An interaction between OTULIN and SCRIB uncovers roles for linear ubiquitination in planar cell polarity.

Planar cell polarity (PCP) plays critical roles in developmental and homeostatic processes. Membrane presentation of PCP complexes containing Van Gogh-like (VANGL) transmembrane proteins is central to PCP and can be directed by the scaffold protein scribble (SCRIB). The role atypical linear ubiquitin (Met1-Ub) chains might play in PCP is unknown. Here, HEK293 cell-based interactomic analyses of the Met1-Ub deubiquitinase OTULIN revealed that OTULIN can interact with SCRIB. Moreover, Met1-Ub chains associated with VANGL2 and PRICKLE1, but not SCRIB, can direct VANGL2 surface presentation. Mouse embryos lacking Otulin showed variable neural tube malformations, including rare open neural tubes, a deficit associated with PCP disruption in mice. In Madin-Darby canine kidney cells, in which the enrichment of VANGL2-GFP proteins at cell-cell contacts represents activated PCP complexes, endogenous OTULIN was recruited to these sites. In the human MDA-MB-231 breast cancer cell model, OTULIN loss caused deficits in Wnt5a-induced filopodia extension and trafficking of transfected HA-VANGL2. Taken together, these findings support a role for linear (de)ubiquitination in PCP signaling. The association of Met1-Ub chains with PCP complex components offers new opportunities for integrating PCP signaling with OTULIN-dependent immune and inflammatory pathways.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

Autism spectrum disorder: insights into convergent mechanisms from transcriptomics.

Heredity has a major role in autism spectrum disorder (ASD), yet underlying causal genetic variants have been defined only in a fairly small subset of cases. The enormous genetic heterogeneity associated with ASD emphasizes the importance of identifying convergent pathways and molecular mechanisms that are responsible for this disorder. We review how recent transcriptomic analyses have transformed our understanding of pathway convergence in ASD. In particular, deep RNA sequencing coupled with downstream investigations has revealed that a substantial fraction of autistic brains possess distinct transcriptomic signatures. These signatures are in part a consequence of altered neuronal activity and have a particular impact on pre-mRNA alternative splicing patterns.

The tyrosine kinase receptor Tyro3 enhances lifespan and neuropeptide Y (Npy) neuron survival in the mouse anorexia (anx) mutation.

Severe appetite and weight loss define the eating disorder anorexia nervosa, and can also accompany the progression of some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Although acute loss of hypothalamic neurons that produce appetite-stimulating neuropeptide Y (Npy) and agouti-related peptide (Agrp) in adult mice or in mice homozygous for the anorexia (anx) mutation causes aphagia, our understanding of the factors that help maintain appetite regulatory circuitry is limited. Here we identify a mutation (C19T) that converts an arginine to a tryptophan (R7W) in the TYRO3 protein tyrosine kinase 3 (Tyro3) gene, which resides within the anx critical interval, as contributing to the severity of anx phenotypes. Our observation that, like Tyro3-/- mice, anx/anx mice exhibit abnormal secondary platelet aggregation suggested that the C19T Tyro3 variant might have functional consequences. Tyro3 is expressed in the hypothalamus and other brain regions affected by the anx mutation, and its mRNA localization appeared abnormal in anx/anx brains by postnatal day 19 (P19). The presence of wild-type Tyro3 transgenes, but not an R7W-Tyro3 transgene, doubled the weight and lifespans of anx/anx mice and near-normal numbers of hypothalamic Npy-expressing neurons were present in Tyro3-transgenic anx/anx mice at P19. Although no differences in R7W-Tyro3 signal sequence function or protein localization were discernible in vitro, distribution of R7W-Tyro3 protein differed from that of Tyro3 protein in the cerebellum of transgenic wild-type mice. Thus, R7W-Tyro3 protein localization deficits are only detectable in vivo Further analyses revealed that the C19T Tyro3 mutation is present in a few other mouse strains, and hence is not the causative anx mutation, but rather an anx modifier. Our work shows that Tyro3 has prosurvival roles in the appetite regulatory circuitry and could also provide useful insights towards the development of interventions targeting detrimental weight loss.

Misregulation of an Activity-Dependent Splicing Network as a Common Mechanism Underlying Autism Spectrum Disorders.

