Regulation of embryonic and adult neurogenesis by Ars2.

Neural development is controlled at multiple levels to orchestrate appropriate choices of cell fate and differentiation. Although more attention has been paid to the roles of neural-restricted factors, broadly expressed factors can have compelling impacts on tissue-specific development. Here, we describe in vivo conditional knockout analyses of murine Ars2, which has mostly been studied as a general RNA-processing factor in yeast and cultured cells. Ars2 protein expression is regulated during neural lineage progression, and is required for embryonic neural stem cell (NSC) proliferation. In addition, Ars2 null NSCs can still transition into post-mitotic neurons, but fail to undergo terminal differentiation. Similarly, adult-specific deletion of Ars2 compromises hippocampal neurogenesis and results in specific behavioral defects. To broaden evidence for Ars2 as a chromatin regulator in neural development, we generated Ars2 ChIP-seq data. Notably, Ars2 preferentially occupies DNA enhancers in NSCs, where it colocalizes broadly with NSC regulator SOX2. Ars2 association with chromatin is markedly reduced following NSC differentiation. Altogether, Ars2 is an essential neural regulator that interacts dynamically with DNA and controls neural lineage development.

A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice.

Pediatric hydrocephalus is characterized by an abnormal accumulation of cerebrospinal fluid (CSF) and is one of the most common congenital brain abnormalities. However, little is known about the molecular and cellular mechanisms regulating CSF flow in the developing brain. Through whole-genome sequencing analysis, we report that a homozygous splice site mutation in coiled-coil domain containing 39 (Ccdc39) is responsible for early postnatal hydrocephalus in the progressive hydrocephalus (prh) mouse mutant. Ccdc39 is selectively expressed in embryonic choroid plexus and ependymal cells on the medial wall of the forebrain ventricle, and the protein is localized to the axoneme of motile cilia. The Ccdc39prh/prh ependymal cells develop shorter cilia with disorganized microtubules lacking the axonemal inner arm dynein. Using high-speed video microscopy, we show that an orchestrated ependymal ciliary beating pattern controls unidirectional CSF flow on the ventricular surface, which generates bulk CSF flow in the developing brain. Collectively, our data provide the first evidence for involvement of Ccdc39 in hydrocephalus and suggest that the proper development of medial wall ependymal cilia is crucial for normal mouse brain development.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Cilia in hereditary cerebral anomalies.

Ciliopathies are complex genetic multi-system disorders causally related to abnormal assembly or function of motile or non-motile cilia. While most human cells possess a non-motile sensory/primary cilium (PC) during development and/or in adult tissues, motile cilia are restricted to specialised cells. As a result, PC-associated ciliopathies are characterised by high phenotypic variability with extensive clinical and genetic overlaps. In the present review, we have focused on cerebral developmental anomalies, which are commonly found in PC-associated ciliopathies and which have mostly been linked to Hedgehog signalling defects. In addition, we have reviewed emerging evidence that PC dysfunctions could be directly or indirectly involved in the mechanisms underlying malformations of cerebral cortical development including primary microcephaly.

Evaluation of Hemodynamic Changes in Fetuses With Isolated Mild-to-Moderate Ventriculomegaly by Transabdominal Ultrasound.

OBJECTIVES: To investigate fetal hemodynamic alterations using transabdominal ultrasound in fetuses with isolated mild-to-moderate ventriculomegaly (VM). METHODS: Fetuses diagnosed with isolated mild-to-moderate VM by transabdominal ultrasound were evaluated for hemodynamic changes, including changes in fetal cardiac function, the umbilical artery, the ductus venosus, and the middle cerebral artery. The fetuses with isolated mild-to-moderate VM were divided into 2 groups, namely, before 32 weeks' gestation (20 weeks-31 weeks 6 days) and after 32 weeks' gestation (32-38 weeks), and matched to corresponding healthy control fetuses. RESULTS: The 53 fetuses with VM before 32 weeks had a longer mean isovolumetric relaxation time (IRT; mean ± SD, 42.9 ± 6.8 versus 40.4 ± 5.0 milliseconds; P < .05) and an apparently higher modified myocardial performance index 0.46 ± 0.06 versus 0.43 ± 0.05; P < .01) than the healthy control fetuses. The 43 fetuses with VM after 32 weeks had a significantly longer mean IRT (45.5 ± 6.7 versus 40.9 ± 7.2 milliseconds; P < .01) and a lower UA pulsatility index (0.81 ± 0.13 versus 0.89 ± 0.11; P < .01). The optimal cutoff levels for the IRT in the prediction of adverse perinatal outcomes were 40 and 43 milliseconds before and after 32 weeks, respectively (sensitivity, 100% versus 100%; specificity, 40.4% versus 50.0%; area under the curve, 0.601 versus 0.748; 95% confidence interval, 0.457-0.733 versus 0.590-0.869; P = .291 versus .005). CONCLUSIONS: Some fetuses with isolated mild-to-moderate VM may have impaired cardiac function, characterized by a higher modified myocardial performance index or longer IRT. This finding might be useful for improving fetal surveillance.

