Biology subjects

Kingsley, D. M.

Publications and source records attributed to Kingsley, D. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Linked origins but distinct roles for extreme length and sequence variation at a tandem repeat in CACNA1C

Length variation in tandem repeats is a well-established driver of disease risk and is commonly assumed to arise from persistent genomic instability. Here, we characterize TRACT, a 30-bp variable number tandem repeat (VNTR) intronic to the calcium channel gene CACNA1C. TRACT exhibits extreme length variation (3-30+ kb) and has been previously linked to risk for bipolar disorder and schizophrenia. By examining multiple human cohorts, we find that TRACT alleles are strikingly bimodal in both length and sequence composition. Short alleles (TRACTS, [~]6 kb) and long alleles (TRACTL, [~]24 kb) are enriched for distinct 30-bp variants and are found on separate haplotypes that arose prior to the human migration out of Africa. Our data suggest that these ancient alleles expanded via perfect repeat tracts that were disrupted by accumulated mutations to result in relative length stability in extant humans, where there is no evidence for overt germline or somatic instability. Interestingly, neuropsychiatric disease risk is associated with specific 30-bp variants within TRACTS alleles, but not with overall TRACT length or with 30-bp variants enriched in TRACTL alleles. Instead, TRACTL alleles are associated with decreased gene expression in fibroblasts and testis. Together, these findings motivate joint examination of both sequence composition and length variation to fully understand the effects of VNTRs on evolution, trait variation, and disease risk.

genetics↗

bioRxiv2026

Genetic analysis of bone morphometry and ivory vertebrae in threespine stickleback

Previous genetic studies of skeletal variation in threespine stickleback fish (Gasterosteus aculeatus) have focused primarily on striking morphological differences. Here, we examine the largely unexplored genetic architecture of internal bone microstructural variation between marine and freshwater stickleback. {micro}CT X-ray analysis revealed differences in the porosity, bone thickness, and bone volume fraction within armor plates and vertebrae from a marine and freshwater stickleback. Quantitative trait locus mapping in F2 progeny from a marine x freshwater stickleback cross identified a significant locus on chromosome 4 influencing multiple aspects of armor plate internal microstructure. This locus overlaps the well-characterized Eda region previously known to control armor plate number and size. Co-mapping of bone microstructure could be due to pleiotropic effects of Eda on multiple aspects of plate development or to changes in closely linked genes including Itm2a, which also plays a role in bone formation. Most bone microstructure traits in vertebrae showed weak or no genetic signal, consistent with a polygenic architecture. However, we identified a highly significant locus on chromosome 17 that is strongly associated with abnormally thickened "ivory vertebrae" that occurred in 8.4% of F2 offspring. This phenotype resembles Pagets disease in humans, and the major locus region contains Tnfrsf1b, the stickleback ortholog of a human Pagets disease susceptibility gene TNFRSF11A. Together, our findings identify genetic loci underlying natural variation in bone microstructure in wild fish and reveal a candidate gene associated with a disease-like skeletal phenotype, highlighting stickleback as a model for studying both evolutionary and pathological bone biology.

genetics↗

bioRxiv2025

Rapid and repeated evolution of myosin copy number in threespine stickleback

Copy number variants at genomic loci evolve at a high rate, are linked to many different diseases, and play a role in adaptive evolution in humans and other organisms. Here we show that stickleback fish from freshwater environments have rapidly and repeatedly evolved an expanded number of copies of a gene family involved in muscle development, Myosin Heavy Chain 3 Cluster C (MYH3C), compared to marine populations. Differences in copy number between marine and freshwater fish are maintained even in the presence of gene flow, suggesting that MYH3C changes represent adaptive divergence between ecotypes. Copy number expansion occurs by tandem duplication of MYH3C coding and regulatory regions on the stickleback sex chromosome. We identify a muscle regulatory enhancer within the expanded MYH3C region and show that elevated copy number is associated with developmental and tissue-specific increases in corresponding mRNA expression levels in skeletal muscle. Common MYH3C clusters include 3-, 4-, 5-, and 6-copy variants that likely evolved through a combination of microhomology-mediated break repair and non-allelic homologous recombination. Our results provide a new example of copy number changes in a wild species and identify CNVs as potential "hotspots" of repeated adaptive evolution.

