Viviparity has evolved multiple times within squamates. Eggshells are reduced in viviparous forms, which reduces calcium available to embryos during development. This study tested the hypothesis that reduced calcium impacts neonates of viviparous forms. Developmental series from oviparous and viviparous populations of Zootoca vivipara (reproductively bimodal) were cleared & stained to reveal bone development. Photographs (high magnification, calibrated to size) were used to obtain measurements of lengths of the body, humerus, femur, skull and lower jaw, and of ossified portions of limb bones. Percent ossification was scored for targeted skull bones. Results were analyzed using general linear models and revealed no differences in ossification in either limbs or skull. Overall size of oviparous neonates was significantly larger. Findings do not support our hypothesis and indicate that reduction in eggshell calcium in embryos of viviparous populations does not negatively impact limb or skull ossification during development but may influence overall size.

xvii, 125 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / MicroRNAs are small regulatory RNAs that control various developmental and physiological processes in animals and plants. To study the involvement of microRNAs in skeletal development, I manipulated the expression of miR-140, which is strongly expressed in the developing skeleton, and miR-196, which is located among the body patterning Hox cluster genes. I found that miR-140 regulates zebrafish palate formation by interfering with neural crest cell migration through the inhibition of the expression of the platelet derived growth factor receptor alpha ( pdgfra ) gene. I also found that miR196 regulates zebrafish pectoral fin initiation by regulating the expression of the retinoic acid receptor alpha b ( rarab ) gene and that miR-196 is involved in the patterning of zebrafish pharyngeal arches and vertebrae. These results illuminate previously unknown regulatory mechanisms of skeletal development. I also reviewed current knowledge concerning microRNAs in skeletal development and evolution and discussed potential relationships between microRNAs and skeletal disease. This dissertation includes previously published and unpublished coauthored material. / Committee in charge: Judith Eisen, Chairperson, Biology; John Postlethwait, Advisor, Biology; Charles Kimmel, Member, Biology; William Cresko, Member, Biology; J. Andrew Berglund, Outside Member, Chemistry

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population. The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. The gene Dual-specificity tyrosine-regulated kinase 1a (Dyrk1a) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic Dyrk1a is related to the emergence of trabecular defects at P30, expression of Dyrk1a in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30. The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background. These data led to the hypothesis that reduction of Dyrk1a gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce Dyrk1a gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic Dyrk1a does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.

