Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

Laminating the mammalian cortex during development: cell polarity protein function and Hippo signaling.

During mammalian brain development, radial glial progenitors balance between proliferation and differentiation to generate the laminated cortical layers in a temporally precise fashion. Defects in the individual steps going into this complex organogenesis can result in cortical malformations and human nervous system disorders. In this issue of Genes & Development, Liu and colleagues (pp. 763-780) present experimental evidence that an evolutionarily conserved cellular polarity gene, Pard3 (partitioning-defective 3), controls the balance of radial glial proliferation and differentiation through interaction with the Hippo signal transduction pathway. Conditional deletion of Pard3 in the developing rodent cortex resulted in striking subcortical band heterotopia, reminiscent of a severe form of human cortical malformation.

PARD3 dysfunction in conjunction with dynamic HIPPO signaling drives cortical enlargement with massive heterotopia.

Proper organization and orderly mitosis of radial glial progenitors (RGPs) drive the formation of a laminated mammalian cortex in the correct size. However, the molecular underpinnings of the intricate process remain largely unclear. Here we show that RGP behavior and cortical development are controlled by temporally distinct actions of partitioning-defective 3 (PARD3) in concert with dynamic HIPPO signaling. RGPs lacking PARD3 exhibit developmental stage-dependent abnormal switches in division mode, resulting in an initial overproduction of RGPs located largely outside the ventricular zone at the expense of deep-layer neurons. Ectopically localized RGPs subsequently undergo accelerated and excessive neurogenesis, leading to the formation of an enlarged cortex with massive heterotopia and increased seizure susceptibility. Simultaneous removal of HIPPO pathway effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) suppresses cortical enlargement and heterotopia formation. These results define a dynamic regulatory program of mammalian cortical development and highlight a progenitor origin of megalencephaly with ribbon heterotopia and epilepsy.

Grey matter heterotopia subtypes show specific morpho-electric signatures and network dynamics.

Grey matter heterotopia (GMH) are neurodevelopmental disorders associated with abnormal cortical function and epilepsy. Subcortical band heterotopia (SBH) and periventricular nodular heterotopia (PVNH) are two well-recognized GMH subtypes in which neurons are misplaced, either forming nodules lining the ventricles in PVNH, or forming bands in the white matter in SBH. Although both PVNH and SBH are commonly associated with epilepsy, it is unclear whether these two GMH subtypes differ in terms of pathological consequences or, on the contrary, share common altered mechanisms. Here, we studied two robust preclinical models of SBH and PVNH, and performed a systematic comparative assessment of the physiological and morphological diversity of heterotopia neurons, as well as the dynamics of epileptiform activity and input connectivity. We uncovered a complex set of altered properties, including both common and distinct physiological and morphological features across heterotopia subtypes, and associated with specific dynamics of epileptiform activity. Taken together, these results suggest that pro-epileptic circuits in GMH are, at least in part, composed of neurons with distinct, subtype-specific, physiological and morphological properties depending on the heterotopia subtype. Our work supports the notion that GMH represent a complex set of disorders, associating both shared and diverging pathological consequences, and contributing to forming epileptogenic networks with specific properties. A deeper understanding of these properties may help to refine current GMH classification schemes by identifying morpho-electric signatures of GMH subtypes, to potentially inform new treatment strategies.

Patient-specific mutation of Dync1h1 in mice causes brain and behavioral deficits.

