The cortical hem is a signaling center at the midline of the embryonic telencephalon that provides instructive cues to the adjacent cortical primordium and serves as the hippocampal organizer, inducing multiple distinct hippocampal field identities. The cortical hem consists of neuroepithelial progenitors, and gives rise to diverse neuronal and non-neuronal cell types, including Cajal-Retzius cells, hippocampal pyramidal neurons, and choroid plexus epithelial cells. Here, we use two independent Cre drivers, Wnt3aCre and Lmx1aCre, to examine the lineages from the cortical hem. We report that these lineages also contribute pyramidal neurons to the neocortex, and we describe their spatiotemporal distribution. Together, our results extend the known lineages of the cortical hem.

In the mouse, two telencephalic signaling centers orchestrate embryonic patterning of the cerebral cortex. From the rostral patterning center in the telencephalon, the Fibroblast Growth Factor, FGF8, disperses as a morphogen to establish the rostral to caudal axis of the neocortical area map. FGF8 coordinates with Wnt3a from the cortical hem to regulate graded expression of transcription factors that position neocortical areas, and control hippocampal development. Whether similar signaling centers pattern the much larger cortices of carnivore and primate species, however, is unclear. The limited dispersion range of FGF8 and Wnt3a is inconsistent with patterning larger cortical primordia. Yet the implication that different mechanisms organize cortex in different mammals flies in the face of the tenet that developmental patterning mechanisms are conserved across vertebrate species. In the present study, both signaling centers were identified in the ferret telencephalon, as were expression gradients of the patterning transcription factor genes regulated by FGF8 and Wnt3a. Notably, at the stage corresponding to the peak period of FGF8 signaling in the mouse neocortical primordium (NP), the NP was the same size in ferret and mouse, which would allow morphogen patterning of the ferret NP. Subsequently, the size of ferret neocortex shot past that of the mouse. Images from online databases further suggest that NP growth in humans, too, is slowed in early cortical development. We propose that if early growth in larger brains is held back, mechanisms that pattern the neocortical area map in the mouse could be conserved across mammalian species.

The cerebral cortex controls our unique higher cognitive abilities. Modifications to gene expression, progenitor behavior, cell lineage, and neural circuitry have accompanied evolution of the cerebral cortex. This chapter considers the progress made over the past thirty years in defining potential mechanisms that contribute to cortical development and evolution. It discusses the value of model systems for understanding elaboration of cortical organization in humans, with an emphasis on recent technical and conceptual advances. It then examines our current understanding of the molecular and cellular basis for cortical development and evolution; discusses how neuronal fates are specified and organized in lamina, columns, and areas; and revisits the radial unit and protomap hypotheses. Finally, it considers our current understanding of the development, stability, and plasticity of cortical circuitry. Throughout, it highlights the profound impact that new technological advances have made at the molecular and cellular level, and how this has changed our understanding of cortical development and evolution. The authors conclude by identifying critical and tractable research directions to address gaps in our understanding of cortical development and evolution.

Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5. Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon. SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.

