Article1 July 2004free access Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways Carol Schuurmans Corresponding Author Carol Schuurmans IGBMC, Illkirch, CU de Strasbourg, France Genes and Development Research Group, University of Calgary, Calgary, AB, Canada Search for more papers by this author Olivier Armant Olivier Armant IGBMC, Illkirch, CU de Strasbourg, France Division of Molecular Neurobiology, NIMR, The Ridgeway, Mill Hill, London, UK Search for more papers by this author Marta Nieto Marta Nieto Beth Israel Deaconess Medical Center, HHMI, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jan M Stenman Jan M Stenman Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH, USA Search for more papers by this author Olivier Britz Olivier Britz IGBMC, Illkirch, CU de Strasbourg, France Division of Molecular Neurobiology, NIMR, The Ridgeway, Mill Hill, London, UK Search for more papers by this author Natalia Klenin Natalia Klenin Genes and Development Research Group, University of Calgary, Calgary, AB, Canada Search for more papers by this author Craig Brown Craig Brown Department of Psychology, University of Calgary, Calgary, AB, Canada Search for more papers by this author Lisa-Marie Langevin Lisa-Marie Langevin Genes and Development Research Group, University of Calgary, Calgary, AB, Canada Search for more papers by this author Julie Seibt Julie Seibt INSERM U371, Bron, France Search for more papers by this author Hua Tang Hua Tang Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author James M Cunningham James M Cunningham Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Richard Dyck Richard Dyck Department of Psychology, University of Calgary, Calgary, AB, Canada Search for more papers by this author Christopher Walsh Christopher Walsh Beth Israel Deaconess Medical Center, HHMI, Harvard Medical School, Boston, MA, USA Search for more papers by this author Kenny Campbell Kenny Campbell Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH, USA Search for more papers by this author Franck Polleux Franck Polleux INSERM U371, Bron, France Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author François Guillemot Corresponding Author François Guillemot IGBMC, Illkirch, CU de Strasbourg, France Division of Molecular Neurobiology, NIMR, The Ridgeway, Mill Hill, London, UK Search for more papers by this author Carol Schuurmans Corresponding Author Carol Schuurmans IGBMC, Illkirch, CU de Strasbourg, France Genes and Development Research Group, University of Calgary, Calgary, AB, Canada Search for more papers by this author Olivier Armant Olivier Armant IGBMC, Illkirch, CU de Strasbourg, France Division of Molecular Neurobiology, NIMR, The Ridgeway, Mill Hill, London, UK Search for more papers by this author Marta Nieto Marta Nieto Beth Israel Deaconess Medical Center, HHMI, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jan M Stenman Jan M Stenman Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH, USA Search for more papers by this author Olivier Britz Olivier Britz IGBMC, Illkirch, CU de Strasbourg, France Division of Molecular Neurobiology, NIMR, The Ridgeway, Mill Hill, London, UK Search for more papers by this author Natalia Klenin Natalia Klenin Genes and Development Research Group, University of Calgary, Calgary, AB, Canada Search for more papers by this author Craig Brown Craig Brown Department of Psychology, University of Calgary, Calgary, AB, Canada Search for more papers by this author Lisa-Marie Langevin Lisa-Marie Langevin Genes and Development Research Group, University of Calgary, Calgary, AB, Canada Search for more papers by this author Julie Seibt Julie Seibt INSERM U371, Bron, France Search for more papers by this author Hua Tang Hua Tang Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author James M Cunningham James M Cunningham Department of Medicine, Harvard Medical School, Boston, MA, USA Search for more papers by this author Richard Dyck Richard Dyck Department of Psychology, University