, we generated cortex-specific Baf155 knockout mice by crossing floxed Baf155 (Baf155fl/fl) mice (Choi et al., 2012) with the Emx1-Cre line, in which Cre-recombinase is driven in cortical progenitors starting from E9.5 and reaching full recombination activity before E12.5 (Gorski et al., 2002). After performing IHC in E15.5 brain sections using an anti-BAF155 antibody, a complete loss of BAF155 protein in the pallium of Baf155fl/fl;Emx1-Cre embryos was observed, thus confirming Baf155 knockout (Figure S1A). The term “BAF155cKO” is used for all subsequent references to Baf155fl/fl;Emx1-Cre embryos. The BAF155cKO mice were viable, healthy, and fertile and reached adulthood. Although from our previous study we observed that the activation and inactivation of Baf170 profoundly affect the expression of BAF155 in cortical tissues, the expression of BAF170 is largely preserved in the BAF155cKO cortex (Figure S1A). To confirm that there is no increase in apoptosis, we performed IHC using antibody against cleaved-caspase3. The results indicated that BAF155cKO mice do not show an increase in apoptosis in the cortex, including in the PAX6+ RG cell population (Figure S1B).

BAF155 Controls Expression of a Large Set of PAX6-Target Genes by Potentiating PAX6 Transcriptional Activity

Previous findings indicated that PAX6 interacts with BAF subunits, including BRM, BAF170, and BAF155 in cortical cells (Ninkovic et al., 2013, Tuoc et al., 2013a). To investigate whether the interaction between BAF155 and PAX6 influences PAX6-dependent transcriptional activity, we used a PAX6-dependent reporter vector (pCON/P3) (Epstein et al., 1994, Tuoc and Stoykova, 2008) and primary cortical neural stem cell (NSC) culture from BAF155cKO and control embryos. The NSCs were nucleofected with pCON/P3 plus eGFP plasmids, and the expression level of luciferase was examined by western blot (WB) analysis after 2 days in vitro (DIV). We found that without affecting the expression of PAX6, the loss of BAF155 in NSCs led to a significantly reduced level of luciferase as compared with those from control embryos (Figures 2A and 2B). To determine if BAF155 regulates PAX6-dependent transcriptional activity in vivo, E13.5 brains of BAF155cKO and control embryos were electroporated with pCON/P3 plus eGFP plasmids. One day post-electroporation, cortical tissue was examined by WB for luciferase expression. Protein quantification revealed that BAF155cKO cortices contained only 23% of the amount of luciferase protein found in the control (Figures 2A and 2B), thus suggesting that BAF155 is required for normal PAX6-dependent transcriptional activity.

Figure 2.

Figure 2

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BAF155 and PAX6 Co-regulate Global Gene Expression Program in Developing Cerebral Cortex

(A and B) Luciferase reporter assay was performed using pCON/P3 construct containing PAX6 binding motifs that drives luciferase expression. The construct was transfected to cortical NSCs or electroporated into E13.5 brain, and samples were collected 2 DIV or 1 DPE, respectively, for western blotting (A). In both cases, the loss of BAF155 does not affect the expression of PAX6 (left chart in B). There is, however, a significant reduction of luciferase activity in BAF155cKO compared with control (right chart, B).

(C and D) RNA-seq was performed using cortical samples from E15.5 BAF155cKO and PAX6cKO embryos. Graphs depict the number of both down- and up-regulated genes in BAF155cKO (C) and PAX6cKO (D).

(E and F) The overlap between genes down- (E) and up-regulated (F) in BAF155cKO and PAX6cKO embryos.

(G and H) Gene ontology analysis with the top ten pathways revealed that both BAF155 and PAX6 regulate key genes involved in forebrain development, neurogenesis, and neuron differentiation. Values are presented as Means ± SEMs (***p < 0.001) Abbreviations: NSC, neural stem cell; DIV, days in vitro; DPE, days post-electroporation. See also Tables S1, S2, S3, S4, and S5.

To gain further insights into the functional interaction between BAF155 and PAX6, we determined gene expression changes by applying transcriptomic analysis of E15.5 BAF155cKO and PAX6cKO cortices (Figures 2C and 2D). In the PAX6cKO cortex, there were 2,956 down- and 2,415 up-regulated genes, whereas in the BAF155cKO cortex those numbers were 1,179 and 939, respectively (Figures 2C and 2D, Tables S1 and S2, p value < 0.01 & |fold change| > 1.2). Furthermore, we noticed considerable overlap between BAF155- and PAX6-regulated genes, with altered expression (up/down) in the corresponding mutants (Figures 2E and 2F, Table S3). Gene ontology (GO) analysis of the RNA-seq data of the down-regulated genes in PAX6cKO and BAF155cKO (Figures 2G and 2H) revealed that both BAF155 and PAX6 control the expression of genes related to forebrain development, neurogenesis, and neuron differentiation, among others (Figures 2G and 2H, Tables S4 and S5). Taken together, these findings suggested that BAF155 controls the expression of a large set of PAX6-regulated genes, possibly by potentiating PAX6 transcriptional activity.

BAF155 Controls Genesis of Basal Intermediate Progenitors

Earlier studies have identified TF PAX6 as an intrinsic determinant of the RG-cell fate that controls the choice of direct versus indirect mode of cortical neurogenesis, the latter depending specifically on bIP production (Georgala et al., 2011, Gotz et al., 1998, Quinn et al., 2006, Sansom et al., 2009, Thakurela et al., 2016, Tuoc et al., 2009, Tuoc et al., 2013a). In our RNA-seq experiment, the expression of over ten genes with known role in bIP genesis (such as Eomes/Tbr2, Neurog1, Neurog2, Neurod1, Unc5d, Cxcl12, Sstr2, Pou3f2, Pou3f3, Cux1, Cux2) was down-regulated not only in PAX6cKO (Figure 3A, Table S1), but also in the BAF155cKO cortex (Figure 3B, Table S2). We then tested whether BAF155 also controls the promoter activity of bIP genes, which are known to be direct targets of PAX6, such as Ngn2 (Scardigli et al., 2003), Tbr2, Cux1 (Sansom et al., 2009, Tuoc et al., 2013a). Indeed, we found that the knock down (KD) of BAF155 reduces PAX6-dependent activation of those promoters in the luciferase reporter assay (Figure 3C). Furthermore, we quantitatively studied bIP generation in the BAF155cKO cortex by examining the expression of the bIP marker TBR2 (Hevner et al., 2006), as well as NGN2 and AP2ϫ, both of which label progenitors transitioning between aRGs and bIPs (Arnold et al., 2008, Ochiai et al., 2009, Pinto et al., 2009, Sessa et al., 2008). We found that in BAF155cKO embryos, the number of TBR2+ bIPs and AP2ϫ+/NGN2+ cells (bIPs and aRGs) were substantially diminished (Figures 3D–3G, S2A, and S2B), suggesting that BAF155 cooperates with TF PAX6 to regulate bIP genesis in the developing cerebral cortex.