Natural History of Ventriculomegaly in Fetal Agenesis of the Corpus Callosum.

OBJECTIVES: To assess the natural evolution of the size of the fetal lateral ventricles throughout pregnancy in fetuses with callosal anomalies. METHODS: Cases of fetal callosal anomalies were retrospectively classified as isolated or complex based on the presence of other structural or genetic anomalies. Longitudinal ultrasound studies were reviewed, and postnatal outcomes were retrieved for isolated cases. RESULTS: In 135 fetuses, those who first presented after 24 weeks' gestation were more likely to have ventriculomegaly (n = 58 of 68 [85%]) than those who presented before 24 weeks (n = 39 of 67 [58%]; P < .001). In 79 cases that had longitudinal follow-up, the mean increase in ventricular width was 0.6 mm/wk, without a significant difference between isolated and complex cases (mean ± SD, 0.6 ± 1.5 versus 0.6 ± 1.1 mm; P = .45). CONCLUSIONS: Callosal anomalies are associated with progressive ventriculomegaly on prenatal ultrasound imaging, without a difference between isolated and complex anomalies. This feature should be considered part of the disease spectrum. The consequence of progressive ventriculomegaly on the long-term neurodevelopmental outcome is still unknown, and further studies should be aimed at obtaining long-term follow-up of these cases.

Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells.

Mucociliary clearance and fluid transport along epithelial surfaces are carried out by multiciliated cells (MCCs). Recently, human mutations in Cyclin O (CCNO) were linked to severe airway disease. Here, we show that Ccno expression is restricted to MCCs and the genetic deletion of Ccno in mouse leads to reduced numbers of multiple motile cilia and characteristic phenotypes of MCC dysfunction including severe hydrocephalus and mucociliary clearance deficits. Reduced cilia numbers are caused by compromised generation of centrioles at deuterosomes, which serve as major amplification platform for centrioles in MCCs. Ccno-deficient MCCs fail to sufficiently generate deuterosomes, and only reduced numbers of fully functional centrioles that undergo maturation to ciliary basal bodies are formed. Collectively, this study implicates CCNO as first known regulator of deuterosome formation and function for the amplification of centrioles in MCCs.

Functional antagonism of voltage-gated K+ channel α-subunits in the developing brain ventricular system.

The brain ventricular system is essential for neurogenesis and brain homeostasis. Its neuroepithelial lining effects these functions, but the underlying molecular pathways remain to be understood. We found that the potassium channels expressed in neuroepithelial cells determine the formation of the ventricular system. The phenotype of a novel zebrafish mutant characterized by denudation of neuroepithelial lining of the ventricular system and hydrocephalus is mechanistically linked to Kcng4b, a homologue of the 'silent' voltage-gated potassium channel α-subunit Kv6.4. We demonstrated that Kcng4b modulates proliferation of cells lining the ventricular system and maintains their integrity. The gain of Kcng4b function reduces the size of brain ventricles. Electrophysiological studies suggest that Kcng4b mediates its effects via an antagonistic interaction with Kcnb1, the homologue of the electrically active delayed rectifier potassium channel subunit Kv2.1. Mutation of kcnb1 reduces the size of the ventricular system and its gain of function causes hydrocephalus, which is opposite to the function of Kcng4b. This demonstrates the dynamic interplay between potassium channel subunits in the neuroepithelium as a novel and crucial regulator of ventricular development in the vertebrate brain.

Yap and Taz play a crucial role in neural crest-derived craniofacial development.

