Biology subjects

Kwon, R. Y.

Publications and source records attributed to Kwon, R. Y..

14 recordsLinked to original sources

bioRxiv2025

Automatic Body Morphometric Analysis of Adult Zebrafish Using MicroCT

Zebrafish (Danio rerio) are an important model for the study of musculoskeletal genetics, development, disease, regeneration, and evolution. While micro-computed tomography (microCT) allows for the assessment of quantitative 3D morphometric measures related to body size and shape and lean mass in adult zebrafish, computing these measures is typically a labor- intensive process. The purpose of this study was to develop fully automatic methods for the calculation of lean volume, fineness ratio, anterior and posterior swim bladder length, and standard length from microCT scans of adult zebrafish. Here, we show that built-in functions in the open-source software FIJI/ImageJ can be combined into fully automated methods to compute these measures. We show that measures computed by automated methods compare favorably to those computed using manual and semi-automated methods while significantly improving the throughput of data collection and eliminating inter-observer variability. We have implemented these methods as ImageJ macros, providing an accessible tool to facilitate the use of microCT for body morphometric analyses in adult zebrafish.

genetics↗

bioRxiv2024

Loss of cped1 does not affect bone and lean mass in zebrafish

Human genetic studies have nominated Cadherin-like and PC-esterase Domain-containing 1 (CPED1) as a candidate target gene mediating bone mineral density (BMD) and fracture risk heritability. Recent efforts to define the role of CPED1 in bone in mouse and human models have revealed complex alternative splicing and inconsistent results arising from gene targeting, making its function in bone difficult to interpret. To better understand the role of CPED1 in adult bone mass and morphology, we conducted a comprehensive genetic and phenotypic analysis of cped1 in zebrafish, an emerging model for bone and mineral research. We analyzed two different cped1 mutant lines and performed deep phenotyping to characterize more than 200 measures of adult vertebral, craniofacial, and lean tissue morphology. We also examined alternative splicing of zebrafish cped1 and gene expression in various cell/tissue types. Our studies fail to support an essential role of cped1 in adult zebrafish bone. Specifically, homozygous mutants for both cped1 mutant alleles, which are expected to result in loss-of-function and impact all cped1 isoforms, exhibited no significant differences in the measures examined when compared to their respective wildtype controls, suggesting that cped1 does not significantly contribute to these traits. We identified sequence differences in critical residues of the catalytic triad between the zebrafish and mouse orthologs of CPED1, suggesting that differences in key residues, as well as distinct alternative splicing, could underlie different functions of CPED1 orthologs in the two species. Our studies fail to support a requirement of cped1 in zebrafish bone and lean tissue, adding to evidence that variants at 7q31.31 can act independently of CPED1 to influence BMD and fracture risk. Lay summaryBone mineral density (BMD) is a key indicator for predicting and diagnosing osteoporosis and fracture risk, and it has been estimated that up to 89% of variation in BMD is determined by genetics. Multiple human genetics studies have nominated CPED1 as a potential gene underlying BMD and fracture risk heritability, however the function of CPED1 remains poorly understood. In this study, we examined the role of cped1 in bone by quantifying over 200 morphological measures of vertebral and craniofacial bone size, shape, and density in two different mutant lines of zebrafish in which cped1 function was reduced or eliminated. We also examined lean tissue mass because co-heritability of this trait with BMD has also been hypothesized to involve CPED1. Surprisingly, despite the loss of cped1 function, there were no significant differences between the mutant zebrafish and their respective controls. Our study therefore fails to support a role for cped1 in bone and lean tissue, suggesting that hereditary influence on BMD and fracture risk can occur independently of CPED1.

genetics↗

bioRxiv2024

Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility

Heritable Fragile Bone Disorders (FBDs) encompass a spectrum of conditions, from widespread multifactorial to rare monogenic diseases, all characterized by an elevated risk of fractures. The process of validating causative genes and elucidating their pathogenic mechanisms remains a daunting and resource-intensive task. In this study, we evaluated the feasibility of a semi-high throughput zebrafish screening platform for rapid validation and in vivo functional testing and validation of candidate disease-causing genes for a wide range of heritable FBDs. Six genes associated with severe recessive forms of Osteogenesis Imperfecta (OI) and four genes associated with bone mineral density (BMD), a key osteoporosis indicator, identified through genome-wide association studies (GWAS) were selected. The crispant screening approach, based on CRISPR/Cas9 technology, was used to phenotype directly in F0 mosaic founder zebrafish. Next-Generation Sequencing (NGS) analysis revealed a mean indel efficiency of 88% across ten different crispants, indicating a high proportion of knock-out alleles and thus resembling stable knock-out models. We applied multiple techniques to evaluate skeletal characteristics at 7, 14 and 90 days post-fertilization (dpf), including microscopy for osteoblast reporter visualization and mineralization by Alizarin Red S staining, and microCT for quantitative skeletal analysis. While larval crispants exhibited variable differences in osteoblast-positive and mineralized surface areas, adult-stage crispants displayed more pronounced and consistent skeletal phenotypes. Notably, all crispants developed malformed neural and haemal arches, with a majority presenting vertebral fractures and fusions, and some showing significant alterations in vertebral bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR analysis of osteoblast differentiation and bone formation markers at larval stages indicated differential expression of osteogenic markers bglap and col1a1a in a substantial portion of the crispants, hinting at their utility as biomarkers for FBD crispant screening. In summary, our findings demonstrate that crispant screening in zebrafish offers a viable and efficient strategy for the functional assessment of FBD genes. We advocate for a novel comprehensive approach that integrates various techniques and evaluates distinct skeletal and molecular profiles across different developmental and adult stages. This methodology has the potential to provide new insights into the role of these genes in skeletal biology.

