AUTS2 Regulates RNA Metabolism and Dentate Gyrus Development in Mice.

Human AUTS2 mutations are linked to a syndrome of intellectual disability, autistic features, epilepsy, and other neurological and somatic disorders. Although it is known that this unique gene is highly expressed in developing cerebral cortex, the molecular and developmental functions of AUTS2 protein remain unclear. Using proteomics methods to identify AUTS2 binding partners in neonatal mouse cerebral cortex, we found that AUTS2 associates with multiple proteins that regulate RNA transcription, splicing, localization, and stability. Furthermore, AUTS2-containing protein complexes isolated from cortical tissue bound specific RNA transcripts in RNA immunoprecipitation and sequencing assays. Deletion of all major functional isoforms of AUTS2 (full-length and C-terminal) by conditional excision of exon 15 caused breathing abnormalities and neonatal lethality when Auts2 was inactivated throughout the developing brain. Mice with limited inactivation of Auts2 in cerebral cortex survived but displayed abnormalities of cerebral cortex structure and function, including dentate gyrus hypoplasia with agenesis of hilar mossy neurons, and abnormal spiking activity on EEG. Also, RNA transcripts that normally associate with AUTS2 were dysregulated in mutant mice. Together, these findings indicate that AUTS2 regulates RNA metabolism and is essential for development of cerebral cortex, as well as subcortical breathing centers.

Reelin Mediates Hippocampal Cajal-Retzius Cell Positioning and Infrapyramidal Blade Morphogenesis.

We have previously described hypomorphic reelin (Reln) mutant mice, RelnCTRdel, in which the morphology of the dentate gyrus is distinct from that seen in reeler mice. In the RelnCTRdel mutant, the infrapyramidal blade of the dentate gyrus fails to extend, while the suprapyramidal blade forms with a relatively compact granule neuron layer. Underlying this defect, we now report several developmental anomalies in the RelnCTRdel dentate gyrus. Most strikingly, the distribution of Cajal-Retzius cells was aberrant; Cajal-Retzius neurons were increased in the suprapyramidal blade, but were greatly reduced along the subpial surface of the prospective infrapyramidal blade. We also observed multiple abnormalities of the fimbriodentate junction. Firstly, progenitor cells were distributed abnormally; the "neurogenic cluster" at the fimbriodentate junction was absent, lacking the normal accumulation of Tbr2-positive intermediate progenitors. However, the number of dividing cells in the dentate gyrus was not generally decreased. Secondly, a defect of secondary glial scaffold formation, limited to the infrapyramidal blade, was observed. The densely radiating glial fibers characteristic of the normal fimbriodentate junction were absent in mutants. These fibers might be required for migration of progenitors, which may account for the failure of neurogenic cluster formation. These findings suggest the importance of the secondary scaffold and neurogenic cluster of the fimbriodentate junction in morphogenesis of the mammalian dentate gyrus. Our study provides direct genetic evidence showing that normal RELN function is required for Cajal-Retzius cell positioning in the dentate gyrus, and for formation of the fimbriodentate junction to promote infrapyramidal blade extension.

A dual-fluorescence reporter in the Eomes locus for live imaging and medium-term lineage tracing.

The T-box transcription factor Eomes (also known as Tbr2) shows short-lived expression in various localized domains of the embryo, including epiblast cells during gastrulation and intermediate progenitor cells in the cerebral cortex. In these tissues Eomes fulfills crucial roles for lineage specification of progenitors. To directly observe Eomes-dependent cell lineages in the living embryo, we generated a novel dual-fluorescence reporter allele that expresses a membrane-bound tdTomato protein for investigation of cell morphology and a nuclear GFP for cell tracing. This allele recapitulates endogenous EOMES protein expression and is suitable for live imaging. We found that the allele can also be used as a short-to-medium-term lineage tracer, as GFP persists in cells longer than EOMES protein and marks Eomes-dependent lineages with a timeframe of days to weeks depending on the proliferation rate. In summary, we present a novel genetic tool for investigation of Eomes-dependent cell types by live imaging and lineage tracing.

Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity.

Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

Neuropathologic features of pontocerebellar hypoplasia type 6.

