Increased Anxiety-Related Behavior, Impaired Cognitive Function and Cellular Alterations in the Brain of Cend1-deficient Mice

Cend1 is a neuronal-lineage specific modulator involved in coordination of cell cycle exit and differentiation of neuronal precursors. We have previously shown that Cend1−/− mice show altered cerebellar layering caused by increased proliferation of granule cell precursors, delayed radial granule cell migration and compromised Purkinje cell differentiation, leading to ataxic gait and deficits in motor coordination. To further characterize the effects of Cend1 genetic ablation we determined herein a range of behaviors, including anxiety and exploratory behavior in the elevated plus maze (EPM), associative learning in fear conditioning, and spatial learning and memory in the Morris water maze (MWM). We observed significant deficits in all tests, suggesting structural and/or functional alterations in brain regions such as the cortex, amygdala and the hippocampus. In agreement with these findings, immunohistochemistry revealed reduced numbers of γ amino butyric acid (GABA) GABAergic interneurons, but not of glutamatergic projection neurons, in the adult cerebral cortex. Reduced GABAergic interneurons were also observed in the amygdala, most notably in the basolateral nucleus. The paucity in GABAergic interneurons in adult Cend1−/− mice correlated with increased proliferation and apoptosis as well as reduced migration of neuronal progenitors from the embryonic medial ganglionic eminence (MGE), the origin of these cells. Further we noted reduced GABAergic neurons and aberrant neurogenesis in the adult dentate gyrus (DG) of the hippocampus, which has been previously shown to confer spatial learning and memory deficits. Our data highlight the necessity of Cend1 expression in the formation of a structurally and functionally normal brain phenotype

Bmp7 Regulates the Survival, Proliferation, and Neurogenic Properties of Neural Progenitor Cells during Corticogenesis in the Mouse

Bone morphogenetic proteins (BMPs) are considered important regulators of neural development. However, results mainly from a wide set of in vitro gain-of-function experiments are conflicting since these show that BMPs can act either as inhibitors or promoters of neurogenesis. Here, we report a specific and non-redundant role for BMP7 in cortical neurogenesis in vivo using knockout mice. Bmp7 is produced in regions adjacent to the developing cortex; the hem, meninges, and choroid plexus, and can be detected in the cerebrospinal fluid. Bmp7 deletion results in reduced cortical thickening, impaired neurogenesis, and loss of radial glia attachment to the meninges. Subsequent in vitro analyses of E14.5 cortical cells revealed that lack of Bmp7 affects neural progenitor cells, evidenced by their reduced proliferation, survival and self-renewal capacity. Addition of BMP7 was able to rescue these proliferation and survival defects. In addition, at the developmental stage E14.5 Bmp7 was also required to maintain Ngn2 expression in the subventricular zone. These data demonstrate a novel role for Bmp7 in the embryonic mouse cortex: Bmp7 nurtures radial glia cells and regulates fundamental properties of neural progenitor cells that subsequently affect Ngn2-dependent neurogenesis

Integrin signalling regulates the expansion of neuroepithelial progenitors and neurogenesis via Wnt7a and Decorin

Development of the cerebral cortex requires regulation of proliferation and differentiation of neural stem cells and a diverse range of progenitors. Recent work suggests a role for extracellular matrix (ECM) and the major family of ECM receptors, the integrins. Here we show that enhancing integrin beta-1 signalling, by expressing a constitutively active integrin beta-1 (CA*β1) in the embryonic chick mesencephalon, enhances neurogenesis and increases the number of mitotic cells dividing away from the ventricular surface, analogous to sub-apical progenitors in mouse. Only non-integrin-expressing neighbouring cells (lacking CA*β1) contributed to the increased neurogenesis. Transcriptome analysis reveals upregulation of Wnt7a within the CA*β1 cells and upregulation of the ECM protein Decorin in the neighbouring non-expressing cells. Experiments using inhibitors in explant models and genetic knock-downs in vivo reveal an integrin-Wnt7a-Decorin pathway that promotes proliferation and differentiation of neuroepithelial cells, and identify Decorin as a novel neurogenic factor in the central nervous system

Increased anxiety-related behavior, impaired cognitive function and cellular alterations in the brain of cend1-deficient mice

