Biology subjects

Smith, J. J.

Publications and source records attributed to Smith, J. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Dissecting functional elements in giant genomes using the first generation of high quality salamander assemblies

Salamanders are an enduring and historically significant group that helped originate multiple scientific fields of study, including regeneration, cytogenetics, experimental embryology, cloning, evolutionary developmental biology and evolution of extreme genome size. We generated a high-quality, single haplotype assembly for the 32 Gb axolotl (Ambystoma mexicanum) genome using an F1 hybrid between axolotl and a divergent relative the tiger salamander (A. tigrinum). Using multi-genome alignments to other salamanders that meet VGP metrics, we leveraged strong conservation of karyotypic structure to identify >523,000 highly conserved non-coding elements within massively expanded intronic and intergenic regions, the vast majority of which are unique to the salamander lineage. Orthogonal measures of chromatin state and enhancer activity confirmed that many conserved elements correspond to cis-regulatory sequences and identified several thousand that are utilized during regeneration. The conserved elements identified here will enable robust functional genomic analyses of tissue regeneration and other traits that uniquely empower salamander models in biological research.

genomics↗

bioRxiv2026

stMCP: Spatial Transcriptomics with a Model Context Protocol Server

Spatial transcriptomics enables high-resolution mapping of gene expression in intact tissues but remains challenging due to complex computational workflows that limit accessibility and reproducibility. Here, we present a Model Context Protocol (MCP) framework enabling natural language-driven spatial transcriptomics analysis. By executing analytical tools locally, this architecture eliminates the need to upload massive datasets to large language models, bypassing high token costs and mitigating data privacy and training risks. The MCP orchestrator interprets intent, dynamically routes requests, maintains session state, and verifies input integrity to ensure reproducible execution. Benchmarking across biological discovery, orchestration accuracy, token usage, and execution time demonstrates robust performance. This architecture establishes a scalable template for AI-native research by standardizing the interface between models and local analytical engines. Rather than replacing bioinformaticians, this framework empowers biologists to independently and comprehensively explore their data, accelerating hypothesis testing, and unlocking broader biological discoveries.

bioinformatics↗

bioRxiv2026

Functional Analyses of Histone Methyltransferases in Sea Lamprey Embryos Undergoing Programmed DNA Elimination

During early embryogenesis, the sea lamprey (Petromyzon marinus) undergoes a dramatic form of genome reprogramming wherein specific chromosomes are selectively eliminated from somatic progenitor cells. These programmatic elimination events effectively silence all genes on these chromosomes in all somatic cells. Previous studies in lamprey and other eliminating species have shown that epigenetic silencing marks are enriched on germline-specific chromosomes during programmed elimination. These silencing marks include the histone marks H3K9me3 and H4K20me3, which are respectively deposited by KMT1A/SUV39 and KMT5/SUV420 methyltransferases. To test whether lamprey homologs of these methyltransferases contribute to deposition of silencing marks on eliminated (micronucleated) chromatin and whether these marks contribute to the highly coordinated process of DNA elimination in sea lamprey, we used Cas9 gene editing, lightsheet imaging and RNA sequencing to investigate their potential roles in DNA elimination and more generally during early development. Analysis of knockout embryos for four histone methyltransferases show that these genes contribute to the deposition of repressive histone marks on elimination micronuclei, but are not essential for programmed DNA elimination per se. Analysis of later embryogenic stages suggests that these marks may contribute to interim silencing of germline-specific chromosomes, and reveals major impacts on post-blastula survival and development.

evolutionary biology↗

bioRxiv2025

Intrinsic and non-cell autonomous roles for a neurodevelopmental syndrome-linked transcription factor

Transcription factors (TFs) are essential for neuronal identity, yet their potential non-cell-autonomous functions remain largely unexplored. Here, we uncover both cell- and non-cell-autonomous roles for the conserved terminal selector UNC-3 in C. elegans motor neurons (MNs). UNC-3 is an ortholog of human EBF3, mutations in which cause a severe neurodevelopmental syndrome. Single-cell RNA sequencing of cholinergic MNs, which express unc-3, and downstream GABA MNs, which do not, revealed that unc-3 loss disrupts neuronal identity in distinct ways across MN classes. Four cholinergic MN classes lose their molecular identity entirely, whereas the AS class retains it partially, illuminating terminal selector-driven neuronal diversification processes. Integrated transcriptomic and genomic analyses uncovered a dual cell-autonomous role for UNC-3 as both a direct activator and repressor of neuron-type- specific genes in cholinergic MNs, including repression of alternate neurotransmitter programs. Unexpectedly, unc-3 loss also caused widespread transcriptional, morphological, and connectivity defects in downstream GABA MNs. Mechanistically, these non-cell-autonomous effects are mediated by cholinergic neurotransmission and include activation of the pro-regenerative bZIP TF CEBP-1 (C/EBP) and dysregulation of UNC-6/Netrin signaling. These findings redefine terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, offering a mechanistic framework for understanding EBF3 syndrome pathogenesis.