Figure 3.

Figure 3

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BAF155 Deficiency Leads to a Decreased Number of Basal Intermediate Progenitors in SVZ

(A and B) RNA-seq analysis of samples from E15.5 PAX6cKO (A) and BAF155cKO (B) cortices revealed transcriptional down-regulation of genes with known functions in bIP genesis.

(C) Luciferase reporter assay in Neuro2A cells indicates that BAF155 controls PAX6-dependent activation of bIP lineage-specific gene promoters (Ngn2, Tbr2, Cux1).

(D and E) IHC for bIP markers TBR2, NGN2 on coronal sections of E15.5 control (left) and BAF155cKO (right) cortices. Higher magnification is presented in the middle panels.

(F and G) Statistical analysis of the number of TBR2+ (F) and NGN2+ (G) cells in E15.5 cortices revealed diminished numbers of total TBR2+ and NGN2+ cells in the BAF155cKO compared with control.

Note in D and E the presence of TBR2+, NGN2+ cells in ectopic basal locations (white arrows). Values are presented as means ± SEMs (*0.01 < p < 0.05, **0.001 < p < 0.01, ***p < 0.001). Abbreviations: SVZ, subventricular zone; IZ, intermediate zone; bIP, basal intermediate progenitor. Scale bar, 100 μm. See also Figures S2–S5.

Ectopic Distribution of Cortical Progenitors in BAF155-Deficient Cortex

Although the total number of TBR2+/AP2ϫ+/NGN2+ bIPs is lower in the BAF155cKO mutant as compared with the control, an extensive ectopic presence of TBR2+/AP2ϫ+/NGN2+ cells (bIPs) was seen in the intermediate zone (IZ) and cortical plate (CP) (Figures 3D–3G, S2A, and S2B, white arrow). Intrigued by this finding, we next quantified the RG cell populations in the developing pallium by using both nuclear (PAX6, SOX2, AP2ϫ) and cytoplasmic (GLAST, BLBP) RG markers (Hartfuss et al., 2001). Interestingly, we found significantly more ectopic RGs in the IZ/CP of the BAF155cKO cortex that are PAX6+ (Figures 4A–4C), SOX2+/GLAST+, and AP2ϫ+/BLBP+ (Figure S2). This visibly enlarged set of PAX6+ and TBR2+ cells in the IZ/CP of the BAF155cKO cortex are cycling progenitors, because many of them are immunoreactive for KI67 as well as BrdU antibody upon 1 hr pulse-labeling (Figure 4D, filled arrows) but negative for NeuN staining (Figure 4D, empty arrows). Some of those RG in the IZ/CP were positive for phosphorylated Vimentin (pVIM, a cytoplasmic marker for dividing RG) and were also labeled with DiI, displaying their clear pia-directed basal process characteristic of bRGs (Figures 4E and 4F, white arrow) (Fietz et al., 2010, Hansen et al., 2010, Wang et al., 2011). Interestingly, the results from our RNA-seq analysis (Figure S3A) indicated that the expression of most of the recently identified bRG-specific genes, such as Tnc, Ptprz1, Fam107a, and Hopx (Pollen et al., 2015, Thomsen et al., 2016), was altered in the cortex of PAX6cKO and BAF155cKO mutants. Thus, both TF PAX6- and BAF155-dependent chromatin remodeling seem to play a role in controlling the gene expression program of bRGs.

Figure 4.

Figure 4

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BAF155-Deficient Cortex Displays Increased Number of Basal Radial Glial Progenitors

(A) IHC for RG marker PAX6 on coronal sections of control (left) and BAF155cKO (right) cortices at E15.5 and E16.5. Note the presence of more PAX6+ RG in ectopic basal locations (white arrows) in BAF155 mutant cortices.

(B and C) Statistical analysis of the total number of PAX6+ RG comparing BAF155cKO and control cortices across rostral, medial, and caudal regions at E15.5 (B) and dorsal cortical region at E16.5 (C). Although the total number of PAX6+ RG remains unaffected, a diminished number of PAX6+ RG in the VZ and an increased number of PAX6+ RG in the IZ was found in the BAF155cKO cortex as compared with control.

(D) IHC analysis indicated that these PAX6+, TBR2+ cells are NSCs, as many of them are immunoreactive for KI67 and BrdU (filled arrows) and are negative for NeuN (empty arrows).

(E and F) bRGs with a long basal but missing apical process in the BAF155cKO cortex shows double labeling after IHC with PAX6 and phosphorylated Vimentin (pVIM) antibodies (E) or DiI labeling (F). High magnification of PAX6+/pVIM+ bRG cells (marked by arrow) is shown in the lower panels.

(G and H) To compare RG mitotic cleavage plane orientation in control and BAF155cKO mutants, E15.5 cortices were stained for pVIM and phosphorylated Histone H3 (pHH3) to mark mitotic cells and mitotic chromatin, respectively (G). The observed angle between the cleavage plane and the apical surface of the cortex were grouped into three classes, 60–90° scored as vertical, 30–60° as oblique, and 0–30° as horizontal cell divisions. Note that in the BAF155cKO cortex, there is a shift from vertical to non-vertical divisions (H).