AIMS: Cytoplasmic dynein heavy chain (DYNC1H1) is a multi-subunit protein complex that provides motor force for movement of cargo on microtubules and traffics them back to the soma. In humans, mutations along the DYNC1H1 gene result in intellectual disabilities, cognitive delays, and neurologic and motor deficits. The aim of the study was to generate a mouse model to a newly identified de novo heterozygous DYNC1H1 mutation, within a functional ATPase domain (c9052C > T(P3018S)), identified in a child with motor deficits, and intellectual disabilities. RESULTS: P3018S heterozygous (HET) knockin mice are viable; homozygotes are lethal. Metabolic and EchoMRI™ testing show that HET mice have a higher metabolic rate, are more active, and have less body fat compared to wildtype mice. Neurobehavioral studies show that HET mice perform worse when traversing elevated balance beams, and on the negative geotaxis test. Immunofluorescent staining shows neuronal migration abnormalities in the dorsal and lateral neocortex with heterotopia in layer I. Neuron-subtype specific transcription factors CUX1 and CTGF identified neurons from layers II/III and VI respectively in cortical layer I, and abnormal pyramidal neurons with MAP2+ dendrites projecting downward from the pial surface. CONCLUSION: The HET mice are a good model for the motor deficits seen in the child, and highlights the importance of cytoplasmic dynein in the maintenance of cortical function and dendritic orientation relative to the pial surface. Our results are discussed in the context of other dynein mutant mice and in relation to clinical presentation in humans with DYNC1H1 mutations.

Characterisation of Kürsteiner canals of parathyroid: imparting relevance to a one-and-a-quarter-century-old concept.

AIMS: Kürsteiner canals (KC) were described at least 125 years ago as pharyngeal pouch embryological remnants of parathyroid and thymic development. While considered precursors for a subset of parathyroid cysts and salivary heterotopias (SH), they remain enigmatic. We now define a comprehensive phenotype of KC remnants and investigate their role in a spectrum of parathyroid lesions. METHODS AND RESULTS: `Sixty-two cystic and 22 non-cystic parathyroid lesions (73 patients) were retrieved from our institutional archive (2011-23) and evaluated for the presence of KC and prevalence of KC phenotype in parathyroid hormone (PTH)-positive and PTH-negative cysts. KC phenotype was defined as: cysts and tubules with surrounding sclerosis; bland, unilayered lining with frequent nuclear indentation of lumina; vesicular chromatin relative to chief cells; attenuated eosinophilic to 'hyper-cleared' cytoplasm; and staining pattern PTH-negative, SOX-10-positive, CK7-positive, GATA-3-positive and PAX-9 dim, a subset with oestrogen/progesterone receptor (ER/PR) positivity. Thirty PTH-negative cysts were identified in the neck/mediastinum; 14 of this group also showed SH. Thirty-two PTH-positive cysts included: 11 cystic parathyroid adenomas, 17 hyperplastic parathyroids, and four carcinomas. KC showed two distinct subtypes and were often found near PTH-negative cysts. PTH-negative cysts were associated with inferior parathyroids, SOX-10 positivity, fibrosclerosis, vesicular nuclei indenting cyst lumina and hyper-cleared or attenuated eosinophilic cytoplasm. CONCLUSIONS: KC are common in parathyroids and show a distinct histological and immunohistochemical profile, with an inferior predilection favouring branchial cleft III distribution. Diagnostically, the high prevalence of this phenotype in PTH-negative cysts and salivary heterotopia supports derivation of non-functioning cysts from KC. Conversely, PTH-positive cysts are more compatible with cystic change within hyperfunctioning glands.

Cerebellar Heterotopia: Broadening the Neuroradiological Spectrum of KBG Syndrome.

KBG syndrome is a rare genetic disorder caused by heterozygous pathogenic variants in ANKRD11. Affected individuals have developmental delay, short stature, characteristic facial features, and other dysmorphic findings. To date, a spectrum of unspecific neuroradiological defects has been reported in KBG patients, such as cortical defects, white matter abnormalities, corpus callosum, and cerebellar vermis hypoplasia.Deep clinical and neuroradiological phenotyping and genotype of a patient presenting with mild cognitive and behavioral problems were obtained after written informed consent.We herein describe the first KBG patient presenting with cerebellar heterotopia, a heterogeneous malformation characterized by the presence of clusters of neurons within the white matter of cerebellar hemispheres.This novel association broadens the neuroradiological spectrum of KBG syndrome, and further prompts to investigate the potential functions of ANKRD11 in cerebellar development.

Cerebellar heterotopia in an 11-year-old child with KDM6B-related neurodevelopmental disorder: A case report and review of the literature.

