胰臟內分泌細胞分化的調控事件的研究揭示了胰島素分泌細胞的形成。這一原理既有利於體外誘導用於移植的胰島素分泌細胞，又可應用于糖尿病病人自體胰島素分泌細胞的再生。正在發育的組織和器官中，發現了腎素血管緊張素（RAS）成員，揭示了他們在發育過程中的潛在調控作用。另外，對 RAS 信號系統做出應答的活性氧化物質（ROS），被認為是第二信使，通過對轉錄調控因子的氧化還原的修飾促進分化。作為 ROS 的主要來源，NADPH 已被證實在各類細胞和組織中參與了祖細胞的分化。儘管如此，依賴於 NADPH 氧化酶的 ROS對于胰腺內分泌細胞分化的調控作用仍不清楚。基於這個背景，本研究致力於揭示 RAS 和 NADPH 氧化酶依賴性 ROS 在胰腺內分泌細胞分化中的作用。本實驗將在小鼠胰臟原基培養物和尿鏈黴素（STZ）誘導的新生大鼠上進行。 結果顯示，經典 RAS 成員中的血管緊張素 2 型受體（AT₂R）分佈於內分泌祖細胞的細胞核，之後穿梭定位於胰島素分泌細胞的細胞質。阻斷 AT₂R 功能抑制了Ngn3，胰島素的表達以及 β 細胞的增值。在不同的胚胎期 ROS 的水平發生了改變。對于培養的胰臟原基施加適當的外源 ROS，刺激了內分泌細胞的分化。同時，ROS 清除劑減弱了胰島細胞分化和成熟的標記基因的表達。NOX4 以及其相關的亞基 p22phox 是 NADPH 氧化酶成員，其在胰臟發育過程中的變化同 ROS 水平的變化相似，並且持續表達與內分泌細胞系統。在 NGN3 高表達的胚胎期15.5 天，它們定位于表達 NGN3 的細胞；在 NGN3 表達下調，且胰島素表達升高的胚胎期 17.5 天，它們分佈於胰島素表達細胞。而且，NADPH 氧化酶的抑製劑 DPI 削弱了胰臟培養物中的內分泌祖細胞的分化, 外源 H₂O₂ 的加入扭轉了這一現象。 / 另一方面，在 STZ 誘導的新生大鼠的研究中，DPI 負調節 β 細胞的再生。血糖失調，胰島結構毀壞以及血清胰島素匱乏的現象發生在了 DPI 處理組。另外，DPI 減弱了 NGN3 的表達而並非 Ki67, 顯示 β 細胞的分化而並非增值對於 ROS 的刺激進行了應答。在體內和體外的實驗中，DPI 也抑制了 NGN3 的轉綠調控因子 SOX9 在胰腺祖細胞中的表達。有趣的是，過表達 SOX9 可以恢復 DPI 引發的對於 NGN3 的抑制 。結合以上數據，本研究顯示 NADPH 氧化酶依賴性ROS 誘導的信號通路參與了胰腺祖細胞到胰島素分泌細胞的分化。 / Investigations into the regulatory events that modulate pancreatic endocrine cell differentiation shed light on the generation of sufficient insulin-producing cells in vitro for transplantation or regeneration of β cells in patients with diabetes. The expression of renin-angiotensin system (RAS) components has been detected in development tissue and organs, implicating their regulatory role in developmental processes. On the other hand, reactive oxygen species (ROS) are responsive to RAS signaling pathways and act as second messengers to promote differentiation through redox modification of transcriptional factors essential for differentiation. As a major source of ROS, NADPH oxidase has been shown to participate in the progenitor differentiation in a variety of cells and tissues. Despite this finding, the role of NADPH oxidase-dependent ROS in regulating pancreatic endocrine cell differentiation remains ambiguous. Against this background, the study was aimed at elucidating the roles of RAS components and NADPH oxidase-derived ROS during differentiation of pancreatic endocrine cells using mouse pancreatic rudiments and streptozotocin-treated neonatal rats. / Results showed that angiotensin II type 2 receptor (AT₂R), a major component of the classical RAS, was localized within the nuclei of endocrine progenitors in the cultured pancreatic rudiments; following the differentiation of endocrine progenitors into insulin producing cells, it translocated to cytoplasm. Blockade of AT₂R impeded the expression of Ngn3 and insulin as well as proliferation of β-cells. In addition, the dynamic changes of ROS levels were found in mouse pancreata at different embryonic days, concomitant with induction of endocrine cell differentiation induced by modest exogenous ROS in pancreatic rudiment cultures. Moreover, scavenger of ROS diminished the expression of islet cell markers for differentiation and maturation. NOX4 and its associated subunit p22phox, which are the member of NADPH oxidase, exhibited similar changes of expression to that of ROS levels during pancreas development and persisted in the endocrine lineage; they were located in NGN3⁺ cells at E15.5 during the burst of NGN3 expression and then distributed in insulin⁺ cell at E17.5, the latter being the phase that has a decline in NGN3 expression with an increase of insulin. Furthermore, administration of NADPH oxidase inhibitor, diphenylene iodonium (DPI) attenuated the differentiation of endocrine progenitors in rudiment cultures, while exogenous ROS reversed this effect. / On the other hand, studies performed in