Pontocerebellar hypoplasia is a group of severe developmental disorders with prenatal onset affecting the growth and function of the brainstem and cerebellum. The rarity and genetic heterogeneity of this group of disorders can make molecular diagnosis challenging. We report 3 siblings who were born to nonconsanguineous parents, were hypotonic at birth, developed seizures, had repeated apneic spells, and died within 2 months of life. Neuroimaging showed that all had profound cerebellar hypoplasia and simplified cortical gyration. Genetic analysis by whole-exome sequencing demonstrated compound heterozygous mutations in the mitochondrial arginyl transfer RNA synthetase gene RARS2, indicating that the children had pontocerebellar hypoplasia type 6. Autopsies on the younger twin siblings revealed small and immature cerebella at an approximate developmental age of less than 18 weeks. The basis pontis showed regressive changes, and the medulla had marked inferior olivary hypoplasia. The brains of both twins were microencephalic and had simplified gyri; cortices were immature, and deep white matter had extensive astrocytosis. The findings suggest a near-normal embryologic period followed by midgestation developmental slowing or cessation and later regression in select anatomic regions. This is the first detailed description of neuropathologic findings associated with pontocerebellar hypoplasia type 6 and demonstrates the profound effects of RARS2 disruption during early neurodevelopment.

Neuropathology of brain and spinal malformations in a case of monosomy 1p36.

Monosomy 1p36 is the most common subtelomeric chromosomal deletion linked to mental retardation and seizures. Neuroimaging studies suggest that monosomy 1p36 is associated with brain malformations including polymicrogyria and nodular heterotopia, but the histopathology of these lesions is unknown. Here we present postmortem neuropathological findings from a 10 year-old girl with monosomy 1p36, who died of respiratory complications. The findings included micrencephaly, periventricular nodular heterotopia in occipitotemporal lobes, cortical dysgenesis resembling polymicrogyria in dorsolateral frontal lobes, hippocampal malrotation, callosal hypoplasia, superiorly rotated cerebellum with small vermis, and lumbosacral hydromyelia. The abnormal cortex exhibited "festooned" (undulating) supragranular layers, but no significant fusion of the molecular layer. Deletion mapping demonstrated single copy loss of a contiguous 1p36 terminal region encompassing many important neurodevelopmental genes, among them four HES genes implicated in regulating neural stem cell differentiation, and TP73, a monoallelically expressed gene. Our results suggest that brain and spinal malformations in monosomy 1p36 may be more extensive than previously recognized, and may depend on the parental origin of deleted genes. More broadly, our results suggest that specific genetic disorders may cause distinct forms of cortical dysgenesis.

Generation and characterization of a tamoxifen-inducible Eomes(CreER) mouse line.

Transgenic mouse lines expressing inducible forms of Cre-recombinase in a tissue-specific manner are powerful genetic tools for studying aspects of development and various processes in the adult. The T-box transcription factor eomesodermin (Eomes) plays critical roles for maintenance and differentiation of different pools of stem and progenitor cells from early embryonic stages to adulthood. These include trophoblast stem cells, epiblast cells during the generation of the primary germ layers, neurogenic intermediate progenitor cells in embryonic and adult cortical neurogenesis, and maturing natural killer and T cells. Here, we report on the generation and analysis of an Eomes(CreER) -targeted allele by placing the tamoxifen-activatable Cre-recombinase (CreER) under the control of the Eomes genomic locus. We demonstrate that CreER expression recapitulates endogenous Eomes transcription within different progenitor cell populations. Tamoxifen administration specifically labels Eomes-expressing cells and their progeny as demonstrated by crossing Eomes(CreER) animals to different Cre-inducible reporter strains. In summary, this novel Eomes(CreER) allele can be used as elegant genetic tool that allows to follow the fate of Eomes-positive cells and to genetically manipulate them in a temporal specific manner.

The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map.

The cortical area map is initially patterned by transcription factor (TF) gradients in the neocortical primordium, which define a "protomap" in the embryonic ventricular zone (VZ). However, mechanisms that propagate regional identity from VZ progenitors to cortical plate (CP) neurons are unknown. Here we show that the VZ, subventricular zone (SVZ), and CP contain distinct molecular maps of regional identity, reflecting different gene expression gradients in radial glia progenitors, intermediate progenitors, and projection neurons, respectively. The "intermediate map" in the SVZ is modulated by Eomes (also known as Tbr2), a T-box TF. Eomes inactivation caused rostrocaudal shifts in SVZ and CP gene expression, with loss of corticospinal axons and gain of corticotectal projections. These findings suggest that cortical areas and connections are shaped by sequential maps of regional identity, propagated by the Pax6 → Eomes → Tbr1 TF cascade. In humans, PAX6, EOMES, and TBR1 have been linked to intellectual disability and autism.

