Biology subjects

GRAY, R.

Publications and source records attributed to GRAY, R..

5 recordsLinked to original sources

bioRxiv2026

Genetic interaction between Adgrg6 and Sox9 reveals a feedforward mechanism for postnatal spinal stability

Adolescent idiopathic scoliosis (AIS) is the most common spinal disorder in children and is best understood as a complex polygenic condition. Genome-wide association studies (GWAS) have identified several AIS risk loci, including regions near ADGRG6 and SOX9, yet the functional mechanisms underlying AIS heritability remain poorly defined. Here, we use spatial transcriptomics to characterize altered gene expression in the spine of a conditional Adgrg6 mutant mouse model of AIS (Adgrg6-cKO), revealing reduced expression of Sox9 and several components of extracellular matrix organization in the intervertebral disc. We further show that SOX9 occupies regions of open chromatin within the Adgrg6 locus in cells isolated from the mouse intervertebral disc. Finally, we demonstrate a strong genetic interaction between Adgrg6-cKO and a hypomorphic Sox9 allele that increases both the penetrance and severity of AIS-like pathology in mice. Collectively, these findings support a self-reinforcing feedforward regulatory circuit, where Adgrg6 and Sox9 are co-regulated to maintain extracellular matrix gene expression in the annulus fibrosus and paraspinal tissues. These findings provide mechanistic insight into the functional significance of AIS-associated GWAS loci near ADGRG6 and SOX9 and establish combined Adgrg6-Sox9 insufficiency as a tractable model of polygenic scoliosis susceptibility. O_LIAdgrg6 maintains Sox9 and extracellular matrix gene expression in the intervertebral disc C_LIO_LISOX9 directly occupies open chromatin at the Adgrg6 locus in SOX9 skeletal cells C_LIO_LIAdgrg6 and Sox9 genetically interact to increase scoliosis incidence and severity C_LIO_LICombined loss of Adgrg6 and Sox9 drives ectopic cartilage formation and reduced vertebral bone mineral density in the thoracic spine C_LI

developmental biology↗

bioRxiv2024

Discovery and Engineering of Retrons for Precise GenomeEditing

Retrons are promising gene editing tools because they can produce multi-copy single-stranded DNA in cells via self-primed reverse transcription. However, their potential for inserting genetic cargos in eukaryotes remains largely unexplored. Here we report the discovery and engineering of highly efficient retron-based gene editors for mammalian cells and vertebrates. Through bioinformatic analysis of metagenomic data and functional screening, we identified novel retron reverse transcriptases (RTs) that are highly active in mammalian cells. Rational design further improved the editing efficiency to levels comparable with conventional single-stranded oligodeoxynucleotide donors but from a genetically encoded cassette. Small molecule inhibitors of non-homologous end joining factors and Cas9-DNA repair protein fusions further increase homology-directed repair. Retron editors also exhibited robust activity with Cas12a nuclease and Cas9 nickase, expanding the genomic target scope and bypassing the need for a DNA double-stranded break. Using a rationally engineered retron editor, we incorporate a split GFP epitope tag for live cell imaging. Finally, we develop an all-RNA delivery strategy to enable DNA-free gene editing in cells and vertebrate embryos. This work establishes retron editors as a versatile and efficient tool for precise gene editing, offering new opportunities for biotechnology and biomedical research.

molecular biology↗

bioRxiv2021

A G protein-coupled receptor is required in cartilaginous and dense connective tissues to maintain spine alignment

Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs and dense connective tissues (e.g., ligaments and tendons), are essential for maintenance of spinal alignment. We show that the G-coupled protein receptor Adgrg6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine morphology. We show that Adgrg6 regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with an cAMP agonist was able to mirror aspects of receptor function in culture defining core pathways for regulation of these axial connective tissues. As ADGRG6 is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis. HighlightsO_LIKnockout mice lacking Adgrg6 function in the tendons and ligaments of the spine develop perinatal-onset thoracic scoliosis. C_LIO_LILoss of Adgrg6 function in cartilaginous tissues of the discs contribute to the incidence and severity of scoliosis. C_LIO_LIThe loss of Adgrg6 function in spine tissues provide a model of construct validity for human adolescent idiopathic scoliosis C_LIO_LIFine tuning of the biomechanical properties of dense connective tissues is essential for maintaining spine alignment. C_LI

developmental biology↗

bioRxiv2020

Postembryonic screen for mutations affecting spine development in zebrafish

The spinal vertebral column gives structural support for the adult body plan, protects the spinal cord, and provides muscle attachment and stability, which allows the animal to move within its environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10{degrees} around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant mutant zebrafish, displaying overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1,229 F1 animals, and subsequently bred to homozygosity in F3 families, from these, 314 haploid genomes were screened for recessive phenotypes. We cumulatively found 39 adult-viable (3 dominant and 36 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing within this phenotypic group revealed at least 16 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report several recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Many of the alleles characterized thus far are non-synonymous mutations in known essential scospondin and adamts9 genes. Our results provide evidence of monogenic traits that are critical for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.

developmental biology↗

bioRxiv2019

A Comparative Study of the Turnover of Multiciliated Cells in the Mouse Trachea, Oviduct and Brain

In mammals, multiciliated cells (MCCs) line the lumen of the trachea, oviduct, and brain ventricles, where they drive fluid flow across the epithelium. Each MCC population experiences vastly different local environments that may dictate differences in their lifetime and turnover rates. However, with the exception of MCCs in the trachea, the turnover rates of these multiciliated epithelial populations at extended time scales are not well described. Here, using genetic lineage-labeling techniques we provide a direct comparison of turnover rates of MCCs in these three different tissues. We find that oviduct turnover is similar to that in the airway ([~]6 months), while multiciliated ependymal cells turnover more slowly.

developmental biology↗