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The size of the cerebral cortex increases dramatically across amniotes, from reptiles to great apes. This is primarily due to different numbers of neurons and glial cells produced during embryonic development. The evolutionary expansion of cortical neurogenesis was linked to changes in neural stem and progenitor cells, which acquired increased capacity of self-amplification and neuron production. Evolution works via changes in the genome, and recent studies have identified a small number of new genes that emerged in the recent human and primate lineages, promoting cortical progenitor proliferation and increased neurogenesis. However, most of the mammalian genome corresponds to noncoding DNA that contains gene-regulatory elements, and recent evidence precisely points at changes in expression levels of conserved genes as key in the evolution of cortical neurogenesis. Here, we provide an overview of basic cellular mechanisms involved in cortical neurogenesis across amniotes, and discuss recent progress on genetic mechanisms that may have changed during evolution, including gene expression regulation, leading to the expansion of the cerebral cortex., European Research Council grant 309633; Spanish State Research Agency “Severo Ochoa” Programme for Centers of Excellence in R&D grant SEV-2017-0723; Spanish State Research Agency grant PGC2018-102172-B-I00

[Significance]: The present study highlights a fundamental role of GABAergic interneuron-derived Reelin in neuronal migration, in addition to CR cell–expressed Reelin. Further, we observed transient migratory deficits, indicating that Reelin expressed by either neuronal population is sufficient to reverse some lamination defects. We propose a model of Reelin action in corticogenesis based on the spatial and cell-specific expression of this key protein. Because several neuropsychiatric disorders are linked to Reelin deficits in interneurons, this study may provide a better understanding of the mechanisms associated with human brain disorders related to Reelin deficits., The extracellular protein Reelin, expressed by Cajal–Retzius (CR) cells at early stages of cortical development and at late stages by GABAergic interneurons, regulates radial migration and the “inside-out” pattern of positioning. Current models of Reelin functions in corticogenesis focus on early CR cell–derived Reelin in layer I. However, developmental disorders linked to Reelin deficits, such as schizophrenia and autism, are related to GABAergic interneuron–derived Reelin, although its role in migration has not been established. Here we selectively inactivated the Reln gene in CR cells or GABAergic interneurons. We show that CR cells have a major role in the inside-out order of migration, while CR and GABAergic cells sequentially cooperate to prevent invasion of cortical neurons into layer I. Furthermore, GABAergic cell–derived Reelin compensates some features of the reeler phenotype and is needed for the fine tuning of the layer-specific distribution of cortical neurons. In the hippocampus, the inactivation of Reelin in CR cells causes dramatic alterations in the dentate gyrus and mild defects in the hippocampus proper. These findings lead to a model in which both CR and GABAergic cell–derived Reelin cooperate to build the inside-out order of corticogenesis, which might provide a better understanding of the mechanisms involved in the pathogenesis of neuropsychiatric disorders linked to abnormal migration and Reelin deficits., This work was supported by grants from the Spanish Ministerio de Ciencia y Competitividad and Ministerio de Ciencia e Innovación (SAF2016-76340R and PID2019-106764RB-C21, Excellence Unit 629, María de Maeztu/Institute of Neurosciences) and by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (Instituto de Salud Carlos III, Spanish Ministry of Health) (to E.S.).

Preview., How the patterns of cortex folding are implemented during embryonic development is poorly understood. In this issue of Neuron, Han et al. (2021) establish that a population of neural progenitor cells co-expressing Neurog2 and Ascl1 are key in this process.

Description, Expression of miR-3607 in embryonic mammalian cerebral cortex was lost in rodents, limiting progenitor cell proliferation., The evolutionary expansion and folding of the mammalian cerebral cortex resulted from amplification of progenitor cells during embryonic development. This process was reversed in the rodent lineage after splitting from primates, leading to smaller and smooth brains. Genetic mechanisms underlying this secondary loss in rodent evolution remain unknown. We show that microRNA miR-3607 is expressed embryonically in the large cortex of primates and ferret, distant from the primate-rodent lineage, but not in mouse. Experimental expression of miR-3607 in embryonic mouse cortex led to increased Wnt/β-catenin signaling, amplification of radial glia cells (RGCs), and expansion of the ventricular zone (VZ), via blocking the β-catenin inhibitor APC (adenomatous polyposis coli). Accordingly, loss of endogenous miR-3607 in ferret reduced RGC proliferation, while overexpression in human cerebral organoids promoted VZ expansion. Our results identify a gene selected for secondary loss during mammalian evolution to limit RGC amplification and, potentially, cortex size in rodents.

The human brain is characterized by the large size and intricate folding of its cerebral cortex, which are fundamental for our higher cognitive function and frequently altered in pathological dysfunction. Cortex folding is not unique to humans, nor even to primates, but is common across mammals. Cortical growth and folding are the result of complex developmental processes that involve neural stem and progenitor cells and their cellular lineages, the migration and differentiation of neurons, and the genetic programs that regulate and fine-tune these processes. All these factors combined generate mechanical stress and strain on the developing neural tissue, which ultimately drives orderly cortical deformation and folding. In this review we examine and summarize the current knowledge on the molecular, cellular, histogenic, and mechanical mechanisms that are involved in and influence folding of the cerebral cortex, and how they emerged and changed during mammalian evolution. We discuss the main types of pathological malformations of human cortex folding, their specific developmental origin, and how investigating their genetic causes has illuminated our understanding of key events involved. We close our review by presenting the animal and in vitro models of cortex folding that are currently used to study these devastating developmental brain disorders in children, and what are the main challenges that remain ahead of us to fully understand brain folding.