of Calgary, Calgary, AB, Canada Search for more papers by this author Christopher Walsh Christopher Walsh Beth Israel Deaconess Medical Center, HHMI, Harvard Medical School, Boston, MA, USA Search for more papers by this author Kenny Campbell Kenny Campbell Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH, USA Search for more papers by this author Franck Polleux Franck Polleux INSERM U371, Bron, France Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author François Guillemot Corresponding Author François Guillemot IGBMC, Illkirch, CU de Strasbourg, France Division of Molecular Neurobiology, NIMR, The Ridgeway, Mill Hill, London, UK Search for more papers by this author Author Information Carol Schuurmans 1,2, Olivier Armant1,3, Marta Nieto4, Jan M Stenman5, Olivier Britz1,3, Natalia Klenin2, Craig Brown6, Lisa-Marie Langevin2, Julie Seibt7, Hua Tang8, James M Cunningham8, Richard Dyck6, Christopher Walsh4, Kenny Campbell5, Franck Polleux7,9 and François Guillemot 1,3 1IGBMC, Illkirch, CU de Strasbourg, France 2Genes and Development Research Group, University of Calgary, Calgary, AB, Canada 3Division of Molecular Neurobiology, NIMR, The Ridgeway, Mill Hill, London, UK 4Beth Israel Deaconess Medical Center, HHMI, Harvard Medical School, Boston, MA, USA 5Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH, USA 6Department of Psychology, University of Calgary, Calgary, AB, Canada 7INSERM U371, Bron, France 8Department of Medicine, Harvard Medical School, Boston, MA, USA 9Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA *Corresponding authors. Genes and Development Research Group, University of Calgary, 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N 4N1. Tel.: +1 403 220 3025; Fax: +1 403 270 2211; E-mail: [email protected] of Molecular Neurobiology, NIMR, Mill Hill, London. Tel.: +44 20 8816 2740; Fax: +44 20 8816 2109; E-mail: [email protected] The EMBO Journal (2004)23:2892-2902https://doi.org/10.1038/sj.emboj.7600278 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons. Introduction Neuronal diversity in the neocortex is striking, with the human neocortex subdivided into more than 40 tangential areas and six radial layers, each characterized by unique neuronal morphologies, cytoarchitectures, axonal projections and molecular identities (Job and Tan, 2003). During development, neocortical neurons are generated sequentially, with multipotent progenitors in the dorsal telencephalon initially giving rise to neurons in the cortical preplate, followed by lower-layer (layers V/VI) and finally upper-layer (layers II–IV) neurons of the cortical plate (Caviness, 1982). In addition to their unique properties, cortical projection neurons share several essential properties, including their regional identity, as defined by common gene-expression profiles in progenitors and neurons, and use of glutamate as an excitatory neurotransmitter. They can be distinguished from cortical interneurons, which are born and differentiate in the ventral telencephalon, reach the neocortex by tangential migration, express distinct ventral-specific regional markers and use GABA as a neurotransmitter (Parnavelas et al, 2000). A question that remains unanswered is whether a single genetic pathway specifies all common features of neocortical neurons, or whether distinct genetic programs are sequentially activated to specify the features that are both common and unique to projection neuron populations in each cortical layer. The known molecular determinants of cortical identity include three homeodomain (HD) transcription factors, Lhx2, Emx2 and Pax6, that act either alone or in combination to pattern the telencephalon and establish a cortical territory (Bulchand et al, 2001; Monuki et al, 2001; Muzio et al, 2002). Pax6 and Emx2 are also required to establish regional identities along the tangential axis of the neocortex, setting up territories that are thought to prefigure the formation of cortical areas (Bishop et al, 2000; Mallamaci