京都大学 / 新制・論文博士 / 博士(医学) / 乙第13466号 / 論医博第2253号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 中島 貴子, 教授 藤田 恭之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Dlx homeobox genes encode homeodomain transcription factors that are involved in multiple developmental aspects. In the brain, these genes take part in neuronal migration and differentiation, more precisely in the migration and differentiation of GABAergic neurons. Dysfunctions in the GABAergic system can lead to various pathological conditions, where impaired inhibitory function is one of the main causes of several neuropathies characterized by neuronal hyperexcitability. The Dlx genes are organized as bi-gene clusters and highly conserved cis-regulatory elements have been previously characterized to be fundamental for the regulation of Dlx expression in developing embryos of different vertebrates. The activity of these regulatory elements and the Dlx genes has been well studied in developmental stages of mice and zebrafish, but little is known about their activity in the adult brain. The extensive neurogenesis that takes place in the adult zebrafish brain provides an ideal platform for the visualization of mechanisms involving dlx genes during adulthood and their possible involvement in adult neurogenesis. Here we show novel information concerning the expression of dlx1a, dlx2a, dlx5a and dlx6a in the adult zebrafish brain and provide insight into the identity of cells that express dlx. We also demonstrate the involvement of dlx genes in brain regeneration and through lineage tracing, their fate determination in the adult zebrafish brain. Analyses in the adult zebrafish has revealed that all four dlx paralogs are expressed in the forebrain and midbrain throughout adulthood and expression is found in almost all areas presenting continuous proliferation. Most dlx-expressing cells present GABAergic neuronal identity in the adult forebrain where, in some areas they were identified as the Calbindin subtype. In some areas of the midbrain, especially within the hypothalamus, many dlxexpressing cell co-localized with a marker for neural stem cells. However, cells expressing dlx iii genes did not co-localize with markers for proliferating cells or for glia. Investigations during brain regeneration in response to injury in the adult zebrafish brain has revealed that dlx5a expression decreases shortly after lesion and that the dlx5a/6a bi-gene cluster, more specifically, dlx5a, is up regulated during the peak of regeneration response proposing a possible role for dlx during regeneration in adults. Studies of lineage tracing have shown the progeny of dlx1a/2a-expressing cells in adults are located within small clusters in different areas of the adult brain where they seem to become mature neurons. Our observations provide a better understanding about the role of dlx genes during adulthood, further contributing to the general knowledge of the molecular pathways involved in adult neurogenesis and regeneration in the zebrafish adult brain.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18510号 / 医博第3930号 / 新制||医||1006(附属図書館) / 31396 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Houses and lineages are both named, corporate units of social organization defined in part on the connection between people and place. They are distinguished from one another by the relative emphasis on biological descent in societies organized on the basis of corporate group membership. Over the past 15 years, researchers have debated whether ancient Maya social organization was characterized by house or lineage organization. Drawing on ethnographic, epigraphic, and archaeological data, researchers have concluded that the ancient Maya had some characteristics of house societies, but that biological descent was an important principle. One relevant line of evidence conspicuously absent from this debate is biological distance analysis as a means of identifying patterns of biological relatedness within sites. In this chapter we review intracemetery analyses from Mesoamerica, focusing on the Maya area, and discuss what, if any, insight such analyses of biological spatial organization might bring to bear on the house versus lineage debate. We suggest that the use of biological distance analysis will not resolve debates about the relevant importance of house and biological lineage in ancient Maya society, but increasing incorporation of intracemetery analyses within existing research programs will help identify those the circumstances in which biological kinship was emphasized in Maya society. As such, intracemetery biodistance adds an important and independent line of evidence that is currently underutilized in studies of ancient Maya social organization.

