The growth and evolutionary expansion of the cerebral cortex are defined by the spatial-temporal production of neurons, which itself depends on the decision of radial glial cells (RGCs) to self-amplify or to switch to neurogenic divisions. The mechanisms regulating these RGC fate decisions are still incompletely understood. Here, we describe a novel and evolutionarily conserved role of the canonical BMP transcription factors SMAD1/5 in controlling neurogenesis and growth during corticogenesis. Reducing the expression of both SMAD1 and SMAD5 in neural progenitors at early mouse cortical development caused microcephaly and an increased production of early-born cortical neurons at the expense of late-born ones, which correlated with the premature differentiation and depletion of the pool of cortical progenitors. Gain- and loss-of-function experiments performed during early cortical neurogenesis in the chick revealed that SMAD1/5 activity supports self-amplifying RGC divisions and restrains the neurogenic ones. Furthermore, we demonstrate that SMAD1/5 stimulate RGC self-amplification through the positive post-transcriptional regulation of the Hippo signalling effector YAP. We anticipate this SMAD1/5-YAP signalling module to be fundamental in controlling growth and evolution of the amniote cerebral cortex.

The cerebral cortex is the region of the human brain responsible for higher cognitive functions. Errors during its formation provoke a vast array of brain disorders that affect intellectual ability and social behaviour (Hu et al., 2014; Jayaraman et al., 2018). This highlights the relevance of identifying the mechanisms that govern cortical development, in particular those controlling its growth and the production of neurons from cortical stem and progenitor cells.

Emerging in the dorsal part of the telencephalon (pallium), the mammalian cerebral cortex first consists of a pseudo-stratified epithelial layer (also called the ventricular zone, VZ) formed by primitive neural stem cells, the neuroepithelial cells, that rapidly mature into radial glial cells (RGCs) (Taverna et al., 2014). Like neuroepithelial cells, RGCs contact both the ventricle and the basal lamina, they divide at the apical membrane and they can expand through self-amplifying divisions that produce two daughter RGCs (De Juan Romero and Borrell, 2015; Taverna et al., 2014). This self-amplification of neuroepithelial cells and RGCs is the only mode of division that can ensure the tangential growth (i.e. along the rostral-caudal and medial-lateral axes) of the cortical VZ (De Juan Romero and Borrell, 2015; Taverna et al., 2014). Neurogenesis is initiated when RGCs start producing daughter cells committed to the neuronal lineage. Clonally-related cortical neurons (originating from a same original RGC) migrate basally with little tangential dispersion and appear to mature in a radial column, the position of which reflects that of their mother RGC, thereby ensuring the radial growth of the developing cerebral cortex (De Juan Romero and Borrell, 2015; Taverna et al., 2014). Neurogenesis can occur directly, when an RGC divides asymmetrically to generate another RGC and a daughter cell that differentiates directly into a neuron (De Juan Romero and Borrell, 2015; Taverna et al., 2014). Alternatively, neurogenesis can be indirect, whereby an RGC gives rise to an RGC and a basal progenitor (BP) that harbours a restricted lineage potential, delaminates from the VZ and divides basally. These BPs will increase the neuronal output, possibly self-amplifying for several rounds of divisions before generating two neurons through a final self-consuming division (Lui et al., 2011). Although intermediate progenitor cells (IPCs) possess a very limited self-amplifying potential and represent the vast majority of cortical BPs in lissencephalic species, basal (or outer) RGCs harbour a considerable self-amplifying potential and they are responsible for the tremendous production of neurons and the larger neocortex observed in gyrencephalic species (Lui et al., 2011; Shitamukai et al., 2011; Wang et al., 2011). Therefore, the decision of an RGC to self-amplify or to give rise to neurons, either directly or indirectly, has a huge impact on the final number of cortical neurons and represents the primary event that defines the tangential and radial growth of the tissue.

As highlighted by the gene mutations causing primary microcephaly, a variety of intracellular events regulate cerebral cortical size, including centrosome behaviour and centriole biogenesis, DNA replication and repair, cytokinesis and apical-basal polarity (Jayaraman et al., 2018; Saade et al., 2018). Moreover, the balance between RGC self-amplification and neurogenesis appears to be regulated by extrinsic cues emanating from the ventricular fluid, meninges, blood vessels and neighbouring cells (Llinares-Benadero and Borrell, 2019; Martynoga et al., 2012; Taverna et al., 2014). Thus, the molecular events regulating RGC fate are complex and they are still not fully understood.

Seminal studies performed on cortical progenitors in vitro suggested that bone morphogenetic protein (BMP) signalling regulates their neurogenic potential (Li et al., 1998; Mabie et al., 1999). The microcephaly described in Bmp7 mutant mice, and the overproliferation and premature differentiation reported in the brains of transgenic mice expressing constitutively active forms of the type-1 BMP receptors Alk3 (also known as Bmpr1a) or Alk6 (Bmpr1b) supported this idea (Panchision et al., 2001; Segklia et al., 2012). However, definitive proofs of an instructive role for BMP signalling in cortical RGC fate decision in vivo are still lacking.

Here, we found that the activity of the transcription factors (TFs) SMAD1/5, two canonical effectors of BMP signalling, promotes cortical RGC self-amplification in both chick and mouse embryos, preventing their premature neurogenic switch and exhaustion, and thereby ensuring the appropriate production of the distinct classes of cortical excitatory neurons. In searching for the effectors of SMAD1/5 activity, we show that this role depends on the post-transcriptional regulation of YAP, a core component of the Hippo signalling pathway known to regulate cell growth and organ size. Together, our findings reveal an instructive and evolutionarily conserved role of the canonical BMP effectors SMAD1/5 in the control of RGC self-amplification in the developing cerebral cortex.

SMAD1/5 activity is required for brain growth and cortical neurogenesis in mouse

To determine the role played by the canonical BMP pathway during mammalian cerebral cortex development, we focused on the function of its canonical effectors SMAD1, SMAD5 and SMAD9 (human SMAD8) during mouse corticogenesis. In situ hybridization revealed that mSmad1 and mSmad5 transcripts are expressed throughout the rostral-caudal axis of the developing mouse cerebral cortex at embryonic day (E) 14.5 and are particularly enriched in the VZ, whereas mSmad8/9 transcripts were not detected (Fig. 1A). In agreement with previous observations (Saxena et al., 2018), immunostaining at mid-corticogenesis with an antibody that specifically recognizes the active carboxy-terminal phosphorylated form of SMAD1/5/8 (pSMAD1/5/8) revealed activity of these canonical BMP effectors in differentiating neurons as well as in both apically- and basally-dividing cortical progenitors (Fig. 1B). When quantified in phospho-Histone 3+ (pH3+) mitotic nuclei, pSMAD1/5/8 immunostaining revealed SMAD1/5 activity to be stronger in apically-dividing mouse RGCs than in basally-dividing IPCs (Fig. 1C), suggesting a correlation between high SMAD1/5 activity and RGC maintenance.

Fig. 1.

The SmadNes mutant mice present severe microcephaly and growth retardation. (A) Sagittal sections of the developing mouse dorsal telencephalon showing mSmad1, mSmad5 and mSmad8 transcripts at E14.5, obtained from Genepaint (https://gp3.mpg.de). (B,C) The pSMAD1/5/8 immunoreactivity a t E13.5 (B) and its mean intensity±s.d. measured in 144 apical and 64 basal mitoses obtained from three mouse E13.5 embryos (C). Magnification of boxed area (left) shown in right panels. Dotted lines indicate pH3+ mitotic nuclei. (D) Weight±s.d. of the SmadNes mutant mice (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and their control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) calculated from 17, 10, 12, 5 +/+ and 27, 9, 5, 3 +/− animals at E17.5, P0, P7 and P60, respectively. (E) Brain weight±s.d. calculated from 6, 10, 5, 5 +/+ and 16, 9, 3, 3 +/− animals at E17.5, P0, P7 and P60, respectively. (F,G) Dorsal view (F) and mean volume±s.d. (G) of the brain of the SmadNes mutants and their control littermates at P60, obtained from 3 +/+ and 3 +/− animals. (H,I) Rostral-caudal brain level (H) at which the distinct parameters were analysed at P60 (I). (J-O) Coronal sections (J) obtained at the fimbria level (as shown in H) and the corresponding mean area±s.d. of the whole brain (K) and of the cerebral cortex (L), the length±s.d. of the cortical VZ (M) and of the cortical pia (N) and the thickness of the corpus callosum relative to whole dorsal-ventral brain thickness (O), all obtained from 5 +/+ and 3 +/− animals. Significance was assessed using the non-parametric Mann–Whitney test (C,K-O) or a two-way ANOVA+Sidak's test (D,E). *P<0.05, ***P<0.001; n.s, P>0.05. See also Figs S1 and S2. Scale bars: 50 µm (A,B); 5 mm (F); 250 µm (J).

Fig. 1.

The SmadNes mutant mice present severe microcephaly and growth retardation. (A) Sagittal sections of the developing mouse dorsal telencephalon showing mSmad1, mSmad5 and mSmad8 transcripts at E14.5, obtained from Genepaint (https://gp3.mpg.de). (B,C) The pSMAD1/5/8 immunoreactivity a t E13.5 (B) and its mean intensity±s.d. measured in 144 apical and 64 basal mitoses obtained from three mouse E13.5 embryos (C). Magnification of boxed area (left) shown in right panels. Dotted lines indicate pH3+ mitotic nuclei. (D) Weight±s.d. of the SmadNes mutant mice (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and their control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) calculated from 17, 10, 12, 5 +/+ and 27, 9, 5, 3 +/− animals at E17.5, P0, P7 and P60, respectively. (E) Brain weight±s.d. calculated from 6, 10, 5, 5 +/+ and 16, 9, 3, 3 +/− animals at E17.5, P0, P7 and P60, respectively. (F,G) Dorsal view (F) and mean volume±s.d. (G) of the brain of the SmadNes mutants and their control littermates at P60, obtained from 3 +/+ and 3 +/− animals. (H,I) Rostral-caudal brain level (H) at which the distinct parameters were analysed at P60 (I). (J-O) Coronal sections (J) obtained at the fimbria level (as shown in H) and the corresponding mean area±s.d. of the whole brain (K) and of the cerebral cortex (L), the length±s.d. of the cortical VZ (M) and of the cortical pia (N) and the thickness of the corpus callosum relative to whole dorsal-ventral brain thickness (O), all obtained from 5 +/+ and 3 +/− animals. Significance was assessed using the non-parametric Mann–Whitney test (C,K-O) or a two-way ANOVA+Sidak's test (D,E). *P<0.05, ***P<0.001; n.s, P>0.05. See also Figs S1 and S2. Scale bars: 50 µm (A,B); 5 mm (F); 250 µm (J).

