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 difficult to interpret. To better understand the role of CPED1 in adult bone mass and morphology, we turned to zebrafish, an emerging model for orthopaedic 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, and discuss how these differences, as well as distinct alternative splicing, could underlie different functions of CPED1 orthologs in the two species. Our studies demonstrate that cped1 is not required for normal adult zebrafish bone mass, lean mass, or bone and lean mass morphology, adding to evidence that variants at 7q31.31 can act independently of CPED1 to influence BMD and fracture risk.

wnt16 regulates spine and muscle morphogenesis through parallel signals from notochord and dermomyotome.

Bone and muscle are coupled 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.

Single-cell resolution of MET- and EMT-like programs in osteoblasts 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.

Phenomics-Based Quantification of CRISPR-Induced Mosaicism in Zebrafish.

Genetic mosaicism can manifest as spatially variable phenotypes that vary from site to site within an organism. Here, we use imaging-based phenomics to quantitate phenotypes at many sites within the axial skeleton of CRISPR-edited G0 zebrafish. Through characterization of loss-of-function cell clusters in the developing skeleton, we identify a distinctive size distribution shown to arise from clonal fragmentation and merger events. We quantitate the phenotypic mosaicism produced by somatic mutations of two genes, plod2 and bmp1a, implicated in human osteogenesis imperfecta. Comparison of somatic, CRISPR-generated G0 mutants to homozygous germline mutants reveals phenotypic convergence, suggesting that CRISPR screens of G0 animals can faithfully recapitulate the biology of inbred disease models. We describe statistical frameworks for phenomic analysis of spatial phenotypic variation present in somatic G0 mutants. In sum, this study defines an approach for decoding spatially variable phenotypes generated during CRISPR-based screens.

Using zebrafish to study skeletal genomics.

While genome-wide association studies (GWAS) have revolutionized our understanding of the genetic architecture of skeletal diseases, animal models are required to identify causal mechanisms and to translate underlying biology into new therapies. Despite large-scale knockout mouse phenotyping efforts, the skeletal functions of most genes residing at GWAS-identified loci remain unknown, highlighting a need for complementary model systems to accelerate gene discovery. Over the past several decades, zebrafish (Danio rerio) has emerged as a powerful system for modeling the genetics of human diseases. In this review, our goal is to outline evidence supporting the utility of zebrafish for accelerating our understanding of human skeletal genomics, as well as gaps in knowledge that need to be filled for this purpose. We do this by providing a basic foundation of the zebrafish skeletal morphophysiology and phenotypes, and surveying evidence of skeletal gene homology and the use of zebrafish for post-GWAS analysis in other tissues and organs. We also outline challenges in translating zebrafish mutant phenotypes. Finally, we conclude with recommendations of future directions and how to leverage the large body of tools and knowledge of skeletal genetics in zebrafish for the needs of human skeletal genomic exploration. Due to their amenability to rapid genetic approaches, as well as the large number of conserved genetic and phenotypic features, there is a strong rationale supporting the use of zebrafish for human skeletal genomic studies.

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. In this study, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using prefilled screening plates, the system enables dosing of 96 fish in ∼3 min, with a 10-fold 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 postembryonic models of development, disease, and regeneration.

MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system.

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 phenotypic measures comprised of morphological and densitometric traits at a large number of sites within 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 system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish.

Long-term in vivo imaging in adult zebrafish (i.e., 1-24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 h.

Osteogenic programs during zebrafish fin regeneration.

Recent advances in genomic, screening and imaging technologies have provided new opportunities to examine the molecular and cellular landscape underlying human physiology and disease. In the context of skeletal research, technologies for systems genetics, high-throughput screening and high-content imaging can aid an unbiased approach when searching for new biological, pathological or therapeutic pathways. However, these approaches necessitate the use of specialized model systems that rapidly produce a phenotype, are easy to manipulate, and amenable to optical study, all while representing mammalian bone physiologies at the molecular and cellular levels. The emerging use of zebrafish (Danio rerio) for modeling human disease highlights its potential to accelerate therapeutic and pathway discovery in the mammalian skeleton. In this review, we consider the potential value of zebrafish fin ray regeneration (a rapid, genetically tractable and optically transparent model of intramembranous ossification) as a translational model for such studies.

Changes in cochlear PMCA2 expression correlate with the maturation of auditory sensitivity.

The plasma membrane Ca(2+) ATPase 2 (PMCA2) is necessary for auditory transduction and serves as the primary Ca(2+) extrusion mechanism in auditory stereocilia bundles. To date, studies examining PMCA2 in auditory function using mutant mice have focused on the phenotype of late adolescent and adult mice. Here, we focus on the changes of PMCA2 in the maturation of auditory sensitivity by comparing auditory responses to RNA and protein expression levels in haploinsufficient PMCA2 and wild-type mice from P16 into adulthood. Auditory sensitivity in wild-type mice improves between P16 and 3 weeks of age, when it becomes stable through adolescence. In haploinsufficient mice, there are frequency-dependent loss of sensitivity and subsequent recovery of thresholds between P16 and adulthood. RNA analysis demonstrates that α-Atp2b2 transcript levels increase in both wild-type and heterozygous cochleae between P16 and 5 weeks. The increases reported for the α-Atp2b2 transcript type during this stage in development support the requisite usage of this transcript for mature auditory transduction. PMCA2 expression also increases in wild-type cochleae between P16 and 5 weeks suggesting that this critical auditory protein may be involved in normal maturation of auditory sensitivity after the onset of hearing. We also characterize expression levels of two long noncoding RNA genes, Gm15082 (lnc82) and Gm15083 (lnc83), which are transcribed on the opposite strand in the 5' region of Atp2b2 and propose that the lnc83 transcript may be involved in regulating α-Atp2b2 expression.

A new Atp2b2 deafwaddler allele, dfw(i5), interacts strongly with Cdh23 and other auditory modifiers.

Tight regulation of calcium (Ca2+) concentrations in the stereocilia bundles of auditory hair cells of the inner ear is critical to normal auditory transduction. The plasma membrane Ca2+ ATPase 2 (PMCA2), encoded by the Atp2b2 gene, is the primary mechanism for clearance of Ca2+ from auditory stereocilia, keeping intracellular levels low, and also contributes to maintaining adequate levels of extracellular Ca2+ in the endolymph. This study characterizes a novel null Atp2b2 allele, dfw(i5), by examining cochlear anatomy, vestibular function and auditory physiology in mutant mice. Loss of auditory function in PMCA2 mutants can be attributed to dysregulation of intracellular Ca2+ inside the stereocilia bundles. However, extracellular Ca2+ ions surrounding the stereocilia are also required for rigidity of cadherin 23, a component of the stereocilia tip-link encoded by the Cdh23 gene. This study further resolves the interaction between Atp2b2 and Cdh23 in a gene dosage and frequency-dependent manner, and finds that low frequencies are significantly affected by the interaction. In +/dfw(i5) mice, one mutant copy of Cdh23 is sufficient to cause broad frequency hearing impairment. Additionally, we report another modifying interaction with Atp2b2 on auditory sensitivity, possibly caused by an unidentified hearing loss gene in mice.