A common single nucleotide variant in the cytokine receptor-like factor-3 (CRLF3) gene causes neuronal deficits in human and mouse cells.

Single nucleotide variants in the general population are common genomic alterations, where the majority are presumed to be silent polymorphisms without known clinical significance. Using human induced pluripotent stem cell (hiPSC) cerebral organoid modeling of the 1.4 megabase Neurofibromatosis type 1 (NF1) deletion syndrome, we previously discovered that the cytokine receptor-like factor-3 (CRLF3) gene, which is co-deleted with the NF1 gene, functions as a major regulator of neuronal maturation. Moreover, children with NF1 and the CRLF3L389P variant have greater autism burden, suggesting that this gene might be important for neurologic function. To explore the functional consequences of this variant, we generated CRLF3L389P-mutant hiPSC lines and Crlf3L389P-mutant genetically engineered mice. While this variant does not impair protein expression, brain structure, or mouse behavior, CRLF3L389P-mutant human cerebral organoids and mouse brains exhibit impaired neuronal maturation and dendrite formation. In addition, Crlf3L389P-mutant mouse neurons have reduced dendrite lengths and branching, without any axonal deficits. Moreover, Crlf3L389P-mutant mouse hippocampal neurons have decreased firing rates and synaptic current amplitudes relative to wild type controls. Taken together, these findings establish the CRLF3L389P variant as functionally deleterious and suggest that it may be a neurodevelopmental disease modifier.

Mecp2 deletion results in profound alterations of developmental and adult functional connectivity.

As a regressive neurodevelopmental disorder with a well-established genetic cause, Rett syndrome and its Mecp2 loss-of-function mouse model provide an excellent opportunity to define potentially translatable functional signatures of disease progression, as well as offer insight into the role of Mecp2 in functional circuit development. Thus, we applied widefield optical fluorescence imaging to assess mesoscale calcium functional connectivity (FC) in the Mecp2 cortex both at postnatal day (P)35 in development and during the disease-related decline. We found that FC between numerous cortical regions was disrupted in Mecp2 mutant males both in juvenile development and early adulthood. Female Mecp2 mice displayed an increase in homotopic contralateral FC in the motor cortex at P35 but not in adulthood, where instead more posterior parietal regions were implicated. An increase in the amplitude of connection strength, both with more positive correlations and more negative anticorrelations, was observed across the male cortex in numerous functional regions. Widespread rescue of MeCP2 protein in GABAergic neurons rescued none of these functional deficits, nor, surprisingly, the expected male lifespan. Altogether, the female results identify early signs of disease progression, while the results in males indicate MeCP2 protein is required for typical FC in the brain.

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.

Functions of Gtf2i and Gtf2ird1 in the developing brain: transcription, DNA binding and long-term behavioral consequences.

Gtf2ird1 and Gtf2i are two transcription factors (TFs) among the 28 genes deleted in Williams syndrome, and prior mouse models of each TF show behavioral phenotypes. Here we identify their genomic binding sites in the developing brain and test for additive effects of their mutation on transcription and behavior. GTF2IRD1 binding targets were enriched for transcriptional and chromatin regulators and mediators of ubiquitination. GTF2I targets were enriched for signal transduction proteins, including regulators of phosphorylation and WNT. Both TFs are highly enriched at promoters, strongly overlap CTCF binding and topological associating domain boundaries and moderately overlap each other, suggesting epistatic effects. Shared TF targets are enriched for reactive oxygen species-responsive genes, synaptic proteins and transcription regulators such as chromatin modifiers, including a significant number of highly constrained genes and known ASD genes. We next used single and double mutants to test whether mutating both TFs will modify transcriptional and behavioral phenotypes of single Gtf2ird1 mutants, though with the caveat that our Gtf2ird1 mutants, like others previously reported, do produce low levels of a truncated protein product. Despite little difference in DNA binding and transcriptome-wide expression, homozygous Gtf2ird1 mutation caused balance, marble burying and conditioned fear phenotypes. However, mutating Gtf2i in addition to Gtf2ird1 did not further modify transcriptomic or most behavioral phenotypes, suggesting Gtf2ird1 mutation alone was sufficient for the observed phenotypes.

Altered neuronal physiology, development, and function associated with a common chromosome 15 duplication involving CHRNA7.