To understand the role played by SMAD1/5 during mouse cerebral cortex development, we crossed Smad1fl/fl;Smad5fl/fl mice (Moya et al., 2012) with a Nestin:cre transgenic line that produces Cre-mediated recombination in neural progenitor cells and somites as early as E8.5 (Petersen et al., 2002), earlier than the more commonly used Nestin:cre transgenic line that produces efficient Cre-mediated recombination in cortical progenitors from mid-embryogenesis (Liang et al., 2012). In agreement with the early Cre-mediated recombination expected from this Nestin:cre transgenic line (Petersen et al., 2002), SMAD1 and SMAD5 protein levels were reduced by 54% and 32% in telencephalic extracts from E11.5 Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0 heterozygous embryos relative to their control Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0 littermates (Fig. S1). These Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0 heterozygous mutant mice were viable and born following Mendelian ratios but were sterile, precluding the study of the homozygous compound mutants. Compared with their Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0 littermates (hereafter referred to as controls or +/+), the Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0 heterozygous mutant mice (hereafter referred to as SmadNes mutants or +/−) presented an overall growth retardation, including a reduction in brain weight, detected from postnatal day (P)7 (Fig. 1D,E). The brain of the adult (P60) SmadNes mutants showed a 25% reduction in weight (+/−: 0.407 g±0.006 versus +/+: 0.544 g±0.021; mean±s.d.; Fig. 1E), that correlated to a 29% decrease in volume (Fig. 1F,G) and an 18% reduction of its surface area in coronal sections (+/−: 42.91 mm2±2.302 versus +/+: 52.46 mm2±1.18, Fig. 1H-K). These reductions in brain weight and size were both greater than three standard deviations implying, according to clinical standards (Passemard et al., 2013), that the SmadNes heterozygous mutants suffer a severe microcephaly. The coronal areas of the whole brain and cerebral cortex were similarly reduced in adult SmadNes mutants (18% and 19%, respectively; Fig. 1K,L), and this reduction was constant across the rostral-caudal axis (Fig. S2). Constant decreases in the length of the cortical VZ and pia along this axis were also observed in the SmadNes mutant brain (Fig. 1M,N and Fig. S2). Apart from these growth defects and a thinner corpus callosum (Fig. 1O), the brain of the SmadNes mutants did not present any major neuro-anatomical defects (Fig. 1J and Fig. S2B), thereby suggesting that SMAD1/5 inhibition impaired growth equally in all brain regions. Importantly, the brain area, cortical area and length of the cortical VZ were also reduced in E17.5 SmadNes mutant embryos (Fig. S3), indicating that these brain growth defects have an embryonic origin.

The cerebral cortices of the adult SmadNes mutants and their control littermates showed comparable thicknesses and presented similar densities of NeuN+ (also known as Rbfox3) neurons (Fig. 2A-C). They also presented similar densities of macroglial cells, including SOX9+ astrocytes (Fig. S4A,B; Sun et al., 2017), and oligodendroglial cells [SOX10+ or OLIG2+;CC1+ (APC) oligodendrocytes and OLIG2+;CC1 progenitors, Fig. S4C-F; Bhat et al., 1996; Claus Stolt et al., 2002]. Taken together, these findings suggested that SMAD1/5 inhibition affects the tangential growth of the brain and the generation of its radial columns rather than its radial growth and the number of cells per radial unit. Nevertheless, we observed that the relative proportions of the different neuronal layers forming the cerebral cortex were altered in the adult SmadNes mutant (Fig. 2A,C). The number of early-born neurons forming the deep layer L6 and its thickness were increased, whereas these parameters were diminished in the superficial layer L2/3 containing late-born callosal projection neurons (Fig. 2A,C,D), consistent with the reduced thickness of the corpus callosum (Fig. 1O). This phenotype was observed at the early postnatal stage P7 (Fig. 2E-G), supporting the idea that these cortical defects originated during the embryonic phase.

Fig. 2.

Inhibiting SMAD1/5 activity in mouse neural progenitors causes an increase in early-born neocortical neurons at the expense of late-born ones. (A,B) NeuN+ neurons present in coronal sections of the brains of SmadNes mutant mice (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and their control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) at P60 (A), and their mean number±s.d. quantified in a 100 µm-wide cortical area, obtained from 3 +/+ and 4 +/− animals (B). (C,D) Mean thickness of the cortical neuronal layers±s.e.m. (C) and mean number of NeuN+ neurons±s.e.m. in the different layers in a 100 µm-wide cortical area at P60 (D), obtained from 4 +/+ and 3 +/− animals. (E-G) Early- and late-born neocortical neurons present in coronal sections of the brains of SmadNes mutant pups and their control littermates at P7. Early-born L6 (TBR1+), L5 (CTIP2+) and late-born L4-2/3 (CUX1+) projection neurons (E), mean thickness of the layers±s.e.m. (F) and mean number of neurons±s.e.m. in the different layers (G) in a 100 µm-wide cortical area, obtained from 5-7 +/+ and 5 +/− animals. Significance was assessed using the non-parametric Mann–Whitney test (B; C and F for the total cumulated thickness) or a two-way ANOVA+Sidak's test (C,D,F,G). *P<0.05, **P<0.01, ***P<0.001; n.s, P>0.05. See also Fig. S3. L1-6, cortical neuronal layers 1-6; SP, subplate; C.C, corpus callosum. Scale bars: 100 µm.

Fig. 2.

Inhibiting SMAD1/5 activity in mouse neural progenitors causes an increase in early-born neocortical neurons at the expense of late-born ones. (A,B) NeuN+ neurons present in coronal sections of the brains of SmadNes mutant mice (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and their control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) at P60 (A), and their mean number±s.d. quantified in a 100 µm-wide cortical area, obtained from 3 +/+ and 4 +/− animals (B). (C,D) Mean thickness of the cortical neuronal layers±s.e.m. (C) and mean number of NeuN+ neurons±s.e.m. in the different layers in a 100 µm-wide cortical area at P60 (D), obtained from 4 +/+ and 3 +/− animals. (E-G) Early- and late-born neocortical neurons present in coronal sections of the brains of SmadNes mutant pups and their control littermates at P7. Early-born L6 (TBR1+), L5 (CTIP2+) and late-born L4-2/3 (CUX1+) projection neurons (E), mean thickness of the layers±s.e.m. (F) and mean number of neurons±s.e.m. in the different layers (G) in a 100 µm-wide cortical area, obtained from 5-7 +/+ and 5 +/− animals. Significance was assessed using the non-parametric Mann–Whitney test (B; C and F for the total cumulated thickness) or a two-way ANOVA+Sidak's test (C,D,F,G). *P<0.05, **P<0.01, ***P<0.001; n.s, P>0.05. See also Fig. S3. L1-6, cortical neuronal layers 1-6; SP, subplate; C.C, corpus callosum. Scale bars: 100 µm.

Although the SmadNes mutant embryos did not present any obvious defect in telencephalic patterning (Fig. S5A), their programme of cortical neurogenesis did appear to be altered (Fig. 3). Around the onset of cortical neurogenesis (E11.5), the SmadNes mutant embryos presented RGCs [PAX6+;TBR2 (EOMES) cells] in correct numbers per radial area and of a normal cell size (Fig. 3A-C and Fig. S5B). At this early neurogenic stage, the SmadNes mutant embryos also presented correct numbers of TBR2+ IPCs and a germinal zone of normal thickness (Fig. 3B,D,E), yet their neuronal output was increased (Fig. 3F,G). Accordingly, there were more early-born differentiating TBR1+ neurons in the SmadNes mutant cortex than in their control littermates from E11.5 onwards (Fig. 3H,I; Bulfone et al., 1995). The developing cerebral cortex of the SmadNes mutant embryos contained fewer IPCs from E13.5 onwards and fewer RGCs at E17.5 (Fig. 3B-D). This decrease in cortical progenitors at E17.5 was associated with a reduced thickness of the germinal zones (Fig. 3E), a lower neuronal output (Fig. 3F,G) and fewer late-born CUX1+ and SATB2+ neurons (Fig. 3J,K and Fig. S6; Britanova et al., 2008; Nieto et al., 2004). Together, these data confirmed the developmental origin of the alterations in cortical projection neurons seen in the cerebral cortex of P7 and P60 SmadNes mutants (Fig. 2). The mitotic indices of the cortical RGCs and IPCs were comparable in SmadNes mutants and controls at all stages examined (Fig. S7), suggesting that the proliferation rate of the cortical progenitors is not severely affected in SmadNes mutants. We thus reasoned that the increased production of early-born neurons, the premature reduction in the number of RGCs and IPCs, and the decreased production of late-born neurons observed in the cerebral cortex of SmadNes heterozygous mutant embryos might likely be the result of a premature switch of RGCs from self-amplifying to neurogenic divisions during early corticogenesis.

Fig. 3.

Inhibiting SMAD1/5 activity in mouse cortical progenitors causes premature neurogenesis and depletion of RGCs and IPCs. (A) Timeline of cortical neurogenesis and BrdU injections during mouse embryonic development. (B-J) Analysis of corticogenesis in the developing cerebral cortex of SmadNes mutant embryos (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and their control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) at early (E11.5), mid (E13.5) and late (E17.5) embryonic stages. (B-D) Immunostaining of cortical progenitors and mean number±s.d. of PAX6+;TBR2 RGCs (C) and TBR2+ IPCs (D) quantified in a 100 µm-wide cortical area, obtained from 5, 4, 4 +/+ and 5, 3, 4 +/− animals analyzed at E11.5, E13.5 and E17.5, respectively. (E) Mean thickness of VZ+SVZ±s.d., obtained from 5, 4, 4 +/+ and 5, 4, 5 +/− animals at E11.5, E13.5 and E17.5, respectively. (F,G) Neuronal output defined as the mean percentage±s.d. of BrdU+;Tuj1+/BrdU+ cells quantified in a 100 µm-wide cortical area 24 h after a BrdU pulse (see A), obtained from 5, 4, 3 +/+ and 5,4,4 +/− animals at E11.5, E13.5 and E17.5, respectively. (H-K) Immunostaining and mean number±s.d. of early-born (TBR1; H,I) and late-born (CUX1; J,K) projection neurons quantified in a 100 µm-wide cortical area, obtained from 5, 4, 4 +/+ and 5, 3, 4 +/− animals at E11.5, E13.5 and E17.5, respectively (I), and 5 +/+ and 5 +/− animals at E17.5 (K). Each dot represents the value of one animal. Significance was assessed using the two-sided unpaired t-test (C,D,G,I), the non-parametric Mann–Whitney test (K) or a two-way ANOVA+Sidak's test (E). *P<0.05, **P<0.01, ***P<0.01; n.s, P>0.05. CP, cortical plate; IZ, intermediate zone; L1-6, cortical neuronal layers 1-6; SP, subplate; SVZ, sub-ventricular zone; VZ, ventricular zone. See also Figs S4-S6. Scale bars: 50 µm.

Fig. 3.

Inhibiting SMAD1/5 activity in mouse cortical progenitors causes premature neurogenesis and depletion of RGCs and IPCs. (A) Timeline of cortical neurogenesis and BrdU injections during mouse embryonic development. (B-J) Analysis of corticogenesis in the developing cerebral cortex of SmadNes mutant embryos (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and their control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) at early (E11.5), mid (E13.5) and late (E17.5) embryonic stages. (B-D) Immunostaining of cortical progenitors and mean number±s.d. of PAX6+;TBR2 RGCs (C) and TBR2+ IPCs (D) quantified in a 100 µm-wide cortical area, obtained from 5, 4, 4 +/+ and 5, 3, 4 +/− animals analyzed at E11.5, E13.5 and E17.5, respectively. (E) Mean thickness of VZ+SVZ±s.d., obtained from 5, 4, 4 +/+ and 5, 4, 5 +/− animals at E11.5, E13.5 and E17.5, respectively. (F,G) Neuronal output defined as the mean percentage±s.d. of BrdU+;Tuj1+/BrdU+ cells quantified in a 100 µm-wide cortical area 24 h after a BrdU pulse (see A), obtained from 5, 4, 3 +/+ and 5,4,4 +/− animals at E11.5, E13.5 and E17.5, respectively. (H-K) Immunostaining and mean number±s.d. of early-born (TBR1; H,I) and late-born (CUX1; J,K) projection neurons quantified in a 100 µm-wide cortical area, obtained from 5, 4, 4 +/+ and 5, 3, 4 +/− animals at E11.5, E13.5 and E17.5, respectively (I), and 5 +/+ and 5 +/− animals at E17.5 (K). Each dot represents the value of one animal. Significance was assessed using the two-sided unpaired t-test (C,D,G,I), the non-parametric Mann–Whitney test (K) or a two-way ANOVA+Sidak's test (E). *P<0.05, **P<0.01, ***P<0.01; n.s, P>0.05. CP, cortical plate; IZ, intermediate zone; L1-6, cortical neuronal layers 1-6; SP, subplate; SVZ, sub-ventricular zone; VZ, ventricular zone. See also Figs S4-S6. Scale bars: 50 µm.

