Biology subjects

Gray, J. M.

Publications and source records attributed to Gray, J. M..

7 recordsLinked to original sources

bioRxiv2026

In vivo reconstruction of the cell lineage history of a developing mouse with DNA Typewriter, from zygote to late organogenesis

The complete cell lineage of C. elegans, mapped over four decades ago, was tractable because the animal is small, transparent, and lineage invariant1. Most animals are none of these, having orders of magnitude more cells, being opaque, and developing with substantial stochasticity2. Since 2016, genome editing-based lineage tracing has opened the door to dense cell lineage reconstruction in such organisms3-8, but delivering on that promise has proven technically challenging. Here we apply DNA Typewriter9-11, a prime editing-based recorder that writes stochastic symbolic insertions to an engineered genomic TAPE in strictly sequential order, to trace mouse development from zygote (E0) to late organogenesis (E13.5). We introduce constructs encoding a prime editor, engineered prime editing guide RNAs (epegRNAs), and Pol-III-driven circularized TAPE RNA (circTAPE) into wildtype zygotes by pronuclear injection (PNI), then assay embryos by single-nucleus transcriptional profiling (sci-RNA-seq3)12 with paired circTAPE recovery. From one E13.5 embryo bearing [~]7 integrations of a constitutively expressed prime editor and 11 integrations of 6-unit circTAPE, we recover [~]1.75M single-nucleus transcriptomes and reconstruct a time-calibrated phylogeny of 1,340,794 annotated cells with parsimony-based node support. The first cell division is marked unequivocally, and although the resulting blastomeres contribute asymmetrically to the embryo proper, they are fate-neutral and serve as internal replicates that reproduce every finding. A modest cohort of pre-gastrulation founders dominates the embryo, with inequality exceeding neutral expectation within one to two cell cycles of founder allocation, yet these founders remain broadly multipotent; a second phase of clonal dominance arises in specific lineages during organogenesis. At the finest scale, sibling cells share cell type 9-fold in excess of chance, reaching 68- to 107-fold for cell types arising from spatially restricted founder pools, while the recent differentiations of organogenesis are legible in the heterotypic structure of terminal clades. From clade co-occurrence alone, we recover germ-layer organization and a dated hierarchy of cell-type couplings with branch points from E8.5 onwards. Finally, by integrating these data with our single-cell time-series of mouse development13, we impute transcriptional states and annotations for the majority of internal nodes and recover established state histories for diverse cell types. All data are made freely available, together with NextCell, an interactive browser for this annotated cellular phylogeny of mouse development from zygote to late organogenesis.

developmental biology↗

bioRxiv2024

Asymmetric Interactions Shape Survival During Population Range Expansions

An organism that is newly introduced into an existing population has a survival probability that is dependent on both the population density of its environment and the competition it experiences with the members of that population. Expanding populations naturally form regions of high and low density, and simultaneously experience ecological interactions both internally and at the boundary of their range. For this reason, systems of expanding populations are ideal for studying the combination of density and ecological effects. Conservation ecologists have been studying the ability of an invasive species to establish for some time, attributing success to both ecological and spatial factors. Similar behaviors have been observed in spatially structured cell populations, such as those found in cancerous tumors and bacterial biofilms. In these scenarios, invasion may be the introduction of a new mutation or bacterial species with some form of drug resistance, leading to the possibility of treatment failure. In order to gain insight into the relationship between population density and ecological interactions, we study an expanding population of interacting wild-type cells and mutant cells. We simulate these interactions in time and study the spatially dependent probability for a mutant to survive or to take over the front of the population wave (gene surfing). Additionally, we develop a mathematical model that describes this survival probability and find agreement when the payoff for the mutant is positive (corresponding to cooperation, exploitation, or commensalism). Given the types of interactions, our model provides insight into the spatial distribution of survival probability. Conversely, given a spatial distribution of survival probabilities, our model provides insight into the types of interactions that were involved to generate it.

evolutionary biology↗

bioRxiv2024

Diversified, miniaturized and ancestral parts for mammalian genome engineering and molecular recording