Yes / The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle. / This work was supported by EDCTP2 grant DRIA2014-326—DIAMA of the European Union, the Belgian General Directorate for Development Cooperation (PhD fellowship to J.C.S.N.), Grant ANR-16-CE35-0009 from Agence Nationale de la Recherche, the Swiss National Science Foundation (Grants 310030_188888, IZRJZ3_164171, IZLSZ3_170834 and CRSII5_177163), and the European Research Council (309540-EVODRTB). The views and opinions of authors expressed herein do not necessarily state or reflect those of EDCTP. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Embryonen von Höheren Krebsen (Malacostraca) zeigen ein stereotypes Zellteilungsmuster im Ektoderm des Rumpfes, im Verlaufe dessen paarige seitliche Reihen von Zellen und eine unpaare mittlere Reihe von Zellen gebildet werden. Das Muster der seitlichen Zellen ist von Dohle (1970, 1976) und Mitarbeitern geklärt worden. Die vorliegende Arbeit untersucht die cell lineage und Zelldifferenzierung der Mittellinienzellen im Thorax. Diese Zellen sind von besonderem Interesse, weil sie bei Insekten bereits intensiv erforscht wurden. (i) Die DiI Markierungen von Mittellinienzellen von Orchestia cavimana zeigen: Die Bildung der Mittellinie beginnt mit einer Zelle, die sich zweimal in Längsrichtung teilt. Die resultierenden vier Zellen werden mit a0, b0, c0 und d0 bezeichtet. Aus den Zellen a0, b0 und c0 gehen Paare von Gliazellen hervor. Die Tochterzellen von a0 und c0 umhüllen die Kommissuren. Die Zelle d0 ist ein medianer Neuroblast, aus dem mehrere Neurone hervorgehen, unter anderem ein unpaares Neuron im medianen Fasertrakt, interneurone und ein Motoneuron. (ii)BrdU Markierungen von Porcellio scaber zeigen: In den Ganglienanlagen liegt in der Mittellinie je eine Zelle, die größer ist als die benachbarten, schneller proliferiert und deshalb vermutlich ein medianer Neuroblast ist. (iii) Die Expression von engrailed setzt bei Orchestia und Porcellio ein in der Zelle a0 und wird in den zwei Tochterzellen fortgesetzt. Für Orchestia wird gezeigt, daß diese Expression zurückgeht und die Tochterzellen der Zelle d0 de novo mit einer Expression von engrailed beginnen.Aus den Ergebnissen kann abgeleitet werden, daß der gemeinsame Vorfahr von Insekten und Höheren Krebsen eine Mittellinie differenziert, die Vorläufer für Glia der Kommissuren und einen medianen Neuroblasten umfaßt und eine Expression von engrailed in den Tochterzellen des Neuroblasten zeigt. / Embryos of higher crustaceans (Malacostraca) show a highly stereotypic cell division pattern in the ectoderm of the trunk region while forming paired rows of lateral cells and an unpaired median row of midline cells. By using nuclear dyes, the pattern of the lateral cells has been determined by Dohle (1970, 1976) an co-workers. This study addresses the cell lineage and cell differentiation of the midline cells in the thorax. These kind of cells are of particular interest as they have been investigated extensively in insects. (i) The DiI labelling of midline cells in Orchestia cavimana reveals: Formation of the midline starts with a single midline cell that divides twice in longitudinal direction. The resulting four cells are termed a0, b0, c0, and d0. The cells a0, b0, and c0 give rise to pairs of glial cells. The progeny of a0 and c0 enwrap the commissures. The cell d0 is a median neuroblast that gives rise to several neurons, among them an unpaired neuron in the median fibre tract, interneurons and probably a single motoneuron. (ii) BrdU labelling in Porcellio scaber shows: there is a single larger and faster dividing cell in the midline in each segmental ganglion anlage that is a putative median neuroblast. (iii)The expression of engrailed starts in Orchestia and Porcellio the cell a0 and continues in the two daughter cells during segmentation. In Orchestia it can be shown that this expression ceases and progenies of the cell d0 start de novo with the expression of engrailed. From the results can be concluded that the common ancestor of insects and higher crustaceans differentiated an unpaired midline comprising precursors for glial cells enwrapping the commissures and a single median neuroblast whose derivatives express engrailed.