Endochondral ossification is the process of skeletal bone growth via the formation of a cartilage template that subsequently undergoes mineralization to form trabecular bone. Genetic mutations affecting the proliferation or differentiation of chondrocytes result in skeletal abnormalities. Activating transcription factor-2 (ATF-2) modulates expression of cell cycle regulatory genes in chondrocytes, and mutation of ATF-2 results in a dwarfed phenotype. Here we investigate the regulatory role that ATF-2 plays in expression of the pocket proteins, cell cycle regulators important in cellular proliferation and differentiation. The spatial and temporal pattern of pocket protein expression was identified in wild type and mutant growth plates. Expression of retinoblastoma (pRb) mRNA and protein were decreased in ATF-2 mutant primary chondrocytes. pRb mRNA expression was coordinated with chondrogenic differentiation and cell cycle exit in ATDC5 cells. Type X collagen immunohistochemistry was performed to visualize a delay in differentiation in response to loss of ATF-2 signaling. Chondrocyte proliferation was also affected by loss of ATF-2. These studies suggest pRb plays a role in chondrocyte proliferation, differentiation and growth plate development by modulating cell cycle progression. ATF-2 regulates expression of pRb within the developing growth plate, contributing to the skeletal phenotype of ATF-2 mutant mice through the regulation of chondrocyte proliferation and differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20047号 / 理博第4232号 / 新制||理||1608(附属図書館) / 京都大学大学院理学研究科地球惑星科学専攻 / (主査)准教授 生方 貴男, 教授 酒井 治孝, 教授 平島 崇男 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Because the ancestry of extant amphibians remains highly controversial, under traditional perspectives, amphibians and amniotes often are distinguished by differences in developmental mode rather than their evolutionary relationships. Resolution of relationships is important, however, because phylogeny affects interpretations of biology, including the evolution of development. To address those issues, I documented the growth and development of two extinct lepospondyls, Microbrachis pelikani and Hyloplesion longicostatum, and compared the patterns in those taxa to data from other tetrapods. I quantified allometry in the skeleton using both measurement-based and geometric morphometric analyses. I applied Ontogenetic Sequence Analysis (OSA), a size-independent method, to the reconstruction of ossification sequences based on fossils. I also documented skeletal morphogenesis and used Parsimov Analysis and Parsimov-based Genetic Inference of ossification sequence data to evaluate the three hypotheses of extant amphibian ancestry, the Lepospondyl (LH), Temnospondyl (TH), and Polyphyletic (PH) hypotheses. Skeletal growth in Microbrachis pelikani and Hyloplesion longicostatum is primarily isometric. Comparisons with data from other Paleozoic taxa suggest that isometry was the ancestral pattern of growth in tetrapods. All regression analyses had a linear fit indicating lack of an abrupt metamorphosis. Absence of metamorphosis is also supported by the possession of lateral lines in both taxa throughout ontogeny, and Microbrachis pelikani additionally retained gills. However, ossification of the skeleton was completed at small body size. The greatest resolution in ossification sequence reconstruction was achieved with OSA, but results from all reconstruction methods indicated advanced ossification of the pubis and delayed ossification of the scapula in the lepospondyls. In terms of total number of sequence shifts optimized across each hypothesis of amphibian relationships, the TH had the shortest tree length. However, the values for the three hypotheses did not differ significantly, demonstrating that none was supported strongly. Based on my synthesis of new developmental data, I propose that Microbrachis pelikani and Hyloplesion longicostatum expressed a mosaic pattern of skeletal development. That pattern included a gradual transition to an adult morphology, and a lack of an amphibian-like metamorphosis. A similar pattern is common to most early tetrapods and Eusthenopteron, supporting the hypothesis that metamorphosis is not ancestral for Tetrapoda. / text

A longitudinal study was designed to characterize developmental patterns of plasma (PL) and synovial fluid (SF) total insulin-like growth factor-I (IGF-I) concentrations, as well as their association with measurements of skeletal growth in Thoroughbred horses. Horses were randomly assigned to one of two dietary treatment groups and fed diets with either a high or low starch content to examine the effects of dietary energy source on PL and SF IGF-I. At 3, 6, 9, 12 and 15 mo of age, PL and carpal SF samples were collected for analysis of total IGF-I. Body weight gain, wither height gain and forearm length gain were calculated for the 90 day periods between SF and PL sampling. No influence of diet on PL or SF IGF-I was detected (P > 0.05). Average SF IGF-I concentrations were 30.1 ± 1.8% of that found in PL, and PL and SF IGF-I were positively correlated (r = 0.48, P = 0.0003) There was an effect of month of age on both PL and SF IGF-I concentrations (P < 0.05). There was a positive correlation between all measures of gain except forearm length gain with PL and SF IGF-I (r = 0.41 to 0.55, P < 0.05). In our second study, we evaluated the use of a liquid-nitrogen cooled mortar and pestle, motorized freezer mill and rotor-stator homogenizer for homogenization of small (<50mg) articular cartilage samples. The rotor-stator homogenizer produced quanitfiable RNA yields, and was used to evaluate three different RNA isolation protocols. Two of the protocols were commercially available RNA extraction kits, with the third a modified guanidinium isothiocyanate/acid-phenol extraction procedure. The combined average yield for all protocols was 91.9 ng RNA/mg of cartilage. All protocols yielded a sufficient quantity of quality RNA suitable for gene expression analysis. / Master of Science

Yes / The skeletal remains of infants and children are a poignant reminder of the perilous nature of childhood in the past, yet they offer valuable insight into the life histories of individuals and into the health of populations. Many osteoarchaeological and bioarchaeological analyses are dependent on two vital pieces of information: the age-at-death and sex of the individual(s) under study. This chapter will outline how age-at-death and sex can be estimated from the skeletal remains and dental development of non-adults, and how these are easier or more difficult to determine than for adults, and will discuss the complexities and controversies surrounding different methods.