As the synthetic biology and genome engineering fields mature and converge, there is a clear need for a "parts list" of components that are diversified with respect to both functional activity (to facilitate design) and primary sequence (to facilitate assembly). Here we designed libraries composed of extant, ancestral, mutagenized or miniaturized variants of Pol III promoters or guide RNA (gRNA) scaffolds and quantified their ability to mediate precise edits to the mammalian genome via multiplex prime editing. We identified thousands of parts that reproducibly drive a range of editing activities in human and mouse stem cells and cancer cell lines, including hundreds exhibiting similar or greater activity than the sequences used in conventional genome engineering constructs. We further conducted saturation mutagenesis screens of canonical Pol III promoters (U6p, 7SKp, H1p) and the prime editing guide RNA (pegRNA) scaffold, which identified tolerated variants that can be superimposed on baseline parts to further enhance sequence diversity. While characterizing thousands of orthologous promoters from hundreds of extant or ancestral genomes, we incidentally mapped the functional landscape of mammalian Pol III promoter evolution. Finally, to showcase the usefulness of these parts, we designed a "ten key" molecular recording array that lacks repetitive subsequences in order to facilitate its one-step assembly in yeast. Upon delivering this 15.8 kb tandem array of promoters and guides to mammalian cells, individual pegRNAs exhibited balanced activities as predicted by the activity of component parts, despite their relocation to a single locus. Looking forward, we anticipate that the diversified parts and variant effect maps reported here can be leveraged for the design, assembly and deployment of synthetic loci encoding arrays of gRNAs exhibiting predictable, differentiated levels of activity, which will be useful for multiplex perturbation, advanced biological recorders and complex genetic circuits.

synthetic biology↗

bioRxiv2023

The evolution of diverse antimicrobial responses in vancomycin-intermediate Staphylococcus aureus and its therapeutic implications

Staphylococcus aureus bacteremia is typically treated empirically with vancomycin, with therapy later tailored based on susceptibility results. However, these tests occur before vancomycin exposure and do not account for adaptation during empiric treatment that can alter S. aureus susceptibility to first-line drugs. To investigate these collateral drug responses, we experimentally evolved 18 methicillin-susceptible S. aureus (MSSA) populations under increasing vancomycin concentrations until they achieved intermediate resistance. Genomic sequencing revealed two distinct adaptive pathways characterized by mutations in the WalKR regulon, affecting cell wall metabolism, or rpsU, impacting translational stress responses. These pathways correlated with divergent collateral sensitivity profiles to first-line antibiotics. By developing a Collateral Response Score (CRS), we quantified the probability and magnitude of these responses, demonstrating that evolutionary dynamics critically influence resistance outcomes. Our findings suggest a probabilistic approach to antimicrobial therapy, advocating for rapid genomic diagnostics alongside susceptibility testing to better anticipate and respond to evolutionary changes. SignificanceAntibiotic treatment can influence bacterial evolution, altering the effectiveness of subsequent therapies by inducing collateral resistance or sensitivity. This study reveals that evolution toward vancomycin-intermediate resistance in the pathogen Staphylococcus aureus proceeds through at least two distinct evolutionary pathways: one characterized by alterations in cell wall metabolism and another by changes in global stress response. These adaptive trajectories result in contrasting collateral sensitivities to first-line antibiotics. By introducing the Collateral Response Score, we assess the uncertainty in these outcomes, providing a probabilistic framework to evaluate how past antibiotic exposure shapes future treatment responses. Further validation studies are needed; however, we believe that improved forecasting of pathogen evolution can enhance antibiotic stewardship, inform therapeutic decisions, and ultimately improve patient outcomes.

evolutionary biology↗

bioRxiv2019

Firing rate homeostasis occurs in the absence of neuronal activity-regulated transcription

