Biology subjects

McKinney, S.

Publications and source records attributed to McKinney, S..

16 recordsLinked to original sources

bioRxiv2026

In situ identification of pre-hematopoietic stem cells and their niche

Definitive hematopoietic stem cells (HSCs) emerge predominantly along the ventral surface of the dorsal aorta in the embryonic aorta-gonad-mesonephros (AGM) region, yet the spatial organization of the niche underlying this developmental asymmetry remains poorly understood. Defining the native niche of pre-HSCs has also been hindered by their rarity, transient nature and lack of a single definitive marker for their identification in situ. Here, we integrated single-cell and spatial transcriptomics to construct a spatiotemporal atlas of the niche in the AGM. Neural cells and chondrogenic mesenchyme predominated dorsally, whereas N-cadherin mesenchymal stromal cells (N-cad+ MSCs) formed a ventral layer between the aortic endothelium and mesonephric cells. BMP and SHH signaling showed overall dorsal enrichment, whereas WNT, NOTCH and KIT signaling were enriched ventrally, with these cellular and signaling patterns dynamically remodeled across HSC ontogeny. Imaging-based spatial transcriptomics validated the findings and further enabled the identification of individual pre-HSCs in situ and revealed a multicellular niche comprising endothelial cells, N-cad+ MSCs, intra-aortic hematopoietic cluster cells and macrophages. TGF{beta} and NOTCH signals were primarily supplied locally, whereas major sources of BMP and WNT were more distant. Functional perturbation identified niche-derived CD200 as an immune-regulatory signal promoting HSC development, validating a spatially predicted niche interaction. Together, our findings link tissue-scale spatial organization with the local multicellular niche supporting the preferential development of HSCs along the ventral aorta.

developmental biology↗

bioRxiv2026

Minicollagen expression dynamics reveal a transcriptional program for cnidogenesis in the sea anemone Nematostella vectensis

Cnidae are explosive harpoon-like organelles localized within stinging cells, or cnidocytes, of the phylum Cnidaria (jellyfish, hydroids, sea anemones, and corals). These unique Golgi-derived vesicular structures define the phylum and are prominent examples of an evolutionary cellular novelty. While recent studies have focused on the developmental specification and regulation of cnidocytes more broadly, less is understood about gene expression patterns, structural variations, and toxin repertoires within distinct cnidae subtypes. Here, we determine the transcriptional profile of two major cnidae subtypes in the sea anemone Nematostella vectensis, nematocytes and spirocytes, using the cnidae-specific structural family of proteins called minicollagens. We first define the in vivo expression patterns for three known and three uncharacterized minicollagen orthologs. We show that four minicollagens are broadly expressed throughout ectodermal cnidocytes in developing larvae and primary polyps while two others are restricted to tentacular cnidocytes. Leveraging whole adult scRNA-seq data and two novel transgenic reporter lines, we then demonstrate that the tentacle-restricted cnidocytes are developing spirocytes that are distinguished by expression of the minicollagen NvNcol5. To deepen our analysis of cnidocyte gene expression, we used a customized RNA-FACS-seq pipeline to determine global transcriptional differences between these two subtypes. This approach identified a suite of differentially expressed genes, illuminating spatial and temporal gene expression dynamics across both developing nematocytes and spirocytes. Altogether, our experiments provide fundamental and novel insights into the specialization of cnidarian stinging cells while establishing a rich set of resources for further investigation.

evolutionary biology↗

bioRxiv2026

The consequences of array size for human centromere performance in mitosis

Long-read sequencing and assembly of human genomes have revealed extreme variation in centromeric alpha-satellite array sizes across chromosomes and individuals. However, assessing the impact of centromeric array size on centromere function remains challenging. In this study, using quantitative FISH and microscopy assays, we established a framework for the systematic functional evaluation of naturally occurring variations in centromeric array size during mitosis. By comparing 8 pairs of homologous chromosomes exhibiting 1.24 - 4.6 -fold variation in centromeric array size, we uncovered size-based differences in function. We found that between a pair of homologous chromosomes, the chromosome harboring the smaller centromeric array is more prone to chromosome missegregation. Centromere size-based differences in chromosome missegregation rates cannot be explained by differential enrichment of molecular factors like centromeric histone CENP-A, CENP-B, or most kinetochore proteins. However, the smaller centromere out of a homologous pair consistently exhibits increased cohesion fatigue, suggesting the involvement of the cohesin complex in size-based differences in centromere cohesion. Consistent with our hypothesis, complete deprotection of the cohesin complex by depletion of shugoshin-1, removes the size-based bias in centromere cohesion. These results suggest that while CENP-A-enriched core centromeres are essential for kinetochore assembly, variation in overall centromeric array size has a significant impact on centromere performance and the fidelity of chromosome segregation during mitosis.

