Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 118-136).

Temperature is arguably the most important abiotic factor influencing the embryonic development in ectothermic species. Incubation temperature has demonstrated effects on offspring phenotypes in ectotherms, including traits such as sex, size, shape, colouration and post-hatch growth and survival. However, in endotherms the influence temperature has on development is relatively unexplored due to the narrow range of temperatures that embryonic endotherms are exposed to during develop. Megapode birds utilize environmental heat sources to incubate their eggs and therefore provide a potential model to test how temperature influences embryonic development and offspring phenotypes in endotherms. I used the Australian Brush-turkey (Alectura lathami), a megapode bird that incubates its eggs in mounds of soil and leaf litter to investigate the effects of temperature on embryonic development and chick morphology. Previous reports of Australian Brush-turkey incubation mound temperatures relied on spot measurements and theoretical modeling and thus have not provided a comprehensive examination of the range of temperatures Brush-turkey embryos are exposed to and how these might vary during the course of incubation. Therefore to examine the range of temperatures experienced by developing embryos I continuously recorded the temperature of eggs and mound material at naturally occurring positions within incubation mounds over the full developmental period. As in previous studies I found the average incubation temperature to be about 34°C, however egg temperatures typically fluctuated more than previously reported or predicted from modeling. The thermal tolerance of Brush-turkeys is remarkable compared to non-megapode birds, with embryos developing successfully despite prolonged exposure to sub-optimal temperatures over the range 25-40°C. I also demonstrated that the incubation period was negatively correlated with mean incubation temperature. To simplify the examination of temperature effects on embryonic development, constant temperature artificial incubation of Brush-turkey eggs was used to determine influence of incubation temperature on the energetics of embryonic development and the sex ratio, morphology and chemical composition of chicks. Because initial investigation of mound temperatures determined the mean incubation temperature in Brush-turkeys to be 34°C this was used as the preferred temperature for constant temperature incubation with 32°C and 36°C representing low and high temperatures respectively. Previously, the sex ratio of Brush-turkey chicks at hatching was shown to be temperature dependent. A thermally sensitive period early in development resulted in more females hatching from high temperature and more males hatching from low temperatures with an equal ratio at the preferred temperature. Using molecular sexing techniques to determine the sex of both failed embryos and chicks that hatched, I established that at laying the sex ratio of eggs was 50:50, and that temperature-dependent sex-biased embryo mortality was the mechanism behind the skewed sex ratio of chicks hatching from non-preferred temperatures. Low incubation temperature increased female embryonic mortality and high incubation temperature increased male embryonic mortality. This represents a novel mechanism operating to alter sex ratios in a bird species and offers an unparalleled system to explore sex allocation theory. It is well established that temperature influences the rate of development and the morphology of offspring in reptilian species. Also, in a previous study using artificially incubated Brush-turkey eggs, temperature was found to affect the mass of chicks but not their size (linear dimensions). This finding suggests that at different incubation temperatures the amount of yolk converted into tissue during embryonic development is influenced by incubation temperature. I tested this hypothesis by incubating eggs at different constant temperatures and found high incubation temperatures produce chicks with lighter yolk-free bodies and heavier residual yolks but similar linear dimensions compared to chicks hatching from lower temperatures. Because eggs incubated at low temperatures have longer incubation periods, I hypothesized the proportion of lipid in the yolk-free body would be higher in chicks emerging from eggs incubated at low temperature because more time is available for the conversion of yolk to fat bodies during embryonic development. This hypothesis was not supported as the composition of yolk-free chicks (total water, lipid, protein and ash) was not temperature dependent. A previous study in Malleefowl (Leipoa ocellata), another megapode bird, found that the total energetic cost of production was influenced by incubation temperature. Such that embryos developing at low temperatures required 72% more energy than embryos developing at high temperatures. However these findings were contrary to expectation from studies of reptilian incubation where the energetic cost of development is independent of temperature. Therefore I tested the hypothesis that the total energetic cost of development is temperature dependent in the Australian Brush-turkey. I used bomb calorimetry to measure the energy content of freshly laid eggs and of chicks (both the yolk-free body and residual yolk) that had hatched from eggs incubated 32oC, 34oC and 36oC. I found that the total energy content of chicks at hatching was greater in chicks emerging from eggs incubated at 34oC and 36oC compared to eggs incubated at 32oC. My thesis work demonstrated that incubation temperature is more variable for Brush-turkey embryos than for non-megapode birds and that even a small difference in temperature can have important effects on chick sex ratios, morphology and energy reserves. I have shown that incubation under artificial constant temperature conditions can significantly alter the developmental trajectories and phenotypic outcomes for chicks. In addition to laboratory based work, future studies should continue to examine how embryonic development and chick attributes are influenced by temperatures experienced under natural incubation conditions. Furthermore, investigation is required to determine how incubation temperature induced differences in hatchling phenotypes influence the post-hatch grow and fitness of chicks.