(I) RNA-seq transcriptome profiling between control and BAF155cKO cortices showing down-regulated transcripts, among which numerous genes belong to cleavage plane orientation-related pathways (such as Par3, Plk1, Eml1, Map4, Dctn1, and Arhgef2).

Values are presented as means ± SEMs (**0.001 < p < 0.01, ***p < 0.001). Abbreviations: VZ, ventricular zone; IZ, intermediate zone; RG, radial glia; bRG, basal radial glia. Scale bar, 100 μm (A and D), 25 μm (E and F). See also Figures S2, S3, and S5.

Reminiscent to reported characteristics of the mouse VZ/SVZ (Hevner et al., 2006) and human oSVZ (Hansen et al., 2010), we observed that the IZ/CP in the BAF155cKO cortex at E15.5 contained PAX6+/SOX2+, NGN2+, and TBR2+ progenitors, which could be cataloged through triple IHC analyses into distinct populations: SOX2+/NGN2+/TBR2+, SOX2+, SOX2+/NGN2+, NGN2+/TBR2+, and TBR2+ cells. Notably, BPs that were immunoreactive only for NGN2 or SOX2/TBR2 were very rare. These findings are suggestive of a sequential expression of TFs during bRGs to bIPs differentiation in the BAF155cKO cortex (Figures S3B–S3D).

To further characterize the abundance of bRGs during the neurogenic phase of cortical development, we quantified the number of PAX6+ cells in the BAF155cKO cortex between E13.5 and E16.5. TAU-1 or TUJ1 immunoreactivity was used to distinguish the boundaries between the VZ/SVZ and IZ/CP (Martinez-Cerdeno et al., 2012) (Figure S4A). We found that the ectopic PAX6+ cells appear in the BAF155cKO cortex from E14.5, 1 day after the preplate splitting known to initiate the formation of cortical layering at E13.5 (Figure S4B). Our analysis revealed that the total number of PAX6+ RGs was not significantly different in the control and BAF155cKO cortices; however, the number of PAX6+ aRGs was strongly diminished, together with a remarkable increase of bRGs in the IZ/CP (Figures 4A–4C).

Earlier studies have established that the mitotic cleavage plane of aRG influences the fate of its progenies (Asami et al., 2011, Estivill-Torrus et al., 2002, Postiglione et al., 2011, Sanada and Tsai, 2005). Given the presence of bRGs in the BAF155cKO cortex, we were curious to know whether BAF155 has a similar role in regulating the mitotic cleavage plane of aRG. We therefore performed a double IHC for pVIM (cytoplasmic marker for dividing RG) and pHH3 (nuclear marker for dividing cells) (Figure 4G). We found that loss of BAF155 resulted in a shift in mitotic cleavage planes from vertical to orientations with reduced angle (Figure 4H). It is particularly interesting to note that in the BAF155cKO cortex, there is a clear increase in the number of aRGs having a horizontal cleavage plane orientation, which is known to predominantly generate bRGs (Asami et al., 2011, Estivill-Torrus et al., 2002, Postiglione et al., 2011). In addition, the expression of many genes, which belong to cleavage plane orientation-related pathways (e.g., mitotic spindle, such as Par3, Plk1, Eml1, Map4, Dctn1, Arhgef2), was down-regulated in the PAX6cKO and BAF155cKO cortices in our RNA-seq GO analysis (Table S2, Figure 4I). To ascertain that these findings are progenitor-specific, we created a cortical neuron-specific knockout of BAF155 by using Nex-Cre (Goebbels et al., 2006). The selective deletion of BAF155 in post-mitotic neurons did not cause the abnormal distribution of cortical progenitors in the IZ/CP (Figure S4C), indicating that post-mitotic expression of BAF155 plays no demonstrable role in the genesis of BPs.

Taken together, these results indicate that the loss of BAF155 during corticogenesis induces the genesis of basal progenitors with distinct basal processes that express RG and IP molecular markers. These bRGs appear to be generated, at least in part, through a change in the mitotic cleavage plane of aRGs.

Conditional Ablation of BAF155 Has No Significant Effect on Cortical Layer Formation

Given the significant loss of bIPs and ectopic distribution of cortical progenitors in the IZ/CP of the BAF155cKO mutant, we examined if this had any consequence on cortical size and thickness. To this end, we performed IHC in coronal sections of E18.5 control and BAF155cKO cortices using antibodies against CTIP2 (marker for early-born neurons/lower layer marker, LL) (Figures S5A–S5C) and SATB2 (marker for late-born neurons/upper layer marker, UL) (Figures S5B and S5C). The results indicated that there is no significant change in the number of both LL and UL neurons in the rostral and caudal regions of the BAF155cKO cortex compared with control (Figures S5A–S5C). Quantifications of Tbr1+ UL and LL neurons at stage P7 in control and BAF155cKO cortex sagittal sections (at medial and lateral levels) did not reveal any significant defects in the layering in BAF155 loss of function (LOF) (Figure S5D). To assess more precisely eventual defects in cortical size and thickness upon deletion of the BAF155 subunit, we employed ex vivo the structural magnetic resonance imaging (MRI) approach. The total thickness, size, and surface area of the cerebral cortex were not significantly different between adult (P56) BAF155cKO and control mice at any level (Figures S5E–S5G). Collectively, these findings suggest that, despite the alteration in pool size of bIP (diminished) and bRGs (increased), the BAF155cKO cortex displayed normal surface area and thickness.