Cell Biology↗

bioRxiv2026

Nucleolar Dynamics During Oogenesis

Ribosome biogenesis is a conserved and highly regulated process that starts in the nucleolus, a membrane-less multi-phase organelle. Although the architecture of the nucleolus is known to change due to perturbations, how nucleolar organization is modulated during physiological processes to meet changing translational demands remains unclear. Here, we use zebrafish oogenesis as a developmental context requiring a rapid expansion of translational capacity to investigate the regulation of nucleolar architecture. We show nucleoli undergo coordinated changes in number, size, subnuclear localization, and layering throughout oogenesis. We further demonstrate that nucleoli form around extrachromosomal DNA circles that contain the rDNA locus. Notably, mouse oocytes undergo similar developmental changes in nucleolar layering and phase organization, indicating that remodeling of nucleolar condensates is a conserved feature of oogenesis. These findings reveal previously unexplored regulation of nucleolar architecture as developmental adaptations to changing biosynthetic needs.

developmental biology↗

bioRxiv2025

Complete genomes of a multi-generational pedigree to expand studies of genetic and epigenetic inheritance

Pedigree analysis remains the gold standard for rare disease diagnostics, yet whole genome sequencing studies typically omit critical regions like centromeres, telomeres, and acrocentric chromosome p-arms. Here, we present telomere-to-telomere (T2T) reference genomes for four self-identified African American individuals of admixed ancestry spanning three generations. Our parent-of-origin assigned, chromosome-level assemblies revealed precise meiotic recombination breakpoints in previously inaccessible regions, including recombination events across acrocentric and subtelomeric sequences. Centromeric regions were highly stable, with multi-megabase arrays inherited intact across three generations, while the position of kinetochore assembly sites remained consistent and predominantly associated with the p-arm proximal region. The relative lengths of telomeres on individual chromosomes were maintained across generations. Using a targeted rDNA assembly approach, we reconstructed a complete megabase-scale ribosomal DNA (rDNA) array corresponding to the paternal chromosome 14. This openly available pedigree provides a benchmark dataset for studying recombination and genetic and epigenetic variation across the complete genome.

genomics↗

bioRxiv2025

Loss of SPECC1L in cranial neural crest cells results in increased hedgehog signaling and frontonasal dysplasia

SPECC1L encodes a cytoskeletal scaffolding protein that interacts with filamentous actin, microtubules, and cell junctional components. In humans, autosomal dominant mutations in SPECC1L cause a syndrome characterized by craniofrontonasal anomalies including broad nasal bridge, ocular hypertelorism, prominent forehead, and cleft lip/palate. Complete loss of SPECC1L in mice on a homogenous genetic background results in perinatal lethality, accompanied by subtle cranial differences and incompletely penetrant cleft palate. This lethality limits postnatal analysis of craniofacial development. Because cranial neural crest cells (CNCCs) contribute extensively to the formation of anterior craniofacial structures, we investigated whether disruption of SPECC1L in CNCCs contributes to the craniofrontonasal phenotypes observed in SPECC1L-related syndrome. We generated a Specc1l-floxed allele and crossed it with the Wnt1-Cre2 deleter strain, which drives Cre recombinase expression in the dorsal neuroectoderm and NCCs. Most homozygous mutant Specc1l{Delta}CNCC mutants survived postnatally and exhibited hallmark features of the human SPECC1L-related syndrome, including shortened skulls, reduced frontal bone area, nasal defects, and midface hypoplasia. The cranial mesenchyme of Specc1l{Delta}CNCC mice displayed shortened primary cilia and increased Hedgehog (Hh) signaling activity at E13.5, as evidenced by enhanced GLI1 immunostaining. These defects were also observed early in E9.5 facial prominences, indicating that they are etiologic in nature. Collectively, Specc1l{Delta}CNCC mice provide a novel model for investigating the roles of CNCCs, primary cilia, and Hh signaling in frontonasal prominence and midfacial development.