streptozotocin-induced neonatal rats showed that β cell regeneration was negatively affected by DPI treatments; consistently, impaired blood glucose control, disturbed islet architecture and deficient serum insulin were observed in DPI-treated groups. In addition, DPI treatments blunted NGN3 expression, but not Ki67-labeling beta-cells, suggesting that differentiation beyond proliferation of β-cells was accountable in response to ROS stimulation. Administration of DPI also suppressed the levels of SOX9, a transcriptional regulator of NGN3, in pancreatic progenitor cells, as evidenced by both in vivo and in vitro studies. Interestingly, over-expression of SOX9 could restore the repression of NGN3 induced by DPI. Taken all these data together, our results indicate that NADPH oxidase-dependent ROS-induced signaling pathway is involved in the differentiation of pancreatic endocrine progenitors into insulin-producing β cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Juan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 171-205). / Abstracts also in Chinese.

Normal germ cell development in the human testis is crucial for subsequent fertility and reproductive health. Disruption of testis development in fetal life can result in deleterious health consequences such as testicular dysgenesis syndrome (TDS), which includes disorders, such as cryptorchidism, hypospadias, infertility and testicular germ cell tumours (TGCT). A rat model of TDS in which rats are exposed to phthalates in utero has been validated, but does result in the development of TGCT. In humans, TGCTs result from transformation of pre-neoplastic carcinoma in-situ (CIS) cells and these CIS cells are believed to arise from human fetal germ cells during their transition from gonocyte to spermatogonia, based on their morphology and protein expression profile. It has been proposed asynchronous differentiation of germ cells in the human fetal testis may predispose fetal germ cells to become CIS cells. Studying the development of these tumours in humans is difficult because of their fetal origins and prolonged duration from initiation of impaired development to invasive disease. For this reason the use of relevant animal models that can mimic normal and abnormal germ cell development may provide new insight into how TGCT develop. The Common Marmoset monkey, a New World primate exhibits many similarities to the human in terms of reproductive biology and could represent such a model. This thesis aimed to further characterise the origins of CIS cells in the human testis by investigating the protein expression profile of CIS cells in patients with TGCT and comparing them to established markers of human fetal germ cell types using immunohistochemistry and immunofluorescence. Quantification of the various subpopulations of CIS and proliferation within these populations was performed. The thesis also investigated the Common Marmoset monkey as a potential model of normal testis and germ cell development by comparing the differentiation and proliferation profile of germ cells with those of the human during fetal and early postnatal life. During the present studies methods were successfully developed that enabled us to use testicular xenografts to recapitulate normal development of immature testes from marmoset and human. This involved grafting pieces of testis tissue subcutaneously under the dorsal skin of immunodeficient mice and retrieving them several weeks later to investigate their development during the grafting period. Xenografts using tissue from fetal, neonatal and juvenile marmosets were performed in addition to testes from first and second trimester