Summary In vivo electroporation has become a key technique to study genetic mechanisms of brain development. However, electroporation of the embryonic pallium in oviparous species, interesting for evolutionary studies but distinct from in utero electroporation, is quite infrequent. Here, we detail the in ovo electroporation of the developing pallium in chick and snake embryos. This protocol allows gene manipulation through introducing exogenous DNA into brain progenitor cells and can be adapted to any type of gene manipulation of the embryonic telencephalon. For complete information on the use and execution of this protocol, please refer to Cárdenas et al. (2018)., Graphical abstract, Highlights • In ovo electroporation of dorsal telencephalon in chick and snake embryos • Maximal viability of the embryos makes the protocol highly efficient • The simplicity of the procedure makes it accessible to non-expert researchers • Adaptable to any type of gene manipulation of the embryonic telencephalon, In vivo electroporation has become a key technique to study genetic mechanisms of brain development. However, electroporation of the embryonic pallium in oviparous species, interesting for evolutionary studies but distinct from in utero electroporation, is quite infrequent. Here, we detail the in ovo electroporation of the developing pallium in chick and snake embryos. This protocol allows gene manipulation through introducing exogenous DNA into brain progenitor cells and can be adapted to any type of gene manipulation of the embryonic telencephalon.

The size and organization of the brain are determined by the activity of progenitor cells early in development. Key mechanisms regulating progenitor cell biology involve miRNAs. These small noncoding RNA molecules bind mRNAs with high specificity, controlling their abundance and expression. The role of miRNAs in brain development has been studied extensively, but their involvement at early stages remained unknown until recently. Here, recent findings showing the important role of miRNAs in the earliest phases of brain development are reviewed, and it is discussed how loss of specific miRNAs leads to pathological conditions, particularly adult and pediatric brain tumors. Let-7 miRNA downregulation and the initiation of embryonal tumors with multilayered rosettes (ETMR), a novel link recently discovered by the laboratory, are focused upon. Finally, it is discussed how miRNAs may be used for the diagnosis and therapeutic treatment of pediatric brain tumors, with the hope of improving the prognosis of these devastating diseases.

The mammalian cerebral cortex is the pinnacle of brain evolution, reaching its maximum complexity in terms of neuron number, diversity and functional circuitry. The emergence of this outstanding complexity begins during embryonic development, when a limited number of neural stem and progenitor cells manage to generate myriads of neurons in the appropriate numbers, types and proportions, in a process called neurogenesis. Here we review the current knowledge on the regulation of cortical neurogenesis, beginning with a description of the types of progenitor cells and their lineage relationships. This is followed by a review of the determinants of neuron fate, the molecular and genetic regulatory mechanisms, and considerations on the evolution of cortical neurogenesis in vertebrates leading to humans. We finish with an overview on how dysregulation of neurogenesis is a leading cause of human brain malformations and functional disabilities.

The mammalian cerebral cortex is the pinnacle of brain evolution, reaching its maximum complexity in terms of neuron number, diversity and functional circuitry. The emergence of this outstanding complexity begins during embryonic development, when a limited number of neural stem and progenitor cells manage to generate myriads of neurons in the appropriate numbers, types and proportions, in a process called neurogenesis. Here we review the current knowledge on the regulation of cortical neurogenesis, beginning with a description of the types of progenitor cells and their lineage relationships. This is followed by a review of the determinants of neuron fate, the molecular and genetic regulatory mechanisms, and considerations on the evolution of cortical neurogenesis in vertebrates leading to humans. We finish with an overview on how dysregulation of neurogenesis is a leading cause of human brain malformations and functional disabilities., Work in our lab is supported by grants to V.B. from the ERC Consolidator Grant CORTEXFOLDING, the Spanish State Research Agency (PGC2018-102172-B-I00) and through the “Severo Ochoa" Programme for Centers of Excellence in R&D (ref. SEV-2017-0723), and to M.G. from the ERC Advanced Grant ChroNeuroRepair.

Editorial on the Research Topic., YH was supported by grants from the Japan Society for thePromotion of Science (KAKENHI JP20K06670), the Ichiro Kanehara Foundation, SENSHIN Medical Research Foundation, and Keio Gijuku Academic Development Funds. VB was supported by grants from the European Research Council (309633) and the Spanish Research Agency (SAF2015-69168-R, PGC2018-102172-B-I00, and through the Severo Ochoa Program for Centers of Excellence in R&D, ref. SEV-2017-0723).

