The Polycomb group protein Ring1 regulates dorsoventral patterning of the mouse telencephalon.

Dorsal-ventral patterning of the mammalian telencephalon is fundamental to the formation of distinct functional regions including the neocortex and ganglionic eminence. While Bone morphogenetic protein (BMP), Wnt, and Sonic hedgehog (Shh) signaling are known to determine regional identity along the dorsoventral axis, how the region-specific expression of these morphogens is established remains unclear. Here we show that the Polycomb group (PcG) protein Ring1 contributes to the ventralization of the mouse telencephalon. Deletion of Ring1b or both Ring1a and Ring1b in neuroepithelial cells induces ectopic expression of dorsal genes, including those for BMP and Wnt ligands, as well as attenuated expression of the gene for Shh, a key morphogen for ventralization, in the ventral telencephalon. We observe PcG protein-mediated trimethylation of histone 3 at lysine-27 and binding of Ring1B at BMP and Wnt ligand genes specifically in the ventral region. Furthermore, forced activation of BMP or Wnt signaling represses Shh expression. Our results thus indicate that PcG proteins suppress BMP and Wnt signaling in a region-specific manner and thereby allow proper Shh expression and development of the ventral telencephalon.

Telencephalic Flexure and Malformations of the Lateral Cerebral (Sylvian) Fissure.

After sagittal division of the prosencephalon at 4.5 weeks of gestation, the early fetal cerebral hemisphere bends or rotates posteroventrally from seven weeks of gestation. The posterior pole of the telencephalon thus becomes not the occipital but the temporal lobe as the telencephalic flexure forms the operculum and finally the lateral cerebral or Sylvian fissure. The ventral part is infolded to become the insula. The frontal and temporal lips of the Sylvian fissure, as well as the insula, all derive from the ventral margin of the primitive telencephalon, hence may be influenced by genetic mutations with a ventrodorsal gradient of expression. The telencephalic flexure also contributes to a shift of the hippocampus from a dorsal to a ventral position, the early rostral pole of the hippocampus becoming caudal and dorsal becoming ventral. The occipital horn is the most recent recess of the lateral ventricle, hence most vulnerable to anatomic variations that affect the calcarine fissure. Many major malformations include lack of telencephalic flexure (holoprosencephaly, extreme micrencephaly) or dysplastic Sylvian fissure (lissencephalies, hemimegalencephaly, schizencephaly). Although fissures and sulci are genetically programmed, mechanical forces of growth and volume expansion are proposed to be mainly extrinsic (including ventricles) for fissures and intrinsic for sulci. In fetal hydrocephalus, the telencephalic flexure is less affected because ventricular dilatation occurs later in gestation. Flexures can be detected prenatally by ultrasound and fetal magnetic resonance imaging and should be described neuropathologically in cerebral malformations.

Crumbs 2 prevents cortical abnormalities in mouse dorsal telencephalon.

The formation of a functionally integrated nervous system is dependent on a highly organized sequence of events that includes timely division and differentiation of progenitors. Several apical polarity proteins have been shown to play crucial roles during neurogenesis, however, the role of Crumbs 2 (CRB2) in cortical development has not previously been reported. Here, we show that conditional ablation of Crb2 in the murine dorsal telencephalon leads to defects in the maintenance of the apical complex. Furthermore, within the mutant dorsal telencephalon there is premature expression of differentiation proteins. We examined the physiological function of Crb2 on wild type genetic background as well as on background lacking Crb1. Telencephalon lacking CRB2 resulted in reduced levels of PALS1 and CRB3 from the apical complex, an increased number of mitotic cells and expanded neuronal domain. These defects are transient and therefore only result in rather mild cortical abnormalities. We show that CRB2 is required for maintenance of the apical polarity complex during development of the cortex and regulation of cell division, and that loss of CRB2 results in cortical abnormalities.

Proliferative defects and formation of a double cortex in mice lacking Mltt4 and Cdh2 in the dorsal telencephalon.

