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

Abstract

Background 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. Results We defined the transcriptomic profiles of ontogenetic and regenerating scales of zebrafish and identified 604 differentially expressed genes (DEGs). These were enriched for extracellular matrix, ossification, and cell adhesion pathways, but not in enamel or dentin formation processes indicating that scales are reminiscent to bone. Hypergeometric tests involving monogenetic skeletal disorders showed that DEGs were strongly enriched for human orthologues that are mutated in low bone mass and abnormal bone mineralisation diseases (P< 2× 10−3). The DEGs were also enriched for human orthologues associated with polygenetic skeletal traits, including height (P< 6× 10−4), and estimated bone mineral density (eBMD, P< 2× 10−5). Zebrafish mutants of two human orthologues that were robustly associated with height (COL11A2, P=6× 10−24) or eBMD (SPP1, P=6× 10−20) showed both exo- and endo- skeletal abnormalities as predicted by our genetic association analyses; col11a2Y228X/Y228X mutants showed exoskeletal and endoskeletal features consistent with abnormal growth, whereas spp1P160X/P160X mutants predominantly showed mineralisation defects. Conclusion We show that scales have a strong osteogenic expression profile comparable to other elements of the dermal skeleton, enriched in genes that favour collagen matrix growth. Despite the many differences between scale and endoskeletal developmental processes, we also show that zebrafish scales express an evolutionarily conserved sub-population of genes that are relevant to human skeletal disease.

Single-cell sequencing provides clues about the developmental genetic basis of evolutionary adaptations in syngnathid fishes

“…In Gulf pipefish, we found the retained genes scpp1 , spp1 , and enam, are expressed in structures outside of tooth development, suggesting developmental pleiotropic constraint. Specifically, we found spp1 and scpp1 expressed in osteoblasts which is consistent with zebrafish ( Bergen et al, 2022 ; Kawasaki, 2009 ; Liu et al, 2016 ) and enam expressed in the epidermis which has not been reported in zebrafish ( Goldsmith et al, 2003 ; Jain et al, 2007 ; Liu et al, 2016 ). Through examining the conservation of the scpp genes in close syngnathid relatives, we found that most scpp gene losses ( scpp4 , scpp7 , and scpp9 and a functional scpp5 ) are shared with a tooth-bearing outgroup to the family, and likely occurred prior to the loss of teeth in syngnathids.…”

Single-cell sequencing provides clues about the developmental genetic basis of evolutionary adaptations in syngnathid fishes

“…In Gulf pipefish, we found the retained genes scpp1 , spp1 , and enam, are expressed in structures outside of tooth development, suggesting developmental pleiotropic constraint. Specifically, we found spp1 and scpp1 expressed in osteoblasts which is consistent with zebrafish ( Bergen et al, 2022 ; Kawasaki, 2009 ; Liu et al, 2016 ) and enam expressed in the epidermis which has not been reported in zebrafish ( Goldsmith et al, 2003 ; Jain et al, 2007 ; Liu et al, 2016 ). Through examining the conservation of the scpp genes in close syngnathid relatives, we found that most scpp gene losses ( scpp4 , scpp7 , and scpp9 and a functional scpp5 ) are shared with a tooth-bearing outgroup to the family, and likely occurred prior to the loss of teeth in syngnathids.…”

Expression of secretory calcium-binding phosphoprotein (scpp) genes in medaka during the formation and replacement of pharyngeal teeth

“…In ISH, transcripts for scpp7 were detected in the epithelial cells adjacent to the n ray bone (Additional le 1). In addition, scpp5, scpp7, spp1, and sparc are expressed over time during scale regeneration in zebra sh [27]. These results suggest that scpp7 expression during skin and scale formation and regeneration might be critical.…”

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

“…At the posterior edge, the epidermis cell layer separates the scale from direct contact with the water outside 10 . When viewed in cross-section (XZ view) around the radius (grooves), the scale is organized in 3 compositionally different layers: i) a mineralized collagen layer (external layer), ii) a non-mineralized collagen layer (elasmoid), and iii) a layer of cells that produce and maintain the extracellular matrix (elasmoblasts) 8 . In this work, the cross-sectional plane perpendicular to the direction of the radius will be denoted as the XZ plane, and the cross-sectional plane parallel to the direction of the radius as the YZ plane, regardless of the imaging technique used (Fig.…”

“…The zebrafish scale has been proposed as model system for studying bone regeneration 8, 9 . Its greatest advantage is the fish’s ability to generate a new scale when needed, providing a unique opportunity to study the structural and developmental aspects of a newly generated hybrid material tissue.…”

