Biology subjects

Auer, D.

Publications and source records attributed to Auer, D..

8 recordsLinked to original sources

bioRxiv2022

Cardiac muscle-restricted partial loss of Nos1ap expression has limited impact on electro- and echo-cardiographic features

Genome-wide association studies (GWAS) of QT interval variation have identified common noncoding variants at the NOS1AP gene as the most common genetic regulators of trait variation in the general population. Invoking a cis-regulatory mechanistic hypothesis, we have reported identification of a functional enhancer variant underlying the GWAS signal that influenced human cardiac NOS1AP expression. Functional studies based on in vitro overexpression in murine cardiomyocytes and ex vivo knockdown in zebrafish embryonic hearts, by us and others, have demonstrated that NOS1AP expression levels can alter cellular electrophysiology. Here, to explore the role of NOS1AP in cardiac electrophysiology at an organismal level, we generated and characterized constitutive and heart muscle-restricted Nos1ap knockout mice to assess whether NOS1AP disruption alters the QT interval in vivo. Constitutive loss of Nos1ap led to genetic background-dependent variable lethality at or right before birth. Heart muscle-restricted Nos1ap knockouts generated using cardiac specific alpha-myosin heavy chain promoter-driven tamoxifen-inducible Cre resulted in tissue-level Nos1ap expression reduced by half. This partial loss of expression had no detectable effect on the QT interval, but led to a small yet significant reduction in the QRS interval. Given that challenges associated with defining the end of T wave on murine electrocardiogram can limit identification of subtle effects on QT interval, and that common noncoding NOS1AP variants are also associated with QRS interval, our findings support the role of NOS1AP in regulation of the cardiac electrical cycle.

genetics↗

bioRxiv2021

Ret loss-of-function decreases enteric neural crest progenitor proliferation and restricts developmental fate potential during enteric nervous system development

The receptor tyrosine kinase gene RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. Pathogenic RET loss of function (LoF) alleles are associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. ENCDCs invade the developing GI tract, proliferate, migrate caudally, and differentiate into all of the major ENS cell types. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, its cell type and state-specific effects are unknown. Consequently, we performed single- cell RNA sequencing (scRNA-seq) on an enriched population of ENCDCs isolated from the developing GI tract of Ret null heterozygous and homozygous mouse embryos at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: (1) Ret-expressing ENCDCs are a heterogeneous population composed of ENS progenitors as well as glial and neuronal committed cells; (2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; (3) HSCR-associated and Ret gene regulatory network genes exhibit distinct expression patterns across Ret-expressing ENCDC with their expression impacted by Ret LoF; and (4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes associated with the proper development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment. Summary StatementRet LoF affects proper development of the mouse ENS through multiple distinct cellular phenotypes including restriction of neuronal fate potential, disruption of ENCDC migration, and modulation of progenitor proliferation rate.

developmental biology↗

bioRxiv2020

Comprehensive label-free characterization of extracellular vesicles and their surface proteins

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Preclinical studies show that MSC-EVs have anti-apoptotic and neuroprotective effects, boost wound healing, and improve the integration of allogeneic grafts through immunomodulation. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers and gauge their prevalence. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and non-vesicular particles (NVPs) in a therapeutic formulation. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV formulations and opens the door to improved quality control.

molecular biology↗

bioRxiv2020

Multiple, independent, common variants overlapping known and putative gut enhancers at RET, SEMA3 and NRG1 underlie Hirschsprung disease risk in European ancestry subjects

PurposeHirschsprung disease (HSCR) is a developmental disorder of the enteric nervous system (ENS) characterized by congenital aganglionosis, and where individual cases harbor coding risk variants in ENS genes. Low-penetrance, common, noncoding variants at RET, SEMA3 and NRG1 loci have been associated in HSCR as well, implicating variable gene expression mediated by cis-regulatory element (CRE) variants as the causal mechanism. However, the extent and combinatorial effects of all putative CRE variants within and across these loci on HSCR is unknown. MethodsUsing 583 HSCR subjects, one of the largest samples of European ancestry studied, and genotyping 56 tag variants we evaluated association of all common variants overlapping putative gut CREs and fine-mapped variants at RET, SEMA3 and NRG1. ResultsWe demonstrate that 28 and 8 tag variants, several of which are independent, overlapping putative-enhancers at the RET and SEMA3 loci, respectively, and, 2 fine-mapped tag variants at NRG1 locus, are associated with HSCR. We demonstrate that risk increases with increasing risk allele dosage from multiple variants within and across these loci and varies >25- fold. ConclusionGene regulatory networks in HSCR-relevant cell types quantify the total burden of risk alleles through sensing reduced gene expression of multiple genes on disease.

genetics↗

bioRxiv2020

Identifying the spatial and temporal dynamics of molecularly-distinct glioblastoma sub-populations