human fetuses. Finally the present studies aimed to use the marmoset and the xenografting approach as systems in which to examine the effects of gonadotrophin suppression and phthalate treatment on germ cell differentiation and proliferation, with particular attention to the potential for development of CIS and TGCT. Heterogeneous phenotypes of CIS cells were identified, mostly consistent with those seen in the normal human fetal testis, however some of these CIS cells did not exhibit the same phenotype as germ cells identified in normal fetal testes. In addition it was shown that some of the proteins considered to be ‘classical’ markers of CIS cells, such as the pluripotent transcription factor OCT4, were not expressed in a proportion of the CIS cells. The proliferation index of CIS cells is also significantly higher in those subpopulations with the most ‘undifferentiated’ phenotype (i.e. OCT4+/VASA-). The present studies have generated novel data showing that the marmoset is a good model of fetal and neonatal germ cell development, with similarities to the human in terms of an asynchronous and prolonged period of differentiation and proliferation of germ cells from gonocyte to spermatogonia. This feature is also common to the human, but not a characteristic of the rodent. Fetal, neonatal and pre-pubertal germ cell development can be re-capitulated by xenografting tissue from marmoset and human testes into nude mouse hosts. Human fetal testis grafts produced testosterone and were responsive to hCG stimulation. First trimester human testis xenografts that have not developed fully formed seminiferous cords prior to grafting can complete the process of cord formation whilst grafted in host mice. In addition, germ cells in fetal human and marmoset xenografts can differentiate and proliferate in a similar manner to that seen in the intact non-grafted testis. In the intact neonatal marmoset, suppression of gonadotrophins resulted in a 30% decrease in proliferation, however differentiation of gonocytes is not affected. In-utero treatment of neonatal marmosets with mono-n-butyl phthalate was associated with unusual ‘gonocyte’ clusters, however, di-n-butyl phthalate treatment of mice carrying fetal marmoset xenografts resulted in no visible effects on germ cell differentiation or proliferation and did not result in the development of CIS or TGCT. In conclusion, this thesis has shown that there are many subpopulations of CIS cells of which many have not been previously described. These subpopulations have different characteristics, such as variable proliferation rates and this may indicate the potential for progression or invasiveness. These subpopulations have similar protein expression phenotypes to normal human fetal germ cells although the present studies have identified some CIS cells with phenotypes that are not found in the normal human testis. This thesis has demonstrated that the marmoset is a comparable model to the human in terms of asynchronous fetal germ cell development, which may predispose this species to the development of CIS/TGCT. In addition to the use of intact marmosets, these studies have also demonstrated for the first time that testis xenografting provides a comparable system for testis cord formation, germ cell differentiation and proliferation in fetal/postnatal marmosets and fetal human testis. In addition the marmoset and xenografting models have indicated that phthalates may have minor effects on testis development in the human and marmoset but do not result in CIS or TGCT. These model systems are suitable for further investigation of normal and disrupted testis development.