BACKGROUND: Copy number variants (CNVs) linked to genes involved in nervous system development or function are often associated with neuropsychiatric disease. While CNVs involving deletions generally cause severe and highly penetrant patient phenotypes, CNVs leading to duplications tend instead to exhibit widely variable and less penetrant phenotypic expressivity among affected individuals. CNVs located on chromosome 15q13.3 affecting the alpha-7 nicotinic acetylcholine receptor subunit (CHRNA7) gene contribute to multiple neuropsychiatric disorders with highly variable penetrance. However, the basis of such differential penetrance remains uncharacterized. Here, we generated induced pluripotent stem cell (iPSC) models from first-degree relatives with a 15q13.3 duplication and analyzed their cellular phenotypes to uncover a basis for the dissimilar phenotypic expressivity. RESULTS: The first-degree relatives studied included a boy with autism and emotional dysregulation (the affected proband-AP) and his clinically unaffected mother (UM), with comparison to unrelated control models lacking this duplication. Potential contributors to neuropsychiatric impairment were modeled in iPSC-derived cortical excitatory and inhibitory neurons. The AP-derived model uniquely exhibited disruptions of cellular physiology and neurodevelopment not observed in either the UM or unrelated controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, and increased endoplasmic reticulum (ER) stress. Both the neuronal migration deficit and elevated ER stress could be selectively rescued by different pharmacologic agents. Neuronal gene expression was also dysregulated in the AP, including reduced expression of genes related to behavior, psychological disorders, neuritogenesis, neuronal migration, and Wnt, axonal guidance, and GABA receptor signaling. The UM model instead exhibited upregulated expression of genes in many of these same pathways, suggesting that molecular compensation could have contributed to the lack of neurodevelopmental phenotypes in this model. However, both AP- and UM-derived neurons exhibited shared alterations of neuronal function, including increased action potential firing and elevated cholinergic activity, consistent with increased homomeric CHRNA7 channel activity. CONCLUSIONS: These data define both diagnosis-associated cellular phenotypes and shared functional anomalies related to CHRNA7 duplication that may contribute to variable phenotypic penetrance in individuals with 15q13.3 duplication. The capacity for pharmacological agents to rescue some neurodevelopmental anomalies associated with diagnosis suggests avenues for intervention for carriers of this duplication and other CNVs that cause related disorders.

Gtf2i and Gtf2ird1 mutation do not account for the full phenotypic effect of the Williams syndrome critical region in mouse models.

Williams syndrome (WS) is a neurodevelopmental disorder caused by a 1.5-1.8 Mbp deletion on chromosome 7q11.23, affecting the copy number of 26-28 genes. Phenotypes of WS include cardiovascular problems, craniofacial dysmorphology, deficits in visual-spatial cognition and a characteristic hypersocial personality. There are still no genes in the region that have been consistently linked to the cognitive and behavioral phenotypes, although human studies and mouse models have led to the current hypothesis that the general transcription factor 2 I family of genes, GTF2I and GTF2IRD1, are responsible. Here we test the hypothesis that these two transcription factors are sufficient to reproduce the phenotypes that are caused by deletion of the WS critical region (WSCR). We compare a new mouse model with loss of function mutations in both Gtf2i and Gtf2ird1 to an established mouse model lacking the complete WSCR. We show that the complete deletion (CD) model has deficits across several behavioral domains including social communication, motor functioning and conditioned fear that are not explained by loss of function mutations in Gtf2i and Gtf2ird1. Furthermore, transcriptome profiling of the hippocampus shows changes in synaptic genes in the CD model that are not seen in the double mutants. Thus, we have thoroughly defined a set of molecular and behavioral consequences of complete WSCR deletion and shown that genes or combinations of genes beyond Gtf2i and Gtf2ird1 are necessary to produce these phenotypic effects.

Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation.

Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that ~60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.

Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion of Ctcf in Camk2a-Cre-Expressing Neurons.

CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying 3D chromatin structure. Human mutations in CTCF cause intellectual disability and autistic features. Knocking out Ctcf in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out Ctcf postnatally in glutamatergic forebrain neurons under the control of Camk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+ (Ctcf CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor coordination, and decreased sociability by 4 months of age. Ctcf CKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA from Ctcf CKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically from Ctcf CKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia in Ctcf CKO hippocampus. Finally, we found that microglia in Ctcf CKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm that Ctcf KO in postnatal neurons causes a neurobehavioral phenotype in mice and provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction.SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations in CTCF cause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lacking Ctcf in Camk2a-expressing neurons (Ctcf CKO mice) have spatial learning/memory deficits, impaired fine motor skills, subtly altered social interactions, and decreased dendritic spine density. We demonstrate that Ctcf CKO mice overexpress inflammation-related genes in the brain and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron-microglia interactions are factors in the pathology of CTCF deficiency.

Using animal models to evaluate the functional consequences of anesthesia during early neurodevelopment.

Fifteen years ago Olney and colleagues began using animal models to evaluate the effects of anesthetic and sedative agents (ASAs) on neurodevelopment. The results from ongoing studies indicate that, under certain conditions, exposure to these drugs during development induces an acute elevated apoptotic neurodegenerative response in the brain and long-term functional impairments. These animal models have played a significant role in bringing attention to the possible adverse effects of exposing the developing brain to ASAs when few concerns had been raised previously in the medical community. The apoptotic degenerative response resulting from neonatal exposure to ASAs has been replicated in many studies in both rodents and non-human primates, suggesting that a similar effect may occur in humans. In both rodents and non-human primates, significantly increased levels of apoptotic degeneration are often associated with functional impairments later in life. However, behavioral deficits following developmental ASA exposure have not been consistently reported even when significantly elevated levels of apoptotic degeneration have been documented in animal models. In the present work, we review this literature and propose a rodent model for assessing potential functional deficits following neonatal ASA exposure with special reference to experimental design and procedural issues. Our intent is to improve test sensitivity and replicability for detecting subtle behavioral effects, and thus enhance the translational significance of ASA models.

The disruption of Celf6, a gene identified by translational profiling of serotonergic neurons, results in autism-related behaviors.

The immense molecular diversity of neurons challenges our ability to understand the genetic and cellular etiology of neuropsychiatric disorders. Leveraging knowledge from neurobiology may help parse the genetic complexity: identifying genes important for a circuit that mediates a particular symptom of a disease may help identify polymorphisms that contribute to risk for the disease as a whole. The serotonergic system has long been suspected in disorders that have symptoms of repetitive behaviors and resistance to change, including autism. We generated a bacTRAP mouse line to permit translational profiling of serotonergic neurons. From this, we identified several thousand serotonergic-cell expressed transcripts, of which 174 were highly enriched, including all known markers of these cells. Analysis of common variants near the corresponding genes in the AGRE collection implicated the RNA binding protein CELF6 in autism risk. Screening for rare variants in CELF6 identified an inherited premature stop codon in one of the probands. Subsequent disruption of Celf6 in mice resulted in animals exhibiting resistance to change and decreased ultrasonic vocalization as well as abnormal levels of serotonin in the brain. This work provides a reproducible and accurate method to profile serotonergic neurons under a variety of conditions and suggests a novel paradigm for gaining information on the etiology of psychiatric disorders.

Antisense reduction of tau in adult mice protects against seizures.

Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS--brain and spinal cord tissue, interstitial fluid, and CSF--while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability.

Characterization of early markers of disease in the mouse model of mucopolysaccharidosis IIIB.