evolutionary biology↗

bioRxiv2025

Human-specific tandem repeat in CACNA1C modulates responses to neuronal stimulation

The recent development of long-read sequencing has made it possible to catalog variable number tandem repeats (VNTRs) in the human genome. However, little is known about their functional consequences. Here, we characterized the effect of TRACT, a VNTR that is unique to humans and that has sequence variants linked to risk for bipolar disorder and schizophrenia. By adding or removing this VNTR in both mouse models and human neural organoids, we find that TRACT, which is intronic to the L-type voltage-gated calcium channel gene CACNA1C, increases intracellular calcium after neuronal stimulation and leads to widespread changes in activity-dependent transcription programs in neurons. TRACT-dependent changes are enriched for genes associated with synapse formation and plasticity, and partially recapitulate evolutionary changes in activity-dependent transcription between species. These findings demonstrate that a single, human-specific, non-coding element can strongly affect the neuronal response to stimulation, and motivate the study of VNTRs as a genetic source of phenotypic variation in both evolution and disease.

genetics↗

bioRxiv2025

FOS binding sites are a hub for the evolution of activity-dependent gene regulatory programs in human neurons

After birth, sensory inputs to neurons trigger the induction of activity-dependent genes (ADGs) that mediate many aspects of neuronal maturation and plasticity. To identify human-specific ADGs, we characterized these genes in human-chimpanzee tetraploid neurons. We identified 235 ADGs that are differentially expressed between human and chimpanzee neurons and found that their nearby regulatory sites are species-biased in their binding of the transcription factor FOS. An assessment of these sites revealed that many are enriched for single nucleotide variants that promote or eliminate FOS binding in human neurons. Disrupting the function of individual species-biased FOS-bound enhancers diminishes expression of nearby genes and affects the firing dynamics of human neurons. Our findings indicate that FOS-bound enhancers are frequent sites of evolution and that they regulate human-specific ADGs that may contribute to the unusually protracted and complex process of postnatal human brain development.

evolutionary biology↗

bioRxiv2025

Human-chimpanzee tetraploid system defines mechanisms of species-specific neural gene regulation

A major challenge in human evolutionary biology is to pinpoint genetic differences that underlie human-specific traits, such as increased neuron number and differences in cognitive behaviors. We used human-chimpanzee tetraploid cells to distinguish gene expression changes due to cis-acting sequence variants that change local gene regulation, from trans expression changes due to species differences in the cellular environment. In neural progenitor cells, examination of both cis and trans changes -- combined with CRISPR inhibition and transcription factor motif analyses -- identified cis-acting, species-specific gene regulatory changes, including to TNIK, FOSL2, and MAZ, with widespread trans effects on neurogenesis-related gene programs. In excitatory neurons, we identified POU3F2 as a key cis-regulated gene with trans effects on synaptic gene expression and neuronal firing. This study identifies cis-acting genomic changes that cause cascading trans gene regulatory effects to contribute to human neural specializations, and provides a general framework for discovering genetic differences underlying human traits.

evolutionary biology↗

bioRxiv2024

The fourspine stickleback (Apeltes quadracus) has an XY sex chromosome system with polymorphic inversions on both X and Y chromosomes