The role of the Hippo signaling pathway in cranial neural crest (CNC) development is poorly understood. We used the Wnt1(Cre) and Wnt1(Cre2SOR) drivers to conditionally ablate both Yap and Taz in the CNC of mice. When using either Cre driver, Yap and Taz deficiency in the CNC resulted in enlarged, hemorrhaging branchial arch blood vessels and hydrocephalus. However, Wnt1(Cre2SOR) mutants had an open cranial neural tube phenotype that was not evident in Wnt1(Cre) mutants. In O9-1 CNC cells, the loss of Yap impaired smooth muscle cell differentiation. RNA-sequencing data indicated that Yap and Taz regulate genes encoding Fox transcription factors, specifically Foxc1. Proliferation was reduced in the branchial arch mesenchyme of Yap and Taz CNC conditional knockout (CKO) embryos. Moreover, Yap and Taz CKO embryos had cerebellar aplasia similar to Dandy-Walker spectrum malformations observed in human patients and mouse embryos with mutations in Foxc1. In embryos and O9-1 cells deficient for Yap and Taz, Foxc1 expression was significantly reduced. Analysis of Foxc1 regulatory regions revealed a conserved recognition element for the Yap and Taz DNA binding co-factor Tead. ChIP-PCR experiments supported the conclusion that Foxc1 is directly regulated by the Yap-Tead complex. Our findings uncover important roles for Yap and Taz in CNC diversification and development.

The third ventricle of the human fetal brain: Normative data and pathologic correlation. A 3D transvaginal neurosonography study.

OBJECTIVE: The objective of the study are to describe (a) the technical aspects and (b) the anatomical boundaries of the fetal third ventricle (3V) on the midsagittal sonographic view and to assess (c) different biometric parameters in normal and abnormal fetuses and (d) and their reproducibility. METHODS: This study included 67 normal and 50 CNS anomalies fetuses which include (1) obstructive severe ventriculomegaly (SVM; atrial width ≥ 15 mm), (2) moderate ventriculomegaly (10-14.9 mm), and (3) corpus callosum agenesis (ACC). All underwent transvaginal 3D neurosonography of the midsagittal view of the 3V. The following parameters were measured: area, perimeter, craniocaudal and anteroposterior (AP) diameters, interthalamic adhesion diameter (ITAD), wedge angle, and the ratio between the last 2 variables (ITAD/WA). Repeatability was also assessed. RESULTS: The ITAD and the ITAD/WA are significantly different between normal fetuses and the SVM (P ≤ .001). Interthalamic adhesion diameter of ≤7.1 mm is able to identify SVM with 98.6% accuracy (CI: 0.92-0.99). In ACC cases, the AP diameter is significantly shorter than both normal fetuses and ventriculomegaly. Intraobserver/interobserver reliability was good for most variables. CONCLUSIONS: Transvaginal neurosonography enables visualization of the normal and abnormal fetal third ventricle. An ITAD <7.1 identifies aqueductal stenosis as the likely etiology of severe ventriculomegaly with an accuracy of 98.6%.

Use of high-frequency ultrasound to study the prenatal development of cranial neural tube defects and hydrocephalus in Gldc-deficient mice.

OBJECTIVE: We used non-invasive high-frequency ultrasound (HFUS) imaging to investigate embryonic brain development in a mouse model for neural tube defects (NTDs) and non-ketotic hyperglycinemia (NKH). METHOD: Using HFUS, we imaged embryos carrying loss of function alleles of Gldc encoding glycine decarboxylase, a component of the glycine cleavage system in mitochondrial folate metabolism, which is known to be associated with cranial NTDs and NKH in humans. We serially examined the same litter during the second half of embryonic development and quantified cerebral structures. Genotype was confirmed using PCR. Histology was used to confirm ultrasound findings. RESULTS: High-frequency ultrasound allowed in utero detection of two major brain abnormalities in Gldc-deficient mouse embryos, cranial NTDs (exencephaly) and ventriculomegaly (corresponding with the previous finding of post-natal hydrocephalus). Serial ultrasound allowed individual embryos to be analysed at successive gestational time points. From embryonic day 16.5 to 18.5, the lateral ventricle volume reduced in wild-type and heterozygous embryos but increased in homozygous Gldc-deficient embryos. CONCLUSION: Exencephaly and ventriculomegaly were detectable by HFUS in homozygous Gldc-deficient mouse embryos indicating this to be an effective tool to study CNS development. Longitudinal analysis of the same embryo allowed the prenatal onset and progression of ventricle enlargement in Gldc-deficient mice to be determined. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Association between Fetal Cerebral Ventriculomegaly and Platelet Alloimmunisation.

INTRODUCTION: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare condition that may lead to intracerebral haemorrhage (ICH) in the fetus or neonate. Platelet alloimmunisation causing FNAIT has been described in association with fetal cerebral ventriculomegaly (VM), presumably due to subclinical ICH. The objective of this study was to assess the association between fetal VM and platelet alloimmunisation. METHODS: This is a case series of pregnancies with fetal VM screened for platelet alloantibodies from 2003 to 2012. Cases of multiple pregnancies, structural anomalies, aneuploidies, or congenital infection were excluded. RESULTS: Of 45 pregnancies with fetal VM that were screened for platelet alloantibodies, 5 (11%) were positive. There was only one antenatal ICH, with confirmed fetal severe thrombocytopenia before termination of pregnancy. The other cases were treated with intravenous immunoglobulins without prior fetal blood sampling. No other case of neonatal thrombocytopenia was confirmed. CONCLUSIONS: The prevalence of platelet alloimmunisation was high in this series of fetal VM. Prospective large studies are needed to confirm the role of platelet alloimmunisation in fetal VM.

