Biology subjects

Patton, E. E.

Publications and source records attributed to Patton, E. E..

12 recordsLinked to original sources

bioRxiv2026

A Chemical-Genetic Interaction Matrix Reveals Drug Mechanism and Genetic Architecture

To probe drug mechanism of action (MOA) and interrogate the genetic architecture of human cells, we carried out isogenic genome-wide CRISPR/Cas9 knockout screens against 310 diverse drugs, bioactive compounds, and stress conditions. Stringent statistical correction for gene knockout fitness defects yielded a large-scale matrix of >12,000 high confidence chemical-genetic interactions (CGIs). This dataset revealed many previously unappreciated off-target effects for well-characterized compounds and novel MOAs for uncharacterized compounds. The CGI matrix uncovered dense genetic modules that yielded new biological insights into phospholipidosis, mitotic regulation, metabolism, the DNA damage response, and mTOR signaling. The dataset allowed identification of multi-drug sensitization and resistance mechanisms, inference of gene function, elaboration of cross-process connectivity, evaluation of the cell type specificity of CGIs, prediction of chemical synergism, and extensive annotation of understudied genes. This resource provides a map of the genetic landscape in human cells and a framework to help guide drug discovery.

systems biology↗

bioRxiv2025

Loss of MITF activity leads to emergent cell states from the melanocyte stem cell lineage

How embryonic cells generate large clones of cells in the adult represents a fundamental question in biology. Here using melanocyte stem cells (McSCs) in the zebrafish as a model we explore the function of the master melanocyte transcription factor (MITF) in safeguarding McSCs in embryonic development and their potential to pigment large clones in the adult. MITF is well known is for its role in the specification of melanoblasts from the neural crest (NC) and their differentiation into melanocytes, yet little is known about how this activity shapes the stem cell lineages. Here, we use live imaging coupled with single-cell transcriptomics and lineage tracing to show that MITF (mitfa in zebrafish) protects the melanocyte stem cell (McSC) fate in zebrafish. Utilizing a temperature sensitive mitfavc7 mutant, we show that loss of Mitfa activity leads to a surprising premature and aberrant expansion of McSC progeny at the niche during embryogenesis, coupled with novel emergent transcriptional cell states. Linage tracing of McSCs from the embryonic to juvenile stages reveals Mitfa activity is subsequently required in regeneration by Schwann cell-like and melanocyte stem cell progenitors that serve as a reservoir for fast-responding pigment progenitors. Thus, the impact of Mitfa loss on the melanocyte lineage is cell-state and stage-specific. The emergent cell states resulting from mitfa loss may have important implications for understanding how reduced MITF activity contributes to human genetic disease and melanoma.

developmental biology↗

bioRxiv2025

Mosaic PIK3CA-related overgrowth spectrum (PROS) mutations cause non-cell autonomous vascular overgrowth and pan-lineage dysregulation at disease onset

Post-zygotic gain-of-function PIK3CA mutations arising during embryonic development cause disorders collectively known as the PIK3CA-related overgrowth spectrum (PROS). This ranges from minor, localized overgrowth to devastating multi-tissue overgrowth. Disease severity is widely attributed to a combination of PIK3CA genotype, affected cell type, and developmental timing of mutation acquisition. However, in PROS this explains neither the biased pattern of overgrowth - disproportionately affecting mesoderm and neuroectoderm-derived tissues - nor the typical low mutation burden within areas of extensive tissue overgrowth. Indeed, growing evidence suggests PROS mutations cause overgrowth non-cell-autonomously, although mechanisms of this are poorly understood. Here, we develop mosaic zebrafish models of PROS with overexpression of orthologous hotspot pik3ca mutations (pik3caPROS) to visualize and examine the effects of mutated cells on early development, in whole live animals. Reminiscent of PROS, we observe a spectrum of embryonic vasculature malformations (VMs), accompanied by larval muscle and bone overgrowth. Surprisingly, VMs only rarely expressed pik3caPROS in constituent endothelial cells, with pik3caPROS-expressing cells often closely abutting malformations instead. Single-cell transcriptomics of pik3caPROSmosaic zebrafish prior to VM onset revealed that most pik3caPROS cells are relatively immature and developmentally inert or constitute a small minority of mesodermal-derived cell types. Despite this constriction, global changes to cell fate were evident, alongside pervasive, pan-lineage abnormalities of gene expression, and rewiring of predicted ligand-receptor communication between lineages. We propose that targeting signals that indirectly propagate overgrowth through non-cell autonomous mechanisms - as well as PI3K activation itself - is worthy of therapeutic investigation. Significance statementPatchy, or mosaic, activating mutations in PIK3CA cause asymmetric human overgrowth due to aberrant hyperactivation of phosphoinositide 3-kinase (PI3K) signaling. Overgrowth prominently affects blood vessels and may be severely debilitating. While causation by PIK3CA mutations is clear, the explanation for the extent and pattern of associated overgrowth is not. Leveraging the power of zebrafish models for observation of early development, we now provide evidence for widely pervasive developmental effects of PIK3CA mutations extending beyond transcriptionally and phenotypically affected cells and lineages. This suggests potential therapeutic value of targeting secondary effects of PIK3CA activation as well as the activated PI3K itself.

