The Immune Signaling Adaptor LAT Contributes to the Neuroanatomical Phenotype of 16p11.2 BP2-BP3 CNVs

Copy-number changes in 16p11.2 contribute significantly to neuropsychiatric traits. Besides the 600 kb BP4-BP5 CNV found in 0.5%–1% of individuals with autism spectrum disorders and schizophrenia and whose rearrangement causes reciprocal defects in head size and body weight, a second distal 220 kb BP2-BP3 CNV is likewise a potent driver of neuropsychiatric, anatomical, and metabolic pathologies. These two CNVs are engaged in complex reciprocal chromatin looping, intimating a functional relationship between genes in these regions that might be relevant to pathomechanism. We assessed the drivers of the distal 16p11.2 duplication by overexpressing each of the nine encompassed genes in zebrafish. Only overexpression of LAT induced a reduction of brain proliferating cells and concomitant microcephaly. Consistently, suppression of the zebrafish ortholog induced an increase of proliferation and macrocephaly. These phenotypes were not unique to zebrafish; Lat knockout mice show brain volumetric changes. Consistent with the hypothesis that LAT dosage is relevant to the CNV pathology, we observed similar effects upon overexpression of CD247 and ZAP70, encoding members of the LAT signalosome. We also evaluated whether LAT was interacting with KCTD13, MVP, and MAPK3, major driver and modifiers of the proximal 16p11.2 600 kb BP4-BP5 syndromes, respectively. Co-injected embryos exhibited an increased microcephaly, suggesting the presence of genetic interaction. Correspondingly, carriers of 1.7 Mb BP1-BP5 rearrangements that encompass both the BP2-BP3 and BP4-BP5 loci showed more severe phenotypes. Taken together, our results suggest that LAT, besides its well-recognized function in T cell development, is a major contributor of the 16p11.2 220 kb BP2-BP3 CNV-associated neurodevelopmental phenotypes

Protein subnetwork of human orthologs of genes parasitized by EnSpm-N6_DR elements in zebrafish classified by their involvement in specific developmental pathways, such as neurogenesis, synaptic transmission and regulation of programmed cell death.

<p>The network is visualized in STRING action view with lines and arrows of different colors indicating diverse types of interaction: binding (blue), activation (green), inhibition (red), post-translational modifications (violet) and co-expression (yellow). Of note the recently published interaction between TRIM8 and p53 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046642#pone.0046642-Caratozzolo1" target="_blank">[34]</a> was added explicitly as it is not yet annotated within the STRING database.</p

Functional p53 responsive elements map within the zebrafish-specific EnSpm-N6_DR transposon.

<p>(<b>A</b>) Predicted p53 responsive elements (REs) and their respective sequences in the first intron of <i>Danio rerio trim8a</i> long allele, <i>trim8a</i> short allele and <i>trim8b</i>. The indicated positions for REs are calculated from the annotated transcription start site. (<b>B</b>) Responsiveness to <i>Danio rerio</i> p53 transactivation of the first intron of <i>trim8a</i> engineered to contain different combinations of REs. The assessed mutant constructs are schematically represented on the left. (<b>C</b>) Zebrafish p53-dependent transactivation assessment in luciferase reporter assays of <i>Danio rerio trim8a</i> long allele, <i>trim8a</i> short allele, <i>trim8b</i> and <i>Homo sapiens p21</i> and <i>TRIM8</i>.</p

Transposon-embedded p53REs show p53-mediated transactivation.

<p>(<b>A</b>) Zebrafish p53-dependent transactivation assessment in luciferase reporter assays of 27 sequences containing EnSpm-N6_DR elements and mapping in close proximity to the indicated genes. Tested sequences are detailed in <b>Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046642#pone.0046642.s010" target="_blank">Table S2</a></b>, They are categorized by mapping location within gene loci, i.e. 5′, 3′, within the first or other more downstream exons. The names of the human orthologs are used for simplicity. Note that <i>atp2b1</i>, <i>cacna1d</i>, <i>dach2</i>, <i>drd2</i>, <i>drd4</i>, <i>fibcd1</i>, <i>grin2a</i>, <i>itga8</i>, <i>lamb2</i>, <i>pgbd1</i>, <i>rics</i>, <i>sema3a</i>, <i>sh2d3c</i>, <i>ski</i>, <i>tnfsf10</i>, <i>ttll9</i> and <i>wfikkn2</i> correspond to zebrafish <i>atp2b1a</i>, <i>cacna1da</i>, <i>dacha</i>, <i>drd4-rs</i>, <i>dkeyp-51b7.3</i>, <i>grin2aa</i>, <i>zgc:172265</i>, <i>lamb2l</i>, <i>si:ch73-353p21.4</i>, <i>dkey-269g4.4</i>, <i>sema3aa</i>, <i>sh2d3ca</i>, <i>skia</i>, <i>tnfsf10l4</i>, <i>si:dkey-211h10.2</i> and LOC564992, respectively. The percentage of transactivated targets per mapping class is shown in panel (<b>B</b>).</p

trim8a and trim8b expression in zebrafish embryos.