Loss of BAF155 or PAX6 Induces Genesis of Basal Progenitors Preferably in Non-Cell-Autonomous Manner

As the loss of BAF155 (Figures 3 and 4) and PAX6 (Asami et al., 2011) triggered the ectopic distribution of cortical progenitors in the IZ, we were interested in determining if BAF155 and PAX6 act within a common genetic pathway in basal progenitor specification. Owing to the severe down-regulation of GLAST expression in the PAX6cKO cortex (Figure S6A), we sought to compare the number of cortical aRGs and bRGs (SOX2+/GLAST+) in the cortex-specific single (BAF155cKO) and double (BAF155, PAX6cKO; dcKO) mutants. The number of SOX2+/GLAST+ aRG in BAF155cKO is similar to that of the control, whereas the additional loss of Pax6 in dcKO resulted in a significant reduction of this cell population (Figures S5A–S5C). On the other hand, both the BAF155cKO and dcKO cortices showed significantly higher numbers of SOX2+/GLAST+ bRGs in the IZ as compared with the control (Figures S5A–S5C). In addition, we examined the TBR2+ bIP population in mutants, which lost either BAF155 or PAX6 or both at E16.5 (Figures S6B–S6D). Compared with the control, a depleted pool of TBR2+ bIPs was observed in the BAF155cKO cortex and more severely in PAX6cKO mutants. Notably, an almost equal number of TBR2+ bIPs was seen in PAX6cKO and PAX6_BAF155cKO mutants (Figures S6B–S6D). Thus, our data suggested that PAX6 has a lead role, whereas BAF155 acts as a PAX6 cofactor in bIP genesis.

To analyze more directly the role of BAF155 and PAX6 in fate choice of aRG, we performed in utero electroporation (IUE) using Cre-ires-eGFP (Hand et al., 2005) plasmid to achieve deletion of either Baf155 or Pax6 from aRGs and their progenies in the E13.5 cortex of Baf155fl/fl or Pax6fl/fl embryos (Figure 5A). The cortical tissue was collected 30 hr after electroporation and immunostained with GFP, SOX2, TBR2 antibodies. Statistical analysis revealed no significant difference in the number of GFP+/SOX2+ aRGs in Cre-injected Baf155fl/fl and control hemispheres (Figures 5A and 5B). However, the number of GFP+/TBR2+ cells (bIP fate) in the SVZ was diminished in Baf155-ablated cortices as compared with the control (Figures 5A and 5C), indicating that BAF155 controls the genesis of bIPs in a cell-autonomous manner. As expected, the loss of BAF155 following IUE also led to an increase in the number of progenitors in the IZ/CP (Figure 5A, white filled arrows) consisting of both GFP+/SOX2+ and GFP+/TBR2+ cells (Figures 5B and 5C). These results favor the idea that BAF155 LOF shifts the output of dividing aRGs toward BP production in the IZ rather than the generation of bIPs in the SVZ, a phenotype that is even stronger upon PAX6 LOF (Figures 5B and 5C). Remarkably, more GFP(−) BPs (Figure 5A; white empty arrows) than GFP(+) BPs (Figure 5A; white filled arrows) were detected in the IZ/CP of Cre plasmid-injected Baf155fl/fl and Pax6fl/fl cortices (Figures 5B and 5C). These results suggest that in contrast to the cell-autonomous effect of both BAF155 and PAX6 on the genesis of SVZ bIPs, these two genes control the genesis of BPs in the IZ/CP preferably in a non-cell-autonomous manner.

Figure 5.

Figure 5

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Loss of PAX6 and Competition between BAF155 and BAF170 Promote Genesis of Basal Progenitors Predominantly in Non-Cell-Autonomous Manner

(A–D) Manipulation of endogenous expression level of Pax6, Baf155, and Baf170 in aRG via in utero electroporation (IUE). E13.5 cortices of WT, Pax6fl/fl, or Baf155fl/fl embryos were electroporated with the indicated plasmids. Thirty hours post-electroporation, sections of isolated brains were examined by IHC staining for expression of GFP, RG (SOX2, PAX6), bIPs (TBR2) markers. (B and C) The total number of GFP+ cells in the electroporated cortical areas was used as a reference (as 100%) with which other categories of cells (GFP+/SOX2+, GFP-/SOX2+, and GFP+/TBR2+, GFP-/TBR2+) were compared. Statistical analyses revealed that the loss of PAX6 and BAF155 in IUE experiments led to decrease in the number of GFP+/TBR2+ bIPs in the SVZ, but not GFP+/SOX2+ RG in the VZ, accompanied by an increase in GFP+/TBR2+ and GFP+/SOX2+ basal progenitors in the IZ/CP. Remarkably, the basal progenitors in the IZ/CP were predominantly GFP− (white empty arrows in A) than GFP+ (white filled arrows in A).

(E) Statistical comparisons indicated that overexpression of BAF170, which causes diminished expression of BAF155 (Tuoc et al., 2013a, Tuoc et al., 2013b), leads also to augmentation of the number of PAX6+ bRGs in the IZ/CP. This phenotype is partially rescued upon co-electroporation with both BAF170/BAF155 expression plasmids.

(F) The scheme illustrates the temporal complementary expression patterns of BAF155 and BAF170 in aRG in early and mid-corticogenesis as well as the effect of BAF155cKO or BAF170 overexpression in BP generation.

Values are presented as means ± SEMs (***p < 0.001). Abbreviations: VZ, ventricular zone; SVZ, subventricular zone. Scale bar, 100 μm (A), 25 μm (D). See also Figures S5, S6, and S7.

Competition between BAF155 and BAF170 Subunit Modulates BP Genesis

Our previous data indicated that the composition of BAF complex in cortical progenitors in terms of stoichiometry of BAF170 and BAF155 subunits influenced bIP production (Tuoc et al., 2013a). We sought to investigate whether such interplay might also affect bRG genesis. We examined the phenotypic consequence of overexpression (OE) of BAF170 by means of IUE, which is known to cause diminished expression of BAF155 (Tuoc et al., 2013a, Tuoc et al., 2013b). As expected, the activation of BAF170 also resulted in an increased number of PAX6+ bRGs in the IZ in non-cell-autonomous mode as seen in the Baf155-ablated cortex (Figures 5D and 5E). To ascertain that this phenotype results specifically from the altered stoichiometry between BAF170 and BAF155 within the BAF complex, we co-electroporated both BAF170/BAF155 expression plasmids into the E14.5 cortex and analyzed cortical phenotype after 30 hr. Remarkably, the phenotype observed upon BAF170 overexpression was partially rescued (Figures 5D and 5E), indicating that the balance between BAF170 and BAF155 plays a crucial role in the genesis of both bIPs and bRGs (Figure 5F) (Tuoc et al., 2013a).