Radial glial cells (RGCs) in the ventricular neuroepithelium of the dorsal telencephalon are the progenitor cells for neocortical projection neurons and astrocytes. Here we show that the adherens junction proteins afadin and CDH2 are critical for the control of cell proliferation in the dorsal telencephalon and for the formation of its normal laminar structure. Inactivation of afadin or CDH2 in the dorsal telencephalon leads to a phenotype resembling subcortical band heterotopia, also known as "double cortex," a brain malformation in which heterotopic gray matter is interposed between zones of white matter. Adherens junctions between RGCs are disrupted in the mutants, progenitor cells are widely dispersed throughout the developing neocortex, and their proliferation is dramatically increased. Major subtypes of neocortical projection neurons are generated, but their integration into cell layers is disrupted. Our findings suggest that defects in adherens junctions components in mice massively affects progenitor cell proliferation and leads to a double cortex-like phenotype.

Exposure to benzidine caused apoptosis and malformation of telencephalon region in zebrafish.

Exposure to benzidine has been known to induce human cancers, particularly bladder carcinomas. In this study, the zebrafish model was used to investigate the developmental toxicity of benzidine. Embryos at 6 h postfertilization (hpf) that were exposed to benzidine exhibited embryonic death in a dose- and time-dependent manner. Benzidine induced malformations in zebrafish, such as small brain development, shorter axes, and a slight pericardial edema. High concentrations (50, 100, and 200 µM) of benzidine triggered widespread apoptosis in the brain and dorsal neurons, as evidenced by acridine orange and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays. Real-time polymerase chain reaction analysis also showed that benzidine treatment affected p53, bax, and noxa expression. Decreases in specific brain markers, such as emx1 in the telencephalon, ngn1 in differentiated neurons, and otx2 in the midbrain, were observed in benzidine-treated embryos at 24 hpf. Conversely, no overt changes to pax2.1 expression in the midbrain-hindbrain boundary were found. Moreover, the use of Tg(HuC:GFP) zebrafish showed that benzidine caused a malformation of the telencephalon region. Our findings show that benzidine exposure triggers widespread apoptosis in the zebrafish brain and dorsal neurons, resulting in the development of an abnormal telencephalon.

Bilateral cavitations of ganglionic eminence: a fetal MR imaging sign of halted brain development.

SUMMARY: Ganglionic eminence is the main transitory proliferative structure of the ventral telencephalon in human fetal brain and it contributes for at least 35% to the population of cortical interneurons; however data on the human GE anomalies are scarce. We report 5 fetal MR imaging observations with bilateral symmetric cavitations in their GE regions resembling an inverted open C shape and separating the GE itself form the deeper parenchyma. Imaging, neuropathology, and follow-up features suggested a malformative origin. All cases had in common characteristics of lissencephaly with agenesis or severe hypoplasia of corpus callosum of probable different genetic basis. From our preliminary observation, it seems that GE cavitations are part of conditions which are also accompanied by severe cerebral structure derangement.

PTB deficiency causes the loss of adherens junctions in the dorsal telencephalon and leads to lethal hydrocephalus.

Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre-mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.

Disrupted-in-Schizophrenia 1 (DISC1) is necessary for the correct migration of cortical interneurons.

Disrupted-in-Schizophrenia 1 (DISC1) is a prominent susceptibility gene for major psychiatric disorders. Previous work indicated that DISC1 plays an important role during neuronal proliferation and differentiation in the cerebral cortex and that it affects the positioning of radial migrating pyramidal neurons. Here we show that in mice, DISC1 is necessary for the migration of the cortical interneurons generated in the medial ganglionic eminence (MGE). RT-PCR, in situ hybridizations, and immunocytochemical data revealed expression of DISC1 transcripts and protein in MGE-derived cells. To study the possible functional role of DISC1 during tangential migration, we performed in utero and ex utero electroporation to suppress DISC1 in the MGE in vivo and in vitro. Results indicate that after DISC1 knockdown, the proportion of tangentially migrating MGE neurons that reached their cortical target was strongly reduced. In addition, there were profound alterations in the morphology of DISC1-deficient neurons, which exhibited longer and less branched leading processes than control cells. These findings provide a possible link between clinical studies reporting alterations of cortical interneurons in schizophrenic patients and the current notion of schizophrenia as a neurodevelopmental disorder.