Thesis (Ph.D.)--Medical University of Ohio, 2005. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Joseph F. Margiotta. Includes abstract. Document formatted into pages: iii, 226 p. Title from title page of PDF document. Bibliography: pages 80-92,130-139,149-225.

During amniote embryogenesis, the segmented pattern characteristic of the vertebral column appears early during development through the sequential formation of multipotent structures called somites. Somites differentiate subsequently into dermomyotome (giving rise later to skin and skeletal muscles) and sclerotome (giving rise to vertebral bone structures and cartilage). In addition, sclerotomes subdivide following their rostro-caudal intrasegmental boundary into an axon growth-permissive region (anterior half) and an axon growth-repulsive region (posterior half). This binary system instructs motor and sensory axon navigation, as well as neural crest cell migration, to ensure that the peripheral nervous system develops without obstruction by the future cartilage and bones of the vertebral column. Repellent cues are expressed in posterior half-sclerotomes in order to exclude navigating axons from “no-go” areas and restrict their growth to specific exit points of the future vertebral column. Interestingly, similar repellent cues (e.g. Eph/Ephrins) are expressed in the adult central nervous system (CNS) and have been shown to control connectivity and plasticity throughout life. Following brain or spinal cord injury, these repellent molecules are upregulated by reactive astrocytes accumulating at the lesion site, and may impede axon regeneration in this region. In this dissertation, I am presenting the results of a differential gene expression analysis of anterior and posterior half-sclerotomes, based on RNA-sequencing data and using the chick embryo as a model organism. This study led to the identification of molecules, previously uncharacterized in this system, that may play a role in adhesive and mechanical properties of somites and in axon guidance and fasciculation. I focused on the functional analysis of one molecule of the posterior half-sclerotome, the extracellular matrix protein Fibulin-2. To look at its role in the segmentation of spinal axons, I used ectopic misexpression in a subset of segments based on somite electroporation. The width of spinal nerve bundle growth was restricted by Fibulin-2 overexpression in posterior and anterior half-sclerotomes, suggesting a role in sharpening/controlling the path of spinal axon growth. In addition, I showed that this could occur via an interaction with the axon growth repellent Semaphorin 3A. Then I looked at the expression of Fibulin-2 in two models of CNS injury: mouse cerebral cortical stab injury and rat dorsal crush spinal cord injury. In both cases, I observed an increase in Fibulin-2 protein level compared to control. I also used primary cultures of rat cortical astrocytes to show that the expression of Fibulin-2 after inflammatory cytokine-induced activation is increased. Finally, I studied a candidate axon growth repellent previously identified in the laboratory. I explored the hypothesis that this repellent molecule is an O-glycosylated, spliced variant form of a known protein. To characterize this repellent molecule, I used RNA-sequencing data from chick embryonic somites and 2D gel electrophoresis of an astrocytic cell line protein extract. Together, these results suggested that the developing vertebral column and the adult CNS share molecular features to control axon growth and plasticity. This type of study could lead to the characterization of molecular systems that regulate axon growth, and to the identification of novel therapeutic targets in brain or spinal cord injury.