A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.

Essential roles for the splicing regulator nSR100/SRRM4 during nervous system development.

Alternative splicing (AS) generates vast transcriptomic complexity in the vertebrate nervous system. However, the extent to which trans-acting splicing regulators and their target AS regulatory networks contribute to nervous system development is not well understood. To address these questions, we generated mice lacking the vertebrate- and neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4). Loss of nSR100 impairs development of the central and peripheral nervous systems in part by disrupting neurite outgrowth, cortical layering in the forebrain, and axon guidance in the corpus callosum. Accompanying these developmental defects are widespread changes in AS that primarily result in shifts to nonneural patterns for different classes of splicing events. The main component of the altered AS program comprises 3- to 27-nucleotide (nt) neural microexons, an emerging class of highly conserved AS events associated with the regulation of protein interaction networks in developing neurons and neurological disorders. Remarkably, inclusion of a 6-nt, nSR100-activated microexon in Unc13b transcripts is sufficient to rescue a neuritogenesis defect in nSR100 mutant primary neurons. These results thus reveal critical in vivo neurodevelopmental functions of nSR100 and further link these functions to a conserved program of neuronal microexon splicing.

A highly conserved program of neuronal microexons is misregulated in autistic brains.

Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide "microexons" display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.

Abnormal interneuron development in disrupted-in-schizophrenia-1 L100P mutant mice.

BACKGROUND: Interneuron deficits are one of the most consistent findings in post-mortem studies of schizophrenia patients and are likely important in the cognitive deficits associated with schizophrenia. Disrupted-in-Schizophrenia 1 (DISC1), a strong susceptibility gene for schizophrenia and other mental illnesses, is involved in neurodevelopment, including that of interneurons. However, the mechanism by which DISC1 regulates interneuron development remains unknown. In this study, we analyzed interneuron histology in the Disc1-L100P single point mutation mouse, that was previously shown to have behavioral abnormalities and cortical developmental defects related to schizophrenia. RESULTS: We sought to determine whether a Disc1-L100P point mutation in the mouse would alter interneuron density and location. First, we examined interneuron position in the developing mouse cortex during embryonic days 14-16 as an indicator of interneuron tangential migration, and found striking migration deficits in Disc1-L100P mutants. Further analysis of adult brains revealed that the Disc1-L100P mutants have selective alterations of calbindin- and parvalbumin-expressing interneurons in the cortex and hippocampus, decreased GAD67/PV co-localization and mis-positioned interneurons across the neocortex when compared to wild-type littermates. CONCLUSION: Our results are consistent with the anomalies seen in post-mortem schizophrenia studies and other Disc1 mutant mouse models. Future research is required to determine the specific mechanisms underlying these cellular deficits. Overall, these findings provide further evidence that DISC1 participates in interneuron development and add to our understanding of how DISC1 variants can affect susceptibility to psychiatric illness.

The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis.

A complex interaction of signalling events, including the Wnt pathway, regulates sprouting of blood vessels from pre-existing vasculature during angiogenesis. Here we show that two distinct mutations in the (uro)chordate-specific gumby (also called Fam105b) gene cause an embryonic angiogenic phenotype in gumby mice. Gumby interacts with disheveled 2 (DVL2), is expressed in canonical Wnt-responsive endothelial cells and encodes an ovarian tumour domain class of deubiquitinase that specifically cleaves linear ubiquitin linkages. A crystal structure of gumby in complex with linear diubiquitin reveals how the identified mutations adversely affect substrate binding and catalytic function in line with the severity of their angiogenic phenotypes. Gumby interacts with HOIP (also called RNF31), a key component of the linear ubiquitin assembly complex, and decreases linear ubiquitination and activation of NF-κB-dependent transcription. This work provides support for the biological importance of linear (de)ubiquitination in angiogenesis, craniofacial and neural development and in modulating Wnt signalling.

Disc1 point mutations in mice affect development of the cerebral cortex.

