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No description available.

These studies present a series of novel roles for glucocorticoid signaling in the developing zebrafish embryo. The best-characterized roles of cortisol, the primary circulating corticosteroid in teleost fish, are known to occur by the activation of the glucocorticoid receptor (GR) in the post-hatch animal to mobilize energy reserves for response and recovery from stressful situations. For the first time, evidence is presented that GR and cortisol are key developmental regulators in the pre-hatch zebrafish embryo and that glucocorticoid signaling modulates multiple critical developmental pathways and affects embryogenesis in diverse ways. Prior to these experiments, very little was known regarding the developmental role of glucocorticoids in lower vertebrates. In mammalian models, there has been extensive study of the action of these steroids in late-stage organ maturation, and they have a variety of clinical and biomedical applications. However, in fish, there was a relative dearth of information regarding the basic dynamics and potential functional roles of cortisol and GR in embryogenesis. Zebrafish are a popular model for developmental study, with optically transparent embryos that allow for reliable observation. Additionally, the zebrafish genome is fully sequenced and extensively annotated, and a variety of molecular biology techniques are well-established in the existing literature. The zebrafish is also now recognized as an advantageous model for endocrine and stress axis studies, as it expresses a single GR gene, unique among teleosts but comparable to mammals. Preliminary studies published in the literature described cortisol and GR as deposited in the zebrafish embryo prior to fertilization, and showed their expression declining prior to hatch, then rising significantly as larvae approach the stage of first feeding. This dynamic expression of both ligand and receptor during embryogenesis, combined with knowledge from mammalian models, led to the hypothesis that glucocorticoid signaling may be functionally relevant in zebrafish development. A variety of techniques were used to examine the roles of cortisol and GR in the zebrafish embryo. Morpholino oligonucleotides were injected into one-cell embryos to block GR protein translation, allowing for the identification of GR-responsive developmental events and putative GR target genes. High-density microarray analysis of GR morphants presented numerous novel genes and pathways that are modulated by glucocorticoid signaling in the embryo. The ability to microinject molecules into a newly-fertilized zygote also allowed for other manipulations, including the addition of exogenous cortisol or the use of a cortisol-specific antibody to sequester maternally deposited cortisol. These studies provided the first evidence regarding the functional importance of the maternal cortisol deposition in the zebrafish oocyte prior to fertilization. The detailed temporal and spatial expression of GR mRNA and protein in the developing embryo has been characterized for the first time. GR expression is widespread, especially in developing mesoderm, and de novo GR transcription in the zebrafish embryo begins within 12 hours post fertilization. Lack of GR protein expression in the developing zebrafish embryo causes reduced growth, delayed somitogenesis, altered myogenesis, and severely reduces post-hatch survival. Additionally, GR modulates the expression of bone morphogenetic proteins, a family of morphogens that are involved in major developmental events including dorsoventral patterning, somitogenesis, myogenesis, and organogenesis. Reduction in GR protein content in the developing embryo is also linked to other major developmental processes including neurogenesis, eye formation, cellular adhesion, and development and function of the hypothalamic-pituitary-interrenal (HPI) axis. Cortisol in the early embryo, which is contributed entirely by maternal deposition prior to fertilization, is an important regulator of cardiogenesis and development of the HPI axis. Modulation of cortisol content in the early embryo causes an impairment of the post-hatch response to a physical stressor, as larvae exposed to increased cortisol during embryogenesis displayed an inability to increase heart rate in response to an acute physical stress, and did not display the classical increase in cortisol that follows a stressor challenge. Embryos that experience lowered glucocorticoid signaling in development tend to have a heightened post-hatch response to stress, further supporting the conclusion that HPI axis development is regulated by glucocorticoid signaling. These studies have identified key cardiogenic and HPI axis genes that are GR-responsive, providing mechanistic explanations for these phenotypic changes. Together these findings indicate that maternal deposition of cortisol in the embryo can pattern the post-hatch larva and has definitive impacts on performance as the offspring begin locomotion and approach feeding stages. In total, these studies demonstrate that glucocorticoid signaling is critically important to zebrafish embryogenesis, defining novel roles that are completely independent of the classical vertebrate stress response. These functions have significant effects on diverse developmental pathways and processes, and with the potential applicability of the zebrafish model to studies in higher vertebrates, may have important biomedical applications.