Dataflow systems have become the norm for developing data-intensive computing applications. These systems provide transparent scalability and fault tolerance. For fault tolerance, many dataflow-system adopt a snapshotting approach which persists the state of an operator once it has received a snapshot marker on all its input channels. This approach requires channels to be blocked for potentially prolonged durations until all other input channels have received their markers to guarantee that no events from the future make it into the operator’s present state snapshot. Alignment can for this reason have a severe performance impact. In particular, for black-box user-defined operators, the system has no knowledge about how events from different channels affect the operator’s state. Thus, the system must conservatively assume that all events affect the same state and align all channels. In this thesis, we argue that alignment between two channels is unnecessary if messages from those channels are not written to the same output channel. We propose a snapshotting approach for the fault tolerance and call it partial approach. The partial approach does not require alignment when an operator’s input channels are independent. Two input channels are independent if their events do not affect the same state and are never written to the same output channel. We propose the use of static code analysis to identify such dependencies. To enable this analysis, we translate operators into finite state machines that make the operator’s state explicit. As a proof of concept, we extend the implementation of Arc-Lang, an existing dataflow language, so that applications written in it transparently execute with fault tolerance. We evaluate our approach by comparing it to a baseline eager approach that always requires alignment between the input channels. The conducted experiments’ results show that the partial approach performs about 47 % better than the eager approach when the streaming sources are producing data at different velocities. / Dataflödessystem har blivit normen för utveckling av dataintensiva datorapplikationer. Dessa system erbjuder transparent skalbarhet och felhantering. För felhantering adopterar många dataflödessystem en snapshot-approach som sparar en operatörs tillstånd när den har fått en snapshot-markör på alla sina ingångskanaler. Denna metod kräver att kanalerna blockeras under möjligen förlängda tidsperioder tills alla andra ingångskanaler har fått sina markörer, vilket görs för att garantera att inga händelser från framtiden når operatörens nuvarande tillstånd. Synkronisering mellan kanaler kan därför ha en allvarlig prestandapåverkan. Särskilt för black-box användardefinierade operatörer där systemet inte har kunskap om hur händelser från olika kanaler påverkar operatörens tillstånd. Systemet måste därför konservativt anta att alla händelser påverkar samma tillstånd och synkronisera alla kanaler. I denna avhandling argumenterar vi för att synkroniseringen mellan två kanaler inte är nödvändig om meddelanden från de kanalerna inte skrivs till samma utgångskanal. Vi föreslår en snapshot-approach för felhantering och kallar den för partial-approach. Partial-approach kräver inte justering när en operatörs ingångskanaler är oberoende. Två ingångskanaler är oberoende om deras händelser inte påverkar samma tillstånd och aldrig skrivs till samma utgångskanal. Vi föreslår användning av statisk kodanalys för att identifiera sådana beroenden. För att möjliggöra denna analys översätter vi operatörer till finite state machines som gör operatörens tillstånd explicit. För att bevisa konceptet utökar vi implementeringen av Arc-Lang, vilket är en existerande dataflödesspråk, så att program skrivna i den transparent körs med felhantering. Vi utvärderar vår approach genom att jämföra den med en baseline eager-approach som alltid kräver justering mellan ingångskanalerna. Resultaten från de genomförda experimenten visar att partial-approach presterar cirka 47 % bättre än eager-approach när sourcestreams producerar data i otakt.