Specification of dorsal/ventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsal/ventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence such as Gsx2 are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5. Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2; Dmrt5 double KO show a phenotype similar to Dmrt3; Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5 and EMX2 in dividing dorsal from ventral in the telencephalon. SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double but not the single knockout produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the PSB boundary by antagonizing the ventral homeobox transcription factor GSX2. INTRODUCTION The mammalian telencephalon is the largest and most complex region of the mammalian brain, controlling cognitive processes and purposeful actions. It comprises the cerebral cortex dorsally and the amygdala and basal ganglia structures ventrally. Defects in telencephalon development are associated with many human neuropsychiatric and neurological disorders (Gaitanis and Walsh, 2004; Hu et al., 2014). Related to this study, a loss-of-function mutation in the human DMRT5/DMRTA2 gene has been found to be associated with microcephaly (Urquhart et al., 2016). Specification of dorsoventral (DV) regional identity in progenitors of the developing telencephalon is a pivotal step in the development of the cerebral cortex and basal ganglia. In the developing telencephalon, as in the spinal cord, Bone Morphogenetic Proteins (BMPs) and Wingless-Int proteins (WNTs) produced dorsally and Sonic Hedgehog (SHH) secreted from ventral sources are implicated in DV specification of the telencephalon (Ericson et al., 1995; Chiang et al., 1996; Backman et al., 2005; Fernandes et al., 2007). Opposition between these morphogens alone does not establish DV telencephalic identity. Rather, interactions among the transcription factor GLI3, Fibroblast Growth Factor (FGF) signaling from the rostral telencephalic patterning center (RTPC), and ventral sources of SHH, regulate DV patterning (Ohkubo et al., 2002; Shimogori et al., 2004; Hasenpusch-Theil et al., 2017). GLI3 is a transcriptional activator in the presence of high levels of SHH, and a repressor, GLI3R, when levels of SHH are low (Grove et al., 1998; Theil et al., 1999; Tole et al., 2000a; Aoto et al., 2002; Kuschel et al., 2003). SHH promotes ventral identity by maintaining FGF signaling at the RTPC in part by suppressing formation of Gli3R which represses Fgf8 expression (Ohkubo et al., 2002; Rallu et al., 2002; Rash and Grove, 2007). FGFs at the RTPC are in turn required to establish or maintain Shh expression in the ventral telencephalon (Storm et al., 2006). FGF signaling further promotes ventral telencephalon development independently of SHH, through regulating expression of the Foxg1 transcription factor gene (Gutin et al., 2006; Tole and Hébert, 2013). Pax6 and other homeobox genes such as Emx2 (Muzio et al., 2002b, a) expressed throughout the pallium, and the homeobox gene Gsx2 expressed in the subpallium are also involved in positioning the pallium-subpallium boundary (PSB) (Stoykova et al., 2000; Toresson et al., 2000; Yun et al., 2001; Kroll and O'Leary, 2005; Carney et al., 2009). How these different transcription factors function together to control telencephalon DV patterning and whether there are other players involved remains unknown. Dmrt3 and Dmrt5 (Dmrta2) encode related transcription factors expressed in a similar high caudomedial to low rostrolateral gradient in the cortical primordium. Their loss leads to a similar phenotype, more severe in Dmrt5 than in Dmrt3 mutants. In either single null mutant, Wnt and BMP expression at the cortical hem is decreased and adjacent hippocampus and caudal neocortical areas are reduced in size. In conditional Dmrt5 mouse models, Dmrt5 loss or gain of function after hem formation also leads to a reduction of hippocampal size and alters neocortical area map formation, indicating that DMRT5 is not only required