Linker histone H1 plays a key role in facilitating folding of higher order chromatin structure. Previous studies have shown that deletion of three somatic H1 subtypes together leads to embryonic lethality and that H1c/H1d/H1e triple knockout (TKO) embryonic stem cells (ESCs) display bulk chromatin decompaction. Following this initial work, we investigated the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, as well as the regulation of Hox genes expression. We find that H1 TKO ESCs are more resistant to spontaneous differentiation, impaired in embryoid body differentiation, and largely blocked in neural differentiation. We present evidence that H1 contributes to efficient repression of the expression of pluripotency factors, Oct4 and Nanog, and participates in establishment and maintenance of DNA methylation and histone modification necessary for silencing pluripotency genes during stem cell differentiation and embryogenesis. In addition, we find reduced expression of a distinct set of Hox genes in embryos and ESCs, respectively. Furthermore, by characterizing H1c−/−; H1d−/−; and H1e−/− single-H1 null ESCs established in this study, we showed that individual H1 subtypes regulated specific Hox genes in ESCs. Finally, we demonstrate that the levels of H3K4me3 were significantly diminished at the affected Hox genes in H1 TKO- and single-H1 KO- ESCs, whereas H3K27me3 occupancy is modestly increased at specific Hox genes. Our results suggest that marked reduction of H1 levels and decondensation of bulk chromatin affect the expression of pluripotency genes and Hox genes in embryos and ESCs, which may be in part mediated through establishment and maintenance of epigenetic marks.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) hold great promise in the fields of drug development and regenerative medicine. If iPSCs reprogrammed from patient cells replicate what is seen in vivo they may be used as a model of disease. A process that is disrupted in many neurodegenerative diseases is mitochondrial biogenesis. One of these diseases is amyotrophic lateral sclerosis (ALS), which is characterized by loss of motor neurons in the brain and spinal cord. Differentiation of hPSCs into motor neurons offers a way to study a previous unavailable cell type and may further our understanding of human motor neuron biology. The aims of the present study were to differentiate motor neurons from hESCs and iPSCs in low oxygen conditions and to explore mitochondrial biogenesis and electrical maturation during this process. After three weeks of treatment with retinoic acid and purmorphamine, a sonic hedgehog agonist, cells increased expression of post mitotic spinal motor neuron markers. One week later electrophysiological analysis revealed voltage-gated currents and action potential generation. Mitochondrial biogenesis signaling and expression of respiratory chain proteins increased with motor neuron differentiation. Respiration analysis revealed a decrease in glycolysis in motor neurons compared to neural stem cells. Interestingly, this was not accompanied by an increase in basal respiration or mitochondrial mass. These findings enhance our understanding of motor neuron mitochondrial biogenesis, a process impaired in ALS.

Alternative splicing (AS) is involved in expansion of transcriptome and proteome during cell growth, cell death, pluripotency, cell differentiation and development. There is increasing evidence to suggest that splicing decisions are made when the nascent RNA is still associated with chromatin. Here, I studied regulation of AS via chromatin modification with main focus on histone acetylation. First, we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection in 700 genes. We provided evidence that HDAC inhibition induces histone H4 acetylation and increases RNA Polymerase II (RNA Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduces co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. Further we showed that histone acetylation reader, Brd2 protein, affect transcription of 1450 genes. Besides, almost 290 genes change their AS pattern upon Brd2 depletion. We study distribution of Brd2 along the target and control genes and find that Brd2 is specifically localized at promoters of target genes only. Surprisingly, Brd2 interaction with chromatin cannot be explained solely by histone acetylation, which suggests that other protein-domains (in addition to bromodomains) are important for...

