287 research outputs found
La Treviri di Oswald Mathias Ungers
No full textTrier, characterized by the presence of numerous UNESCO World Heritage monuments, was considered by Oswald Mathias Ungers as his adoptive city. Here, the author had the opportunity to realise three build-ings that engage with the city’s historical heritage. The project for the redevelopment of Konstantinplatz, in front of the Basilica (1981-83), the Museum of the Thermen am Forum (1988-1996), and the entrance to the Kaiserthermen (2003-2007) are works that engage with the city’s historical legacy, making them exemplary witnesses of the relationship between architectural forms, history, and place. The paper proposes an interpre-tation of the works that not only aims to identify their characteristics and their relationship with history, but also and above all seeks to investigate the system of relationships that links Ungers to Trier. For this investi-gation, the author’s projects embody the value of a paradigm that outlines a possible contemporary attitude towards history in architectural design
Step-wise differentiation of cerebral organoids towards hippocampal and choroid plexus progeny by sustained expression of early NSC stage-specific microRNA-20b
No full textPluripotent stem cells (PSCs) have the ability to undergo indefinite self-renewal while giving rise to the three germ layers. This remarkable capacity has turned PSCs to be a fundamental cell source for regenerative medicine research and applications. The derivation of induced pluripotent stem cells (iPSCs) over a decade ago has sparked a widespread enthusiasm and opportunity for personalized autologous cell-based therapies in a wide array of diseases. However, the development of optimized disease models and iPSC-derived products heavily relies on the generation of homogenous culture systems for the cell type of interest, which currently presents one of the major hindrances towards the use of PSC derivatives in therapeutic applications.
The cerebral cortex is an excellent example of a tissue with enormous heterogeneity that introduces immense challenges for in vitro disease models and therapeutic applications. Our lab mainly focuses on studying the development of the cerebral cortex, particularly investigating the development of cortical neural stem cells (NSCs). Our goal is to devise approaches for the differentiation of PSCs into founder NSC building blocks. We put particular emphasis on achieving high purity that will enable studying authentic cell fate decisions that are associated with regional patterning, self-renewal and differentiation processes that shape the cortex. A homogeneous early cortical population will also enable extracting a more reliable molecular identity of those cells, which can then be used for meaningful disease modeling and for devising sophisticated approaches to induce or maintain self-renewal of such populations in vitro.
However, recent research from our lab has shown that current methods to derive early cortical progenitors from PSCs are highly diverse and yield heterogeneous populations containing both cortical and non-cortical cell types. This results in heterogeneous founder NSC populations, limiting their use as a universal pure NSC source. To overcome this hurdle, our lab established a streamlined method known as Triple-i paradigm to derive homogenous starting cortical progenitors both in neural rosettes (2D) and in organoids (3D) platforms.
The main objectives of this thesis are to identify early signals that modulate temporal and regional fates within developing founder cortical NSC populations, focusing on small non-coding RNA (miRNAs), which are known to be key regulators of many developmental processes including stem cell proliferation and lineage specifications. These miRNAs guide the early development in a spatiotemporal manner by regulating the expression of multiple gene networks at the post-transcriptional level. They are abundantly expressed in the developing neural tube that eventually gives rise to different regions of central nervous system, including the cerebral cortex. However, it is unknown whether they have the capacity to specify a particular brain region in the cortex such as archicortex that consists of cortical hem, hippocampus and choroid plexus.
In this study, we have identified a miRNA important for the specification of archicortex and interrogated its role in cell fate specification using a battery of molecular and cellular studies. First, we employed in vitro human embryonic stem cell-based models (2D monolayer and 3D cerebral organoid) for the derivation of founder neural stem cell and their progression. We then identified a list of miRNAs that are specifically expressed in this early neural stem cell stage, among which we found hsa-miR-20b-5p to be one of the highly expressed microRNAs in early NSCs derived under both monolayer culture and cerebral organoids.