21 páginas, 11 figuras, Development of the cerebral cortex depends critically on the regulation of progenitor cell proliferation and fate. Cortical progenitor cells are remarkably diverse with regard to their morphology as well as laminar and areal position. Extrinsic factors, such as thalamic axons, have been proposed to play key roles in progenitor cell regulation, but the diversity, extent and timing of interactions between extrinsic elements and each class of cortical progenitor cell in higher mammals remain undefined. Here we use the ferret to demonstrate the existence of a complex set of extrinsic elements that may interact, alone or in combination, with subpopulations of progenitor cells, defining a code of extrinsic influences. This code and its complexity vary significantly between developmental stages, layer of residence and morphology of progenitor cells. By analyzing the spatial-temporal overlap of progenitor cell subtypes with neuronal and axonal populations, we show that multiple sets of migrating neurons and axon tracts overlap extensively with subdivisions of the Subventricular Zones, in an exquisite lamina-specific pattern. Our findings provide a framework for understanding the feedback influence of both intra- and extra-cortical elements onto progenitor cells to modulate their dynamics and fate decisions in gyrencephalic brains., The research leading to a part of these results received funding from MINECO (Spanish Ministry of Economy and Competitiveness) (BFU2012-33473, SAF2015-69168-R) and European Research Council (309633). V.B. acknowledges financial support from the Spanish State Research Agency, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (ref. SEV-2013-0317).

Structural integrity and cellular homeostasis of the embryonic stem cell niche are critical for normal tissue development. In the telencephalic neuroepithelium, this is controlled in part by cell adhesion molecules and regulators of progenitor cell lineage, but the specific orchestration of these processes remains unknown. Here, we studied the role of microRNAs in the embryonic telencephalon as key regulators of gene expression. By using the early recombiner Rx‐Cre mouse, we identify novel and critical roles of miRNAs in early brain development, demonstrating they are essential to preserve the cellular homeostasis and structural integrity of the telencephalic neuroepithelium. We show that Rx‐Cre;DicerF/F mouse embryos have a severe disruption of the telencephalic apical junction belt, followed by invagination of the ventricular surface and formation of hyperproliferative rosettes. Transcriptome analyses and functional experiments in vivo show that these defects result from upregulation of Irs2 upon loss of let‐7 miRNAs in an apoptosis‐independent manner. Our results reveal an unprecedented relevance of miRNAs in early forebrain development, with potential mechanistic implications in pediatric brain cancer., V.F. was recipient of an FPI fellowship from the Spanish State Research Agency (AEI), and A.P.‐C. was recipient of a predoctoral fellowship from Fundación Tatiana Pérez de Guzmán el Bueno. This work was supported by grants to V.B. from AEI (SAF2015‐69168‐R, PGC2018‐102172‐B‐I00) and European Research Council (309633). V.B. acknowledges financial support from the AEI, through the “Severo Ochoa” Program for Centers of Excellence in R&D (Ref. SEV‐2017‐0723).

The neocortex is the largest part of the mammalian brain and is the seat of our higher cognitive functions. This outstanding neural structure increased massively in size and complexity during evolution in a process recapitulated today during the development of extant mammals. Accordingly, defects in neocortical development commonly result in severe intellectual and social deficits. Thus, understanding the development of the neocortex benefits from understanding its evolution and disease and also informs about their underlying mechanisms. Here, I briefly summarize the most recent and outstanding advances in our understanding of neocortical development and focus particularly on dorsal progenitors and excitatory neurons. I place special emphasis on the specification of neural stem cells in distinct classes and their proliferation and production of neurons and then discuss recent findings on neuronal migration. Recent discoveries on the genetic evolution of neocortical development are presented with a particular focus on primates. Progress on all these fronts is being accelerated by high-throughput gene expression analyses and particularly single-cell transcriptomics. I end with novel insights into the involvement of microglia in embryonic brain development and how improvements in cultured cerebral organoids are gradually consolidating them as faithful models of neocortex development in humans.

The evolutionary expansion of the mammalian cerebral cortex is recapitulated during embryonic development in large mammals, but the underlying genetic mechanisms remain mostly unknown. Previous transcriptomic analyses of the developing ferret cortex identify candidate genes related to the expansion of germinal layers and cortex size. Here we focused onMIR3607, a microRNA differentially expressed between germinal layers of the large human and ferret cortex, not expressed in the small mouse cortex. Expression ofMIR3607in mouse embryos at E14.5 leads to increased progenitor cell proliferation. This is reflected in transcriptomic changes, which also reveal increased Wnt/βCatenin signaling. Expression ofMIR3607at E12.5, when progenitor cells expand, causes amplification and severe delamination of apical progenitors, leading to rosette formation. This is rescued by co-expressing Adenomatous Polyposis Coli, inhibitor of canonical Wnt signaling. A similar phenotype is produced in human cerebral organoids. Our findings demonstrate thatMIR3607expands and delaminates apical progenitor cells via activating Wnt/βCatenin, and suggest that a secondary loss of expression in mouse may underlie their reduction in cortex size during recent evolution.