Heterozygous pathogenic variants in KDM6B have recently been associated to a rare neurodevelopmental disorder referred to as "Neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities" and characterized by non-pathognomonic facial and body dysmorphisms, a wide range of neurodevelopmental and behavioral disorders and nonspecific neuroradiological findings. KDM6B encodes a histone demethylase, expressed in different tissues during development, which regulates gene expression through the modulation of chromatin accessibility by RNA polymerase. We herein describe a 11-year-old male patient carrying a novel de novo pathogenic variant in KDM6B exhibiting facial dysmorphisms, dysgraphia, behavioral traits relatable to oppositional defiant, autism spectrum, and attention deficit hyperactivity disorders, a single seizure episode, and a neuroimaging finding of a single cerebellar heterotopic nodule, never described to date in this genetic condition. These findings expand the phenotypic spectrum of this syndrome, highlighting the potential role for KDM6B in cerebellar development and providing valuable insights for genetic counseling.

Periventricular nodular heterotopia is coupled with the neocortex during resting and task states.

Periventricular nodular heterotopia (PVNH) is a well-defined developmental disorder characterized by failed neuronal migration, which forms ectopic neuronal nodules along the ventricular walls. Previous studies mainly focus on clinical symptoms caused by the PVNH tissue, such as seizures. However, little is known about whether and how neurons in the PVNH tissue functionally communicate with neurons in the neocortex. To probe this, we applied magnetoencephalography (MEG) and stereo-electroencephalography (sEEG) recordings to patients with PVNH during resting and task states. By estimating frequency-resolved phase coupling strength of the source-reconstructed neural activities, we found that the PVNH tissue was spontaneously coupled with the neocortex in the α-ß frequency range, which was consistent with the synchronization pattern within the neocortical network. Furthermore, the coupling strength between PVNH and sensory areas effectively modulated the local neural activity in sensory areas. In both MEG and sEEG visual experiments, the PVNH tissue exhibited visual-evoked responses, with a similar pattern and latency as the ipsilateral visual cortex. These findings demonstrate that PVNH is functionally integrated into cognition-related cortical circuits, suggesting a co-development perspective of ectopic neurons after their migration failure.

Periventricular nodular heterotopia in patients with a prenatal diagnosis of myelomeningocele/myeloschisis: associations with seizures and neurodevelopmental outcomes during early childhood.

PURPOSE: Historically, the presence of gray matter heterotopia was a concern for adverse postnatal neurocognitive status in patients undergoing fetal closure of open spinal dysraphism. The purpose of this study was to evaluate neurodevelopmental outcomes and the onset of seizures during early childhood in patients with a prenatal diagnosis of myelomeningocele/myeloschisis (MMC) and periventricular nodular heterotopia (PVNH). METHODS: All patients evaluated at the Center for Fetal Diagnosis and Treatment with a diagnosis of MMC between June 2016 to March 2023 were identified. PVNH was determined from prenatal and/or postnatal MRI. The Bayley Scales of Infant and Toddler Development (edition III or IV) were used for neurodevelopmental assessments. Patients were screened for seizures/epilepsy. RESULTS: Of 497 patients evaluated with a prenatal diagnosis of MMC, 99 were found to have PVNH on prenatal MRI, of which 35 had confirmed PVNH on postnatal imaging. From the 497 patients, 398 initially did not exhibit heterotopia on prenatal MRI, but 47 of these then had confirmed postnatal PVNH. The presence of PVNH was not a significant risk factor for postnatal seizures in early childhood. The average neurodevelopmental scores were not significantly different among heterotopia groups for cognitive, language, and motor domains. CONCLUSION: The presence of PVNH in patients with a prenatal diagnosis of MMC does not indicate an increased risk for neurodevelopmental delay at 1 year of age. We did not demonstrate an association with seizures/epilepsy. These findings can aid clinicians in prenatal consultation regarding fetal repair of open spinal dysraphism. Long-term follow-up is required to discern the true association between PVNH seen on prenatal imaging and postnatal seizures/epilepsy and neurodevelopmental outcomes.

