Biology subjects

Reinholdt, L. G.

Publications and source records attributed to Reinholdt, L. G..

28 records · Page 2Linked to original sources

bioRxiv2022

Lesion environments direct transplanted neural progenitors towards a wound repair astroglial phenotype

Neural progenitor cells (NPC) represent potential cell transplantation therapies for CNS injuries. To understand how lesion environments influence transplanted NPC fate in vivo, we derived NPC expressing a ribosomal protein-hemagglutinin tag (RiboTag) for transcriptional profiling of transplanted NPC. Here, we show that NPC grafted into uninjured CNS generate cells that are transcriptionally similar to healthy astrocytes and oligodendrocyte lineages. In striking contrast, NPC transplanted into serum-exposed CNS lesions after stroke or spinal cord injury generate cells that share transcriptional, morphological and functional features with newly proliferated host astroglia that restrict inflammation and fibrosis and thereby protect adjacent neural tissue. Our findings reveal overlapping differentiation potentials of grafted NPC and proliferating host astrocytes; and show that in the absence of other interventions, non-cell autonomous cues in CNS lesions direct the differentiation of grafted NPC predominantly towards a naturally occurring neuroprotective wound repair astroglial phenotype.

neuroscience↗

bioRxiv2022

An in vitro neurogenetics platform for precision disease modeling in the mouse

The power and scope of disease modeling can be markedly enhanced through the incorporation of broad genetic diversity. In the the mouse, the introduction of pathogenic mutations into a single inbred strain sometimes fails to mimic human disease. We describe a cross-species precision disease modeling platform that exploits mouse genetic diversity to bridge cell-based modeling with whole organism analysis. We developed a universal protocol that permitted robust and reproducible neural differentiation of genetically diverse human and mouse pluripotent stem cell lines, then carried out a proof of concept study of the neurodevelopmental gene DYRK1A. The results in vitro reliably predicted the effects of genetic background on DYRK1A loss of function phenotypes and identified optimal mouse strains for in vivo disease modeling. Transcriptomic comparison of responsive and unresponsive strains identified novel molecular pathways conferring sensitivity or resilience to DYRK1A loss, and highlighted differential mRNA isoform usage as an important genetic determinant of response This cross-species strategy will be an powerful tool in the functional analysis of candidate disease variants identified through human genetic studies. Teaser: Genetically diverse mouse embryonic stem cells provide a rapid approach to precision modeling of human disease.

neuroscience↗

bioRxiv2021

Trisomy 21 induces pericentrosomal crowding disrupting early stages of primary ciliogenesis and mouse cerebellar development

Primary cilia are signaling organelles essential for development and homeostasis. Loss of primary cilia is lethal, and decreased or defective cilia cause multisystemic conditions called ciliopathies. Down syndrome shares clinical overlap with ciliopathies. We previously showed that trisomy 21 diminishes primary cilia formation and function due to elevated Pericentrin, a centrosome protein encoded on chromosome 21. Pericentrin is mislocalized, creating aggregates that disrupt pericentrosomal trafficking and microtubule organization. Here, we examine the cilia-related molecules and pathways disrupted in trisomy 21 and their in vivo phenotypic relevance. Utilizing ciliogenesis time course experiments, we reveal how Pericentrin, microtubule networks, and components of ciliary vesicles are reorganized for ciliogenesis in euploid cells. Early in ciliogenesis, chromosome 21 polyploidy results in elevated Pericentrin and microtubule networks away from the centrosome that ensnare MyosinVA and EHD1, blocking mother centriole uncapping that is essential for ciliogenesis. Ciliated trisomy 21 cells have persistent trafficking defects that reduce transition zone protein localization, which is critical for Sonic hedgehog signaling. Sonic hedgehog signaling is decreased and anticorrelates with Pericentrin levels in trisomy 21 primary mouse embryonic fibroblasts. Finally, we observe decreased ciliation in vivo. A mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers. Our work reveals that elevated Pericentrin in trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding results in signaling defects consistent with the neurological deficits found in individuals with Down syndrome.

cell biology↗

bioRxiv2021

Heritable Variation in Locomotion, Reward Sensitivity, and Impulsive Action, Choice, and Waiting in a Genetically Diverse Inbred Mouse Panel

Drugs of abuse, including alcohol and stimulants like cocaine, produce effects that are subject to individual variability, and genetic variation accounts for at least a portion of those differences. Notably, research in both animal models and human subjects point towards reward sensitivity and impulsivity as being trait characteristics that predict relatively greater positive subjective responses to stimulant drugs. Here we describe use of the eight Collaborative Cross (CC) founder strains and multiple CC strains to examine the heritability of reward sensitivity and impulsivity traits, as well as genetic correlations between these measures and existing addiction-related phenotypes. Methods. Strains were all tested for activity in an open field and reward sensitivity (intake of chocolate BOOST(R)). Mice were then divided into two counterbalanced groups and underwent reversal learning (impulsive action and waiting impulsivity) or delay discounting (impulsive choice). Results. CC and founder mice demonstrate significant heritability for impulsive action, impulsive choice, waiting impulsivity, locomotor activity, and reward sensitivity, with each impulsive phenotype determined to be non-correlating, independent traits. This research was conducted within the broader, inter-laboratory effort of the Center for Systems Neurogenetics of Addiction (CSNA) to characterize CC and DO mice for multiple, cocaine abuse related traits. These data will facilitate the discovery of genetic correlations between predictive traits, which will then guide discovery of genes and genetic variants that contribute to addictive behaviors.

neuroscience↗

bioRxiv2021

Genetic Control of Pluripotency Epigenome Determines Differentiation Bias in Mouse Embryonic Stem Cells

Genetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naive networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naive pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

genomics↗

bioRxiv2020

Micronuclei arising due to loss of KIF18A form stable micronuclear envelopes and do not promote tumorigenesis

Micronuclei, whole or fragmented chromosomes which are spatially separated from the main nucleus, are strongly associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to unaligned chromosomes in vivo but do not develop spontaneous tumors, raising questions about whether all micronuclei contribute similarly to genomic instability and cancer. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. To further explore micronuclear envelope stability in KIF18A KO cells, we compared micronuclei induced via different insults in cultured cells. Micronuclei in KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of core and non-core nuclear envelope components and successful expansion of decondensing chromatin compared to those induced by microtubule drug washout or exposure to radiation. We also observed that lagging chromosomes, which lead to micronucleus formation, were positioned closer to the main chromatin masses, and further from the central spindle, in KIF18A KO cells. Our studies provide in vivo support to models suggesting that micronuclear fate depends on the sub-cellular location of late lagging chromosomes and suggest that not all micronuclei actively promote tumorigenesis.

cell biology↗

bioRxiv2020

Genetic variation and sex differences are missed opportunities for addiction biology.

Concerns about external validity of rodent models and translation of findings across species are often based on narrow investigations of populations with limited diversity. Sources of individual variation - including genetics and sex - are only infrequently encompassed in model organism studies. As with most complex diseases, risk for cocaine use disorder is subject to considerable inter-individual variation. Explicit inclusion of individual differences in rodent research may reveal conserved phenotypes and molecular systems relevant to human addiction. We surveyed cocaine-related traits in both males and females of eight inbred mouse strains whose genomes collectively capture 90% of the genetic diversity of the mouse species. Across these strains, individual differences explained a substantial proportion of variance in cocaine-responsive or cocaine response-predictive behavioral and physiological phenotypes. Wild-derived mouse strains often extended the phenotypic ranges of these behaviors beyond what is observed in conventional laboratory strains. Striatum transcriptional responses to cocaine were also highly dependent upon strain and sex differences; most cocaine-responsive genes were differentially expressed in a manner moderated by strain, sex, or their combination. We compared the strain- and sex-mediated transcriptional responses to cocaine in mice to transcriptomic analysis of people with cocaine use disorder and found that mouse similarity to humans was highly dependent upon mouse genetic background and sex. Specifically, male WSB/EiJ mice and female NOD/ShiLtJ mice exhibited the greatest degree of neural transcriptional consilience with humans with cocaine use disorder. Model organism diversity thus represents a crucial source of biological information that can substantially improve external validity of neuropsychiatric research. Significance StatementLaboratory mice are widely used in research on neurobiological mechanisms of addiction, but most studies use a single strain and often sex of mice. To assess how individual differences in mice modulate addiction-related traits and how this impacts comparative analysis with humans, we studied cocaine-relevant behaviors and brain molecular correlates in both males and females of genetically diverse mouse strains. In this population, individual differences related to sex and/or genetics explain large proportions of differences in cocaine-related traits. Importantly, brain gene expression data demonstrated that some strains mimic human genomic states more readily than others. Individual differences thus represent a crucial and underdeveloped source of biological information about addiction mechanisms that may influence the translational utility of such studies.

neuroscience↗

bioRxiv2019

Genetic variation influences pluripotent ground state stability in mouse embryonic stem cells through a hierarchy of molecular phenotypes

Mouse embryonic stem cells (mESCs) cultured under controlled conditions occupy a stable ground state where pluripotency-associated transcriptional and epigenetic circuitry are highly active. However, mESCs from some genetic backgrounds exhibit metastability, where ground state pluripotency is lost in the absence of ERK1/2 and GSK3 inhibition. We dissected the genetic basis of metastability by profiling gene expression and chromatin accessibility in 185 genetically heterogeneous mESCs. We mapped thousands of loci affecting chromatin accessibility and/or transcript abundance, including eleven instances where distant QTL co-localized in clusters. For one cluster we identified Lifr transcript abundance as the causal intermediate regulating 122 distant genes enriched for roles in maintenance of pluripotency. Joint mediation analysis implicated a single enhancer variant ~10kb upstream of Lifr that alters chromatin accessibility and precipitates a cascade of molecular events affecting maintenance of pluripotency. We validated this hypothesis using reciprocal allele swaps, revealing mechanistic details underlying variability in ground state metastability in mESCs.

genomics↗

bioRxiv2018

Mitotic chromosome alignment is required for proper nuclear envelope reassembly

Chromosome alignment at the equator of the mitotic spindle is a highly conserved step during cell division, however, its importance to genomic stability and cellular fitness are not understood. Normal mammalian somatic cells lacking Kif18A function complete cell division without aligning chromosomes. These alignment-deficient cells display normal chromosome copy numbers in vitro and in vivo, suggesting that chromosome alignment is largely dispensable for maintenance of euploidy. However, we find that loss of chromosome alignment leads to interchromosomal compaction defects during anaphase, abnormal organization of chromosomes into a single nucleus at mitotic exit, and the formation of micronuclei in vitro and in vivo. These defects slow cell proliferation and reduce postnatal growth and survival with variable penetrance in mice. Our studies support a model in which the alignment of mitotic chromosomes promotes proper nuclear envelope reassembly and continued proliferation by ensuring that chromosomes segregate as a compact mass during anaphase.

cell biology↗

bioRxiv2017

Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis

Transgenesis has been a mainstay of mouse genetics for over 30 years, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported due in part to limitations in the discovery tools. Targeted Locus Amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events, and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

genetics↗