developmental biology↗

bioRxiv2025

Aging-dependent dysregulation of EXOSC2 is maintained in cancer as a dependency

Reprogramming of aged donor tissue cells into induced pluripotent stem cells (A-iPSC) preserved the epigenetic memory of aged-donor tissue, defined as genomic instability and poor tissue differentiation in our previous study. The unbalanced expression of RNA exosome subunits affects the RNA degradation complex function and is associated with geriatric diseases including premature aging and cancer progression. We hypothesized that the age-dependent progressive subtle dysregulation of EXOSC2 (exosome component 2) causes the aging traits (abnormal cell cycle and poor tissue differentiation). We used embryonic stem cells as a tool to study EXOSC2 function as the aging trait epigenetic memory determined in A-iPSC because these aging traits could not be studied in senesced aged cells or immortalized cancer cells. We found that the regulatory subunit of PP2A phosphatase, PPP2R5E, is a key target of EXOSC2 and this regulation is preserved in stem cells and cancer.

cell biology↗

bioRxiv2024

A genetically-encoded fluorescent biosensor for visualization of acetyl-CoA in live cells

Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA production and consumption are highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. In this work, we engineer an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). We biochemically characterize the sensor and demonstrate its selectivity for acetyl-CoA over other CoA species. We then deploy the biosensor in E. coli and HeLa cells to demonstrate its utility in living cells. In E. coli, we show that the biosensor enables detection of rapid changes in acetyl-CoA levels. In human cells, we show that the biosensor enables subcellular detection and reveals the compartmentalization of acetyl-CoA metabolism.

biochemistry↗

bioRxiv2023

A molecular atlas of adult C. elegans motor neurons reveals ancient diversity delineated by conserved transcription factor codes

Motor neurons (MNs) constitute an ancient cell type targeted by multiple adult-onset diseases. It is therefore important to define the molecular makeup of adult MNs in animal models and extract organizing principles. Here, we generated a comprehensive molecular atlas of adult Caenorhabditis elegans MNs and a searchable database (http://celegans.spinalcordatlas.org). Single-cell RNA-sequencing of 13,200 cells revealed that ventral nerve cord MNs cluster into 29 molecularly distinct subclasses. All subclasses are delineated by unique expression codes of either neuropeptide or transcription factor gene families. Strikingly, we found that combinatorial codes of homeodomain transcription factor genes define adult MN diversity both in C. elegans and mice. Further, molecularly defined MN subclasses in C. elegans display distinct patterns of connectivity. Hence, our study couples the connectivity map of the C. elegans motor circuit with a molecular atlas of its constituent MNs, and uncovers organizing principles and conserved molecular codes of adult MN diversity.

developmental biology↗

bioRxiv2023

Cytoplasmic Male Sterility and Abortive Seed Traits Generated through Mitochondrial Genome Editing Coupled with Allotopic Expression of atp1 in Tobacco

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated ({Delta}atp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/{Delta}atp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/{Delta}atp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent {Delta}atp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

plant biology↗

bioRxiv2023

Structural basis and functional roles for Toll-like receptor binding to Latrophilin adhesion-GPCR in embryo development

Latrophilins/ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects, lethality, and sterility across multiple model organisms. However, their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown. Here, we identified a cell-surface receptor, TOL-1, the sole Toll-like receptor in C. elegans, as a novel ligand for the C. elegans Latrophilin, LAT-1. The extracellular lectin domain of LAT-1 directly binds to the second leucine-rich repeat domain of TOL-1. The highresolution crystal structure and the cryo-EM density map of the LAT-1-TOL-1 ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by a large interface. CRISPR/Cas9-mediated mutation of key interface residues selectively disrupted the endogenous LAT-1-TOL-1 interaction in C. elegans, leading to partial sterility, lethality, and malformed embryos. Thus, TOL-1 binding to LAT-1 represents a receptor-ligand axis essential for animal morphogenesis.

biochemistry↗

bioRxiv2022

Mediator Kinase Inhibition Impedes Transcriptional Plasticity and Prevents Resistance to ERK/MAPK-Targeted Therapy in KRAS-Mutant Cancers

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this novel class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and tumor-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved population-level process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, tumor-specific resistance programs.

cancer biology↗

bioRxiv2022

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults

Light sheet fluorescence microscopy (LSFM) has become a method of choice for live imaging because of its fast acquisition and reduced photobleaching and phototoxicity. Despite the strengths and growing availability of LSFM systems, no generalized LSFM mounting protocol has been adapted for live imaging of post-embryonic stages of C. elegans. A major challenge has been to develop methods to limit animal movement using a mounting media that matches the refractive index of the optical system. Here, we describe a simple mounting and immobilization protocol using a refractive-index matched UV-curable hydrogel within fluorinated ethylene propylene (FEP) tubes for efficient and reliable imaging of larval and adult C. elegans stages.