6 ABSTRAKT Kardiovaskulární onemocnění představují celosvětově nejčastější příčinu úmrtí. Arytmie neboli poruchy srdečního rytmu k této mortalitě přispívají nemalou měrou. Výzkum v oblasti morfologie převodního systému srdečního má více jak stoletou historii, ale oblast ontogeneze není ani v dnešní době dostatečně prozkoumána. Přitom pochopení mechanismů indukce pracovního myokardu ve specializovanou tkáň by mohl být jeden z klíčů k otevření dveří nových možností diagnostiky a terapie onemocnění v této oblasti. Cílem této práce je pochopení vlivu mechanické zátěže na vznik a vývoj převodního systému srdečního. Chci prokázat, že mechanické zatížení srdce krevním oběhem hraje v embryonálním vývoji nezastupitelnou roli v diferenciaci převodního systému srdečního (PSS). Jako model pozorování jsem si vybral kuřecí srdce. Při porovnání dat získaných na srdci ptáků a savců můžeme najít drobné odlišnosti v detailech, nicméně hlavní principy a mechanismy se zdají být stejné. Kuřecí embryo se vyvíjí 21 dní, přičemž toto časové údobí lze podle Hamburgera a Hamiltona rozdělit do 46 stádií. Zpočátku představuje srdce pouze primitivní trubici, která se stáčí v srdeční kličku. Posléze je přeměněna na zralý čtyřoddílový orgán charakteristický pro vyšší obratlovce. Tyto morfologické změny korelují i se změnami šíření...

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The overall objectives of this thesis were to develop a short-term culture system and to examine the effects of vitrification and short-term culture on the viability of fresh and vitrified bovine ovarian tissue and the follicles within. The first objective was to compare the health and development of preantral follicles in bovine ovarian tissue, as well as the neovascularization of these tissues, subjected to avian chorioallantoic membrane (CAM) culture with the traditional in vitro culture system. We hypothesized that the chorioallantoic membrane (CAM) of the chicken embryo is a 
more suitable culture system than traditional in vitro culture. Bovine ovaries were retrieved from a local abattoir and cortical pieces (1-2mm3) were randomly assigned to one of the following groups; control (fixed immediately), CAM or in in vitro culture. Ovarian tissue fragments from both groups were removed on D1, D3 and D5 of culture, fixed, sectioned (5μm) and stained with H&E. The numbers of healthy and degenerated follicles, primordial and activated preantral (primary and secondary), and the number of infiltrated bovine and avian blood vessels were determined using standard stereological procedures. All grafts placed on the traumatized CAM demonstrated increased neovascularization over time. The healthy primordial follicle density decreased over time concomitant with an increase in degenerated (primordial and activated preantral) follicles in both treatment groups. Healthy activated preantral follicle density did not differ between the two culture systems at a given time. In CAM group, blood vessel density increased over time (p = 0.015). The second objective of this thesis was to develop a suitable vitrification protocol for bovine ovarian tissue. The viability of bovine ovarian tissue vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryotop and cryovial) was assessed. We hypothesized that during vitrification the higher cooling rate on the cryotop (open vitrification method) will yield better post-thaw viability of bovine ovarian tissue as compared to the cryovial (closed vitrification method). We also hypothesized that trehalose is a superior non-permeating cryoprotectant to sucrose for vitrification of bovine ovarian tissue. The ovarian tissue was fragmented (1-2mm3) and divided into 6 different treatment groups. Tissues were vitrified in TCM199 supplemented with 15% EG, 15% DMSO, 20% calf serum and 0.5M sucrose or trehalose then placed in a cryovial or on a cryotop. After warming, the vitrified tissues were either immediately placed in 10% formalin (control) or on the chorioallantoic membrane of a 10-day old chicken embryo for 5 days. Follicles from control and vitrified tissue were observed under a light microscope for normal morphology and the total, normal and degenerated follicle densities were determined by standard stereological procedures. Sucrose and trehalose did not differ, nor was a difference observed between the cryovial and the cryotop for total, healthy or degenerated follicle density. Proportion of healthy follicles was higher in the control than all treatment tissues grafted to the CAM. All grafts placed on the traumatized CAM demonstrated presence of avian erythrocytes in the blood vessels after 5 days, but no difference was observed for blood vessel density among treatments. Lastly, the cooling rate of bovine ovarian tissue subjected to open and closed system devices for vitrification was evaluated. A thermocouple wire was used to determine the cooling velocity of 1-2mm3 fragments of bovine ovarian tissue placed on a cryotop (open system) or in a sealed cryovial (closed system). The cooling rate of tissues on the cryotop and in the cryovial was 7481±205.9° C/min and 664±26.0° C/min respectively. In conclusion, the CAM supported the bovine ovarian tissue, thus the CAM culture system may be considered an acceptable alternative to traditional in vitro culture system for bovine ovarian tissue. Furthermore, angiogenesis may be an additional indication of ovarian tissue health. The hypotheses of our second study were refuted. Results indicated that sucrose and trehalose, and the cryotop and cryovial were equally effective in vitrifying bovine ovarian tissue.

