Engrailed-2 (En2) deletion produces multiple neurodevelopmental defects in monoamine systems, forebrain structures and neurogenesis and behavior.

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 ß-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.

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 Gpr161(vl) 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 Gpr161(vl-C3H) was crossed to different inbred backgrounds including MOLF/EiJ and the Gpr161(vl) mutant phenotypes were rescued. Five modifiers were mapped (Modvl: Modifier of vl) including Modvl5(MOLF). In this study we demonstrate the Modvl5(MOLF) congenic rescues the Gpr161(vl)-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 Gpr161(vl/vl) but rescued by the Modvl5(MOLF) congenic during neurulation. Intraperitoneal RA injection restores expression of canonical Wnt markers and rescues Gpr161(vl/vl) NTDs. These results establish the RA and canonical Wnt as pathways downstream of Gpr161 during neurulation, and suggest that Modvl5(MOLF) bypasses the Gpr161(vl) mutation by restoring the activity of these pathways.

Cut-like homeobox 1 and nuclear factor I/B mediate ENGRAILED2 autism spectrum disorder-associated haplotype function.

Both common and rare variants contribute to autism spectrum disorder (ASD) risk, but few variants have been established as functional. Previously we demonstrated that an intronic haplotype (rs1861972-rs1861973 A-C) in the homeobox transcription factor ENGRAILED2 (EN2) is significantly associated with ASD. Positive association has also been reported in six additional data sets, suggesting EN2 is an ASD susceptibility gene. Additional support for this possibility requires identification of functional variants that affect EN2 regulation or activity. In this study, we demonstrate that the A-C haplotype is a transcriptional activator. Luciferase (luc) assays in mouse neuronal cultures determined that the A-C haplotype increases expression levels (50%, P < 0.01, 24 h; 250%, P < 0.0001, 72 h). Mutational analysis indicates that the A-C haplotype activator function requires both associated A and C alleles. A minimal 202-bp element is sufficient for function and also specifically binds a protein complex. Mass spectrometry identified these proteins as the transcription factors, Cut-like homeobox 1 (Cux1) and nuclear factor I/B (Nfib). Subsequent antibody supershifts and chromatin immunoprecipitations demonstrated that human CUX1 and NFIB bind the A-C haplotype. Co-transfection and knock-down experiments determined that both CUX1 and NFIB are required for the A-C haplotype activator function. These data demonstrate that the ASD-associated A-C haplotype is a transcriptional activator, and both CUX1 and NFIB mediate this activity. These results provide biochemical evidence that the ASD-associated A-C haplotype is functional, further supporting EN2 as an ASD susceptibility gene.

The orphan G protein-coupled receptor, Gpr161, encodes the vacuolated lens locus and controls neurulation and lens development.

The vacuolated lens (vl) mouse mutant causes congenital cataracts and neural tube defects (NTDs), with the NTDs being caused by abnormal neural fold apposition and fusion. Our positional cloning of vl indicates these phenotypes result from a deletion mutation in an uncharacterized orphan G protein-coupled receptor (GPCR), Gpr161. Gpr161 displays restricted expression to the lateral neural folds, developing lens, retina, limb, and CNS. Characterization of the vl mutation indicates that C-terminal tail of Gpr161 is truncated, leading to multiple effects on the protein, including reduced receptor-mediated endocytosis. We have also mapped three modifier quantitative trait loci (QTL) that affect the incidence of either the vl cataract or NTD phenotypes. Bioinformatic, sequence, genetic, and functional data have determined that Foxe3, a key regulator of lens development, is a gene responsible for the vl cataract-modifying phenotype. These studies have extended our understanding of the vl locus in three significant ways. One, the cloning of the vl locus has identified a previously uncharacterized GPCR-ligand pathway necessary for neural fold fusion and lens development, providing insight into the molecular regulation of these developmental processes. Two, our QTL analysis has established vl as a mouse model for studying the multigenic basis of NTDs and cataracts. Three, we have identified Foxe3 as a genetic modifier that interacts with Gpr161 to regulate lens development.

Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects.

The vacuolated lens (vl) mouse mutant arose spontaneously on the C3H/HeSn background and exhibits neural tube defects (NTDs), congenital cataract, and occasionally a white belly spot. We previously reported that 1) the vl phenotypes are due to a mutation in an orphan G protein-coupled receptor (GPCR), Gpr161; 2) the penetrance of the vl NTD and cataract phenotypes are affected by genetic background, allowing three unlinked quantitative trait loci (QTL) to be mapped (modifiers of vacuolated lens, Modvl1-3); and 3) phenotype-based bioinformatics followed by genetic and molecular analysis identified a lens-specific transcription factor that contributes to the cataract-modifying effect of Modvl3. We now extend this analysis in three ways. First, using the Gpr161 mutation to unequivocally identify mutant adults and embryos, we determined that approximately 50% of vl/vl NTD-affected embryos die during development. Second, the MOLF/Ei genetic background suppresses this embryonic lethality but increases the incidence of the adult belly spot phenotype. Additional QTL analysis was performed, and two novel modifiers were mapped [Modvl4, logarithm of odds ratio (LOD) 4.4; Modvl5, LOD 5.0]. Third, phenotype-based bioinformatics identified candidate genes for these modifiers including two GPCRs that cause NTD or skin/pigmentation defects (Modvl4: Frizzled homolog 6; Modvl5: Melanocortin 5 receptor). Because GPCRs form oligomeric complexes, these genes were resequenced and nonsynonymous coding variants were identified. Bioinformatics and protein modeling suggest that these variants may be functional. Our studies further establish vl as a multigenic mouse model for NTDs and identify additional QTL that interact with Gpr161 to regulate neurulation.