et al, 2000). In contrast, the molecules involved in specifying laminar fates and a glutamatergic neurotransmitter phenotype remain virtually unexplored. Given the central role that Pax6 plays in cortical development, it is of particular interest that Pax6 directly activates Ngn1 and Ngn2, two highly related basic–helix–loop–helix (bHLH) transcription factors (Scardigli et al, 2003). Ngns have been implicated in multiple cell fate choices in the nervous system, including the selection of neural progenitors, specification of neuronal phenotype at the expense of glial cell fates, and choice of neuronal differentiation programs (Bertrand et al, 2002). In the telencephalon, Ngns are specifically expressed in cortical and not subcortical progenitors, where they specify the regional identity of the earliest-born preplate neurons in the neocortex (Fode et al, 2000). Here we examine the function of Ngn1, Ngn2, Pax6 and Tlx in specifying the cortical regional identity, glutamatergic neurotransmitter phenotype and laminar-specific properties of neurons in the cortical plate. Results Ngn1 and Ngn2 specify a glutamatergic, cortical phenotype and repress GABAergic, subcortical genes We analyzed the role of Ngn1 and Ngn2 in specifying the identity of neocortical neurons. To evaluate specification defects resulting from Ngn mutations at the early stage of corticogenesis, we profiled and compared gene expression in wild-type versus Ngn1 and Ngn2 single-, and Ngn1;Ngn2 double-mutant cortices at embryonic day (E) 13.5. Hybridization of total cortical cDNA to Affymetrix microarrays revealed a significant number of up- and downregulated genes in all genotypes, except in Ngn1 mutants, which were not investigated further (Figure 1A). Figure 1.Gene profiling reveals a global shift of neuronal phenotype from cortical, glutamatergic to subcortical, GABAergic in E13.5 Ngn mutants. (A) Expression profiling in E13.5 cortices using Affymetrix microarrays, showing fold differences in gene expression, comparing Ngn2 (blue bar), Ngn1;Ngn2 (green bar) and Ngn2;Mash1 (red bar) mutants to wild type. (B–E) Expression of Math2 in PP and early-born CP neurons was reduced in Ngn2 and Ngn1;Ngn2 mutants (arrowheads, C, D). Expression of VGLUT1 protein in the PP (F–I) and VGLUT2 transcripts (J–M) in the SVZ was reduced in Ngn2, Ngn1;Ngn2 and Pax6 mutants (arrowheads, G-I, K–M). (N–Q) GAD1 was ectopically expressed in the PP/SVZ of Ngn2 and Ngn1;Ngn2 mutants (arrowheads, O, P). (R–T) Double staining of GAD1 RNA (blue) and VGLUT1 protein (brown) in wild-type (R, S) and Ngn1;Ngn2 (T, U) mutants showing that most cells express either the glutamatergic (arrows) or the GABAergic (arrowheads) marker. PP, preplate; SVZ, subventricular zone. Download figure Download PowerPoint Affymetrix data and RNA in situ hybridization for nonrepresented genes revealed no deregulation of genes normally expressed in dorsal telencephalic progenitors (Gli3, Pax6, Emx2, Lhx2, Foxg1, Otx1, Tlx, COUP-TFI) in Ngn mutants (Figure 1A, Supplementary Figure S1). In contrast, several transcription factors specifically expressed by cortical neurons (Math2, Nscl1, NeuroD, NeuroD2, Tbr1, Tbr2) were reduced in Ngn2 mutant cortices, and more severely downregulated in Ngn1;Ngn2 mutants (Figure 1A–D). Vesicular glutamate transporter1 (VGLUT1) and VGLUT2, which load glutamate into synaptic vesicles (Fremeau et al, 2001), were also downregulated in Ngn mutants (Figure 1A). In E13.5 telencephalic sections, VGLUT genes were specifically expressed in dorsal neurons that use glutamate as a neurotransmitter, with VGLUT1 protein predominant in preplate (PP) and cortical plate (CP) neurons (Figure 1F), whereas VGLUT2 was transiently expressed in differentiating cortical neurons in the subventricular zone (SVZ; Figure 1J). In Ngn2 mutants, VGLUT1 protein and VGLUT2 transcript levels were reduced in dorsomedial and not lateral cortical neurons, likely due to compensation by Ngn1, which persists in lateral domains (Fode