Dystroglycan on radial glia end feet is required for pial basement membrane integrity and columnar organization of the developing cerebral cortex.

Interactions between the embryonic pial basement membrane (PBM) and radial glia (RG) are essential for morphogenesis of the cerebral cortex because disrupted interactions cause cobblestone malformations. To elucidate the role of dystroglycan (DG) in PBM-RG interactions, we studied the expression of DG protein and Dag1 mRNA (which encodes DG protein) in developing cerebral cortex and analyzed cortical phenotypes in Dag1 CNS conditional mutant mice. In normal embryonic cortex, Dag1 mRNA was expressed in the ventricular zone, which contains RG nuclei, whereas DG protein was expressed at the cortical surface on RG end feet. Breaches of PBM continuity appeared during early neurogenesis in Dag1 mutants. Diverse cellular elements streamed through the breaches to form leptomeningeal heterotopia that were confluent with the underlying residual cortical plate and contained variably truncated RG fibers, many types of cortical neurons, and radial and intermediate progenitor cells. Nevertheless, layer-specific molecular expression seemed normal in heterotopic neurons, and axons projected to appropriate targets. Dendrites, however, were excessively tortuous and lacked radial orientation. These findings indicate that DG is required on RG end feet to maintain PBM integrity and suggest that cobblestone malformations involve disturbances of RG structure, progenitor distribution, and dendrite orientation, in addition to neuronal "overmigration."

Multiple autism-like behaviors in a novel transgenic mouse model.

Autism spectrum disorder (ASD) diagnoses are behaviorally based with no defined universal biomarkers, occur at a 1:110 ratio in the population, and predominantly affect males compared to females at approximately a 4:1 ratio. One approach to investigate and identify causes of ASD is to use organisms that display abnormal behavioral responses that model ASD-related impairments. This study describes a novel transgenic mouse, MALTT, which was generated using a forward genetics approach. It was determined that the transgene integrated within a non-coding region on the X chromosome. The MALTT line exhibited a complete repertoire of ASD-like behavioral deficits in all three domains required for an ASD diagnosis: reciprocal social interaction, communication, and repetitive or inflexible behaviors. Specifically, MALTT male mice showed deficits in social interaction and interest, abnormalities in pup and juvenile ultrasonic vocalization communications, and exhibited a repetitive stereotypy. Abnormalities were also observed in the domain of sensory function, a secondary phenotype prevalently associated with ASD. Mapping and expression studies suggested that the Fam46 gene family may be linked to the observed ASD-related behaviors. The MALTT line provides a unique genetic model for examining the underlying biological mechanisms involved in ASD-related behaviors.

Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex.

Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. Here, we report that Tbr1, a transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, our findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons.

Autism susceptibility candidate 2 (Auts2) encodes a nuclear protein expressed in developing brain regions implicated in autism neuropathology.

Autism susceptibility candidate 2 (Auts2) is a gene associated with autism and mental retardation, whose function is unknown. Expression of Auts2 mRNA and protein were studied in the developing mouse brain by in situ hybridization, immunohistochemistry, and western blotting. Auts2 mRNA was highly expressed in the developing cerebral cortex and cerebellum, regions often affected by neuropathological changes in autism, and a few other brain regions. On embryonic day (E) 12, Auts2 mRNA was expressed in the cortical preplate, where it colocalized with Tbr1, a transcription factor specific for postmitotic projection neurons. From E16 to postnatal day 21, Auts2 was expressed most abundantly in frontal cortex, hippocampus and cerebellum, including Purkinje cells and deep nuclei. High levels of Auts2 were also detected in developing dorsal thalamus, olfactory bulb, inferior colliculus and substantia nigra. Auts2 protein showed similar regional expression patterns as the mRNA. At the cellular level, Auts2 protein was localized in the nuclei of neurons and some neuronal progenitors.

Intermediate neuronal progenitors (basal progenitors) produce pyramidal-projection neurons for all layers of cerebral cortex.