Phenotypic Variability in Novel Doublecortin Gene Variants Associated with Subcortical Band Heterotopia.

Doublecortin, encoded by the DCX gene, plays a crucial role in the neuronal migration process during brain development. Pathogenic variants of the DCX gene are the major causes of the "lissencephaly (LIS) spectrum", which comprehends a milder phenotype like Subcortical Band Heterotopia (SBH) in heterozygous female subjects. We performed targeted sequencing in three unrelated female cases with SBH. We identified three DCX-related variants: a novel missense (c.601A>G: p.Lys201Glu), a novel nonsense (c.210C>G: p.Tyr70*), and a previously identified nonsense (c.907C>T: p.Arg303*) variant. The novel c.601A>G: p.Lys201Glu variant shows a mother-daughter transmission pattern across four generations. The proband exhibits focal epilepsy and achieved seizure freedom with a combination of oxcarbazepine and levetiracetam. All other affected members have no history of epileptic seizures. Brain MRIs of the affected members shows predominant fronto-central SBH with mixed pachygyria on the overlying cortex. The two nonsense variants were identified in two unrelated probands with SBH, severe drug-resistant epilepsy and intellectual disability. These novel DCX variants further expand the genotypic-phenotypic correlations of lissencephaly spectrum disorders. Our documented phenotypic descriptions of three unrelated families provide valuable insights and stimulate further discussions on DCX-SBH cases.

Optimal testing time for cerebral heterotopia formation in the rat comparative thyroid assay, a downstream indicator for perinatal thyroid hormone insufficiency.

In a past study, we proposed a modified Comparative Thyroid Assay (CTA) with additional examinations of brain thyroid hormone (TH) concentrations and brain histopathology but with smaller group sizes. The results showed that the modified CTA in Sprague Dawley rats detected 10 ppm 6-propylthiouracil (6-PTU)-induced significant suppressions of serum/brain TH concentrations in offspring. To confirm the reliability of qualitative brain histopathology and identify the optimal testing time for heterotopia (a cluster of ectopic neurons) in the modified CTA, brain histopathology together with serum/brain TH concentrations were assessed in GD20 fetuses and PND2, 4, 21, and 28 pups using a similar study protocol but with a smaller number of animals (N=3-6/group/time). Significant hypothyroidism was observed and brain histopathology revealed cerebral heterotopia formation in PND21 and PND28 pups, with likely precursor findings in PND2 and PND4 pups but not in GD20 fetuses. This study confirmed that the optimal testing time for cerebral heterotopia in rat CTA was PND21 and thereafter. These findings suggest that cerebral heterotopia assessment at appropriate times may be a useful alternative to the original CTA design.

Early suppression of excitability in subcortical band heterotopia modifies epileptogenesis in rats.

Malformations of cortical development represent a major cause of epilepsy in childhood. However, the pathological substrate and dynamic changes leading to the development and progression of epilepsy remain unclear. Here, we characterized an etiology-relevant rat model of subcortical band heterotopia (SBH), a diffuse type of cortical malformation associated with drug-resistant seizures in humans. We used longitudinal electrographic recordings to monitor the age-dependent evolution of epileptiform discharges during the course of epileptogenesis in this model. We found both quantitative and qualitative age-related changes in seizures properties and patterns, accompanying a gradual progression towards a fully developed seizure pattern seen in adulthood. We also dissected the relative contribution of the band heterotopia and the overlying cortex to the development and age-dependent progression of epilepsy using timed and spatially targeted manipulation of neuronal excitability. We found that an early suppression of neuronal excitability in SBH slows down epileptogenesis in juvenile rats, whereas epileptogenesis is paradoxically exacerbated when excitability is suppressed in the overlying cortex. However, in rats with active epilepsy, similar manipulations of excitability have no effect on chronic spontaneous seizures. Together, our data support the notion that complex developmental alterations occurring in both the SBH and the overlying cortex concur to creating pathogenic circuits prone to generate seizures. Our study also suggests that early and targeted interventions could potentially influence the course of these altered developmental trajectories, and favorably modify epileptogenesis in malformations of cortical development.