for hem formation but also directly controls cortical progenitor proliferation and specification. DMRT3 and DMRT5are thus crucial regulators of cortical development, acting at different steps of its formation (Konno et al., 2012; Saulnier et al., 2013; Young et al., 2017; De Clercq et al., 2018). DMRT3 and DMRT5 have similar DNA binding properties (Murphy et al., 2007) suggesting they act redundantly in telencephalic development, implying that analysis of single KO embryos did not reveal their full function. We therefore generated double KO and compared their telencephalic development with that of single KO embryos. Further, we conditionally overexpressed Dmrt5 in the telencephalon. As Emx2 is coexpressed with Dmrt3 and Dmrt5 in cortical progenitors, we also generated Dmrt5;Emx2 double KO embryos. Our new findings reveal that DMRT3, DMRT5 and EMX2 cooperate to repress Gsx2 and maintain DV patterning in the telencephalon. MATERIAL AND METHODS Animals. All mice were maintained on a C57/Bl6 or CD1/C57Bl6 mixed background and mice of either sex were used. Midday of the day of the vaginal plug discovery was defined as embryonic day (E) 0.5.Animal care was in accordance with Institutional guidelines, and the policies of the US National Institutes of Health. Dmrt3 (Saulnier et al., 2013), Dmrt5 (De Clercq et al., 2018), Emx2 (Pellegrini et al., 1996) and Gsx2EGFP mice (Wang et al., 2009) were genotyped by polymerase chain reaction (PCR) as described respectively in these articles. Dmrt3+/or Dmrt3-/animals, which are viable, were crossed with Dmrt5+/mice to obtain Dmrt3+/;Dmrt5+/mice. These double heterozygotes were then crossed to obtain Dmrt3/;Dmrt5-/homozygous double KO embryos. Dmrt5-/-;Emx2-/homozygous double mutants were obtained by intercrossing Dmrt5+/heterozygous mutants with Emx2+/heterozygous mutants. Dmrt5 conditional transgenic (Dmrt5Tg) mice were maintained and genotyped as described (De Clercq et al., 2018) and crossed to Foxg1-IRES-Cre (Kawaguchi et al., 2016) mice to overexpress Dmrt5 throughout the telencephalon or to Gsx2-Cre-IRES-EGFP (Gsx2-CIE) (Qin et al., 2016) to overexpress it in ventral telencephalon progenitors. tetO-Gsx2-IRES-EGFP mice were crossed to Foxg1TA/+ mice to obtain Foxg1TA/+;tetO-Gsx2-IRES-EGFP embryos overexpressing Gsx2 throughout the telencephalon (Waclaw et al., 2009). Histology, immunofluorescence and in situ hybridization. Standard hematoxylin and eosin (H&E) staining was performed on 6–8 μm sections of embryosor brains fixed overnight in 4% paraformaldehyde/PBS, dehydrated and paraffinembedded. For immunofluorescence, embryos were fixed overnight at 4°C in 4% paraformaldehyde/PBS, infused in 30% sucrose/PBS overnight, frozen in gelatin (7.5% gelatin, 15% sucrose/PBS) or NEG-50 and cryosectioned (12-20 μm). Antigen retrieval was performed by boiling the sections in Target Retrieval Solution Citrate pH 6.0 (DAKO®). Slides were the blocked with 10% normal goat serum, 0.3% Triton X-100 in PBS and incubated with primary antibodies O/N at 4°C. The incubation with secondary antibodies was carried out for 2h at RT. Samples were then mounted in DAKO® mounting medium. The following primary antibodies were used: rabbit anti-TH (1:500, Immunostar), rabbit anti-DMRT5 (1:2000, (De Clercq et al., 2018)), rabbit anti-GSX2 (1:500, (Toresson et al., 2000)); mouse anti-ASCL1 (1 :100, gift from C. Parras laboratory); goat anti-PAX6, (1:200, Santa Cruz); rabbit anti-TBR2 (1/500, Abcam) and chicken anti-GFP (1:1000, Aves Labs). The following secondary antibodies were used: anti-Mouse AlexaFluor 488 (1 :400, Invitrogen), anti-Mouse AlexaFluor 594 (1 :400,Invitrogen), antiRabbit AlexaFluor 488 (1 :400, Invitrogen), antiRabbit AlexaFluor 488 (1 :400, Invitrogen), antiRabbit AlexaFluor 594 (1 :400, Invitrogen). Sections were counterstained with DAPI. Images were acquired with a Zeiss LSM 70 confocal microscope using ZenBlack® software or Nikon A1R GaAsP inverted Confocal Microscope and processed using ImageJ and Photoshop® softwares. In sit