The amnioserosa (AS) is an extraembryonic tissue that undergoes programmed cell death (PCD) during the normal course of Drosophila embryogenesis. AS degeneration involves morphological evidence of autophagy as well as caspase activation, but the relationship between these two processes is not well defined. While the bulk of the AS tissue dies at the conclusion of the morphogenetic process of dorsal closure (DC), approximately 10% of AS cells are actively extruded from the epithelium during DC. Using live imaging confocal microscopy and various fluorescent protein sensors, I have been able to observe caspase activation as well as autophagy upregulation in the context of epithelial extrusion events as well as overall AS degeneration. The data show that epithelial extrusion events are caspase-dependent but are also associated with localized onset of autophagy. Furthermore, extensive characterization of loss of function mutants of the key Drosophila regulator Atg1 kinase indicates that autophagy is not required for the normal degeneration of AS, contrary to earlier studies. This thesis also introduces new relationships between caspase activation and autophagic cell death. In addition, new data suggest that the InR/TOR and EGFR/Ras/MAPK signaling pathways interact with the pro-apoptotic protein Head involution defective (Hid) and Atg1 kinase to regulate the progression of programmed cell death in the AS.

The amnioserosa (AS) is an extraembryonic tissue that undergoes programmed cell death (PCD) during the normal course of Drosophila embryogenesis. AS degeneration involves morphological evidence of autophagy as well as caspase activation, but the relationship between these two processes is not well defined. While the bulk of the AS tissue dies at the conclusion of the morphogenetic process of dorsal closure (DC), approximately 10% of AS cells are actively extruded from the epithelium during DC. Using live imaging confocal microscopy and various fluorescent protein sensors, I have been able to observe caspase activation as well as autophagy upregulation in the context of epithelial extrusion events as well as overall AS degeneration. The data show that epithelial extrusion events are caspase-dependent but are also associated with localized onset of autophagy. Furthermore, extensive characterization of loss of function mutants of the key Drosophila regulator Atg1 kinase indicates that autophagy is not required for the normal degeneration of AS, contrary to earlier studies. This thesis also introduces new relationships between caspase activation and autophagic cell death. In addition, new data suggest that the InR/TOR and EGFR/Ras/MAPK signaling pathways interact with the pro-apoptotic protein Head involution defective (Hid) and Atg1 kinase to regulate the progression of programmed cell death in the AS.

These studies present a series of novel roles for glucocorticoid signaling in the developing zebrafish embryo. The best-characterized roles of cortisol, the primary circulating corticosteroid in teleost fish, are known to occur by the activation of the glucocorticoid receptor (GR) in the post-hatch animal to mobilize energy reserves for response and recovery from stressful situations. For the first time, evidence is presented that GR and cortisol are key developmental regulators in the pre-hatch zebrafish embryo and that glucocorticoid signaling modulates multiple critical developmental pathways and affects embryogenesis in diverse ways. Prior to these experiments, very little was known regarding the developmental role of glucocorticoids in lower vertebrates. In mammalian models, there has been extensive study of the action of these steroids in late-stage organ maturation, and they have a variety of clinical and biomedical applications. However, in fish, there was a relative dearth of information regarding the basic dynamics and potential functional roles of cortisol and GR in embryogenesis. Zebrafish are a popular model for developmental study, with optically transparent embryos that allow for reliable observation. Additionally, the zebrafish genome is fully sequenced and extensively annotated, and a variety of molecular biology techniques are well-established in the existing literature. The zebrafish is also now recognized as an advantageous model for endocrine and stress axis studies, as it expresses a single GR gene, unique among teleosts but comparable to mammals. Preliminary studies published in the literature described cortisol and GR as deposited in the zebrafish embryo prior to fertilization, and showed their expression declining prior to hatch, then rising significantly as larvae approach the stage of first feeding. This dynamic expression of both ligand and receptor during embryogenesis, combined with knowledge from mammalian models, led to the hypothesis that glucocorticoid signaling may be functionally relevant in zebrafish development. A variety of techniques were used to examine the roles of cortisol and GR in the zebrafish embryo. Morpholino oligonucleotides were injected into one-cell embryos to block GR protein translation, allowing for the identification of GR-responsive developmental events and putative GR target genes. High-density microarray analysis of GR morphants presented numerous novel genes and pathways that are modulated by glucocorticoid signaling in the embryo. The ability to microinject molecules into a newly-fertilized zygote also allowed for other