genetics↗

bioRxiv2024

Dominant nonsense mutations in efemp1 alter vertebral and craniofacial characteristics in adult zebrafish

Heritable Disorders of Connective Tissues (HDCT) are a heterogenous, pleiotropic group of conditions that broadly affect connective tissues. EFEMP1 is a member of the fibulin family of extracellular matrix (ECM) glycoproteins which is expressed in various human tissues. Individuals with EFEMP1 variants have recently been identified and appear to have Marfan-like characteristics. Clinical phenotypes of these individuals include hernias, advanced bone age, tall stature, myopia, joint laxity, and thin skin. EFEMP1-associated HDCTs have been identified in individuals with biallelic and monoallelic variants. There is an urgent need to better understand the role of EFEMP1 in regulating connective tissues including bone, and the pathophysiological mechanisms underlying the spectrum of genotype-phenotype relationships seen in EFEMP1-associated HDCTs. To investigate the role of EFEMP1 in developing and adult bone, we used CRISPR-based editing to generate two efemp1 zebrafish alleles encoding for premature termination codons (PTCs) predicted to delete or severely alter the fibulin-type domain. Both alleles exhibited similar phenotypes in juvenile and adult fish. In juvenile fish, we did not identify changes in body size or vertebral development. In adults, we found significant changes in body length, bone microarchitecture, and craniofacial measurements in both heterozygous and homozygous mutant fish. These results expand our understanding of the role of efemp1 in the skeleton and highlight the potential for dominant nonsense variants to play a role in manifestation of clinical phenotypes in EFEMP1-associated HDCTs.

genetics↗

bioRxiv2022

Examining craniofacial variation among crispant and mutant zebrafish models of human skeletal diseases

Genetic diseases affecting the skeletal system present with a wide range of symptoms that make diagnosis and treatment difficult. Genome-wide association and sequencing studies have identified genes linked to human skeletal diseases. Gene editing of zebrafish models allows researchers to further examine the link between genotype and phenotype, with the long-term goal of improving diagnosis and treatment. While current automated tools enable rapid and in-depth phenotyping of the axial skeleton, characterizing the effects of mutations on the craniofacial skeleton has been more challenging. The objective of this study was to evaluate a semi-automated screening tool can be used to quantify craniofacial variations in zebrafish models using four genes that have been associated with human skeletal diseases (meox1, plod2, sost, and wnt16) as test cases. We used traditional landmarks to ground truth our dataset and pseudolandmarks to quantify variation across the 3D cranial skeleton between the groups (somatic crispant, germline mutant, and control fish). The proposed pipeline identified variation between the crispant or mutant fish and control fish for four genes. Variation in phenotypes parallel human craniofacial symptoms for two of the four genes tested. This study demonstrates the potential as well as the limitations of our pipeline as a screening tool to examine multi-dimensional phenotypes associated with the zebrafish craniofacial skeleton.