SMAD1/5 activity is required for RGC self-amplification during chick cortical neurogenesis

There is increasing evidence that the basic gene regulatory networks, progenitor cell types and cellular events governing the generation of neurons during corticogenesis are evolutionarily conserved between mammals and sauropsids, particularly birds (Cardenas et al., 2018; Le Dreau et al., 2018; Nomura et al., 2013; Suzuki et al., 2012; Yamashita et al., 2018). To assess whether SMAD1/5 regulate the mode of division of cortical RGCs, we turned to the developing chick cerebral cortex, as this avian model offered greater possibilities than the mouse to accurately manipulate SMAD1/5 activity at the onset of cortical neurogenesis.

The production of neurons in the developing chick cerebral cortex spans from E3 to E8 (Fig. 4A,B; Suzuki et al., 2012). As in mammals, cortical neurogenesis in the chick is initiated by the onset of neurogenic divisions of PAX6+;TBR2 RGCs that divide apically in the VZ (Fig. 4A,B). From E5 onwards, the production of cortical neurons is enhanced by symmetric neurogenic divisions of basally-dividing TBR2+ IPCs (Fig. 4A,B; Suzuki et al., 2012). In situ hybridization revealed that both cSmad1 and cSmad5 transcripts are expressed throughout the neurogenic period, mostly in the VZ where cSmad8 transcripts were essentially absent (Fig. S8). Immunostaining with the pSMAD1/5/8 antibody at E5 revealed activity of SMAD1/5 in both apically- and basally-dividing cortical progenitors as well as in differentiating neurons (Fig. 4C), as previously observed in the developing mouse cortex (Fig. 1B). When quantified in pH3+ mitotic nuclei, SMAD1/5 activity was weaker in basal IPC divisions than in RGC divisions (Fig. 4D). When quantified after in ovo electroporation of a pTis21(Btg2):RFP reporter that is specifically activated during neurogenic divisions (Fig. S9; Le Dreau et al., 2014; Saade et al., 2013; Saade et al., 2017), SMAD1/5 activity was diminished in pTis21:RFP+ neurogenic divisions relative to pTis21:RFP self-amplifying divisions (Fig. 4E,F). Therefore, a positive correlation exists between SMAD1/5 activity and the potential for RGC self-amplification during chick cortical neurogenesis.

Fig. 4.

Inhibiting SMAD1/5 activity in chick cortical RGCs increases neurogenic divisions and causes their premature depletion and differentiation. (A) Schematic showing cortical neurogenesis in the chick, its main cortical progenitor subtypes and their modes of division. (B) Coronal sections from 3, 5 and 8 day-old chick embryos showing the PAX6+ developing cerebral cortex, formed of SOX2+ neural progenitors, differentiating HuC/D+ and TBR1+ neurons, PAX6+;TBR2 RGCs undergoing mitosis at the apical surface (black arrowheads) and TBR2+ IPCs dividing mostly basally (white arrowheads). Panels on right show magnification of boxed areas in left panels. (C,D) The active, phosphorylated form of SMAD1/5/8 (pSMAD1/5/8) immunoreactivity at E5 (C), and its mean intensity±s.d. measured in 276 apical and 93 basal mitoses obtained from five embryos (D). Panels on right show magnification of boxed area in left panel. Dotted circles indicate pH3+ mitotic nuclei. (E,F) The pSMAD1/5/8 immunoreactivity in mitotic cortical progenitors 24 h after in ovo electroporation (IOE) with the pTis21:RFP reporter along with a control H2B-GFP-producing plasmid (E), and its mean intensity±s.d. quantified in 137 pTis21:RFP+ and 107 pTis21:RFP divisions derived from eight embryos (F). Dotted circles indicate pH3+ mitotic nuclei. (G,H) The mean proportion±s.d. of electroporated (H2B-GFP+) cortical progenitors undergoing pTis21:RFP+ divisions (white arrowheads) or pTis21:RFP divisions (black arrowheads) after IOE of shRNAs targeting cSmad1 or cSmad5 (sh-S1/5, n=12 embryos) or their control (n=9). (I,J) Representative images (I) and mean proportions±s.e.m. (J) of electroporated (H2B-GFP+) cells marked as SOX2±;HuC/D+/- (top), PAX6+/−;TBR2+/− (middle) and HuC/D+/−;TBR1+/− (bottom), assessed 48 h after IOE with sh-S1 (n=8, 7, 5 embryos), sh-S5 (n=12, 5, 6) or their control (n=13, 8, 9). (K) The mean proportion±s.d. of electroporated cells identified as PAX6+;TBR2- RGCs (top), TBR2+ committed cells (middle) and HuC/D+ neurons (bottom), assessed 24 (E4), 48 (E5) and 72 (E6) h after IOE and obtained from n≥6 embryos per condition and stage. Significance was assessed using the non-parametric Mann–Whitney test (D,F), the two-sided unpaired t-test (H) or a two-way ANOVA+Tukey's (J) or Sidak's (K) test. **P<0.01, ***P<0.001; ns, not significant. D, dorsal; L, lateral; M, medial; MZ: mantle zone; N, neuron; NN, symmetric neurogenic division; PN, asymmetric division; PP, self-amplifying division; SVZ, sub-ventricular zone; V, ventral; VZ, ventricular zone. See also Figs S7 and S8. Scale bars: 50 µm (B,C,I); 10 µm (E,G).

Fig. 4.

Inhibiting SMAD1/5 activity in chick cortical RGCs increases neurogenic divisions and causes their premature depletion and differentiation. (A) Schematic showing cortical neurogenesis in the chick, its main cortical progenitor subtypes and their modes of division. (B) Coronal sections from 3, 5 and 8 day-old chick embryos showing the PAX6+ developing cerebral cortex, formed of SOX2+ neural progenitors, differentiating HuC/D+ and TBR1+ neurons, PAX6+;TBR2 RGCs undergoing mitosis at the apical surface (black arrowheads) and TBR2+ IPCs dividing mostly basally (white arrowheads). Panels on right show magnification of boxed areas in left panels. (C,D) The active, phosphorylated form of SMAD1/5/8 (pSMAD1/5/8) immunoreactivity at E5 (C), and its mean intensity±s.d. measured in 276 apical and 93 basal mitoses obtained from five embryos (D). Panels on right show magnification of boxed area in left panel. Dotted circles indicate pH3+ mitotic nuclei. (E,F) The pSMAD1/5/8 immunoreactivity in mitotic cortical progenitors 24 h after in ovo electroporation (IOE) with the pTis21:RFP reporter along with a control H2B-GFP-producing plasmid (E), and its mean intensity±s.d. quantified in 137 pTis21:RFP+ and 107 pTis21:RFP divisions derived from eight embryos (F). Dotted circles indicate pH3+ mitotic nuclei. (G,H) The mean proportion±s.d. of electroporated (H2B-GFP+) cortical progenitors undergoing pTis21:RFP+ divisions (white arrowheads) or pTis21:RFP divisions (black arrowheads) after IOE of shRNAs targeting cSmad1 or cSmad5 (sh-S1/5, n=12 embryos) or their control (n=9). (I,J) Representative images (I) and mean proportions±s.e.m. (J) of electroporated (H2B-GFP+) cells marked as SOX2±;HuC/D+/- (top), PAX6+/−;TBR2+/− (middle) and HuC/D+/−;TBR1+/− (bottom), assessed 48 h after IOE with sh-S1 (n=8, 7, 5 embryos), sh-S5 (n=12, 5, 6) or their control (n=13, 8, 9). (K) The mean proportion±s.d. of electroporated cells identified as PAX6+;TBR2- RGCs (top), TBR2+ committed cells (middle) and HuC/D+ neurons (bottom), assessed 24 (E4), 48 (E5) and 72 (E6) h after IOE and obtained from n≥6 embryos per condition and stage. Significance was assessed using the non-parametric Mann–Whitney test (D,F), the two-sided unpaired t-test (H) or a two-way ANOVA+Tukey's (J) or Sidak's (K) test. **P<0.01, ***P<0.001; ns, not significant. D, dorsal; L, lateral; M, medial; MZ: mantle zone; N, neuron; NN, symmetric neurogenic division; PN, asymmetric division; PP, self-amplifying division; SVZ, sub-ventricular zone; V, ventral; VZ, ventricular zone. See also Figs S7 and S8. Scale bars: 50 µm (B,C,I); 10 µm (E,G).

Endogenous SMAD1/5 activity was inhibited from the onset of cortical neurogenesis by in ovo electroporation of sh-RNA plasmids that specifically target cSmad1 or cSmad5 and reduced their transcript levels by 40% and 60%, respectively (sh-S1/5; Le Dreau et al., 2012). Inhibiting SMAD1 or SMAD5 activity resulted in a similar phenotype, nearly doubling the proportion of electroporated RGCs undergoing neurogenic pTis21:RFP+ divisions (Fig. 4G,H). This precocious switch to neurogenic divisions provoked a premature and accelerated depletion of electroporated PAX6+;TBR2 RGCs, their accelerated progression towards a committed TBR2+ fate and, ultimately, their differentiation into SOX2;HuC/D+ (Elavl3/4) and TBR1+ neurons (Fig. 4I-K). Thus, full SMAD1/5 activity is required to support RGC self-amplification during chick cortical neurogenesis.

Conversely, enhancing SMAD5 activity through in ovo electroporation of a constitutively active SMAD5 mutant (SMAD5-S/D; Le Dreau et al., 2012) produced the opposite phenotype and reduced by 50% the proportion of electroporated RGCs undergoing neurogenic pTis21:RFP+ divisions (Fig. 5A,B). This reduction in neurogenic divisions was associated with the electroporated cells remaining as PAX6+;TBR2 RGCs and it impeded their transition into the neuronal lineage (Fig. 5C,D). Notably, the SMAD5-S/D construct rescued the phenotype caused by sh-S5 (Fig. 5E,F). In 5 out of 20 electroporated embryos, SMAD5-S/D electroporation itself caused the abnormal generation of ectopic rosettes of cortical progenitors, which developed an apical-basal polarity (Fig. S10). Together, these data revealed that the fine tuning of SMAD1/5 activity is required to properly balance the self-amplification of RGCs with the production of cortical excitatory neurons.

Fig. 5.

Increasing SMAD1/5 activity in chick cortical RGCs impedes neurogenic divisions and restrains differentiation. (A,B) Representative images (A) and mean proportion±s.d. (B) of electroporated (H2B-GFP+) cortical progenitors undergoing pTis21:RFP+ divisions (white arrowheads) or pTis21:RFP divisions (black arrowheads) after in ovo electroporation (IOE) of a constitutively active SMAD5 mutant (5-S/D, n=8 embryos) or its control (n=9). (C,D) Representative images (C) and mean proportion±s.e.m. (D) of electroporated (H2B-GFP+) cells marked as PAX6+/−;TBR2+/− and SOX2+/−;HuC/D+/− cells 48 h after IOE of 5-S/D (n=15 and 14 embryos) or its control (n=7 and 10). (E,F) Representative images (E) and mean proportion±s.e.m. (F) of electroporated (H2B-GFP+) cells marked as SOX2+/−;HuC/D+/− cells 48 h after IOE of a control plasmid (n=13), or sh-S5 combined with a control plasmid (sh-S5, n=12) or with the constitutively active SMAD5-S/D mutant (sh-S5+5-S/D, n=8). Significance was assessed using the two-sided unpaired t-test (B), a two-way ANOVA+Sidak's (D) or +Tukey's (F) tests. **P<0.01, ***P<0.001. See also Fig. S9. Scale bars: 10 µm (A), 50 µm (C,E).

