Mutations in the catalytic subunit of phosphoinositide 3-kinase (PIK3CA) and other PI3K-AKT pathway components have been associated with cancer and a wide spectrum of brain and body overgrowth. In the brain, the phenotypic spectrum of PIK3CA-related segmental overgrowth includes bilateral dysplastic megalencephaly, hemimegalencephaly and focal cortical dysplasia, the most common cause of intractable pediatric epilepsy. We generated mouse models expressing the most common activating Pik3ca mutations (H1047R and E545K) in developing neural progenitors. These accurately recapitulate all the key human pathological features including brain enlargement, cortical malformation, hydrocephalus and epilepsy, with phenotypic severity dependent on the mutant allele and its time of activation. Underlying mechanisms include increased proliferation, cell size and altered white matter. Notably, we demonstrate that acute 1 hr-suppression of PI3K signaling despite the ongoing presence of dysplasia has dramatic anti-epileptic benefit. Thus PI3K inhibitors offer a promising new avenue for effective anti-epileptic therapy for intractable pediatric epilepsy patients.

The Pax6 transcription factor is expressed in cerebellar granule cells and when mutated, as in the Sey/Sey mouse, produces granule cells with disturbed survival and migration and with defects in neurite extension. The impact of Pax6 on other genes in the context of cerebellar development has not been identified. In this study, we performed transcriptome comparisons between wildtype and Pax6-null whole cerebellar tissue at embryonic day (E) 13.5, 15.5 and 18.5 using Affymetrix arrays (U74Av2). Statistical analyses identified 136 differentially regulated transcripts (FDR 0.05, 1.2-fold change cutoff) over time in Pax6-null cerebellar tissue. In parallel we examined the Math1-null granuloprival cerebellum and identified 228 down-regulated transcripts (FDR 0.05, 1.2-fold change cutoff). The intersection of these two microarray datasets produced a total of 21 differentially regulated transcripts. For a subset of the identified transcripts, we used qRT-PCR to validate the microarray data and demonstrated the expression in the rhombic lip lineage and differential expression in Pax6-null cerebellum with in situ hybridisation analysis. The candidate genes identified in this way represent direct or indirect Pax6-downstream genes involved in cerebellar development.

Loss of Foxc1 is associated with Dandy-Walker malformation, the most common human cerebellar malformation characterized by cerebellar hypoplasia and an enlarged posterior fossa and fourth ventricle. Although expressed in the mouse posterior fossa mesenchyme, loss of Foxc1 non-autonomously induces a rapid and devastating decrease in embryonic cerebellar ventricular zone radial glial proliferation and concurrent increase in cerebellar neuronal differentiation. Subsequent migration of cerebellar neurons is disrupted, associated with disordered radial glial morphology. In vitro, SDF1α, a direct Foxc1 target also expressed in the head mesenchyme, acts as a cerebellar radial glial mitogen and a chemoattractant for nascent Purkinje cells. Its receptor, Cxcr4, is expressed in cerebellar radial glial cells and conditional Cxcr4 ablation with Nes-Cre mimics the Foxc1-/- cerebellar phenotype. SDF1α also rescues the Foxc1-/- phenotype. Our data emphasizes that the head mesenchyme exerts a considerable influence on early embryonic brain development and its disruption contributes to neurodevelopmental disorders in humans.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

In this study, we developed and evaluated a novel text-mining approach, using non-negative tensor factorization (NTF), to simultaneously extract and functionally annotate transcriptional modules consisting of sets of genes, transcription factors (TFs), and terms from MEDLINE abstracts. A sparse 3-mode term × gene × TF tensor was constructed that contained weighted frequencies of 106,895 terms in 26,781 abstracts shared among 7,695 genes and 994 TFs. The tensor was decomposed into sub-tensors using non-negative tensor factorization (NTF) across 16 different approximation ranks. Dominant entries of each of 2,861 sub-tensors were extracted to form term-gene-TF annotated transcriptional modules (ATMs). More than 94% of the ATMs were found to be enriched in at least one KEGG pathway or GO category, suggesting that the ATMs are functionally relevant. One advantage of this method is that it can discover potentially new gene-TF associations from the literature. Using a set of microarray and ChIP-Seq datasets as gold standard, we show that the precision of our method for predicting gene-TF associations is significantly higher than chance. In addition, we demonstrate that the terms in each ATM can be used to suggest new GO classifications to genes and TFs. Taken together, our results indicate that NTF is useful for simultaneous extraction and functional annotation of transcriptional regulatory networks from unstructured text, as well as for literature based discovery. A web tool called Transcriptional Regulatory Modules Extracted from Literature (TREMEL), available at http://binf1.memphis.edu/tremel, was built to enable browsing and searching of ATMs.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability in males and the most common genetic cause of autism. Although executive dysfunction is consistently found in humans with FXS, evidence of executive dysfunction in Fmr1 KO mice, a mouse model of FXS, has been inconsistent. One possible explanation for this is that executive dysfunction in Fmr1 KO mice, similar to humans with FXS, is only evident when cognitive demands are high. Using touchscreen operant conditioning chambers, male Fmr1 KO mice and their male wildtype littermates were tested on the acquisition of a pairwise visual discrimination followed by four serial reversals of the response rule. We assessed reversal learning performance under two different conditions. In the first, the correct stimulus was salient and the incorrect stimulus was non-salient. In the second and more challenging condition, the incorrect stimulus was salient and the correct stimulus was non-salient; this increased cognitive load by introducing conflict between sensory-driven (i.e., bottom-up) and task-dependent (i.e., top-down) signals. Fmr1 KOs displayed two distinct impairments relative to wildtype littermates. First, Fmr1 KOs committed significantly more learning-type errors during the second reversal stage, but only under high cognitive load. Second, during the first reversal stage, Fmr1 KOs committed significantly more attempts to collect a reward during the timeout following an incorrect response. These findings indicate that Fmr1 KO mice display executive dysfunction that, in some cases, is only evident under high cognitive load.

