Zebrafish Scale Regeneration <em>In Toto</em> and <em>Ex Vivo</em> Culture of Scales

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new ...

University of Bristol

Sustained forward migration through a fibrillar extracellular matrix requires localization of protrusive signals. Contact with fibronectin at the tip of a cell protrusion activates Rac1, and for linear migration it is necessary to dampen Rac1 activity in off-axial positions and redistribute Rac1 from non-protrusive membrane to the leading edge. Here, we identify interactions between coronin-1C (Coro1C), RCC2 and Rac1 that focus active Rac1 to a single protrusion. Coro1C mediates release of inactive Rac1 from non-protrusive membrane and is necessary for Rac1 redistribution to a protrusive tip and fibronectin-dependent Rac1 activation. The second component, RCC2, attenuates Rac1 activation outside the protrusive tip by binding to the Rac1 switch regions and competitively inhibiting GEF action, thus preventing off-axial protrusion. Depletion of Coro1C or RCC2 by RNA interference causes loss of cell polarity that results in shunting migration in 1D or 3D culture systems. Furthermore, morpholinos against Coro1C or RCC2, or mutation of any of the binding sites in the Rac1-RCC2-Coro1C complex delays the arrival of neural crest derivatives at the correct location in developing zebrafish, demonstrating the crucial role in migration guidance in vivo.

Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure.

The Golgi is essential for glycosylation of newly synthesised proteins including almost all cell-surface and extracellular matrix proteoglycans. Giantin, encoded by the  gene, is a member of the golgin family of proteins that reside within the Golgi stack, but its function remains elusive. Loss of function of giantin in rats causes osteochondrodysplasia; knockout mice show milder defects, notably a cleft palate. , giantin has been implicated in Golgi organisation, biosynthetic trafficking, and ciliogenesis. Here we show that loss of function of giantin in zebrafish, using either morpholino or knockout techniques, causes defects in cilia function. Giantin morphants have fewer cilia in the neural tube and those remaining are longer. Mutants have the same number of cilia in the neural tube but these cilia are also elongated. Scanning electron microscopy shows that loss of giantin results in an accumulation of material at the ciliary tip, consistent with a loss of function of retrograde intraflagellar transport. Mutants show milder defects than morphants consistent with adaptation to loss of giantin.

The Golgi matrix protein giantin is required for normal cilia function in zebrafish.

The Golgi is the cellular hub for complex glycosylation, controlling accurate processing of complex proteoglycans, receptors, ligands and glycolipids. Its structure and organisation are dependent on golgins, which tether cisternal membranes and incoming transport vesicles. Here, we show that knockout of the largest golgin, giantin, leads to substantial changes in gene expression but only limited effects on Golgi structure. Notably, 22 Golgi-resident glycosyltransferases, but not glycan-processing enzymes or the ER glycosylation machinery, are differentially expressed following giantin ablation. This includes near-complete loss of function of GALNT3 in both mammalian cell and zebrafish models. Giantin-knockout zebrafish exhibit hyperostosis and ectopic calcium deposits, recapitulating phenotypes of hyperphosphatemic familial tumoral calcinosis, a disease caused by mutations in GALNT3. These data reveal a new feature of Golgi homeostasis: the ability to regulate glycosyltransferase expression to generate a functional proteoglycome.

Giantin-knockout models reveal a feedback loop between Golgi function and glycosyltransferase expression.

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies, which are characterised by debilitating developmental defects that affect many tissues. Here, we report a new role for regulator of calcineurin 2 (RCAN2) in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia after serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in the control of cilia length that are seen upon RNAi of giantin itself. Together, these data define RCAN2 as a regulator of cilia function that can compensate for the loss of giantin function.

Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control.

Osteoporosis is metabolic bone disease caused by an altered balance between bone anabolism and catabolism. This dysregulated balance is responsible for fragile bones that fracture easily after minor falls. With an aging population, the incidence is rising and as yet pharmaceutical options to restore this imbalance is limited, especially stimulating osteoblast bone-building activity. Excitingly, output from large genetic studies on people with high bone mass (HBM) cases and genome wide association studies (GWAS) on the population, yielded new insights into pathways containing osteo-anabolic players that have potential for drug target development. However, a bottleneck in development of new treatments targeting these putative osteo-anabolic genes is the lack of animal models for rapid and affordable testing to generate functional data and that simultaneously can be used as a compound testing platform. Zebrafish, a small teleost fish, are increasingly used in functional genomics and drug screening assays which resulted in new treatments in the clinic for other diseases. In this review we outline the zebrafish as a powerful model for osteoporosis research to validate potential therapeutic candidates, describe the tools and assays that can be used to study bone homeostasis, and affordable (semi-)high-throughput compound testing.

