During vertebrate gastrulation, an elaborate series of cellular motility events occur that define the three germ layers, and establishes the primary body axis. While it is known that non-canonical Wnt signaling plays a crucial role during this period of development, a complete picture of the molecular mechanisms controlling this pathway has yet to be established. Previous studies has shown that one essential component of the pathway linking the Wnt ligands to the cytoskeletal changes that occur during vertebrate development is Dishevelled-associated activator of morphogenesis (Daam1). Daam1 bridges the gap between Dishevelled (Dvl) and the small GTPase RhoA and is required for Wnt-dependent RhoA activation, but the biochemical details of this process are to date still undefined. To identify additional factors that might be involved in this process, a yeast two-hybrid screen using a C-terminal region of Daam1 was performed. During this screen SLIT-ROBO Rho GTPase-activating protein 2 (SRGAP2), was identified. The studies presented here were designed to establish a functional interaction between SRGAP2 and Daam1 during non-canonical Wnt signaling and to characterize the function of SRGAP2 during early vertebrate development. My studies uncover that SRGAP2 and Daam1 do indeed interact and that this interaction is positively influenced by Wnt stimulation. I also uncover via immunocytochemistry, that these two proteins share common sub-cellular localization patterns in HeLa cells that is responsive to Wnt stimulation. Further, I show that ectopic expression of SRGAP2 in HeLa cells has adverse effects on stress fiber formation in Wnt5a treated cells. And lastly, micro-injection experiments in Xenopus laevis show that over- or under-expression of SRGAP2 produces severe gastrulation defects in the developing embryos. These studies together demonstrate that SRGAP2 plays a critical role in regulating non-canonical Wnt signaling through its interaction with Daam1 and through regulation of the monomeric GTPases Rho. It also shows that SRGAP2 plays a functional role for gastrulation during early vertebrate development. / Biology

Most vertebrates have separate sexes, and sex-specific traits that are regulated by genes with sex-biased expression patterns. In many species with genetic sex determination system, genetic recombination is suppressed in genomic regions linked to the master regulator of sex determination – the gene or set of linked loci that orchestrate sexual differentiation. Natural selection may favour alleles with sex-specific effects - including those with sexually antagonistic (SA) fitness effects (e.g., beneficial to females but harmful to males) – to become fixed in or be translocated to these non-recombining regions of sex chromosomes, because sex-specific or sex-biased modes of inheritance can resolve genomic conflict associated with SA. Sexually antagonism may also be resolve by sex-biased gene expression, and in theory these two mechanism (sex-linkage and sex-biased gene expression) could operate synergistically. However, there are relatively few empirical studies that test whether genes with sex biased expression patterns are indeed more abundant on sex chromosomes – and especially on newly evolved sex chromosomes. We explored this question with an African frog species Xenopus borealis, whose sex chromosome evolved within the last 25 million years (my) and have a large (~50Mbp) region of suppressed recombination, making it a young sex chromosome system compared to many other intensively studied systems, such as the sex chromosomes of mammals. We tested the possibility that a higher proportion of genes with sex-biased expression would be located on the sex-linked region of the sex chromosome of this species. By examining gene expression in adult liver and gonad and also tadpole gonad/mesenephros at two developmental stages, we found that the sex-linked region of these sex chromosome do have a higher proportion of sex biased genes compared to the non-sex-linked region of the same sex chromosomes, compared to (i) a homeologous genomic region in the tetraploid genome of X. borealis, and also (ii) the autosomes of this species. We did not observe the same pattern in a closely related frog species, Xenopus laevis, which has sex chromosome that are not homologous to those of X. borealis and, unlike X. borealis, lacks a large region of suppressed recombination on its sex chromosome. Using Brownian Motion model, we found as well that expression divergence evolution of genes in the sex-linked region of X. borealis is faster compared to its non-sex-linked homeologs (within X. borealis), and also compared to orthologous regions that are also non-sex-linked. One possible explanation for these observations is that natural selection favoured an expansion of recombination suppression (via unknown mechanisms) on chromosome such that polymorphic regulatory regions became linked (or unlinked) to the sex determining locus in such a way to resolve SA. Alternatively, it is possible that these sex-biased expression pattern evolved rapidly after recombination suppression. / Thesis / Master of Science (MSc) / Sexual selection favours the evolution of distinctive traits in each sex in order to optimize the reproductive success of each one. However, because most of the genome is shared between the sexes, sexual selection may result in genomic conflict when mutations are beneficial to one sex but harmful to the other; this conflict is known as sexual antagonism. Genomic conflict associated with alleles with sexually antagonistic (SA) fitness effects can be resolved via the origin of sex-biased expression patterns and this may be catalyzed by genetic linkage to a sex-determining locus on a sex chromosome. Consequently, one might predict there to be an enrichment of genes with sex-biased expression patterns on the sex chromosome as compared to the autosomes. We tested this expectation in an African frog species Xenopus borealis, which has a relatively young sex chromosomes and a large region of recombination suppression on the female-specific W-chromosome. We found enrichment of sex-biased genes on the nonrecombining region of the sex chromosomes of this species in adult liver and gonad tissue and also tadpole mesenephros/gonads at two developmental stages. Additionally, we found that expression divergence of genes in the non-recombining region have a faster rate of evolution as compared to the rate of expression divergence of genes in other genomic regions. One possible explanation for these observations is that natural selection favours an expansion of recombination suppression (via unknown mechanisms) on sex chromosome such that polymorphic regulatory region become linked (or unlinked) to the sex determining locus in such a way as to resolve SA.