developmental biology↗

bioRxiv2025

Simplified In Vitro Generation of Human Gastruloids for Modelling Early Development

The purpose of this study was to optimize the efficiency and cost of human gastruloid formation by testing and adjusting individual parameters using as examples two distinct human pluripotent stem cell lines, both available from the UK Stem Cell Bank. For the first step, commercially sourced culture medium was replaced with a home-made defined recipe, known as N2B27, into which specific reagents can be titrated. By reducing the concentration of Activin A to 15% of the original protocol, efficient elongation of aggregated embryoid bodies was achieved. Also, titrating initial cell density and delaying the brief culture in GSK3 inhibitor until the onset of cell aggregation in individual wells was advantageous. Efficiency of formation of early gastruloids exhibiting the expected regionalization of the three embryonic germ layers was further enhanced by addition of TGF{beta}-inhibitor. The optimization steps presented here thus provide a simplified, robust and relatively economical protocol for consistent generation of elongated gastruloids from human pluripotent stem cells. This streamlined method improves accessibility and reproducibility, also providing a standardized platform to investigate fundamental principles of early human development. Summary StatementWe present an optimized protocol for human gastruloid production that should enhance efficiency, reproducibility and affordability for future in vitro studies into early human post-implantation development.

developmental biology↗

bioRxiv2023

ALDH1A3-acetaldehyde re-wires neural crest stem cell and high metabolism states to potentiate melanoma heterogeneity

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-CoA synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest lineage and glucose metabolism genes. Importantly, we show acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3, dependent manner providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish model of melanoma residual disease, a subpopulation of ALDH1-high cells emerges following BRAF inhibitor treatment and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma. HighlightsO_LIALDH1A3-high melanomas are in a high glucose metabolic flux and neural crest stem cell dual state. C_LIO_LINuclear ALDH1A3 partners with ACSS2 to promote selective acetyl-histone H3. C_LIO_LIAcetaldehyde is an acetyl source for ALDH1A3 dependent histone H3 acetylation. C_LIO_LIALDH1A3 is a master regulator and pharmaceutical target for melanoma heterogeneity. C_LI

cancer biology↗

bioRxiv2022

Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish

Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we apply tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage tracing methodology in adult zebrafish cancer models. Summary statementWe fate map melanoma cells from the primary tumour into a persister cell state and show that persister cells directly contribute to recurrent disease.

cancer biology↗

bioRxiv2021

Long-term non-invasive drug treatments for adult zebrafish

Zebrafish embryos are widely used for drug-discovery however administering drugs to adult zebrafish is limited by current protocols that can cause stress. Here, we develop a drug formulation and administration method for adult zebrafish by producing food-based drug pellets which are consumed voluntarily. We apply this to zebrafish with BRAF-mutant melanoma, a model that has significantly advanced our understanding of melanoma progression, but not of drug resistance due to the limitations of current treatment methods. Short-term, precise, and daily dosing with drug-pellets made with the BRAFV600E inhibitor, vemurafenib, led to tumour regression. On-target drug efficacy was determined by phospho-ERK staining. Continued drug treatment led to the emergence, for the first time in zebrafish, of acquired drug resistance and melanoma relapse, modelling the responses seen in melanoma patients. This method presents a controlled, non-invasive approach that permits long-term drug studies, and can be widely applied to any adult zebrafish model. SUMMARY STATEMENTThis drug-pellet approach is a precise dosing method to administer short or long-term drug treatments to adult zebrafish, that is stress-free for the fish and requires minimal animal handling. We use this method to develop new drug-resistant models of melanoma in zebrafish, opening new doors for modelling and screening drug treatments in adult zebrafish.

cancer biology↗

bioRxiv2021

Aldh2 dependent formaldehyde metabolism fuels purine demands in melanocyte stem cells

Melanocyte stem cells (McSCs) in zebrafish serve as an on-demand source of melanocytes during growth and regeneration, but metabolic programs associated with their activation and regenerative processes are not well known. Here, using live imaging coupled with scRNA-sequencing, we discovered that quiescent McSCs during regeneration activate a dormant embryonic neural crest transcriptional program followed by an aldehyde dehydrogenase (Aldh) 2 metabolic switch to generate progeny. Unexpectedly, while ALDH2 is well known for its aldehyde clearing mechanisms we find that in regenerating McSCs, Aldh2 activity is required to generate formate - the one-carbon (1C) building block for nucleotide biosynthesis - through formaldehyde metabolism. Consequently, we find that disrupting the 1C cycle with low-doses of methotrexate caused melanocyte regeneration defects. In the absence of Aldh2, we find that purines (but not pyrimidines) are the metabolic end product sufficient for activated McSCs to generate progeny. Together, our work reveals McSCs undergo a two-step cell state transition during regeneration, and that the reaction products of Aldh2 enzymes have tissue-specific stem cell functions that meet metabolic demands in regeneration. SUMMARY STATEMENTIn melanocyte regeneration, quiescent McSCs respond by re-expressing a neural crest identity, followed by an Aldh2-dependent metabolic switch to generate progeny.