The signalling lipid PI(3,5)P(2) is generated on endosomes and regulates retrograde traffic to the trans-Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P(2) levels. Mutations that lower PI(3,5)P(2) cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P(2) was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P(2) regulatory complex by direct contact with the known regulators of PI(3,5)P(2): Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P(2) regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P(2). Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse.

Deletions of chromosome 1q42-q44 have been reported in a variety of developmental abnormalities of the brain, including microcephaly (MIC) and agenesis of the corpus callosum (ACC). Here, we describe detailed mapping studies of patients with unbalanced structural rearrangements of distal 1q4. These define a 3.5-Mb critical region extending from RP11-80B9 to RP11-241M7 that we hypothesize contains one or more genes that lead to MIC and ACC when present in only one functional copy. Next, mapping of a balanced reciprocal t(1;13)(q44;q32) translocation in a patient with postnatal MIC and ACC demonstrated a breakpoint within this region that is situated 20 kb upstream of AKT3, a serine-threonine kinase. The murine orthologue Akt3 is required for the developmental regulation of normal brain size and callosal development. Whereas sequencing of AKT3 in a panel of 45 patients with ACC did not demonstrate any pathogenic variations, whole-mount in situ hybridization confirmed expression of Akt3 in the developing central nervous system during mouse embryogenesis. AKT3 represents an excellent candidate for developmental human MIC and ACC, and we suggest that haploinsufficiency causes both postnatal MIC and ACC.

DEAD-Box Helicase 3 X-Linked (DDX3X) is frequently mutated in the Wingless (WNT) and Sonic hedghog (SHH) subtypes of medulloblastoma-the commonest malignant childhood brain tumor, but whether DDX3X functions as a medulloblastoma oncogene or tumor suppressor gene is not known. Here, we show that Ddx3x regulates hindbrain patterning and development by controlling Hox gene expression and cell stress signaling. In mice predisposed to Wnt- or Shh medulloblastoma, Ddx3x sensed oncogenic stress and suppressed tumor formation. WNT and SHH medulloblastomas normally arise only in the lower and upper rhombic lips, respectively. Deletion of Ddx3x removed this lineage restriction, enabling both medulloblastoma subtypes to arise in either germinal zone. Thus, DDX3X is a medulloblastoma tumor suppressor that regulates hindbrain development and restricts the competence of cell lineages to form medulloblastoma subtypes.

Fibrosis-driven solid organ failure is an enormous burden on global health. Spiny mice (Acomys) are terrestrial mammals that can regenerate severe skin wounds without scars to avoid predation. Whether spiny mice also regenerate internal organ injuries is unknown. Here, we show that despite equivalent acute obstructive or ischemic kidney injury, spiny mice fully regenerate nephron structure and organ function without fibrosis, whereas C57Bl/6 or CD1 mice progress to complete organ failure with extensive renal fibrosis. Two mechanisms for vertebrate regeneration have been proposed that emphasize either extrinsic (pro-regenerative macrophages) or intrinsic (surviving cells of the organ itself) controls. Comparative transcriptome analysis revealed that the Acomys genome appears poised at the time of injury to initiate regeneration by surviving kidney cells, whereas macrophage accumulation was not detected until about day 7. Thus, we provide evidence for rapid activation of a gene expression signature for regenerative wound healing in the spiny mouse kidney.

