Engrailed2 modulates cerebellar granule neuron precursor proliferation, differentiation and insulin-like growth factor 1 signaling during postnatal development

BACKGROUND: The homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar development and evidence of cerebellar pathology in autism spectrum disorder (ASD), we previously examined an ENGRAILED2 association and found evidence to support EN2 as a susceptibility gene, a finding replicated by several other investigators. However, its functions at the cell biological level remain undefined. In the mouse, En2 gene is expressed in granule neuron precursors (GNPs) just as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may modulate these developmental processes. METHODS: To define En2 functions, we examined proliferation, differentiation and signaling pathway activation in En2 knockout (KO) and wild-type (WT) GNPs in response to a variety of extracellular growth factors and following En2 cDNA overexpression in cell culture. In vivo analyses of cerebellar GNP proliferation as well as responses to insulin-like growth factor-1 (IGF1) treatment were also conducted. RESULTS: Proliferation markers were increased in KO GNPs in vivo and in 24-h cultures, suggesting En2 normally serves to promote cell cycle exit. Significantly, IGF1 stimulated greater DNA synthesis in KO than WT cells in culture, a finding associated with markedly increased phospho-S6 kinase activation. Similarly, there was three-fold greater DNA synthesis in the KO cerebellum in response to IGF1 in vivo. On the other hand, KO GNPs exhibited reduced neurite outgrowth and differentiation. Conversely, En2 overexpression increased cell cycle exit and promoted neuronal differentiation. CONCLUSIONS: In aggregate, our observations suggest that the ASD-associated gene En2 promotes GNP cell cycle exit and differentiation, and modulates IGF1 activity during postnatal cerebellar development. Thus, genetic/epigenetic alterations of EN2 expression may impact proliferation, differentiation and IGF1 signaling as possible mechanisms that may contribute to ASD pathogenesis

mTORC1 activity oscillates throughout the cell cycle, promoting mitotic entry and differentially influencing autophagy induction

Summary: Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that is active in nearly all proliferating eukaryotic cells; however, it is unclear whether mTORC1 activity changes throughout the cell cycle. We find that mTORC1 activity oscillates from lowest in mitosis/G1 to highest in S/G2. The interphase oscillation is mediated through the TSC complex but is independent of major known regulatory inputs, including Akt and Mek/Erk signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex. mTORC1 has long been known to promote progression through G1. We find that mTORC1 also promotes progression through S and G2 and is important for satisfying the Chk1/Wee1-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 consequences for proliferating cells

Congenital Cataract in <i>Gpr161^vl/vl</i> Mice Is Modified by Proximal Chromosome 15

<div><p>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, <i>vacuolated lens</i> (<i>vl</i>), arose on the C3H/HeSnJ background. The mutation disrupts secondary lens fiber development by E16.5, leading to full penetrance of congenital cataract. The <i>vl</i> locus was mapped to a frameshift deletion in the orphan G protein-coupled receptor, <i>Gpr161</i>, which is expressed in differentiating lens fiber cells. When <i>Gpr161</i>^<i>vl/vl</i> 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 (<i>Modvl3-5</i>: <b>Mod</b>ifier of <b><i>vl</i></b>) and in this study we characterized <i>Modvl4</i> (Chr15; LOD = 4.4). A <i>Modvl4</i>^MOLF 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 <i>Gpr161</i>^<i>vl/vl</i> and subcongenic E16.5 eyes, bioinformatics analysis of C3H/MOLF polymorphisms, and the biological relevancy of the genes in the interval identified three genes (<i>Cdh6</i>, <i>Ank</i> and <i>Trio</i>) that likely contribute to the rescue of the lens phenotype. These studies demonstrate that modification of the <i>Gpr161</i>^<i>vl/vl</i> cataract phenotype is likely due to genetic variants in at least one of three closely linked candidate genes on proximal Chr15.</p></div

The orphan GPCR, Gpr161, regulates the retinoic acid and canonical Wnt pathways during neurulation

The vacuolated lens (vl) mouse mutation arose on the C3H/HeSnJ background and results in lethality, neural tube defects (NTDs) and cataracts. The vl phenotypes are due to a deletion/frameshift mutation in the orphan GPCR, Gpr161. A recent study using a null allele demonstrated that Gpr161 functions in primary cilia and represses the Shh pathway. We show the hypomorphic Gpr161vl allele does not severely affect the Shh pathway. To identify additional pathways regulated by Gpr161 during neurulation, we took advantage of naturally occurring genetic variation in the mouse. Previously Gpr161vl-C3H was crossed to different inbred backgrounds including MOLF/EiJ and the Gpr161vl mutant phenotypes were rescued. Five modifiers were mapped (Modvl: Modifier of vl) including Modvl5MOLF. In this study we demonstrate the Modvl5MOLF congenic rescues the Gpr161vl-associated lethality and NTDs but not cataracts. Bioinformatics determined the transcription factor, Cdx1, is the only annotated gene within the Modvl5 95% CI co-expressed with Gpr161 during neurulation and not expressed in the eye. Using Cdx1 as an entry point, we identified the retinoid acid (RA) and canonical Wnt pathways as downstream targets of Gpr161. QRT-PCR, ISH and IHC determined that expression of RA and Wnt genes are down-regulated in Gpr161vl/vl but rescued by the Modvl5MOLF congenic during neurulation. Intraperitoneal RA injection restores expression of canonical Wnt markers and rescues Gpr161vl/vl NTDs. These results establish the RA and canonical Wnt as pathways downstream of Gpr161 during neurulation, and suggest that Modvl5MOLF bypasses the Gpr161vl mutation by restoring the activity of these pathways

Autism-relevant social abnormalities and cognitive deficits in engrailed-2 knockout mice.

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

Autism-relevant social abnormalities and cognitive deficits in engrailed-2 knockout mice.

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

Autism Associated Gene, <i>ENGRAILED2</i>, and Flanking Gene Levels Are Altered in Post-Mortem Cerebellum

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