During the early development of Xenopus laevis embryos, cells divide without checkpoints. At the midblastula transition (MBT), the cell cycle is remodeled as the division time lengthens and checkpoints are acquired. Initiation of the MBT depends upon the degradation of maternally supplied cyclin E, which is the regulatory partner of the cyclin dependent kinase, Cdk2. To study the program that drives cyclin E degradation and cell cycle remodeling at the MBT, embryos were treated with two cell cycle inhibitors, GST-D34Xic1 and XChk1. Injection of embryos with GST-D34Xic1, a stoichiometric inhibitor of cyclin E/Cdk2, delays degradation of cyclin E and onset of the MBT. GST-D34Xic1 lowers Wee1 level, a kinase that maintains Cdks in an inactivate state. Eventual degradation of cyclin E is preceded by degradation of GST-D34Xic1. The mathematical modelers, Andrea Ciliberto and John Tyson, incorporated the data into a kinetic model and set of ordinary differential equations. The model accurately described the experimental data and made additional predictions, which were tested experimentally. Additionally, embryos were injected with mRNA encoding XChk1, a kinase that activates Wee1 and inhibits Cdc25, the phosphatase opposing Wee1. Like GST-D34Xic1, XChk1 inhibits cyclin E/Cdk2 and delays the degradation of cyclin E. In contrast to GST-D34Xic1, XChk1 elevates the level of Wee1 at a time when sibling controls begin the MBT, despite cell cycle arrest. Since XChk1 inhibits both Cdk1 and Cdk2, and GST-D34Xic1 inhibits only Cdk2, we propose Cdk1 destabilizes Wee1, whereas Cdk2 elevates Wee1 level. Prior to the MBT, when cyclin E/Cdk2 is active, Wee1 is maintained. After cyclin E/Cdk2 is destroyed at the MBT, Wee1 is degraded. / Master of Science

In somatic cells, checkpoint pathways trigger cell cycle arrest in response to unreplicated or damaged DNA by inhibiting the activity of cyclin-dependent kinases (Cdks). In the Xenopus laevis embryo, checkpoints are not operational until the midblastula transition (MBT). Studies in cell-free egg extracts indicate that a threshold concentration of nuclei, which approximates the MBT concentration, is required to elicit a checkpoint. The checkpoint response to unreplicated DNA in the extract prevents transition into mitosis by inhibiting Cdk1/cyclin B, causing an increase in the minimum amount of cyclin B necessary to enter mitosis, termed the cyclin threshold. Once the threshold of cyclin is maintained or exceeded, the system will proceed into mitosis after a lag time. We have investigated the relationship between nuclear concentration and cell cycle regulation in the extract. By precisely regulating the concentration of cyclin B and nuclear content in extract samples, we have found 1) the concentration of nuclei affects cyclin B thresholds and lag time of entry into mitosis, 2) elevated cyclin thresholds caused by DNA replication blocks are further increased by increasing the concentration of nuclei, and 3) double-stranded DNA breaks in the extract system do not affect cyclin thresholds or lag time of entry into mitosis within the range of nuclear concentrations that can be efficiently replicated. This data provides evidence of the importance of the nucleocytoplasmic ratio in normal cell cycle progression and its importance for checkpoint acquisition during early Xenopus laevis development. / Master of Science

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Endocrine disrupting contaminants (EDCs) coming from households, industrial parks, wastewater (sewage) treatment and agricultural areas have been shown to pollute our freshwater systems. These contaminants may disrupt early development and reproductive systems in freshwater organisms (fish, frogs and crocodile species) as well as humans. Agricultural pesticides are shown as a large contributor to endocrine disruption activity in water catchment areas through spray drift, runoff, and/or groundwater leeching. Although South Africa is recognized as the largest consumer of agricultural pesticides in Africa, few studies have been undertaken to assess the prevalence and impact of endocrine disorders activities of pesticides in local freshwater systems. Recent studies have suggested that various agricultural pesticides, especially fungicides, might