manipulations, including the addition of exogenous cortisol or the use of a cortisol-specific antibody to sequester maternally deposited cortisol. These studies provided the first evidence regarding the functional importance of the maternal cortisol deposition in the zebrafish oocyte prior to fertilization. The detailed temporal and spatial expression of GR mRNA and protein in the developing embryo has been characterized for the first time. GR expression is widespread, especially in developing mesoderm, and de novo GR transcription in the zebrafish embryo begins within 12 hours post fertilization. Lack of GR protein expression in the developing zebrafish embryo causes reduced growth, delayed somitogenesis, altered myogenesis, and severely reduces post-hatch survival. Additionally, GR modulates the expression of bone morphogenetic proteins, a family of morphogens that are involved in major developmental events including dorsoventral patterning, somitogenesis, myogenesis, and organogenesis. Reduction in GR protein content in the developing embryo is also linked to other major developmental processes including neurogenesis, eye formation, cellular adhesion, and development and function of the hypothalamic-pituitary-interrenal (HPI) axis. Cortisol in the early embryo, which is contributed entirely by maternal deposition prior to fertilization, is an important regulator of cardiogenesis and development of the HPI axis. Modulation of cortisol content in the early embryo causes an impairment of the post-hatch response to a physical stressor, as larvae exposed to increased cortisol during embryogenesis displayed an inability to increase heart rate in response to an acute physical stress, and did not display the classical increase in cortisol that follows a stressor challenge. Embryos that experience lowered glucocorticoid signaling in development tend to have a heightened post-hatch response to stress, further supporting the conclusion that HPI axis development is regulated by glucocorticoid signaling. These studies have identified key cardiogenic and HPI axis genes that are GR-responsive, providing mechanistic explanations for these phenotypic changes. Together these findings indicate that maternal deposition of cortisol in the embryo can pattern the post-hatch larva and has definitive impacts on performance as the offspring begin locomotion and approach feeding stages. In total, these studies demonstrate that glucocorticoid signaling is critically important to zebrafish embryogenesis, defining novel roles that are completely independent of the classical vertebrate stress response. These functions have significant effects on diverse developmental pathways and processes, and with the potential applicability of the zebrafish model to studies in higher vertebrates, may have important biomedical applications.

Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008. / Committee Chair: Dr. Sheldon May; Committee Member: Dr. Donald Doyle; Committee Member: Dr. Gerald Pullman; Committee Member: Dr. James Powers; Committee Member: Dr. Nicholas Hud.

Morphogenesis in walled organisms represents a highly controlled process by which the variability of shapes arises through changes in the structure and mechanics of the cell wall. Despite taking different evolutionary paths, land plants and some brown algae exhibit great developmental and morphological similarities. In two brown algal model systems: the Sargassum muticum apex and the Fucus serratus embryo, I have used a combination of imaging techniques, growth analyses, surgical and pharmacological treatments, as well as molecular, biochemical and mechanical approaches to characterise the growth patterns and the cell wall contribution to shape change. To understand how the adult algal body is formed, I examined the branching strategy (phyllotaxis) in S. muticum. My results suggest that in S. muticum the spiral phyllotactic pattern and the apical cell division pattern are not linked. The phytohormone auxin and the biochemical changes of the cell wall do not seem to be correlated with the bud outgrowth, contrary to observations in plants. In summary, these results suggest Sargassum convergently developed a distinct growth mechanism with similar shape outcome as observed in plants. This dissertation is one of the first attempts to explore cell wall mechanics in brown algal development and its correlation with underlying cell wall biochemistry utilising the Fucus embryo as a known system. The results suggest a correlation between the wall mechanics and alginate biochemistry with the growing and non-growing regions of the embryo. In addition, altering cell wall deposition or composition has a strong effect on embryo rhizoid elongation and is, in certain cases, accompanied by significant increase in cell wall stiffness and reduction of alginate epitopes. Furthermore, preliminary results exploring transcriptomic changes during development indicate differential expression of particular alginate biosynthesis enzymes (mannuronan C5 epimerases) during development, suggesting alginate conformational modifications might be stage specific. These results contribute to the current knowledge addressing the importance of cell walls in brown algal development using novel tools and approaches. Understanding developmental processes in brown algae will provide a better insight how similar morphogenetic traits are established using different body-building mechanisms.

