High affinity enhancer-promoter interactions can bypass CTCF/cohesin-mediated insulation and contribute to phenotypic robustness

Transcriptional control by distal enhancers is an integral feature of gene regulation. To understand how enhancer-promoter interactions arise and assess the impact of disrupting 3D chromatin structure on gene expression, we generated an allelic series of mouse mutants that perturb the physical structure of the Sox2 locus. We show that in the epiblast and in neuronal tissues, CTCF-mediated loops are neither required for the interaction of the Sox2 promoter with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs between Sox2 and its distal enhancers generated ectopic loops with varying degrees of insulation that directly correlated with reduced transcriptional output. Yet, even the mutants exhibiting the strongest insulation, with six CTCF motifs in divergent orientation, could not fully abolish activation by distal enhancers, and failed to disrupt implantation and neurogenesis. In contrast, cells of the anterior foregut were more susceptible to chromatin structure disruption with no detectable SOX2 expression in mutants with the strongest CTCF-mediated boundaries. These animals phenocopied loss of SOX2 in the anterior foregut, failed to separate trachea from esophagus and died perinatally. We propose that baseline transcription levels and enhancer density may influence the tissue-specific ability of distal enhancers to overcome physical barriers and maintain faithful gene expression. Our work suggests that high affinity enhancer-promoter interactions that can overcome chromosomal structural perturbations, play an essential role in maintaining phenotypic robustness.

Evolutionarily conserved genetic interactions between nphp-4 and bbs-5 mutations exacerbate ciliopathy phenotypes

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.

ABNORMAL LYMPHATIC PHENOTYPE IN A CRISPR MOUSE MODEL OF THE HUMAN LYMPHEDEMA-CAUSING CONNEXIN47 R260C POINT MUTATION

Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p<0.01). Associated lymphatic channels were increased in Cx47R259C mice and mesenteric lymph reflux occurred in homozygous Cx47R259C mice but not in wildtype. Contractility of superficial cervical lymphatics, assessed by pressure myography, was reduced in homozygous Cx47R259C mice compared to wildtype. In conclusion, our data are the first to demonstrate a role for the Cx47 protein in lymphatic anatomy and function. This phenotype is similar to that found with other valve deficient mouse mutants, e.g., in Foxc2. Of significance, this study is the first to use CRISPR technology to develop a pre-clinical model of primary lymphedema and demonstrates the importance of distinguishing between lack of and presence of mutant protein when developing clinically relevant animal models for translation of pre-clinical findings.

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

Brain mapping across 16 autism mouse models reveals a spectrum of functional connectivity subtypes

Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches.

Conditional Loss of BAF (mSWI/SNF) Scaffolding Subunits Affects Specification and Proliferation of Oligodendrocyte Precursors in Developing Mouse Forebrain

Oligodendrocytes are responsible for axon myelination in the brain and spinal cord. Generation of oligodendrocytes entails highly regulated multistage neurodevelopmental events, including proliferation, differentiation and maturation. The chromatin remodeling BAF (mSWI/SNF) complex is a notable regulator of neural development. In our previous studies, we determined the indispensability of the BAF complex scaffolding subunits BAF155 and BAF170 for neurogenesis, whereas their role in gliogenesis is unknown. Here, we show that the expression of BAF155 and BAF170 is essential for the genesis of oligodendrocytes during brain development. We report that the ablation of BAF155 and BAF170 in the dorsal telencephalic (dTel) neural progenitors or in oligodendrocyte-producing progenitors in the ventral telencephalon (vTel) in double-conditional knockout (dcKO) mouse mutants, perturbed the process of oligodendrogenesis. Molecular marker and cell cycle analyses revealed impairment of oligodendrocyte precursor specification and proliferation, as well as overt depletion of oligodendrocytes pool in dcKO mutants. Our findings unveil a central role of BAF155 and BAF170 in oligodendrogenesis, and thus substantiate the involvement of the BAF complex in the production of oligodendrocytes in the forebrain.