Das Herz eines Zebrafishs ist bemerkenswert, da es sich nach einer Verletzung vollständig regenerieren kann. Der Regenerationsprozess wird von Fibrose begleitet - der Bildung von überschüssigem Gewebe der extrazellulären Matrix (ECM). Anders als bei Säugetieren ist die Fibrose im Zebrafish nur transient. Viele Signalwege wurden identifiziert, die an der Herzregeneration beteiligt sind. Allerdings sind die Zelltypen, insbesondere Nicht-Kardiomyozyten, die für die Regulation des Regenerationsprozesses verantwortlich sind, weitgehend unbekannt. In dieser Arbeit haben wir systematisch alle Zelltypen des gesunden und des verletzten Zebrafischherzens mithilfe einer auf Mikrofluidik basierenden Hoch-Durchsatz- Einzelzell-RNA-Sequenzierung bestimmt. Wir fanden eine große Heterogenität von ECM-produzierenden Zellen, einschließlich einer Reihe neuer Fibroblasten, die nach einer Verletzung mit unterschiedlicher Dynamik auftreten. Wir konnten aktivierte Fibroblasten beschreiben und Fibroblasten-Subtypen mit einer pro-regenerativen Funktion identifizieren. Darüber hinaus haben wir eine Methode entwickelt, um die Transkriptomanalyse und die Rekonstruktion von Zell-Verwandtschaften auf Einzelzellebene zu kombinieren. Unter Verwendung der CRISPR-Cas9-Technologie führten wir zufällige Mutationen in bekannte und ubiquitär transkribierte DNA-Loci während der Embryonalentwicklung von Zebrafischen ein. Diese Mutationen dienten als zellspezifische, permanente und vererbbare “Barcodes”, die zu einem späteren Zeitpunkt erfasst werden konnten. Mit maßgeschneiderten Analysealgorithmen konnten wir dann Stammbäume der sequenzierten Einzelzellen erstellen. Mit dieser neuen Methode haben wir gezeigt, dass im sich regenerierenden Zebrafischherz ECM-produzierende Zellpopulationen entweder mit dem Epi- oder mit dem Endokardium verwandt sind. Zusätzlich entdeckten wir, dass vom Endokardium abgeleitete Zelltypen vom Wnt-Signalweg abhängig sind. / The zebrafish heart has the remarkable capacity to fully regenerate after injury. The regeneration process is accompanied by fibrosis - the formation of excess extracellular matrix (ECM) tissue, at the injury site. Unlike in mammals, the fibrosis of the zebrafish heart is only transient. While many pathways involved in heart regeneration have been identified, the cell types, especially non-myocytes, responsible for the regulation of the regenerative process have largely remained elusive. Here, we systematically determined all different cell types of both the healthy and cryo-injured zebrafish heart in its regeneration process using microfluidics based high-throughput single-cell RNA sequencing. We found a considerable heterogeneity of ECM producing cells, including a number of novel fibroblast cell types which appear with different dynamics after injury. We could describe activated fibroblasts that extensively switch on gene modules for ECM production and identify fibroblast sub- types with a pro-regenerative function. Furthermore, we developed a method that is capable of combining transcriptome analysis with lineage tracing on the single-cell level. Using CRISPR-Cas9 technology, we introduced random mutations into known and ubiquitously transcribed DNA loci during the zebrafish embryonic development. These mutations served as cell-unique, permanent, and heritable barcodes that could be captured at a later stage simultaneously with the transcriptome by high-throughput single-cell RNA sequencing. With custom tailored analysis algorithms, we were then able to build a developmental lineage tree of the sequenced single cells. Using this new method, we revealed that in the regenerating zebrafish heart, ECM contributing cell populations derive either from the epi- or the endocardium. Additionally, we discovered in a functional experiment that endocardial derived cell types are Wnt signaling dependent.