Lrp12/Mig13a reveals changing patterns of preplate neuronal polarity during corticogenesis that are absent in reeler mutant mice.

During corticogenesis, the earliest generated neurons form the preplate, which evolves into the marginal zone and subplate. Lrp12/Mig13a, a mammalian gene related to the Caenorhabditis elegans neuroblast migration gene mig-13, is expressed in a subpopulation of preplate neurons that undergo ventrally directed tangential migrations in the preplate layer and pioneer axon projections to the anterior commissure. As the preplate separates, Lrp12/Mig13a-positive neurons polarize in the radial plane and form a pseudocolumnar pattern, prior to moving to a deeper position within the emerging subplate layer. These changes in neuronal polarity do not occur in reeler mutant mice, revealing the earliest known defect in reeler cortical patterning and suggesting that the alignment of preplate neurons into a pseudolayer facilitates the movement of later-born radially migrating neurons into the emerging cortical plate.

Growth defects in the dorsal pallium after genetically targeted ablation of principal preplate neurons and neuroblasts: a morphometric analysis.

The present study delineates the large-scale, organic responses of growth in the dorsal pallium to targeted genetic ablations of the principal PP (preplate) neurons of the neocortex. Ganciclovir treatment during prenatal development [from E11 (embryonic age 11) to E13] of mice selectively killed cells with shared S-phase vulnerability and targeted expression of a GPT [golli promoter transgene; GPT linked to HSV-TK (herpes simplex virus-thymidine kinase), τ-eGFP and lacZ reporters] localized in PP neurons and their intermediate progenitor neuroblasts. The volume, area and thickness of the pallium were measured in an E12-P4 (postnatal age 4) longitudinal study with comparisons between ablated (HSV-TK(+/0)) and control (HSV-TK(0/0)) littermates. The extent of ablations was also systematically varied, and the effect on physical growth was assessed in an E18 cross-sectional study. The morphological evidence obtained in the present study supports the conclusion that genetically targeted ablations delay the settlement of the principal PP neurons of the dorsal pallium. This leads to progressive and substantial reductions of growth, despite compensatory responses that rapidly replace the ablated cells. These growth defects originate from inductive cellular interactions in the proliferative matrix of the ventricular zone of the pallium, but are amplified by subsequent morphogenic and trophic cellular interactions. The defects persist during the course of prenatal and postnatal development to demonstrate a constrained dose-response relationship with the extent of specific killing of GPT neurons. The defects propagate simultaneously in both the horizontal and vertical cytoarchitectural dimensions of the developing pallium, an outcome that produces a localized shortfall of volume in the telencephalic vesicles.

Molecular regulation of the developing commissural plate.

Coordinated transfer of information between the brain hemispheres is essential for function and occurs via three axonal commissures in the telencephalon: the corpus callosum (CC), hippocampal commissure (HC), and anterior commissure (AC). Commissural malformations occur in over 50 human congenital syndromes causing mild to severe cognitive impairment. Disruption of multiple commissures in some syndromes suggests that common mechanisms may underpin their development. Diffusion tensor magnetic resonance imaging revealed that forebrain commissures crossed the midline in a highly specific manner within an oblique plane of tissue, referred to as the commissural plate. This specific anatomical positioning suggests that correct patterning of the commissural plate may influence forebrain commissure formation. No analysis of the molecular specification of the commissural plate has been performed in any species; therefore, we utilized specific transcription factor markers to delineate the commissural plate and identify its various subdomains. We found that the mouse commissural plate consists of four domains and tested the hypothesis that disruption of these domains might affect commissure formation. Disruption of the dorsal domains occurred in strains with commissural defects such as Emx2 and Nfia knockout mice but commissural plate patterning was normal in other acallosal strains such as Satb2(-/-). Finally, we demonstrate an essential role for the morphogen Fgf8 in establishing the commissural plate at later developmental stages. The results demonstrate that correct patterning of the commissural plate is an important mechanism in forebrain commissure formation.