have adverse effects on the male endocrine system. There is therefore a need to test for a wider range of endocrine disrupting activities (mechanisms) in environmental waters other than conventional estrogenic (feminising) activities. Furthermore, there is a need to establish biomarkers in endemic species (bio-indicators) to show endocrine disruption in vertebrates (therefore also apply to humans). The specific objectives of the study were to: (1) describe and confirm the use of androgen-controlled breeding glands in male African clawed frogs (Xenopus laevis) as a biomarker for (anti)androgenic endocrine disruption activity (Chapter 2), (2) to investigate the premature development of breeding glands in X. laevis tadpoles (pre-metamorphic) and young froglets (post-metamorphic) (Chapter 2), (3) to investigate the disruption of male reproductive traits in adult X. laevis frogs by exposure to substances disrupting two different anti-androgenic endocrine disruption pathways (Chapter 3), (4) screen for (anti)androgenic activity of individual and binary mixtures of pesticides, which are regularly used in agricultural areas in the Western Cape Province of South Africa (Chapter 4), and (5) to test for (anti)androgenic and estrogenic endocrine disrupting activities by making use of in vitro assays as well as adult male X. laevis frogs collected from selected ponds surrounded by vineyards and fruit orchards in the Stellenbosch Winelands. The present study confirmed that male breeding glands can serve as biomarkers for (anti)androgenic endocrine disruption and that male reproductive and secondary sexual characteristics can be disrupted through two different biochemical control pathways. The study also confirmed that the expression of androgen-regulated breeding glands can be stimulated in pre-metamorphic tadpoles and immature, post-metamorphic frogs, and can thus be used for (anti)androgenic testing. The rapid testing and predictive value of an in vitro recombinant yeast screen for androgen receptor binding inhibition of selected individual or binary mixtures of pesticides was also confirmed. However, the current study showed that the predicted in vitro (anti)androgenic activity did not always correspond with in vivo (anti)androgenic biomarker outcomes. This It also confirmed that single-cell in vitro assays can be used as a first-level prediction for (anti)androgenic activities of individual or mixtures of agricultural pesticides. This study provides a better understanding for potential mixture interactions of commonly used agricultural pesticides, the hormonal control of secondary sexual characteristics in male frogs and the use of reproduction biomarkers to study long-term effects of endocrine disruptors in local water supplies. / AFRIKAANSE OPSOMMING: Endokriene versteurings-kontaminante (EVKe) wat vanaf huishoudings, industriële parke, afvalwater(riool)-behandeling en landbougebiede kom, besoedel ons varswaterstelsels. Hierdie kontaminante mag versteuring van vroeë ontwikkeling- en voorplantingstelsels in varswater-organismes (vis-, padda- en krokodil-spesies) sowel as die mens inhou. Landbou-plaagdoders word uitgesonder as ’n bydraer van endokriene versteuring-aktiwiteite in wateropvangs-gebiede deur spuitnewel, afloop-water en/of grondwater-deurvloei. Hoewel Suid-Afrika erken word as die grootste verbruiker van landbou-plaagdoders in Afrika, word min studies onderneem om die voorkoms en impak van endokriene versteurings-aktiwiteite van plaagdoders in plaaslike varswaterstelsels te ondersoek. Onlangse studies het voorgestel dat verskeie landbou-plaagdoders, veral swamdoders, nadelige uitwerkings kan hê op die manlike endokriene stelsel. Daar bestaan dus 'n behoefte om te toets vir 'n wyer verskeidenheid van endokriene versteurings-aktiwiteite (meganismes) in omgewingswater anders as konvensionele estrogeniese (vervroulikings) aktiwiteite. Verder bestaan daar ’n behoefte om biomerkers in endemiese spesies te gebruik as bio-indikators van endokriene versteuring in werweldiere (daarom ook van toepassing op die mens). Die spesifieke doelwitte van die studie het ingesluit om: (1) die gebruik van androgeen-beheerde parings- velkliere (“breeding glands”) in manlike platannas (Xenopus laevis) as 'n biomerker vir (anti)androgeniese