Disrupted-in-Schizophrenia 1 (DISC1) is a strong candidate gene for schizophrenia and other mental disorders. DISC1 regulates neurodevelopmental processes including neurogenesis, neuronal migration, neurite outgrowth, and neurotransmitter signaling. Abnormal neuronal morphology and cortical architecture are seen in human postmortem brain from patients with schizophrenia. However, the etiology and development of these histological abnormalities remain unclear. We analyzed the histology of two Disc1 mutant mice with point mutations (Q31L and L100P) and found a relative reduction in neuron number, decreased neurogenesis, and altered neuron distribution compared to wild-type littermates. Frontal cortical neurons have shorter dendrites and decreased surface area and spine density. Overall, the histology of Disc1 mutant mouse cortex is reminiscent of the findings in schizophrenia. These results provide further evidence that Disc1 participates in cortical development, including neurogenesis and neuron migration.

A tryptophan hydroxlyase 1 reporter that directs Cre recombinase extinguishable placental alkaline phosphatase expression in serotonergic (5-HT) neurons and peripheral tissues.

The serotonergic (5-HT) system modulates many behaviors and has been implicated in psychiatric disorders, but the density of 5-HT processes has complicated analyses. We have used regulatory regions from the Tryptophan hydroxylase 1 (Tph1) gene to drive expression of LoxP-flanked placental alkaline phosphatase (PLAP) to generate the Tph1-Lox-PLAP reporter mouse line. In these mice, PLAP is expressed in the hindbrain raphe nuclei and in peripheral tissues known to express Tph1. Tph1 is expressed at low levels in neurons. While, in Tph1-Lox-PLAP mice, most PLAP-expressing neurons are monoaminergic, PLAP was expressed in only 5-10% of neurons expressing the predominant neuronal 5-HT biosynthetic enzyme Tph2, serotonin transporter (SERT) or aromatic amino acid decarboxylase (AADC). To test this reporter further, we examined the brains of mice carrying the anorexia (anx) mutation, in which increased overall density of 5-HT immunoreactivity had been previously observed at P21. PLAP-labeling of processes in anx/anx and anx/+ mice was reduced at P0. By P10, distribution of PLAP-labeled processes in anx/+ and +/+ cortices was indistinguishable, but differed markedly from that seen in the cortical layers of anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter revealed a dosage sensitive role of the anx mutation in the early 5-HT system and later cortical layer-specific differences in 5-HT process distribution in anx/anx mice. Thus, the Tph1-LoxP-PLAP reporter provides a sensitive indicator for analyses of serotonergic cells in the brain and periphery.

Cdx1 refines positional identity of the vertebrate hindbrain by directly repressing Mafb expression.

An interplay of transcription factors interprets signalling pathways to define anteroposterior positions along the vertebrate axis. In the hindbrain, these transcription factors prompt the position-appropriate appearance of seven to eight segmental structures, known as rhombomeres (r1-r8). The evolutionarily conserved Cdx caudal-type homeodomain transcription factors help specify the vertebrate trunk and tail but have not been shown to directly regulate hindbrain patterning genes. Mafb (Kreisler, Krml1, valentino), a basic domain leucine zipper transcription factor, is required for development of r5 and r6 and is the first gene to show restricted expression within these two segments. The homeodomain protein vHnf1 (Hnf1b) directly activates Mafb expression. vHnf1 and Mafb share an anterior expression limit at the r4/r5 boundary but vHnf1 expression extends beyond the posterior limit of Mafb and, therefore, cannot establish the posterior Mafb expression boundary. Upon identifying regulatory sequences responsible for posterior Mafb repression, we have used in situ hybridization, immunofluorescence and chromatin immunoprecipitation (ChIP) analyses to determine that Cdx1 directly inhibits early Mafb expression in the neural tube posterior of the r6/r7 boundary, which is the anteriormost boundary of Cdx1 expression in the hindbrain. Cdx1 dependent repression of Mafb is transient. After the 10-somite stage, another mechanism acts to restrict Mafb expression in its normal r5 and r6 domain, even in the absence of Cdx1. Our findings identify Mafb as one of the earliest direct targets of Cdx1 and show that Cdx1 plays a direct role in early hindbrain patterning. Thus, just as Cdx2 and Cdx4 govern the trunk-to-tail transition, Cdx1 may regulate the hindbrain-to-spinal cord transition.

A vertebrate Polycomb response element governs segmentation of the posterior hindbrain.