Second, by overexpressing hsa-miR-20b-5p in cerebral organoids, we provided evidence that this miRNA is involved in the stepwise specification of choroid plexus of archicortex, by caudalizing the early cortical NSC, due to modulation of WNT and BMP signalling components. In addition, cellular immunostainings displayed a characteristic presence of giant but thin single- layer vesicles positive for choroid plexus markers in miR-20b overexpressing organoids, as opposed to pseudostratified cortical neural rosettes in wildtype organoids. Furthermore, we showed that co-overexpression of miR-20b with of its main target CCND1, lead to the generation of cortical hem/hippocampus organoids.
Altogether, through an extensive miRNA-sequencing, single cell RNA-sequencing and cellular immunofluorescence studies, we revealed the expression of hsa-miR-20b-5p in early neural stem cells, its role in the specification of the archicortex lineages and its part as a cell fate modulator for converting cortical organoids towards choroid plexus structures.Pluripotente Stammzellen (PSCs) haben die Fähigkeit, sich auf unbestimmte Zeit selbst zu erneuern und dabei die drei Keimblätter hervorzubringen. Diese bemerkenswerte Fähigkeit hat PSCs zu einer grundlegenden Zellquelle für die Forschung und Anwendungen der regenerativen Medizin gemacht. Die Gewinnung von induzierten pluripotenten Stammzellen (iPSCs) vor über einem Jahrzehnt hat eine weit verbreitete Begeisterung geweckt und Möglichkeiten für personalisierte autologe zellbasierte Therapien bei einer Vielzahl von Krankheiten aufgedeckt. Die Entwicklung optimierter Krankheitsmodelle und von iPSC abgeleiteter Produkte hängt jedoch stark von der Erzeugung homogener Kultursysteme für den bestimmten Zelltyp ab, was derzeit eines der Haupthindernisse für den Einsatz von PSC-Derivaten in therapeutischen Anwendungen darstellt.
Die Großhirnrinde ist ein hervorragendes Beispiel für ein Gewebe mit enormer Heterogenität, das für In-vitro-Krankheitsmodelle und therapeutische Anwendungen immense Herausforderungen mit sich bringt. Unser Labor konzentriert sich hauptsächlich auf die Untersuchung der Entwicklung der Großhirnrinde, insbesondere der Entwicklung von kortikalen neuralen Stammzellen (NSCs). Unser Ziel ist es, Ansätze zur Differenzierung von PSCs in Gründer-NSC-Bausteine zu entwickeln. Wir legen besonderen Wert darauf, eine hohe Reinheit zu erreichen, die es ermöglicht, authentische Entscheidungen über das Zellschicksal zu untersuchen, die mit den regionalen Mustern, Selbsterneuerung und Differenzierungsprozessen verbunden sind, die den Kortex formen. Eine homogene frühe kortikale Population wird es auch ermöglichen, eine zuverlässigere molekulare Identität dieser Zellen zu bestimmen, die dann für aussagekräftige Krankheitsmodelle und für die Entwicklung fortgeschrittener Ansätze zur Induktion oder Aufrechterhaltung der Selbsterneuerung solcher Populationen in vitro verwendet werden kann.
Jüngste Forschungen aus unserem Labor haben jedoch gezeigt, dass aktuelle Methoden zur Ableitung früher kortikaler Vorläufer aus PSCs sehr vielfältig sind und heterogene Populationen ergeben, die sowohl kortikale als auch nicht-kortikale Zelltypen enthalten. Dies führt zu heterogenen Gründer-NSC-Populationen, die nur beschränkt als universelle reine NSC-Quelle dienen können. Um diese Hürde zu überwinden, hat unser Labor eine optimierte Methode namens Triple-i-Paradigma entwickelt, um homogene kortikale Ausgangsvorläufer sowohl in neuronalen Rosetten (2D) als auch in Organoiden (3D) abzuleiten.
Die Hauptziele dieser Arbeit sind die Identifizierung von frühen Signalen, die zeitliche und regionale Schicksale innerhalb sich entwickelnder kortikaler NSC-Populationen modulieren. Dabei liegt der Fokus auf kleinen nicht-kodierenden RNAs (miRNAs), die wichtige Regulatoren vieler Entwicklungsprozesse sind, wie Stammzellproliferation und Zelltyp-Spezifizierung. Diese miRNAs steuern die frühe Entwicklung in Raum und Zeit, indem sie die Expression mehrerer Gennetzwerke auf posttranskriptioneller Ebene regulieren. Sie werden reichlich im sich entwickelnden Neuralrohr exprimiert, aus dem schließlich verschiedene Regionen des zentralen Nervensystems, einschließlich der Großhirnrinde, hervorgehen. Es ist jedoch nicht bekannt, ob sie in der Lage sind, eine bestimmte Hirnregion im Kortex zu spezifizieren, wie z.B. den Archicortex, der aus kortikalem Saum, Hippocampus und Plexus choroideus besteht.