In addition to the cortical phenotype analysis of the above-mentioned mutants (BAF170cKO, BAF170_OE, BAF155cKO), we characterized the cortical phenotypes of BAF155_OE mutants, which express an exogenous BAF155 cDNA together with a Myc tag sequence (BAF155-Myc) (Han et al., 2008, Lee et al., 2007) (Figure S7A). Although the transgenic BAF155-Myc is driven by the ubiquitously active CMV/β-actin promoter, notably, the increased level of BAF155 protein was not observed in entire BAF155_OE embryos (e.g., in the developing heart, but not in lung) (Han et al., 2008, Lee et al., 2007). By applying WB and qPCR analyses to quantify the overexpression level of BAF155 protein and mRNA in the BAF155_OE cortex at E13.5 (Figures S7B–S7D), we found that the Baf155 mRNA level is highly increased (Figure S7D), whereas the total BAF155 protein level is unaltered in the mutant cortex as compared with the control (Figures S7B and S7C). The IHC analysis indicated that there is no difference in the number and distribution of TBR2+ bIPs and PAX6+ RGs between control and BAF155_OE cortices at E15.5 (Figures 7E and 7F). Furthermore, comparable cortical size was seen in mutants and control at P9 and also at adult stage (Figure 7G).

Figure 7.

Figure 7

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BAF155 Regulates Basal Progenitor Genesis through a PAX6-Independent Mechanism Mediated by FOXN4

(A) RNA-seq transcriptome profiling between control and BAF155cKO cortices showing up-regulated transcripts, among which three genes (Foxn4, Lrig3 and C8orf4) were previously identified as specific to hRGs.

(B) qPCR confirmation of the indicated hRG genes up-regulated in BAF155cKO (but not in PAX6cKO) cortices at E15.5.

(C) Immunohistochemical analysis of E15.5 wild-type cortex electroporated with FOXN4 expression vectors. The FOXN4 GOF resulted in massive delamination of PAX6+ RG and TBR2+ bIPs 48 hr after electroporation compared with the non-injected hemisphere, thus generating abundant populations of bRGs and bIPs in the IZ. Notably, the majority of these basal progenitors were GFP− (white empty arrow) than GFP+ (white filled arrow).

(D) Statistical analysis of the number of PAX6+ and TBR2+ progenitors in the IZ in the same experiment revealed that there is a significant increase in basal progenitors upon FOXN4 GOF compared with the non-electroporated (control) cortex.

Values are presented as Mean ± SEMs (**0.001 < p < 0.01, ***p < 0.001). Abbreviations: VZ, ventricular zone; IZ, intermediate zone; RG, radial glia; bRG, basal radial glia; bIP, basal intermediate progenitor; hRG, human radial glia; GOF, gain of function. Scale bar, 100 μm. See also Figure S9.

Together, our data imply that the BAF170 subunit is able to compete with the BAF155 subunit within BAF complexes. Accordingly, the loss of BAF170 in cortex-specific BAF170cKO mutants leads to the incorporation of an additional BAF155 subunit(s) into the BAF complex, causing a dramatic over-production of bIPs. In contrast, overexpression of BAF170 results in competition of BAF170 with BAF155 for occupancy in the BAF complex, leading to a severely depleted bIP pool (Tuoc et al., 2013a) and an increased number of PAX6+ bRGs as observed in the BAF155cKO cortex. BAF170, however, does not substitute for the loss of BAF155 in the BAF155cKO cortex (Figure S1A). Moreover, in the presence of BAF170, exogenous BAF155 molecules are not able to integrate into BAF complexes and the excess BAF155 molecules in the BAF155_OE cortex are possibly degraded post-translationally.

BAF155 Suppresses Progenitor Delamination by Regulating Adherens Junction and Cell-Cell Interaction Machinery

To further investigate the determinants possibly involved in the non-cell-autonomous mechanism by which PAX6 and BAF155 control the genesis of BPs, we examined genes markedly down-regulated in the PAX6cKO and BAF155cKO cortices and having relevance in the maintenance of VZ integrity. The GO analyses of the RNA-seq data showed a significant enrichment of genes involved in cell-cell interaction and cell morphology, including the cell-cell adherens junction (AJ), apical junction complex, basement membrane, actin cytoskeleton, regulation of cell shape, and Rho protein signal transduction, among others (Figures 6A–6C, Tables S4 and S5). For some selected candidates, we also confirmed their down-regulation by qPCR (Figure 6D). To provide additional evidence that loss of BAF155 as well as PAX6 impairs AJ formation, we examined the expression of AJ molecules (ZO1 and α-catenin), which shows a down-regulated expression in our RNA-seq analysis. The expression of these AJ molecules is highly enriched at the apical surface (Figure S8). Consistent with the RNA-seq data, the protein level of ZO1 and α-catenin was apparently lower as revealed by fainter fluorescent intensity in the PAX6cKO, BAF155cKO cortices as compared with the control (Figure S8).

Figure 6.

Figure 6

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BAF155 Regulates Basal Progenitor Genesis through PAX6-Dependent Mechanism Mediated by CEP4

(A) Analysis of gene ontology (GO) from RNA-seq data indicated that many gene pathways that are important for cell-cell interactions were similarly altered in BAF155 and PAX6KO mutants.

(B and C) RNA-seq transcriptome profiling between control and PAX6cKO (B) or BAF155cKO (C) cortices showing down-regulated transcripts, among which numerous genes play important role for cell-cell interaction and cell adhesion.

(D) qPCR confirmation of the selected cell adhesion-related genes down-regulated in PAX6cKO, BAF155cKO cortices at E15.5.

(E) Luciferase reporter assay revealed that PAX6 activates promoter activity of several target genes that are crucial for cell-cell interaction (Luc+CMV-PAX6 condition). Upon silencing of BAF155, this promoter activity tends to reduce (Luc+CMV-PAX6+shBAF155 condition), more significantly for the genes Cdc42ep1 (Cep1) and Cdc42ep4 (Cep4).