et al, 2000), whereas defects extended throughout the cortex of Ngn1;Ngn2 double mutants (Figure 1F–H and J–L). Ngns are thus required to activate cortical- and glutamatergic-specific differentiation programs in early-born CP neurons, likely acting downstream of cortical patterning genes, which are normally expressed in Ngn mutants, and cannot compensate for the loss of Ngn activity. Mash1 is upregulated in Ngn mutant cortical progenitors, and was previously linked to the ectopic expression of subcortical genes Dlx1 and GAD1 in PP neurons (Fode et al, 2000). Gene profiling at E13.5 revealed a more extensive upregulation of subcortical genes in Ngn2 and Ngn1;Ngn2 mutants that included ventral telencephalic transcription factors (Mash1, Dlx1, Dlx2, Dlx5, Brn4), biosynthetic enzymes for GABA (glutamic acid decarboxylase 1 (GAD1), GAD2) and GABA transporters (GABA transporter 1 (GABA-T1), GABA and glycine transporter (GABA/glyT)), suggesting that many, and possibly all, components of a subcortical, GABAergic differentiation program were ectopically activated in Ngn mutant cortical neurons (Figure 1A). Consistent with a switch of neurotransmitter phenotype, GAD1 was ectopically expressed in the PP/CP and SVZ of Ngn2 and Ngn1;Ngn2 mutants (Figure 1N–P). The ectopic ventral-like neurons were misspecified neurons of cortical origin, and not subcortical neurons that had inappropriately migrated into the cortex, based on previous explant and migration studies (Fode et al, 2000; Chapouton et al, 2001). Moreover, GAD1 transcripts and VGLUT1 protein were for the most part detected in complementary sets of cortical neurons in both wild-type and Ngn1;Ngn2 mutant cortices (Figure 1R–U), suggesting that cortical neurons choose between a glutamatergic and GABAergic phenotype. To determine the extent to which ectopic Mash1 expression was responsible for specification defects in Ngn mutant cortices, we profiled gene expression in E13.5 Ngn2;Mash1 double mutants. A similar reduction in transcription of cortical-specific neuronal markers was observed in Ngn2 and Ngn2;Mash1 mutants, with the exception of Math3 (Figure 1A; compare Ngn2 versus WT (blue bars) against Ngn2;Mash1 versus WT (red bars)). In Ngn2;Mash1 mutants, the loss of Math2 transcripts was restricted to rostromedial domains (Figure 2L), where Ngn1 was no longer expressed. Thus, the loss of neurons with a cortical character in Ngn mutants occurs independently of the upregulation of Mash1, suggesting that the Ngns directly activate a cortical, glutamatergic differentiation pathway. Figure 2.Change in regional identity and neurotransmitter phenotype of early-born CP neurons in E15.5 Ngn mutants. E15.5 expression of cortical neuronal markers Id2 (A, B), Tbr1 (C, D), Robo1 (E, F), Slit1 (G, H) and Math2 (I–L), showing a correctly specified upper layer in Ngn2 and Ngn1;Ngn2 mutants above distinct gaps in lower CP expression (arrowheads, B, D, F, H, J, K) and in the medial cortex of Ngn2;Mash1 mutants (arrowheads, L). (M–P) The glutamatergic marker VGLUT2 was appropriately expressed in newly born neurons in the SVZ of wild-type (M) and Ngn2 mutants (N), whereas GluR2 was expressed in the IZ and CP of wild-type cortices (O), but displayed distinct gaps in the lower CP of Ngn2 mutants (arrowheads, P). (Q–T) Dlx1 transcripts and protein (insets) were detected in marginal zone and SVZ interneurons, and were ectopic in lower CP clusters in Ngn2 and Ngn1;Ngn2 mutants (arrowheads, R, S), and in medial clusters in Ngn2;Mash1 mutants (arrowheads, T). (U–W) Mash1 transcript and protein (insets) levels were moderately and strongly upregulated in Ngn2 (V) and Ngn2KIM1 mutants (W), respectively. (X) Ectopic Dlx1+ clusters were in the deep CP in Ngn2KIM1 mutants (asterisks). uc, upper CP; lc, lower CP; iz, intermediate zone; svz, subventricular zone. Download figure Download PowerPoint Dlx1, Dlx2 and Brn4 remained elevated to varying extents in Ngn2;Mash1 cortices as compared to wild type (Figure 1A), with