endokriene versteuring-aktiwiteit te beskryf en bevestig (Hoofstuk 2); (2) ondersoek in te stel na die voortydige ontwikkeling van parings-kliere in X. laevis paddavisse (pre-metamorfose) asook jong paddas (post-metamorfose) as biomerkers van androgeniese (vermanlikheids) aktiwiteite (Hoofstuk 2); (3) ondersoek in te stel na die versteuring van manlike geslags-eienskappe in volwasse X. laevis paddas deur middel van blootstelling aan stowwe wat twee verskillende androgeniese endokrien reaksie-weë verteenwoordig (Hoofstuk 3); (4) toets vir (anti)androgeniese aktiwiteit van individuele en binêre mengsels van landbou-plaagdoders wat gereeld in die Westelike Provinsie van Suid Afrika gebruik word (Hoofstuk 4) en (5) te toets vir (anti)androgeniese en estrogeniese endokriene versteurings aktiwiteite deur gebruik te maak van in vitro toetse asook volwasse manlike X. laevis paddas wat uit geselekteerde damme (omring deur wingerde en vrugte boorde in die Stellenbosch wynland distrik) versamel was. Die huidige studie het bevestig dat die manlike parings-velkliere as biomerkers vir (anti)androgeniese versteuring kan dien en dat manlike voortplanting en sekondêre geslagskenmerke deur twee verskillende biochemiese beheer-weë ontwrig kan word. Die studie het verder bevestig dat die uitdrukking van androgeen-gereguleerde parings-velkliere voortydig gestimuleer kan word in pre-metamorfose paddavissies asook onvolwasse, post-metamorfose paddas. Die vinnige toetsing en voorspellingswaarde van 'n rekombinante in vitro gis toets om binding-inhibisie van die androgeen reseptor deur geselekteerde individuele of binêre mengsels van plaagdoders aan te toon is ook bevestig. Alhoewel, die huidige studie het getoon dat die voorspelde in vitro (anti)androgeniese aktiwiteit nie altyd ooreenstem met in vivo (anti)androgeniese biomerker uitkomstes nie. Hierdie studie bevestig dat enkel-sel in vitro toetse aangewend kan word as eerste vlak- en voorspelling-toetse vir (anti)androgeniese aktiwiteite van enkel of mengsels van landbou-plaagdoders. Sodoende is 'n beter begrip verkry vir potensiële mengsel-interaksies van algemeen-gebruikte landbou plaagdoders, die hormonale beheer van sekondêre geslagskenmerke in manlike paddas asook die aanwending van voortplantingsbiomerkers om langtermyn effekte van endokriene versteurders in plaaslike waterbronne te ondersoek.

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La plasticité du système nerveux central face aux contraintes environnementales ou morphologiques est un processus fondamental mis en place afin de permettre à l’animal de maintenir des comportements adaptés. Parce que le comportement locomoteur est essentiel à la survie de l'animal, les mécanismes neuronaux permettant sa genèse doivent s’adapter aux modifications morphologiques de l’organisme pendant son développement. Pour aborder cette question, nous avons développé un nouveau modèle expérimental pour lequel les modifications morphologiques au cours du développement sont extrêmes et impliquent des reconfigurations à long terme du système nerveux. L'amphibien Xenopus laevis lors de sa métamorphose est, en effet, un modèle pertinent pour étudier (par des approches comportementales, neuroanatomiques, électro-physiologiques et pharmacologiques), les mécanismes impliqués dans la réorganisation des réseaux neuronaux locomoteurs de la moelle épinière face à des modifications extrêmes du schéma corporel. En effet, pendant sa métamorphose, l'animal passe d'un mode de locomotion ondulatoire mettant en jeu sa musculature axiale, à un mode de locomotion appendiculaire grâce aux membres néo-formés. Il existe de plus des stades intermédiaires où les deux modes de locomotion coexistent et expriment des relations fonctionnelles variables. Nos expériences d’électrophysiologie extracellulaire nous ont permis de dégager la dynamique temporelle de l’émergence du réseau de neurones commandant la locomotion appendiculaire adulte et de ses relations fonctionnelles avec le réseau locomoteur commandant la nage larvaire lorsque ces deux réseaux coexistent. D’après les résultats présentés, il apparaît un changement de l’équilibre fonctionnel et des interactions entre les commandes locomotrices ondulatoire et appendiculaire, faisant des stades intermédiaires de la métamorphose