BACKGROUND: Mucopolysaccharidosis (MPS) IIIB, also known as Sanfilippo Syndrome B, is a devastating childhood disease. Unfortunately, there are currently no available treatments for MPS IIIB patients. Yet, animal models of lysosomal storage diseases have been valuable tools in identifying promising avenues of treatment. Enzyme replacement therapy, gene therapy, and bone marrow transplant have all shown efficacy in the MPS IIIB model systems. A ubiquitous finding across rodent models of lysosomal storage diseases is that the best treatment outcomes resulted from intervention prior to symptom onset. Therefore, the aim of the current study was to identify early markers of disease in the MPS IIIB mouse model as well as examine clinically-relevant behavioral domains not yet explored in this model. METHODS: Using the MPS IIIB mouse model, we explored early developmental trajectories of communication and gait, and later social behavior, fear-related startle and conditioning, and visual capabilities. In addition, we examined brain structure and function via magnetic resonance imaging and diffusion tensor imaging. RESULTS: We observed reduced maternal isolation-induced ultrasonic vocalizations in MPS IIIB mice relative to controls, as well as disruption in a number of the spectrotemporal features. MPS IIIB also exhibited disrupted thermoregulation during the first two postnatal weeks without any differences in body weight. The developmental trajectories of gait were largely normal. In early adulthood, we observed intact visual acuity and sociability yet a more submissive phenotype, increased aggressive behavior, and decreased social sniffing relative to controls. MPS IIIB mice showed greater inhibition of startle in response to a pretone with a decrease in overall startle response and reduced cued fear memory. MPS IIIB also weighed significantly more than controls throughout adulthood and showed larger whole brain volumes and normalized regional volumes with intact tissue integrity as measured with magnetic resonance and diffusion tensor imaging, respectively. CONCLUSIONS: Together, these results indicate disease markers are present as early as the first two weeks postnatal in this model. Further, this model recapitulates social, sensory and fear-related clinical features. Our study using a mouse model of MPS IIIB provides essential baseline information that will be useful in future evaluations of potential treatments.

Oxytocin-induced birth causes sex-specific behavioral and brain connectivity changes in developing rat offspring.

Despite six decades of the use of exogenous oxytocin for management of labor, little is known about its effects on the developing brain. Motivated by controversial reports suggesting a link between oxytocin use during labor and autism spectrum disorders (ASDs), we employed our recently validated rat model for labor induction with oxytocin to address this important concern. Using a combination of molecular biological, behavioral, and neuroimaging assays, we show that induced birth with oxytocin leads to sex-specific disruption of oxytocinergic signaling in the developing brain, decreased communicative ability of pups, reduced empathy-like behaviors especially in male offspring, and widespread sex-dependent changes in functional cortical connectivity. Contrary to our hypothesis, social behavior, typically impaired in ASDs, was largely preserved. Collectively, our foundational studies provide nuanced insights into the neurodevelopmental impact of birth induction with oxytocin and set the stage for mechanistic investigations in animal models and prospective longitudinal clinical studies.

Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties.

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. We recently reported a family with a paternally inherited intragenic ASTN2 duplication with a range of neurodevelopmental disorders, including autism spectrum disorder (ASD), learning difficulties, and speech and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity and repetitive behaviors, altered social behaviors, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors were also prominent in Astn2 cKO animals. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrates a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs) in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

Antecedent glycemic control reduces severe hypoglycemia-induced neuronal damage in diabetic rats.

Brain damage due to severe hypoglycemia occurs in insulin-treated people with diabetes. This study tests the hypothesis that chronic insulin therapy that normalizes elevated blood glucose in diabetic rats would be neuroprotective against brain damage induced by an acute episode of severe hypoglycemia. Male Sprague-Dawley rats were split into three groups: 1) control, non-diabetic; 2) STZ-diabetic; and 3) insulin-treated STZ-diabetic. After 3 wk of chronic treatment, unrestrained awake rats underwent acute hyperinsulinemic severe hypoglycemic (10-15 mg/dl) clamps for 1 h. Rats were subsequently analyzed for brain damage and cognitive function. Severe hypoglycemia induced 15-fold more neuronal damage in STZ-diabetic rats compared with nondiabetic rats. Chronic insulin treatment of diabetic rats, which nearly normalized glucose levels, markedly reduced neuronal damage induced by severe hypoglycemia. Fortunately, no cognitive defects associated with the hypoglycemia-induced brain damage were observed in any group. In conclusion, antecedent blood glucose control represents a major modifiable therapeutic intervention that can afford diabetic subjects neuroprotection against severe hypoglycemia-induced brain damage.

A comprehensive assay of social motivation reveals sex-specific roles of autism-associated genes and oxytocin.

Social motivation is critical to the development of typical social functioning. Social motivation, specifically one or more of its components (e.g., social reward seeking or social orienting), could be relevant for understanding phenotypes related to autism. We developed a social operant conditioning task to quantify effort to access a social partner and concurrent social orienting in mice. We established that mice will work for access to a social partner, identified sex differences, and observed high test-retest reliability. We then benchmarked the method with two test-case manipulations. Shank3B mutants exhibited reduced social orienting and failed to show social reward seeking. Oxytocin receptor antagonism decreased social motivation, consistent with its role in social reward circuitry. Overall, we believe that this method provides a valuable addition to the assessment of social phenotypes in rodent models of autism and the mapping of potentially sex-specific social motivation neural circuits.