L'acide ascorbic acid (AA) a été considéré, pendant longtempss, comme une molecule devantêtre absorbée dans la nutrition, et prévenant le scorbut. Notre hypothèse, fondé sur desrésulats de notre groupe, suggèrent de nouvelles fonctions.Parmi celles-ci, nous nous sommes posé la question de l'AA molècule de signalling, durantl'embryogenèse et chez l'adulte, commme l'acide rétinoique (principe actif de la vitamine A)l'est. A cet effet, nous avons utilisé deux modèles cellulaires : des cellules souchesembryonnaires murines et des lignées de cellules souches/progénétrices adultes. Nous avonsainsi montré que l'AA stimule la différentiation de ces cellules en cellules musculairessquelettiques et en osteoblastes et inhibe l'adipogenèse et la neurogenèse. Cet effet passe parle transporteur de l'AA SVCT2 et implique la voie p38/MAPK. D'autre part, nous avonsdemontré que l'AA agit en compétition avec le RA, sur la neurogenèse et la myogenèse.Enfin, dans des cellules mésenchymateuses adultes, nous avons montré que l'AA inhibel'adipogenèse et stimule l'ostéogenèse. Cette action, comme chez l'embryon implique SVCT2et une modulation du pool du cAMP.En conclusion, l'AA pousse les cellules à se différencier en cellule musculaire squelettique eten ostéoblste et inhibie l'adipogenèse et la neurogenèse. / AA has been considered for a long time as a molecule involved in nutrition, to prevent scurvy. Our hypothesis is that AA could also be involved in development during embryogenesis, as well as in cell differentiation in adults. The aim of this study is to evaluate the potential implication of AA in cell differentiation, especially of mesenchyme cells, and to propose potential pathways that could be involved in these processes. Using murine ESCs we observed that AA markedly enhance the differentiation of ESCs toward muscle cells. Furthermore, we demonstrated that induction of myocytes by AA involves p38MAPK pathway and p-CREB. Moreover, we demonstrated that AA acts in mirror with retinoic acid. ESCs treated with RA mainly differentiate into neuronal cells, but AA compete, in a dosage dependent way to this differentiation. AA induces differentiation of ESCs into cardiac myocytes and could probably acts through p38MAPK pathway. Regarding adipocyte we revealed that SVCT2 expression significantly decreased as preadipocytes cells differentiate to adipocytes. This data suggests that mature adipocytes could not receive signals from AA. In addition, our results show that the expression of SVCT2 is increased in cells treated with AA and without IBMX. Moreover, we demonstrated that AA evolves in decreasing of cells containing lipids. Finally, we demonstrated that AA is not only involved in muscle differentiation of mesenchyme but is also involved in adipose tissue as a negative inducer. In conclusion, AA drives differentiation of ESCs toward muscle cells and osteoblast, incompetition with RA, and has a negative effect on adipogenesis and neurogenesis differentiation.

Although skeletal myogenesis is largely controlled by myogenic regulatory factors, epigenetic modifications have recently emerged as an essential regulatory mechanism of gene expression. Molecular regulation of stem cell differentiation is exerted through both genetic and epigenetic factors over distal enhancer regions. Understanding the mechanistic action of active or poised enhancers is therefore, imperative for the control of stem cell differentiation. Based on the genome-wide co-occurrence of different epigenetic marks in proliferating myoblasts, we have generated a chromatin state model to profile differentiation- and rexinoid-responsive histone acetylation in early myoblast differentiation. Here, we delineate the functional mode of transcription regulators during early myogenic differentiation using genome-wide chromatin state association. We define a role of transcriptional coactivator p300, when recruited by muscle master regulator MyoD, in the establishment and regulation of myogenic loci at the onset of myoblast differentiation. In addition, we reveal an enrichment of loci-specific histone acetylation at p300 associated active or poised enhancers, mainly when enlisted by MyoD. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Hence, we investigated the genome-wide impact of rexinoids on myogenic differentiation and uncovered a new mechanism of rexinoid action, which is mediated by the nuclear receptor and largely reconciled through direct regulation of MyoD gene expression. In addition, we determined rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated with MyoD and myogenin. Finally, through ChIP-seq and RNA-seq analyses, we have identified dystroglycan (Dag1) as a differentiation-dependent and a rexinoid-responsive model target, and we revealed a possible co-regulation of Dag1 by p300 and MyoD accompanied by enrichment of loci-specific histone acetylation. Taken together, we provide novel molecular insights into the regulation of myogenic enhancers by p300 in concert with MyoD. Furthermore, we provide novel mechanistic perceptions into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation. Our studies present a valuable insight for driving condition-specific chromatin state or enhancers pharmacologically to treat muscle-related diseases and for the identification of additional myogenic targets and molecular interactions for therapeutic development.