les témoins privilégiés du passage de relais progressif entre les deux systèmes locomoteurs. Nos travaux ont également démontré que l’activité de chaque réseau ainsi que leurs relations fonctionnelles sont sujettes à modulation glutamatergique et aminergique destinées à adapter la locomotion aux besoins de l'animal. Nous montrons que certains modulateurs (tels que le glutamate, la sérotonine et la noradrénaline) exercent des effets opposés sur les réseaux locomoteurs larvaires et adultes, alors qu'à l'inverse, la dopamine conserve les mêmes propriétés modulatrices sur ces réseaux malgré les profonds bouleversements subis pendant le développement. Outre leur rôle modulateur, nos résultats suggèrent aussi un rôle des afférences aminergiques dans la maturation des réseaux locomoteurs et ouvrent de nombreuses interrogations quant aux mécanismes impliqués dans la plasticité des afférences neuromodulatrices elles-mêmes au cours de la métamorphose. L’apparition et la disparition de neurones sérotoninergiques intraspinaux concomitantes avec la croissance des membres postérieurs, et précédant la régression de l'appendice caudal laissent envisager un rôle de la sérotonine dans la maturation du réseau locomoteur appendiculaire ou dans la chronologie de la régression du réseau axial. / Plasticity of the central nervous system is fundamental to an animal's capacity to adapt to continually changing biomechanical and environmental demands. Although the neuronal mechanisms underlying such essential behaviours as locomotion must adapt to an organism's morphological modifications during growth and development, the associated changes that occur in central nervous function remain poorly understood. To address this issue, we have developed a new experimental model - the amphibian Xenopus laevis during its metamorphosis - in which the extreme biomechanical modifications occurring during this critical period necessitate a correspondingly extensive and long-term reorganisation of locomotor neural circuitry within the animal's spinal cord. During metamorphosis, the locomotory strategy of Xenopus shifts from undulatory swimming involving axial tail-based movements, to appendicular propulsion that uses the newly formed limbs. At intermediate metamorphic stages, moreover, the two locomotor strategies coexist within the same animal as the secondary limb-based motor circuitry is progressively replaces the primary axial network as the limbs are added and the tail regresses. By making extracellular recordings of spontaneous "fictive" locomotor patterns generated by isolated brainstem/spinal cord preparations, we have charted the temporal dynamics of the emergence of the appendicular neuronal network and determined its functional relationship with larval axial locomotor circuitry through the metamorphic period. Our results have shown that the limb circuitry is initially present but not functional, functional but subordinate to the embryonic axial network, functionally independent from the axial network, and ultimately alone after axial circuitry disappears with tail resorption. Furthermore, the use of pharmacological approaches established that during the metamorphic transition, the coexisting spinal locomotory networks and their functional interactions are subject to glutamatergic and aminergic modulation in order to adapt locomotory performance to the immediate behavioural needs of the animal. Interestingly, the neuromodulators glutamate, serotonin and noradrenaline exert directly opposing influences on the larval and adult locomotor networks, while dopamine preserves a similar modulatory action on the two circuits in spite of their profound remodelling during metamorphic development. Finally, in addition to a short-term modulatory role, our immunocytochemical evidence suggested that descending aminergic systems may contribute to the long-term maturation of spinal locomotor circuitry during metamorphosis in parallel with their own developmental reconfiguration. Specifically, the appearance and disappearance of a population of intraspinal serotonergic neurons concomitant with hindlimb growth and preceding tail regression suggested a role of serotonin in the maturation of the appendicular locomotor network and/or in the chronology of axial network regression.