In dieser Studie haben wir eine miRNA identifiziert, die für die Spezifikation des Archicortex wichtig ist, und ihre Rolle bei der Spezifikation des Zellschicksals mit einer Reihe von molekularen und zellulären Studien untersucht. Zunächst verwendeten wir in vitro-Modelle auf der Basis von humanen embryonalen Stammzellen (2D-Monolayer und 3D-zerebrales Organoid) zur Ableitung von neuralen Gründerstammzellen und deren Progression. Wir identifizierten eine Reihe miRNAs, die spezifisch in diesem frühen neuralen Stammzellstadium exprimiert werden. Unter anderem identifizierten wir hsa-miR-20b-5p als eine der hochexprimierten miRNAs in frühen NSCs, die entweder aus Monolayer-Kulturen oder zerebralen Organoiden stammen.
Zweitens lieferten wir durch die Überexpression von hsa-miR-20b-5p in zerebralen Organoiden den Nachweis, dass diese miRNA an der schrittweisen Spezifizierung des Plexus choroideus des Archicortex beteiligt ist, indem sie die frühen kortikalen NSC durch die Modulation von WNT- und BMP-Signalkomponenten kaudalisiert. Darüber hinaus zeigten zelluläre Immunfärbungen ein charakteristisches Vorhandensein von riesigen, aber dünnen einschichtigen Vesikeln, die – im Gegensatz zu pseudostratifizierten kortikalen neuralen Rosetten in Wildtyp-Organoiden – positiv für Plexus choroideus-Marker in miR-20b-überexprimierenden Organoiden waren. Darüber hinaus haben wir gezeigt, dass die Ko-Überexpression von miR-20b mit seinem Hauptziel CCND1 zur Bildung von kortikalen Hem-/Hippocampus-Organoiden führt.
Insgesamt haben wir durch umfangreiche miRNA-Sequenzierung, Einzelzell-RNA-Sequenzierung und zelluläre Immunfluoreszenzstudien die Expression von hsa-miR-20b-5p in frühen neuralen Stammzellen, seine Rolle bei der Spezifizierung der Archicortex-Linien und seine Rolle als Zellschicksalsmodulator zur Umwandlung von kortikalen Organoiden in Strukturen des Plexus choroideus aufgedeckt
Pattern formation as a neural wiring strategy during development of the Drosophila visual system
No full textNatural patterns are everywhere around us and can greatly support the developmental progress
of complex organs. The eye of a fruit fly (Drosophila melanogaster ) is such a perfectly patterned
organ. The 800 small eyes, called ommatidia, contain six motion-vision photoreceptors
(PRs) each. Via axons, PRs transfer environmental information to the first optic ganglion
in the brain; the lamina. The six photoreceptors from neighboring ommatidia that see one
point in the environment are connected to one post-synaptic target in the lamina, to form a
retinotopic map of the surroundings. Neural superposition sorting systematically sorts PR
axons during a series of distinct developmental steps. Initially, PR axons extend in bundles
from the ommatidia to the lamina plexus (LP) and form a scaffold, or pre-pattern. Scaffold
formation is an essential step in creating a functional visual map, yet, how it establishes
is largely unknown. From this scaffold, all six PRs from one ommatidium extend their axonal
growth cones in different angles during a lateral extension phase towards their different
post-synaptic targets. How PR growth cones determine this sub type specific angle is largely
unknown. Finally, extended growth cones adhere and connect with Lamina Neurons (LNs)
at the target location to form synapses and a functional visual circuit. Whether LNs are
required for axon extension and/or adherence at the target location is unknown.
