Biology subjects

Krupa, O.

Publications and source records attributed to Krupa, O..

9 recordsLinked to original sources

bioRxiv2026

CellPheno: A High-throughput Computational Platform for Quantifying Cellular Resolution Whole Brain Microscopy Images

Advances in tissue clearing and light-sheet microscopy enable cellular resolution whole-brain 3D imaging. However, whole-brain quantification tools do not yet meet demands for efficiency or assess morphometry. Here we present CellPheno, a 3D nuclei instance segmentation framework for high-throughput cellular phenotyping. CellPheno quantifies an entire P4 mouse brain within 15 hours. We showcase whole-brain morphometry, enhanced stitching, and co-localization across multiple cell types in 53 brains.

neuroscience↗

bioRxiv2026

Chd8 haploinsufficiency leads to molecular layer heterotopias and age-dependent cortical expansion

Mutations in the chromatin remodeler CHD8 are associated with autism and macrocephaly. While mouse models of Chd8 haploinsufficiency recapitulate brain overgrowth, the specific cellular mechanisms and developmental timing that lead to these anatomical abnormalities remain poorly understood. Here, we conducted 3D imaging of Chd8V986*/+ mouse brains using magnetic resonance imaging followed by tissue clearing and cellular resolution light-sheet microscopy across embryonic and postnatal developmental stages. We found that brain overgrowth occurs postnatally, driven by an increase in non-neuronal cells prior to volumetric expansion. Unexpectedly, we identified prevalent molecular layer heterotopias (MLH) within the frontal cortex of Chd8V986*/+ mice composed of neurons breaking through the pial surface during embryonic development and persisting throughout life. Increased incidence of MLH, previously identified in individuals with idiopathic autism, was replicated across three independent Chd8+/- mouse models and present across multiple genomic backgrounds, establishing aberrant neuronal migration as a core feature of Chd8 haploinsufficiency.

neuroscience↗

bioRxiv2025

Massively parallel assessment of gene regulatory activity at human cortical structure associated variants

Genetic association studies have identified hundreds of largely non-coding loci associated with inter-individual differences in the structure of the human cortex, though the specific genetic variants that impact regulatory activity are unknown. We implemented a Massively Parallel Reporter Assay (MPRA) to measure the regulatory activity of 9,092 cortical structure associated DNA variants in human neural progenitor cells during Wnt stimulation and at baseline. We identified 918 variants with regulatory potential from 150 cortical structure associated loci (76% of loci studied), of which >50% showed allelic effects. Wnt stimulation modified regulatory activity at a subset of loci that functioned as condition-dependent enhancers. Regulatory activity in MPRA was largely induced by Alu elements that were hypothesized to contribute to cortical expansion. The regionally specific impact of genetic variants that disrupt motifs is likely mediated through the levels of transcription factor expression during development, further clarifying the molecular mechanisms altering cortical structure.

genetics↗

bioRxiv2023

Genetics of cell-type-specific post-transcriptional gene regulation during human neurogenesis

The function of some genetic variants associated with brain-relevant traits has been explained through colocalization with expression quantitative trait loci (eQTL) conducted in bulk post-mortem adult brain tissue. However, many brain-trait associated loci have unknown cellular or molecular function. These genetic variants may exert context-specific function on different molecular phenotypes including post-transcriptional changes. Here, we identified genetic regulation of RNA-editing and alternative polyadenylation (APA), within a cell-type-specific population of human neural progenitors and neurons. More RNA-editing and isoforms utilizing longer polyadenylation sequences were observed in neurons, likely due to higher expression of genes encoding the proteins mediating these post-transcriptional events. We also detected hundreds of cell-type-specific editing quantitative trait loci (edQTLs) and alternative polyadenylation QTLs (apaQTLs). We found colocalizations of a neuron edQTL in CCDC88A with educational attainment and a progenitor apaQTL in EP300 with schizophrenia, suggesting genetically mediated post-transcriptional regulation during brain development lead to differences in brain function.

genomics↗

bioRxiv2022

MicroRNA-eQTLs in the developing human neocortex link miR-4707-3p expression to brain size

Expression quantitative trait loci (eQTL) data have proven important for linking non-coding loci to protein-coding genes. But eQTL studies rarely measure microRNAs (miRNAs), small non-coding RNAs known to play a role in human brain development and neurogenesis. Here, we performed small-RNA sequencing across 212 mid-gestation human neocortical tissue samples, measured 907 expressed miRNAs, discovering 111 of which were novel, and identified 85 local-miRNA-eQTLs. Colocalization of miRNA-eQTLs with GWAS summary statistics yielded one robust colocalization of miR-4707-3p expression with educational attainment and brain size phenotypes, where the miRNA expression increasing allele was associated with decreased brain size. Exogenous expression of miR-4707-3p in primary human neural progenitor cells decreased expression of predicted targets and increased cell proliferation, indicating miR-4707-3p modulates progenitor gene regulation and cell fate decisions. Integrating miRNA-eQTLs with existing GWAS yielded evidence of a miRNA that may influence human brain size and function via modulation of neocortical brain development.