genetics↗

bioRxiv2021

Wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish

Bone and muscle interact through developmental, mechanical, paracrine, and autocrine signals. Genetic variants at the CPED1-WNT16 locus are dually associated with bone- and muscle-related traits. While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at this locus. Here, we show wnt16 is required for spine and muscle morphogenesis in zebrafish. In embryos, wnt16 is expressed in dermomyotome and developing notochord, and contributes to larval myotome morphology and notochord elongation. Later, wnt16 is expressed at the ventral midline of the notochord sheath, and contributes to spine mineralization and osteoblast recruitment. Morphological changes in wnt16 mutant larvae are mirrored in adults, indicating that wnt16 impacts bone and muscle morphology throughout the lifespan. Finally, we show that wnt16 is a gene of major effect on lean mass at the CPED1-WNT16 locus. Our findings indicate that Wnt16 is secreted in structures adjacent to developing bone (notochord) and muscle (dermomyotome) where it affects the morphogenesis of each tissue, thereby rendering wnt16 expression into dual effects on bone and muscle morphology. This work expands our understanding of wnt16 in musculoskeletal development and supports the potential for variants to act through WNT16 to influence bone and muscle via parallel morphogenetic processes. AUTHOR SUMMARYIn humans, genetic variants (DNA sequences that vary amongst individuals) have been identified that appear to influence bone and skeletal muscle mass. However, how single genes and genetic variants exert dual influence on both tissues is not well understood. In this study, we found a gene called wnt16 is necessary for specifying the size and shape of muscle and bone during development in zebrafish. Moreover, we disentangled how wnt16 affects both tissues: distinct cellular populations adjacent to muscle and bone secrete Wnt16, where it acts as a signal this guides the size and shape of each tissue. This is important because in humans, genetic variants near the WNT16 gene have effects on both bone- and muscle-related traits. This study expands our understanding of the role of WNT16 in bone and muscle development, and helps to explain how genetic variants near WNT16 affect traits for both tissues. Moreover, WNT16 is actively being explored as a target for osteoporosis therapies; our study could have implications with regard to the potential to target WNT16 to treat bone and muscle simultaneously.

genetics↗

bioRxiv2021

Single-cell resolution of MET and EMT programs during zebrafish fin regeneration

Zebrafish regenerate fin rays following amputation through epimorphic regeneration, a process that has been proposed to involve the epithelial-to-mesenchymal transition (EMT). We performed single-cell RNA-sequencing (scRNA-seq) to elucidate osteoblastic transcriptional programs during zebrafish caudal fin regeneration. We show that osteoprogenitors are enriched with components associated with EMT and its reverse, mesenchymal-to-epithelial transition (MET), and provide evidence that the EMT markers cdh11 and twist2 are co-expressed in dedifferentiating cells at the amputation stump at 1 dpa, and in differentiating osteoblastic cells in the regenerate, the latter of which are enriched in EMT signatures. We also show that esrp1, a regulator of alternative splicing in epithelial cells that is associated with MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a single cell resource for the study of osteoblastic cells during zebrafish fin regeneration, and supports the contribution of MET- and EMT-associated components to this process.

developmental biology↗

bioRxiv2021

Computational anatomy and geometric shape analysis enables analysis of complex craniofacial phenotypes in zebrafish mutants

Due to the complexity of fish skulls, previous attempts to classify craniofacial phenotypes have relied on qualitative features or 2D landmarks. In this work we aim to identify and quantify differences in 3D craniofacial phenotypes in adult zebrafish mutants. We first estimate a synthetic normative zebrafish template using microCT scans from a sample pool of wildtype animals using the Advanced Normalization Tools (ANTs). We apply a computational anatomy (CA) approach to quantify the phenotype of zebrafish with disruptions in bmp1a, a gene implicated in later skeletal development and whose human ortholog when disrupted is associated with Osteogenesis Imperfecta. Compared to controls, the bmp1a fish have larger otoliths and exhibit shape differences concentrated around the operculum, anterior frontal, and posterior parietal bones. Moreover, bmp1a fish differ in the degree of asymmetry. Our CA approach offers a potential pipeline for high throughput screening of complex fish craniofacial phenotypes, especially those of zebrafish which are an important model system for testing genome to phenome relationships in the study of development, evolution, and human diseases. Summary statementA computational anatomy approach offers a potential pipeline for high throughput screening of complex zebrafish craniofacial phenotypes, an important model system for the study of development, evolution, and human diseases.

developmental biology↗

bioRxiv2019

The Reissner Fiber is Highly Dynamic in vivo and Controls Morphogenesis of the Spine

Spine morphogenesis requires the integration of multiple musculoskeletal tissues with the nervous system. Cerebrospinal fluid (CSF) physiology is important for development and homeostasis of the central nervous system and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an enigmatic glycoprotein thread called the Reissner fiber, which is secreted from the subcomissural organ (SCO) in the brain and extends caudally through the central canal to where it terminates at the base of the spinal cord. In zebrafish, scospondin null mutants are unable to assemble the Reissner fiber and fail to extend a straight body axis during embryonic development [3]. Here, we describe zebrafish hypomorphic missense alleles, which assemble the Reissner fiber and straighten the body axis during early embryonic development, yet progressively lose the fiber, concomitant with the emergence of body curvature, alterations in neuronal gene expression, and scoliosis in adults. Using an endogenously tagged scospondin-GFP zebrafish knock-in line, we directly visualized Reissner fiber dynamics during the normal development and during the progression of scoliosis, and demonstrate that the Reissner fiber is critical for the morphogenesis of the spine. Our study establishes a framework for future investigations of mechanistic roles of the Reissner fiber including its dynamic properties, molecular interactions, and how these processes are involved in the regulation of spine morphogenesis and scoliosis. HighlightsO_LIHypomorphic mutations in zebrafish scospondin result in progressive scoliosis C_LIO_LIThe disassembly of the Reissner fiber in scospondin hypomorphic mutants results in the strong upregulation of neuronal receptors and synaptic transport components C_LIO_LIAn endogenous fluorescent knock-in allele of scospondin reveals dynamic properties of the Reissner fiber during zebrafish development C_LIO_LILoss of the Reissner fiber during larval development is a common feature of zebrafish scoliosis models C_LI

genetics↗

bioRxiv2018

Deep Phenotyping Enhances Somatic Mutant Detection and Mosaic Pattern Recognition in the Zebrafish Skeleton