Le processus de formation des cils mobiles multiples (multiciliogénèse) est composé de nombreuses étapes. Récemment, nous avons démontré que les microARNs de la famille miR-449 contrôlent plusieurs de ces étapes. Au cours de mon travail, je me suis concentré sur le rôle joué par miR-449 dans deux aspects du développement de l'épithélium multicilié. Dans les cellules multiciliées, un réseau dense d'actine sous-jacent l'aspect apicale de la membrane cellulaire (coiffe d'actine) est nécessaire pour l'ancrage des multiples corps basaux, et donc pour une ciliogenèse approprié. Dans le cadre de mon travail, j'ai participé à l' identification de la petite GTPase R-Ras comme une des véritables cibles de miR-449. J'ai démontré que la réorganisation de la coiffe d'actine et l'ensemble du processus de multiciliogénèse étaient compromis lorsque l'ARN messager de R-Ras se trouve protégé de la liaison avec miR-449. J'ai aussi contribué à identifier une nouvelle cible de miR-449, le gène Steel, qui code pour le ligand du récepteur transmembranaire à tyrosine-kinase KIT. La repression de Steel par miR449 est impliquée dans le processus par lequel les cellules multiciliées atteignent leur position finale dans l'épiderme embryonnaire de Xenopus. STEEL, qui agit probablement comme une molécule de guidage pour les cellules multiciliées qui expriment KIT, doit être réprimé par miR-449 dans ces mêmes cellules en cours de migration pour assurer leur deplacement directionnel approprié. En conclusion, mon travail a contribué à élucider le rôle complexe joué par le miARN miR-449 dans le processus de multiciliogénèse chez les vertébrés. / The process of multiple motile cilia formation (multiciliogenesis) is composed of many different steps. Recently, we demonstrated that microRNAs of the miR-449 family control several of these steps. During my work, I focused on the role played by miR-449 in two aspects of the development of the mucociliary epithelium. In multiciliated cells, a dense actin network underlying the apical aspect of the cell membrane (actin cap) is required for the anchoring of the multiple basal bodies, and therefore for proper ciliogenesis. Small GTPases play important role in the formation of the actin cap. In the course of my work, I took part in the identification of transcripts coding the small GTPase R-Ras as bona fide targets of miR-449. I demonstrated that apical and subapical actin network reorganization and multiciliogenesis were impaired when R-Ras mRNA was protected from miR-449 binding. Moreover, the actin cap formation and multiciliogenesis were rescued when the translation of protected R-Ras transcripts was prevented. I also contributed to the finding that a new miR-449 target, the KIT receptor tyrosin kinase ligand STEEL, is involved in the process through which the multiciliated cells reach their final position within the developing frog epidermis. STEEL, which likely acts as a guidance molecule for the KIT-expressing multiciliated cells, needs to be repressed by miR-449 within the migrating cells to ensure their proper directional migration. Altogether, my work contributed to elucidate the complex role played by the miR-449 miRNA in the process of vertebrate multiciliogenesis.

糖調節蛋白78 (Glucose-regulated protein 78),也稱之Bip,是70kDa熱休克蛋白家族成员之一。已有的研究表明，Bip 是一個具有多功能的蛋白，參與眾多的生物調控過程，包括蛋白折疊，調節鈣平衡，以及作為內質網緊張（ER stress) 的感應器。有研究表明，Bip可以在細胞膜上定位，作為Nodal信號通路的一個輔助受體發揮作用。大量的研究表明，Bip在疾病和代謝方面也發揮重要作用。它參與胰島素的生物合成，並可以提高長期高血糖下β細胞的功能。同時具有抗細胞凋亡的作用。然而Bip在胚胎髮育中的生物功能卻知之甚少。 / 高等脊椎動物腎臟發育中經歷形成3種腎臟形式：前腎，中腎和後腎。腎單位是這3種形式的基本結構和功能單位。在兩棲類，前腎在胚胎時期發揮作用，在胚胎的兩側各只有一個腎單位。這使得爪蟾成為前腎研究的一個非常好的模型。 / 在此項研究中，我們採用非洲爪蛙作為動物模型來研究Bip在胚胎髮育過程中，尤其是在前腎發育中的生物功能。Bip是一個母性因子，在尾芽期，Bip 表達在粘液腺，前腎，肝以及耳囊。 Bip在前腎清晰明確的表達，表明Bip可能在前腎的發育中發揮作用。我們利用BipMO來進行敲低功能實驗，免疫印記顯示BipMO能阻斷帶Flag標記Bip的翻譯。通過原位雜交技術檢測前腎的不同標記基因的表達發現，敲低Bip抑制前腎的形成，表明Bip的正常表達是前腎發育所必須的。 / 為了研究Bip調節前腎的發育的分子機制， 我們使用Affmetrix基因芯片分析在Bip敲低情況下的不同時期胚胎中基因的表達譜，發現在Bip敲低表達的胚胎中，視黃酸信號通路的一些重要的組分的表達受到抑制。爪蛙胚胎原腸胚的動物帽細胞具有多能性， 使用激活素和視黃酸一同處理動物帽細胞可以誘導其分化成為原腎組織。在此體外分化體系中敲低Bip表達，前腎標記基因表達降低，顯示在這一體外系統中前腎的分化受到抑制。該實驗結果與體內實驗結果一致。