Cend1 is a neuronal-lineage specific modulator involved in coordination of cell cycle exit and differentiation of neuronal precursors. We have previously shown that Cend1−/− mice show altered cerebellar layering caused by increased proliferation of granule cell precursors, delayed radial granule cell migration and compromised Purkinje cell differentiation, leading to ataxic gait and deficits in motor coordination. To further characterize the effects of Cend1 genetic ablation we determined herein a range of behaviors, including anxiety and exploratory behavior in the elevated plus maze (EPM), associative learning in fear conditioning, and spatial learning and memory in the Morris water maze (MWM). We observed significant deficits in all tests, suggesting structural and/or functional alterations in brain regions such as the cortex, amygdala and the hippocampus. In agreement with these findings, immunohistochemistry revealed reduced numbers of γ amino butyric acid (GABA) GABAergic interneurons, but not of glutamatergic projection neurons, in the adult cerebral cortex. Reduced GABAergic interneurons were also observed in the amygdala, most notably in the basolateral nucleus. The paucity in GABAergic interneurons in adult Cend1−/− mice correlated with increased proliferation and apoptosis as well as reduced migration of neuronal progenitors from the embryonic medial ganglionic eminence (MGE), the origin of these cells. Further we noted reduced GABAergic neurons and aberrant neurogenesis in the adult dentate gyrus (DG) of the hippocampus, which has been previously shown to confer spatial learning and memory deficits. Our data highlight the necessity of Cend1 expression in the formation of a structurally and functionally normal brain phenotype. © 2019 Segklia, Stamatakis, Stylianopoulou, Lavdas and Matsas

Image_1_Increased Anxiety-Related Behavior, Impaired Cognitive Function and Cellular Alterations in the Brain of Cend1-deficient Mice.TIF

Cend1 is a neuronal-lineage specific modulator involved in coordination of cell cycle exit and differentiation of neuronal precursors. We have previously shown that Cend1−/− mice show altered cerebellar layering caused by increased proliferation of granule cell precursors, delayed radial granule cell migration and compromised Purkinje cell differentiation, leading to ataxic gait and deficits in motor coordination. To further characterize the effects of Cend1 genetic ablation we determined herein a range of behaviors, including anxiety and exploratory behavior in the elevated plus maze (EPM), associative learning in fear conditioning, and spatial learning and memory in the Morris water maze (MWM). We observed significant deficits in all tests, suggesting structural and/or functional alterations in brain regions such as the cortex, amygdala and the hippocampus. In agreement with these findings, immunohistochemistry revealed reduced numbers of γ amino butyric acid (GABA) GABAergic interneurons, but not of glutamatergic projection neurons, in the adult cerebral cortex. Reduced GABAergic interneurons were also observed in the amygdala, most notably in the basolateral nucleus. The paucity in GABAergic interneurons in adult Cend1−/− mice correlated with increased proliferation and apoptosis as well as reduced migration of neuronal progenitors from the embryonic medial ganglionic eminence (MGE), the origin of these cells. Further we noted reduced GABAergic neurons and aberrant neurogenesis in the adult dentate gyrus (DG) of the hippocampus, which has been previously shown to confer spatial learning and memory deficits. Our data highlight the necessity of Cend1 expression in the formation of a structurally and functionally normal brain phenotype.</p

Image_2_Increased Anxiety-Related Behavior, Impaired Cognitive Function and Cellular Alterations in the Brain of Cend1-deficient Mice.TIF

Cend1 is a neuronal-lineage specific modulator involved in coordination of cell cycle exit and differentiation of neuronal precursors. We have previously shown that Cend1−/− mice show altered cerebellar layering caused by increased proliferation of granule cell precursors, delayed radial granule cell migration and compromised Purkinje cell differentiation, leading to ataxic gait and deficits in motor coordination. To further characterize the effects of Cend1 genetic ablation we determined herein a range of behaviors, including anxiety and exploratory behavior in the elevated plus maze (EPM), associative learning in fear conditioning, and spatial learning and memory in the Morris water maze (MWM). We observed significant deficits in all tests, suggesting structural and/or functional alterations in brain regions such as the cortex, amygdala and the hippocampus. In agreement with these findings, immunohistochemistry revealed reduced numbers of γ amino butyric acid (GABA) GABAergic interneurons, but not of glutamatergic projection neurons, in the adult cerebral cortex. Reduced GABAergic interneurons were also observed in the amygdala, most notably in the basolateral nucleus. The paucity in GABAergic interneurons in adult Cend1−/− mice correlated with increased proliferation and apoptosis as well as reduced migration of neuronal progenitors from the embryonic medial ganglionic eminence (MGE), the origin of these cells. Further we noted reduced GABAergic neurons and aberrant neurogenesis in the adult dentate gyrus (DG) of the hippocampus, which has been previously shown to confer spatial learning and memory deficits. Our data highlight the necessity of Cend1 expression in the formation of a structurally and functionally normal brain phenotype.</p

