tissue-collection-of-bats-for-omics-analyses-and-primary-cell-culture

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Max Planck Institute for Psycholinguistics

Retinoic acid-related orphan receptor alpha gene (RORa) and the microRNA MIR137 have both recently been identified as novel candidate genes for neuropsychiatric disorders. RORa encodes a ligand-dependent orphan nuclear receptor that acts as a transcriptional regulator and miR-137 is a brain enriched small non-coding RNA that interacts with gene transcripts to control protein levels. Given the mounting evidence for RORa in autism spectrum disorders (ASD) and MIR137 in schizophrenia and ASD, we investigated if there was a functional biological relationship between these two genes. Herein, we demonstrate that miR-137 targets the 3'UTR of RORa in a site specific manner. We also provide further support for MIR137 as an autism candidate by showing that a large number of previously implicated autism genes are also putatively targeted by miR-137. This work supports the role of MIR137 as an ASD candidate and demonstrates a direct biological link between these previously unrelated autism candidate genes. 

A direct molecular link between the autism candidate gene RORa and the schizophrenia candidate MIR137.

FOXP2 was the first gene shown to cause a Mendelian form of speech and language disorder. Although developmentally expressed in many organs, loss of a single copy of FOXP2 leads to a phenotype that is largely restricted to orofacial impairment during articulation and linguistic processing deficits. Why perturbed FOXP2 function affects specific aspects of the developing brain remains elusive. We investigated the role of FOXP2 in neuronal differentiation and found that FOXP2 drives molecular changes consistent with neuronal differentiation in a human model system. We identified a network of FOXP2 regulated genes related to retinoic acid signaling and neuronal differentiation. FOXP2 also produced phenotypic changes associated with neuronal differentiation including increased neurite outgrowth and reduced migration. Crucially, cells expressing FOXP2 displayed increased sensitivity to retinoic acid exposure. This suggests a mechanism by which FOXP2 may be able to increase the cellular differentiation response to environmental retinoic acid cues for specific subsets of neurons in the brain. These data demonstrate that FOXP2 promotes neuronal differentiation by interacting with the retinoic acid signaling pathway and regulates key processes required for normal circuit formation such as neuronal migration and neurite outgrowth. In this way, FOXP2, which is found only in specific subpopulations of neurons in the brain, may drive precise neuronal differentiation patterns and/or control localization and connectivity of these FOXP2 positive cells. 

FOXP2 drives neuronal differentiation by interacting with retinoic acid signaling pathways.

Mutations of FOXP2 in 7q31 cause a rare disorder involving speech apraxia, accompanied by expressive and receptive language impairments. A recent report described a child with speech and language deficits, and a genomic rearrangement affecting chromosomes 7 and 11. One breakpoint mapped to 7q31 and, although outside its coding region, was hypothesised to disrupt FOXP2 expression. We identified an element 2 kb downstream of this breakpoint with epigenetic characteristics of an enhancer. We show that this element drives reporter gene expression in human cell-lines. Thus, displacement of this element by translocation may disturb gene expression, contributing to the observed language phenotype. 

A chromosomal rearrangement in a child with severe speech and language disorder separates FOXP2 from a functional enhancer.

Neurodevelopmental disorders have a strong genetic component, but despite widespread efforts, the specific genetic factors underlying these disorders remain undefined for a large proportion of affected individuals. Given the accessibility of exome sequencing, this problem has thus far been addressed from a protein-centric standpoint; however, protein-coding regions only make up ∼1% to 2% of the human genome. With the advent of whole genome sequencing we are in the midst of a paradigm shift as it is now possible to interrogate the entire sequence of the human genome (coding and noncoding) to fill in the missing heritability of complex disorders. These new technologies bring new challenges, as the number of noncoding variants identified per individual can be overwhelming, making it prudent to focus on noncoding regions of known function, for which the effects of variation can be predicted and directly tested to assess pathogenicity. The 3'UTRome is a region of the noncoding genome that perfectly fulfills these criteria and is of high interest when searching for pathogenic variation related to complex neurodevelopmental disorders. Herein, we review the regulatory roles of the 3'UTRome as binding sites for microRNAs or RNA binding proteins, or during alternative polyadenylation. We detail existing evidence that these regions contribute to neurodevelopmental disorders and outline strategies for identification and validation of novel putatively pathogenic variation in these regions. This evidence suggests that studying the 3'UTRome will lead to the identification of new risk factors, new candidate disease genes, and a better understanding of the molecular mechanisms contributing to neurodevelopmental disorders.