cell biology↗

bioRxiv2021

Tfap2b specifies an embryonic melanocyte stem cell population that retains adult multi-fate potential

Melanocytes, our pigment producing cells, are replenished from multiple stem cell niches in adult tissues. Although pigmentation traits are known risk-factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs, and lack key lineage markers with which to identify McSCs and study their function. Here, we discover that Tfap2b, and a select set of its target genes, specifies an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and instead is essential for McSC-dependent melanocyte regeneration. Fate- mapping data reveal that tfap2b-expressing McSCs have multi-fate potential, and are the cell-of- origin for large patches of adult melanocytes, and two other pigment cell types, iridophores and xanthophores. Hence, Tfap2b confers McSC identity in early development, thereby distinguishing McSCs from other neural crest and pigment cell lineages, and retains multi-fate potential in the adult zebrafish. HighlightsO_LITfap2b and its target genes specify McSCs with mixed pigment cell identities C_LIO_LIFunctional dependence on Tfap2b for melanocyte regeneration from the McSC C_LIO_LItfap2b specifies ErbB-dependent McSCs at the stem cell niche C_LIO_LIFate mapping reveals Tfap2b-McSCs have multi-fate potential for adult pigment cells C_LI

developmental biology↗

bioRxiv2020

MITF reprograms the extracellular matrix and focal adhesion in melanoma

The microphthalmia associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation. It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states. Here we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial to mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions. Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model. The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug resistant melanomas. Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas. Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.

cancer biology↗

bioRxiv2018

Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependant pigment progenitors in zebrafish

Skin pigment patterns are important, being under strong selection for multiple roles including camouflage and UV protection. Pigment cells underlying these patterns form from adult pigment stem cells (APSCs). In zebrafish, APSCs derive from embryonic neural crest cells, but sit dormant until activated to produce pigment cells during metamorphosis. The APSCs are set-aside in an ErbB signaling dependent manner, but the mechanism maintaining quiescence until metamorphosis remains unknown. Mutants for a pigment pattern gene, parade, exhibit ectopic pigment cells localised to the ventral trunk. We show that parade encodes Endothelin receptor Aa, expressed in the blood vessels. Using chemical genetics, coupled with analysis of cell fate studies, we show that the ectopic pigment cells derive from APSCs. We propose that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels.\n\nLay AbstractPigment patterns are crucial for the many aspects of animal biology, for example, providing camouflage, enabling mate selection and protecting against UV irradiation. These patterns are generated by one or more pigment cell-types, localised in the skin, but derived from specialised stem cells (adult pigment stem cells, APSCs). In mammals, such as humans, but also in birds and fish, these APSCs derive from a transient population of multipotent progenitor cells, the neural crest. Formation of the adult pigment pattern is perhaps best studied in the zebrafish, where the adult pigment pattern is formed during a metamorphosis beginning around 21 days of development. The APSCs are set-aside in the embryo around 1 day of development, but then remain inactive until that metamorphosis, when they become activated to produce the adult pigment cells. We know something of how the cells are set-aside, but what signals maintain them in an inactive state is a mystery. Here we study a zebrafish mutant, called parade, which shows ectopic pigment cells in the embryo. We clone the parade gene, identifying it as ednraa encoding a component of a cell-cell communication process, which is expressed in blood vessels. By characterising the changes in the neural crest and in the pigment cells formed, and by combining this with an innovative assay identifying drugs that prevent the ectopic cells from forming, we deduce that the ectopic cells in the larva derive from precocious activation of APSCs to form pigment cells. We propose that a novel population of APSCs are associated with the blood vessels, that these are held in a quiescent state by signals coming from these vessels, and that these signals depend upon ednraa. Together this opens up an exciting opportunity to identify the signals maintaining APSC quiescence in zebrafish.

developmental biology↗

bioRxiv2017

Wilms Tumor 1b defines a wound-specific sheath cell subpopulation associated with notochord repair

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify a novel and unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury. Using live imaging in zebrafish, we show that localized damage leads to Wt1b expression in the sheath, and that wt1b+ cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. At the wound wt1b+ and entpd5+ cells constitute distinct subpopulations, and mark the site of an extra vertebra that forms in an untypical manner via a cartilage intermediate. Surprisingly, wt1b+ cells become closely associated with the chordacentra and sustain wt1b expression for over 35 days during vertebra formation. Given that remnants of notochord cells remain in the adult intervertebral disc, the identification of novel subpopulations may have important implications for regenerative treatments for spine disorders.\n\nHighlightsO_LINotochord injury triggers wound-specific expression of wt1b in novel sheath subpopulation\nC_LIO_LIWT1b notochord sheath cells fill injury site and form stopper-like structure\nC_LIO_LIWT1b subpopulation marks site of a new vertebra that forms via a cartilage intermediate\nC_LIO_LIWT1b wound-specific subpopulation perdures throughout and after vertebra repair\nC_LI

developmental biology↗