the ectopic expression of ventral markers restricted to rostromedial domains (Figure 2T). Ngns therefore repress ventral-specific genes in at least a partially Mash1-independent manner. In contrast, ectopic expression of Dlx5, GAD1, GAD2, GABA-T1 and GABA/glyT in Ngn2 mutant cortices was mostly or strictly dependent on the presence of Mash1 (Figure 1A), suggesting that ectopic activation of these genes was largely due to derepression of Mash1. Ngn1/2 thus specify regional and neurotransmitter phenotypes via multiple mechanisms, including activation of dorsal, cortical-specific genetic pathway(s), and repression of ventral telencephalic programs that are both Mash1 dependent and independent (Figure 8). Ngns are required to specify early- and not later-born CP neurons We extended our analysis of Ngn mutants to mid-corticogenesis (E15.5), when lower-layer neurons have differentiated and are migrating to the CP. As at earlier stages, telencephalic patterning genes (Emx2, Lhx2, Tlx, Pax6) were normally expressed in Ngn2 and Ngn1;Ngn2 mutants, suggesting a correct regional identity of progenitors (data not shown). To assess neuronal identities, we analyzed the expression of CP-specific markers Id2, Robo1, Slit1, Math2 and Tbr1 (Figure 2A–K). In the rostral cortex of Ngn2 mutants, where Ngn1 expression was selectively lost, and throughout the cortex of Ngn1;Ngn2 mutants, gaps in expression of all cortical markers were observed in the deep CP (Figure 2B, D, F, H, J and K). The loss of cortical-specific gene expression was not due to a loss of neurons, as pan-neuronal markers such as SCG10 were unperturbed (data not shown), and cell death, as assessed by TUNEL, was not elevated in E15.5 Ngn mutants (Supplementary Figure S2). Strikingly, gaps in cortical-specific gene expression were not observed in the superficial CP or intermediate zone (IZ) of Ngn mutants, zones that contain more recently generated CP neurons, suggesting that only early-born CP neurons are misspecified. Consistent with this, CP neurons differentiating at E15.5 in Ngn2 and Ngn1;Ngn2 mutants acquired their correct glutamatergic phenotype, as assessed by normal levels of VGLUT2, which transiently labels glutamatergic neurons migrating through the SVZ (Figure 2M–N). Transcripts for the glutamate receptor GluR2 were also maintained in the IZ and upper CP of E15.5 Ngn2 mutants, and were only lost in clusters of lower-layer CP neurons (Figure 2O and P). In a complementary manner, ectopic expression of Dlx1 (Figure 2Q–S) and GAD1 (data not shown) was confined to large aggregates in the lower CP of Ngn mutants (Figure 2A–K), and was excluded from the more recently generated, superficial layer of the CP. Thus, in contrast with early-born CP neurons, neurons differentiating during mid-corticogenesis acquire a normal identity in the absence of Ngn function. Changing Mash1 responsiveness of cortical progenitors The misspecification of early- and not later-born CP neurons in Ngn mutants was surprising, given that Ngn expression persists throughout corticogenesis (data not shown). Since derepression of Mash1 contributes to cortical misspecification in Ngn mutants at E13.5 (Fode et al, 2000), we speculated that Mash1 might not be sufficiently upregulated at later stages to alter the differentiation of cortical progenitors. We tested this using Ngn2KIM1 homozygous mutant embryos in which Ngn2 coding sequences were replaced with Mash1 (Fode et al, 2000; Parras et al, 2002). At E15.5, Mash1 transcript and protein levels were low in wild-type cortical progenitors, slightly elevated in Ngn2 mutants and very significantly elevated in Ngn2KIM1 mutants (Figure 2U–W). However, Dlx1 was ectopically expressed only in small clusters deep within the rostral CP of Ngn2KIM1 mutants (Figure 2X). Thus, increasing Mash1 expression levels was not sufficient to respecify cortical neurons born after E14.5, suggesting that both the dependency of cortical neurons on Ngn1/2 and the responsiveness of cortical