SORBS2 is a genetic factor contributing to cardiac malformation of 4q deletion syndrome patients

Chromosome 4q deletion is one of the most frequently detected genomic imbalance events in congenital heart disease (CHD) patients. However, a portion of CHD-associated 4q deletions without known CHD genes suggests unknown CHD genes within these intervals. Here, we have shown that knockdown of SORBS2, a 4q interval gene, disrupted sarcomeric integrity of cardiomyocytes and caused reduced cardiomyocyte number in human embryonic stem cell differentiation model. Molecular analyses revealed decreased expression of second heart field (SHF) marker genes and impaired NOTCH and SHH signaling in SORBS2-knockdown cells. Exogenous SHH rescued SORBS2 knockdown-induced cardiomyocyte differentiation defects. Sorbs2-/- mouse mutants had atrial septal hypoplasia/aplasia or double atrial septum (DAS) derived from impaired posterior SHF with a similar expression alteration. Rare SORBS2 variants were significantly enriched in a cohort of 300 CHD patients. Our findings indicate that SORBS2 is a regulator of SHF development and its variants contribute to CHD pathogenesis. The presence of DAS in Sorbs2-/- hearts reveals the first molecular etiology of this rare anomaly linked to paradoxical thromboembolism.

In vivo 5-ethynyluridine (EU) labelling detects reduced transcription in Purkinje cell degeneration mouse mutants, but can itself induce neurodegeneration

Fluorescent staining of newly transcribed RNA via metabolic labelling with 5-ethynyluridine (EU) and click chemistry enables visualisation of changes in transcription, such as in conditions of cellular stress. Here, we tested whether EU labelling can be used to examine transcription in vivo in mouse models of nervous system disorders. We show that injection of EU directly into the cerebellum results in reproducible labelling of newly transcribed RNA in cerebellar neurons and glia, with cell type-specific differences in relative labelling intensities, such as Purkinje cells exhibiting the highest levels. We also observed EU-labelling accumulating into cytoplasmic inclusions, indicating that EU, like other modified uridines, may introduce non-physiological properties in labelled RNAs. Additionally, we found that EU induces Purkinje cell degeneration nine days after EU injection, suggesting that EU incorporation not only results in abnormal RNA transcripts, but also eventually becomes neurotoxic in highly transcriptionally-active neurons. However, short post-injection intervals of EU labelling in both a Purkinje cell-specific DNA repair-deficient mouse model and a mouse model of spinocerebellar ataxia 1 revealed reduced transcription in Purkinje cells compared to controls. We combined EU labelling with immunohistology to correlate altered EU staining with pathological markers, such as genotoxic signalling factors. These data indicate that the EU-labelling method provided here can be used to identify changes in transcription in vivo in nervous system disease models.

Early loss of Scribble affects cortical development, interhemispheric connectivity and psychomotor activity

Neurodevelopmental disorders arise from combined defects in processes including cell proliferation, differentiation, migration and commissure formation. The evolutionarily conserved tumor-suppressor protein Scribble (Scrib) serves as a nexus to transduce signals for the establishment of apicobasal and planar cell polarity during these processes. Human SCRIB gene mutations are associated with neural tube defects and this gene is located in the minimal critical region deleted in the rare Verheij syndrome. In this study, we generated brain-specific conditional cKO mouse mutants and assessed the impact of the Scrib deletion on brain morphogenesis and behavior. We showed that embryonic deletion of Scrib in the telencephalon leads to cortical thickness reduction (microcephaly) and partial corpus callosum and hippocampal commissure agenesis. We correlated these phenotypes with a disruption in various developmental mechanisms of corticogenesis including neurogenesis, neuronal migration and axonal connectivity. Finally, we show that Scrib cKO mice have psychomotor deficits such as locomotor activity impairment and memory alterations. Altogether, our results show that Scrib is essential for early brain development due to its role in several developmental cellular mechanisms that could underlie some of the deficits observed in complex neurodevelopmental pathologies.

Fibroblast Growth Factor Signalling in the Diseased Nervous System

Fibroblast growth factors (FGFs) act as key signalling molecules in brain development, maintenance, and repair. They influence the intricate relationship between myelinating cells and axons as well as the association of astrocytic and microglial processes with neuronal perikarya and synapses. Advances in molecular genetics and imaging techniques have allowed novel insights into FGF signalling in recent years. Conditional mouse mutants have revealed the functional significance of neuronal and glial FGF receptors, not only in tissue protection, axon regeneration, and glial proliferation but also in instant behavioural changes. This review provides a summary of recent findings regarding the role of FGFs and their receptors in the nervous system and in the pathogenesis of major neurological and psychiatric disorders.