Fig. 5.

Increasing SMAD1/5 activity in chick cortical RGCs impedes neurogenic divisions and restrains differentiation. (A,B) Representative images (A) and mean proportion±s.d. (B) of electroporated (H2B-GFP+) cortical progenitors undergoing pTis21:RFP+ divisions (white arrowheads) or pTis21:RFP divisions (black arrowheads) after in ovo electroporation (IOE) of a constitutively active SMAD5 mutant (5-S/D, n=8 embryos) or its control (n=9). (C,D) Representative images (C) and mean proportion±s.e.m. (D) of electroporated (H2B-GFP+) cells marked as PAX6+/−;TBR2+/− and SOX2+/−;HuC/D+/− cells 48 h after IOE of 5-S/D (n=15 and 14 embryos) or its control (n=7 and 10). (E,F) Representative images (E) and mean proportion±s.e.m. (F) of electroporated (H2B-GFP+) cells marked as SOX2+/−;HuC/D+/− cells 48 h after IOE of a control plasmid (n=13), or sh-S5 combined with a control plasmid (sh-S5, n=12) or with the constitutively active SMAD5-S/D mutant (sh-S5+5-S/D, n=8). Significance was assessed using the two-sided unpaired t-test (B), a two-way ANOVA+Sidak's (D) or +Tukey's (F) tests. **P<0.01, ***P<0.001. See also Fig. S9. Scale bars: 10 µm (A), 50 µm (C,E).

SMAD1/5 regulate RGC self-amplification and early cortical neurogenesis through YAP

To identify the gene regulatory networks controlled by SMAD1/5 during cortical neurogenesis, cortical RGCs from SmadNes mutant and control E12.5 embryos were purified by fluorescence-activated cell sorting (FACS) based on their prominin 1 expression (Corti et al., 2007), and their transcriptomes were compared by genome-wide RNA-sequencing (RNA-seq; Fig. 6A). A shortlist of differentially expressed transcripts (DETs) was identified (90 DETs with adjusted P<0.05 and 128 with adjusted P<0.1: Fig. 6B and Table S1). A gene ontology (GO) term enrichment analysis correlated this DET signature especially to biological processes related to the regulation of neurogenesis and cell biosynthesis (Fig. 6C and Table S2), these two categories containing 33 and 44 genes, respectively, including 15 in common (Table S2). The genes retrieved in these two GO categories code for proteins playing various functions, the most frequent one being related to DNA-binding and the regulation of transcription (Fig. 6D and Table S2). A Transfac/Jaspar analysis revealed that the promoter regions of these DETs are enriched in binding motifs for TFs of the TEAD and SP families (Fig. 6E and Table S3). We considered particularly interesting these latter results pointing to an altered TEAD activity in response to SMAD1/5 inhibition, as the TEAD TFs indeed represent the transcriptional effectors of the Hippo signalling pathway, which regulates cell growth and organ size (Yu et al., 2015). Of the 42 DETs found to be related to either TEAD2 or TEAD4, 15 and 20 were retrieved in the GO terms related to neurogenesis and biosynthesis, respectively (Table S3). Using the genepaint database, we observed that the genes encoding these DETs are indeed expressed in the mouse developing cerebral cortex at mid-corticogenesis, mainly in the VZ (such as Camk2g, Cct3, Cd2bp2, Glce, Hmgn2, Phf21a, Spata13 and Trim24), the cortical plate (Ctnna2, Islr2, Nav1, Ndn and Reln) or in both (Agrn, Arid1a, Ehmt2, Hnrnpk, Klhl25, Spire1 and Slc25a51; Fig. S11).

Fig. 6.

The transcriptional program regulated by SMAD1/5 in early mouse cortical RGCs correlates with an alteration of TEAD activity. (A) Methodology used to compare the transcriptome of cortical RGCs from E12.5 SmadNes mutant embryos (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and control littermate (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) embryos. dT, dorsal telencephalon. (B) Spearman rank correlation and heat map representing the differentially expressed transcripts (DETs; with adjusted P<0.05) between mutant (m) and control (c) cortical RGCs. (C-E) Top 10 GO biological processes (C), molecular function of the genes included in these biological processes (D) and top 10 TF binding motifs associated with the DETs (with adjusted P<0.1) (E). (F) Upon BMP activity, SMAD1/5 are activated and can physically interact and act synergistically with YAP (left). When SMAD1/5 activity is reduced or abrogated, YAP is inactivated and primed for proteosomal degradation (right). (G) Mean activity±s.d. of a TEAD-responsive p8xGTIIC:luciferase reporter 24 h after in ovo electroporation of E3 chick telencephalons with a control plasmid (n=8 embryos), a wild-type YAP1 construct (YAP, n=8), sh-S1 alone (n=11) or combined with YAP (n=9). Significance was assessed using a one-way ANOVA+Tukey's test. ***P<0.001. See also Fig. S10 and Tables S1-S4.

Fig. 6.

The transcriptional program regulated by SMAD1/5 in early mouse cortical RGCs correlates with an alteration of TEAD activity. (A) Methodology used to compare the transcriptome of cortical RGCs from E12.5 SmadNes mutant embryos (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−) and control littermate (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+) embryos. dT, dorsal telencephalon. (B) Spearman rank correlation and heat map representing the differentially expressed transcripts (DETs; with adjusted P<0.05) between mutant (m) and control (c) cortical RGCs. (C-E) Top 10 GO biological processes (C), molecular function of the genes included in these biological processes (D) and top 10 TF binding motifs associated with the DETs (with adjusted P<0.1) (E). (F) Upon BMP activity, SMAD1/5 are activated and can physically interact and act synergistically with YAP (left). When SMAD1/5 activity is reduced or abrogated, YAP is inactivated and primed for proteosomal degradation (right). (G) Mean activity±s.d. of a TEAD-responsive p8xGTIIC:luciferase reporter 24 h after in ovo electroporation of E3 chick telencephalons with a control plasmid (n=8 embryos), a wild-type YAP1 construct (YAP, n=8), sh-S1 alone (n=11) or combined with YAP (n=9). Significance was assessed using a one-way ANOVA+Tukey's test. ***P<0.001. See also Fig. S10 and Tables S1-S4.

The activity of TEADs depends directly on the availability of their co-factors YAP/TAZ, which are themselves regulated by upstream kinases of the Hippo pathway (Yu et al., 2015). Cortical RGCs from the SmadNes embryos did not present any alteration in the transcript levels of the TEAD TFs, nor of other factors known to participate in Hippo signalling (Table S4). Thus, SMAD1/5 does not appear to regulate any member of the Hippo signalling pathway at the transcriptional level. However, previous findings established that YAP can physically interact with SMAD1/5 and that its activity is required for optimal SMAD1/5 activity in several cellular contexts, including cultured mouse embryonic stem cells, the Drosophila wing imaginal disc and mouse astrocyte differentiation during postnatal development (Fig. 6F; Alarcón et al., 2009; Huang et al., 2016). On the other hand, BMP-induced SMAD1/5 signalling has been reported to stimulate YAP protein stability, hence its activity (Fig. 6F; Huang et al., 2016). We thus reasoned that YAP might participate together with SMAD1/5 in controlling RGC self-amplification during cortical neurogenesis. A luciferase assay performed after in ovo electroporation of a TEAD-responsive reporter (p8xGTIIC; Dupont et al., 2011) in the chick dorsal telencephalon confirmed that the ability of YAP overexpression to stimulate TEAD transcriptional activity was markedly impaired when SMAD1 activity was concomitantly inhibited (Fig. 6G).

Therefore, we analysed YAP expression and activity relative to SMAD1/5 activity during cortical neurogenesis. In agreement with recent reports (Kostic et al., 2019; Saito et al., 2018), immunostaining for the YAP protein revealed that the active (nuclear) YAP was more intensely expressed in mitotic apical RGCs than in basally-dividing IPCs, and its expression was strongly correlated with SMAD1/5 activity during both chick and mouse cortical neurogenesis (Fig. 7A-D and Fig. S12A-D). Inhibiting SMAD1/5 during chick cortical neurogenesis impaired YAP activity, as witnessed through the reduced nuclear YAP intensity in mitotic cortical RGCs and the global increase in the proportion of the phosphorylated form of YAP (pYAP), which is primed for proteasomal degradation (Fig. 7E,F and Fig. S12E,F). Similarly, the pYAP/YAP ratio increased in the cortex of E11.5 SmadNes mutant embryos, as shown by immunohistochemistry and western blotting (Fig. 7G,H and Fig. S12G,H). These latter findings indicate that SMAD1/5 positively regulate YAP protein levels and activity both during mouse and chick cortical neurogenesis.

Fig. 7.

SMAD1/5 regulate early cortical neurogenesis through YAP. (A) YAP expression during early mouse corticogenesis (E11.5) relative to SMAD1/5 activity (pSMAD1/5/8). Dotted circles indicate pH3+ mitotic nuclei. (B-D) The mean intensity±s.d. of YAP (B) and pSMAD1/5/8 (C) immunoreactivities, quantified in 70 apical and 28 basal pH3+ mitotic nuclei (n=3 embryos), and their Pearson's correlation coefficient r (D). (E,F) Total YAP immunostaining in the developing chick cerebral cortex (E) and its ratio±s.d. (F) quantified in electroporated pH3+ mitotic cells relative to non-electroporated mitotic cells 24 h after in ovo electroporation (IOE) with a control (Ctrl: 147 pH3+;GFP+ cells, 154 pH3+;GFP cells, n=7 embryos) or sh-S1/5 (sh-S1/5: 162 pH3+;GFP+ cells and 140 pH3+;GFP cells, n=7 embryos). Right panels show magnification of boxed area in left panels. Dotted circles indicate pH3+ mitotic nuclei. (G,H) Immunostaining of pYAP and total YAP (G) and the quantification of the pYAP/YAP intensity ratio±s.d. (H) in the developing cerebral cortex of E11.5 SmadNes mutants (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−, n=4 embryos) and control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+, n=4). (I-M) Representative sections (I) and mean proportion±s.e.m. (J-M) of electroporated (GFP+) cells identified as SOX2+/−;HuC/D+/− (J), PAX6+;TBR2 RGCs (K), TBR2+ (L) and ectopic SOX2+ (M) 48 h after IOE of sh-S1 or its control electroporated alone (Ctrl, n=9 embryos; sh-S1, n=8) or together with a wild-type YAP1 construct (Ctrl+YAP, n=8; sh-S1+YAP, n=8). In I, dashed lines delimitate the ventricular zone and black arrowheads highlight ectopic SOX2+ cortical progenitors. (N) Model proposing that, upon BMP signalling, the activated SMAD1/5 recruit YAP to promote RGC self-amplification. When SMAD1/5 activity is reduced or abrogated and YAP is inactivated and primed for degradation, their transcriptional activity is suppressed, enabling RGCs to undergo neurogenic divisions. N, neuron; S1, SMAD1; S4, SMAD4; S5, SMAD5; VZ, ventricular zone. Significance was assessed using the non-parametric Mann–Whitney test (B,C,F,H) or a two-way ANOVA+Tukey's multiple comparisons test (J-M). *P<0.05, ***P<0.001; ns, P>0.05. See also Fig. S11. Scale bars: 25 µm.