progenitors to ectopic Mash1 change over time. A subset of Ngn mutant early-born neurons contribute to deep cortical layers while others segregate out of the CP To further examine the fate of early-born, misspecified neurons, we analyzed Ngn mutant cortices at E18.5, a stage when neurogenesis is complete, although CP neurons are still migrating to their final destination. Strikingly, the cortical-specific marker Math2 was for the most part uniformly expressed throughout the CP in E18.5 Ngn2 and Ngn1;Ngn2 mutant cortices (Figure 3A–D), in contrast to defects at E15.5 (Figure 2I–K). Moreover, in E18.5 Ngn mutants, misspecified Dlx1+ (Figure 3E–J) and GAD1+ (data not shown) neurons segregated out of the CP, aggregating instead in small clusters in the germinal zone (GZ), rostral marginal zone (MZ) and flanking the IZ/lower CP border. Thus, by E18.5, the Ngn mutant CP was comprised almost exclusively of neurons with their correct regional identities. Figure 3.Misspecification of lower-layer CP neurons in E18.5 Ngn mutants. (A–D) Math2 was uniformly expressed in the CP and IZ of wild-type (A), Ngn2 (B) and Ngn1;Ngn2 (C) mutants, with the exception of small superficial gaps in Ngn1;Ngn2 mutants (uc, arrowheads, D). (E–H) Dlx1 was expressed in cortical interneurons in the GZ, MZ and CP (E), and in ectopic clusters in the IZ (asterisks) and superficial CP (arrowheads) of Ngn2 (F) and Ngn1;Ngn2 (G, H) mutants. (I) DAPI and (J) Dlx immunostaining showed that abnormal cellular aggregates (asterisks) beneath the CP were Dlx+. (K–L) Retrograde labeling of P0 cortical neurons in layers II/III (K, L) and V/VI (K′, L′) revealed similar immature neuronal morphologies in wild-type (K, K′) and Ngn2 mutant (L, L′) cortices, with sparsely branched apical and basal dendritic processes (also see Supplementary Figure S4). (M–O) Layer VI expression of Tbr1 was reduced in the rostral CP in Ngn2 mutants (N), and throughout the Ngn1;Ngn2 mutant cortex (O). (P–R) ER81 expression was reduced and disorganized (arrowheads) in rostral layer V in Ngn2 mutants (Q), and throughout the Ngn1;Ngn2 mutant layer V (R). (S–U) Layer IV expression of RORβ was normal in Ngn2 (T) and Ngn1;Ngn2 (U) mutants. Ectopic RORβ (U) and ER81 (Q, R) were observed in clusters in the IZ and MZ of Ngn2 and Ngn1;Ngn2 mutants, correlating with migration defects of a subset of early-born neurons. Oct6 transcripts (V–X) and Cux1 protein (Y, Z, AA) were expressed in layers II–IV in all genotypes. (BB, CC) Anterograde tracing revealed fewer corticofugal projections in E16.5 Ngn2 mutants (arrows, CC). uc, upper CP; lc, lower CP; iz, intermediate zone. Download figure Download PowerPoint To determine whether any early-born cortical neurons remained in the CP of Ngn mutants, neuronal lamination was assessed with histological stains at P0, revealing that lower layers were indeed present in Ngn2 and Ngn1;Ngn2 mutants, but were significantly thinner in double mutants (Figure 4A–F). To determine whether histologically identifiable lower layers were composed of neurons born at the correct time, we used birthdating (Caviness, 1982; Caviness et al, 1995). BrdU was administered at different embryonic stages (Supplementary Figure S3), followed by an assessment at P0 of the laminar position of darkly labeled nuclei, marking neurons born at the time of BrdU injection (black bars, Figure 4G), and lightly labeled nuclei, representing neurons derived from progenitors that had undergone additional cell divisions (gray bars, Figure 4G). Labeling at E12 revealed a peak accumulation of strong BrdU+ nuclei in layer VI of wild-type cortices (top row, Figure 4G). In Ngn mutants, there was an increased number of heavily labeled neurons blocked in the GZ/IZ, and in the MZ of Ngn1;Ngn2 mutants (top row, Figure 4G), corresponding to sites of aggregation of misspecified Dlx1+/GAD1+ neurons (Figure 3F–J), and confirming that many early-born, misspecified neurons were not integrated in the CP. However, not all Ngn mutant