Region-specific changes in brain diffusivity in fetal isolated mild ventriculomegaly.

OBJECTIVES: To evaluate the impact of symmetric and asymmetric isolated mild ventriculomegaly (IMVM, atrial width 10-15 mm) on apparent diffusion coefficient (ADC) values in fetal brain areas. METHODS: Sixty-seven sequential fetal head magnetic resonance imaging scans (feMRI) of VM cases performed between 2009 and 2014 were compared to 38 normal feMRI scans matched for gestational age (controls). Ultrasound- and MRI-proven IMVM cases were divided into asymmetrical (AVM, ≥2 mm difference in atrial width), symmetrical (SVM, <2 mm difference in atrial width), and asymmetrical IMVM with one normal-sized ventricle (AV1norm). RESULTS: ADC values were significantly elevated in the basal ganglia (BG) of the SVM and AV1norm groups compared to controls (p < 0.004 and p < 0.013, respectively). High diffusivity was constantly detected in the BG ipsilateral to the enlarged atria relative to the normal-sized atria in the AV1norm group (p < 0.03). Frontal lobe ADC values were significantly reduced in the AVM and SVM groups (p < 0.003 and p < 0.003 vs. controls). Temporal lobe ADC values were significantly reduced in the AVM group (p < 0.001 vs. controls). CONCLUSION: Isolated mild ventriculomegaly is associated with distinct ADC value changes in different brain regions. This phenomenon could reflect the pathophysiology associated with different IMVM patterns. KEY POINTS: Various ventriculomegaly patterns are associated with distinct diffusional changes. Frontal and temporal lobe ADC values are altered bilaterally, even in asymmetric ventriculomegaly. Basal ganglia ADC values are elevated ipsilateral to the enlarged ventricle.

Cortical overgrowth in fetuses with isolated ventriculomegaly.

Mild cerebral ventricular enlargement is associated with schizophrenia, autism, epilepsy, and attention-deficit/hyperactivity disorder. Fetal ventriculomegaly is the most common central nervous system (CNS) abnormality affecting 1% of fetuses and is associated with cognitive, language, and behavioral impairments in childhood. Neurodevelopmental outcome is partially predictable by the 2-dimensional size of the ventricles in the absence of other abnormalities. We hypothesized that isolated fetal ventriculomegaly is a marker of altered brain development characterized by relative overgrowth and aimed to quantify brain growth using volumetric magnetic resonance imaging (MRI) in fetuses with isolated ventriculomegaly. Fetal brain MRI (1.5 T) was performed in 60 normal fetuses and 65 with isolated ventriculomegaly, across a gestational age range of 22-38 weeks. Volumetric analysis of the ventricles and supratentorial brain structures was performed on 3-dimensional reconstructed datasets. Fetuses with isolated ventriculomegaly had increased brain parenchyma volumes when compared with the control cohort (9.6%, P < 0.0001) with enlargement restricted to the cortical gray matter (17.2%, P = 0.002). The extracerebral cerebrospinal fluid and third and fourth ventricles were also enlarged. White matter, basal ganglia, and thalamic volumes were not significantly different between cohorts. The presence of relative cortical overgrowth in fetuses with ventriculomegaly may represent the neurobiological substrate for cognitive, language, and behavioral deficits in these children.

Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome.

Meckel-Gruber syndrome (MKS) is an embryonic lethal ciliopathy resulting from mutations in genes encoding proteins localising to the primary cilium. Mutations in the basal body protein MKS1 account for 7% of cases of MKS. The condition affects the development of multiple organs, including brain, kidney and skeleton. Here we present a novel Mks1(tm1a(EUCOMM)Wtsi) knockout mouse which accurately recapitulates the human condition, consistently developing pre-axial polydactyly, complex posterior fossa defects (including the Dandy-Walker malformation), and renal cystic dysplasia. TOPFlash Wnt reporter assays in mouse embryonic fibroblasts (MEFs) showed general de-regulated high levels of canonical Wnt/ß-catenin signalling in Mks1(-/-) cells. In addition to these signalling defects, we also observed ectopic high proliferation in the brain and kidney of mutant animals at mid- to late-gestation. The specific role of Mks1 in regulating cell proliferation was confirmed in Mks1 siRNA knockdown experiments which showed increased levels of proliferation after knockdown, an effect not seen after knockdown of other ciliopathy genes. We suggest that this is a result of the de-regulation of multiple signalling pathways (Wnt, mTOR and Hh) in the absence of functional Mks1. This novel model system offers insights into the role of MKS1 in Wnt signalling and proliferation, and the impact of deregulation of these processes on brain and kidney development in MKS, as well as expanding our understanding of the role of Mks1 in multiple signalling pathways.