Teleost fish are well-known for possessing a diversity of sex chromosomes and for undergoing frequent turnovers of these sex chromosomes. However, previous studies have mainly focused on variation between species, while comparatively little attention has been given to sex chromosome polymorphisms within species, which may capture early stages of sex chromosome changes. To better understand the evolution of sex chromosomes, we used the fourspine stickleback (Apeltes quadracus) as a model organism. Previously, it was believed that females of this species possessed a ZW heteromorphic sex chromosome system. However, genetic crosses and our whole-genome sequencing of wild populations revealed that A. quadracus has an XY sex chromosome on chromosome 23. This chromosome has not previously been identified as a sex chromosome in other stickleback species, indicating a recent sex chromosome turnover. We also identified two genes - rxfp2a and zar1l - as novel candidate sex determination genes. Notably, we observed inversions on both the X and Y chromosomes in different populations, resulting in distinctive patterns of differentiation between the X and Y chromosomes across populations. We propose that the inversion on the X chromosome may have been favored by sexually antagonistic selection. The new sex chromosome and intraspecies inversion polymorphisms observed in A. quadracus provide an excellent system for studying the evolution of sex chromosomes and their turnovers. Author SummaryAs compared to mammals and birds, teleost fish exhibit a very high level of diversity in their sex chromosomes, even among closely related species. Thus far, little attention has been paid to variation within species, although it offers a valuable opportunity to advance our understanding of the mechanisms underlying the formation and turnover of sex chromosomes. Through a quantitative trait locus (QTL) cross and sequencing diverse populations, we determined that instead of the previously reported ZW system, A. quadracus has an XY sex determination system on chromosome 23. Within the sex determining region, we identified rxfp2a and zar1l as putative sex determining genes. Notably, we also observed polymorphic inversions present on both the X and Y chromosomes that differ among populations. Based on our findings, we hypothesize that the X-linked inversions are favored by selection that differs between males and females. These observations represent a rare situation in which sex chromosomes are still polymorphic for sex-linked inversions, which offers important insights into the early stages of sex chromosome evolution.

evolutionary biology↗

bioRxiv2023

Evolution of novel sensory organs in fish with legs

How do animals evolve new traits? Sea robins are unusual "walking" fishes that use leg-like appendages to navigate the seafloor. Here, we show that legs are bona fide sense organs that mediate the unique ability to localize and uncover buried prey. We then probe the developmental and physiological basis of these novel sense organs as a striking example of a major trait gain in evolution. We find certain sea robin species have legs with unique end-organs called papillae that mediate enhanced mechanical and chemical sensitivity to enable predatory digging behavior. Papillae exhibit dense innervation from touch-sensitive neurons, noncanonical epithelial taste receptors, and chemical sensitivity that drives predatory digging behavior. Using a combination of developmental analyses, crosses between species with and without papillae, and interspecies comparisons of sea robins from around the world, we demonstrate that papillae represent a key evolutionary innovation associated with behavioral niche expansion on the seafloor. These discoveries provide a conceptual framework for understanding how molecular, cellular, and tissue-scale adaptations integrate to produce novel organismic traits and behavior.

evolutionary biology↗

bioRxiv2023

The genetic basis of novel trait gain in walking fish

A major goal in biology is to understand how organisms evolve novel traits. Multiple studies have identified genes contributing to regressive evolution, the loss of structures that existed in a recent ancestor. However, fewer examples exist for genes underlying constructive evolution, the gain of novel structures and capabilities in lineages that previously lacked them. Sea robins are fish that have evolved enlarged pectoral fins, six mobile locomotory fin rays (legs) and six novel macroscopic lobes in the central nervous system (CNS) that innervate the corresponding legs. Here, we establish successful husbandry and use a combination of transcriptomics, CRISPR-Cas9 editing, and behavioral assays to identify key transcription factors that are required for leg formation and function in sea robins. We also generate hybrids between two sea robin species with distinct leg morphologies and use allele-specific expression analysis and gene editing to explore the genetic basis of species-specific trait diversity, including a novel sensory gain of function. Collectively, our study establishes sea robins as a new model for studying the genetic basis of novel organ formation, and demonstrates a crucial role for the conserved limb gene tbx3a in the evolution of chemosensory legs in walking fish.

evolutionary biology↗

bioRxiv2023

Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development

While long-range enhancers and their target promoters are frequently contained within a TAD, many developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling such cross-TAD interactions remain to be assessed. To test these hypotheses, we use Optical Reconstruction of Chromatin Architecture (ORCA) to characterize the conformations of the Pitx1 locus on thousands of single chromosomes in developing mouse limbs. Our data supports a model in which neighboring boundaries are stacked with each other as a result of loop-extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also explains the appearance of architectural stripes in the population average maps (e.g. Hi-C data). Through molecular dynamics simulations, we further propose that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders function, both facilitating as well as preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.