The cerebral cortex varies dramatically in size and complexity between amniotes due to differences in neuron number and composition. These differences emerge during embryonic development as a result of variations in neurogenesis, which are thought to recapitulate modifications occurred during evolution that culminated in the human neocortex. Here, we review work from the last few decades leading to our current understanding of the evolution of neurogenesis and size of the cerebral cortex. Focused on specific examples across vertebrate and amniote phylogeny, we discuss developmental mechanisms regulating the emergence, lineage, complexification and fate of cortical germinal layers and progenitor cell types. At the cellular level, we discuss the fundamental impact of basal progenitor cells and the advent of indirect neurogenesis on the increased number and diversity of cortical neurons and layers in mammals, and on cortex folding. Finally, we discuss recent work that unveils genetic and molecular mechanisms underlying this progressive expansion and increased complexity of the amniote cerebral cortex during evolution, with a particular focus on those leading to human-specific features. Whereas new genes important in human brain development emerged the recent hominid lineage, regulation of the patterns and levels of activity of highly conserved signaling pathways are beginning to emerge as mechanisms of central importance in the evolutionary increase in cortical size and complexity across amniotes., Work in our lab is funded by the Spanish Ministry of Science, Innovation and Universities (SAF2015-69168-R; SAF2017-92781-EXP; PGC2018-102172-B-I00), Consejo Superior de Investigaciones Científicas (201820E129) and the “Severo Ochoa” Programme for Centers of Excellence in Research and Development (Reference SEV-2017-0723).

This is an open access article under theterms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License., One of the most prominent features of the human brain is the fabulous size of the cerebral cortex and its intricate folding. Cortical folding takes place during embryonic development and is important to optimize the functional organization and wiring of the brain, as well as to allow fitting a large cortex in a limited cranial volume. Pathological alterations in size or folding of the human cortex lead to severe intellectual disability and intractable epilepsy. Hence, cortical expansion and folding are viewed as key processes in mammalian brain development and evolution, ultimately leading to increased intellectual performance and, eventually, to the emergence of human cognition. Here, we provide an overview and discuss some of the most significant advances in our understanding of cortical expansion and folding over the last decades. These include discoveries in multiple and diverse disciplines, from cellular and molecular mechanisms regulating cortical development and neurogenesis, genetic mechanisms defining the patterns of cortical folds, the biomechanics of cortical growth and buckling, lessons from human disease, and how genetic evolution steered cortical size and folding during mammalian evolution., V.F. was recipient of a Formación de Personal Investigador fellowship from the Spanish Ministry of Economy and Competitivity (MINECO), and C.L. was supported by the European Union Seventh Framework Programme FP7/2007–2013 under the project DESIRE (Grant Agreement No. 602531). Work in our laboratory is also supported by grants from MINECO (BFU2012-33473) and European Research Council (ERC StG309633) to V.B. The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa”.

Folding of the cerebral cortex is a fundamental milestone of mammalian brain evolution and is associated with dramatic increases in size and complexity. New animal models, genetic tools and bioengineering materials have moved the study of cortical folding from simple phenomenological observation to sophisticated experimental testing. Here, we provide an overview of how genetics, cell biology and biomechanics shape this complex and multifaceted process and affect each other. We discuss the evolution of cortical folding and the genomic changes in the primate lineage that seem to be responsible for the advent of larger brains and cortical folding. Emerging technologies now provide unprecedented tools to analyse and manipulate cortical folding, with the promise of elucidating the mechanisms underlying the stereotyped folding of the cerebral cortex in its full complexity., The authors’ research was supported by a European Research Grant (CORTEXFOLDING-309633), a Spanish Ministry of Economy and Competitiveness Grant (SAF2015-69168-R), the European Union Seventh Framework Programme (FP7/2007-2013, under the project DESIRE-602531) and the Spanish State Research Agency through the ‘Severo Ochoa’ Programme for Centers of Excellence in Research and Development (reference SEV-2017-0723).

Excitatory neurons of the cerebral cortex migrate radially from their place of birth to their final position in the cortical plate during development. Radially‐migrating neurons display a single leading process that establishes the direction of movement. This leading process has been described as being unbranched, and the occurrence of branches proposed to impair radial migration. Here we have analyzed the detailed morphology of leading process in radially‐migrating pyramidal neurons and its impact on radial migration. We have compared ferret and mouse to identify differences between cortices that undergo folding or not. In mouse, we find that half of radially‐migrating neurons exhibit a branched leading process, this being even more frequent in ferret. Branched leading processes are less parallel to radial glia fibers than those unbranched, suggesting some independence from radial glia fibers. Two‐photon videomicroscopy revealed that a vast majority of neurons branch their leading process at some point during radial migration, but this does not reduce their migration speed. We have tested the functional impact of exuberant leading process branching by expressing a dominant negative Cdk5. We confirm that loss of Cdk5 function significantly impairs radial migration, but this is independent from increased branching of the leading process. We propose that excitatory neurons may branch their leading process as an evolutionary mechanism to allow cells changing their trajectory of migration to disperse laterally, such that increased branching in gyrencephalic species favors the tangential dispersion of radially‐migrating neurons, and cortical folding., Ministerio de Ciencia e Innovación, Grant/Award Numbers: SAF2011‐28845, SAF2015‐69168‐R

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination., Funding was provided by the DFG (GO 640/12-1, SFB 870 A06 to M.G.; JU 2957/1-1, SFB 1032 A11 to R.J.; INST86/1581-1FUGG, IRTG 2290 to S.R.; SFB 1089 to F.B.), MINECO (SAF2015-69168-R to V.B.), Fundación Francisco Cobos (fellowship to C.D.J.R.), the ERC (Chroneurorepair to M.G.; Cortexfolding – 306933 to V.B.; MolMap – 680241 to R.J.), ERANET (AXON REPAIR and RATER SCI to F.B.; STEM-MCD and NEUROTALK to L.N.), the F.R.S.-FNRS (EOS O019118F-RG36 to L.N.), and NIH (R01DA024681 and R01NS085004 to S.-H.S.).