<p>The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly <italic>in vivo</italic>. The gonad is an ideal place to study this process because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. The early gonad is composed of four lineages: supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. Each lineage in the early gonad consists of bipotential progenitors capable of adopting either a male or female fate, which they do in a coordinated manner to form a functional testis or ovary. The supporting cell lineage is of particular interest because the decision of these cells to adopt the male or female fate dictates the fate of the gonad as a whole. </p><p><p>To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells of the four lineages from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. Our analysis identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We also determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. Multipotent progenitors that show lineage priming express markers of the various fates into which they can differentiate and subsequently silence genes associated with the fate not adopted as they differentiate. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cell progenitors were primed with a female bias. This yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. </p><p><p>We also used a genetic approach to investigate how individual factors contribute to the adoption of the male supporting cell fate. We previously demonstrated that <italic>Fgf9</italic> and <italic>Wnt4</italic> act as mutually antagonistic factors to promote male or female development of the bipotential mammalian gonad. <italic>Fgf9</italic> is necessary to maintain <italic>Sox9</italic> expression, which drives male development. However, whether FGF9 acted directly on <italic>Sox9</italic> or indirectly through repression of <italic>Wnt4</italic>, was unknown. <italic>Wnt4</italic> is a female-primed gene, and is therefore repressed during male development. To determine how <italic>Fgf9</italic> functioned, we generated double <italic>Fgf9/Wnt4</italic> and <italic>Fgfr2/Wnt4</italic> mutants. While single XY <italic>Fgf9</italic> and <italic>Fgfr2</italic> mutants showed partial or complete male-to-female sex reversal, loss of <italic>Wnt4</italic> in an <italic>Fgf9</italic> or <italic>Fgfr2</italic> mutant background rescued normal testis development. We also found that <italic>Wnt4</italic> and another female-associated gene (<italic>Rspo1</italic>) were derepressed in <italic>Fgf9</italic> mutants prior to the down-regulation of <italic>Sox9</italic>. Thus, the primary function of <italic>Fgf9</italic> is the repression of female genes, including <italic>Wnt4</italic>. We also tested the reciprocal possibility: that de-repression of <italic>Fgf9</italic> was responsible for the aspects of male development observed in XX <italic>Wnt4</italic> mutants. However, we show that loss of <italic>Fgf9</italic> in XX <italic>Wnt4<super>-/-</super></italic> gonads does not rescue the partial female-to-male sex reversal. </p><p><p>Based on the <italic>Fgf9/Wnt4</italic> double mutant studies, we propose a two part model of male sex determination in which both the activation of male genes and repression of female genes is required. Also, this work demonstrates that the repression of the female-primed gene <italic>Wnt4</italic> is required for male development, and <italic>Fgf9</italic> is one factor that leads to the repression of female-primed genes.</p> / Dissertation

The purposes of this research were to determine (1) the extent to which faculty are employed by the types of institutions from which they earned their doctorates in the United States, (2) the extent to which faculty have higher professional rank at employing institutions tat are the same type of institutions as those from which they earned their doctorates, (3) the extent to which female faculty are employed by the types of institutions from which they earned their doctorates, (4) the extent to which female faculty have higher professional rank at employing institutions that are the same type of institutions as those from which they received their doctorates, and (5) the extent of variability across academic disciplines in which faculty are employed by types of institutions from which they earned their doctorates. An exhaustive review of the literature on academic lineage was used to develop this research. All stratified random sample of 260 institutions from 2,873 colleges and universities was selected by Carnegie Foundation classification categories. Institutions were selected at random until the number of faculty members in each category corresponded to the estimated national distribution of faculty across Carnegie classification categories (n=3,940). The analyses revealed that the majority of faculty (74%) employed at all types of higher education institutions obtained their doctoral degrees fromresearch I institutions. Professional academic rank is an interaction between doctoral-granting institution and employing institution combined with gender. Male faculty are following the traditional "trickle down" theory of academic lineage while women faculty appear to be charting a different career path. Another unique finding was that there was not significant variability in the findings across academic disciplines. Academic success is a complex phenomenon that is not singularly explainable by academic lineage. As more women are entering into the academic ranks of higher education, changes in academic lineage are beginning to appear. The inflexibility and segmentation discussed in previous research is undergoing subtle but statistically noticeable modifications.