developmental biology↗

bioRxiv2023

Whole-genome comparisons identify repeated regulatory changes underlying convergent appendage evolution in diverse fish lineages

Fins are major functional appendages of fish that have been repeatedly modified in different lineages. To search for genomic changes underlying natural fin diversity, we compared the genomes of 36 wild fish species that either have complete or reduced pelvic and caudal fins. We identify 1,614 genomic regions that are well-conserved in fin-complete species but missing from multiple fin-reduced lineages. Recurrent deletions of conserved sequences (CONDELs) in wild fin-reduced species are enriched for functions related to appendage development, suggesting that convergent fin reduction at the organismal level is associated with repeated genomic deletions near fin-appendage development genes. We used sequencing and functional enhancer assays to confirm that PelA, a Pitx1 enhancer previously linked to recurrent pelvic loss in sticklebacks, has also been independently deleted and may have contributed to the fin morphology in distantly related pelvic-reduced species. We also identify a novel enhancer that is conserved in the majority of percomorphs, drives caudal fin expression in transgenic stickleback, is missing in tetraodontiform, syngnathid, and synbranchid species with caudal fin reduction, and which alters caudal fin development when targeted by genome editing. Our study illustrates a general strategy for mapping phenotypes to genotypes across a tree of vertebrate species, and highlights notable new examples of regulatory genomic hotspots that have been used to evolve recurrent phenotypes during 100 million years of fish evolution.

evolutionary biology↗

bioRxiv2022

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy Against 2D and 3D Breast Cancer Models

Sophorolipids are biosurfactants derived from the non-pathogenic yeast Starmerella bombicola, with potential efficacy in anti-cancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drugscreening. Drug screening typically utilizes 2D cell monolayers due to their simplicity and potential for high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment, and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. We screened two sophorolipid candidates and clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing Optical Coherence Tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.

pharmacology and toxicology↗

bioRxiv2021

Evolution of stickleback spines through independent cis-regulatory changes at HOXDB

Understanding the genetic mechanisms leading to new traits is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different stickleback fish species to alter the length and number of bony dorsal spines. In Gasterosteus aculeatus, a variant HOXDB allele is genetically linked to shortening an existing spine and adding a spine. In Apeltes quadracus, a variant allele is associated with lengthening an existing spine and adding a spine. The alleles alter the same conserved non-coding HOXDB enhancer by diverse molecular mechanisms, including SNPs, deletions, and transposable element insertions. The independent cis-acting regulatory changes are linked to anterior expansion or contraction of HOXDB expression. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key morphological patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/472698v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@8e6675org.highwire.dtl.DTLVardef@e89e13org.highwire.dtl.DTLVardef@1e1f1dcorg.highwire.dtl.DTLVardef@cdb6f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2021

Genetic studies of human-chimpanzee divergence using stem cell fusions

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee auto-tetraploids and allo-tetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or auto-tetraploid iPSCs. Analysis of gene expression patterns in inter-specific allo-tetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves, and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allo-tetraploid cells. We successfully generated derivative cells with nested deletions or inter-specific recombination on the X chromosome. These studies identify a long distance cis-regulatory domain of the Fragile X-associated gene (FMR1), confirm an important role for the X chromosome in trans-regulation of other expression differences, and illustrate the potential of this system for more detailed mapping of the molecular basis of human and chimpanzee evolution. Significance StatementComparative studies of humans and chimpanzees have revealed many anatomical, physiological, behavioral, and molecular differences. However, it has been challenging to map these differences to particular chromosome regions. Here, we develop a genetic approach in fused stem cell lines that makes it possible to map human-chimpanzee molecular and cellular differences to specific regions of the genome. We illustrate this approach by mapping chromosome regions responsible for species-specific gene expression differences in fused tetraploid cells. This approach is general, and could be used in the future to map the genomic changes that control many other humanchimpanzee differences in various cell types or organoids in vitro.