(F and G) Immunohistochemical (F) and statistical (G) analyses of E15.5 wild-type cortices electroporated with control (GFP), shCEP4 (silencing), or shCEP4+CEP4 (rescue) vectors. Loss of function of CEP4 led to an increase in PAX6+ bRGs and TBR2+ bIPs in the IZ, which are predominantly GFP− (white empty arrow in F) than GFP+ (white filled arrow in F). (G) Statistical analysis of the number of PAX6+ and TBR2+ progenitors in the IZ in the above experiment revealed that there is a significant increase in basal progenitors upon CEP4 silencing compared with the control electroporated cortex. In addition, the co-expression of CEP4 rescued this phenotype.

Values are presented as means ± SEMs (*/#0.01 < p < 0.05, **/##0.001 < p < 0.01, ***p < 0.001). Abbreviations: bRG, basal radial glia; bIP, basal intermediate progenitor; IZ, intermediate zone. Scale bar, 25 μm. See also Figures S8 and S9.

Aiming at identifying target genes of PAX6 and BAF155 that control cell-cell interaction and AJ, we focused on the down-regulated genes from GO terms related to these processes (Figure 6A) in our RNA-seq data for PAX6cKO (Figure 6B) and BAF155cKO cortices (Figure 6C). Moreover, promoters of many genes in these categories, such as Ssx2ip, Wnt5a, Fgfr1, Celsr1, Cdc42ep1, and Cdc42ep4, were found to be bound by PAX6 in the previously published PAX6 ChIP-seq data (Xie et al., 2013). Promoter regions of these candidate genes were cloned into vector driving luciferase expression and transiently transfected into Neuro2A cells together with a combination of PAX6-expression and BAF155-silencing vectors. After lysate collection and luminescence quantification, the results from the reporter assay confirmed that compared with the control (in Luc+ CMV-EV condition), PAX6 (in Luc+ CMV-PAX6 condition) significantly regulates the promoter activity of these genes (Figure 6E). However, upon Baf155 knockdown using a short hairpin RNA (shRNA) construct, the level of luciferase reduced (Luc+ CMV-PAX6+shBAF155 condition), specifically in the case of Cdc42ep1 and Cdc42ep4 genes, implying that both PAX6 and BAF155 are required for maintaining the promoter activity of these genes (Figure 6E).

We chose for further functional characterization the two CDC42 effector proteins 1 and 4 (CDC42EP1/CEP1 and CDC42EP4/CEP4), members of the Rho family of guanosine triphosphatases (GTPases), which are known key players in cytoskeletal remodeling acting downstream of CDC42 to induce actin filament organization (Hirsch et al., 2001, Joberty et al., 2001). Because Cep4 but not Cep1 is specifically expressed in the E14.5 cortical VZ (Figure S9A) (Visel et al., 2004), we focused on understanding whether Cep4 is important for the genesis of BPs. We designed shRNA construct against Cep4 (Figure S9B) and electroporated it into E13.5 WT cerebral cortices (Figure 6F). Interestingly, 48 hr post-electroporation, knockdown of Cep4 resulted in an obvious increase in numbers of both PAX6+ bRGs and TBR2+ bIPs in the IZ compared with the control (GFP-only electroporated cortex) (Figures 6F and 6G). Similar to the data from our in vivo LOF experiments for BAF155 and PAX6 through IUE, a majority of bIPs and bRGs in Cep4 shRNA electroporated cortex were GFP(−) cells (Figure 6F, white empty arrow). In addition, co-electroporation of Cep4 shRNA and pCIG2-CEP4 overexpression plasmid was able to rescue the effect of Cep4 knockdown (Figures 6E and 6F). Taken together, our findings strongly support the idea that BAF155 controls the genesis of bRGs in a non-cell-autonomous manner, at least in part, by directly regulating PAX6 target genes (such as Cep4) involved in cell shape dynamics and cell-cell interaction.

BAF155 Regulates the Expression of Human RG-Specific Genes

Both BAF155 LOF (this study) and PAX6 LOF (Asami et al., 2011) cause ectopic location of basal progenitors in the IZ of the developing cortex. However, although the generation of distinct neuronal identities were unaffected in the BAF155cKO cortex, a lack of PAX6 has been reported to result in an expansion of LL at the expense of UL neurons (Georgala et al., 2011, Gotz et al., 1998, Quinn et al., 2006, Sansom et al., 2009, Thakurela et al., 2016, Tuoc et al., 2009, Tuoc et al., 2013a). In addition, the analysis of the transcriptome data of BAF155cKO and PAX6cKO cortices indicated that despite the significant overlap in differentially expressed genes, a set of genes appeared to be independently regulated by BAF155 (Figures 2E and 2F).

Recently, evidence has been presented for genes showing specific expression in the RG of the human cortex (hRG) (Lui et al., 2014). Comparing our RNA-seq data of cortical samples from the BAF155cKO mutant with those of Lui et al. (2014), we identified that three such hRG-specific genes, Foxn4, Lrig3 and C8orf4, were up-regulated in BAF155cKO, but not in the PAX6cKO cortex (Figures 7A and 7B). Interestingly, Foxn4, although present in the mouse genome, is not expressed in the mouse cortex (Figure S9C) (Visel et al., 2004). To get insight into its function, we generated the Foxn4 cDNA expression vector (Figure S9D) and performed GOF in vivo through IUE into E13.5 WT cerebral cortices. Interestingly, 2 days post-electroporation, we observed increased delamination of both PAX6+ RG and TBR2+ bIPs, giving rise to abundant BP population (Figures 7C and 7D), the majority of which were GFP(−) cells (Figure 7C, white empty arrow). Together, these findings suggest that during mouse cortical development, chromatin remodeler BAF155 seems to suppress the expression of hRG-specific genes (such as Foxn4), which otherwise promotes abundant generation of BPs in the primate brain.

