Chromatin organizer CTCF in brain development and behaviour

Chromatin architecture is an important regulator of gene expression, which dictates development. Mutations in one copy of the CTCF chromatin organizer gene cause intellectual disability and autism. Polymorphisms in CTCF have also been associated with increased risk for schizophrenia, a condition that overlaps in biological etiology with autism and intellectual disability. In this thesis, we sought to understand the role of CTCF in neurodevelopment using brain-specific conditional knockout and heterozygote mouse models. Using the Ctcf-null animals, we identify a cell-autonomous role for CTCF in regulating cortical interneuron development in the medial ganglionic eminence (MGE) through the transcriptional control of Lhx6. In the absence of CTCF, MGE-derived cortical interneuron subtypes are inappropriately specified such that their cortical laminar position is altered and there is a reduction in the number of cells expressing PV and SST. These features are rescued with viral-mediated re-expression of Lhx6. In addition, there is a concomitant increase in the expression of Lhx8, which specifies ventral telencephalic cell types in the MGE, indicating CTCF is an important regulator of cell fate choice in the MGE. To model the human condition associated with CTCF mutation, we generated mice heterozygous for Ctcf deletion in the developing brain (CtcfNestinHet). These mice had spontaneous hyperactivity and impaired spatial learning on behavioural testing. In addition to these behaviours, male mice had decreased sociability, altered aggression, and decreased anxiety. Together, this constellation of behaviours is reminiscent of other mouse models of schizophrenia, autism and intellectual disability. In addition, structural MRI revealed that CtcfNestinHet mouse brains had decreased white matter volume, suggestive of hypoconnectivity, a feature commonly attributed to the pathophysiology of autism. There were also significant volume decreases in the cerebellar nuclei, and an increase in the anterior cerebellar lobe. These findings provide further evidence for the emerging role of the cerebellum in cognition and in neurodevelopmental disorders. In summary, this work addresses the consequence of reduced CTCF expression in the developing brain at cellular, structural and behaviour levels, and thus significantly furthers our understanding of chromatin architecture regulation in neurodevelopmental disease

Dual effect of CTCF loss on neuroprogenitor differentiation and survival

An increasing number of proteins involved in genome organization have been implicated in neurodevelopmental disorders, highlighting the importance of chromatin architecture in the developing CNS. The CCCTC-binding factor (CTCF) is a zinc finger DNA binding protein involved in higher-order chromatin organization, and mutations in the human CTCF gene cause an intellectual disability syndrome associated with microcephaly. However, information on CTCF function in vivo in the developing brain is lacking. To address this gap, we conditionally inactivated the Ctcf gene at early stages of mouse brain development. Cre-mediated Ctcf deletion in the telencephalon and anterior retina at embryonic day 8.5 triggered upregulation of the p53 effector PUMA (p53 upregulated modulator of apoptosis), resulting in massive apoptosis and profound ablation of telencephalic structures. Inactivation of Ctcf several days later at E11 also resulted in PUMA upregulation and increased apoptotic cell death, and the Ctcf-null forebrain was hypocellular and disorganized at birth. Although deletion of both Ctcf and Puma in the embryonic brain efficiently rescued Ctcf-null progenitor cell apoptosis, it failed to improve neonatal hypocellularity due to decreased proliferative capacity of rescued apical and outer radial glia progenitor cells. This was exacerbated by an independent effect of CTCF loss that resulted in depletion of the progenitor pool due to premature neurogenesis earlier in development. Our findings demonstrate that CTCF activities are required for two distinct events in early cortex formation: first, to correctly regulate the balance between neuroprogenitor cell proliferation and differentiation, and second, for the survival of neuroprogenitor cells, providing new clues regarding the contributions of CTCF in microcephaly/intellectual disability syndrome pathologies. © 2014 the authors