Understanding Neurodevelopmental Disorders: The Promise of Regulatory Variation in the 3'UTRome.

Genes including FOXP2, FOXP1, and CNTNAP2, have been implicated in human speech and language phenotypes, pointing to a role in the development of normal language-related circuitry in the brain. Although speech and language are unique to humans a comparative approach is possible by addressing language-relevant traits in animal systems. One such trait, vocal learning, represents an essential component of human spoken language, and is shared by cetaceans, pinnipeds, elephants, some birds and bats. Given their vocal learning abilities, gregarious nature, and reliance on vocalizations for social communication and navigation, bats represent an intriguing mammalian system in which to explore language-relevant genes. We used immunohistochemistry to detail the distribution of FoxP2, FoxP1, and Cntnap2 proteins, accompanied by detailed cytoarchitectural histology in the brains of two vocal learning bat species; Phyllostomus discolor and Rousettus aegyptiacus. We show widespread expression of these genes, similar to what has been previously observed in other species, including humans. A striking difference was observed in the adult P. discolor bat, which showed low levels of FoxP2 expression in the cortex that contrasted with patterns found in rodents and nonhuman primates. We created an online, open-access database within which all data can be browsed, searched, and high resolution images viewed to single cell resolution. The data presented herein reveal regions of interest in the bat brain and provide new opportunities to address the role of these language-related genes in complex vocal-motor and vocal learning behaviors in a mammalian model system.

Mapping the distribution of language related genes FoxP1, FoxP2, and CntnaP2 in the brains of vocal learning bat species.

Mutations of the  gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with  mutations have structural and functional alterations affecting brain circuits that overlap with sites of  expression, including regions of the cortex, striatum, and cerebellum.  displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of  or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human  locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the  promoter and enhancer regions by candidate regulators - FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human . Understanding the upstream regulatory pathways controlling  expression will bring new insight into the molecular networks contributing to human language and related disorders.

Mapping of Human  Enhancers Reveals Complex Regulation.

Intellectual disability (ID) is a severe neurodevelopmental disorder with genetically heterogeneous causes. Large-scale sequencing has led to the identification of many gene-disrupting mutations; however, a substantial proportion of cases lack a molecular diagnosis. As such, there remains much to uncover for a complete understanding of the genetic underpinnings of ID. Genetic variants present in non-coding regions of the genome have been highlighted as potential contributors to neurodevelopmental disorders given their role in regulating gene expression. Nevertheless the functional characterization of non-coding variants remains challenging. We describe the identification and characterization of de novo non-coding variation in 3'UTR regulatory regions within an ID cohort of 50 patients. This cohort was previously screened for structural and coding pathogenic variants via CNV, whole exome and whole genome analysis. We identified 44 high-confidence single nucleotide non-coding variants within the 3'UTR regions of these 50 genomes. Four of these variants were located within predicted miRNA binding sites and were thus hypothesised to have regulatory consequences. Functional testing showed that two of the variants interfered with miRNA-mediated regulation of their target genes, AMD1 and FAIM. Both these variants were found in the same individual and their functional consequences may point to a potential role for such variants in intellectual disability.

Article	Year
A direct molecular link between the autism candidate gene RORa and the schizophrenia candidate MIR137. Scientific reports\| PubMed ID: 24500708	2014
FOXP2 drives neuronal differentiation by interacting with retinoic acid signaling pathways. Frontiers in cellular neuroscience\| PubMed ID: 25309332	2014
A chromosomal rearrangement in a child with severe speech and language disorder separates FOXP2 from a functional enhancer. Molecular cytogenetics\| PubMed ID: 26300977	2015
Understanding Neurodevelopmental Disorders: The Promise of Regulatory Variation in the 3'UTRome. Biological psychiatry\| PubMed ID: 29289333	2018
Mapping the distribution of language related genes FoxP1, FoxP2, and CntnaP2 in the brains of vocal learning bat species. The Journal of comparative neurology\| PubMed ID: 29297931	2018
Mapping of Human Enhancers Reveals Complex Regulation. Frontiers in molecular neuroscience\| PubMed ID: 29515369	2018
Genome-wide investigation of an ID cohort reveals de novo 3'UTR variants affecting gene expression. Human genetics\| PubMed ID: 30097719	2018

Paolo Devanna

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