In this thesis, several techniques were combined to optimize, evaluate and characterize craniofacial development in Xenopus, with additional focus on understanding the alterations made during maturation in the craniofacial region and the cartilage. Three important techniques used were: confocal microscopy in conjunction with Acridine Orange (AO) labeling, Alcian Blue (AB) labeling, and geometric morphometric analysis. I found that facial width increased across all techniques used to evaluate it. Included within this focus was the study of the development of the ceratohyal (CH) cartilage, which supported the mouth and snout. This was also found to increase width wise, in unison with facial and orofacial growth. This data may suggest a link between the face, mouth and CH growth, in which the developing cartilage elongates and widens causing the increase seen in the width and distension of the mouth.

La crête neurale (CN) est une structure transitoire apparaissant en bordure de la plaque neurale chez les embryons de vertébrés. Au cours du développement embryonnaire, les cellules de la CN prolifèrent, subissent une transition épithélio-mésenchymateuse, migrent et se différencient en de nombreux types cellulaires tels que des neurones et cellules gliales du système nerveux périphérique, des mélanocytes, des cellules musculaires lisses ou des élements du squelette cranio-facial. Afin de mieux comprendre les mécanismes moléculaires contrôlant la prolifération et la spécification des cellules de la CN, nous avons étudié le rôle de deux facteurs de transcription, Hairy2 et Stat3, via des expériences de perte et gain de fonction chez l’embryon de xénope. Le gène Hairy2 code pour un facteur de transcription bHLH-O répresseur. Il est exprimé précocement au niveau de la bordure de la plaque neurale incluant la CN présomptive. Nous avons montré que Hairy2 est requis pour la prolifération des cellules de la CN en aval de signaux FGFs et qu’il maintient les cellules dans un état indifférencié en réprimant l’expression précoce des gènes spécifiques de la CN. Hairy2 réprime aussi la transcription du gène Id3 codant pour un facteur HLH essentiel à la prolifération des cellules de la CN. Id3 affecte également Hairy2. Nous avons observé que la protéine Id3 interagit physiquement avec Hairy2 et bloque son activité, démontrant que les interactions entre Hairy2 et Id3 jouent un rôle important dans la prolifération et la spécification des cellules de la CN. Afin de comprendre le mode d’action de Hairy2 dans la CN, nous avons comparé les propriétés de la protéine Hairy2 sauvage à celle d’une version mutée de la protéine incapable de lier l’ADN. Nos résultats ont montré que Hairy2 fonctionne selon deux mécanismes distincts. La capacité de Hairy2 à promouvoir la survie et la maintenance des cellules progénitrices de la CN dans un état non spécifié et indifférencié est dépendante de sa liaison à l’ADN. A l’inverse, sa capacité à stimuler la prolifération cellulaire et l’expression des gènes spécifiques de la CN est indépendante de sa liaison à l’ADN mais nécessite l’activation du ligand du récepteur Notch, Delta1. De plus, nous avons également montré que la capacité de Hairy2 d’induire Delta1 dans la CN requiert Stat3. Le gène Stat3 code pour un facteur de transcription latent dans le cytoplasme pouvant être activé par de nombreux signaux extracellulaires. Nos résultats ont montré que Stat3 joue un rôle crucial dans la prolifération cellulaire et dans l’expression des gènes de la bordure de la plaque neurale et de la CN. Stat3 est phosphorylé directement par la voie de signalisation FGF via FGFR4 et est requis in vivo en aval de FGFR4. Nous avons aussi montré que Hairy2 et Id3 sont des régulateurs positifs et négatifs de l’activité de Stat3 qui facilite et inhibe la formation du complexe Stat3-FGFR4, respectivement. De plus, Stat3 contrôle la transcription des gènes Hairy2 et Id3 de manière dose dépendante. Nous avons observé que Hairy2 est activé à faible dose et Id3 à forte dose de Stat3, suggérant que Stat3 s’auto-régule de manière indirecte via l’activation d’une boucle de rétro-contrôle positive (Hairy2) et une négative (Id3). Stat3 régule également de manière dose dépendante la prolifération et la différenciation des cellules de la CN. Une faible activité de Stat3 stimule la prolifération cellulaire et l’expression des gènes spécifiques de la CN tandis qu’une forte activité de Stat3 ralentit le cycle cellulaire, inhibe l’expression des gènes de la CN et maintient les cellules de l’ectoderme dans un état non spécifié et indifférencié. En conclusion, nous montrons pour la première fois que Stat3, en aval des FGFs et sous le rétro-contrôle de Hairy2 et Id3, joue un rôle essentiel dans la coordination de la progression du cycle cellulaire et de la spécification de la CN au cours du développement embryonnaire du xénope.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Early embryonic development in vertebrates is directed in part by maternal mRNAs expressed in oocytes and stored in cytoplasmic messenger ribonucleoprotein particles (mRNPs). Abundant evidence demonstrates the importance of mRNPs in embryonic development and in post-embryonic cellular function; however their characterization has been hampered by lack of suitable methodologies. The Xenopus oocyte has been the primary model system for studies of mRNPs. YY1 is a well-studied transcriptional regulatory factor that is sequestered in the oocyte cytoplasm and present entirely in cytoplasmic oocyte mRNPs. The objective of this thesis was to examine the biochemistry of YY1 association with maternal mRNA molecules in order to shed light on the role of YY1 in development and the poorly understood biology of oocyte mRNPs. The initial working hypotheses were that association of YY1 with mRNPs is dependent on sequence-specific RNA-binding activity and, therefore, that YY1 associates with a definite subset of maternal mRNA. A number of unique methods were developed in this study to address these hypotheses. RNA immunoprecipitation-DNA microarray (RIP-CHIP) analysis establishes that YY1 associates with a subset of mRNAs in the oocyte pool. A novel sequence-specific RNA-binding activity of the YY1 protein is demonstrated, and the RNA-binding activity of YY1 is shown to be required for its association with oocyte mRNPs in vivo. The functional roles of YY1 mRNA substrates are discussed in the context of embryological development and the biological function of YY1 in oocyte mRNPs. Extension of the experimental approaches developed in this thesis to the entire set of mRNP proteins would significantly advance our understanding of mRNP composition and heterogeneity, as well as the biological function of maternal mRNAs and mRNPs in development.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Neurulation is critical for the proper development of the central nervous system during embryogenesis. This process requires coordinated morphogenetic movements driven by localized cell movements. The key morphogenetic process responsible for lengthening the neural plate is convergent extension. During convergent extension medially oriented cell polarity, protrusive activity, and motility are thought to generate forces through cell intercalation resulting in stiffer elongating tissues. My research determines that forces that help shape the neural plate arise from morphogenetic movements in the neural tissue and determines PCP signaling regulates tissue stiffness in the neural ectoderm. We have established an experimental system sensitive enough to evaluate the stiffness of Xenopus neural tissue. Stiffness is measured by gluing two fine wires onto neural explants from an early gastrula stage Xenopus laevis embryo. The wires stretch the tissue at a constant strain rate using a real-time image-based feedback system and stiffness is determined by measuring the deflection of one wire. Measurements obtained from control embryos prior to neurulation estimate tissue stiffness at approximately 12.7 ± 0.53 mN/m in both mediolateral and anteroposterior directions. Stiffness measurements double in early neurula embryos (P < 0.05). Mediolateral stiffness, 24.9 ±6.2 mN/m, is significantly greater than anteroposterior stiffness, 21.4 ±5.3 mN/m (P < 0.05). These trends are strengthened in normalized data to reduce clutch-to-clutch variation. Expressions of dominant-negative Wnt11, Fz7, and Dsh constructs successfully disrupt neurulation by interfering with the PCP pathway. Changes in stiffness of the neural plate were measured and show reduced stiffness at early neurula stage in both mediolateral and anteroposterior directions suggesting mechanical forces are generated within the neural plate.