Discussion

During cerebral cortex development, glutamatergic neurons are sequentially produced in an inside-out fashion—first the LL neurons followed by UL neurons. The aRGs in the VZ undergo different modes of neurogenesis, either giving rise predominantly to LL neurons (direct neurogenesis) or to UL neurons (indirect neurogenesis) produced by several types of BP descendants located in the SVZ (Gotz and Huttner, 2005, Kriegstein et al., 2006, Leone et al., 2008, Molyneaux et al., 2007, O'Leary et al., 2007, Tuoc et al., 2014). In lissencephalic species, such as mouse, the SVZ is mainly populated by bIPs, which account for the generation of UL neurons and contribute to the radial expansion of the cortex. However, in gyrencephalic species, such as primates and humans, the pool of BPs becomes expanded comprising not only of bIPs but also bRGs, assumed to underlie an exponential increase in neuronal output, expansion and folding of the cortex (Dehay et al., 2015, Fernandez et al., 2016, Florio and Huttner, 2014, Lui et al., 2011, Sun and Hevner, 2014, Taverna et al., 2014, Tuoc et al., 2014). In this study, we have shown data suggesting that the BAF155 subunit of chromatin remodeling BAF complex possibly regulates BP genesis involving PAX6-dependent and PAX6-independent mechanisms.

BAF155 and PAX6 Regulate bRG Genesis by Maintaining Adherens Junction Formation and VZ Integrity

Evidences have been shown that the commitment of the aRG to generate bIP fate occurs upon activation of the expression of PAX6 target genes (Ngn2, Tbr2) (Arnold et al., 2008, Sansom et al., 2009, Schuurmans et al., 2004, Sessa et al., 2008, Tuoc et al., 2013a). Similar to the cortical phenotype of the PAX6 LOF mutants, we found that depletion of BAF155 expression caused a diminished bIP pool. In addition, the results from the reporter assay indicate that BAF155 is needed for the activation of PAX6-dependent transcription in cortical progenitors. Furthermore, the RNA sequencing of BAF155cKO and PAX6cKO cortices indicated a significant overlap between differentially expressed (up- or down-regulated) genes in both mutants. Together, these data implicate a possible existence of a link between the PAX6-dependent transcriptional control of bIP genesis and BAF155-dependent chromatin remodeling. However, additional confirmatory experiments (such as ChIP-seq for PAX6, BAF155, epigenetic marks) will be necessary to get a deeper insight into the mechanism of interaction between these two factors.

The loss of PAX6 results in a reduction in the expression of cell adhesion proteins (Asami et al., 2011, Stoykova et al., 1997) known to trigger detachment of aRG from the apical VZ surface to ectopic basal location and displaying bRG identity (Asami et al., 2011). Similarly, in the BAF155cKO cortex we found an increased frequency of progenitor delamination from the VZ and a significant down-regulation of the expression of genes regulating cell adhesion and AJ. In the ferret developing cortex, a substantial reduction of Cdh1 expression, alongside change in the orientation of aRG mitotic division plane, resulted in the generation of pioneer bRGs, highlighting the significance of aRG delamination for bRG genesis (Martinez-Martinez et al., 2016). It is plausible therefore to suggest that both BAF155 and PAX6 could be also involved in the regulation of cell adhesion and AJ formation contributing to anchor aRG in the VZ. Although the sustained expression of PAX6 in bIPs in the mouse cortex resulted in a primate-like mode of generation of bRGs suggesting that PAX6 alone is able to reprogram bIPs to bRGs (Wong et al., 2015), our findings highlight an additional role of TF PAX6 in regulating aRG delamination through cell adhesion.

Another interesting finding was that the majority of bRGs found both in the BAF155 and PAX6 LOF cortices seem to arise through non-cell-autonomous mechanism. Upon loss of BAF155 and PAX6, we observed a down-regulation of the expression of CEP4, a CDC42 effector protein, which is specifically expressed in the VZ during corticogenesis. Indeed, we found that BAF155 directly regulates the expression of Cep4 in a PAX6-dependent manner and that the loss of CEP4 function causes delamination of progenitors in a predominantly non-cell-autonomous manner. Being a multifaceted regulator of cellular signaling, the Rho GTPase CDC42 activates Par complex that maintains the apico-basal polarity of aRG and AJ formation in the developing cortex, thereby regulating apical vs basal fate of the progenitors (Cappello et al., 2006). It is therefore tempting to speculate that CEP4 likely regulates key components of the AJ, cell-cell interaction, and signaling machineries. Although further experiments are needed to study the underlying mechanisms, these results suggest that CEP4 has a role in progenitor delamination participating either directly or through its interaction with CDC42.

Dynamic Expression of BAF155 and BAF170 Has a Functional Relevance in BP Genesis during Cortical Development

During cortical development, both BAF155 and BAF170 are detectable in the progenitor cells of the VZ; however, they show temporal complementary expression patterns (Figures 1 and 5F) (Tuoc et al., 2013a). The expression of BAF155 in aRG peaks at mid-corticogenesis (E15.5-E16.5), whereas a higher level of BAF170 is found in early corticogenesis (E10.5-E13.5). The results presented here suggest that the expression dynamics of the chromatin remodeling subunits BAF155 and BAF170 have a functional relevance in the temporal genesis of BPs during cortical development (Figure 5F).

We have shown previously that TF PAX6 interacts with both BAF155 and BAF170, the latter recruiting REST-corepressor complexes to promoters of PAX6 target genes involved in specification of bIPs (Tuoc et al., 2013a). Loss of BAF170 leads to replacement of BAF170 by an additional BAF155 subunit, elimination of the REST-co-repressor complex, and induction of a euchromatin state that relieves the repression of PAX6 target genes involved in bIP specification. Thus, the biological outcome of BAF subunit exchange is a heterochronic, premature expression of genes that specify bIPs. Conversely, over-expression of BAF170 results in competition with BAF155 for occupancy of the BAF complex that leads (opposite to the BAF170 LOF condition) to an increase of bIP generation. In agreement with these findings, we showed in this study that the loss of BAF155 causes repression of PAX6 target genes involved in bIP specification likely inducing a heterochromatin state on their regulatory regions (Figure 3C).

In addition to the effect on bIP generation, the balance between BAF170 and BAF155 within the BAF complex in cortical progenitors is also essential for bRG genesis. Accordingly, loss of BAF155 achieved by either knockout of BAF155 or overexpression of BAF170 causes delamination of aRG to generate bRGs. Taken together, the results suggest that changes in the equilibrium between BAF170 and BAF155 in aRG result in chromatin re-arrangement on the genomic loci of PAX6 target genes, which are important for bIP specification and delamination of progenitors.