developmental biology↗

bioRxiv2025

Reproductive Adaptation of Astyanax mexicanus Under Nutrient Limitation

Reproduction is a fundamental biological process for the survival and continuity of species. Examining changes in reproductive strategies offers valuable insights into how animals have adapted their life histories to different environments. Since reproduction is one of the most energy-intensive processes in female animals, nutrient scarcity is expected to interfere with the ability to invest in gametes. Lately, a new model to study adaptation to nutrient limitation has emerged; the Mexican tetra Astyanax mexicanus. This fish species exists as two different morphs, a surface river morph and a cave-dwelling morph. The cave-dwelling morph has adapted to the dark, biodiversity, and nutrient-limited cave environment and consequently evolved an impressive starvation resistance. However, how reproductive strategies have adapted to nutrient limitations in this species remains poorly understood. Here, we compared breeding activities and maternal contributions between laboratory-raised surface fish and cavefish. We found that cavefish produce different clutch sizes of eggs with larger yolk compared to surface fish, indicating a greater maternal nutrient deposition in cavefish embryos. To systematically characterize yolk compositions, we used untargeted proteomics and lipidomics approaches to analyze protein and lipid profiles in 2-cell stage embryos and found an increased proportion of sphingolipids in cavefish compared to surface fish. Additionally, we generated transcriptomic profiles of surface fish and cavefish ovaries using a combination of single cell and bulk RNA sequencing to examine differences in maternal contribution. We found that genes essential for hormone regulation were upregulated in cavefish follicular somatic cells compared to surface fish. To evaluate whether these differences contribute to their reproductive abilities under natural-occurring stress, we induced breeding in starved female fish. Remarkably, cavefish maintained their ability to breed under starvation, whereas surface fish largely lost this ability. We identified insulin-like growth factor 1a receptor (igf1ra) as a potential candidate gene mediating the downregulation of ovarian development genes, potentially contributing to the starvation-resistant fertility of cavefish. Taken together, we investigated the female reproductive strategies in Astyanax mexicanus, which will provide fundamental insights into the adaptations of animals to environments with extreme nutrient deficit.

evolutionary biology↗

bioRxiv2024

Characterization of Multicellular Niches Supporting Hematopoietic Stem Cells Within Distinct Zones

Previous studies of hematopoietic stem cells (HSCs) primarily focused on single cell-based niche models, yielding fruitful but conflicting findings1-5. Here we report our investigation on the fetal liver (FL) as the primary fetal hematopoietic site using spatial transcriptomics. Our study reveals two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. The PV niche, composing N-cadherin (N-cad)HiPdgfr+ mesenchymal stromal cells (MSCs), endothelial cells (ECs), and N-cadLoAlbumin+ hepatoblasts, maintains quiescent and multipotential FL-HSCs. Conversely, the sinusoidal niche, comprising ECs, hepatoblasts and hepatocytes, as well as potential macrophages and megakaryocytes, supports proliferative FL-HSCs biased towards myeloid lineages. Unlike prior reports on the role of Cxcl12, with its depletion from vessel-associated stromal cells leading to 80% of HSCs reduction in the adult bone marrow (BM)6,7, depletion of Cxcl12 via Cdh2CreERT (encoding N-cad) induces altered localization of HSCs from the PV to the sinusoidal niches, resulting in an increase of HSC number but with myeloid-bias. Similarly, we discovered that adult BM encompasses two niches within different zones, each composed of multi-cellular components: trabecular bone area (TBA, or metaphysis) supporting deep-quiescent HSCs, and central marrow (CM, or diaphysis) fostering heterogenous proliferative HSCs. This study transforms our understanding of niches by shifting from single cell-based to multicellular components within distinct zones, illuminating the intricate regulation of HSCs tailored to their different cycling states.

