Musculoskeletal Development in Jawed Vertebrates : Gene function, cis-regulation, and 3D phenotypes in zebrafish

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhancer with different species-dependent activity patterns. In summary, this thesis contributes to our understanding of the derived and conserved functions of Nkx3.2, Gdf5, and Mkx in the development of the vertebrate skeleton and associated connective tissues, and provides a novel high-resolution 3D imaging method for future studies

Characterisation of Transcription Factor Regulation During the Development of Zebrafish Craniofacial Structures

The appearance of jaws approximately 420 million years ago was a major event in vertebrate evolution. This new structure allowed gnathostomes to take on roles as active predators and increase their energy intake, facilitating their success as a group. Craniofacial musculoskeletal structures evolved in concert with the jaw to support its function, including the connective tissues that connect muscles and bones. Understanding the development of these tendons and ligaments in extant gnathostomes will shed light on how they originated and diversified. The expression patterns of Scleraxis and Mohawk, two transcription factors known to be embryonic markers of tendon and ligament tissues, were determined in the early stages of zebrafish development using whole mount in situ hybridization and mapped to specific connective elements. In order to progress our understanding of the regulation of Mohawk expression, a bioinformatic pipeline involving searches for evolutionarily conserved elements with relevant transcription factor binding motifs was undertaken. This led to the identification of CNS.8 as a promising enhancer candidate. Orthologous sequences of CNS.8 from eight different vertebrate species were isolated and cloned into plasmids containing a fluorescent reporter gene for functional validation using transgenesis in the future. The regenerative abilities of the jaw joint between Meckel’s cartilage and the palatoquadrate are another intriguing unknown, so an expression clone containing a Nitroreductase-based chemical ablation system was constructed under the control of a previously-identified conserved enhancer of the nkx3.2 transcription factor gene that marks the joint interzone tissue. A stable transgenic zebrafish line was generated containing this construct and preliminary experiments indicate the promise of this system for specifically ablating the jaw joint interzone tissue to study its regeneration. Together, these results extend our knowledge of the regulation and expression of transcription factors in developing craniofacial connective tissues, and provide a potentially powerful zebrafish model for joint regeneration

Musculoskeletal Development in Jawed Vertebrates : Gene function, cis-regulation, and 3D phenotypes in zebrafish

Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhancer with different species-dependent activity patterns. In summary, this thesis contributes to our understanding of the derived and conserved functions of Nkx3.2, Gdf5, and Mkx in the development of the vertebrate skeleton and associated connective tissues, and provides a novel high-resolution 3D imaging method for future studies

Characterisation of Transcription Factor Regulation During the Development of Zebrafish Craniofacial Structures

The appearance of jaws approximately 420 million years ago was a major event in vertebrate evolution. This new structure allowed gnathostomes to take on roles as active predators and increase their energy intake, facilitating their success as a group. Craniofacial musculoskeletal structures evolved in concert with the jaw to support its function, including the connective tissues that connect muscles and bones. Understanding the development of these tendons and ligaments in extant gnathostomes will shed light on how they originated and diversified. The expression patterns of Scleraxis and Mohawk, two transcription factors known to be embryonic markers of tendon and ligament tissues, were determined in the early stages of zebrafish development using whole mount in situ hybridization and mapped to specific connective elements. In order to progress our understanding of the regulation of Mohawk expression, a bioinformatic pipeline involving searches for evolutionarily conserved elements with relevant transcription factor binding motifs was undertaken. This led to the identification of CNS.8 as a promising enhancer candidate. Orthologous sequences of CNS.8 from eight different vertebrate species were isolated and cloned into plasmids containing a fluorescent reporter gene for functional validation using transgenesis in the future. The regenerative abilities of the jaw joint between Meckel’s cartilage and the palatoquadrate are another intriguing unknown, so an expression clone containing a Nitroreductase-based chemical ablation system was constructed under the control of a previously-identified conserved enhancer of the nkx3.2 transcription factor gene that marks the joint interzone tissue. A stable transgenic zebrafish line was generated containing this construct and preliminary experiments indicate the promise of this system for specifically ablating the jaw joint interzone tissue to study its regeneration. Together, these results extend our knowledge of the regulation and expression of transcription factors in developing craniofacial connective tissues, and provide a potentially powerful zebrafish model for joint regeneration

The broad role of Nkx3.2 in the development of the zebrafish axial skeleton

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements

The role of Gdf5 in the development of the zebrafish fin endoskeleton

BackgroundThe development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota. ResultsWe generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. ConclusionsOur study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes

A novel cis-regulatory element drives early expression of Nkx3.2 in the gnathostome primary jaw joint

The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element of Nkx3.2 that is deeply conserved in most gnathostomes but undetectable in the jawless hagfish and lamprey. This enhancer is active in the developing jaw joint region of the zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 results in a significant reduction of early gene expression of nkx3.2 and leads to a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws

Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including mu CT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SR mu CT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2(-/-) mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2(-/-) mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2(-/-) zebrafish, and demonstrate the promise of DICE-PPC-SR mu CT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly

Video_3_Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants.mov

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including µCT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRµCT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2-/- mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2-/- mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2-/- zebrafish, and demonstrate the promise of DICE-PPC-SRµCT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.</p

Video_1_Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants.mov

Detailed histological analyses are desirable for zebrafish mutants that are models for human skeletal diseases, but traditional histological techniques are limited to two-dimensional thin sections with orientations highly dependent on careful sample preparation. On the other hand, techniques that provide three-dimensional (3D) datasets including µCT scanning are typically limited to visualizing the bony skeleton and lack histological resolution. We combined diffusible iodine-based contrast enhancement (DICE) and propagation phase-contrast synchrotron radiation micro-computed tomography (PPC-SRµCT) to image late larval and juvenile zebrafish, obtaining high-quality 3D virtual histology datasets of the mineralized skeleton and surrounding soft tissues. To demonstrate this technique, we used virtual histological thin sections and 3D segmentation to qualitatively and quantitatively compare wild-type zebrafish and nkx3.2-/- mutants to characterize novel soft-tissue phenotypes in the muscles and tendons of the jaw and ligaments of the Weberian apparatus, as well as the sinus perilymphaticus associated with the inner ear. We could observe disrupted fiber organization and tendons of the adductor mandibulae and protractor hyoideus muscles associated with the jaws, and show that despite this, the overall muscle volumes appeared unaffected. Ligaments associated with the malformed Weberian ossicles were mostly absent in nkx3.2-/- mutants, and the sinus perilymphaticus was severely constricted or absent as a result of the fused exoccipital and basioccipital elements. These soft-tissue phenotypes have implications for the physiology of nkx3.2-/- zebrafish, and demonstrate the promise of DICE-PPC-SRµCT for histopathological investigations of bone-associated soft tissues in small-fish skeletal disease models and developmental studies more broadly.</p