The aim of this thesis is to describe underlying mechanisms involved in pattern formation and
neural superposition sorting during development of the visual map, as well as to investigate
the role of LNs during superposition sorting. The results presented in this thesis show that
PR growth cones can form a scaffold in absence of LNs. Furthermore, the extension of
photoreceptors is independent of the presence of LNs the presentation of adhesion proteins
on their cell membrane. Strikingly, it is possible for extending growth cones to find their
target correctly and, in rare cases, form photoreceptor clusters where they would normally
form a cartridge with LNs. This suggests that photoreceptor and LN dynamics are largely
independent and that neural superposition sorting is remarkably robust. I conclude that LNs
are required in late development of the visual map for robust circuit formation, but do not
contribute to PR sorting. This is a novel finding for the wiring of the Drosophila visual
system.Widerkehrende Strukturen und Muster sind überall in der Natur zu finden und können
während der Entwicklung einen wichtigen Einfluss spielen. Ein perfekt strukturiertes Organ
ist das Auge der Fruchtfliege, Drosophila melanogaster. Es besteht aus etwa 800 repetitiven
Untereinheiten, den so genannten Ommatidien, welche jeweils sechs bewegungssensitive
Photorezeptoren (PRs) besitzen. Diese leiten Umgebungsinformationen über Axone zum ersten
optischen Ganglion, der Lamina, weiter. Die sechs Photorezeptoren von benachbarten
Ommatidien, die denselben Raumpunkt wahrnehmen, sind mit dem selben postsynaptischen
Zellen in der Lamina verbunden um eine retinotope Karte der Umgebung zu arrangieren. Die
Axone von Photorezeptoren werden durch neuronale Superposition-Sortierung in einer Reihe
von Entwicklungsschritten geordnet. Zunächst wachsen die Axone der Photorezeptoren in
Bündeln von den Ommatidien zum Lamina Plexus (LP) und formen dabei ein eindeutiges
Grundgerüst oder ‘pre-pattern‘. Die Bildung dieses Grundgerüsts stellt einen essenziellen
Schritt in der Formierung einer funktionellen visuellen Karte dar. Wie genau das Gerüst
etabliert wird, ist jedoch größtenteils unbekannt. Nach Bildung des Grundgerüsts wachsen
alle sechs PRs eines Ommatidiums in einer lateralen Wachstums-Phase. In dieser wachsen
die Wachstumskegel der PRs in unterschiedlichen, subtyp-spezifischen Winkeln zu deren
jeweiligen neuronalen Partnern. Wie hierbei die Wachstumskegel der Photorezeptoren ihre
spezifischen Winkel bestimmen ist weitestgehend unbekannt. Letztlich verbinden sich Wachstumskegel
der Photorezeptoren mit Lamina Neuronen (LNs) an ihren Zielpositionen, formen
Synapsen und so einen funktionellen visuellen Schaltplan. Ob LNs für axonales Wachstum
und/oder Adhäsion von Wachstumskegeln an ihrer Zielposition nötig sind, ist nicht bekannt.
Das Ziel dieser Studie ist die grundlegenden Mechanismen, die in der Entwicklung einer funktionellen
visuellen Karte notwendig sind, zu untersuchen. Hierbei werden die Mechanismen,
die für die Grundgerüstformierung und neuronale Superpositions-Sortierung notwendig sind,
sowie die Rolle von LNs während dieser, analysiert. Die hier präsentierten Ergebnisse zeigen,
dass Wachstumskegel von Photorezeptoren sogar in Abwesenheit von Lamina Neuronen ein
Grundgerüst bilden können. Zudem ist das laterale Wachstum der Photorezeptoren unabhängig
von der Laminar Neuronen Präsenz oder deren Membran-Adhäsionsproteinen. Überraschenderweise
ist es ausdehnenden Wachstumskegeln möglich ihr Ziel korrekt zu finden und
in seltenen Fällen sogar Photorezeptor-Cluster zu bilden, wo normalerweise “cartridges” mit
LNs geformt würden. Diese Ergebnisse legen nahe, dass Photorezeptoren und LN-Dynamiken
größtenteils voneinander unabhängig sind und neuronale Superpositions-Sortierung erstaunlich
robust ist. Ich schlussfolgere, dass LNs während der späteren Entwicklung einer visuellen
Karte für robuste Schaltkreisformierung notwendig sind, jedoch keinen Einfluss auf die Photorezeptor-
Sortierung haben. Diese Erkenntnisse stellen neue Forschungsergebnisse bezüglich der neuronalen
Verschaltung des visuellen Systems von Drosophila melanogaster dar.Natuurlijke patronen zijn wijdverspreid en kunnen bijdragen aan de ontwikkeling van complexe
organen. Het visuele systeem van een fruitvliegje (Drosophila melanogaster) is zo’n
orgaan dat ontwikkelt met gebruik van patronen. Eén oog bevat ongeveer 800 individuele
minioogjes, zogenaamde ommatidia, die elk zes bewegingssensitieve fotoreceptoren bevatten.