The objectives of this thesis were to trace the developmental pathways of somatic embryos of an abscisic-acid independent line of Larix x eurolepis. to catalogue the phenotypes of mature embryos, to determine critical stages of development and to attempt to increase the number of maturing somatic embryos. The low rate of maturation could not be entirely explained by differences in phenotypes of early embryos, critical stages of development, or the lack of plant growth regulators in the medium. In addition, the shape and epidermal type of the mature embryo did not always determine the type of epicotyl produced, nor did it affect the rooting and mortality rates. Six types of embryonal structures were evident in the aggregates of line 2086: (1) a smooth (SEMLS) or (2) rough (REMLS) embryonal mass subtended by a cylindrical, compact, long suspensor. (3) a rough embryonal mass subtended by a long, loose suspensor (REMLLS). (4) a rough embryonal mass subtended by a suspensor arising from greater than one quarter of the surface area of the embryonal mass (REMST). (5) a rough embryonal mass subtended by a short, compact, cylindrical suspensor (REMSS). and (6) a cluster of meristematic cells which may or may not have single suspensor cells attached (MC). For isolated embryonal structures of all types, to continue development into a nodule or a mature embryo was the least common fate, while proliferation and developmental arrest were more common. In general, the more organized embryonal structure types (SEMLS and REMLS) had higher rates of maturation compared to the other 4 types but the most common fate was still developmental arrest (74% SEMLS. 62% REMLS), followed by proliferation (10% SEMLS. 30% REMLS), and nodule or embryo development (16% SEMLS. 9% REMLS). REMLLS and REMST embryonal structures became developmentally arrested or proliferated (43-47%) while the rate of nodules/mature embryos production was 9-11%. Neither individual REMSS nor MC structures produced any nodules or mature embryos, but REMSS had a lower rate of developmental arrest (81%) and a higher rate of proliferation (19%) than MC (89% and 11% respectively). Embryos at more advanced stages of development were less likely to die, become developmentally arrested or become nodules, but more likely to become mature embryos than embryos at less advanced stages of development. A critical stage of development appeared to be the focal zone stage at the formation of a complete polyphenol band around the basal end of the embryonal mass. At this stage, the majority of immature embryos became mature embryos (61%) while only 3% of the embryos died. 10% became developmentally arrested, and 20% became nodules. The majority of mature somatic embryos were normally proportioned with a smooth epidermis (43%) rather than vitrified (12%). normal with a rough epidermis (12%) or misshapen (smooth or rough. 33%). The shape of the mature embryo was associated with the type of epidermis, with mature somatic embryos with normal proportions more likely to have smooth epidermis (78%) than a rough epidermis (22%) while mature embryos with abnormal proportions were as likely to have a smooth epidermis as a rough epidermis. The shape of the mature embryo was associated with the shape of the epicotyl produced. Normal-smooth, mature embryos were more likely to produce normal-smooth epicotyls (73%) than twin epicotyls (21%), vitrified epicotyls (2%) or misshapen epicotyls (5%) compared to vitrified mature embryos (42% normal-smooth epicotyls, 34% twin epicotyls, 23% vitrified epicotyls, 1% misshapen epicotyls) or misshapen mature embryos (22% normal-smooth epicotyls, 47% twin epicotyls, 7% vitrified epicotyls, 24% misshapen smooth/rough embryos). The number of mature embryos which germinated or died was not associated with either the epidermal quality or the shape of the mature embryo. Few SEMLS or REMLS embryonal structures responded to auxin and cytokinin treatments. There appeared to be a trend towards less developmental arrest and proliferation and more nodules/mature embryos produced on media with no auxin compared to media with 2,4-D and a trend towards more developmental arrest and fewer nodules/mature embryos on media without BA compared to media with BA. Only nodules on media without plant growth regulators produced roots or cotyledons. There was no effect of embryonal structure type (SEMLS or REMLS), or sucrose concentration (58 μM or 174 μM) on the maturation of immature embryos, but on media without ABA, fewer immature embryos proliferated or became developmently arrested and more embryos became nodules or mature embryos than on medium with 6-24 μM ABA. / Graduate