This report presents the first comprehensive database that specifically compiles genes critical for cerebellar development and function. The Cerebellar Gene Database details genes that, when perturbed in mouse models, result in a cerebellar phenotype according to available data from both Mouse Genome Informatics and PubMed, as well as references to the corresponding studies for further examination. This database also offers a compilation of human genetic disorders with a cerebellar phenotype and their associated gene information from the Online Mendelian Inheritance in Man (OMIM) database. By comparing and contrasting the mouse and human datasets, we observe that only a small proportion of human mutant genes with a cerebellar phenotype have been studied in mouse knockout models. Given the highly conserved nature between mouse and human genomes, this surprising finding highlights how mouse genetic models can be more frequently employed to elucidate human disease etiology. On the other hand, many mouse genes identified in the present study that are known to lead to a cerebellar phenotype when perturbed have not yet been found to be pathogenic in the cerebellum of humans. This database furthers our understanding of human cerebellar disorders with yet-to-be-identified genetic causes. It is our hope that this gene database will serve as an invaluable tool for gathering background information, generating hypotheses, and facilitating translational research endeavors. Moreover, we encourage continual inputs from the research community in making this compilation a living database, one that remains up-to-date with the advances in cerebellar research.

Spiny mice (Acomys cahirinus) are terrestrial mammals that evolved unique scar-free regenerative wound-healing properties. Myofibroblasts (MFs) are the major scar-forming cell type in skin. We found that following traumatic injury to ear pinnae, MFs appeared rapidly in both Acomys and mouse yet persisted only in mouse. The timing of MF loss in Acomys correlated with wound closure, blastema differentiation, and nuclear localization of the Hippo pathway target protein Yap. Experiments in vitro revealed an accelerated PP2A-dependent dephosphorylation activity that maintained nuclear Yap in Acomys dermal fibroblasts (DFs) and was not detected in mouse or human DFs. Treatment of Acomys in vivo with the nuclear Yap-TEAD inhibitor verteporfin prolonged MF persistence and converted tissue regeneration to fibrosis. Forced Yap activity prevented and rescued TGF-β1-induced human MF formation in vitro. These results suggest that Acomys evolved modifications of Yap activity and MF fate important for scar-free regenerative wound healing in vivo.

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

Characterization of neurochemical and behavioral responses to ethanol in phenotypically distinct mouse strains can provide insight into the mechanisms of ethanol stimulant actions. Increases in striatal dopamine (DA) levels have often been linked to ethanol-induced hyperactivity. We examined the functional status of the DA system and behavioral responsiveness to ethanol, cocaine, and a DA-receptor agonist in an N-ethyl-N-nitrosourea-mutagenized mouse strain, 22-TNJ, generated by the Integrative Neuroscience Initiative on Alcoholism Consortium. The 22-TNJ mouse strain exhibited greater locomotor responses to 2.25g/kg ethanol and 10mg/kg cocaine, compared with control mice. In vivo microdialysis showed low-baseline DA levels and a larger DA increase with both 2.25g/kg ethanol and 10mg/kg cocaine. In in vitro voltammetry studies, the 22-TNJ mice displayed increased V(max) rates for DA uptake, possibly contributing to the low-baseline DA levels found with microdialysis. Finally, 22-TNJ mice showed enhanced in vitro autoreceptor sensitivity to the D2/D3 agonist, quinpirole, and greater locomotor responses to both autoreceptor-selective and postsynaptic receptor-selective doses of apomorphine compared with controls. Taken together, these results indicate that the dopaminergic system of the 22-TNJ mouse is low functioning compared with control, with consequent receptor supersensitivity, such that mutant animals exhibit enhanced behavioral responses to DA-activating drugs, such as ethanol. Thus, the 22-TNJ mouse represents a model for a relatively hypodopaminergic system, and could provide important insights into the mechanisms of hyper-responsiveness to ethanol's stimulant actions.

The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo.

Mouse early transposon insertions are responsible for ~10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd), a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-β early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5' LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development.

Numerous studies have identified the roof plate as an embryonic signaling center critical for dorsal central nervous system patterning, but little is known about mechanisms that control its formation and its separation from clonally related neural crest cells and dI1 sensory interneurons. We demonstrate that the LIM homeodomain transcription factor, Lmx1a, mutated in the dreher mouse, acts to withdraw dorsal spinal cord progenitors from the cell cycle and simultaneously direct their differentiation into functional roof plate cells. Lmx1a cell-autonomously represses the dI1 progenitor fate, distinguishing the roof plate and dI1 interneuron programs, two major developmental programs of the dorsal neural tube. Lmx1a is not directly involved in neural crest development. We establish that Bmp signaling from epidermal ectoderm is necessary and sufficient for inducing Lmx1a and other co-factors that also regulate the extent of roof plate induction. We conclude that Lmx1a controls multiple aspects of dorsal midline patterning and is a major mediator of early Bmp signaling in the developing spinal cord.