Neurulation is critical for the proper development of the central nervous system during embryogenesis. This process requires coordinated morphogenetic movements driven by localized cell movements. The key morphogenetic process responsible for lengthening the neural plate is convergent extension. During convergent extension medially oriented cell polarity, protrusive activity, and motility are thought to generate forces through cell intercalation resulting in stiffer elongating tissues. My research determines that forces that help shape the neural plate arise from morphogenetic movements in the neural tissue and determines PCP signaling regulates tissue stiffness in the neural ectoderm. We have established an experimental system sensitive enough to evaluate the stiffness of Xenopus neural tissue. Stiffness is measured by gluing two fine wires onto neural explants from an early gastrula stage Xenopus laevis embryo. The wires stretch the tissue at a constant strain rate using a real-time image-based feedback system and stiffness is determined by measuring the deflection of one wire. Measurements obtained from control embryos prior to neurulation estimate tissue stiffness at approximately 12.7 ± 0.53 mN/m in both mediolateral and anteroposterior directions. Stiffness measurements double in early neurula embryos (P < 0.05). Mediolateral stiffness, 24.9 ±6.2 mN/m, is significantly greater than anteroposterior stiffness, 21.4 ±5.3 mN/m (P < 0.05). These trends are strengthened in normalized data to reduce clutch-to-clutch variation. Expressions of dominant-negative Wnt11, Fz7, and Dsh constructs successfully disrupt neurulation by interfering with the PCP pathway. Changes in stiffness of the neural plate were measured and show reduced stiffness at early neurula stage in both mediolateral and anteroposterior directions suggesting mechanical forces are generated within the neural plate.