Genetic mosaicism manifests as spatially variable phenotypes, whose detection and interpretation remains challenging. This study identifies biological factors influencing spatial phenotypic patterns in the skeletons of somatic mutant zebrafish, and tests methods for their analysis using deep phenotyping. We explore characteristics of loss-of-function clusters in the skeleton of CRISPR-edited G0 (\"crispant\") zebrafish, and identify a distinctive size distribution shown to arise from clonal fragmentation and merger events. Using microCT-based phenomics, we describe diverse phenotypic manifestations in somatic mutants for genes implicated in monogenic (plod2 and bmp1a) and polygenic (wnt16) bone diseases, each showing convergence with germline mutant phenomes. Finally, we describe statistical frameworks for phenomic analysis which confers heightened sensitivity in discriminating somatic mutant populations, and quantifies spatial phenotypic variation. Our studies provide strategies for decoding spatially variable phenotypes which, paired with CRISPR-based screens, can identify genes contributing to skeletal disease.

genetics↗

bioRxiv2018

Developmental tuning of mineralization drives morphological diversity of gill cover bones in sculpins and their relatives

The role of osteoblast placement in skeletal morphological variation is relatively wellunderstood, but alternative developmental mechanisms affecting bone shape remain largely unknown. Specifically, very little attention has been paid to variation in later mineralization stages of intramembranous ossification as a driver of morphological diversity. We discover the occurrence of specific, sometimes large regions of nonmineralized osteoid within bones that also contain mineralized tissue. We show through a variety of histological, molecular, and tomographic tests that this \"extended\" osteoid material is most likely nonmineralized bone matrix. This tissue type is a significant determinant of gill cover bone shape in the teleostean suborder Cottoidei. We demonstrate repeated evolution of extended osteoid in Cottoidei through ancestral state reconstruction and test for an association between its presence and habitat differences among species. Through measurement of extended osteoid at various stages of gill cover development in species across the phylogeny, we gain insight into possible evolutionary developmental origins of the trait. We conclude that this finetuned developmental regulation of bone matrix mineralization reflects heterochrony at multiple biological levels and is a novel mechanism for the evolution of diversity in skeletal morphology. This research lays the groundwork for a new model in which to study bone mineralization and evolutionary developmental processes, particularly as they may relate to adaptation during a prominent evolutionary radiation of fishes.

evolutionary biology↗

bioRxiv2018

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of Osteogenesis Imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and the first zebrafish model of human EDS, which harbors characteristic defects in the soft connective tissues. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen related disease models. Our studies reveal a high degree of inter-genotype variability in phenotypic expressivity that closely correlates with associated OI severity. Further, we demonstrate the potential for select mutations to give rise to variable phenotypic penetrance, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention\n\nSIGNIFICANCE STATEMENTType I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders.

genetics↗

bioRxiv2017

ScreenCube: A 3D Printed System For Rapid and Cost-Effective Chemical Screening in Adult Zebrafish

Phenotype-based small molecule screens in zebrafish embryos and larvae have been successful in accelerating pathway and therapeutic discovery for diverse biological processes. Yet, the application of chemical screens to adult physiologies has been relatively limited due to additional demands on cost, space, and labor associated with screens in adult animals. Here, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using pre-filled screening plates, the system enables dosing of 96 fish in {small tilde}3min, with a tenfold-reduction in drug quantity compared to that used in previous chemical screens in adult zebrafish. We characterize water quality kinetics during immersion in the system, and use these kinetics to rationally design intermittent dosing regimens that result in 100% fish survival. As a demonstration of system fidelity, we show the potential to identify two known chemical inhibitors of adult tail fin regeneration, cyclopamine and dorsomorphin. By developing methods for rapid and cost-effective chemical administration in adult zebrafish, this study expands the potential for small molecule discovery in post-embryonic models of development, disease, and regeneration.

pharmacology and toxicology↗

bioRxiv2017

microCT-Based Skeletal Phenomics in Zebrafish Reveals Virtues of Deep Phenotyping at the Whole-Organism Scale

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of measures comprised of morphological and densitometric traits from a large number sites in the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ. Analyzing phenotypic patterns may increase productivity in genetic screens, and could facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

genetics↗