在體外培養的HEK293T細胞中敲低Bip，抑制視黃酸處理後視黃酸信號通路螢光素報告的活性。 lhx1是前腎發育早期表達標記之一，對於前腎原基的初始化具有重要的作用，同是它也是視黃酸信號通路的靶基因。共同註射BipMO和lhx1表明，前腎的異常可以明顯降低，顯示lhx1可以部份拯救由於Bip缺失所造成的腎臟發育缺陷。該實驗表明Bip通過調節視黃酸信號通路，來調控lhx1的表達前腎的形成。我們進一不發現，敲低Bip後，前腎異常形成的區域內，細胞凋亡增加，增殖減少。該結果在細胞水平上解釋了Bip敲低表達時前腎形成異常的一個原因。 / 综述所述，Bip正確表達对胚胎前肾的发育極為重要。它胚胎发育过程中通过視黃酸信号通路調控lhx1的表達，從而对前肾的形成发挥重要作用。 / Glucose-regulated protein 78 (Grp78), also known as Bip, belongs to heat shock protein 70kDa family. It has been implicated in various biological processes including protein folding, regulation of calcium homeostasis, and serving as a sensor of ER (Endoplasmic Reticulum) stress. Moreover, it can localize in cell membrane, acting as co-receptor of nodal signaling. It is essential for insulin biosynthesis. In addition, Bip plays important roles in a number of diseases. For example, BIP can improve β-cell function in the prolonged hyperglycemia. Knockdown of BIP in β-cell can induce apoptosis. However, little is known about its function during embryonic development. / In high vertebrate, three sets of nephric forms develop successively during embryonic kidney development. They are pronephros, mesonephros, and metanephros. Nephron is the basic structural and functional unit of all these three forms. In amphibian, the pronephros performs function at the embryonic stages, which has only one nephron on either side of the body. It makes Xenopus a very good model for pronephros study. / In this study, we took advantage of Xenopus leavis as an animal model to investigate Bip function during embryonic development, especially its role in pronephros development. We first examined the expression of Bip in developing embryos. Whole mount in situ hybridization showed that Bip was expressed in the cement gland, pronephros, liver and ear vesicle during tailbud stages. It was expressed in the pronephros strongly and clearly which suggested that Bip might play roles in pronephros development. We performed loss-of-function experiment by using morpholino oligonucleotide (MO) knock down translation of endogenous Bip expression. Depletion of Bip impaired formation of pronephros revealed by reduction expression of different pronephros maker genes. The pluripotent animal caps can differentiate into pronephros tissue when treated with activin and all-trans retinoic acid (atRA) in vitro kidney induction assay. In line with our in vivo observation, knockdown of Bip inhibited pronephros differentiation that can normally achieved by combined effects of activin and atRA in animal cap assay. / In order to investigate the molecular mechanisms as how Bip regulated pronephros development, we performed Affymetrix DNA microarray assay to generate gene expression profile in Bip morphants. We found that some components of RA signaling were inhibited when Bip was knockdown. Moreover, knockdown of Bip caused reduction of RA target genes expression after treatment with RA. Consistent with above observations, luciferase activities of RA signaling reporter was reduced in HEK293T cells when BIP expression was depleted by RNAi. lhx1 is one of RA target genes and has been implicated playing essential roles in pronephros development. The inhibition of pronephros formation induced by Bip depletion can be partially rescued by co-overpression, suggesting 1) lhx1 is downstream of Bip in the regulatory network of pronephros formation; and 2) Bip regulates pronephros formation through RA signaling via lhx1. We also found increased apoptosis and decreased cell proliferation at pronephros-forming region in Bip morphants. That could explain the reason of pronephros malformation when Bip is downregulated. / Taken together, Bip is essential for pronephros development. It functions through RA signaling during the complex developmental processes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shi, Weili. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 125-143). / Abstracts also in Chinese.

Thesis (M.Sc.), Memorial University of Newfoundland, 1998. / Restricted until June 1999. Bibliography: leaves 85-97.