Zebrafish as an Emerging Model for Osteoporosis: A Primary Testing Platform for Screening New Osteo-Active Compounds.

Novel anabolic drug targets are needed to treat osteoporosis. Having established a large national cohort with unexplained high bone mass (HBM), we aimed to identify a novel monogenic cause of HBM and provide insight into a regulatory pathway potentially amenable to therapeutic intervention. We investigated a pedigree with unexplained HBM in whom previous sequencing had excluded known causes of monogenic HBM. Whole exome sequencing identified a rare (minor allele frequency 0.0023), highly evolutionarily conserved missense mutation in SMAD9 (c.65T>C, p.Leu22Pro) segregating with HBM in this autosomal dominant family. The same mutation was identified in another two unrelated individuals both with HBM. In silico protein modeling predicts the mutation severely disrupts the MH1 DNA-binding domain of SMAD9. Affected individuals have bone mineral density (BMD) Z-scores +3 to +5, mandible enlargement, a broad frame, torus palatinus/mandibularis, pes planus, increased shoe size, and a tendency to sink when swimming. Peripheral quantitative computed tomography (pQCT) measurement demonstrates increased trabecular volumetric BMD and increased cortical thickness conferring greater predicted bone strength; bone turnover markers are low/normal. Notably, fractures and nerve compression are not found. Both genome-wide and gene-based association testing involving estimated BMD measured at the heel in 362,924 white British subjects from the UK Biobank Study showed strong associations with SMAD9 (P = 6 × 10 ; P = 8 × 10 ). Furthermore, we found Smad9 to be highly expressed in both murine cortical bone-derived osteocytes and skeletal elements of zebrafish larvae. Our findings support SMAD9 as a novel HBM gene and a potential novel osteoanabolic target for osteoporosis therapeutics. SMAD9 is thought to inhibit bone morphogenetic protein (BMP)-dependent target gene transcription to reduce osteoblast activity. Thus, we hypothesize SMAD9 c.65T>C is a loss-of-function mutation reducing BMP inhibition. Lowering SMAD9 as a potential novel anabolic mechanism for osteoporosis therapeutics warrants further investigation. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

A Rare Mutation in SMAD9 Associated With High Bone Mass Identifies the SMAD-Dependent BMP Signaling Pathway as a Potential Anabolic Target for Osteoporosis.

Osteoporosis and other conditions associated with low bone density or quality are highly prevalent, are increasing as the population ages and with increased glucocorticoid use to treat conditions with elevated inflammation. There is an unmet need for therapeutics which can target skeletal precursors to induce osteoblast differentiation and osteogenesis. Genes associated with high bone mass represent interesting targets for manipulation, as they could offer ways to increase bone density. A damaging mutation in  has recently been associated with high bone mass. Here we show that Smad9 labels groups of osteochondral precursor cells, which are not labelled by the other Regulatory Smads: Smad1 or Smad5. We show that Smad9 cells are proliferative, and that the Smad9 pocket expands following osteoblast ablation which induced osteoblast regeneration. We further show that treatment with retinoic acid, prednisolone, and dorsomorphin all alter Smad9 expression, consistent with the effects of these drugs on the skeletal system. Taken together these results demonstrate that Smad9 cells represent an undifferentiated osteochondral precursor population, which can be manipulated by commonly used skeletal drugs. We conclude that Smad9 represents a target for future osteoanabolic therapies.

Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations.

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

Giantin is required for intracellular N-terminal processing of type I procollagen.

Genetic disorders of the skeleton encompass a diverse group of bone diseases differing in clinical characteristics, severity, incidence and molecular etiology. Of particular interest are the monogenic rare bone mass disorders, with the underlying genetic defect contributing to either low or high bone mass phenotype. Extensive, deep phenotyping coupled with high-throughput, cost-effective genotyping is crucial in the characterization and diagnosis of affected individuals. Massive parallel sequencing efforts have been instrumental in the discovery of novel causal genes that merit functional validation using  and  cell-based techniques, and  models, mainly mice and zebrafish. These translational models also serve as an excellent platform for therapeutic discovery, bridging the gap between basic science research and the clinic. Altogether, genetic studies of monogenic rare bone mass disorders have broadened our knowledge on molecular signaling pathways coordinating bone development and metabolism, disease inheritance patterns, development of new and improved bone biomarkers, and identification of novel drug targets. In this comprehensive review we describe approaches to further enhance the innovative processes taking discoveries from clinic to bench, and then back to clinic in rare bone mass disorders. We highlight the importance of cross laboratory collaboration to perform functional validation in multiple model systems after identification of a novel disease gene. We describe the monogenic forms of rare low and high rare bone mass disorders known to date, provide a roadmap to unravel the genetic determinants of monogenic rare bone mass disorders using proper phenotyping and genotyping methods, and describe different genetic validation approaches paving the way for future treatments.