Single-cell sequencing provides clues about the developmental genetic basis of evolutionary adaptations in syngnathid fishes

“…In Gulf pipefish, we found the retained genes scpp1 , spp1 , and enam, are expressed in structures outside of tooth development, suggesting developmental pleiotropic constraint. Specifically, we found spp1 and scpp1 expressed in osteoblasts which is consistent with zebrafish ( Bergen et al, 2022 ; Kawasaki, 2009 ; Liu et al, 2016 ) and enam expressed in the epidermis which has not been reported in zebrafish ( Goldsmith et al, 2003 ; Jain et al, 2007 ; Liu et al, 2016 ). Through examining the conservation of the scpp genes in close syngnathid relatives, we found that most scpp gene losses ( scpp4 , scpp7 , and scpp9 and a functional scpp5 ) are shared with a tooth-bearing outgroup to the family, and likely occurred prior to the loss of teeth in syngnathids.…”

Expression of secretory calcium-binding phosphoprotein (scpp) genes in medaka during the formation and replacement of pharyngeal teeth

“…In ISH, transcripts for scpp7 were detected in the epithelial cells adjacent to the n ray bone (Additional le 1). In addition, scpp5, scpp7, spp1, and sparc are expressed over time during scale regeneration in zebra sh [27]. These results suggest that scpp7 expression during skin and scale formation and regeneration might be critical.…”

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

“…At the posterior edge, the epidermis cell layer separates the scale from direct contact with the water outside 10 . When viewed in cross-section (XZ view) around the radius (grooves), the scale is organized in 3 compositionally different layers: i) a mineralized collagen layer (external layer), ii) a non-mineralized collagen layer (elasmoid), and iii) a layer of cells that produce and maintain the extracellular matrix (elasmoblasts) 8 . In this work, the cross-sectional plane perpendicular to the direction of the radius will be denoted as the XZ plane, and the cross-sectional plane parallel to the direction of the radius as the YZ plane, regardless of the imaging technique used (Fig.…”

“…The zebrafish scale has been proposed as model system for studying bone regeneration 8, 9 . Its greatest advantage is the fish’s ability to generate a new scale when needed, providing a unique opportunity to study the structural and developmental aspects of a newly generated hybrid material tissue.…”

Single-cell sequencing provides clues about the developmental genetic basis of evolutionary adaptations in syngnathid fishes

“…In Gulf pipefish, we found the retained genes scpp1 , spp1 , and enam, are expressed in structures outside of tooth development, suggesting developmental pleiotropic constraint. Specifically, we found spp1 and scpp1 expressed in osteoblasts which is consistent with zebrafish ( Bergen et al, 2022 ; Kawasaki, 2009 ; Liu et al, 2016 ) and enam expressed in the epidermis which has not been reported in zebrafish ( Goldsmith et al, 2003 ; Jain et al, 2007 ; Liu et al, 2016 ). Through examining the conservation of the scpp genes in close syngnathid relatives, we found that most scpp gene losses ( scpp4 , scpp7 , and scpp9 and a functional scpp5 ) are shared with a tooth-bearing outgroup to the family, and likely occurred prior to the loss of teeth in syngnathids.…”

Expression of secretory calcium-binding phosphoprotein (scpp) genes in medaka during the formation and replacement of pharyngeal teeth

“…In ISH, transcripts for scpp7 were detected in the epithelial cells adjacent to the n ray bone (Additional le 1). In addition, scpp5, scpp7, spp1, and sparc are expressed over time during scale regeneration in zebra sh [27]. These results suggest that scpp7 expression during skin and scale formation and regeneration might be critical.…”

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

“…At the posterior edge, the epidermis cell layer separates the scale from direct contact with the water outside 10 . When viewed in cross-section (XZ view) around the radius (grooves), the scale is organized in 3 compositionally different layers: i) a mineralized collagen layer (external layer), ii) a non-mineralized collagen layer (elasmoid), and iii) a layer of cells that produce and maintain the extracellular matrix (elasmoblasts) 8 . In this work, the cross-sectional plane perpendicular to the direction of the radius will be denoted as the XZ plane, and the cross-sectional plane parallel to the direction of the radius as the YZ plane, regardless of the imaging technique used (Fig.…”

“…The zebrafish scale has been proposed as model system for studying bone regeneration 8, 9 . Its greatest advantage is the fish’s ability to generate a new scale when needed, providing a unique opportunity to study the structural and developmental aspects of a newly generated hybrid material tissue.…”