Chromatin remodeling by Polycomb group (PcG) and trithorax group (trxG) proteins regulates gene expression in all metazoans. Two major complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), are thought to mediate PcG-dependent repression in flies and mammals. In Drosophila, PcG/trxG protein complexes are recruited by PcG/trxG response elements (PREs). However, it has been unclear how PcG/trxG are recruited in vertebrates. Here we have identified a vertebrate PRE, PRE-kr, that regulates expression of the mouse MafB/Kreisler gene. PRE-kr recruits PcG proteins in flies and mouse F9 cells and represses gene expression in a PcG/trxG-dependent manner. PRC1 and 2 bind to a minimal PRE-kr region, which can recruit stable PRC1 binding but only weak PRC2 binding when introduced ectopically, suggesting that PRC1 and 2 have different binding requirements. Thus, we provide evidence that similar to invertebrates, PREs act as entry sites for PcG/trxG chromatin remodeling in vertebrates.

Generation of a transgenic mouse line expressing GFP-Cre protein from a Hoxb4 neural enhancer.

Here, we describe a transgenic mouse line, in which expression of green fluorescent protein fused to Cre recombinase (GFP-Cre) is directed by the early neuronal enhancer (ENE) of Hoxb4. In E9.0-13.5 transgenic embryos, Cre activity coincided with endogenous Hoxb4 throughout the neural tube up to the r6/r7 boundary in the hindbrain, the dorsal root ganglia, and the Xth cranial ganglia. Unexpectedly, Cre activity was also consistently detected in the trigeminal (Vth) cranial nerve, which is devoid of endogenous Hoxb4 expression. Strong GFP dependent fluorescence appeared slightly later in E9.5-E11.5 embryos, and reflected the later expression pattern expected for Hoxb4-ENE directed expression in the neural tube up to the r7/r8 not r6/r7 boundary. Thus, with the exception of the trigeminal nerve, this reporter faithfully reproduces endogenous embryonic neural Hoxb4 expression, and provides an excellent reagent for in vivo gene manipulations in neuronal Hoxb4 positive cells as well as the developing trigeminal nerve. This transgenic mouse line should prove especially useful for determining the fate map of neuronal populations arising in rhombomeres 7 and 8 on its own and in combination with the small set of other existing rhombomere-specific Cre recombinase expressing lines.

A genetic screen for mutations that affect cranial nerve development in the mouse.

Cranial motor and sensory nerves arise stereotypically in the embryonic hindbrain, act as sensitive indicators of general and region-specific neuronal development, and are directly or indirectly affected in many human disorders, particularly craniofacial syndromes. The molecular genetic hierarchies that regulate cranial nerve development are mostly unknown. Here, we describe the first mouse genetic screen that has used direct immunohistochemical visualization methods to systematically identify genetic loci required for cranial nerve development. After screening 40 pedigrees, we recovered seven new neurodevelopmental mutations. Two mutations model human genetic syndromes. Mutation 7-1 causes facial nerve anomalies and a reduced lower jaw, and is located in a region of conserved synteny with an interval associated with the micrognathia and mental retardation of human cri-du-chat syndrome. Mutation 22-1 is in the Pax3 gene and, thus, models human Waardenburg syndrome. Three mutations cause global axon guidance deficits: one interferes with initial motor axon extension from the neural tube, another causes overall axon defasciculation, and the third affects general choice point selection. Another two mutations affect the oculomotor nerve specifically. Oculomotor nerve development, which is disrupted by six mutations, appears particularly sensitive to genetic perturbations. Phenotypic comparisons of these mutants identifies a "transition zone" that oculomotor axons enter after initial outgrowth and in which new factors govern additional progress. The number of interesting neurodevelopmental mutants revealed by this small-scale screen underscores the promise of similar focused genetic screens to contribute significantly to our understanding of cranial nerve development and human craniofacial syndromes.

The vHNF1 homeodomain protein establishes early rhombomere identity by direct regulation of Kreisler expression.