A Roadmap to Gene Discoveries and Novel Therapies in Monogenic Low and High Bone Mass Disorders.

The availability of large human datasets for genome-wide association studies (GWAS) and the advancement of sequencing technologies have boosted the identification of genetic variants in complex and rare diseases in the skeletal field. Yet, interpreting results from human association studies remains a challenge. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary. Multiple unknowns exist for putative causal genes, including cellular localization of the molecular function. Intermediate traits ("endophenotypes"), e.g. molecular quantitative trait loci (molQTLs), are needed to identify mechanisms of underlying associations. Furthermore, index variants often reside in non-coding regions of the genome, therefore challenging for interpretation. Knowledge of non-coding variance (e.g. ncRNAs), repetitive sequences, and regulatory interactions between enhancers and their target genes is central for understanding causal genes in skeletal conditions. Animal models with deep skeletal phenotyping and cell culture models have already facilitated fine mapping of some association signals, elucidated gene mechanisms, and revealed disease-relevant biology. However, to accelerate research towards bridging the current gap between association and causality in skeletal diseases, alternative  platforms need to be used and developed in parallel with the current -omics and traditional  resources. Therefore, we argue that as a field we need to establish resource-sharing standards to collectively address complex research questions. These standards will promote data integration from various -omics technologies and functional dissection of human complex traits. In this mission statement, we review the current available resources and as a group propose a consensus to facilitate resource sharing using existing and future resources. Such coordination efforts will maximize the acquisition of knowledge from different approaches and thus reduce redundancy and duplication of resources. These measures will help to understand the pathogenesis of osteoporosis and other skeletal diseases towards defining new and more efficient therapeutic targets.

Perspective of the GEMSTONE Consortium on Current and Future Approaches to Functional Validation for Skeletal Genetic Disease Using Cellular, Molecular and Animal-Modeling Techniques.

Scales are mineralised exoskeletal structures that are part of the dermal skeleton. Scales have been mostly lost during evolution of terrestrial vertebrates whilst bony fish have retained a mineralised dermal skeleton in the form of fin rays and scales. Each scale is a mineralised collagen plate that is decorated with both matrix-building and resorbing cells. When removed, an ontogenetic scale is quickly replaced following differentiation of the scale pocket-lining cells that regenerate a scale. Processes promoting de novo matrix formation and mineralisation initiated during scale regeneration are poorly understood. Therefore, we performed transcriptomic analysis to determine gene networks and their pathways involved in dermal scale regeneration.

Regenerating zebrafish scales express a subset of evolutionary conserved genes involved in human skeletal disease.

Monogenic high bone mass (HBM) disorders are characterized by an increased amount of bone in general, or at specific sites in the skeleton. Here, we describe 59 HBM disorders with 50 known disease-causing genes from the literature, and we provide an overview of the signaling pathways and mechanisms involved in the pathogenesis of these disorders. Based on this, we classify the known HBM genes into HBM (sub)groups according to uniform Gene Ontology (GO) terminology. This classification system may aid in hypothesis generation, for both wet lab experimental design and clinical genetic screening strategies. We discuss how functional genomics can shape discovery of novel HBM genes and/or mechanisms in the future, through implementation of omics assessments in existing and future model systems. Finally, we address strategies to improve gene identification in unsolved HBM cases and highlight the importance for cross-laboratory collaborations encompassing multidisciplinary efforts to transfer knowledge generated at the bench to the clinic. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Dylan Bergen

School of Physiology,
Pharmacology and Neuroscience,
Biomedical Science Building,
School of Physiology, Pharmacology and Neuroscience, Biomedical Science Building

University of Bristol

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

Dylan Bergen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Physiology,Pharmacology and Neuroscience,Biomedical Science Building,School of Physiology, Pharmacology and Neuroscience, Biomedical Science Building

University of Bristol

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

School of Physiology,
Pharmacology and Neuroscience,
Biomedical Science Building,
School of Physiology, Pharmacology and Neuroscience, Biomedical Science Building