Dual effect of CTCF loss on neuroprogenitor differentiation and survival

An increasing number of proteins involved in genome organization have been implicated in neurodevelopmental disorders, highlighting the importance of chromatin architecture in the developing CNS. The CCCTC-binding factor (CTCF) is a zinc finger DNA binding protein involved in higher-order chromatin organization, and mutations in the human CTCF gene cause an intellectual disability syndrome associated with microcephaly. However, information on CTCF function in vivo in the developing brain is lacking. To address this gap, we conditionally inactivated the Ctcf gene at early stages of mouse brain development. Cre-mediated Ctcf deletion in the telencephalon and anterior retina at embryonic day 8.5 triggered upregulation of the p53 effector PUMA (p53 upregulated modulator of apoptosis), resulting in massive apoptosis and profound ablation of telencephalic structures. Inactivation of Ctcf several days later at E11 also resulted in PUMA upregulation and increased apoptotic cell death, and the Ctcf-null forebrain was hypocellular and disorganized at birth. Although deletion of both Ctcf and Puma in the embryonic brain efficiently rescued Ctcf-null progenitor cell apoptosis, it failed to improve neonatal hypocellularity due to decreased proliferative capacity of rescued apical and outer radial glia progenitor cells. This was exacerbated by an independent effect of CTCF loss that resulted in depletion of the progenitor pool due to premature neurogenesis earlier in development. Our findings demonstrate that CTCF activities are required for two distinct events in early cortex formation: first, to correctly regulate the balance between neuroprogenitor cell proliferation and differentiation, and second, for the survival of neuroprogenitor cells, providing new clues regarding the contributions of CTCF in microcephaly/intellectual disability syndrome pathologies

CTCF Governs the Identity and Migration of MGE-Derived Cortical Interneurons.

The CCCTC-binding factor (CTCF) is a central regulator of chromatin topology recently linked to neurodevelopmental disorders such as intellectual disability, autism, and schizophrenia. The aim of this study was to identify novel roles of CTCF in the developing mouse brain. We provide evidence that CTCF is required for the expression of the LIM homeodomain factor LHX6 involved in fate determination of cortical interneurons (CINs) that originate in the medial ganglionic eminence (MGE). Conditiona

Implantation failure in female Kiss1-/- mice is independent of their hypogonadic state and can be partially rescued by leukemia inhibitory factor.

The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility, a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1(-/-) female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating, and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1(+/-) embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies the implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo, and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, leukemia inhibitory factor (Lif), a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1(-/-) uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1(-/-) female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif levels, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans

Clinical presentation of calmodulin mutations: the International Calmodulinopathy Registry

AIMS: Calmodulinopathy due to mutations in any of the three CALM genes (CALM1-3) causes life-threatening arrhythmia syndromes, especially in young individuals. The International Calmodulinopathy Registry (ICalmR) aims to define and link the increasing complexity of the clinical presentation to the underlying molecular mechanisms. METHODS AND RESULTS: The ICalmR is an international, collaborative, observational study, assembling and analysing clinical and genetic data on CALM-positive patients. The ICalmR has enrolled 140 subjects (median age 10.8 years [interquartile range 5-19]), 97 index cases and 43 family members. CALM-LQTS and CALM-CPVT are the prevalent phenotypes. Primary neurological manifestations, unrelated to post-anoxic sequelae, manifested in 20 patients. Calmodulinopathy remains associated with a high arrhythmic event rate (symptomatic patients, n = 103, 74%). However, compared with the original 2019 cohort, there was a reduced frequency and severity of all cardiac events (61% vs. 85%; P = .001) and sudden death (9% vs. 27%; P = .008). Data on therapy do not allow definitive recommendations. Cardiac structural abnormalities, either cardiomyopathy or congenital heart defects, are present in 30% of patients, mainly CALM-LQTS, and lethal cases of heart failure have occurred. The number of familial cases and of families with strikingly different phenotypes is increasing. CONCLUSION: Calmodulinopathy has pleiotropic presentations, from channelopathy to syndromic forms. Clinical severity ranges from the early onset of life-threatening arrhythmias to the absence of symptoms, and the percentage of milder and familial forms is increasing. There are no hard data to guide therapy, and current management includes pharmacological and surgical antiadrenergic interventions with sodium channel blockers often accompanied by an implantable cardioverter-defibrillator

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Identification of Genes and Putative Regulatory Variants Contributing to Reading Disabilities and Attention Deficit/ Hyperactivity Disorder

Reading Disabilities (RD) and Attention Deficit/Hyperactivity Disorder (ADHD) are common neurodevelopmental disorders with evidence for shared genetic etiology. This study examined four predicted neuronal migration genes in RD (DCDC2, KIAA0319, DCDC2B and NEDD4L), and three of these genes in ADHD (DCDC2, DCDC2B and NEDD4L). Putative regulatory elements of DCDC2 (6p) and KIAA0319 (6p) were screened to identify functional risk variants that explain previous association findings. No statistically significant associations were observed in DCDC2. In KIAA0319, variants with predicted regulatory function showed association with RD. This supports the hypothesis that causal RD risk variants in KIAA0319 alter gene expression. DCDC2B (1p), the homolog of DCDC2, showed suggestive evidence for association to ADHD, but not RD. Previous association findings in NEDD4L (18q) could not be replicated. No pleiotropic gene for RD and ADHD was identified. However, together with previous findings, this study supports that neuronal migration may be a common underlying deficit in both RD and ADHD.MAS