Further characterization of NFIB-associated phenotypes: Report of two new individuals.

Nuclear Factor I B (NFIB) haploinsufficiency has recently been identified as a cause of intellectual disability (ID) and macrocephaly. Here we report on two new individuals carrying a microdeletion in the chromosomal region 9p23-p22.3 containing NFIB. The first is a 7-year 9-month old boy with developmental delays, ID, definite facial anomalies, and brain and spinal cord magnetic resonance imaging findings including periventricular nodular heterotopia, hypoplasia of the corpus callosum, arachnoid cyst in the left middle cranial fossa, syringomyelia in the thoracic spinal cord and distal tract of the conus medullaris, and a stretched appearance of the filum terminale. The second is a 32-year-old lady (the proband' mother) with dysmorphic features, and a history of learning disability, hypothyroidism, poor growth, left inguinal hernia, and panic attacks. Her brain magnetic resonance imaging findings include a dysmorphic corpus callosum, and a small cyst in the left choroidal fissure that marks the hippocampal head. Array-based comparative genomic hybridization identified, in both, a 232 Kb interstitial deletion at 9p23p22.3 including several exons of NFIB and no other known genes. Our two individuals add to the knowledge of this rare disorder through the addition of new brain and spinal cord MRI findings and dysmorphic features. We propose that NFIB haploinsufficiency causes a clinically recognizable malformation-ID syndrome.

Periventricular heterotopia in a male child with USP9X missense variant.

The ubiquitin-specific protease USP9X has been found to play a role in multiple aspects of neural development including processes of neuronal migrations. In males, hemizygous partial loss of function variants in USP9X lead to a clinical phenotype primarily characterized by intellectual disability, hypotonia, speech and language impairment, behavioral disturbances accompanied by additional clinical features with variable expressivity. Structural brain abnormalities are reported in all cases where neuro-imaging was performed. The most common radiological features described include hypoplasia/agenesis of the corpus callosum, widened ventricles, white matter disturbances, and cerebellar hypoplasia. Here we report a child harboring a missense variant in USP9X presenting with the classical neurodevelopmental phenotype and a previously unreported radiological picture of periventricular heterotopia. This case expands the phenotypic landscape of this emergent condition and supports the critical role of USP9X in neuronal migration processes.

Periventricular nodular heterotopia associated with a "transmantle band sign" in patients with epilepsy.

OBJECTIVE: Previous studies using advanced magnetic resonance imaging (MRI) techniques have documented abnormal transmantle bands connecting ectopic nodules to overlying cortex in patients with periventricular nodular heterotopia (PNH). We describe a similar finding using conventional MRI techniques. METHODS: Patients were identified by means of a full-text search of radiological reports. All scanning was performed using conventional sequences at 3 Tesla (3T). Scans were reviewed by three neuroradiologists, and we characterized imaging features based on type of PNH and cortical irregularities associated with the transmantle band. RESULTS: A total 57 PNH patients were reviewed, of whom 41 demonstrated a "transmantle band" connecting the nodule to the overlying cortex. One or more periventricular heterotopic nodules was present in all 41 patients-this was bilateral in 29 of 41 (71%) and unilateral in the remaining 29%. In many cases there was more than one such band, and in some cases this band was nodular. In 19 of the cases, the cortex to which the band connected was abnormal, showing thinning in 4 cases, thickening in 5 cases, and polymicrogyria in another 10. SIGNIFICANCE: The transmantle band can be seen frequently in both unilateral and bilateral cases of PNH and can be visualized with conventional 3T MRI sequences. The band highlights the underlying neuronal migration issues at play in the pathogenesis of this disorder, but its underlying role in the complex, patient-specific epileptogenic networks in this cohort has yet to be determined and warrants further investigation.

Midline cutaneous anomalies of the craniospinal axis.