Plant breeding is focused on selection of new genotypes with improved traits. Conventional methods based on hybridization and those based on biotechnology (somatic hybridization, genetic transformation, ploidy manipulation, etc.) are used to create novel genetic variations. Biotechnology provides powerful tools for plant breeding, for instance, haploid technology allows achievement of homozygous lines from heterozygous parents in one step, which reduces significantly the time required by conventional methods. Concerning woody species, characterized by self-incompatibility, long juvenile period and high degree of heterozygosity, this technique is the only way to get homozygous lines. Haploid plants are of great interest for breeding and genomic studies, being used in mutation research, genetic analysis, genome mapping and gene transfer. Gametic embryogenesis, based on cellular totipotency, produces an embryo from an immature gamete, by switching its developmental pathway from gametophytic to sporophytic. This research is focused on inducing gametic embryogenesis in two important Mediterranean fruit crops: almond (Prunus dulcis Mill.) via in vitro anther culture and mandarin (Citrus reticulata Blanco) via isolation and microspore culture. Also ploidy manipulation was applied to loquat (Eriobotrya japonica (Thunb.) Lindl.) for getting genotypes whit different ploidy levels. The experiments were carried out through years 2014, 2015 and 2016 at the 'Università degli Studi di Palermo' (UNIPA) as well as at the 'Instituto Valenciano de Investigaciones Agrarias' (IVIA). Regarding the almond anther culture, formation of calli and production of embryos was achieved through the direct embryogenesis route. On then other hand, early embryo regeneration is reported, for the first time, from isolated microspore culture of mandarin, 'Mandarino Tardivo di Ciaculli'. Our results report the evidence of gametic embryogenesis and the production of homozygous regenerants in almond and mandarin, two species extremely recalcitrant to microspore embryogenesis. However, the results are affected by many factors that need further studies to better understand the embryogenic development and to increase the rate of embryo achievement. Moreover, another biotechnological tool (ploidy manipulation) was also applied for implementing the IVIA loquat breeding program. Polyploid plants are of great interest in this species, due to its potential for producing seedless genotypes via direct use of triploids or crosses between tetra and diploids. Aimed at obtaining new loquat genotypes, with different ploidy levels (polyploids), colchicine was applied to seeds before germination, to induce chromosome duplication. A total of three triploids (3x) and one tetraploid (4x) were obtained. / La mejora genética tiene como objetivo la selección de nuevos genotipos con mejores características. Los métodos de mejora convencional basados en hibridaciones y aquellos basados en Biotecnología (hibridación somática, transformación genética, manipulación de la ploidía, etc.) se utilizan para obtener nueva variación genética. La Biotecnología proporciona herramientas poderosas en mejora genética, por ejemplo, la obtención de haploides permite obtener líneas homocigotas en un solo paso, disminuyendo significativamente el tiempo requerido usando métodos convencionales. Respecto a especies leñosas, caracterizadas por autoincompatibilidad floral, largo período juvenil y alto grado de heterocigosidad, esta técnica es el único método de obtención de líneas homocigotas. Los genotipos haploides tienen un alto interés en estudios genómicos, siendo utilizados en estudios de mutaciones, análisis genéticos, mapeo genético y transferencia genética. Este estudio tiene como objetivo la inducción de embriogénesis gamética en dos especies mediterráneas muy importantes: el almendro (Prunus dulcis Mill.) por medio de cultivo in vitro de anteras y el mandarino (Citrus reticulata Blanco) por medio de aislamiento de microesporas. Además, se ha estudiado la obtención de poliploides en níspero (Eriobotrya japonica (Thunb.) Lindl.) con el objetivo de obtener genotipos con diversos niveles de ploidía. Los experimentos se llevaron a cabo en los años 2014, 2015 y 2016 en la 'Università degli Studi di