Via axonen geven zij informatie door aan de eerste hersenlaag in het visueel systeem; de
lamina. Zes fotoreceptoren van naburige ommatidia, die allen één punt in de ruimte zien,
worden verbonen met één cluster van post-synaptische partners en vormen een visuele kaart.
Het sorteren van axonen via “neuron superpositie sorteren”, doorloopt verschillende ontwikkelingsstadia.
Eerst sturen fotoreceptoren hun axonen in bundels van het oog naar de lamina
plexus (LP) waar ze een pre-patroon vormen. Deze stap is essentieel voor de vorming van
de visuele kaart, maar hoe het wordt gevormd is grotendeels onbekend. Vanaf het gevormde
pre-patroon groeien zes fotoreceptoren uit één ommatidium in zes verschillende richtingen
naar hun verschillende post-synaptische targets. Hoe ze deze groeirichting bepalen is onbekend.
Tenslotte komen axon extensies samen bij een Lamina Neuron (LN) target en vormen
ze synapsen en daarmee een werkend visueel circuit. Of LNs een rol spelen tijdens extensie,
of voor de vorming van connecties, is onbekend.
Het doel van deze thesis is het beschrijven van onderliggende mechanismen in neuron superpositie
sorteren tijdens de ontwikkeling van het visuele system van Drosophila en te onderzoeken
of LNs nodig zijn voor dit proces. De resultaten laten zien dat photoreceptoren een
pre-patroon kunnen vormen in afwezigheid van LNs en dat hun extensies onafhankelijk zijn
van de aanwezigheid van LNs of van moleculen op het celmembraan. Ook is het voor fotoreceptoren
mogelijk om vanuit correcte extensies in sommige gevallen een cluster te vormen met
andere fotoreceptoren ipv. met LNs. Dit suggereert dat de dynamieken van fotoreceptoren en
LNs voor een groot deel onafhankelijk van elkaar zijn. LNs lijken vereist te zijn gedurende de
laatste fase van sorteren voor de robuste vorming van een functioneel visueel veld, maar niet
voor het eigenlijke sorteren van fotoreceptoren. Dit is een nieuwe vinding in de ontrafeling
van het visuele systeem van Drosophila melanogaster
Funktionelle Rolle Medial Septaler Projektionen zum Parasubiculum
No full textOscillations are a hallmark of brain activity and can be generated by local synchronisation mechanisms. They have been implicated in the communication between brain areas. An important type of oscillations are θ oscillations (4-12 Hz), which are associated with different behaviours, such as movements and navigation, but they also play a crucial role in memory formation and retrieval. One of the major θ rhythm generators in the brain is the medial septum (MS), which with its different types of projecting neurons, innervates many cortical areas and synchronises their activity. I investigated two major projection types of the MS: GABAergic (γ-aminobutyric acid – GABA) and cholinergic (acetylcholine – ACh) projections. Both projections are known to target the medial entorhinal cortex (MEC) and hippocampus. Parvalbumin positive (PV+) projections of the MS, which are GABAergic, are known to synchronise cortical networks via disinhibition often by inhibiting interneurons. In contrast, cholinergic projections of the MS project to a wide range of cell types in the MEC and hippocampus and can have substantially different effects on the target cell (e.g. activation or inhibition). Thus, their function on a network can range from increasing activity through depolarising excitatory cells, to more inhibition of the network by activating interneurons, or even modulating synaptic integration. Previous studies have focussed on identifying projections to the hippocampus and the MEC but did not consider the parasubiculum (PaS), a major input of the MEC. In this study, we electrophysiologically characterised cells in the PaS and demonstrated layer I interneurons to be distinctly different from putative layer II interneurons. The PaS, with its strong θ rhythmic firing cells, was shown to have the highest density of MS PV+ fibres in the parahippocampal formation, suggesting that it is an important target of MS projections and