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Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of β-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders.

ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders.

The Delta-like (Dlk1) gene has been identified in Peromyscus and the laboratory mouse genus Mus. It is paternally expressed and tightly linked in Mus and humans to the maternally expressed Gtl2 gene that encodes an untranslated RNA. The coexpressed Dlk1 and Gtl2 genes have been found to be reciprocally imprinted. They respond reciprocally to loss of DNA methylation and likely are a new example of coordinated imprinting of linked genes. To study the genes, an allelic differential display method using placental mRNA derived from two closely related mouse species was designed.

Mice bearing Robertsonian translocation chromosomes frequently produce aneuploid gametes. They are therefore excellent tools for studying nondisjunction in mammals. Genotypic analysis of embryos from a mouse cross between two different strains of mice carrying a (7, 18) Robertsonian chromosome enabled us to measure the rate of nondisjunction for chromosomes 7 and 18. Embryos (429) were harvested from 76 litters of mice and the parental origin of each chromosome 7 and 18 determined. Genotyping these embryos has allowed us to conclude the following: (1) there were 96 embryos in which at least one nondisjunction event had taken place; (2) the rate of maternal nondisjunction was greater than paternal nondisjunction for the chromosomes sampled in these mice; (3) a bias against chromosome 7 and 18 nullisomic gametes was observed, reflected in a smaller than expected number of uniparental disomic embryos; (4) nondisjunction events did not seem to occur at random throughout the 76 mouse litters, but were clustered into fewer than would be expected by chance; and (5) a deficiency of paternal chromosome 18 uniparental disomic embryos was observed along with a higher than normal rate of developmental retardation at 8.5 days post coitum, raising the possibility that this chromosome has at least one imprinted gene.

The morphology and severity of human congenital cataract varies even among individuals with the same mutation, suggesting that genetic background modifies phenotypic penetrance. The spontaneous mouse mutant, vacuolated lens (vl), arose on the C3H/HeSnJ background. The mutation disrupts secondary lens fiber development by E16.5, leading to full penetrance of congenital cataract. The vl locus was mapped to a frameshift deletion in the orphan G protein-coupled receptor, Gpr161, which is expressed in differentiating lens fiber cells. When Gpr161vl/vl C3H mice are crossed to MOLF/EiJ mice an unexpected rescue of cataract is observed, suggesting that MOLF modifiers affect cataract penetrance. Subsequent QTL analysis mapped three modifiers (Modvl3-5: Modifier of vl) and in this study we characterized Modvl4 (Chr15; LOD = 4.4). A Modvl4MOLF congenic was generated and is sufficient to rescue congenital cataract and the lens fiber defect at E16.5. Additional phenotypic analysis on three subcongenic lines narrowed down the interval from 55 to 15Mb. In total only 18 protein-coding genes and 2 micro-RNAs are in this region. Fifteen of the 20 genes show detectable expression in the E16.5 eye. Subsequent expression studies in Gpr161vl/vl and subcongenic E16.5 eyes, bioinformatics analysis of C3H/MOLF polymorphisms, and the biological relevancy of the genes in the interval identified three genes (Cdh6, Ank and Trio) that likely contribute to the rescue of the lens phenotype. These studies demonstrate that modification of the Gpr161vl/vl cataract phenotype is likely due to genetic variants in at least one of three closely linked candidate genes on proximal Chr15. [ABSTRACT FROM AUTHOR]

Background: Previous genetic studies demonstrated association between the transcription factor ENGRAILED2 (EN2) and Autism Spectrum Disorder (ASD). Subsequent molecular analysis determined that the EN2 ASD-associated haplotype (rs1861972-rs1861973 A-C) functions as a transcriptional activator to increase gene expression. EN2 is flanked by 5 genes, SEROTONIN RECEPTOR5A (HTR5A), INSULIN INDUCED GENE1 (INSIG1), CANOPY1 HOMOLOG (CNPY1), RNA BINDING MOTIF PROTEIN33 (RBM33), and SONIC HEDGEHOG (SHH). These flanking genes are co-expressed with EN2 during development and coordinate similar developmental processes. To investigate if mRNA levels for these genes are altered in individuals with autism, post-mortem analysis was performed. Methods: qRT-PCR quantified mRNA levels for EN2 and the 5 flanking genes in 78 post-mortem cerebellar samples. mRNA levels were correlated with both affection status and rs1861972-rs1861973 genotype. Molecular analysis investigated whether EN2 regulates flanking gene expression. Results: EN2 levels are increased in affected A-C/G-T individuals (p = .0077). Affected individuals also display a significant increase in SHH and a decrease in INSIG1 levels. Rs1861972-rs1861973 genotype is correlated with significant increases for SHH (A-C/G-T) and CNPY1 (G-T/G-T) levels. Human cell line over-expression and knock-down as well as mouse knock-out analysis are consistent with EN2 and SHH being co-regulated, which provides a possible mechanism for increased SHH post-mortem levels. Conclusions: EN2 levels are increased in affected individuals with an A-C/G-T genotype, supporting EN2 as an ASD susceptibility gene. SHH, CNPY1, and INSIG1 levels are also significantly altered depending upon affection status or rs1861972-rs1861973 genotype. Increased EN2 levels likely contribute to elevated SHH expression observed in the post-mortem samples [ABSTRACT FROM AUTHOR]

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that stimulates anabolic cell growth while suppressing catabolic processes such as autophagy. mTORC1 is active in most, if not all, proliferating eukaryotic cells. However, it remains unclear whether and how mTORC1 activity changes from one cell cycle phase to another. Here we tracked mTORC1 activity through the complete cell cycle and uncover oscillations in its activity. We find that mTORC1 activity peaks in S and G2, and is lowest in mitosis and G1. We further demonstrate that multiple mechanisms are involved in controlling this oscillation. The interphase oscillation is mediated through the TSC complex, an upstream negative regulator of mTORC1, but is independent of major known regulatory inputs to the TSC complex, including Akt, Mek/Erk, and CDK4/6 signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex, and instead involves CDK1-dependent control of the subcellular localization of mTORC1 itself. Functionally, we find that in addition to its well-established role in promoting progression through G1, mTORC1 also promotes progression through S and G2, and is important for satisfying the Wee1- and Chk1- dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific functional consequences in proliferating cells., Competing Interests: Declaration of Interests The authors declare no competing interests.