Intrinsic coupling modes (ICMs) provide a framework for describing the interactions of ongoing brain activity at multiple spatial and temporal scales. Two families of ICMs can be distinguished: phase and envelope ICMs. The principles that shape these ICMs remain partly elusive, in particular their relation to the underlying brain structure. Here we explored structure-function relationships in the ferret brain between ICMs quantified from ongoing brain activity recorded with chronically implanted ECoG arrays and structural connectivity (SC) obtained from high-resolution diffusion MRI tractography. Large-scale computational models as well as simple topological ingredients of SC were used to explore the ability to predict both types of ICMs. Importantly, all investigations were conducted with ICM measures that are sensitive or insensitive to volume conduction effects. The results show that both types of ICMs are strongly related to SC, except when using ICM measures removing zero-lag synchronizations. Computational models are challenged to predict these ICM patterns consistently, and simple predictions from SC topological features can sometimes outperform them. Overall, the results demonstrate that patterns of cortical functional coupling as reflected in both phase and envelope ICMs bear a substantial relation to the underlying structural connectivity of the cerebral cortex.

Work performed in our laboratory is funded in part by the Spanish Ministry of Science and Innovation [grant number BFU2012-33473]. The Instituto de Neurociencias is a ‘Centre of Excellence Severo Ochoa’.

Summary Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution., Graphical Abstract, Highlights • Neurogenesis in mammalian neocortex is largely indirect, direct in reptiles and birds • Low Robo and high Dll1 signaling is necessary for indirect neurogenesis • Blocking Robo and increased Dll1 in non-mammals induces indirect neurogenesis and SVZ • High Robo–low Dll1 blocks indirect neurogenesis in human cerebral organoids, Levels of Robo and Notch signaling across amniotes determines their predominant mode of neurogenesis, with consequences on final cerebral cortex size and complexity

The existence of axons extending from one retina to the other has been reported during perinatal development in different vertebrates. However, it has been thought that these axons are either a labelling artefact or misprojections. Here, we unequivocally show that a small subset of retinal ganglion cells (RGCs) project to the opposite retina and that the guidance receptor Unc5c, expressed in the retinal region where the R-R neurons are located, is necessary and sufficient to guide axons to the opposite retina. In addition, Netrin1, an Unc5c ligand, is expressed in the ventral diencephalon in a pattern that is consistent with impeding the growth of Unc5c retinal axons into the brain. We have also generated a mathematical model to explore the formation of retinotopic maps in the presence and absence of a functional connection between both eyes. This model predicts that an R-R connection is required for the bilateral coordination of axonal refinement in species where refinement depends upon spontaneous retinal waves. Consistent with this idea, the retinal expression of Unc5c correlates with the existence and size of an R-R projection in different species and with the extent of axonal refinement in visual targets. These findings demonstrate that active guidance drives the formation of the R-R projection and suggest an important role for these projections in visual mapping to ensure congruent bilateral refinement.

Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type. © The Author 2011., This work was supported by the Strategic Research Program for Brain Sciences from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan to E.S. and H.O. and by ‘‘Funding Program for World-leading Innovative R&D on Science and Technology’’ to E.S. and H.O. W.B.H. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SFB 655, A2; TRR 83, Tp6) and the European Research Council (250197), by the DFG-funded Center for Regenerative Therapies Dresden, and by the Fonds der Chemischen Industrie. V.B. was supported by grants from the International Human Frontier Science Program Organization (CDA0027/2007-C), MICINN (SAF2009-07367), and CONSOLIDER (CSD2007-00023).

Folding of the cerebral cortex is as highly intriguing as poorly understood. At first sight, this may appear as simple tissue crumpling inside an excessively small cranium, but the process is clearly much more complex and developmentally predetermined. Whereas theoretical modeling supports a critical role for biomechanics, experimental evidence demonstrates the fundamental role of specific progenitor cell types, cellular processes, and genetic programs on cortical folding., This work was supported by European Research Council Grant CORTEXFOLDING-309633; Spanish Ministry of Economy and Competitiveness Grant SAF2015-69168-R; and Spanish State Research Agency, through the “Severo Ochoa” Program for Centers of Excellence in R&D (Reference SEV-2013-0317).

This work was completed during OHBM Hackathon Lausanne 2016 and Brainhack Anatomy Paris 2016., Increasingly available high-resolution brain imaging data require specialized processing tools that can leverage their anatomical detail and handle their size. Here, we present user-friendly Python tools for cortical depth resolved analysis in such data. Our implementation is based on the CBS High-Res Brain Processing framework, and aims to make high-resolution data processing tools available to the broader community.

In the last few years, several studies have revisited long-held assumptions in the field of brain development and evolution providing us with a fundamentally new vision on the mechanisms controlling its size and shape, hence function. Among these studies, some described hitherto unforeseeable subtypes of neural progenitors while others reinterpreted long-known observations about their cell cycle in alternative new ways. Most remarkably, this knowledge combined has allowed the generation of mammalian model organisms in which brain size and folding has been selectively increased giving us the means to understand the mechanisms underlying the evolution of the most complex and sophisticated organ. Here we review the key findings made in this area and make a few conjectures about their evolutionary meaning including the likelihood of Martians conquering our planet., VB and FC are supported by the Ministerio de Economía y Competitividad (BFU2012-33473) and European Research Council (309633) (VB), DFG Collaborative Research Center SFB655, Center for Regenerative Therapies and Medical Faculty of the TU–Dresden (FC).