Delamination of aRG Mediated by BAF155 Might Be Relevant for the Expansion of bRGs in the Developing Cortex during Evolution

Since the primate and human cortex contain an expanded population of BPs (Fietz et al., 2010, Hansen et al., 2010, Kelava et al., 2012, Lukaszewicz et al., 2005), we were intrigued to examine whether non-cell-autonomous mechanism of progenitor delamination, as discovered in this study, has acquired evolutionary significance. Interestingly, the transcriptional up-regulation of the expression of Foxn4, Lrig3, and C8orf4 in BAF155cKO mutants as reported here were previously shown as hRG-specific markers (Lui et al., 2014). Among these genes, we focused on understanding the role of FOXN4, a TF with a known role in the determination of cell fate in retinal progenitor cells (Li et al., 2004). By using the in vivo GOF approach, we showed that overexpression of FOXN4 results in a massive progenitor delamination and abundant generation of both bRGs and bIPs (Figure 7) in a non-cell-autonomous manner. Interestingly, a strikingly similar phenotype was previously reported upon overexpression of PDGFRb, another hRG-specific marker (Lui et al., 2014). Together, these findings suggest that, in addition to PAX6-dependent regulation of gene expression, BAF155 appears to control BP genesis independent of PAX6 through regulation of hRG genes, such as Foxn4. It would be interesting in future studies to investigate the homology of BAF155 and PAX6 proteins in mouse and human brain, e.g., in amino acid sequences, their species-specific isoforms, and their dynamic expression pattern during corticogenesis, and how such differences can lead to changes in the abundance of bRGs in primates.

In summary, our data revealed the significance of the interaction between the chromatin remodeling BAF complex and transcriptional machinery in the context of progenitor fate choice and basal progenitor genesis during cortical development. Both BAF155 and PAX6 control the AJ formation between the aRG at the ventricular surface, part of which mechanism is directly dependent on CEP4 that acts downstream of both factors. Furthermore, through the regulation of hRG-specific genes, such as Foxn4, the BAF155 chromatin remodeling subunit seems to exert a PAX6-independent role in progenitor delamination and bRG genesis that might have acquired evolutionary significance during cortical development.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

We acknowledge T. Huttanus and H. Fett for their expert animal care and support. We thank A. Messing, A. Nave, and K. Jones for providing reagents. This work was supported by Research Program, Faculty of Medicine, Georg-August-University Goettingen (T.T.), TU432/1-1, TU432/3-1 DFG grants (T.T.), Schram-Stiftung (T.T.), and DFG-CNMPB (T.T., J.F.S., A.S., A.F.).

Author Contributions

R.N., L.P., P.A.U., G.S., R.C.H., A.S., and T.T. characterized the phenotype of BAF155cKO and dcKO mutants; R.N. performed shCEP4, CEP4, and FOXN4 overexpression in vivo; R.N. and A.B.T. carried out cell division analysis; T.W., R.N., and J.F. contributed to MRI; R.N. and K.A.K. performed luciferase assay; C.K., S.B., R.N., and A.F. contributed to RNA-seq, analysis of genome-wide data; T.T., L.P., G.S., and J.R. carried out shBAF155, BAF155_OE, shPAX6, BAF170 overexpression in vivo; J.F.S., A.S., U.T., and R.H.S. provided transgenic lines and research tools and contributed to discussions. A part of the project was performed in the Stoykova laboratory. T.T. conceived the study.

Declaration of Interests

The authors declare no competing financial interests.

Published: June 29, 2018

Footnotes

Supplemental Information includes Transparent Methods, nine figures, and six tables and can be found with this article online at https://doi.org/10.1016/j.isci.2018.05.014.

Contributor Information

Anastassia Stoykova, Email: astoyko@gwdg.de.

Tran Tuoc, Email: tran.tuoc@med.uni-goettingen.de.

Supplemental Information

Document S1. Transparent Methods and Figures S1–S9
mmc1.pdf (6.2MB, pdf)
Table S1. Genes Regulated by PAX6 in the PAX6cKO Cortex at E15.5, Related to Figure 2
mmc2.xlsx (7MB, xlsx)
Table S2. Genes Regulated by BAF155 in the BAF155cKO Cortex at E15.5, Related to Figure 2
mmc3.xlsx (6.6MB, xlsx)
Table S3. Genes Coregulated by BAF155 and PAX6 in Cortex at E15.5, Related to Figure 2
mmc4.xls (179.5KB, xls)
Table S4. Selected Gene Ontology Categories Significantly Downregulated in E15.5 PAX6cKO Cortex Compared with Controls in Expression Analyses, Related to Figure 2, 6
mmc5.xlsx (7.1MB, xlsx)
Table S5. Selected Gene Ontology Categories Significantly Downregulated in E15.5 BAF155cKO Cortex Compared With controls in Expression Analyses, Related to Figures 2, 6
mmc6.xlsx (3.9MB, xlsx)
Table S6. Statistical Analyses, Related to All Figures
mmc7.xlsx (256.6KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods and Figures S1–S9
mmc1.pdf (6.2MB, pdf)
Table S1. Genes Regulated by PAX6 in the PAX6cKO Cortex at E15.5, Related to Figure 2
mmc2.xlsx (7MB, xlsx)
Table S2. Genes Regulated by BAF155 in the BAF155cKO Cortex at E15.5, Related to Figure 2
mmc3.xlsx (6.6MB, xlsx)
Table S3. Genes Coregulated by BAF155 and PAX6 in Cortex at E15.5, Related to Figure 2
mmc4.xls (179.5KB, xls)
Table S4. Selected Gene Ontology Categories Significantly Downregulated in E15.5 PAX6cKO Cortex Compared with Controls in Expression Analyses, Related to Figure 2, 6
mmc5.xlsx (7.1MB, xlsx)
Table S5. Selected Gene Ontology Categories Significantly Downregulated in E15.5 BAF155cKO Cortex Compared With controls in Expression Analyses, Related to Figures 2, 6
mmc6.xlsx (3.9MB, xlsx)
Table S6. Statistical Analyses, Related to All Figures
mmc7.xlsx (256.6KB, xlsx)

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