developmental biology↗

bioRxiv2022

Maintenance of neurotransmitter identity by Hox proteins through a homeostatic mechanism

Hox transcription factors play fundamental roles during early patterning, but they are also expressed continuously - from embryo through adulthood - in the nervous system. The functional significance of their sustained expression remains unclear. In C. elegans motor neurons (MNs), we find that LIN-39 (Scr/Dfd/Hox4-5) is continuously required during post-embryonic life to maintain neurotransmitter identity, a core element of neuronal function. LIN-39 acts directly to co-regulate genes that define cholinergic identity (e.g., unc-17/VAChT, cho-1/ChT). We further show that LIN-39, MAB-5 (Antp/Hox6-8) and the transcription factor UNC-3 (Collier/Ebf) operate in a positive feedforward loop to ensure continuous and robust expression of cholinergic identity genes. Finally, we identify a two-component, design principle (Hox transcriptional autoregulation counterbalanced by negative UNC-3 feedback) for homeostatic control of Hox gene expression in adult MNs. These findings uncover a noncanonical role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of neurotransmitter identity.

developmental biology↗

bioRxiv2022

Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 12 homeobox genes in 20 distinct neuron classes that are mostly unrelated by location, function and lineage history. 10 of these 20 neuron classes had no homeobox gene function ascribed to them before, while in the other 10 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analysis, 111 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems.

neuroscience↗

bioRxiv2022

Reconstructing the ancestral vertebrate brain using a lamprey neural cell type atlas

The vertebrate brain emerged more than [~]500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey - a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits - based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of the core cell type composition, tissue structures, and gene expression programs of the ancestral brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral vertebrate brain was likely devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors on the jawed vertebrate lineage. Our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.

evolutionary biology↗

bioRxiv2022

A new immunocompetent rectal cancer model to study radiation therapy

We describe a new mouse model of rectal cancer (RC) involving rapid tumor organoid engraftment via orthotopic transplantation; the resulting RC tumors invaded inward from the mucosal surface and metastasized to distant organs. Histologically the tumors closely resemble human RC and mirror remodeling of the tumor microenvirnoment (TME) in response to radiation. This murine RC model thus recapitulates the pathogenesis of human RC, thereby fulfilling the need for a physiologically accurate model for preclinical efficacy studies.

cancer biology↗

bioRxiv2021

The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo

Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors (CRFs) are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and CRFs that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis. SUMMARY STATEMENTCellular invasion through the basement membrane by the C. elegans anchor cell requires both BAF and PBAF SWI/SNF assemblies to arrest the cell cycle and promote the expression of pro-invasive genes.

developmental biology↗

bioRxiv2020

The genomics of trait combinations and their influence on adaptive divergence

Understanding rapid adaptation requires quantifying natural selection on traits and elucidating the genotype-phenotype relationship for those traits. However, recent studies have often failed to predict the direction of adaptive allelic variation in natural populations from laboratory studies. Here, we test for genomic signatures of genetic correlations to illustrate how multifarious, correlational selection may drive counterintuitive patterns of population divergence in the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae). Apple-infesting populations with relatively early emerging adults have recently evolved from hawthorn-infesting populations consisting of relatively later emerging adults. Multiple studies have reported a paradoxical relationship between population differentiation and seasonal timing, as determined by the timing of diapause termination; alleles associated with late termination occur at higher frequencies in the earlier emerging apple-infesting populations compared to hawthorn-infesting populations. We present evidence that historical selection on diapause termination and another trait, initial diapause intensity, over geographic gradients generated genetic correlations between the traits in a direction antagonistic to contemporary selection on apple-infesting populations. Single nucleotide polymorphism in genomic regions of high linkage disequilibrium associated strongly with diapause termination and intensity, population divergence, geography, and evolutionary responses in laboratory selection experiments. These associations were consistent with geographically variable selection and with correlated evolutionary responses driving higher frequencies of late-associated alleles in the early emerging apple race. In contrast, loci associated only with diapause termination showed the expected pattern (more early-associated alleles in the apple race) in half of the population pairs. Our results suggest that selection on loci demonstrating antagonistic pleiotropy may often shape genomic footprints of rapid adaptation.

evolutionary biology↗

bioRxiv2019

Visualizing the metazoan proliferation-differentiation decision in vivo

Cell proliferation and terminal differentiation are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these cell cycle-associated events live in C. elegans and zebrafish. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from terminally differentiated cells in G0, revealing a commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. We also derive a predictive model of future proliferation behavior in C. elegans and zebrafish based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell cycle control in a wide-range of developmental contexts.

developmental biology↗