Fig. 7.

SMAD1/5 regulate early cortical neurogenesis through YAP. (A) YAP expression during early mouse corticogenesis (E11.5) relative to SMAD1/5 activity (pSMAD1/5/8). Dotted circles indicate pH3+ mitotic nuclei. (B-D) The mean intensity±s.d. of YAP (B) and pSMAD1/5/8 (C) immunoreactivities, quantified in 70 apical and 28 basal pH3+ mitotic nuclei (n=3 embryos), and their Pearson's correlation coefficient r (D). (E,F) Total YAP immunostaining in the developing chick cerebral cortex (E) and its ratio±s.d. (F) quantified in electroporated pH3+ mitotic cells relative to non-electroporated mitotic cells 24 h after in ovo electroporation (IOE) with a control (Ctrl: 147 pH3+;GFP+ cells, 154 pH3+;GFP cells, n=7 embryos) or sh-S1/5 (sh-S1/5: 162 pH3+;GFP+ cells and 140 pH3+;GFP cells, n=7 embryos). Right panels show magnification of boxed area in left panels. Dotted circles indicate pH3+ mitotic nuclei. (G,H) Immunostaining of pYAP and total YAP (G) and the quantification of the pYAP/YAP intensity ratio±s.d. (H) in the developing cerebral cortex of E11.5 SmadNes mutants (Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0, +/−, n=4 embryos) and control littermates (Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0, +/+, n=4). (I-M) Representative sections (I) and mean proportion±s.e.m. (J-M) of electroporated (GFP+) cells identified as SOX2+/−;HuC/D+/− (J), PAX6+;TBR2 RGCs (K), TBR2+ (L) and ectopic SOX2+ (M) 48 h after IOE of sh-S1 or its control electroporated alone (Ctrl, n=9 embryos; sh-S1, n=8) or together with a wild-type YAP1 construct (Ctrl+YAP, n=8; sh-S1+YAP, n=8). In I, dashed lines delimitate the ventricular zone and black arrowheads highlight ectopic SOX2+ cortical progenitors. (N) Model proposing that, upon BMP signalling, the activated SMAD1/5 recruit YAP to promote RGC self-amplification. When SMAD1/5 activity is reduced or abrogated and YAP is inactivated and primed for degradation, their transcriptional activity is suppressed, enabling RGCs to undergo neurogenic divisions. N, neuron; S1, SMAD1; S4, SMAD4; S5, SMAD5; VZ, ventricular zone. Significance was assessed using the non-parametric Mann–Whitney test (B,C,F,H) or a two-way ANOVA+Tukey's multiple comparisons test (J-M). *P<0.05, ***P<0.001; ns, P>0.05. See also Fig. S11. Scale bars: 25 µm.

Finally, we tested whether increasing YAP activity could compensate for the phenotype caused by SMAD1/5 inhibition. In ovo electroporation of a wild-type form of YAP rescued the premature exhaustion of RGCs driven by sh-S1, reverting it to control levels and impeding their progression towards differentiation into SOX2;HuC/D+ neurons (Fig. 7I-L). Intriguingly, YAP overexpression forced the vast majority of electroporated cells to remain SOX2+ (Fig. 7I,J), with more than 30% being found ectopically in the mantle zone irrespective of SMAD1 inhibition (Fig. 7I,M and Fig. S12I). Together, these results support a model whereby SMAD1/5 promote RGC self-amplification and orchestrate cortical growth and neurogenesis through YAP (Fig. 7N).

In this study, we identify a novel role for the canonical BMP effectors SMAD1/5 in the regulation of brain growth and cortical neurogenesis. More specifically, our findings demonstrate that SMAD1/5 activity stimulates cortical RGC self-amplification and impedes their premature switch to neurogenic divisions. By altering the balance between these modes of divisions, impairing SMAD1/5 activity during early corticogenesis has two main consequences.

First, the cerebral cortex of adult SmadNes mutant mice is smaller along the medial-lateral and rostral-caudal axes, although its thickness and the cell density are nearly normal. These observations suggest that reducing SMAD1/5 activity affects more severely the generation of radial columns than the number of cells per radial unit, emphasizing the importance of SMAD1/5 activity for cortical RGC self-amplification, which represents the main event ensuring the tangential growth of the cerebral cortex and which is particularly crucial before and during the early stages of cortical neurogenesis (Cardenas and Borrell, 2019). Of note, crossing the Smad1fl/fl;Smad5fl/fl mice with the Nestin:cre line from Jackson Laboratory, which produces an efficient Cre-mediated recombination in cortical progenitors only from late embryogenesis (around E17.5; Liang et al., 2012), did not cause any obvious alteration in cortical neurogenesis nor any apparent brain growth defects (Dr Eve Seuntjens, unpublished observations). These observations further support our conclusion that the role of SMAD1/5 in sustaining RGC self-amplification is especially crucial during the early steps of corticogenesis. As the SmadNes heterozygous mutant mice present an overall reduction in brain size and weight, it is likely that SMAD1/5 play a similar role throughout the developing brain. We previously reported that SMAD1/5 promote neural progenitor self-amplification during spinal neurogenesis (Le Dreau et al., 2014, 2018). Altogether, these data thus suggest that SMAD1/5 promote stem cell maintenance and growth throughout the developing CNS.

Second, the premature switch from RGC self-amplification to neurogenic divisions caused by SMAD1/5 inhibition altered the generation of the distinct classes of cortical projection neurons. Reducing SMAD1/5 activity during early mouse corticogenesis caused cortical progenitors to prematurely enter neurogenesis, enhancing the generation of early-born cortical neurons and prematurely exhausting the RGC and IPC pools, subsequently limiting the production of late-born cortical neurons. Related to this aspect, a recent study revealed that SMAD1/5 activity also orchestrates the transition from an early to late phase of neurogenesis during mouse cerebellum development, by repressing the late-born interneuron fate determinant Gsx1 (Ma et al., 2020). Importantly, our results strongly suggest that the dependence on SMAD1/5 activity to maintain RGC self-amplification and ensure appropriate neuronal production during corticogenesis is evolutionarily conserved in amniotes, at least between mammals and birds. This leads us to hypothesize that the TFs SMAD1/5 are part of the core ancestral gene regulatory network that governs corticogenesis throughout the amniote lineage, together with PAX6, the proneural bHLH proteins, and the NOTCH and SLIT/ROBO signalling pathways (Cardenas et al., 2018; Le Dreau et al., 2018; Nomura et al., 2013; Suzuki et al., 2012; Yamashita et al., 2018).

The endogenous activity of SMAD1/5 was assessed using an antibody that specifically recognizes the active carboxy-terminal phosphorylated form of SMAD1/5/8. This phosphorylation targeting the three carboxy-terminal serine residues is mediated by type-1 BMP receptors, activation of which depends on their interaction with type-2 BMP receptors, which itself depends on the binding of BMP ligands (Massague et al., 2005). Thus, the activity of SMAD1/5 described herein should reflect the activity of BMP ligands. Various members of the BMP family (including Bmp2, Bmp4, Bmp5, Bmp6 and Bmp7) are expressed in the developing mouse cerebral cortex (Mehler et al., 1997). To our knowledge BMP7, deletion of which causes microcephaly in the mouse (Segklia et al., 2012), is the sole BMP ligand with a reported role in cortical neurogenesis. BMP7 is expressed by the hem, the meninges and the choroid plexus and can be detected in the cerebrospinal fluid (Segklia et al., 2012). BMP7 activity might thus be transduced to SMAD1/5 within RGCs through either their apical membrane or basal foot.

In agreement with previous studies (Alarcón et al., 2009; Saxena et al., 2018), the endogenous pSMAD1/5/8 immunoreactivity was particularly obvious in mitotic RGCs and IPCs. Although we do not rule out that the low levels of pSMAD1/5/8 immunoreactivity that we observed in other phases of the cell cycle might correspond to low levels of SMAD1/5 transcriptional activity, our data favour the idea that SMAD1/5 are activated immediately before or during mitosis. A growing body of literature reveals that many, if not most, of the genes transcribed during interphase are also transcribed during mitosis, albeit to low levels (Palozola et al., 2019). This low transcriptional activity occurring during mitosis, termed mitotic bookmarking, is believed to ensure transcriptional memory propagation from a mother cell to its daughters. Such activity has been observed for general promoter TFs, and for a growing list of tissue-specific TFs (Palozola et al., 2019). The detection of active pSMAD1/5 immunoreactivity in mitotic RGCs and IPCs observed herein thus supports the idea that SMAD1/5 could play a role in mitotic bookmarking.

Our RNA-seq approach correlated SMAD1/5 impairment with an altered activity of the Hippo signalling effectors TEADs. However, none of the TEAD family members nor other factors known to participate in Hippo signalling presented an altered transcriptional expression in mouse cortical RGCs in response to SMAD1/5 inhibition (Table S4), thereby pointing to a transcription-independent regulation of TEAD activity by SMAD1/5. To test whether SMAD1/5 and TEADs might cooperatively regulate the same target genes, we analysed in silico the presence of SMAD1/5 binding motifs in the proximal promoter region (−2000bp≥TSS≥+500 bp) of the genes retrieved from our RNA-seq that possess TEAD binding motifs. SMAD1/5 binding motifs were found only in 23% (6 out of 26) of the gene promoters containing TEAD2/4 binding motifs (Table S3). Although these results suggest that SMAD1/5 and TEADs might not directly cooperate at the promoter level, it is worth mentioning that these two families of TFs appear to preferentially regulate transcription by binding to distal enhancers (Morikawa et al., 2011; Stein et al., 2015), thereby leaving the question about their direct cooperation open. By contrast, our findings clearly established that the functional cooperation between SMAD1/5 and YAP, a central actor in the Hippo signalling pathway (Yu et al., 2015), is crucial for the regulation of cortical growth and neurogenesis, whereby SMAD1/5 regulate YAP activity in early cortical RGCs. Accordingly, the tight regulation of YAP activity appears to be crucial for correct brain formation and growth. Cortical and general brain development is affected when YAP activity is enhanced, either directly or indirectly (Lavado et al., 2013, 2018; Liu et al., 2018; Saito et al., 2018). More importantly, an aberrant increase in YAP activity has been linked to various types of cortical heterotopia, such as those observed in the Van Maldergem syndrome (Cappello et al., 2013; Liu et al., 2018). Our observation that YAP overexpression causes ribbon-like heterotopias of SOX2+ and PAX6+ progenitors in the developing chick cerebral cortex is therefore reminiscent of these severe mammalian cortical defects. Conversely, a reduction in YAP activity is plausibly one of the mechanistic events contributing to primordial dwarfism syndromes, the defining features of which include microcephaly and general growth defects (Klingseisen and Jackson, 2011). Of note, the SmadNes heterozygous mutant mice present both microcephaly and growth retardation. Interestingly, the levels of YAP activity also determine the abundance and proliferative ability of neocortical basal progenitors (Kostic et al., 2019), such that its regulation might have contributed to the evolutionary diversification and expansion of the mammalian neocortex. Our findings suggest that the regulation of YAP activity by SMAD1/5 in RGCs might be evolutionarily conserved. It is thus tempting to speculate that modulations of the canonical BMP activity might have influenced the growth and expansion of the cerebral cortex during amniote evolution, a hypothesis that remains to be investigated further.