Generation and functional characterization of mice with a conditional BMP7 allele

Bone Morphogenetic Proteins (BMPs) play multiple and important roles in embryonic development as well as in homeostasis and tissue repair in the adult. Bmp7 has been implicated in developmental disorders and in a variety of diseases, but functional studies to elucidate its role so far have been hampered, since mice deficient in BMP7 die around or just after birth. To facilitate such studies, we generated mice in which the Bmp7 gene has been rendered conditional-null by flanking its first coding exon with loxP sites. To this end, we adapted the two-loxP site strategy to Bacterial Homologous Recombination to create a Bacterial Artificial Chromosome-based vector for direct targeting in mouse embryonic stem cells. Functional analysis showed that in vivo, the conditional-null Bmp7(flx/flx) mice are phenotypically wild type, whereas post Cre-mediated recombination, the resulting Bmp7(delta/delta) mice are phenotypically null. Thus, this study validates the usefulness of the Bmp7(flx/flx) mouse which in turn should empower in vivo studies aimed at elucidating the roles of Bmp7 in postnatal development, homeostasis and disease

Normal layering of the E14.5 neural cortex in <i>Bmp7</i>-deficient mouse embryos.

<p>Comparison of wild-type (wt, panels A, C, E, G, I) and <i>Bmp7</i>^Δ/Δ (panels B, D, F, H, J) cortical sections for expression of <i>Reelin</i> (A, B), presence of Map2 protein (C, D), Svet1 mRNA (E, F), and Tbr2 protein (G, H), and Pax6 mRNA (I, J), demonstrating their respective correct location even in the absence of <i>Bmp7</i>. The cortical plate (CP) may appear somewhat reduced in the Map2-stained sections (C, D).</p

<i>Bmp7</i> null embryos suffer from microencephaly.

<p>(A) <i>Bmp7</i> expression as monitored by lacZ-reporting in the mouse E14.5 cortex. Strong expression is seen in the medial regions, such as hem (H) and choroid plexus (CPl), the pial membrane (Pi) and meninges (Me). No expression is apparent in the ventricular (VZ), the subventricular (SVZ), and intermediate (IZ) zones, and the cortical plate (CP) (inlay). (B) Bmp7 protein (lower panels; the staining with DAPI is shown in the upper panels) is observed in the marginal zone (M), the CP and VZ of wild-type embryos (E14.5) and is lost in the <i>Bmp7</i>-deficient mouse embryo. (C) <i>Bmp7</i>-deletion results in microencephaly with a thinner cortex and a less clearly defined cortical plate, most prominent in medial (A, A′) and lesser in more lateral regions. (D) Other Bmp proteins present in the developing neocortex are not significantly altered following <i>Bmp7</i> deletion. The anti-Bmp6 antibody used here appears to cross-react with Bmp7, resulting in a second upper band, which is lost upon <i>Bmp7</i>^Δ/Δ deletion.</p

BMP7 controls radial glia survival.

<p>(A) Absence of Bmp7 affects RG and neural progenitor cells. Expression of Nestin (i, ii), RC2 (iii, iv), Sox2 (v, vi), Pax6 (vii, viii) in wt or <i>Bmp7</i>^Δ/Δ E14.5 cortices. The <i>Bmp7</i>^Δ/Δ cortex appears less organized (i–iv). The RGC make poor contact to the meninges (iii, iv). Sox2 and Pax6 expression are lost or diminished in the VZ/SVZ. (B) Counts of cell spreads of E14.5 cortices reveal a reduction of NeuN and Pax6-positive cells in <i>Bmp7</i>^Δ/Δ cortical cells (black bar) when compared to wt littermate cells (open bar). (C, D) The number of PCNA-positive cells (C) and cells having incoporated BrdU (D) are reduced in <i>Bmp7</i>^Δ/Δ cortical cells (black bar) when compared to wt littermate cells (open bar). (E) <i>Bmp7</i>^Δ/Δ cortical cells (black bar) showed increased numbers of Caspase-3-positive cells when compared to wt littermate cells, which was corrected following treatment with rBMP7. (F–H) Neurospheres derived from wt, <i>Bmp7^wt/</i>^Δ heterozygote, or <i>Bmp7</i>^Δ/Δ cortical cells (F) are fewer (G) and smaller (H) indicating that Bmp7 affects the survival and self-renewal properties of neural progenitor cells in the developing cortex. *p < 0.05 by Student's t test.</p