Mice that are constitutively null for the zinc finger doublesex and mab-3 related (Dmrt) gene, Dmrt5/Dmrta2, show a variety of patterning abnormalities in the cerebral cortex, including the loss of the cortical hem, a powerful cortical signaling center. In conditional Dmrt5 gain of function and loss of function mouse models, we generated bidirectional changes in the neocortical area map without affecting the hem. Analysis indicated that DMRT5, independent of the hem, directs the rostral-to-caudal pattern of the neocortical area map. Thus, DMRT5 joins a small number of transcription factors shown to control directly area size and position in the neocortex. Dmrt5 deletion after hem formation also reduced hippocampal size and shifted the position of the neocortical/paleocortical boundary. Dmrt3, like Dmrt5, is expressed in a gradient across the cortical primordium. Mice lacking Dmrt3 show cortical patterning defects akin to but milder than those in Dmrt5 mutants, perhaps in part because Dmrt5 expression increases in the absence of Dmrt3. DMRT5 upregulates Dmrt3 expression and negatively regulates its own expression, which may stabilize the level of DMRT5. Together, our findings indicate that finely tuned levels of DMRT5, together with DMRT3, regulate patterning of the cerebral cortex.

A unique population of cells, called "lot cells," circumscribes the path of the lateral olfactory tract (LOT) in the rodent brain and acts to restrict its position at the lateral margin of the telencephalon. Lot cells were believed to originate in the dorsal pallium (DP). We show that Lhx2 null mice that lack a DP show a significant increase in the number of mGluR1/lot cells in the piriform cortex, indicating a non-DP origin of these cells. Since lot cells present common developmental features with Cajal-Retzius (CR) cells, we analyzed Wnt3a- and Dbx1-reporter mouse lines and found that mGluR1/lot cells are not generated in the cortical hem, ventral pallium, or septum, the best characterized sources of CR cells. Finally, we identified a novel origin for the lot cells by combining in utero electroporation assays and histochemical characterization. We show that mGluR1/lot cells are specifically generated in the lateral thalamic eminence and that they express mitral cell markers, although a minority of them express ΔNp73 instead. We conclude that most mGluR1/lot cells are prospective mitral cells migrating to the accessory olfactory bulb (OB), whereas mGluR1+, ΔNp73+ cells are CR cells that migrate through the LOT to the piriform cortex and the OB.

Event Abstract Back to Event DMRT transcription factors are required for cortical development Marc Keruzore1, Sarah De Clercq1, Elodie Desmaris1 and Eric Bellefroid1* 1 University of Brussel, Developmental Genetics, Belgium Patterning the cerebral hemispheres and generating the neocortical area map depend initially on interplay between morphogens secreted by organizing centers and transcription factors expressed in gradients across the cortical primordium. One of the latter, Dmrt5/Dmrta2, a zinc finger doublesex and mab-3 related (Dmrt) gene, is expressed in mouse cortical progenitors in a high caudomedial to low rostrolateral gradient. Dmrt5 is required for the development of caudomedial cerebral cortex but its mode of action remains unclear. In constitutively Dmrt5 null mice, the Wnt-and Bmp-rich cortical hem is missing, suggesting that hem formation relies on DMRT5, and that deletion of Dmrt5 affects cortical patterning indirectly through loss of signalling from the hem (Saulnier et al., 2013). In a positive feedback loop however, WNT signalling upregulates Dmrt5 expression, suggesting a second, direct patterning role for DMRT5. Our recent data indicate that inactivating or overexpressing Dmrt5 conditionally in cortical progenitors close to midgestation still affect cortical patterning without disrupting the function of the hem and that mutation of a related gene, Dmrt3, with a similar expression pattern to Dmrt5, also caused similar, albeit milder cortical patterning defects than that observed in Dmrt5 mutants. Thus, Dmrt5 and Dmrt3 appears to have direct roles in cortical patterning, in addition to their prior role in the establishment of the cortical hem (De Clercq et al., 2015). Analysis of the cortex of Dmrt3-/-;Dmrt5-/- double knock-out mice reveals that the phenotype is more severe than in the single mutants, suggesting that the two genes cooperates to control cortical patterning. RNA-seq and ChIP-seq analyses are underway to identify their direct targets. Résumé en Français Des anomalies de développement du cortex cérébral sont à l’origine de nombreuses maladies neuropsychiatriques et neurologiques chez l’homme. Nos travaux ont montré que chez la souris, les facteurs de transcription Dmrt3/5 jouent un rôle essentiel dans le développement cortical, en particulier dans la formation des aires du néocortex. Chez l’homme, des mutations dans le gène DMRT5 ont été identifiées associées à une microcéphalie. Le but de nos travaux est de mieux comprendre leur mécanisme d'action dans le contrôle de la prolifération et la différenciation des progéniteurs corticaux. Samenvatting in het Nederlands: Ontwikkelingsstoornissen van de hersenschors liggen aan de basis van tal van neuropsychiatrische en neurologische ziekten bij de mens. Ons werk heeft aangetoond dat bij muizen, de transcriptiefactoren Dmrt3/5 een essentiële rol spelen in de ontwikkeling van de hersenschors, meer bijzonder in de vorming van de gebieden van de neocortex. Mutaties van het genDMRT5 bij de mens werden geïdentificeerd en geassocieerd aan een microcefalie. Het doel van ons werk is een beter begrip van hun werking in de controle van de verspreiding en de differentiering van de ontwikkeling van de hersenschors. Acknowledgements Marc Keruzore and Sarah De Clercq contributed equally to the work References Saulnier A. et al. (2013). The doublesex homolog Dmrt5 is required for the development of the caudomedial cerebral cortex in mammals. Cerebral Cortex, 23, 2552-2567. De Clerq S. et al. (2016). Dmrt5 directs neocortical patterning together with Dmrt3 and is controlled by negative autoregulation. Cerebral Cortex, submitted, manuscript under revision. Keywords: cortical hem, Hippocampus, Neocortex, primary visual area, Transcription Factors Conference: 6th Belgian Brain Congress, MONS, Belgium, 8 Oct - 8 Oct, 2016. Presentation Type: Poster Presentation Topic: Brain and brain diseases: between heredity and environment Citation: Keruzore M, De Clercq S, Desmaris E and Bellefroid E (2016). DMRT transcription factors are required for cortical development. Conference Abstract: 6th Belgian Brain Congress. doi: 10.3389/conf.fnagi.2016.03.00034 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Jun 2016; Published Online: 04 Jul 2016. * Correspondence: Prof. Eric Bellefroid, University of Brussel, Developmental Genetics, Gosselies, 6041, Belgium, ebellefr@ulb.ac.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marc Keruzore Sarah De Clercq Elodie Desmaris Eric Bellefroid Google Marc Keruzore Sarah De Clercq Elodie Desmaris Eric Bellefroid Google Scholar Marc Keruzore Sarah De Clercq Elodie Desmaris Eric Bellefroid PubMed Marc Keruzore Sarah De Clercq Elodie Desmaris Eric Bellefroid Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The neocortex undergoes extensive developmental growth, but how its architecture adapts to expansion remains largely unknown. Here, we investigated how early born Cajal-Retzius (CR) neurons, which regulate the assembly of cortical circuits, maintain a dense superficial distribution in the growing neocortex. We found that CR cell density is sustained by an activity-dependent importation of olfactory CR cells, which migrate into the neocortex after they have acted as axonal guidepost cells in the olfactory system. Furthermore, using mouse genetics, we showed that CR cell density severely affects the architecture of layer 1, a key site of input integration for neocortical networks, leading to an excitation/inhibition ratio imbalance. Our study reveals that neurons reenter migration several days after their initial positioning, thereby performing sequential developmental roles in olfactory cortex and neocortex. This atypical process is essential to regulate CR cell density during growth, which in turn ensures the correct wiring of neocortical circuitry.