Perinatal characteristics of fetuses with borderline ventriculomegaly detected by routine ultrasonographic screening of low-risk populations.

AIM: Fetal borderline ventriculomegaly represents a frequent dilemma in perinatal management. The present study aimed to evaluate the clinical significance of fetal borderline ventriculomegaly in a low-risk Japanese population and to identify the risk factors for associated anomalies. METHODS: Data of cases of fetal borderline ventriculomegaly detected at 26-28 weeks of gestation by routine ultrasonographic screening of low-risk singleton pregnancies between 2006 and 2012 were retrospectively collected. Ventricular width, in utero progression, associated anomalies, chromosomal abnormalities, and perinatal and postnatal outcomes were assessed. The ventricular width, in utero progression and other perinatal characteristics were compared between the isolated and non-isolated groups. RESULTS: Among the total 6020 singleton low-risk pregnancies, we noted that 42 had borderline ventriculomegaly. Six (14%) of these cases had other defects by subsequent detailed examination. Ventriculomegaly resolved or regressed in 35 (83%) and progressed in four (10%) cases, of which three were associated with other anomalies. The median ventricular width was 12.8 mm (range, 10.0-14.7) in the six non-isolated cases and 10.5 mm (range, 10.0-13.3) in the 36 isolated cases; the differences were statistically significant. A ventricular width of 12 mm or more and in utero progression were more frequently observed in non-isolated cases than in isolated cases. CONCLUSION: Fetal borderline ventriculomegaly frequently resolves in utero. A ventricular diameter of more than 12 mm and in utero progression are risk factors for additional anomalies. After the initial diagnosis of borderline ventriculomegaly, the pregnancy should be carefully followed up to determine whether the ventricle size is resolved, remains stable or increases.

[Fetal therapy--evaluation of ventriculo-amniotic shunts in the treatment of hydrocephalus]. / Terapia plodu--ocena zastosowania shuntu komorowo-owodniowego w leczeniu wodoglowia.

OBJECTIVE: The aim of the study was to establish optimal diagnostic and therapeutic scheme and to assess the efficacy of intrauterine therapy of hydrocephalus. MATERIAL AND METHODS: The study was carried out between 1992-2012 on the total of 222 fetuses with hydro- cephalus, using Orbis-Sigma and ACCU-Flow valves (168 cases) and Cook8 shunts, according to a strictly defined diagnostic and therapeutic scheme. RESULTS: In the first stage of the study (between 1992-2001), a total of 168 fetuses with prenatally diagnosed hydrocephalus received intrauterine therapy In 91.6% of the cases the therapy resulted in a decreased size of cerebral ventricles. The valve dislocated in 23 cases (13.6%). Preterm delivery occurred in 44% of the affected neonates. Severe mental impairment occurred in 17.76%, average in 36.8%, and slight in 32.9% of the infants. Normal mental development at the age of 3 was observed in 12.5% of the children. A total of 11.2% of chldren did not require further neurosurgical treatment. In the second stage of the study (between 2006-2012) after therapy the size of the right lateral cerebral ventricle decreased by 54.76% (average of27.54 mm to 12.46 mm) and the left lateral cerebral ventricle decreased by 53.12% (average of 26.41 mm to 12.38 mm) (p=0.0018). The maximum and minimum width of the cerebral cortex increased by 23.06% and 27% (average of 9.04 mm to 11.75 mm vs. 3.65 mm to 5 mm), respectively Early complications were observed in 22% of the cases: PROM (6), intrauterine fetal death (4), intrauterine infection (1), and premature detachment of the placenta (1). Average gestational age at delivery was 34 weeks, and 24% of the patients delivered at term. CONCLUSIONS: Implantation of ventriculoamniotic shunts proved to be an effective form of therapy resulting in normalization of intracranial pressure. In both stages of therapy reduction of ventricular size in patients with hydrocephalus and good neurological outcome (45.4% in I stage, 60% in II stage) were observed. In the second stage of therapy the size of lateral brain ventricles after fetal therapy was significantly lower (54%). A total of 18% of the neonates did not require neurosurgical treatment.