Math1 is the defining molecule of the cerebellar rhombic lip and Pax6 is downstream in the Math1 pathway. In the present study, we discover that Wntless (Wls) is a novel molecular marker of the cells in the interior face of the rhombic lip throughout normal mouse cerebellar development. Wls expression is found complementary to the expression of Math1 and Pax6, which are localized to the exterior face of the rhombic lip. To determine the interaction between these molecules, we examine the loss-of-Math1 or loss-of-Pax6 in the cerebellum, i.e., the Math1-null and Pax6-null (Sey) mutant cerebella. The presence of Wls-positive cells in the Math1-null rhombic lip indicates that Wls expression is independent of Math1. In the Sey mutant cerebellum, there is an expansion of Wls-expressing cells into regions that are normally colonized by Pax6-expressing cells. The ectopic expression of Wls in the Pax6-null cerebellum suggests a negative interaction between Wls-expressing cells and Pax6-positive cells. These findings suggest that the rhombic lip is dynamically patterned by the expression of Wls, Math1, and Pax6. We also examine five rhombic lip cell markers (Wls, Math1, Pax6, Lmx1a, and Tbr2) to identify four molecularly distinct compartments in the rhombic lip during cerebellar development. The existence of spatial compartmentation in the rhombic lip and the interplay between Wls, Math1, and Pax6 in the rhombic lip provides novel views of early cerebellar development.

Cerebellar malformations are diverse congenital anomalies frequently associated with developmental disability. Although genetic and prenatal non-genetic causes have been described, no systematic analysis has been performed. Here, we present a large-exome sequencing study of Dandy-Walker malformation (DWM) and cerebellar hypoplasia (CBLH). We performed exome sequencing in 282 individuals from 100 families with DWM or CBLH, and we established a molecular diagnosis in 36 of 100 families, with a significantly higher yield for CBLH (51%) than for DWM (16%). The 41 variants impact 27 neurodevelopmental-disorder-associated genes, thus demonstrating that CBLH and DWM are often features of monogenic neurodevelopmental disorders. Though only seven monogenic causes (19%) were identified in more than one individual, neuroimaging review of 131 additional individuals confirmed cerebellar abnormalities in 23 of 27 genetic disorders (85%). Prenatal risk factors were frequently found among individuals without a genetic diagnosis (30 of 64 individuals [47%]). Single-cell RNA sequencing of prenatal human cerebellar tissue revealed gene enrichment in neuronal and vascular cell types; this suggests that defective vasculogenesis may disrupt cerebellar development. Further, de novo gain-of-function variants in PDGFRB, a tyrosine kinase receptor essential for vascular progenitor signaling, were associated with CBLH, and this discovery links genetic and non-genetic etiologies. Our results suggest that genetic defects impact specific cerebellar cell types and implicate abnormal vascular development as a mechanism for cerebellar malformations. We also confirmed a major contribution for non-genetic prenatal factors in individuals with cerebellar abnormalities, substantially influencing diagnostic evaluation and counseling regarding recurrence risk and prognosis.

Sucrose is recommended for the treatment of pain during minor procedures in preterm infants in the neonatal intensive care unit (NICU) and is currently used worldwide as the standard of care. We recently reported that adult mice repetitively exposed to sucrose compared to water during the first week of life, irrespective of exposure to an intervention, had significantly smaller brain volumes in large white matter, cortical and subcortical structures (e.g., hippocampus, striatum, fimbria). These structures are important for stress regulation and memory formation. Here, we report the effects of repeated neonatal exposure to pain and sucrose on adult behavior in mice. Neonatal C57BL/6J mice (N = 160, 47% male) were randomly assigned to one of two treatments (sucrose, water) and one of three interventions (needle-prick, tactile, handling). Pups received 10 interventions daily from postnatal day 1 (P1) to P6. A single dose of 24% sucrose or water was given orally 2 min before each intervention. At adulthood (P60-85) mice underwent behavioral testing to assess spatial memory, anxiety, motor function, pain sensitivity, and sugar preference. We found that mice that had received sucrose and handling only, had poorer short-term memory in adulthood compared to water/handling controls (p < 0.05). When exposed to pain, mice treated with repetitive sucrose or water did not differ on memory performance (p = 0.1). A sugar preference test showed that adult mice that received sucrose before an intervention as pups consumed less sugar solution compared to controls or those that received water before pain (p < 0.05). There were no significant group differences in anxiety, motor, or pain sensitivity. In a mouse model that closely mimics NICU care, we show for the first time that memory in adulthood was poorer for mice exposed to pain during the first week of life, irrespective of sucrose treatment, suggesting that sucrose does not protect memory performance when administered for pain. In the absence of pain, early repetitive sucrose exposure induced poorer short-term memory, highlighting the importance of accurate pain assessment.