Animals

Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0 embryos and postnatal mice were obtained by crossing Smad1fl/fl;Smad5fl/fl mice (Moya et al., 2012) with transgenic mice that express Cre-recombinase in neural progenitor cells and somites from E8.5 (NesCre8 mice: Petersen et al., 2002). Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0 littermates were used as controls. The days of the vaginal plug and birth were defined as E0.5 and P0, respectively. Smad1fl/fl;Smad5fl/fl and NesCre8 mice were maintained in their original mixed genetic backgrounds (CD1, 129/ola and C57BL6). All the experimental procedures were carried out in accordance with the European Union guidelines (Directive 2010/63/EU) and the protocols were approved by the ethics committee of the Parc Científic de Barcelona.

Fertilized white Leghorn chicken eggs were provided by Granja Gibert, rambla Regueral, S/N, 43850 Cambrils, Spain. Eggs were incubated in a humidified atmosphere at 38°C in a Javier Masalles 240N incubator for the appropriate duration and staged according to the method of Hamburger and Hamilton (HH; Hamburger and Hamilton, 1951). According to EU animal care guidelines, no IACUC approval was necessary to perform the experiments described herein, as the embryos used in this study were always harvested at early stages of embryonic development. Sex was not identified at these stages.

In ovo electroporation

Unilateral in ovo electroporations were performed in the developing chick dorsal telencephalon at stage HH18 (E3, 69-72 h of incubation). Analyses were performed specifically in the dorsal-medial-lateral region of the developing chick cerebral cortex to minimize any possible variability along the medial-lateral axis. Plasmids were diluted in RNAse-free water at the required concentration (0 to 4 µg/µl) and injected into the right cerebral ventricle using a fine glass needle. Electroporation was triggered by applying five pulses of 50 ms at 22.5 V with 50 ms intervals using an Intracel Dual Pulse (TSS10) electroporator. Electroporated chicken embryos were incubated back at 38°C and recovered at the times indicated.

Plasmids

Inhibition of cSmad1 and cSmad5 expression was triggered by electroporation of short-hairpin constructs inserted into the pSuper (Oligoengine) or pSHIN vectors together with a control H2B-GFP-producing plasmid as previously reported (Kojima et al., 2004; Le Dreau et al., 2012, 2018). Electroporation of 2-4 µg/µl of these sh-Smad1 and sh-Smad5 constructs caused specific and reproducible inhibition (40% and 60%, respectively) of the target gene expression (Le Dreau et al., 2012). The pCAGGS_SMAD5-SD_ires_GFP, its control pCAGGS_ires_GFP (pCIG), as well as the pTis21:RFP reporter used to assess the modes of divisions undergone by spinal progenitors, have been previously described in detail (Le Dreau et al., 2012; Megason and McMahon, 2002; Saade et al., 2013). The pCAGGS_Flag-YAP1_ires_GFP construct was obtained by subcloning from a pCDNA:Flag-YAP1 (Addgene plasmid #18881, deposited by Yosef Shaul; Levy et al., 2008) kindly provided by Conchi Estaras (Lewis Katz School of Medicine, Temple University, Philadelphia, PA, USA). The TEAD-responsive p8xGTIIC:luciferase reporter (Addgene plasmid #34615, deposited by Stefano Piccolo; Dupont et al., 2011) was kindly provided by Sebastian Pons (Instituto de Biología Molecular de Barcelona, Barcelona, Spain).

Luciferase assay

TEAD transcriptional activity was assessed following electroporation of the p8xGTIIC:luciferase reporter together with a Renilla luciferase reporter used for normalization, in combination with the indicated plasmids required for experimental manipulation. Embryos were harvested 24 h later, the electroporated telencephalic region carefully dissected and homogenized in a Passive Lysis Buffer on ice (Promega). Firefly- and Renilla-luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega).

In situ hybridization

Chicken embryos were recovered at the indicated stages, fixed overnight at 4°C in 4% paraformaldehyde (PFA), rinsed in PBS and processed for whole-mount RNA in situ hybridization following standard procedures. Probes against chick Smad1 (#chEST899n18) and Smad8 (#chEST222h17) were purchased from the chicken EST project (UK-HGMP RC; www.chick.manchester.ac.uk/). The probe against cSmad5 was kindly provided by Dr Marian Ros (Instituto de Biomedicina y Biotecnología de Cantabria, Santander, Spain). Hybridized embryos were post-fixed in 4% PFA and washed in PBS containing 0.1% Triton X-100 (PBT), and 45 µm-thick vibratome sections (VT1000S, Leica) were mounted and photographed under a microscope (DC300, Leica). The data show representative images obtained from three embryos for each stage and probe. The images of mSmad1, mSmad5 and mSmad8 expression in the developing mouse dorsal telencephalon at E14.5 and those of candidate target genes selected from the RNA-seq analysis were all obtained from the Genepaint database (https://gp3.mpg.de).

Histology and immunohistochemistry

Mouse embryos were recovered at the indicated stages and their heads fixed by immersion in 4% PFA for 24 h at 4°C, cryoprotected with 30% sucrose in PBS, embedded in Tissue-Tek O.C.T. (Sakura Finetek), frozen in isopentane at −30°C and sectioned coronally on a cryostat (Leica). Cryosections (14 μm) were collected on Starfrost pre-coated slides (Knittel Glasser) and distributed serially. Postnatal and adult mice were deeply anaesthetized in a CO2 chamber and transcardially perfused with 4% PFA. The brains were removed, post-fixed and vibratome sections (40 μm) were then distributed serially. Chicken embryos were carefully dissected, fixed for 2 h at room temperature (RT) in 4% PFA, rinsed in PBS and cryoprotected with 30% sucrose in PBS, and 16 µm-thick coronal sections prepared with a cryostat.

For both species, immunostaining was performed following standard procedures. After washing in PBT, the sections were blocked for 1 h at RT in PBT supplemented with 10% bovine serum albumin (BSA). When necessary, sections were submitted to an antigen retrieval treatment before blocking by boiling sections for 10 min in sodium citrate buffer [2 mM citric acid monohydrate, 8 mM tri-sodium citrate dehydrate (pH 6.0)]. For BrdU immunostaining, sections were incubated before blocking in 50% formamide in 2× SSC at 64°C for 10 min followed by an incubation in 2N HCl at 37°C for 30 min and finally 10 min in 0.1 M boric acid (pH 8.5) at RT. Sections were then incubated overnight at 4°C with the appropriate primary antibodies (Table S5) diluted in a solution of PBT supplemented with 10% BSA or sheep serum. After washing in PBT, sections were incubated for 2 h at RT with the appropriate secondary antibodies diluted in PBT supplemented with 10% BSA or sheep serum. Alexa488-, Alexa555-, Alexa633 and Cy5-conjugated secondary antibodies were obtained from Invitrogen (#A-11078; #A-21202; #A-31570; #A-21050; #A-21206; #A-31572; #A-21070; #A-21208; #A-21094) and Jackson Laboratories (#115-175-146; #712-175-150) and all used at dilution 1:1000. Sections were finally stained with 1 μg/ml DAPI and mounted in Mowiol (Sigma-Aldrich).

Cell cycle exit assay

Pregnant female mice received an intra-peritoneal injection of BrdU (100 mg/kg; Sigma-Aldrich) and were sacrificed 24 h later. Embryos were collected and processed as described above. Sections were immunostained for BrdU and Tuj1 and the cell cycle exit rate (neuronal output) was estimated by quantifying the proportion of BrdU+-immunolabelled cells that were Tuj1+ (% of Tuj1+;BrdU+/BrdU+ cells).

Image acquisition and treatment

Optical sections of mouse and chick embryo fixed samples (coronal views) were acquired at RT with the Leica LAS software, in a Leica SP5 confocal microscope using 10× (dry HC PL APO, NA 0.40), 20× (dry HC PL APO, NA 0.70), 40× (oil HCX PL APO, NA 1.25-0.75) or 63× (oil HCX PL APO, NA 1.40-0.60) objective lenses. Maximal projections obtained from 2 µm z-stack images were processed in Photoshop CS5 (Adobe) or ImageJ for image merging, resizing and cell counting. Optical sections of postnatal and adult mouse samples were acquired with a Leica AF7000 motorized wide-field microscope. Cell counting in embryo, postnatal and adult mouse samples was performed in a 100 μm-wide column of the lateral cortical wall, as indicated in the figures. Cell counts were performed in a minimum of three sections of the same rostral-caudal level per embryo or postnatal mouse sample. Cell counting and measurements of the layer thickness in the P7 and P60 mouse cerebral cortex was carried out based on DAPI staining combined with CUX1 (Nieto et al., 2004), CTIP2 (Bcl11b; Arlotta et al., 2005), TBR1 (Bulfone et al., 1995) and NeuN immunostaining. Quantification of pSMAD1/5/8, PAX6, TBR2, YAP and pYAP intensities was assessed using the ImageJ software. Cell nuclei of mitotic pH3+ cells or H2B-GFP+ electroporated and neighbouring non-electroporated cells were delimitated by polygonal selection, and the mean intensity was quantified as mean grey values.

Brain morphometry

Morphometric parameters were estimated from serial coronal sections stained with DAPI, using the ImageJ software. Images were collected in a Leica AF7000 microscope using 5× or 10× objective. The brain volume was calculated according to the Cavalieri principle (Gundersen and Jensen, 1987), which consists of multiplying the distance between sections by their area, using a set of serial consecutive sections spanning from Bregma +1.10 mm to −1.82 mm (Paxinos and Franklin, 2001).

Western blot

Total protein extracts (≈40 µg) were resolved by SDS-PAGE following standard procedures and transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences) that was probed with the primary antibodies (Table S5), binding of which was detected by infra-red fluorescence using the LI-COR Odyssey IR Imaging System V3.0 (LI-COR Biosciences).

Purification of mouse Prominin1+ RGCs

The purification of Prominin1+ cortical RGCs was achieved by FACS of dissociated cells obtained from the forebrain of Smad1wt/fl;Smad5wt/fl;Nestin:Cre+/0 and Smad1wt/fl;Smad5wt/fl;Nestin:Cre0/0 E12.5 embryos. Forebrains of littermates with the same genotype were pooled and incubated in Hanks’ balanced salt solution (HBSS) containing 0.6% glucose and 5 mM EDTA for 5 min at 37°C. Cells were mechanically dissociated by gentle trituration and collected by centrifugation at 300 g for 10 min at 4°C. Cells were resuspended in 200 µl of incubation media (PBS containing 0.6% glucose, 2% foetal bovine serum and 0.02% NaN3) and kept for 10 min at 4°C with mild agitation. Cells were then incubated for 30 min at 4°C with an APC-conjugated anti-Prominin1 antibody (eBioscience, #17-1331-81) diluted at 0.2 mg/ml. Incubation with a rat IgG1-APC antibody (eBioscience, #17-4301-82, diluted at 0.2 mg/ml) was performed in parallel to define non-specific fluorescence. After the incubation, cells were centrifuged at 300 g for 10 min at 4°C and incubated with 1 ml of incubation media. Dissociated cells were then filtered through a falcon tube with a cell strainer cap (BD Biosciences; BD falcon 12×75 mm) in which the filter was previously dampened with 500 µl of incubation media. The number of isolated cells was determined using a Neubauer chamber and cells were diluted to a final concentration of 2-3×106 cells/ml. DAPI (20 ng/ml: Vector Laboratories) was added to identify dead cells. Sorting was performed using a BD FACS Aria™ Fusion (BD Biosciences) cytometer. After sorting, collector tubes were centrifuged at 1000 g for 10 min at 4°C and cell pellets stored at −80°C.

