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Skeletal cell evolution: Comparing an amphibian model to earlier- and later-diverged vertebrates



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In principle, modern-day organisms retain many ancestral traits, some of which can be traced back quantitatively to approximate the order of evolutionary events. Within skeletal research, most studies have focused on more recently diverged vertebrate models, like mammals and birds, or on earlier-diverged fishes. This leaves a gap in the literature as the intermediately positioned amphibian model has been relatively under-explored. As such, amphibian skeletal cells might retain residual information that could account for any evolutionary differences observed between earlier- and later-diverged vertebrate clades. A comparative study of skeletal cell development across clades may reveal how bone and cartilage cells (osteoblasts and chondrocytes, respectively) have evolved over time. With few exceptions, many developmental features of bone and cartilage are currently understood to be fairly conserved among vertebrates. This includes the expression of certain skeletogenic genes that drive and characterize the different phases of skeletal cells as they develop. This thesis investigates and asks two specific questions regarding skeletal development. Is chondrogenic gene expression more common among osteoblasts, as recently discovered in frogs and fish, than previously thought? And is hypertrophy conserved within maturing amphibian chondrocytes as it is in earlier- and later-diverged vertebrates? Generally, we hypothesize that amphibian skeletal cells exhibit molecular and histological characteristics intermediate to those of other vertebrate models, given their position within phylogeny. Testing this hypothesis involved comparing osteoblasts and chondrocytes from skeletal elements in the amphibian frog homologous to those previously examined by the Eames lab in the mouse, chick, and gar. In the western clawed frog, Xenopus tropicalis, these elements were identified as the humerus (upper arm bone), medial angulosplenial (a dermal bone of the lower jaw), and ceratohyal (homologous to the hyoid bone of the larynx). Datasets from all four species were subjected to comparative analyses which were a combination of histology, RNA in situ hybridization, immunohistochemistry, and LCM-RNAseq (laser capture microdissection coupled with RNA sequencing). Preliminary bioinformatic analysis was also performed, but those results still require follow-up examination. Nonetheless, our data suggested amphibian skeletal cells have properties that did not conform to the phenotypic spectra as defined by other vertebrate models. Osteoblasts and hypertrophic chondrocytes of amphibians appeared to express early chondrogenic markers at levels much higher or longer than were typically found in earlier- or later-diverged vertebrates, such as sex determining region Y-box 9 (sox9), collagen type II alpha 1 chain (col2a1), and possibly many other cartilage genes like Aggrecan (acan), sex determining region Y-box 5 (sox5), and sex determining region Y-box 9 (sox6), as well. To the best of our knowledge, these findings would be considered novel discoveries. Chondrogenic expression overall may have increased in skeletal cells of the amphibian lineage before decreasing in later-diverged tetrapods. This could somehow be related to the unusual hypertrophic development of amphibian head cartilages that was also revealed by this project; namely, the rapid hypertrophy of chondrocytes, which had not been previously characterized before, and the persistence of hypertrophic cartilage, a trait seemingly unique to frogs.



Xenopus tropicalis, amphibian, skeletal cell evolution, cartilage, bone



Master of Science (M.Sc.)


Anatomy and Cell Biology


Anatomy and Cell Biology


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