Summary The existence of axons extending from one retina to the other has been reported during perinatal development in different vertebrates. However, it has been thought that these axons are either a labeling artifact or misprojections. Here, we show unequivocally that a small subset of retinal ganglion cells (RGCs) project to the opposite retina and that the guidance receptor Unc5c, expressed in the retinal region where the retinal-retinal (R-R) RGCs are located, is necessary and sufficient to guide axons to the opposite retina. In addition, Netrin1, an Unc5c ligand, is expressed in the ventral diencephalon in a pattern that is consistent with impeding the growth of Unc5c-positive retinal axons into the brain. We also have generated a mathematical model to explore the formation of retinotopic maps in the presence and absence of a functional connection between both eyes. This model predicts that an R-R connection is required for the bilateral coordination of axonal refinement in species where refinement depends upon spontaneous retinal waves. Consistent with this idea, the retinal expression of Unc5c correlates with the existence and size of an R-R projection in different species and with the extent of axonal refinement in visual targets. These findings demonstrate that active guidance drives the formation of the R-R projection and suggest an important role for these projections in visual mapping to ensure congruent bilateral refinement., Highlights • A subset of retinal ganglion cells project to the contralateral retina • Unc5c mediates the formation of the retina-retina projection • Unc5c retinal expression correlates with extent of refinement in visual targets • Congruency of visual maps in species with retinal waves may rely on R-R axons, Murcia-Belmonte et al. demonstrate the existence of an Unc5c-dependent transient projection that connects both retinas in several vertebrate species. Computational modeling suggests this retina-retina projection may be critical for the congruency of visual maps in species that undergo retinal wave-dependent axon refinement during development.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human., We thank the Inserm Avenir program, the French Agence National de la Recherche (ANR-08-MNP-013 (F.F.), ANR-13-BSV4-0008-01 (F.F.), ANR Blanc 1103-01 (J.C.), projet R11039KK, ANR E-Rare Program, convention 2011-RaARE-012-01) (J.C.), the Fondation Bettencourt Schueller and the Federation pour la recherche sur le cerveau (FRC) for grants awarded to F.F., the FRC Rotary for an equipment grant awarded to the Institut du Fer à Moulin, the Swiss National Science Foundation (SNSF 31003A-135574, SPUM-33CM30-124089 and 33CM30-140332) and the Fondation Gianni Biaggi de Blasys for grants awarded to A.C., and the SNSF (31003A-125379) for grants awarded to E.W. The research leading to a part of these results received funding from the European Union Seventh Framework Programme FP7/2007–2013 under the project DESIRE (grant agreement no. 602531), la Fondation pour la Recherche Médicale (FRM, J Chelly–Equipe FRM 2013: DEQ2000326477) la Fondation Jacques Espinasse & Danièle (JED)-Belgique and the Spanish Ministry of Economy and Competitiveness grant (BFU2012-33473) awarded to V.B.

SummaryEvolution of the mammalian brain encompassed a remarkable increase in size of the cerebral cortex, which includes tangential and radial expansion. However, the mechanisms underlying these key features are still largely unknown. Here, we identified the DNA-associated protein Trnp1 as a regulator of cerebral cortex expansion in both of these dimensions. Gain- and loss-of-function experiments in the mouse cerebral cortex in vivo demonstrate that high Trnp1 levels promote neural stem cell self-renewal and tangential expansion. In contrast, lower levels promote radial expansion, with a potent increase of the number of intermediate progenitors and basal radial glial cells leading to folding of the otherwise smooth murine cerebral cortex. Remarkably, TRNP1 expression levels exhibit regional differences in the cerebral cortex of human fetuses, anticipating radial or tangential expansion. Thus, the dynamic regulation of Trnp1 is critical to control tangential and radial expansion of the cerebral cortex in mammals.

Folding of the cerebral cortex during brain development is a complex process that depends on the orchestrated action of a number of factors, including generation and proliferation of basal progenitor cells, and the radial migration of neurons. Patterns of primary cortical folding are stereotyped between individuals and across phylogeny, reflecting a strong genetic regulation of the underlying cellular mechanisms. Here we summarize recent findings on cellular and genetic mechanisms regulating this fascinating process that underlies expansion and functional complexification of the mammalian cerebral cortex., Work in VB's lab is supported by grants from Spanish Ministry of Economy and Competitivity (SAF2015-69168-R), European Research Council (309633) and European Union Seventh Framework Programme FP7/2007-2013 under the project DESIRE (602531).

The folding of the mammalian cerebral cortex into sulci and gyri is thought to be favored by the amplification of basal progenitor cells and their tangential migration. Here, we provide a molecular mechanism for the role of migration in this process by showing that changes in intercellular adhesion of migrating cortical neurons result in cortical folding. Mice with deletions of FLRT1 and FLRT3 adhesion molecules develop macroscopic sulci with preserved layered organization and radial glial morphology. Cortex folding in these mutants does not require progenitor cell amplification but is dependent on changes in neuron migration. Analyses and simulations suggest that sulcus formation in the absence of FLRT1/3 results from reduced intercellular adhesion, increased neuron migration, and clustering in the cortical plate. Notably, FLRT1/3 expression is low in the human cortex and in future sulcus areas of ferrets, suggesting that intercellular adhesion is a key regulator of cortical folding across species.