Disruption of CK2beta in embryonic neural stem cells compromises proliferation and oligodendrogenesis in the mouse telencephalon.

Genetic programs that govern neural stem/progenitor cell (NSC) proliferation and differentiation are dependent on extracellular cues and a network of transcription factors, which can be regulated posttranslationally by phosphorylation. However, little is known about the kinase-dependent pathways regulating NSC maintenance and oligodendrocyte development. We used a conditional knockout approach to target the murine regulatory subunit (beta) of protein kinase casein kinase 2 (CK2beta) in embryonic neural progenitors. Loss of CK2beta leads to defects in proliferation and differentiation of embryonic NSCs. We establish CK2beta as a key positive regulator for the development of oligodendrocyte precursor cells (OPCs), both in vivo and in vitro. We show that CK2beta directly interacts with the basic helix-loop-helix (bHLH) transcription factor Olig2, a critical modulator of OPC development, and activates the CK2-dependent phosphorylation of its serine-threonine-rich (STR) domain. Finally, we reveal that the CK2-targeted STR domain is required for the oligodendroglial function of Olig2. These findings suggest that CK2 may control oligodendrogenesis, in part, by regulating the activity of the lineage-specific transcription factor Olig2. Thus, CK2beta appears to play an essential and uncompensated role in central nervous system development.

6-mercaptopurine (6-MP) induces p53-mediated apoptosis of neural progenitor cells in the developing fetal rodent brain.

6-mercaptopurine (6-MP), a DNA-damaging agent, induces apoptosis of neural progenitor cells, and causes malformation in the fetal brain. The aim of the present study is to clarify the molecular pathway of 6-MP-induced apoptosis of neural progenitor cells in the fetal telencephalon of rats and mice. p53 protein is activated by DNA damage and induces apoptosis through either the intrinsic pathway involving the mitochondria or the extrinsic pathway triggered by death receptors. In this study, the expression of puma and cleaved caspase-9 proteins, which are specific intrinsic pathway factors, increased in the rat telencephalon after 6-MP treatment. 6-MP-induced apoptosis of neural progenitor cells was completely absent in p53-deficient mice. On the other hand, the expression of Fas protein, an extrinsic pathway factor, did not change throughout the experimental period in the rat telencephalon treated with 6-MP. The number of apoptotic neural progenitor cells was similar among Fas-mutated lpr/lpr and wild-type mice, suggesting that the Fas pathway does not play a significant role in 6-MP-induced apoptosis of neural progenitor cells. These results may suggest that the p53-mediated intrinsic pathway is essential for 6-MP-induced apoptosis of neural progenitor cells in the developing telencephalon of rats and mice.

Analysis of early ventral telencephalic defects in mice lacking functional Gli3 protein.

The transcription factor Gli3 is expressed throughout developing telencephalon. Previous studies have focused on Gli3's role in dorsal telencephalon, which is greatly reduced in size in Gli3(Xt/Xt) mutants. We examined the effects of loss of Gli3 on early development of ventral telencephalon. Ventral telencephalon was defined in both wildtypes and Gli3(Xt/Xt) mutants on the basis of its expression of Olig2, Nkx2.1, Mash1, and Foxg1 and its lack of expression of Pax6. We found that at embryonic day (E)10.5 the volume of the ventral telencephalon is about 50% greater in Gli3(Xt/Xt) mutants than in wildtypes. By E12.5, however, the volume of the ventral telencephalon is about 20% lower in Gli3(Xt/Xt) mutants than in wildtypes. We observed a significant increase in the number of both apoptotic cells and newly differentiated neurons in the E10.5 Gli3(Xt/Xt) ventral telencephalon, suggesting that increased cell death and withdrawal of cells from the cell cycle might account for the failure of the Gli3(Xt/Xt) ventral telencephalon to grow normally by E12.5. We found no changes in the lengths of the cell cycles of proliferating ventral telencephalic cells at E10.5. We used marker analysis and optical projection tomography to assess the Gli3(Xt/Xt) forebrain in three dimensions and found that the Gli3(Xt/Xt) diencephalon is shifted relatively rostrally. We conclude that in the absence of Gli3 an abnormally large portion of the newly formed telencephalon is specified to a ventral fate but this then suffers impaired growth, due to defects of cell differentiation and death, contributing to severe distortion of the forebrain.

Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly.

Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, we have now determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. We demonstrate that Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain.

Altered signaling pathway in the dysmorphogenesis of telencephalon in the Gli3 depressed mouse embryo, Pdn/Pdn.

The responsible gene of genetic polydactyly/arhinencephaly mouse (Pdn/Pdn) is Gli3. Pdn/Pdn exhibits absence of the olfactory bulb, suggesting telencephalic dysmorphogenesis. It has been cleared that a transposon was inserted into intron 3 of the Gli3 gene in the Pdn mouse. Adequate PCR primers in the intron 3 and transposon allowed us to discriminate +/+, Pdn/+ and Pdn/Pdn embryos. After genotyping of the Pdn embryos using genomic DNA from the yolk sac membrane, gene expressions in the embryo proper were analyzed by DNA microarray, real-time PCR and whole-mount in situ hybridization (WISH) methods. DNA microarray detected 368 depressed and 425 over-expressed genes in the Pdn/Pdn mouse embryos on day 9 of gestation. In these genes, six signaling pathway and 20 transcription factor genes were included. From these genes, we further investigated Gli3, Emx2, Wnt8b and Wnt7b gene expressions using real-time PCR and WISH, and depression of these gene expression amounts and altered expression patterns were confirmed. Although alterations of Shh and Fgf8 gene expressions were not detected in the DNA microarray, as these genes have been closed up in the telencephalic morphogenesis, we investigated these gene expressions by real-time PCR and WISH. Shh gene expression amount and pattern were not changed. Alteration of Fgf8 gene expression amount was not detected also in the real-time PCR, but altered expression pattern was detected in the Pdn/Pdn embryos by WISH. From the present data, we suggested that Emx2, Wnt8b, Wnt7b and Fgf8 are the important Gli3 signaling pathway in the morphogenesis of telencephalon.

Brain abnormalities prior to the development of bipolar disorder: clues to aid in eventual preventative interventions

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Alternative transcripts of Dclk1 and Dclk2 and their expression in doublecortin knockout mice.

The doublecortin (DCX) gene, mutated in X-linked human lissencephaly, has 2 close paralogs, doublecortin-like kinase 1 and 2 (Dclk1 and 2). In this study we attempted to better understand the dramatic differences between human and mouse DCX/Dcx-deficient phenotypes, focusing on the Dclk genes which are likely to compensate for Dcx function in the mouse. Using sequence database screens, Northern blot analyses and in situ hybridization experiments, we characterized the developmental transcripts of Dclk1 and 2, questioning their conservation between mouse and human, and their similarity to Dcx. Like Dcx, Dcx-like transcripts of the Dclk1 gene are expressed in postmitotic neurons in the developing cortex. No changes of expression were observed at the RNA level for these transcripts in Dcx knockout mice. However, a minor change in expression at the protein level was detected. The Dclk2 gene is less well characterized than Dclk1 and we show here that it is expressed both in proliferating cells and postmitotic neurons, with a notably strong expression in the ventral telencephalon. No major differences in Dclk2 expression at the RNA and protein levels were identified comparing Dcx knockout and wild-type brains. We also analyzed Dclk1 and 2 expression in the hippocampal CA3 region which, unlike the neocortex, is abnormal in Dcx knockout mice. Interestingly, each transcript was expressed in CA3 neurons, including in the heterotopic pyramidal layer of Dcx knockout animals, but is presumably not able to compensate for a lack of Dcx. These results, in addition to characterizing the transcript diversity of an important family of genes, should facilitate further studies of compensation in Dcx-deficient mice.