The early transcriptional hierarchy that subdivides the vertebrate hindbrain into seven to eight segments, the rhombomeres (r1-r8), is largely unknown. The Kreisler (MafB, Krml1, Val) gene is earliest gene expressed in an r5/r6-restricted manner and is essential for r5 and r6 development. We have identified the S5 regulatory element that directs early Kreisler expression in the future r5/r6 domain in 0-10 somite stage embryos. variant Hepatocyte Nuclear Factor 1 (vHNF1/HNF1beta/LF-3B) is transiently expressed in the r5/r6 domain of 0-10 somite stage embryos and a vHNF1binding site within this element is essential but not sufficient for r5/r6-specific expression. Thus, early inductive events that initiate Kreisler expression are clearly distinct from later-acting ones that modulate its expression levels. This site and some of the surrounding sequences are evolutionarily conserved in the genomic DNA upstream of the Kreisler gene among species as divergent as mouse, humans, and chickens. This provides the first evidence of a direct requirement for vHNF1 in initiation of Kreisler expression, suggests that the role of vHNF1 is evolutionarily conserved, and indicates that vHNF1 collaborates with other transcription factors, which independently bind to the S5 regulatory region, to establish the r5/r6 domain.

N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express.

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.

A genetic screen for mouse mutations with defects in serotonin responsiveness.

The serotonergic system plays a key role in regulating basic behaviors. Deficits in serotonergic neurotransmission have been implicated in psychiatric disorders, such as schizophrenia and depression. Here we have optimized a behavioral screen and performed a small scale genetic screen to identify genes involved in serotonin responsiveness in the mouse. Treatment of mice with serotonin, serotonin precursors, or serotonin agonists results in a quantifiable head twitch response (HTR), which is drug dosage-dependent and dependent on the 5-HT2A receptor system. This assay can uncover variation in serotonin responsiveness as shown by our identification of inbred strains with high, medium, and low head twitch responses to administration of the serotonin agonist DOI (+-1-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane). We chose C57Bl/6J mice for our mutagenesis screen, because of their robust HTR and because of the availability of their complete genomic sequence. We optimized this assay by examining dose and age dependence of DOI-induced HTR in 6-week and 3-month-old C57BL/6J mice. HTR decreases only slightly in 3-month-old mice, and a substantial but submaximal HTR is induced by 0.75-1 mg/kg of DOI. We assayed HTR in response to DOI of 247 G1 C57BL/6J progeny from C57BL/6J males, which had been mutagenized with ethylnitrososurea (ENU), and recovered one provisionally heritable hyper-responsive mutation. This and future mutations recovered via this protocol may provide ideal subjects for the study of human psychiatric disorders, such as depression and schizophrenia, and thereby aid in the development of better therapeutic strategies for these disorders. Thus, it is well worth expanding on this genetic screen in its current form and by addition of further pharmacologic assays in the future.

Analysis of hindbrain patterning defects caused by the kreisler(enu) mutation reveals multiple roles of Kreisler in hindbrain segmentation.

The embryonic hindbrain is subdivided into eight subunits, termed rhombomeres (r1-r8). The Kreisler (Krml1/MafB/val) transcription factor is expressed in and essential for patterning rhombomeres 5 and 6. Here, we have shown that in the chemically induced kreisler(enu) (kr(enu)) allele, a point mutation in the DNA binding domain abolishes or severely reduces Kreisler-dependent transcription. Comparison of kr(enu)/kr(enu) embryos with those homozygous for the classic kreisler (kr) mutation has reconciled past discrepancies and revealed multiple roles of Kreisler in hindbrain segmentation. These analyses demonstrate that Kreisler is required for maintenance and expansion but not initiation of the Krox20 expressing r5 domain. The differences in the "r5-like" phenotype of kr(enu)/kr(enu) and kr/kr mouse embryos, and zebrafish carrying mutations in the Kreisler orthologue valentino (val) suggest that Kreisler performs many of its r5-specific functions by associating with other proteins. By contrast, kr/kr and kr(enu)/kr(enu) mouse and val-/- zebrafish embryos all exhibit indistinguishable defects in r6 specification. Thus, transcriptionally active Kreisler is required for r6 specification. Unlike mouse kr(enu)/kr(enu) and zebrafish val-/- embryos, kr/kr embryos exhibited anterior defects. We determined that the kr chromosomal inversion caused ectopic Kreisler expression in r3 of kr/kr and kr/+ embryos. Hence, Kreisler regulates maintenance and expansion of r5 and specification of r6 but is not required for r3 development.