developmental biology↗

bioRxiv2022

Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here we optimized single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the RA response.

developmental biology↗

bioRxiv2022

Metabolic reprogramming underlies cavefish muscular endurance despite loss of muscle mass and contractility

Physical inactivity - specifically the lack of moderate-to-vigorous activity - is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing unique evolutionary insight into adaptive physical inactivity. The Mexican cavefish Astyanax mexicanus lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3-fold slower than their river-dwelling counterpart. We found that this was accompanied by a marked shift in body composition, decreasing muscle mass by 30% and increasing fat mass by 40%. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated this shift in investment is driven by increased expression of ppar{gamma} - the master regulator of adipogenesis - with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming velocity. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omics analysis indicated metabolic reprogramming, specifically increased phosphoglucomutase-1 abundance, phosphorylation, and activity, as contributing mechanisms enhancing cavefish glycogen utilization under metabolically strenuous conditions. Collectively, we reveal broad skeletal muscle reprogramming following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction under fatiguing conditions. SignificanceThe evolutionary consequence of decreased physical activity upon skeletal muscle physiology remains unexplored. Using the Mexican cavefish, we find loss of moderate-to-vigorous swimming following cave colonization has resulted in broad shifts in skeletal muscle investment - away from muscle mass and instead toward fat and sugar accumulation - ultimately decreasing muscle fiber twitch kinetics. Surprisingly though, cavefish possessed marked muscular endurance, reaching maximal swimming speeds rivaling their river-dwelling counterpart. Multi-omics analysis revealed carbohydrate metabolic reprogramming as a contributing component, most notably elevated abundance and phosphorylation of the glycogenolytic enzyme Phosphoglucomutase-1 - a likely adaptation to cave-specific hypoxia. These findings emphasize the impact multiple selective pressures have on skeletal muscle physiology, providing the first evolutionary insight into skeletal muscle adaptation following decreased activity.

evolutionary biology↗

bioRxiv2021

The impact of mutational processes on structural genomic plasticity in cancer cells

Structural genome alterations are determinants of cancer ontogeny and therapeutic response. While bulk genome sequencing has enabled delineation of structural variation (SV) mutational processes which generate patterns of DNA damage, we have little understanding of how these processes lead to cell-to-cell variations which underlie selection and rates of accrual of different genomic lesions. We analysed 309 high grade serous ovarian and triple negative breast cancer genomes to determine their mutational processes, selecting 22 from which we sequenced >22,000 single cell whole genomes across a spectrum of mutational processes. We show that distinct patterns of cell-to-cell variation in aneuploidy, copy number alteration (CNA) and segment length occur in homologous recombination deficiency (HRD) and fold-back inversion (FBI) phenotypes. Widespread aneuploidy through induction of HRD through BRCA1 and BRCA2 inactivation was mirrored by continuous whole genome duplication in HRD tumours, contrasted with early ploidy fixation in FBI. FBI tumours exhibited copy number distributions skewed towards gains, widespread clone-specific variation in amplitude of high-level amplifications, often impacting oncogenes, and break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). SSVs were consistent with a CNA-based molecular clock reflecting a continual and distributed process across clones within tumours. These observations reveal previously obscured genome plasticity and evolutionary properties with implications for cancer evolution, therapeutic targeting and response.

cancer biology↗

bioRxiv2019

An adult brain atlas reveals broad neuroanatomical changes in independently evolved populations of Mexican cavefish

A shift in environmental conditions impacts the evolution of complex developmental and behavioral traits. The Mexican cavefish, Astyanax mexicanus, is a powerful model for examining the evolution of development, physiology, and behavior because multiple cavefish populations can be compared to an extant and ancestral-like surface population of the same species. Many behaviors have diverged in cave populations of A. mexicanus, and previous studies have shown that cavefish have a loss of sleep, reduced stress, an absence of social behaviors, and hyperphagia. Despite these findings, surprisingly little is known about the changes in neuroanatomy that underlie these behavioral phenotypes. Here, we use serial sectioning to generate a brain atlas of surface fish and three independent cavefish populations. Volumetric reconstruction of serial-sectioned brains confirms convergent evolution on reduced optic tectum volume in all cavefish populations tested. In addition, we quantified volumes of specific neuroanatomical loci within several brain regions, which have previously been implicated in behavioral regulation, including the hypothalamus, thalamus, and habenula. These analyses reveal an expansion of the hypothalamus across all three cavefish populations relative to surface fish, as well as subnuclei-specific differences within the thalamus and habenulae. Taken together, these analyses support the notion that changes in environmental conditions are accompanied by neuroanatomical changes in brain structures associated with behavior. This atlas provides a resource for comparative neuroanatomy of additional brain regions and the opportunity to associate brain anatomy with evolved changes in behavior.