genetics↗

bioRxiv2021

Segmentor: A tool for manual refinement of 3D microscopy annotations

BackgroundRecent advances in tissue clearing techniques, combined with high-speed image acquisition through light sheet microscopy, enable rapid three-dimensional (3D) imaging of biological specimens, such as whole mouse brains, in a matter of hours. Quantitative analysis of such 3D images can help us understand how changes in brain structure lead to differences in behavior or cognition, but distinguishing features of interest, such as nuclei, from background can be challenging. Recent deep learning-based nuclear segmentation algorithms show great promise for automated segmentation, but require large numbers of manually and accurately labeled nuclei as training data. ResultsWe present Segmentor, an open-source tool for reliable, efficient, and user-friendly manual annotation and refinement of objects (e.g., nuclei) within 3D light sheet microscopy images. Segmentor employs a hybrid 2D-3D approach for visualizing and segmenting objects and contains features for automatic region splitting, designed specifically for streamlining the process of 3D segmentation of nuclei. We show that editing simultaneously in 2D and 3D using Segmentor significantly decreases time spent on manual annotations without affecting accuracy. ConclusionsSegmentor is a tool for increased efficiency of manual annotation and refinement of 3D objects that can be used to train deep learning segmentation algorithms, and is available at https://www.nucleininja.org/ and https://github.com/RENCI/Segmentor.

neuroscience↗

bioRxiv2020

Genetic influences on cell type specific gene expression and splicing during neurogenesis elucidate regulatory mechanisms of brain traits

Interpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing is generally performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements active during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map expression quantitative trait loci (eQTLs), splicing QTLs (sQTLs), and allele specific expression in primary human neural progenitors (n=85) and their sorted neuronal progeny (n=74), identifying numerous loci not detected in either bulk developing cortical wall or adult cortex. Using colocalization and genetic imputation via transcriptome wide association, we uncover cell-type specific regulatory mechanisms underlying risk for brain-relevant traits that are active during neocortical differentiation. Specifically, we identified a progenitor-specific eQTL for CENPW co-localized with common variant associations of cortical area and educational attainment.

genetics↗

bioRxiv2020

NuMorph: tools for cellular phenotyping in tissue cleared whole brain images

Tissue clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here we introduce NuMorph, a group of image analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after tissue clearing and imaging by a conventional light-sheet microscope. We applied NuMorph to investigate two distinct mouse models: a Topoisomerase 1 (Top1) conditional knockout model with severe neurodegenerative deficits and a Neurofibromin 1 (Nf1) conditional knockout model with a more subtle brain overgrowth phenotype. In each case, we identified differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of 3D whole brain imaging approaches and the tools are widely applicable for studying 3D structural deficits of the brain at cellular resolution in animal models of neuropsychiatric disorders.

neuroscience↗

bioRxiv2020

Cell-type specific effects of genetic variation on chromatin accessibility during human neuronal differentiation

Common genetic risk for neuropsychiatric disorders is enriched in regulatory elements active during cortical neurogenesis. However, the mechanisms mediating the effects of genetic variants on gene regulation are poorly understood. To determine the functional impact of common genetic variation on the non-coding genome longitudinally during human cortical development, we performed a chromatin accessibility quantitative trait loci (caQTL) analysis in neural progenitor cells and their differentiated neuronal progeny from 92 donors. We identified 8,111 caQTLs in progenitors and 3,676 caQTLs in neurons, with highly temporal, cell-type specific effects. A subset ([~]20%) of caQTLs were also associated with changes in gene expression. Motif-disrupting alleles of transcriptional activators generally led to decreases in chromatin accessibility, whereas motif-disrupting alleles of repressors led to increases in chromatin accessibility. By integrating cell-type specific caQTLs and brain-relevant genome-wide association data, we were able to fine-map loci and identify regulatory mechanisms underlying non-coding neuropsychiatric disorder risk variants. HighlightsO_LIGenetic variation alters chromatin architecture during human cortical development C_LIO_LIGenetic effects on chromatin accessibility are highly cell-type specific C_LIO_LIAlleles disrupting TF motifs generally decrease accessibility, except for repressors C_LIO_LIcaQTLs facilitate fine-mapping and inference of regulatory mechanisms of GWAS loci C_LI

genetics↗