yet MS inputs to the PaS are unknown. Using channelrhodopsin (ChR2), a light sensitive ion channel, expressed in the MS of PV-Cre and ChAT-Cre (choline acetyltransferase) mice in-vivo, I identified GABAergic and cholinergic MS connections to the PaS in-vitro and demonstrated cell type specific projection patterns. I found that PV+ MS projections mainly inhibit interneurons in the PaS, including layer I interneurons, representing a novel cortical target of PV+ MS cells. On the other hand, cholinergic projections depolarise layer I interneurons and have multiple effects on deeper cells of the PaS, leading to a depolarisation or hyperpolarisation. To investigate a potential role of GABAergic projections in θ generation, I recorded local field potentials (LFP) in awake head-fixed mice and entrained oscillations in the PaS by stimulating with light in the MS. In contrast, local stimulation of fibres in the PaS could not entrain oscillation, suggesting that increased activity in the PaS might be required for MS PV+ cells to entrain θ. Taken together, stimulation of PV+ cells in the MS is sufficient to drive oscillations in the PaS, likely via disinhibition in line with other areas as the MEC and hippocampus. However, novel targets in layer I could be involved via cholinergic activation and GABAergic entrainment. Whether cholinergic activation by itself can entrain θ remains to be further investigated.Oszillationen sind ein Kennzeichen von Gehirnaktivität und können durch lokale Synchronisationsmechanismen generiert werden. Sie spielen eine wichtige Rolle bei der Kommunikation zwischen Gehirnarealen. Ein wichtiger Typ von Oszillationen sind θ Oszillationen (4 − 12 Hz), welche mit verschiedenen Verhalten wie Bewegung und Navigation assoziiert sind und eine wichtige Rolle in der Gedächtnisbildung und -abrufung spielen. Einer der wichtigen θ Generatoren im Gehirn ist das Mediale Septum (MS), welches mit seinen verschiedenen projizierenden Neuronen viele kortikale Regionen innerviert. Ich habe zwei Typen von Projektionen des MS untersucht: GABAerge (γ-Aminobuttersäure – GABA) und cholinerge (Acetylcholin – ACh) Projektionen. Beide Typen projizieren zum Medialen Entohinalen Kortex (MEC) und zum Hippocampus. Parvalbumin positive (PV+) Projektionen des MS können kortikale Netzwerke via Disinhibition, durch inhibieren von Interneuronen, synchronisieren. Im Gegensatz dazu projizieren cholinerge Projektionen des MS zu verschiedensten Zelltypen des MEC und des Hippocampus und können unterschiedliche weitreichende Effekte auf Zellen haben (z.B. Aktivierung und Inhibierung). Folglich können die Konsequenzen von Aktivierung des Netzwerkes via Depolarisation von exzitatorischen Zellen, über Inhibierung des Netzwerkes via Aktivierung von Interneuronen bis hin zur Modulation von synaptischer Integration reichen. In der Vergangenheit haben Studien sich auf die Identifizierung von Projektionen zum Hippocampus und MECs fokussiert, jedoch nicht zum Parasubiculum (PaS), eines der bedeutendsten Eingänge des MEC. In dieser Studie haben wir elektrophysiologisch Zellen im PaS charakterisiert und konnten herausstellen, dass Schicht I Zellen sich von anderen vermeintlichen Interneuronen in Schicht II unterscheiden. Das PaS, mit seinen im θ Rhythmus feuernden Zellen, hat die höchste Dichte von MS PV+ Fasern im parahippocampalen Netzwerk, was es als besonderes Ziel für MS Projektionen herausstellt. Dennoch sind Projektionen vom MS zum PaS nicht untersucht worden. Mit Hilfe von Channelrhodopsin (ChR2), einem lichtsensitivem Ionenkanal, welcher im MS von PV-Cre und ChAT-Cre Mäusen exprimiert wurde, konnte ich GABAerge und cholinerge MS Verbindungen zum PaS in-vitro detektieren und Zelltyp-speziefische Projektionen identifizieren. Ich konnte herausstellen, dass PV+ MS Projektionen hauptsächlich Interneurone im PaS inhibieren. Insbesondere Schicht I Interneurone stellen ein neues kortikales Ziel von PV+ MS Zellen dar. Im Gegensatz dazu werden Schicht I Interneurone des PaS durch cholinerge