Palermo' (UNIPA) y en el 'Instituto Valenciano de Investigaciones Agrarias' (IVIA). Respecto al cultivo de anteras en almendro, la formación de callos y producción de embriones se obtuvo mediante embriogénesis directa. Por otro lado, se ha conseguido regenerar por primera vez embriones a partir de microesporas aisladas en el cultivar de mandarino 'Mandarino Tardivo di Ciaculli'. Los resultados obtenidos muestran que la embriogénesis gamética y la regeneración de embriones homocigótos en almendro y mandarino, dos especies extremadamente recalcitrantes para la embriogenésis a partir de microesporas, es posible. Sin embargo, los resultados se ven afectados por muchos factores que necesitan estudios adicionales para comprender mejor el desarrollo embriogénico y para aumentar la tasa de obtención del embriones. Además, otra herramienta biotecnológica (manipulación de la ploidía) se aplicó con el objetivo de implementar el programa de mejora de níspero del IVIA. Las plantas poliploides en esta especie tienen un alto interés, pues podrían permitir la obtención de frutos sin semilla, por medio de la obtención directa de triploides o mediante cruzamiento entre tetraploides y diploides. Con el objetivo de obtener nuevos genotipos de níspero con diferentes niveles de ploidía (poliploides), se aplicó colchicina a semillas sin germinar con el fin de inducir la duplicación cromosómica y se obtuvieron 3 triploides (3x) y un tetraploide (4x). / La millora genètica té com objectiu la selecció de nous genotips amb millors característiques. Els mètodes de millora convencional basats en hibridacions i aquells basats en Biotecnologia (hibridació somàtica, transformació genètica, manipulació de la ploïdia, etc.) s'utilitzen per aconseguir nova variació genètica. La Biotecnologia proporciona eines poderoses en millora genètica, per exemple, l'obtenció d'haploides permet obtenir línies homozigòtiques en un sol pas, disminuint significativament el temps requerit usant mètodes convencionals. Pel que fa a espècies llenyoses, caracteritzades per autoincompatibilitat floral, llarg període juvenil i alt grau d'heterozigosi, aquesta tècnica és l'únic mètode d'obtenció de línies homozigòtiques. Els genotips haploides tenen un alt interès en estudis genòmics, sent utilitzats en estudis de mutacions, anàlisis genètics, mapatge genètic i transferència genètica. Aquest estudi té com objectiu la inducció d'embriogènesi gamètica en dos espècies mediterrànies molt importants: l'ametller (Prunus dulcis Mill.) a través del cultiu in vitro d'anteres i el mandariner (Citrus reticulata Blanco) per mitjà d'aïllament de micròspores. A més a més, s'ha estudiat l'obtenció de poliploides en nespra (Eriobotrya japonica (Thunb.) Lindl.) Amb l'objectiu d'aconseguir genotips amb diversos nivells de ploïdia. Els experiments es van dur a terme en els anys 2014, 2015 i 2016 a la 'Università degli Studi di Palermo' (UNIPA) i a 'l'Institut Valencià d'Investigacions Agràries' (IVIA). Respecte al cultiu d'anteres en ametller, la formació de calls i producció d'embrions es va obtenir mitjançant embriogènesi directa. D'altra banda, s'ha aconseguit per primera vegada la regeneració d'embrions a partir de micròspores aïllades en el conrear de mandariner 'Mandarino Tardivo di Ciaculli'. Els resultats obtinguts mostren que l'embriogènesi gamètica i la regeneració d'embrions homozigotics en ametller i mandariner, dues espècies extremadament recalcitrants per l'embriogènesi a partir de micròspores, és possible. No obstant això, els resultats es veuen afectats per molts factors que necessiten estudis addicionals per entendre millor el desenvolupament embriogènic i per augmentar la taxa d'obtenció dels embrions. A més, una altra eina biotecnològica (manipulació de la ploïdia) es va aplicar amb l'objectiu d'implementar el programa de millora de nespra de l'IVIA. Les plantes poliploides en aquesta espècie tenen un alt interès, ja que podrien permetre l'obtenció de fruits sense llavor, per mitjà de l'obtenció directa de triploides o mitjançant encreuament entre tetraploides i diploides. Amb l'objectiu d'aconseguir nous genotips de nespra amb diferents nivells de ploïdia (poliploides), es va aplicar colquicina a llavors sense germinar per tal d'induir la duplicació cromosòmica i es van obtenir 3 triploides (3x) i un tetraploide (4x). / Cimò, G. (2017). Ploidy manipulation for genetic improvement in some Mediterranean fruit crops [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79874 / TESIS