RNA-seq

RNA was extracted from pools of 6×105 FACS-isolated cells using the miRNeasy Micro Kit (Qiagen). Quantification of total RNA was performed by Qubit® RNA BR Assay kit (Thermo Fisher Scientific) and RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent) was used to estimate the total RNA integrity. The three pairs of samples of RNAs from SmadNes mutant and control RGCs presenting the best RNA integrity were used for sequencing. Sample preparation protocol for the RNA-seq libraries was carried out following the manufacturer's recommendations of KAPA Stranded mRNA-Seq Illumina® Platforms Kit (Roche-Kapa Biosystems). The libraries were sequenced on HiSeq2500 (Illumina) in paired-end mode with a read length of 2×100 bp using TruSeq SBS Kit v4 (Illumina). Each sample was sequenced in a fraction of a sequencing v4 flow cell lane, following the manufacturer's protocol. Image analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.18.66.3) and followed by generation of FASTQ sequence files. RNA-seq paired-end reads were mapping against Mus musculus reference genome (GRCm38) using STAR version 2.5.3a with ENCODE parameters for long RNA. Isoforms were quantified using RSEM version 1.3.0 with default parameters for stranded sequencing and the gencode version M15. Differential isoform analysis was performed using DESeq2 version 1.18 with default parameters. We considered differentially expressed transcripts to be those showing an adjusted P-value <0.05 or adjusted P-value <0.1 (extended list). Fold-change values between genotypes (SmadNes mutants over controls) are expressed in Log2 (Table S1). The GO term enrichment analysis (biological process) of the extended DET signature was performed using the PANTHER classification system (http://pantherdb.org) and the Transfac/Jaspar analysis using Enrichr. The binding motifs for SMAD1 and SMAD5 were obtained from Jaspar. The proximal promoter region (−2000bp≥TSS≥+500 bp) of each gene of interest was obtained from the UCSC Genome Browser (https://genome.ucsc.edu/) and the presence of the distinct TF binding motifs in the promoters was determined using the software FIMO (http://meme-suite.org/tools/fimo) (Table S3).

Statistical analyses

No statistical method was used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments. Statistical analyses were performed using the GraphPad Prism 6 software (GraphPad Software, Inc.). Unless noted otherwise (see quantifications), cell counts were typically performed on 3-5 images per embryo and n values correspond to different embryos or animals. The normal distribution of the values was assessed by the Shapiro-Wilk normality test. Significance was then assessed with a two-sided unpaired t-test, one-way ANOVA+Tukey's test or two-way ANOVA+Sidak's or Tukey's test for data presenting a normal distribution, or alternatively with the non-parametric Mann–Whitney test for non-normally distributed data. The following convention was used: n.s.: P>0.05; *P<0.05, **P<0.01, ***P<0.001. The detailed information related to quantifications are detailed in the figure legends.

We thank the members of M.L.A.’s and E.M.’s laboratories for their discussion of this study. We thank E. Rebollo and the IBMB Molecular Imaging platform, J. Comas and the Parc Científic de Barcelona flow cytometry facility, and the CNAG-CRG Sequencing Unit for their assistance. We are grateful to E. J. Robertson and W. Zhong for providing the Smad1fl/fl and Nestin:cre mice, to E. Seuntjens for sharing information, and to C. Estarás, S. Pons, M. Ros and M. Wegner for providing reagents.

Author contributions

Conceptualization: S.N., G.L.D.; Methodology: S.N., I.P., A.E.-C., S.U., J.D.M., G.L.D.; Investigation: S.N., I.P., A.E.-C., G.L.D.; Resources: A.Z., M.L.A., E.M.; Writing - original draft: G.L.D.; Visualization: G.L.D.; Supervision: M.L.A., E.M., G.L.D.; Funding acquisition: M.L.A., E.M.

Funding

The work in M.L.A.’s and E.M.’s laboratories was supported by the Ministerio de Ciencia e Innovacion, Gobierno de España (MCINN; grants SAF2016-77971-R, RED2018-102553 and BFU2016-77498-P). I.P. received a PhD fellowship from the Ministerio de Economía, Industria y Competitividad, Gobierno de España (MINEICO, BES2014-069217). A.E.-C. was supported by Instituto de Salud Carlos III (MINEICO, PT17/0009/0019) and the European Regional Development Fund (FEDER). G.L.D. was supported by the Fundación Científica Asociación Española Contra el Cáncer (AIO14142105LED).

Data availability

RNA-seq data have been deposited in GEO under accession number GSE153751.