Glioblastomas (GBMs) are the most aggressive primary brain tumours and have no known cure. Each individual tumour comprises multiple sub-populations of genetically-distinct cells that may respond differently to targeted therapies and may contribute to disappointing clinical trial results. Image-localized biopsy techniques allow multiple biopsies to be taken during surgery and provide information that identifies regions where particular sub-populations occur within an individual GBM, thus providing insight into their regional genetic variability. These sub-populations may also interact with one another through a competitive or cooperative nature; it is important to ascertain the nature of these interactions, as they may have implications for responses to targeted therapies. We combine genetic information from biopsies with a mechanistic model of interacting GBM sub-populations to characterise the nature of interactions between two commonly occurring GBM sub-populations, those with EGFR and PDGFRA genes amplified. We study population levels found across image-localized biopsy data from a cohort of 25 patients and compare this to model outputs under competitive, cooperative and neutral interaction assumptions. We explore other factors affecting the observed simulated sub-populations, such as selection advantages and phylogenetic ordering of mutations, which may also contribute to the levels of EGFR and PDGFRA amplified populations observed in biopsy data.

cancer biology↗

bioRxiv2019

The gastrointestinal development ‘parts list’: transcript profiling of embryonic gut development in wildtype and Ret-deficient mice

The development of the gut from endodermal tissue to an organ with multiple distinct structures and functions occurs over a prolonged time during embryonic days E10.5-E14.5 in the mouse. During this process, one major event is innervation of the gut by enteric neural crest cells (ENCC) to establish the enteric nervous system (ENS). To understand the molecular processes underpinning gut and ENS development, we generated RNA-seq profiles from wildtype mouse guts at E10.5, E12.5 and E14.5 from both sexes. We also generated these profiles from homozygous Ret null embryos, a model for Hirschsprung disease (HSCR), in whom the ENS is absent. These data reveal four major features: (1) between E10.5 to E14.5 the developmental genetic programs change from expression of major transcription factors (TF) and its modifiers to genes controlling tissue (epithelium, muscle, endothelium) specialization; (2) the major effect of Ret is not only on ENCC differentiation to enteric neurons but also on the enteric mesenchyme and epithelium; (3) a muscle genetic program exerts significant effects on ENS development, and (4) sex differences in gut development profiles are minor. The genetic programs identified, and their changes across development, suggests that both cell autonomous and non-autonomous factors, and interactions between the different developing gut tissues, are important for normal ENS development and its disorders.\n\nSignificance statementThe mammalian gut is a complex set of tissues formed during development by orchestrating the timing of expression of many genes. Here we uncover the identity of these genes, their pathways and how they change during gut organogenesis. We used RNA-seq profiling in the wildtype mouse gut in both sexes during development (E10.5 - E14.5), as well as in a Ret null mouse, a model of Hirschsprung disease (HSCR). These studies have allowed us to expand the universe of genes and developmental processes that contribute to enteric neuronal innervation and to its dysregulation in disease.

genetics↗

bioRxiv2018

Coordinate based meta-analysis of networks in neuroimaging studies

Meta-analysis of published neuroimaging results is commonly performed using coordinate based meta-analysis (CBMA). Most commonly CBMA algorithms detect spatial clustering of reported coordinates across multiple studies by assuming that results relating to the common hypothesis fall in similar anatomical locations. The null hypothesis is that studies report uncorrelated results, which is simulated by random coordinates. It is assumed that multiple clusters are independent yet it is likely that multiple results reported per study are not, and in fact represent a network effect. Here the multiple reported effect sizes (reported peak Z scores) are assumed multivariate normal, and maximum likelihood used to estimate the parameters of the covariance matrix. The hypothesis is that the effect sizes are correlated. The parameters are covariance of effect size, considered as edges of a network, while clusters are considered as nodes. In this way coordinate based meta-analysis of networks (CBMAN) estimates a network of reported meta-effects, rather than multiple independent effects (clusters).\n\nCBMAN uses only the same data as CBMA, yet produces extra information in terms of the correlation between clusters. Here it is validated on numerically simulated data, and demonstrated on real data used previously to demonstrate CBMA. The CBMA and CBMAN clusters are similar, despite the very different hypothesis.

neuroscience↗

bioRxiv2016

Coordinate Based Random Effect Size meta-analysis of neuroimaging studies

1Low power in neuroimaging studies can make them difficult to interpret, and Coordinate based meta- analysis (CBMA) may go some way to mitigating this issue. CBMA has been used in many analyses to detect where published functional MRI or voxel-based morphometry studies testing similar hypotheses report significant summary results (coordinates) consistently. Only the reported coordinates and possibly t statistics are analysed, and statistical significance of clusters is determined by coordinate density.\n\nHere a method of performing coordinate based random effect size meta-analysis and meta-regression is introduced. The algorithm (ClusterZ) analyses both coordinates and reported t statistic or Z score, standardised by the number of subjects. Statistical significance is determined not by coordinate density, but by a random effects meta-analyses of reported effects performed cluster-wise using standard statistical methods and taking account of censoring inherent in the published summary results. Type 1 error control is achieved using the false cluster discovery rate (FCDR), which is based on the false discovery rate. This controls both the family wise error rate under the null hypothesis that coordinates are randomly drawn from a standard stereotaxic space, and the proportion of significant clusters that are expected under the null. Such control is vital to avoid propagating and even amplifying the very issues motivating the meta-analysis in the first place. ClusterZ is demonstrated on both numerically simulated data and on real data from reports of grey matter loss in multiple sclerosis (MS) and syndromes suggestive of MS, and of painful stimulus in healthy controls. The software implementation is available to download and use freely.

neuroscience↗