The function of the amyloid precursor protein (APP), a key molecule in Alzheimer's disease (AD) remains unknown. Among the proteins that interact with the APP cytoplasmic domain in vitro and in heterologous systems is Disabled-1, a signaling molecule of the reelin pathway. The physiological consequence of this interaction is unknown. Here we used an in vitro model of hippocampal neurons grown on a reelin substrate that inhibits neurite outgrowth. Our results show that an excess of APP cytoplasmic domain internalized by a cell permeable peptide, is able to antagonize the neurite outgrowth inhibition of reelin. The APP cytoplasmic domain binds Disabled-1 and retains it in the cytoplasm, preventing it from reaching the plasma membrane and sequesters tyrosine phosphorylated Disabled-1, both of which disrupt reelin signaling. In the context of AD, increased formation of APP cytoplasmic domain in the cytosol released after cleavage of the Aβ peptide, could then inhibit reelin signaling pathway in the hippocampus and thus influence synaptic plasticity.

During the development of the cerebral cortex, neurons generated in the cortical ventricular zone migrate radially toward the marginal zone. Radially migrating neurons are thought to display 1 of 2 morphologies: cells with a long, pia-contacting, apical process utilized for somal translocation early in development, when the cortex is still relatively thin; or cells with a short leading process, abundant at late stages of corticogenesis when neurons need to travel for longer distances. In large convoluted brains, like those of many primates and carnivores, radially migrating neurons must travel distances up to several millimeters before reaching their final destination, often following curvilinear trajectories. Here we analyze modes and morphologies of radially migrating neurons in convoluted brains by studying the visual cortex of developing ferrets. We provide in vivo and in vitro evidence for the existence of late-born cortical neurons that migrate radially by long-distance somal translocation within a long apical process extended to the cortical plate, in contrast to the early somal translocation observed in rodents. Long-distance translocating neurons in the ferret show a discontinuous rhythm of migration, alternating periods of advance with periods of stall. Furthermore, by combining different labeling methods we find the simultaneous presence in the developing ferret cortex of long-distance translocating neurons and neurons migrating within a short leading process., VB was funded by the Human Frontier Science Program. Supported by Packard Foundation and NIH grants EY10742 and MH63912.

Telencephalic inhibitory neurons originate in the ganglionic eminences and migrate to the cerebral cortex following a tangential trajectory, before they differentiate and integrate within the local circuitry. Current studies of interneuron development and function benefit from the use of knock-out and transgenic mice, whereas none take advantage of the versatility of in utero electroporation. Here, we show how in utero electroporation can be directed to the ganglionic eminences to specifically target gene expression to interneurons. Electroporation of GFP-encoding plasmids into the ganglionic eminences results in selective labeling of migrating interneurons during development. In the adult brain of electroporated animals, a wide variety of cortical, hippocampal and olfactory bulb interneurons are labeled. We also show that GFP-expressing interneurons can be visualized in living slices of adult cerebral cortex, where they display normal electrophysiological properties. Photostimulation studies using acute slices show that cortical GFP+ interneurons receive normal, layer-specific synaptic input, indicating that these neurons integrate within the local cortical circuitry. Ganglionic eminence-directed in utero electroporation is therefore an effective, rapid, and versatile method of selectively transfecting telencephalic interneurons, optimal for both developmental studies and adult functional studies.

Spontaneous neural activity is a basic property of the developing brain,which regulates key developmental processes, including migration, neural differentiation and formation and refinement of connections. The mechanisms regulating spontaneous activity are not known. By using transgenic embryos that overexpress BDNF under the control of the nestin promoter, we show here that BDNF controls the emergence and robustness of spontaneous activity in embryonic hippocampal slices. Further, BDNF dramatically increases spontaneous co-active network activity, which is believed to synchronize gene expression and synaptogenesis in vast numbers of neurons. In fact, BDNF raises the spontaneous activity of E18 hippocampal neurons to levels that are typical of postnatal slices.We also show that BDNF overexpression increases the number of synapses at much earlier stages (E18) than those reported previously. Most of these synapses were GABAergic, and GABAergic interneurons showed hypertrophy and a 3-fold increase in GAD expression. Interestingly, whereas BDNF does not alter the expression of GABA and glutamate ionotropic receptors, it does raise the expression of the recently cloned K+/Cl- KCC2 co-transporter, which is responsible for the conversion of GABA responses from depolarizing to inhibitory, through the control of the Cl-potential. Together, results indicate that both the presynaptic and postsynaptic machineries of GABAergic circuits may be essential targets of BDNF actions to control spontaneous activity. The data indicate that BDNF is a potent regulator of spontaneous activity and co-active networks, which is a new level of regulation of neurotrophins. Given that BDNF itself is regulated by neuronal activity, we suggest that BDNF acts as a homeostatic factor controlling the emergence, complexity and networking properties of spontaneous networks.