The developing cerebral cortex contains apical and basal types of neurogenic progenitor cells. Here, we investigated the cellular properties and neurogenic output of basal progenitors, also called intermediate neuronal progenitors (INPs). We found that basal mitoses expressing transcription factor Tbr2 (an INP marker) were present throughout corticogenesis, from embryonic day 10.5 through birth. Postnatally, Tbr2(+) progenitors were present in the dentate gyrus, subventricular zone (SVZ), and posterior periventricle (pPV). Two morphological subtypes of INPs were distinguished in the embryonic cortex, "short radial" in the ventricular zone (VZ) and multipolar in the SVZ, probably corresponding to molecularly defined INP subtypes. Unexpectedly, many short radial INPs appeared to contact the apical (ventricular) surface and some divided there. Time-lapse video microscopy suggested that apical INP divisions produced daughter INPs. Analysis of neurogenic divisions (Tis21-green fluorescent protein [GFP](+)) indicated that INPs may produce the majority of projection neurons for preplate, deep, and superficial layers. Conversely, proliferative INP divisions (Tis21-GFP(-)) increased from early to middle corticogenesis, concomitant with SVZ growth. Our findings support the hypothesis that regulated amplification of INPs may be an important factor controlling the balance of neurogenesis among different cortical layers.

Organotypic slice culture of embryonic brain tissue.

INTRODUCTIONThis protocol describes how to dissect, assemble, and cultivate mouse embryonic (E) brain tissue from age E11.5 to E18.5 (days) for organotypic slice culture. These preparations can be used for a variety of assays and studies including coculture of different brain regions, cell migration assays, axon guidance assays, and DNA electroporation experiments. During electroporation, an electric current is applied to the surface of a specific target area of the brain slice in order to open holes in the plasma membrane and introduce a plasmid of coding DNA. The floating slice-on-membrane construct helps to preserve the structural integrity of the brain slices, while maintaining easy experimental access and optimal viability. Experiments can be monitored in living slices (e.g., with confocal imaging), and further studies can be completed using slices that have been fixed and cryosectioned at the end of the experiment. Any region of embryonic brain or spinal tissue can be used in this protocol.

Unipolar brush cells of the cerebellum are produced in the rhombic lip and migrate through developing white matter.

Unipolar brush cells (UBCs) are glutamatergic interneurons in the cerebellar cortex and dorsal cochlear nucleus. We studied the development of UBCs, using transcription factor Tbr2/Eomes as a marker for UBCs and their progenitors in embryonic and postnatal mouse cerebellum. Tbr2+ UBCs appeared to migrate out of the upper rhombic lip via two cellular streams: a dorsal pathway into developing cerebellar white matter, where the migrating cells dispersed widely before entering the internal granular layer, and a rostral pathway along the cerebellar ventricular zone toward the brainstem. Ablation of the rhombic lip in organotypic slice cultures substantially reduced the production of Tbr2+ UBCs. In coculture experiments, Tbr2+ UBCs migrated from rhombic lip explants directly into the developing white matter of adjacent cerebellar slices. The origin of Tbr2+ UBCs was confirmed by colocalization with beta-galactosidase expressed from the Math1 locus, a molecular marker of rhombic lip lineages. Moreover, the production of Tbr2+ UBCs was Math1 dependent, as Tbr2+ UBCs were severely reduced in Math1-null cerebellum. In reeler mutant mice, Tbr2+ UBCs accumulated near the rhombic lip, consistent with impaired migration through developing white matter. Our results suggest that UBCs arise from the rhombic lip and migrate via novel pathways to their final destinations in the cerebellum and dorsal cochlear nucleus. Our findings support a model of cerebellar neurogenesis, in which glutamatergic and GABAergic neurons are produced from separate progenitor pools located mainly in the rhombic lip and the cerebellar ventricular zone, respectively.

Transcription factors in glutamatergic neurogenesis: conserved programs in neocortex, cerebellum, and adult hippocampus.

Glutamatergic, pyramidal-projection neurons are produced in the embryonic cerebral cortex by a series of genetically programmed fate choices, implemented in large part by developmental transcription factors. Our work has focused on Pax6, Tbr2/Eomes, NeuroD, and Tbr1, which are expressed sequentially during the neurogenesis of pyramidal-projection neurons. Recently, we have found that the same transcription factors are expressed, in the same order, during glutamatergic neurogenesis in the adult dentate gyrus, and (with modifications) in the developing cerebellum. While the precise functional significance of this transcription factor expression sequence is unknown, its common appearance in embryonic and adult neurogenesis, and in different brain regions, suggests it is part of a conserved genetic program that specifies general properties of glutamatergic neurons in these regions. Subtypes of glutamatergic neurons (e.g., layer-specific fates in the cortex) are further determined by combinations of transcription factors, superimposed on general sequential programs. These new perspectives on neurogenesis add to the conceptual framework for strategies to engineer neural stem cells for the repair of specific brain circuits.