Extensive characterization of a Williams Syndrome murine model shows Gtf2ird1 -mediated rescue of select sensorimotor tasks, but no effect on enhanced social behavior.

Williams Syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams Syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1 , which encodes a transcription factor suggested to play a role in the behavioral profile of Williams Syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias, and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A 'Complete Deletion' mouse, heterozygously eliminating the syntenic Williams Syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1 , which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the Complete Deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams Syndrome Critical Region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Extensive characterization of a Williams syndrome murine model shows Gtf2ird1-mediated rescue of select sensorimotor tasks, but no effect on enhanced social behavior.

Williams syndrome is a rare neurodevelopmental disorder exhibiting cognitive and behavioral abnormalities, including increased social motivation, risk of anxiety and specific phobias along with perturbed motor function. Williams syndrome is caused by a microdeletion of 26-28 genes on chromosome 7, including GTF2IRD1, which encodes a transcription factor suggested to play a role in the behavioral profile of Williams syndrome. Duplications of the full region also lead to frequent autism diagnosis, social phobias and language delay. Thus, genes in the region appear to regulate social motivation in a dose-sensitive manner. A "complete deletion" mouse, heterozygously eliminating the syntenic Williams syndrome region, has been deeply characterized for cardiac phenotypes, but direct measures of social motivation have not been assessed. Furthermore, the role of Gtf2ird1 in these behaviors has not been addressed in a relevant genetic context. Here, we have generated a mouse overexpressing Gtf2ird1, which can be used both to model duplication of this gene alone and to rescue Gtf2ird1 expression in the complete deletion mice. Using a comprehensive behavioral pipeline and direct measures of social motivation, we provide evidence that the Williams syndrome critical region regulates social motivation along with motor and anxiety phenotypes, but that Gtf2ird1 complementation is not sufficient to rescue most of these traits, and duplication does not decrease social motivation. However, Gtf2ird1 complementation does rescue light-aversive behavior and performance on select sensorimotor tasks, perhaps indicating a role for this gene in sensory processing or integration.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity is a common feature of monogenic neurodevelopmental disorders that can arise from differential severity of missense variants underlying disease, but how distinct alleles impact molecular mechanisms to drive variable disease presentation is not well understood. Here, we investigate missense mutations in the DNA methyltransferase DNMT3A associated with variable overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity in neurodevelopmental disease. We generate a DNMT3A P900L/+ mouse model mimicking a disease mutation with mild-to-moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. We show that the P900L mutation leads to disease-relevant overgrowth, obesity, and social deficits shared across DNMT3A disorder models, while the R878H mutation causes more extensive epigenomic disruption leading to differential dysregulation of enhancers elements. We identify distinct gene sets disrupted in each mutant which may contribute to mild or severe disease, and detect shared transcriptomic disruption that likely drives common phenotypes across affected individuals. Finally, we demonstrate that core gene dysregulation detected in DNMT3A mutant mice overlaps effects in other developmental disorder models, highlighting the importance of DNMT3A-deposited methylation in neurodevelopment. Together, these findings define central drivers of DNMT3A disorders and illustrate how variable disruption of transcriptional mechanisms can drive the spectrum of phenotypes in neurodevelopmental disease.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity in monogenic neurodevelopmental disorders can arise from differential severity of variants underlying disease, but how distinct alleles drive variable disease presentation is not well understood. Here, we investigate missense mutations in DNA methyltransferase 3A (DNMT3A), a DNA methyltransferase associated with overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity. We generate a Dnmt3aP900L/+ mouse mimicking a mutation with mild to moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. P900L mutants exhibit core growth and behavioral phenotypes shared across models but show subtle epigenomic changes, while R878H mutants display extensive disruptions. We identify mutation-specific dysregulated genes that may contribute to variable disease severity. Shared transcriptomic disruption identified across mutations overlaps dysregulation observed in other developmental disorder models and likely drives common phenotypes. Together, our findings define central drivers of DNMT3A disorders and illustrate how variable epigenomic disruption contributes to phenotypic heterogeneity in neurodevelopmental disease.