Radial glia cells play fundamental roles in the development of the cerebral cortex, acting both as the primary stem and progenitor cells, as well as the guides for neuronal migration and lamination. These critical functions of radial glia cells in cortical development have been discovered mostly during the last 15 years and, more recently, seminal studies have demonstrated the existence of a remarkable diversity of additional cortical progenitor cell types, including a variety of basal radial glia cells with key roles in cortical expansion and folding, both in ontogeny and phylogeny. In this review, we summarize the main cellular and molecular mechanisms known to be involved in cerebral cortex development in mouse, as the currently preferred animal model, and then compare these with known mechanisms in other vertebrates, both mammal and nonmammal, including human. This allows us to present a global picture of how radial glia cells and the cerebral cortex seem to have coevolved, from reptiles to primates, leading to the remarkable diversity of vertebrate cortical phenotypes., Grant sponsor: European Union Seventh Framework Programme FP7/2007–2013 under the project DESIRE; Grant number: 602531; Grant sponsor: European Research Council; Grant number: 309633; Grant sponsor: MINECO; Grant number: BFU2012-33473 (to VB).

Gyrencephalic species develop folds in the cerebral cortex in a stereotypic manner, but the genetic mechanisms underlying this patterning process are unknown. We present a large-scale transcriptomic analysis of individual germinal layers in the developing cortex of the gyrencephalic ferret, comparing between regions prospective of fold and fissure. We find unique transcriptional signatures in each germinal compartment, where thousands of genes are differentially expressed between regions, including ~80% of genes mutated in human cortical malformations. These regional differences emerge from the existence of discrete domains of gene expression, which occur at multiple locations across the developing cortex of ferret and human, but not the lissencephalic mouse. Complex expression patterns emerge late during development and map the eventual location of folds or fissures. Protomaps of gene expression within germinal layers may contribute to define cortical folds or functional areas, but our findings demonstrate that they distinguish the development of gyrencephalic cortices., The research leading to a part of these results received funding from the European Union Seventh Framework Programme FP7/2007–2013 under the project DESIRE (grant agreement no. 602531), MICINN (SAF2009-07367), the Spanish Ministry of Economy and Competitivity (BFU2012-33473, CSD2007-00023) and European Research Council (ERC StG309633) to VB. The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa”.

Severed adult CNS axons can extend over long distances when a permissive 'milieu', such as grafted Schwann cells or ensheathing cells, is provided. Moreover, functional blocking of endogenous inhibitory factors, such as Nogo-A or proteoglycans, enhances the regeneration of axotomized neurons. Here we examine whether guidance cues available during the development of axonal pathways could also potentiate the regeneration of lesioned adult circuits. The Cajal-Retzius cells in the hippocampus are transient pioneer neurons that guide entorhino-hippocampal afferents to their target layers. By using an in vitro model of axotomy of the entorhino-hippocampal pathway we show that Cajal-Retzius cells triggered the regeneration of the axotomized entorhino-hippocampal pathway. Furthermore, the regrowth induced by Cajal-Retzius cells was robust and its pattern was indistinguishable from that of the unlesioned entorhino-hippocampal pathway. Thus, regenerating axons regrew in a layer-specific fashion towards the appropriate target layers, making synaptic contacts with target pyramidal neurons. Interestingly, the ability of lesioned entorhinal axons to regrow was maintained for at least 9 days after axotomy. These results show that the growth-promoting cells controlling the development of neural circuits will be a relevant approach to promoting the regeneration of lesioned adult CNS pathways.

Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.

Reelin is a large, extracellular matrix protein involved in neuronal migration and axonal growth. To analyze the contribution of Reelin to the development of the commissural projection in the hippocampus, we analyzed the ontogeny of this projection in the reeler mutant mouse. Injections of the lipophilic tracer DiI revealed many commissural fibers in the hippocampus of both reeler and control mice at P1-P2. At P5, at P12, and in the adult, the topography of commissural connections was normal in the CA1 region of reeler mice, with axons innervating the stratum radiatum and stratum oriens. In contrast, in the CA3/CA2 region, commissural fibers abnormally innervated the stratum lacunosum-moleculare and, in the dentate gyrus, some fibers were observed in the outer molecular layer. Next, we monitored the distribution of Cajal-Retzius cells in the hippocampus of reeler mutant mice and noted that the stratum lacunosum-moleculare of the CA3/CA2 region was largely devoid of Cajal-Retzius (CR) cells. Taken together, the above results indicate that in the absence of CR cells in the CA3/CA2, commissural axons abnormally grow to the stratum lacunosum-moleculare. To test this hypothesis a series of coculture experiments was performed in collagen gels, in which the CA3 axonal growth was monitored when confronted to the marginal zone. These experiments showed that the marginal zone containing CR cells exerts short-range inhibitory influences for commissural axonal growth.

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents intrkB (−/−) andtrkC (−/−) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10–P16) did not reveal major differences in the topographic patterns of hippocampal connections.In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21–49%) and decreased densities of axonal varicosities (8–17%) in bothtrkB (−/−) andtrkC (−/−) mice. In addition, electron microscopic analyses showed thattrkB (−/−) andtrkC (−/−) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially intrkB (−/−) mice. We conclude that neithertrkB nortrkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNSin vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.