<p>(<b>A</b>) Expression pattern of <i>trim8b</i> at 18 hpf determined by whole mount ISH. Left panel: lateral view; right panel: dorsal view. Of note <i>trim8a</i> is not detected at this stage. (<b>B</b>) Comparison of the expression patterns of <i>trim8a</i> (top row) and <i>trim8b</i> (bottom row) after 30 to 72 hours of development. Embryos are in lateral (first three columns) or dorsal views (far right column). <i>Trim8b</i> expression at 48 hpf and 72 hpf (lateral view) is shown in de-yolked and flat mounted embryos (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046642#s4" target="_blank">Methods</a>). Anterior to the left. AD, anterior diencephalon; C, cerebellum; DD, dorsal diencephalon; DH, dorsal hindbrain; H, hindbrain; OT, optic tectum; R, retina; SCN, ventral spinal chord neurons; T, tegmentum; TG, trigeminal ganglia; VMB ventro-lateral midbrain; VZ, brain ventricular zone.</p

The Immune Signaling Adaptor LAT Contributes to the Neuroanatomical Phenotype of 16p11.2 BP2-BP3 CNVs

Copy-number changes in 16p11.2 contribute significantly to neuropsychiatric traits. Besides the 600 kb BP4-BP5 CNV found in 0.5%-1% of individuals with autism spectrum disorders and schizophrenia and whose rearrangement causes reciprocal defects in head size and body weight, a second distal 220 kb BP2-BP3 CNV is likewise a potent driver of neuropsychiatric, anatomical, and metabolic pathologies. These two CNVs are engaged in complex reciprocal chromatin looping, intimating a functional relationship between genes in these regions that might be relevant to pathomechanism. We assessed the drivers of the distal 16p11.2 duplication by overexpressing each of the nine encompassed genes in zebrafish. Only overexpression of LAT induced a reduction of brain proliferating cells and concomitant microcephaly. Consistently, suppression of the zebrafish ortholog induced an increase of proliferation and macrocephaly. These phenotypes were not unique to zebrafish; Lat knockout mice show brain volumetric changes. Consistent with the hypothesis that LAT dosage is relevant to the CNV pathology, we observed similar effects upon overexpression of CD247 and ZAP70, encoding members of the LAT signalosome. We also evaluated whether LAT was interacting with KCTD13, MVP, and MAPK3, major driver and modifiers of the proximal 16p11.2 600 kb BP4-BP5 syndromes, respectively. Co-injected embryos exhibited an increased microcephaly, suggesting the presence of genetic interaction. Correspondingly, carriers of 1.7 Mb BP1-BP5 rearrangements that encompass both the BP2-BP3 and BP4-BP5 loci showed more severe phenotypes. Taken together, our results suggest that LAT, besides its well-recognized function in T cell development, is a major contributor of the 16p11.2 220 kb BP2-BP3 CNV-associated neurodevelopmental phenotypes.PMC563023

The Immune Signaling Adaptor LAT Contributes to the Neuroanatomical Phenotype of 16p11.2 BP2-BP3 CNVs

Copy-number changes in 16p11.2 contribute significantly to neuropsychiatric traits. Besides the 600 kb BP4-BP5 CNV found in 0.5%–1% of individuals with autism spectrum disorders and schizophrenia and whose rearrangement causes reciprocal defects in head size and body weight, a second distal 220 kb BP2-BP3 CNV is likewise a potent driver of neuropsychiatric, anatomical, and metabolic pathologies. These two CNVs are engaged in complex reciprocal chromatin looping, intimating a functional relationship between genes in these regions that might be relevant to pathomechanism. We assessed the drivers of the distal 16p11.2 duplication by overexpressing each of the nine encompassed genes in zebrafish. Only overexpression of LAT induced a reduction of brain proliferating cells and concomitant microcephaly. Consistently, suppression of the zebrafish ortholog induced an increase of proliferation and macrocephaly. These phenotypes were not unique to zebrafish; Lat knockout mice show brain volumetric changes. Consistent with the hypothesis that LAT dosage is relevant to the CNV pathology, we observed similar effects upon overexpression of CD247 and ZAP70, encoding members of the LAT signalosome. We also evaluated whether LAT was interacting with KCTD13, MVP, and MAPK3, major driver and modifiers of the proximal 16p11.2 600 kb BP4-BP5 syndromes, respectively. Co-injected embryos exhibited an increased microcephaly, suggesting the presence of genetic interaction. Correspondingly, carriers of 1.7 Mb BP1-BP5 rearrangements that encompass both the BP2-BP3 and BP4-BP5 loci showed more severe phenotypes. Taken together, our results suggest that LAT, besides its well-recognized function in T cell development, is a major contributor of the 16p11.2 220 kb BP2-BP3 CNV-associated neurodevelopmental phenotypes

A Potential Contributory Role for Ciliary Dysfunction in the 16p11.2 600 kb BP4-BP5 Pathology

The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.status: publishe

A fish-specific transposable element shapes the repertoire of p53 target genes in zebrafish.

Transposable elements, as major components of most eukaryotic organisms' genomes, define their structural organization and plasticity. They supply host genomes with functional elements, for example, binding sites of the pleiotropic master transcription factor p53 were identified in LINE1, Alu and LTR repeats in the human genome. Similarly, in this report we reveal the role of zebrafish (Danio rerio) EnSpmN6_DR non-autonomous DNA transposon in shaping the repertoire of the p53 target genes. The multiple copies of EnSpmN6_DR and their embedded p53 responsive elements drive in several instances p53-dependent transcriptional modulation of the adjacent gene, whose human orthologs were frequently previously annotated as p53 targets. These transposons define predominantly a set of target genes whose human orthologs contribute to neuronal morphogenesis, axonogenesis, synaptic transmission and the regulation of programmed cell death. Consistent with these biological functions the orthologs of the EnSpmN6_DR-colonized loci are enriched for genes expressed in the amygdala, the hippocampus and the brain cortex. Our data pinpoint a remarkable example of convergent evolution: the exaptation of lineage-specific transposons to shape p53-regulated neuronal morphogenesis-related pathways in both a hominid and a teleost fish