Congenital Cataract in Gpr161vl/vl Mice Is Modified by Proximal Chromosome 15.

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.

Chronic Enzyme Replacement to the Brain of a Late Infantile Neuronal Ceroid Lipofuscinosis Mouse Has Differential Effects on Phenotypes of Disease.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal inherited neurodegenerative disease caused by loss of lysosomal protease tripeptidyl peptidase 1 (TPP1). We have investigated the effects of chronic intrathecal (IT) administration using enzyme replacement therapy (ERT) to the brain of an LINCL mouse model, in which locomotor function declines dramatically prior to early death. Median lifespan was significantly extended from 126 days to >259 days when chronic IT treatment was initiated before the onset of disease. While treated animals lived longer and showed little sign of locomotor dysfunction as measured by stride length, some or all (depending on regimen) still died prematurely. One explanation is that cerebrospinal fluid (CSF)-mediated delivery may not deliver TPP1 to all brain regions. Morphological studies support this, showing delivery of TPP1 to ventral, but not deeper and dorsal regions. When IT treatment is initiated in severely affected LINCL mice, lifespan was extended modestly in most but dramatically extended in approximately one-third of the cohort. Treatment improved locomotor function in these severely compromised animals after it had declined to the point at which animals normally die. This indicates that some pathology in LINCL is reversible and does not simply reflect neuronal death.

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Autism associated gene, engrailed2, and flanking gene levels are altered in post-mortem cerebellum.

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.

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 haplotype affects the regulation of the homeobox gene, ENGRAILED 2.

BACKGROUND: Association analysis identified the homeobox transcription factor, ENGRAILED 2 (EN2), as a possible autism spectrum disorder (ASD) susceptibility gene (ASD [MIM 608636]; EN2 [MIM 131310]). The common alleles (underlined) of two intronic single nucleotide polymorphisms (SNPs), rs1861972 (A/G) and rs1861973 (C/T), are over-transmitted to affected individuals both singly and as a haplotype in three separate datasets (518 families total, haplotype p = .00000035). METHODS: Further support that EN2 is a possible ASD susceptibility gene requires the identification of a risk allele, a DNA variant that is consistently associated with ASD but is also functional. To identify possible risk alleles, additional association analysis and linkage disequilibrium (LD) mapping were performed. Candidate polymorphisms were then tested for functional differences by luciferase (Luc) reporter transfections and electrophoretic mobility shift assays (EMSAs). RESULTS: Association analysis of additional EN2 polymorphisms and LD mapping with Hapmap SNPs identified the rs1861972-rs1861973 haplotype as the most appropriate candidate to test for functional differences. Luciferase reporters for the two common rs1861972-rs1861973 haplotypes (A-C and G-T) were then transfected into human and rat cell lines as well as primary mouse neuronal cultures. In all cases the A-C haplotype resulted in a significant increase in Luc levels (p < .005). The EMSAs were then performed, and nuclear factors were bound specifically to the A and C alleles of both SNPs. CONCLUSIONS: These data indicate that the A-C haplotype is functional and, together with the association and LD mapping results, supports EN2 as a likely ASD susceptibility gene and the A-C haplotype as a possible risk allele.

Identification of a schizophrenia-associated functional noncoding variant in NOS1AP.

OBJECTIVE: The authors previously demonstrated significant association between markers within NOS1AP and schizophrenia in a set of Canadian families of European descent, as well as significantly increased expression in schizophrenia of NOS1AP in unrelated postmortem samples from the dorsolateral prefrontal cortex. In this study the authors sought to apply novel statistical methods and conduct additional biological experiments to isolate at least one risk allele within NOS1AP. METHOD: Using the posterior probability of linkage disequilibrium (PPLD) to measure the probability that a single nucleotide polymorphism (SNP) is in linkage disequilibrium with schizophrenia, the authors evaluated 60 SNPs from NOS1AP in 24 Canadian families demonstrating linkage and association to this region. SNPs exhibiting strong evidence of linkage disequilibrium were tested for regulatory function by luciferase reporter assay. Two human neural cell lines (SK-N-MC and PFSK-1) were transfected with a vector containing each allelic variant of the SNP, the NOS1AP promoter, and a luciferase gene. Alleles altering expression were further assessed for binding of nuclear proteins by electrophoretic mobility shift assay. RESULTS: Three SNPs produced PPLDs >40%. One of them, rs12742393, demonstrated significant allelic expression differences in both cell lines tested. The allelic variation at this SNP altered the affinity of nuclear protein binding to this region of DNA. CONCLUSIONS: The A allele of rs12742393 appears to be a risk allele associated with schizophrenia that acts by enhancing transcription factor binding and increasing gene expression.