Patterning of the cerebral hemispheres and arealization of the neocortex depends initially on interplay between morphogens secreted by organizing centers and transcription factors expressed in gradients across the cortical primordium. One of these, Dmrt5/Dmrta2, a zinc finger doublesex and mab-3 related (Dmrt) gene, is expressed in mouse cortical progenitors in a high caudomedial to low rostrolateral gradient. Dmrt5 is required for the development of the caudomedial part of the cerebral cortex but its mode of action remains unclear. In Dmrt5 null mice, the caudomedial cortical organizing center, the Wnt-and Bmp rich cortical hem, is greatly reduced, implying that hem formation relies on DMRT5 activity, and that loss of Dmrt5 affects caudomedial cortex via decreased hem signalling. In a positive feedback loop however, WNT signalling upregulates Dmrt5 expression, suggesting a downstream patterning role for DMRT5. Here we investigated the latter role by inactivating Dmrt5 conditionally in dorsal telencephalon progenitors, and by generating conditional Dmrt5 gain-of-function transgenic mice. In each mouse line, WNT and BMP signaling at the hem appeared largely unaffected. In these conditional mutants, the hemispheres were however smaller than in controls, and the hippocampus and primary visual area (V1) of the neocortex were sharply reduced. No such defects were observed in Dmrt5 hem specific ablated mice. While heterozygous Dmrt5 null mice show a similar reduction of V1 area, opposite changes are observed when Dmrt5 was overexpressed from midgestation onwards. In each mouse line, expression levels of the cortical patterning genes Emx2, Lhx2, and Pax6 were altered. Dmrt5 expression itself was perturbed revealing that it is controlled by negative feedback autoregulation. Together, our findings reveal that DMRT5 levels are tightly controlled and have autonomous effects on hippocampal development and neocortical arealization.