Development of the deep cerebellar nuclei: transcription factors and cell migration from the rhombic lip.

The deep cerebellar nuclei (DCN) are the main output centers of the cerebellum, but little is known about their development. Using transcription factors as cell type-specific markers, we found that DCN neurons in mice are produced in the rhombic lip and migrate rostrally in a subpial stream to the nuclear transitory zone (NTZ). The rhombic lip-derived cells express transcription factors Pax6, Tbr2, and Tbr1 sequentially as they enter the NTZ. A subset of rhombic lip-derived cells also express reelin, a key regulator of Purkinje cell migrations. In organotypic slice cultures, the rhombic lip was necessary and sufficient to produce cells that migrate in the subpial stream, enter the NTZ, and express Pax6, Tbr2, Tbr1, and reelin. In later stages of development, the subpial stream is replaced by the external granular layer, and the NTZ organizes into distinct DCN nuclei. Tbr1 expression persists to adulthood in a subset of medial DCN projection neurons. In reeler mutant mice, which have a severe cerebellar malformation, rhombic lip-derived cells migrated to the NTZ, despite reelin deficiency. Studies in Tbr1 mutant mice suggested that Tbr1 plays a role in DCN morphogenesis but is not required for reelin expression, glutamatergic differentiation, or the initial formation of efferent axon pathways. Our findings reveal underlying similarities in the transcriptional programs for glutamatergic neuron production in the DCN and the cerebral cortex, and they support a model of cerebellar neurogenesis in which glutamatergic and GABAergic neurons are produced from separate progenitor compartments.

Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex.

The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 --> Tbr2 --> Tbr1 in the differentiation of radial glia --> intermediate progenitor cell --> postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia --> postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.

Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons.

Cortical projection neurons exhibit diverse morphological, physiological, and molecular phenotypes, but it is unknown how many distinct types exist. Many projection cell phenotypes are associated with laminar fate (radial position), but each layer may also contain multiple types of projection cells. We have investigated two hypotheses: (1) that different projection cell types exhibit characteristic molecular expression profiles and (2) that laminar fates are determined primarily by molecular phenotype. We found that several transcription factors were differentially expressed by projection neurons, even within the same layer: Otx1 and Er81, for example, were expressed by different neurons in layer 5. Retrograde tracing showed that Er81 was expressed in corticospinal and corticocortical neurons. In contrast, Otx1 has been detected only in corticobulbar neurons [Weimann et al., Neuron 1999;24:819-831]. Birthdating demonstrated that different molecularly defined types were produced sequentially, in overlapping waves. Cells adopted laminar fates characteristic of their molecular phenotypes, regardless of cell birthday. Molecular markers also revealed the locations of different projection cell types in the malformed cortex of reeler mice. These studies suggest that molecular profiles can be used advantageously for classifying cortical projection cells, for analyzing their neurogenesis and fate specification, and for evaluating cortical malformations.

Cajal-Retzius cells in the mouse: transcription factors, neurotransmitters, and birthdays suggest a pallial origin.

Cajal-Retzius cells are reelin-secreting neurons found in the marginal zone of the mammalian cortex during development. Recently, it has been proposed that Cajal-Retzius cells may be generated both early and late in corticogenesis, and may migrate into the cortex from proliferative zones in the subpallium (lateral ganglionic eminence and medial ganglionic eminence) or cortical hem. In the present study, we used reelin as a marker to study the properties of Cajal-Retzius cells, including their likely origins, neurotransmitters, and birthdates. In double labeling experiments, Cajal-Retzius cells (reelin(+)) expressed transcription factors characteristic of pallial neurons (Tbr1 and Emx2), contained high levels of glutamate, were usually calretinin(+), and were born early in corticogenesis, on embryonic days (E)10.5 and E11.5. Tbr1(+) cells in the marginal zone were almost always reelin(+). The first Cajal-Retzius cells (Tbr1(+)/reelin(+)) appeared in the preplate on E10.5. In contrast, interneurons expressed a subpallial transcription factor (Dlx), contained high levels of GABA, were frequently calbindin(+), and were born throughout corticogenesis (from E10.5 to E16.5). Interneurons (Dlx(+)) first appeared in the cortex on E12.5. Our results suggest that the marginal zone contains two main types of neurons: Cajal-Retzius cells derived from the pallium, and migrating interneurons derived from the subpallium.