Despite dynamic inputs, neuronal circuits maintain relatively stable firing rates over long periods. This maintenance of firing rate, or firing rate homeostasis, is likely mediated by homeostatic mechanisms such as synaptic scaling and regulation of intrinsic excitability. Because some of these homeostatic mechanisms depend on transcription of activity-regulated genes, including Arc and Homer1a, we hypothesized that activity-regulated transcription would be required for firing rate homeostasis. Surprisingly, however, we found that cultured mouse cortical neurons grown on multi-electrode arrays homeostatically adapt their firing rates to persistent pharmacological stimulation even when activity-regulated transcription is disrupted. Specifically, we observed firing rate homeostasis Arc knock-out neurons, as well as knock-out neurons lacking activity-regulated transcription factors, AP1 and SRF. Firing rate homeostasis also occurred normally during acute pharmacological blockade of transcription. Thus, firing rate homeostasis in response to increased neuronal activity can occur in the absence of neuronal-activity-regulated transcription.\n\nSIGNIFICANCE STATEMENTNeuronal circuits maintain relatively stable firing rates even in the face of dynamic circuit inputs. Understanding the molecular mechanisms that enable this firing rate homeostasis could potentially provide insight into neuronal diseases that present with an imbalance of excitation and inhibition. However, the molecular mechanisms underlying firing rate homeostasis are largely unknown.\n\nIt has long been hypothesized that firing rate homeostasis relies upon neuronal activity-regulated transcription. For example, a 2012 review (PMID 22685679) proposed it, and a 2014 modeling approach established that transcription could theoretically both measure and control firing rate (PMID 24853940). Surprisingly, despite this prediction, we found that cortical neurons undergo firing rate homeostasis in the absence of activity-regulated transcription, indicating that firing rate homeostasis is controlled by non-transcriptional mechanisms.

neuroscience↗

bioRxiv2019

Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

Optogenetics is widely used to control diverse cellular functions with light, requiring experimenters to expose cells to bright light. Because extended exposure to visible light can be toxic to cells, it is important to characterize the effects of light stimulation on cellular function in the absence of exogenous optogenetic proteins. Here we exposed cultured mouse cortical neurons that did not express optogenetic proteins to several hours of flashing blue, red, or green light. We found that exposing neurons to as short as one hour of blue, but not red or green, light results in the induction of neuronal-activity-regulated genes without inducing neuronal activity. Our findings suggest blue light stimulation is ill-suited to long-term optogenetic experiments, especially those that measure transcription.\n\nSignificance StatementOptogenetics is widely used to control cellular functions using light. In neuroscience, channelrhodopsins, exogenous light-sensitive channels, are used to achieve light-dependent control of neuronal firing. This optogenetic control of neuronal firing requires exposing neurons to high-powered light. We ask how this light exposure, in the absence of channelrhodopsin, affects the expression of neuronal-activity-regulated genes, i.e., the genes that are transcribed in response to neuronal stimuli. Surprisingly, we find that neurons without channelrhodopsin express neuronal-activity-regulated genes in response to as short as an hour of blue, but not red or green, light exposure. These findings suggest that experimenters wishing to achieve longer-term (an hour or more) optogenetic control over neuronal firing should avoid using systems that require blue light.

neuroscience↗

bioRxiv2017

Distinct neuronal activity patterns induce different gene expression programs

Brief and sustained neuronal activity patterns can have opposite effects on synaptic strength that both require activity-regulated gene (ARG) expression. However, whether distinct patterns of activity induce different sets of ARGs is unknown. In genome-scale experiments, we reveal that a neurons activity-pattern history can be predicted from the ARGs it expresses. Surprisingly, brief activity selectively induces a small subset of the ARG program that that corresponds precisely to the first of three temporal waves of genes induced by sustained activity. These first-wave genes are distinguished by an open chromatin state, proximity to rapidly activated enhancers, and a requirement for MAPK/ERK signaling for their induction. MAPK/ERK mediates rapid RNA polymerase recruitment to promoters, as well as enhancer RNA induction but not histone acetylation at enhancers. Thus, the same mechanisms that establish the multi-wave temporal structure of ARG induction also enable different sets of genes to be induced by distinct activity patterns.

neuroscience↗