The emergence of functional neuronal connectivity in the developing cerebral cortex depends on neuronal migration. This process enables appropriate positioning of neurons and the emergence of ...neuronal identity so that the correct patterns of functional synaptic connectivity between the right types and numbers of neurons can emerge. Delineating the complexities of neuronal migration is critical to our understanding of normal cerebral cortical formation and neurodevelopmental disorders resulting from neuronal migration defects. For the most part, the integrated cell biological basis of the complex behavior of oriented neuronal migration within the developing mammalian cerebral cortex remains an enigma. This review aims to analyze the integrative mechanisms that enable neurons to sense environmental guidance cues and translate them into oriented patterns of migration toward defined areas of the cerebral cortex. We discuss how signals emanating from different domains of neurons get integrated to control distinct aspects of migratory behavior and how different types of cortical neurons coordinate their migratory activities within the developing cerebral cortex to produce functionally critical laminar organization.
Investigating the developmental, structural and functional complexity of mammalian tissues and organs depends on identifying and gaining experimental access to diverse cell populations. Here, we ...describe a set of recombinase-responsive fluorescent indicator alleles in mice that significantly extends our ability to uncover cellular diversity by exploiting the intrinsic genetic signatures that uniquely define cell types. Using a recombinase-based intersectional strategy, these new alleles uniquely permit non-invasive labeling of cells defined by the overlap of up to three distinct gene expression domains. In response to different combinations of Cre, Flp and Dre recombinases, they express eGFP and/or tdTomato to allow the visualization of full cellular morphology. Here, we demonstrate the value of these features through a proof-of-principle analysis of the central noradrenergic system. We label previously inaccessible subpopulations of noradrenergic neurons to reveal details of their three-dimensional architecture and axon projection profiles. These new indicator alleles will provide experimental access to cell populations at unprecedented resolution, facilitating analysis of their developmental origin and anatomical, molecular and physiological properties.
Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is ...the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5′ UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.
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•DDX39B is a potent activator of IL7R exon 6 splicing and a repressor of sIL7R•DDX39B genetic variants are significantly associated with MS risk•The 5′ UTR DDX39B variant reduces protein levels by decreasing translation efficiency•This variant shows strong genetic and functional epistasis with IL7R rs6897932
Two genes interact epistatically in multiple sclerosis risk in humans.
The default mode network (DMN) of the brain is functionally associated with a wide range of behaviors. In this study, we used functional magnetic resonance imaging (fMRI), positron emission ...tomography (PET), and spectral fiber photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)-releasing noradrenergic neurons in the locus coeruleus (LC) on the mouse DMN. Chemogenetic-induced tonic LC activity decreased cerebral blood volume (CBV) and glucose uptake and increased synchronous low-frequency fMRI activity within the frontal cortices of the DMN. Fiber photometry results corroborated these findings, showing that LC-NE activation induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate cortex. These data suggest that LC-NE alters conventional coupling between neuronal activity and CBV in the frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional connectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retrosplenial and hippocampal regions to the association cortices of the DMN.
Contextual fear learning is heavily dependent on the hippocampus. Despite evidence that catecholamines contribute to contextual encoding and memory retrieval, the precise temporal dynamics of their ...release in the hippocampus during behavior is unknown. In addition, new animal models are required to probe the effects of altered catecholamine synthesis on release dynamics and contextual learning.
We generated two new mouse models of altered locus coeruleus norepinephrine (LC-NE) synthesis and utilized them together with GRABNE and GRABDA sensors and in vivo fiber photometry to investigate norepinephrine (NE) and dopamine (DA) release dynamics in dorsal hippocampal CA1 during contextual fear conditioning.
We report that aversive foot-shock increases both NE and DA release in dorsal CA1, while freezing behavior associated with recall of fear memory is accompanied by decreased release. Moreover, we find that freezing at the recent timepoint is sensitive to both partial and complete loss of LC-NE synthesis throughout prenatal and postnatal development, similar to prior observations of mice with global loss of NE synthesis beginning postnatally. In contrast, freezing at the remote timepoint is compromised only by complete loss of LC-NE synthesis beginning prenatally.
Overall, these findings provide novel insights into the role of NE in contextual fear and the precise temporal dynamics of both NE and DA during freezing behavior, and highlight a complex relationship between genotype, sex, and NE signaling.
Alternative splicing is a general mechanism for regulating gene expression that affects the RNA products of more than 90% of human genes. Not surprisingly, alternative splicing is observed among gene ...products of metazoan immune systems, which have evolved to efficiently recognize pathogens and discriminate between "self" and "non-self", and thus need to be both diverse and flexible. In this review we focus on the specific interface between alternative splicing and autoimmune diseases, which result from a malfunctioning of the immune system and are characterized by the inappropriate reaction to self-antigens. Despite the widespread recognition of alternative splicing as one of the major regulators of gene expression, the connections between alternative splicing and autoimmunity have not been apparent. We summarize recent findings connecting splicing and autoimmune disease, and attempt to find common patterns of splicing regulation that may advance our understanding of autoimmune diseases and open new avenues for therapy.
Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage, and its expression is crucial for the development and maintenance of T cells. Alternative splicing of IL7R ...exon 6 results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We identified multiple exonic and intronic cis-acting elements that impact inclusion of exon 6. Moreover, we utilized RNA affinity chromatography followed by mass spectrometry to identify trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein-binding candidates. A consensus polyadenylation signal AAUAAA is present in intron 6 of IL7R directly downstream from the 5' splice site. Mutations to this site and CPSF1 knockdown both resulted in an increase in exon 6 inclusion. We found no evidence that this site is used to produce cleaved and polyadenylated mRNAs, suggesting that CPSF1 interaction with intronic IL7R pre-mRNA interferes with spliceosome binding to the exon 6 5' splice site. Our results suggest that competing mRNA splicing and polyadenylation regulate exon 6 inclusion and consequently determine the ratios of soluble to membrane-bound IL7R. This may be relevant for both T cell ontogeny and function and development of multiple sclerosis.
Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage and its expression is crucial for development and maintenance of T cells. While transcriptional regulation of ...IL7R expression has been widely studied, its posttranscriptional regulation has only recently been uncovered. Alternative splicing of IL7R exon 6, the only exon that encodes the transmembrane domain of the receptor, results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms, respectively. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis, a prototypic demyelinating disease of the central nervous system. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We utilized mutagenesis of exon 6 and surrounding introns to identify multiple exonic and intronic cis-acting regulatory elements that impact inclusion of exon 6. At least two of these elements, one exonic splicing silencer and one exonic splicing enhancer, are located in the direct vicinity of the MS-associated SNP. We also uncovered a consensus polyadenylation signal, AAUAAA in intron 6 of IL7R, 16 nucleotides downstream from exon 6 5' splice site, and showed that mutations to this site resulted in an increase in exon 6 inclusion. Additionally, we determined that the 5' splice site of exon 6 is weak. We propose that this site may be responsible for exon 6 splicing regulation. Using tobramycin RNA affinity chromatography followed by mass spectrometry, we identified trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein binding candidates. siRNA-mediated knockdown of CPSF1 resulted in an increase in exon 6 inclusion, consistent with the results of mutations to the CPSF1 binding site. Correspondingly, CPSF1 depletion had no effect on a minigene with a mutation in the intronic polyadenylation site. Finally, 3'RACE and RT-PCR experiments on RNA from Jurkat cells suggested that the intronic AAUAAA site is utilized at low frequency by the polyadenylation machinery to produce a novel polyadenylated mRNA isoform. Together, our results suggest that competing pre-mRNA splicing and polyadenylation may regulate exon 6 inclusion and resultant levels of functional IL7R produced. Since the intronic polyadenylated isoform of IL7R is predicted to be translated into a membrane-bound protein product with a shortened, signal transduction-incompetent cytoplasmic tail, this may be relevant for both T cell biology and development of multiple sclerosis.
Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage and its expression is crucial for development and maintenance of T cells. While transcriptional regulation of ...IL7R expression has been widely studied, its posttranscriptional regulation has only recently been uncovered. Alternative splicing of IL7R exon 6, the only exon that encodes the transmembrane domain of the receptor, results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms, respectively. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis, a prototypic demyelinating disease of the central nervous system. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing.We utilized mutagenesis of exon 6 and surrounding introns to identify multiple exonic and intronic cis-acting regulatory elements that impact inclusion of exon 6. At least two of these elements, one exonic splicing silencer and one exonic splicing enhancer, are located in the direct vicinity of the MS-associated SNP. We also uncovered a consensus polyadenylation signal, AAUAAA in intron 6 of IL7R, 16 nucleotides downstream from exon 6 5' splice site, and showed that mutations to this site resulted in an increase in exon 6 inclusion. Additionally, we determined that the 5' splice site of exon 6 is weak. We propose that this site may be responsible for exon 6 splicing regulation.Using tobramycin RNA affinity chromatography followed by mass spectrometry, we identified trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein binding candidates. siRNA-mediated knockdown of CPSF1 resulted in an increase in exon 6 inclusion, consistent with the results of mutations to the CPSF1 binding site. Correspondingly, CPSF1 depletion had no effect on a minigene with a mutation in the intronic polyadenylation site. Finally, 3'RACE and RT-PCR experiments on RNA from Jurkat cells suggested that the intronic AAUAAA site is utilized at low frequency by the polyadenylation machinery to produce a novel polyadenylated mRNA isoform. Together, our results suggest that competing pre-mRNA splicing and polyadenylation may regulate exon 6 inclusion and resultant levels of functional IL7R produced. Since the intronic polyadenylated isoform of IL7R is predicted to be translated into a membrane-bound protein product with a shortened, signal transduction-incompetent cytoplasmic tail, this may be relevant for both T cell biology and development of multiple sclerosis.
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