AbstractThe primary cilium was first discovered over 100 years ago but only relatively recently has it been widely regarded as an integral cellular organelle for brain development, maturation and function. Defects in primary cilia contribute to a set of human disorders, ciliopathies, which are multi‐systemic in pathology and often include abnormalCNSarchitecture and intellectual deficits. The intricate structure of the cilium and the molecules that localise to the axoneme all contribute to elegantly orchestrated signalling pathways that influence the whole cell. From their role in neural tube development, neuronal migration and differentiation, to their putative role in adult cognition, primary cilia are critical for diverse aspects of brain function. Yet our current understanding is still very limited: much remains to be discovered about primary cilia biology and ciliary function in the mammalian brain.Key ConceptsPrimary cilia are unusual cell organelles that look similar to antennae protruding from the cell.Almost all brain cell types have primary cilia, which transduce signals from the milieu.Ciliopathies in humans result from primary cilia defects and often include cognitive impairments and cortical malformations.Primary cilia contribute to brain development and function.

Celsr3 and Fzd3, members of "core planar cell polarity" (PCP) genes, were shown previously to control forebrain axon guidance and wiring by acting in axons and/or guidepost cells. Here, we show that Celsr2 acts redundantly with Celsr3, and that their combined mutation mimics that of Fzd3. The phenotypes generated upon inactivation of Fzd3 in different forebrain compartments are similar to those in conditional Celsr2-3 mutants, indicating that Fzd3 and Celsr2-3 act in the same population of cells. Inactivation of Celsr2-3 or Fzd3 in thalamus does not affect forebrain wiring, and joint inactivation in cortex and thalamus adds little to cortical inactivation alone in terms of thalamocortical projections. On the other hand, joint inactivation perturbs strongly the formation of the barrel field, which is unaffected upon single cortical or thalamic inactivation, indicating a role for interactions between thalamic axons and cortical neurons in cortical arealization. Unexpectedly, forebrain wiring is normal in mice defective in Vangl1 and Vangl2, showing that, contrary to epithelial PCP, axon guidance can be Vangl independent in some contexts. Our results suggest that Celsr2-3 and Fzd3 regulate axonal navigation in the forebrain by using mechanisms different from classical epithelial PCP, and require interacting partners other than Vangl1-2 that remain to be identified.

The cortical hem, a source of Wingless-related (WNT) and bone morphogenetic protein (BMP) signaling in the dorsomedial telencephalon, is the embryonic organizer for the hippocampus. Whether the hem is a major regulator of cortical patterning outside the hippocampus has not been investigated. We examined regional organization across the entire cerebral cortex in mice genetically engineered to lack the hem. Indicating that the hem regulates dorsoventral patterning in the cortical hemisphere, the neocortex, particularly dorsomedial neocortex, was reduced in size in late-stage hem-ablated embryos, whereas cortex ventrolateral to the neocortex expanded dorsally. Unexpectedly, hem ablation also perturbed regional patterning along the rostrocaudal axis of neocortex. Rostral neocortical domains identified by characteristic gene expression were expanded, and caudal domains diminished. A similar shift occurs when fibroblast growth factor (FGF) 8 is increased at the rostral telencephalic organizer, yet the FGF8 source was unchanged in hem-ablated brains. Rather we found that hem WNT or BMP signals, or both, have opposite effects to those of FGF8 in regulating transcription factors that control the size and position of neocortical areas. When the hem is ablated a necessary balance is perturbed, and cerebral cortex is rostralized. Our findings reveal a much broader role for the hem in cortical development than previously recognized, and emphasize that two major signaling centers interact antagonistically to pattern cerebral cortex.

Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.

The concept of a morphogen can be traced to the turn of the 20th century, when Morgan postulated the presence of "formative substances" as the basis for different regeneration rates in worms (Morgan, 1901). Very soon thereafter, Boveri entertained this idea for normal development (Boveri, 1901). A seminal event for this field was the discovery of a localized source for morphogens known as the Spemann organizer (Spemann and Mangold, 1924). The term "morphogen" was coined by Turing, who described how uniformly distributed signals made by cells can spread, self-organize, and generate pattern (Turing, 1952). Turing patterns remains highly relevant in development, but for this chapter and the developing forebrain, the more relevant concept is that of nonuniform graded distributions of morphogens, an idea formalized in the famous "French flag" model of Wolpert (Fig. 1.1) (Wolpert, 1969).