Patients with midline cutaneous anomalies of the craniospinal axis can be indicative of underlying embryonic defects, such as neural tube defects. Lack of familiarity with these midline aberrant skin findings may lead to misdiagnosis and delayed treatment. In this review, midline cutaneous anomalies of the craniospinal axis including aplasia cutis congenita, cranial and spinal dysraphism, and other developmental anomalies are explored in detail with emphasis on cutaneous clues to the diagnosis and appropriate workup.

Fetal pharyngeal glial heterotopia manifested as polyhydramnios: a rare case with difficult prenatal diagnosis.

BACKGROUND: Glial heterotopia is a rare congenital developmental malformation that presents as tumor-like lesions of the nerve tissue that grow outside the nervous system, but are not true tumors. At present, most cases are reported in neonates and children and are very rarely found in fetuses. The present report describes a case of fetal pharyngeal glial heterotopia and associated imaging findings to better understand the disease in the future. CASE PRESENTATION: A 32-year-old pregnant woman was admitted to the hospital with polyhydramnios. An ultrasound examination revealed a hypoechoic mass in the neck of the fetus. Magnetic resonance imaging showed a well-defined mass with significant compression of the esophagus and airway. The amniotic fluid index was approximately 40 cm. Considering that difficulty swallowing and breathing may occur due to compression by the mass after birth, tracheotomy and mass resection should be performed immediately. The difficulty of the tumor resection procedure and the nature of the tumor are both factors affecting the prognosis of the fetus. The pregnant woman eventually chose to induce labor. The fetal pharyngeal mass was then resected and its pathological examination indicated pharyngeal glial heterotopia. CONCLUSIONS: Polyhydramnios due to pharyngeal glial heterotopia is extremely rare and accurate prenatal diagnosis is challenging. Clinical diagnosis of glial heterotopia in preterm fetuses is difficult. Therefore, understanding glial heterotopia is helpful to improve clinical treatment options.

Somatic mosaicism of the PI3K-AKT-MTOR pathway is associated with hemimegalencephaly in fetal brains.

It is known that somatic activation of PI3K-AKT-MTOR signaling causes malformations of cortical development varying from hemimegalencephaly to focal cortical dysplasia. However, there have been few reports of fetal cases. Here we report two fetal cases of hemimegalencephaly, one associated with mosaic mutations in PIK3CA and another in AKT1. Both brains showed polymicrogyria, multiple subarachnoidal, subcortical, and subventricular heterotopia resulting from abnormal proliferation of neural stem/progenitor cells, cell differentiation, and migration of neuroblasts. Scattered cell nests immunoreactive for phosphorylated-S6 ribosomal protein (P-RPS6) (Ser240/244) were observed in the polymicrogyria-like cortical plate, intermediate zone, and arachnoid space, suggesting that the PI3K-AKT-MTOR pathway was actually activated in these cells. Pathological analyses could shed light on the mechanisms involved in disrupted brain development in the somatic mosaicism of the PI3K-AKT-MTOR pathway.

Periventricular nodular heterotopias is associated with mutation at the FLNA locus-a case history and a literature review.

BACKGROUND: Periventricular nodular heterotopia (PNH), associated with FLNA mutations, is a rare clinical condition potentially associated with multiple systemic conditions, including cardiac, pulmonary, skeletal, and cutaneous diseases. However, due to a paucity of information in the literature, accurate prognostic advice cannot be provided to patients with the disease. CASE PRESENTATION: We report a 2-year-old female whose PNH was associated with a nonsense mutation in the q28 region of the X chromosome, in exon 31 of FLNA (c.5159dupA). The patient is currently seizure-free and has no congenital heart disease, lung disease or skeletal or joint issues, and her development is normal. CONCLUSIONS: FLNA-associated PNH is a genetically-heterogeneous disease, and the FLNA mutation, c.5159dupA (p.Tyr1720*) is a newly identified pathogenic variant. FLNA characterization will help the clinical diagnosis and treatment of PNH and provide individualized genetic counseling for patients.