Les protéoglycanes (PGs) sont des protéines présentes au niveau de la matrice extracellulaire et à la surface des cellules. Ils sont constitués d’une protéine sur laquelle sont attachées des chaînes de glycosaminoglycanes. Ils jouent un rôle essentiel dans plusieurs processus biologiques. Des mutations au niveau des gènes codant pour la protéine porteuse ou les enzymes impliquées dans la biosynthèse des GAGs sont associées à plusieurs syndromes et pathologies chez l’homme. L’initiation de la synthèse des GAGs est catalysée par la xylosyltransférase I (XT-I). La XT-I joue un rôle clé dans la régulation de la synthèse des PGs au niveau du cartilage et il a été montré récemment que les mutations hypomorphiques de la XT-I sont associées au syndrome du Desbuquois de type II (DBQD2) caractérisé par des anomalies squelettiques (ostéochondrodysplasie). Afin d’élucider le rôle de la XT-I dans le développement ostéoarticulaire, nous avons généré une souris transgénique conditionnelle Col2α1-CreERTM ;XylT1flox/flox permettant l’invalidation de la XT-I au niveau du cartilage. De façon intéressante, l’invalidation de la XT-I induit des anomalies du développement ostéoarticulaire caractérisées par un nanisme important et des défauts des éléments squelettiques. Des études histologiques et la microscopie SHG (génération de seconde harmonique) de la plaque de croissance ont permis de montrer l’importante de la XT-I dans la formation de la matrice extracellulaire (MEC), la fibrillation du collagène II, la maturation des chondrocytes et leur organisation en colonne dans la plaque de croissance. L’analyse des mécanismes moléculaires impliqués indique la perturbation de la voie de signalisation du TGF-β dans la plaque de croissance. D’autre part, des études histomorphométriques et histologiques des os ont révélé que la déficience en XT-I entraîne une accélération du processus d’ossification avec une stimulation de l’activité des ostéoclastes au niveau de l’os spongieux conduisant à une résorption osseuse importante et à une ossification accrue de l’os cortical. Ces travaux ont permis de révéler le rôle de la XT-I dans le développement ostéoarticulaire et dans le maintien de l’homéostasie du cartilage et du tissu osseux et ont mis en évidence le rôle de la voie du TGF-β dans les anomalies du développement. Ces travaux ouvrent également la voie pour le développement de thérapeutiques potentielles pour le traitement des patients atteints du syndrome de Desbuquois type II / Proteoglycans (PGs) are proteins present in the extracellular matrix and on the surface of cells. They consist of a protein to which chains of glycosaminoglycans (GAGs) are attached. PGs play an essential role in many biological processes and in the homeostasis of different tissues including cartilage, bone and skin. Mutations in the genes encoding PG core proteins or the enzymes involved in GAG biosynthesis are associated with several syndromes and pathologies in human. Initiation of GAG synthesis is catalyzed by xylosyltransferase I (XT-I). XT-I plays a key role in the regulation of the synthesis of PGs in cartilage and it has been shown recently that hypomorphic mutations of XT-I are associated with the Desbuquois syndrome type II (DBQD2), characterized by skeletal abnormalities (osteochondrodysplasia). To elucidate the role of XT-I in skeletal development, we generated a conditional transgenic mouse, Col2α1-CreERTM; XylT1flox/flox allowing the invalidation of XT-I gene in the cartilage. Interestingly, the invalidation of XT-I induces skeletal developmental abnormalities characterized by significant dwarfism, and defects in many skeletal elements. Histological studies and SHG microscopy (second harmonic generation) of the growth plate showed the importance of XT-I in extracellular matrix formation, fibrillation of collagen type II, maturation of chondrocytes and their organization in column in the growth plate. The analysis of the molecular mechanisms involved indicates the disruption of the TGF-β signaling pathway in the growth plate. On the other hand, histomorphometric and histological studies of the bones revealed that the XT-I deficiency causes an acceleration of the ossification process with a stimulation of the osteoclasts activity in spongy bone leading to bone resorption, and increased ossification of the cortical bone. This work revealed the role of XT-I in skeletal development and in the maintenance of cartilage and bone homeostasis and highlighted the role of the TGF-β pathway in developmental abnormalities. This work also paves the way for the development of potential therapeutics for the treatment of patients with Desbuquois syndrome type II

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