Chez les vertébrés, les muscles squelettiques du corps sont dérivés de la partie dorsale dessomites, le dermomyotome, structure transitoire mésodermique. Une première étape demyogenèse aboutit à la formation d’un muscle primitif, le myotome primaire, à partir desbordures du dermomyotome : ces cellules constituent les premières fibres musculaires, et formentl’architecture de base du futur muscle. Dans un second temps, une population de progéniteursmusculaires émerge de la région centrale du dermomyotome. Cette population est primordialedans la constitution du muscle. Elle prolifère, et une partie d’entre elle fusionne aux fibresexistantes pour donner les fibres multinucléées adultes. Finalement, une partie des progéniteursmusculaires reste indifférenciée jusqu’à l’âge adulte et compose la population de cellules souchesmusculaires, les cellules satellites. Ainsi, les progéniteurs musculaires contribuent audéveloppement musculaire tout au long du développement embryonnaire et foetal, mais égalementà la myogenèse post-natale avec les cellules satellites.Lors de ma thèse, je me suis intéressé à cette population de progéniteurs musculaires. Deux souspopulationsde progéniteurs musculaires ont précédemment été identifiées dans notre laboratoireau cours de l’embryogénèse précoce de poulet, l’une exprimant le facteur de transcription Pax7,l’autre co-exprimant Pax7 et le facteur de différenciation myogénique précoce Myf5. Face àl’absence de données concernant les progéniteurs musculaires, et à l’importance de cettepopulation pour la myogenèse, j’ai réalisé une étude systématique des progéniteurs musculairestout au long du développement embryonnaire et foetal de deux organismes modèles : le poulet etla souris. J’ai pu montrer que ces deux sous-populations coexistent tout au long dudéveloppement, depuis l’émergence des progéniteurs de la partie centrale du dermomyotome,jusqu’au moment où ces cellules deviennent des cellules satellites à la fin du développementfoetal. De manière très intéressante, j’ai pu montrer qu’au sein des progéniteurs musculaires, lapopulation principale co-exprime Pax7 et Myf5, et prolifère activement, alors que la populationPax7 est mineure et prolifère à un taux moins élevé. Cette dernière entre de manière importanteen quiescence à la fin du développement embryonnaire. Ces caractéristiques sont semblablesentre le poulet et la souris, et montrent que des stratégies cellulaires et moléculaires similairessont conservées au sein des amniotes. / Duringembryonicandfetallife,skeletalmusclegrowthisdependentupontheproliferationandthedifferentiationofapopulationofresidentmuscleprogenitors,fromwhichderivethemusclestemcellsof theadult,thesatellitecells.Underpoorlydefinedextrinsicandintrinsicinfluences,muscleprogenitorsproliferate,differentiateorenteraquiescentstatetobecomereservesatellitecells.Despitetheir primordialrole,surprisinglylittleisknownonthehomeostasisofresidentprogenitorsduringembryogenesis.Preliminarystudiesinchickandmousedescribingthekeyprogenitorpopulationscontributingtomusclegrowthduringembryogenesishaveledtodifferingresultsthatcouldbeduetotechnicalissuesortofundamentaldifferencesbetweenanimalmodels.Toaddressthisquestion,we haveundertakenacomprehensiveanalysisofthestateofdifferentiationandproliferationofmuscleprogenitorcellsfromthetimeoftheiremergencewithinthedermomyotomeuntillatefetallife,whenthey adoptasatellitecell-likepositionunderthebasallamina.Thiswasdonebyimmunostainingagainstkeyplayersofmyogenicdifferentiation,inmuscleschosenfromdifferentregionsofthebodyintwo modelorganisms,thechickandmouse.This studyidentifiedtwoco-existingpopulationsofprogenitorsduringembryonicandfetallifeinboth chickandmouse:aminor,slow-cyclingpoolofundifferentiatedresidentprogenitorswhichexpress Pax7,co-existingwithamajorfast-cyclingpopulationthatco-expressPax7andtheearlymyogenicdifferentiationmarkerMyf5.Wefoundthattheoverallproliferationrateofbothprogenitorsdrasticallydecreasedwithembryonicage,asanincreasinglylargeportionofslowandfast-cyclingprogenitorsenteredquiescenceduringdevelopment.Together,thisdatasuggeststhatthecellularstrategiesthatdrivemusclegrowthduringembryonicand fetallifeareremarkablyconservedinamniotesthroughoutevolution.Theyrelyonthetightregulationofproliferation,entryinquiescence,andmodulationofthecellcycle’slengthforbothoftheco-existingpopulationsofmuscleprogenitorstomaintainthehomeostasisofgrowingmusclesduringdevelopment.