neuroscience↗

bioRxiv2018

Membrane insertion function for SUN-KASH complex revealed by high resolution analysis of yeast centrosomes

Bipolar spindle formation in yeast requires insertion of centrosomes (known as spindle pole bodies (SPBs)) into fenestrated regions of the nuclear envelope (NE). Using structured-illumination microscopy and bimolecular fluorescence complementation, we map protein distribution at SPB fenestra and interrogate protein-protein interactions with high spatial resolution. We find that the Sad1-UNC-84 (SUN) protein Mps3 forms a ring-like structure around the SPB, similar to toroids seen for components of the SPB insertion network (SPIN). Mps3 and the SPIN component Mps2 (a Klarsicht-ANC-1-Syne-1 domain (KASH)-like protein) form a novel non-canonical linker of nucleoskeleton and cytoskeleton (LINC) complex that is connected in both luminal and extraluminal domains. This hairpin-like LINC complex forms during SPB insertion, suggesting it functions in NE reorganization at the pore membrane. The LINC complex also controls the distribution of a soluble SPIN component Bbp1. Taken together our work shows that Mps3 is a fifth SPIN component and suggests both direct and indirect roles for the LINC complex in NE remodeling.

cell biology↗

bioRxiv2018

Synaptonemal complex architecture facilitates the chromosome-specific regulation of recombination in Drosophila

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double strand breaks (DSBs) into crossovers or gene conversions. The removal of any central region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct three in-frame deletions within the predicted coiled-coil region of the C(3)G protein. These three deletion mutants disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, allowing us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently than the autosomes.

genetics↗

bioRxiv2018

Integrated single-nucleotide and structural variation signatures of DNA-repair deficient human cancers

Mutation signatures in cancer genomes reflect endogenous and exogenous mutational processes, offering insights into tumour etiology, features for prognostic and biologic stratification and vulnerabilities to be exploited therapeutically. We present a novel machine learning formalism for improved signature inference, based on multi-modal correlated topic models (MMCTM) which can at once infer signatures from both single nucleotide and structural variation counts derived from cancer genome sequencing data. We exemplify the utility of our approach on two hormone driven, DNA repair deficient cancers: breast and ovary (n=755 cases total). Our results illuminate a new age-associated structural variation signature in breast cancer, and an independently identified substructure within homologous recombination deficient (HRD) tumours in breast and ovarian cancer. Together, our study emphasizes the importance of integrating multiple mutation modes for signature discovery and patient stratification, with biological and clinical implications for DNA repair deficient cancers.

cancer biology↗

bioRxiv2017

An adaptable chromosome preparation methodology for use in invertebrate research organisms

BackgroundThe ability to efficiently visualize and manipulate chromosomes is fundamental to understand the genetic architecture of organisms. This can be challenging for many invertebrates because conventional chromosome preparation protocols deal primarily hypotonic conditions tailored for use with vertebrates and rely extensively on cultured cells which are rarely available for invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate model species is needed.\n\nResultsWe optimized a chromosome preparation protocol and applied it to several planarian species (Phylum: Platyhelminthes), the freshwater apple snail Pomacea canaliculata (Phylum: Mollusca), and the starlet sea anemone Nematostella vectensis (Phylum: Cnidaria). We showed that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with DI water was sufficient for karyotyping. The karyotypes we obtained allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla, this protocol can be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization.\n\nConclusionThe simple and inexpensive chromosome preparation protocol reported here can be readily adapted to new invertebrate research organisms in order to expand and accelerate the discovery of new biology in understudied branches of the tree of life.

developmental biology↗