Alarcón
,
C.
,
Zaromytidou
,
A.-I.
,
Xi
,
Q.
,
Gao
,
S.
,
Yu
,
J.
,
Fujisawa
,
S.
,
Barlas
,
A.
,
Miller
,
A. N.
,
Manova-Todorova
,
K.
,
Macias
,
M. J.
, et al. 
(
2009
).
Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways
.
Cell
139
,
757
-
769
.
Arlotta
,
P.
,
Molyneaux
,
B. J.
,
Chen
,
J.
,
Inoue
,
J.
,
Kominami
,
R.
and
MacKlis
,
J. D.
(
2005
).
Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo
.
Neuron
45
,
207
-
221
.
Bhat
,
R. V.
,
Axt
,
K. J.
,
Fosnaugh
,
J. S.
,
Smith
,
K. J.
,
Johnson
,
K. A.
,
Hill
,
D. E.
,
Kinzler
,
K. W.
and
Baraban
,
J. M.
(
1996
).
Expression of the APC tumor suppressor protein in oligodendroglia
.
Glia
17
,
169
-
174
.
Britanova
,
O.
,
de Juan Romero
,
C.
,
Cheung
,
A.
,
Kwan
,
K. Y.
,
Schwark
,
M.
,
Gyorgy
,
A.
,
Vogel
,
T.
,
Akopov
,
S.
,
Mitkovski
,
M.
,
Agoston
,
D.
, et al. 
(
2008
).
Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex
.
Neuron
57
,
378
-
392
.
Bulfone
,
A.
,
Smiga
,
S. M.
,
Shimamura
,
K.
,
Peterson
,
A.
,
Puelles
,
L.
and
Rubenstein
,
J. L. R.
(
1995
).
T-Brain-1: a homolog of Brachyury whose expression defines molecularly distinct domains within the cerebral cortex
.
Neuron
15
,
63
-
78
.
Cappello
,
S.
,
Gray
,
M. J.
,
Badouel
,
C.
,
Lange
,
S.
,
Einsiedler
,
M.
,
Srour
,
M.
,
Chitayat
,
D.
,
Hamdan
,
F. F.
,
Jenkins
,
Z. A.
,
Morgan
,
T.
, et al. 
(
2013
).
Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical development
.
Nat. Genet.
45
,
1300
-
1308
.
Cardenas
,
A.
and
Borrell
,
V.
(
2019
).
Molecular and cellular evolution of corticogenesis in amniotes
.
Cell. Mol. Life Sci.
77
,
1435
-
1460
.
Cardenas
,
A.
,
Villalba
,
A.
,
de Juan Romero
,
C.
,
Pico
,
E.
,
Kyrousi
,
C.
,
Tzika
,
A. C.
,
Tessier-Lavigne
,
M.
,
Ma
,
L.
,
Drukker
,
M.
,
Cappello
,
S.
, et al. 
(
2018
).
Evolution of cortical neurogenesis in amniotes controlled by robo signaling levels
.
Cell
174
,
590
-
606.e21
.
Claus Stolt
,
C.
,
Rehberg
,
S.
,
Ader
,
M.
,
Lommes
,
P.
,
Riethmacher
,
D.
,
Schachner
,
M.
,
Bartsch
,
U.
and
Wegner
,
M.
(
2002
).
Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10
.
Genes Dev.
16
,
165
-
170
.
Corti
,
S.
,
Nizzardo
,
M.
,
Nardini
,
M.
,
Donadoni
,
C.
,
Locatelli
,
F.
,
Papadimitriou
,
D.
,
Salani
,
S.
,
Del Bo
,
R.
,
Ghezzi
,
S.
,
Strazzer
,
S.
, et al. 
(
2007
).
Isolation and characterization of murine neural stem/progenitor cells based on Prominin-1 expression
.
Exp. Neurol.
205
,
547
-
562
.
De Juan Romero
,
C.
and
Borrell
,
V.
(
2015
).
Coevolution of radial glial cells and the cerebral cortex
.
Glia
63
,
1303
-
1319
.
Dupont
,
S.
,
Morsut
,
L.
,
Aragona
,
M.
,
Enzo
,
E.
,
Giulitti
,
S.
,
Cordenonsi
,
M.
,
Zanconato
,
F.
,
Digabel
,
J.
,
Le, Forcato
,
M.
, et al. 
(
2011
).
Role of YAP / TAZ in mechanotransduction
.
Nature
474
,
179
-
183
.
Gundersen
,
H. J. G.
and
Jensen
,
E. B.
(
1987
).
The efficiency of systematic sampling in stereology and its prediction
.
J. Microsc.
147
,
229
-
263
.
Hamburger
,
V.
and
Hamilton
,
H. L.
(
1951
).
A series of normal stages in the development of chick embryo
.
J. Morphol.
88
,
49
-
92
.
Hu
,
W. F.
,
Chahrour
,
M. H.
and
Walsh
,
C. A.
(
2014
).
The diverse genetic landscape of neurodevelopmental disorders
.
Annu. Rev. Genomics Hum. Genet.
15
,
195
-
213
.
Huang
,
Z.
,
Hu
,
J.
,
Pan
,
J.
,
Wang
,
Y.
,
Hu
,
G.
,
Zhou
,
J.
,
Mei
,
L.
and
Xiong
,
W. C.
(
2016
).
YAP stabilizes SMAD1 and promotes BMP2-induced neocortical astrocytic differentiation
.
Development
143
,
2398
-
2409
.
Jayaraman
,
D.
,
Bae
,
B.-I.
and
Walsh
,
C. A.
(
2018
).
The genetics of primary microcephaly
.
Annu. Rev. Genomics Hum. Genet.
19
,
177
-
200
.
Klingseisen
,
A.
and
Jackson
,
A. P.
(
2011
).
Mechanisms and pathways of growth failure in primordial dwarfism
.
Genes Dev.
25
,
2011
-
2024
.
Kojima
,
S.
,
Vignjevic
,
D.
and
Borisy
,
G. G.
(
2004
).
Improved silencing vector co-expressing GFP and small hairpin RNA
.
BioTechniques
36
,
74
-
79
.
Kostic
,
M.
,
Paridaen
,
J.
,
Long
,
K. R.
,
Kalebic
,
N.
,
Langen
,
B.
,
Grubling
,
N.
,
Wimberger
,
P.
,
Kawasaki
,
H.
,
Namba
,
T.
and
Huttner
,
W. B.
(
2019
).
YAP activity is necessary and sufficient for basal progenitor abundance and proliferation in the developing neocortex
.
Cell Rep.
27
,
1103
-
1118.e6
.
Lavado
,
A.
,
He
,
Y.
,
Pare
,
J.
,
Neale
,
G.
,
Olson
,
E. N.
,
Giovannini
,
M.
and
Cao
,
X.
(
2013
).
Tumor suppressor Nf2 limits expansion of the neural progenitor pool by inhibiting Yap/Taz transcriptional coactivators
.
Development
140
,
3323
-
3334
.
Lavado
,
A.
,
Park
,
J. Y.
,
Pare
,
J.
,
Finkelstein
,
D.
,
Pan
,
H.
,
Xu
,
B.
,
Fan
,
Y.
,
Kumar
,
R. P.
,
Neale
,
G.
,
Kwak
,
Y. D.
, et al. 
(
2018
).
The hippo pathway prevents YAP/TAZ-driven hypertranscription and controls neural progenitor number
.
Dev. Cell
47
,
576
-
591.e8
.
Le Dreau
,
G.
,
Garcia-Campmany
,
L.
,
Rabadan
,
M. A.
,
Ferronha
,
T.
,
Tozer
,
S.
,
Briscoe
,
J.
and
Marti
,
E.
(
2012
).
Canonical BMP7 activity is required for the generation of discrete neuronal populations in the dorsal spinal cord
.
Development
139
,
259
-
268
.
Le Dreau
,
G.
,
Saade
,
M.
,
Gutierrez-Vallejo
,
I.
and
Marti
,
E.
(
2014
).
The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord
.
J. Cell Biol.
204
,
591
-
605
.
Le Dreau
,
G.
,
Escalona
,
R.
,
Fueyo
,
R.
,
Herrera
,
A.
,
Martinez
,
J. D.
,
Usieto
,
S.
,
Menendez
,
A.
,
Pons
,
S.
,
Martinez-Balbas
,
M. A.
and
Marti
,
E.
(
2018
).
E proteins sharpen neurogenesis by modulating proneural bHLH transcription factors' activity in an E-box-dependent manner
.
Elife
7
,
e37267
.
Levy
,
D.
,
Adamovich
,
Y.
,
Reuven
,
N.
and
Shaul
,
Y.
(
2008
).
Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage
.
Mol. Cell
29
,
350
-
361
.
Li
,
W.
,
Cogswell
,
C. A.
and
LoTurco
,
J. J.
(
1998
).
Neuronal differentiation of precursors in the neocortical ventricular zone is triggered by BMP
.
J. Neurosci.
18
,
8853
-
8862
.
Liang
,
H.
,
Hippenmeyer
,
S.
and
Ghashghaei
,
H. T.
(
2012
).
A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors
.
Biol. Open
1
,
1200
-
1203
.
Liu
,
W. A.
,
Chen
,
S.
,
Li
,
Z.
,
Lee
,
C. H.
,
Mirzaa
,
G.
,
Dobyns
,
W. B.
,
Ross
,
M. E.
,
Zhang
,
J.
and
Shi
,
S.-H.
(
2018
).
PARD3 dysfunction in conjunction with dynamic HIPPO signaling drives cortical enlargement with massive heterotopia
.
Genes Dev.
32
,
763
-
780
.
Llinares-Benadero
,
C.
and
Borrell
,
V.
(
2019
).
Deconstructing cortical folding: genetic, cellular and mechanical determinants
.
Nat. Rev. Neurosci.
20
,
161
-
176
.
Lui
,
J. H.
,
Hansen
,
D. V.
and
Kriegstein
,
A. R.
(
2011
).
Development and evolution of the human neocortex
.
Cell
146
,
18
-
36
.
Ma
,
T. C.
,
Vong
,
K. I.
,
Kwan
,
K. M.
,
Ma
,
T. C.
,
Vong
,
K. I.
and
Kwan
,
K. M.
(
2020
).
Spatiotemporal decline of BMP signaling activity in neural progenitors mediates fate transition and safeguards neurogenesis report spatiotemporal decline of BMP signaling activity in neural progenitors mediates fate transition and safeguards neurogenesis
.
CellReports
30
,
3616
-
3624;e4
.
Mabie
,
P. C.
,
Mehler
,
M. F.
and
Kessler
,
J. A.
(
1999
).
Multiple roles of bone morphogenetic protein signaling in the regulation of cortical cell number and phenotype
.
J. Neurosci.
19
,
7077
-
7088
.
Martynoga
,
B.
,
Drechsel
,
D.
and
Guillemot
,
F.
(
2012
).
Molecular control of neurogenesis: a view from the mammalian cerebral cortex
.
Cold Spring Harb. Perspect Biol.
4
,
a008359
.
Massague
,
J.
,
Seoane
,
J.
and
Wotton
,
D.
(
2005
).
Smad transcription factors
.
Genes Dev.
19
,
2783
-
2810
.
Megason
,
S. G.
and
McMahon
,
A. P.
(
2002
).
A mitogen gradient of dorsal midline Wnts organizes growth in the CNS
.
Development
129
,
2087
-
2098
.
Mehler
,
M. F.
,
Mabie
,
P. C.
,
Zhang
,
D.
and
Kessler
,
J. A.
(
1997
).
Bone morphogenetic proteins in the nervous system
.
Trends Neurosci.
20
,
309
-
317
.
Morikawa
,
M.
,
Koinuma
,
D.
,
Tsutsumi
,
S.
,
Vasilaki
,
E.
,
Kanki
,
Y.
,
Heldin
,
C.
,
Aburatani
,
H.
and
Miyazono
,
K.
(
2011
).
ChIP-seq reveals cell type-specific binding patterns of BMP-specific Smads and a novel binding motif
.
Nucleic Acids Res.
39
,
8712
-
8727
.
Moya
,
I. M.
,
Umans
,
L.
,
Maas
,
E.
,
Pereira
,
P. N. G.
,
Beets
,
K.
,
Francis
,
A.
,
Sents
,
W.
,
Robertson
,
E. J.
,
Mummery
,
C. L.
,
Huylebroeck
,
D.
, et al. 
(
2012
).
Stalk cell phenotype depends on integration of Notch and Smad1/5 signaling cascades
.
Dev. Cell
22
,
501
-
514
.
Nieto
,
M.
,
Monuki
,
E. S.
,
Tang
,
H.
,
Imitola
,
J.
,
Haubst
,
N.
,
Khoury
,
S. J.
,
Cunningham
,
J.
,
Gotz
,
M.
and
Walsh
,
C. A.
(
2004
).
Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex
.
J. Comp. Neurol.
479
,
168
-
180
.
Nomura
,
T.
,
Gotoh
,
H.
and
Ono
,
K.
(
2013
).
Changes in the regulation of cortical neurogenesis contribute to encephalization during amniote brain evolution
.
Nat. Commun.
4
,
2206
.
Palozola
,
K. C.
,
Lerner
,
J.
and
Zaret
,
K. S.
(
2019
).
A changing paradigm of transcriptional memory propagation through mitosis
.
Nat. Rev. Mol. Cell Biol.
20
,
55
-
64
.
Panchision
,
D. M.
,
Pickel
,
J. M.
,
Studer
,
L.
,
Lee
,
S. H.
,
Turner
,
P. A.
,
Hazel
,
T. G.
and
McKay
,
R. D.
(
2001
).
Sequential actions of BMP receptors control neural precursor cell production and fate
.
Genes Dev.
15
,
2094
-
2110
.
Passemard
,
S.
,
Kaindl
,
A. M.
and
Verloes
,
A.
(
2013
).
Microcephaly
.
Handb. Clin. Neurol.
111
,
129
-
141
.
Paxinos
,
G.
and
Franklin
,
K. B. J.
(
2001
).
The Mouse Brain in Stereotaxic Coordinates, 2nd ed. Academic Press
Petersen
,
P. H.
,
Zou
,
K.
,
Hwang
,
J. K.
,
Jan
,
Y. N.
and
Zhong
,
W.
(
2002
).
Progenitor cell maintenance requires numb and numblike during mouse neurogenesis
.
Nature
419
,
929
-
934
.
Saade
,
M.
,
Gutiérrez-Vallejo
,
I.
,
LeDréau
,
G.
,
Rabadán
,
M. A.
,
Miguez
,
D. G.
,
Buceta
,
J.
and
Martí
,
E.
(
2013
).
Sonic hedgehog signaling switches the mode of division in the developing nervous system
.
Cell Rep.
4
,
492
-
503
.
Saade
,
M.
,
Gonzalez-Gobartt
,
E.
,
Escalona
,
R.
,
Usieto
,
S.
and
Marti
,
E.
(
2017
).
Shh-mediated centrosomal recruitment of PKA promotes symmetric proliferative neuroepithelial cell division
.
Nat. Cell Biol.
19
,
493
-
503
.
Saade
,
M.
,
Blanco-Ameijeiras
,
J.
,
Gonzalez-Gobartt
,
E.
and
Marti
,
E.
(
2018
).
A centrosomal view of CNS growth
.
Development
145
,
dev170613
.
Saito
,
K.
,
Kawasoe
,
R.
,
Sasaki
,
H.
,
Kawaguchi
,
A.
and
Miyata
,
T.
(
2018
).
Neural progenitor cells undergoing Yap/Tead-mediated enhanced self-renewal form heterotopias more easily in the diencephalon than in the telencephalon
.
Neurochem. Res.
43
,
180
-
189
.
Saxena
,
M.
,
Agnihotri
,
N.
and
Sen
,
J.
(
2018
).
Perturbation of canonical and non-canonical BMP signaling affects migration, polarity and dendritogenesis of mouse cortical neurons
.
Development
145
,
dev147157
.
Segklia
,
A.
,
Seuntjens
,
E.
,
Elkouris
,
M.
,
Tsalavos
,
S.
,
Stappers
,
E.
,
Mitsiadis
,
T. A.
,
Huylebroeck
,
D.
,
Remboutsika
,
E.
and
Graf
,
D.
(
2012
).
Bmp7 regulates the survival, proliferation, and neurogenic properties of neural progenitor cells during corticogenesis in the mouse
.
PLoS ONE
7
,
e34088
.
Shitamukai
,
A.
,
Konno
,
D.
and
Matsuzaki
,
F.
(
2011
).
Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors
.
J. Neurosci.
31
,
3683
-
3695
.
Stein
,
C.
,
Roma
,
G.
,
Bergling
,
S.
,
Clay
,
I.
,
Ruchti
,
A.
,
Agarinis
,
C.
,
Schmelzle
,
T.
,
Bouwmeester
,
T.
,
Schübeler
,
D.
and
Bauer
,
A.
(
2015
).
YAP1 exerts its transcriptional control via TEAD-mediated activation of enhancers
.
PLoS Genet.
11
,
e1005465
.
Sun
,
W.
,
Cornwell
,
A.
,
Li
,
J.
,
Peng
,
S.
,
Joana Osorio
,
M.
,
Aalling
,
N.
,
Wang
,
S.
,
Benraiss
,
A.
,
Lou
,
N.
,
Goldman
,
S. A.
, et al. 
(
2017
).
SOX9 is an astrocyte-specific nuclear marker in the adult brain outside the neurogenic regions
.
J. Neurosci.
37
,
4493
-
4507
.
Suzuki
,
I. K.
,
Kawasaki
,
T.
,
Gojobori
,
T.
and
Hirata
,
T.
(
2012
).
The temporal sequence of the mammalian neocortical neurogenetic program drives mediolateral pattern in the chick pallium
.
Dev. Cell
22
,
863
-
870
.
Taverna
,
E.
,
Götz
,
M.
and
Huttner
,
W. B.
(
2014
).
The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex
.
Annu. Rev. Cell Dev. Biol.
30
,
465
-
502
.
Wang
,
X.
,
Tsai
,
J.-W.
,
LaMonica
,
B.
and
Kriegstein
,
A. R.
(
2011
).
A new subtype of progenitor cell in the mouse embryonic neocortex
.
Nat. Neurosci.
14
,
555
-
561
.
Yamashita
,
W.
,
Takahashi
,
M.
,
Kikkawa
,
T.
,
Gotoh
,
H.
,
Osumi
,
N.
,
Ono
,
K.
and
Nomura
,
T.
(
2018
).
Conserved and divergent functions of Pax6 underlie species-specific neurogenic patterns in the developing amniote brain
.
Development
145
,
dev159764
.
Yu
,
F.-X.
,
Zhao
,
B.
and
Guan
,
K.-L.
(
2015
).
Hippo pathway in organ size control, tissue homeostasis, and cancer
.
Cell
163
,
811
-
828
.

Competing interests

The authors declare no competing or financial interests.

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