Establishment of the biochemical and molecular nature of cardiac development is essential for us to understand the relationship between genetic and morphological aspects of heart formation. The molecular mechanisms that underly heart development are still not clearly defined. To address this issue we have used two approaches to identify genes involved in early chick cardiac development. Differential display previously conducted in our laboratory led to the identification of two gene fragments differentially expressed in the heart that are further described in this thesis. The full-length cDNA sequence of both eukaryotic translation initiation factor-2b (eIF-2b) and NADH cytochrome b5 reductase (b5R) were isolated using library screening. The upreglation of these genes during heart development is expected given the heart is the first functional organ to form in vertebrates and protein synthesis and cell metabolism at this stage of development is maximal. Limitations in the differential display approach led to the development and optimisation of a subtractive hybridisation approach for use with small amounts of cells or tissue. To focus on cardiac gene expression during the initial phases of heart development, subtractive hybridization was performed between the cardiogenic lateral plate mesoderm of Hamburger and Hamilton stage 4 embryos and the heart primordia of stage 9 embryos. Of the 87 independent clones identified by this procedure, 59 matched known sequences with high homology, 25 matched unknown expressed sequence tag (EST) sequences with high homology, and 3 did not match any known sequence on the database. Known genes isolated included those involved in transcription, translation, cell signalling, RNA processing, and energy production. Two of these genes, high mobility group phosphoprotein A2 (HMGA2) and C1-20C, an unknown gene, were chosen for further characterisation. The role of each gene in early chick heart development and indeed development in general, was addressed using techniques such as in situ hybridisation, transfection analysis, in ovo electroporation and RNAi. HMGA2 is a nuclear phosphoprotein commonly referred to as an architectural transcription factor due to its ability to modulate DNA conformation. In keeping with this function, HMGA2/GFP fusion protein was shown to localise to the nucleus and in particular, the nucleolus. In situ hybridisation analysis suggested a role for HMGA2 in heart and somite development. HMGA2 expression was first detected at HH stage 5 in the lateral plate mesoderm, a region synonymous with cells specified to the cardiac fate. HMGA2 was also strongly expressed in the presomitic segmental plate mesoderm and as somites developed from the segmental plate mesoderm, the expression of HMGA2 showed an increasingly more restricted domain corresponding to the level of maturation of the somite. Restriction of HMGA2 expression was first detected in the dorsal region of the epithelial somite, then the dorsomedial lip of the dermomyotome, and finally the migrating epaxial myotome cells. The novel intronless gene, C1-20C, predicts a protein of 148 amino acids containing a putative zinc finger binding domain and prenyl binding motif. Zinc binding assays showed that the zinc finger domain of C1-20C/MBP fusion protein bound over six times the quantity of zinc compared to MBP alone, although not in a 1:1 stoichiometric molar ratio. C1-20C/GFP fusion protein was shown to localise to as yet unidentified intracellular cytoplasmic vesicular compartments. These compartments did not colocalise with the endosome/lysosome pathway, aparently ruling out a role for C1-20C in protein trafficking, recycling or degradation. Expression of C1-20C in the chick embryo suggests a possible role in heart and notochord development and preliminary results using siRNA suggest that C1-20C is involved in normal heart looping.