evolutionary biology↗

bioRxiv2021

Characterization of mouse Bmp5 regulatory injury element in zebrafish wound models

Many key signaling molecules used to build tissues during embryonic development are re-activated at injury sites to stimulate tissue regeneration and repair. Bone morphogenetic proteins provide a classic example, but the mechanisms that lead to reactivation of BMPs following injury are still unknown. Previous studies have mapped a large "injury response element" (IRE) in the mouse Bmp5 gene that drives gene expression following bone fractures and other types of injury. Here we show that the large mouse IRE region is also activated in both zebrafish tail resection and mechanosensory hair cell injury models. Using the ability to test multiple constructs and image temporal and spatial dynamics following injury responses, we have narrowed the original size of the mouse IRE region by over 100 fold and identified a small 142 bp minimal enhancer that is rapidly induced in both mesenchymal and epithelial tissues after injury. These studies identify a small sequence that responds to evolutionarily conserved local signals in wounded tissues and suggest candidate pathways that contribute to BMP reactivation after injury.

developmental biology↗

bioRxiv2020

Predicting future from past: The genomic basis of recurrent and rapid stickleback evolution

Similar forms often evolve repeatedly in nature, raising longstanding questions about the underlying mechanisms. Here we use repeated evolution in sticklebacks to identify a large set of genomic loci that change recurrently during colonization of new freshwater habitats by marine fish. The same loci used repeatedly in extant populations also show rapid allele frequency changes when new freshwater populations are experimentally established from marine ancestors. Dramatic genotypic and phenotypic changes arise within 5-7 years, facilitated by standing genetic variation and linkage between adaptive regions. Both the speed and location of changes can be predicted using empirical observations of recurrence in natural populations or fundamental genomic features like allelic age, recombination rates, density of divergent loci, and overlap with mapped traits. A composite model trained on these stickleback features can also predict the location of key evolutionary loci in Darwins finches, suggesting similar features are important for evolution across diverse taxa.

evolutionary biology↗

bioRxiv2019

Assembly of a young vertebrate Y chromosome reveals convergent signatures of sex chromosome evolution

Heteromorphic sex chromosomes have evolved repeatedly across diverse species. Suppression of recombination between X and Y chromosomes leads to rapid degeneration of the Y chromosome. However, these early stages of degeneration are not well understood, as complete Y chromosome sequence assemblies have only been generated across a handful of taxa with ancient sex chromosomes. Here we describe the assembly of the threespine stickleback (Gasterosteus aculeatus) Y chromosome, which is less than 26 million years old. Our previous work identified that the non-recombining region between the X and the Y spans [~]17.5 Mb on the X chromosome. Here, we combined long-read PacBio sequencing with a Hi-C-based proximity guided assembly to generate a 15.87 Mb assembly of the Y chromosome. Our assembly is concordant with cytogenetic maps and Sanger sequences of over 90 Y chromosome clones from a bacterial artificial chromosome (BAC) library. We found three evolutionary strata on the Y chromosome, consistent with the three inversions identified by our previous cytogenetic analyses. The young threespine stickleback Y shows convergence with older sex chromosomes in the retention of haploinsufficient genes and the accumulation of genes with testis-biased expression, many of which are recent duplicates. However, we found no evidence for large amplicons found in other sex chromosome systems. We also report an excellent candidate for the master sex-determination gene: a translocated copy of Amh (Amhy). Together, our work shows that the same evolutionary forces shaping older sex chromosomes can cause remarkably rapid changes in the overall genetic architecture on young Y chromosomes.

evolutionary biology↗

bioRxiv2018

Characterization of a human-specific tandem repeat associated with bipolar disorder and schizophrenia

Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect over 3% of individuals worldwide. Genomewide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a novel human-specific tandem repeat in this region that is composed of 30 bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution, and may modulate neuropsychiatric disease risk in modern human populations.

genetics↗