neurons born at E12 migrated aberrantly, as a significant number of BrdU+ neurons were correctly positioned in layer VI, likely corresponding to the subset of lower-layer CP neurons with a correct regional identity and neurotransmitter phenotype at E15.5 (Figure 2A–P). Figure 4.Aberrant location of lower-layer CP neurons in Ngn mutants. (A–F) Histological analyses at P0 revealed neurogenesis defects primarily in lower layers of Ngn1;Ngn2 mutants (asterisks, F), a disorganization of layer VI in Ngn2 mutants (asterisks, D), but a correctly laminated CP in both genotypes. (G) Graphical representations of BrdU birthdating studies showing the distribution of P0 cortical neurons labeled with BrdU at different times (E12, E14, E16). Cortices were divided into six bins corresponding to MZ, layers II–IV, layer V, layer VI, IZ and GZ. Darkly labeled nuclei (black bar) and lightly labeled nuclei (gray bars) in each bin were counted. Asterisks label the increased number of cells generated at E12 in the Ngn2 and Ngn1;Ngn2 mutant GZ, and the skewed distribution of neurons labeled at E14 in Ngn2 and Ngn1;Ngn2 mutants. MZ, marginal zone; GZ, germinal zone. Download figure Download PowerPoint A BrdU pulse at E14 primarily marked the genesis of layer IV and V neurons, but in Ngn2 and Ngn1;Ngn2 mutants there was a clear decline in the number of darkly stained nuclei in the lower CP, and instead an accumulation of cells in the GZ (middle row, Figure 4G). This suggested that a subset of CP neurons destined for layers IV–V aggregated in abnormally deep positions in Ngn2 and Ngn1;Ngn2 mutant CPs. In contrast, the laminar position of upper-layer neurons was largely unperturbed in Ngn mutants, as assessed by birthdating at E16, which in mutant as well as control brains resulted in a clear bias towards darkly stained nuclei localizing to upper layers II–IV (bottom row, Figure 4G). Specification defects resulting in abnormal laminar localization in Ngn2 and Ngn1;Ngn2 mutants were thus largely restricted to cortical neurons born between E12 and E14, but a subset of early-born neurons acquired a correct regional identity and populated deep cortical layers. Disruptions in laminar specification restricted to lower layers in Ngn mutants The correct specification and layering of a subset of lower-layer neurons in the Ngn mutant CP allowed us to examine whether these neurons had their correct laminar phenotypes based on molecular markers. At E18.5, Tbr1 was expressed at high levels in layer VI in wild-type cortices, but at diminished levels in the rostral cortex of Ngn2 mutants and throughout the cortex of Ngn1;Ngn2 double mutants (Figure 3M–O). ER81, which is expressed in layer V, was lost in small rostral gaps in layer V of Ngn2 mutants, and was more globally disturbed throughout layer V in Ngn1;Ngn2 mutants (Figure 3P–R). In contrast, layer II–IV markers RORβ (Figure 3S–U), Oct6 (Figure 3V–X) and Cux1 (Figure 3Y, Z and AA) labeled correctly positioned neurons in all genotypes, indicating that upper-layer neurons were correctly specified. Anterograde tracing of descending corticofugal projections of layer V/VI (Koester and O'Leary, 1993) revealed strongly reduced axonal numbers in Ngn2 mutant versus wild-type cortices at E16.5, which were consistent with selective specification defects of lower-layer neurons (Figure 3BB and CC). Lower-layer neurons in the Ngn mutant CP thus present both molecular and axonal projection defects. Neuronal migration in the cortex is not complete until postnatal day (P) 7, such that unequivocal conclusions about laminar phenotypes in Ngn mutants could not be made from embryonic analyses. We thus examined laminar specification in the rare Ngn2 single-mutant pups that survived the first postnatal week, but could not analyze laminar identities in the complete absence of Ngn activity because all Ngn1;Ngn2 mutants died at birth. At P5, the overall size of the Ngn2 mutant neocortex was smaller, but all the six cortical layers were clearly