In eukaryotes, the ubiquitin-proteasome system (UPS) degrades most cellular protein. Inhibition of the UPS has been associated with different disease states and can affect various intracellular processes including the activation of heat shock protein (hsp) gene expression. During cellular stress, HSPs act as molecular chaperones by inhibiting protein aggregation and assisting in their refolding once normal conditions are re-established. In the present study, Withaferin A (WA), a steroidal lactone with possible anti-inflammatory and antitumor properties, was found to inhibit proteasome activity and induce the expression of hsp genes in the amphibian model system, Xenopus laevis. Treatment of Xenopus kidney epithelial A6 cells with WA produced an increase in the accumulation of ubiquitinated protein and a significant decrease in chymotrypsin-like activity. Furthermore, immunoblot analysis revealed that WA induced HSP30 and HSP70 accumulation. For example, cells treated with 5 μM WA for 18 h resulted in the optimal accumulation of HSP30 and HSP70. Northern blot analysis revealed that exposure of cells to 5 μM WA induced hsp30 and hsp70 mRNA accumulation in a time-dependent manner up to 12 h. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in WA-induced hsp gene expression in A6 cells, since pretreatment with the HSF1 inhibitor, KNK437, reduced the accumulation of HSP30 and HSP70. Also, WA acted synergistically with mild heat shock to enhance HSP accumulation to a greater extent than the sum of both stressors individually. In cells recovering from WA, the relative levels of HSP30 and HSP70 accumulation remained elevated from 6 to 12 h after removal of WA. Immuocytochemical analysis and laser scanning confocal microscopy revealed that WA-induced HSP30 accumulation occurred primarily in the cytoplasm with some staining in the nucleus in a granular or punctate pattern. Prolonged exposure to WA resulted in some disorganization of the actin cytoskeleton as well as large cytoplasmic HSP30 staining structures in some cells. Prior exposure of cells to WA treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 °C. In conclusion, this study has shown that WA can induce an inhibition of proteasome activity and an increase hsp gene expression. Activating the heat shock response is a potential avenue for novel drug therapies, which can confer cytoprotection in disease states involving cytotoxic protein aggregation.

Early embryonic development in vertebrates is directed in part by maternal mRNAs expressed in oocytes and stored in cytoplasmic messenger ribonucleoprotein particles (mRNPs). Abundant evidence demonstrates the importance of mRNPs in embryonic development and in post-embryonic cellular function; however their characterization has been hampered by lack of suitable methodologies. The Xenopus oocyte has been the primary model system for studies of mRNPs. YY1 is a well-studied transcriptional regulatory factor that is sequestered in the oocyte cytoplasm and present entirely in cytoplasmic oocyte mRNPs. The objective of this thesis was to examine the biochemistry of YY1 association with maternal mRNA molecules in order to shed light on the role of YY1 in development and the poorly understood biology of oocyte mRNPs. The initial working hypotheses were that association of YY1 with mRNPs is dependent on sequence-specific RNA-binding activity and, therefore, that YY1 associates with a definite subset of maternal mRNA. A number of unique methods were developed in this study to address these hypotheses. RNA immunoprecipitation-DNA microarray (RIP-CHIP) analysis establishes that YY1 associates with a subset of mRNAs in the oocyte pool. A novel sequence-specific RNA-binding activity of the YY1 protein is demonstrated, and the RNA-binding activity of YY1 is shown to be required for its association with oocyte mRNPs in vivo. The functional roles of YY1 mRNA substrates are discussed in the context of embryological development and the biological function of YY1 in oocyte mRNPs. Extension of the experimental approaches developed in this thesis to the entire set of mRNP proteins would significantly advance our understanding of mRNP composition and heterogeneity, as well as the biological function of maternal mRNAs and mRNPs in development.

<p>Cytosolic cytochrome c promotes apoptosis by triggering caspase activation. In healthy cells cytochrome c localizes to mitochondria, where it participates in the electron transport chain. Apoptotic stimuli induce permeabilization of the outer mitochondrial membrane and release of cytochrome c. Once cytosolic, cytochrome c binds Apaf-1, inducing the formation of a protein complex that recruits and activates caspases, which serve to dismantle the dying cell. Although the steps of this signaling pathway have been described, many of the regulatory mechanisms influencing the cellular response to cytosolic cytochrome c remain unclear. Using apoptosis assays and microinjection techniques, we investigated the response of several cell-types to cytosolic cytochrome c.</p> <p>First, we demonstrate that cytosolic cytochrome c kills brain tumor cells but not normal brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in brain tumors compared with negligible Apaf-1 in brain tissue. These differences in Apaf-1 abundance correlate with differences in E2F1, a previously identified activator of Apaf-1 transcription. Chromatin immunoprecipitation assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to Apaf-1 expression in brain tumors. These results demonstrate an unexpected sensitivity of brain tumors to cytochrome c and raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancers.</p> <p>Secondly, we develop a method for monitoring caspase activity in Xenopus laevis oocytes and early embryos. The approach, utilizing microinjection of a near-infrared dye that emits fluorescence only after its cleavage by active caspases, has enabled the elucidation of subtleties in the apoptotic program. We demonstrate that brief caspase activation is sufficient to cause death. We illustrate the presence of a cytochrome c dose threshold, which is lowered by neutralization of inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that eventual death of these oocytes is caspase-independent. Imaging caspase activity in the embryo suggests that apoptosis in early development is not cell-autonomous. Finally, we believe this method presents a useful screening modality for identifying novel apoptotic regulators as well as pro-apoptotic small-molecules that could be useful in treating brain tumors.</p> / Dissertation