MS Projektionen depolarisiert wohingegen Zellen in tieferen Schichten depolarisiert oder hyperpolarisiert werden können. Um zu zeigen, dass man mit GABAergen Projektionen θ generieren kann, nahm ich das lokale Feldpotential (LFP) in Kopffixierten Mäusen auf und fand, dass man Oszillationen mit MS-Stimulation gleichschalten kann, jedoch eine Stimulation der Fasern im PaS nicht ausreichend ist. Das weist darauf hin, dass eine erhöhte PaS-Aktivität notwendig ist, um θ Oszillationen im PaS zu generieren. Zusammenfassend zeigt sich, dass eine Stimulation der PV+ Zellen im MS ausreichend ist, um im PaS Oszillationen zu generieren. Disinhibierung im PaS ist, ähnlich wie auch im MEC und Hippocampus, ein wahrscheinlicher Mechanismus. Weiterhin könnten jedoch neue Ziele von cholinergen und GABAergen Fasern in Schicht I bei der θ Generierung involviert sein. Ob θ Oszillationen durch cholinerge Projektionen gleichgeschaltet werden kann muss jedoch noch durch weitere Studien gezeigt werden
Rab GTPases as mediators of neuronal robustness in Drosophila
No full textOne common feature of all eukaryotic cells is the presence of various specialized organelles,
separated by membranes, which necessitates a coordinated trafficking of materials between
these subcellular membrane-bound compartments. Especially neurons, with their long
lifespan, polarized and often complex morphology, as well as their specialized functions,
have particular requirements for membrane trafficking. Not surprisingly, membrane
trafficking is involved in all aspects of neuronal development, function, and long-term
maintenance. The evolutionary conserved family of small Rab GTPases functions as key
regulator of coordinated vesicular trafficking in the endomembrane system. Expression
profiling efforts revealed that in Drosophila half of all Rab GTPases are enriched in or
specific to the nervous system, in humans it is one-third. However, the exact functions of the
majority of nervous system-enriched Rab proteins are still unknown. Thus, studying the
individual roles of those Rab GTPases more closely provides a great opportunity to gain
more insight into the membrane trafficking networks in neurons. Ultimately, this will surely
contribute to the understanding of what keeps neurons and in particular synapses healthy and functional over extended periods of time. In the nervous system, Rab GTPases and the
membrane trafficking events these mediate have been widely associated with many
neurodegenerative diseases. However, the established relations are often more correlatively
than causatively linked, as discussed in Manuscript 1.
Regarding the importance of an intact intracellular trafficking machinery for the
development as well as neuronal function and maintenance, I primarily focused on the
systematic functional analysis of nervous system-enriched Rab GTPases in Drosophila
during my doctoral work. Previously, no systematic rab mutant characterization in any
multicellular organism had been performed. The analysis, presented in Manuscript 2,
revealed that the homozygous mutants of all nervous system-enriched Rab GTPases, raised
under laboratory conditions, are viable and fertile, whereas, null mutants of ubiquitously
expressed Rabs are all lethal under homozygosity. Thus, suggesting that Rab proteins, with
high expression in the nervous system, serve more modulatory, specialized functions which
are not essential for the survival of the organism. Further, we could show that all viable rab
mutants differentially affect the development or neuronal function under variable, more
challenging environmental conditions, such as temperature and light. This highlights the
evolved robustness of developmental processes and nervous system function towards
varying conditions. Additionally, during the in-depth functional analysis of nervous system-enriched Rab26, we revealed a stimulus-dependent role in the trafficking of the single
acetylcholine receptor subunit Dα4 at cholinergic synapses of outer photoreceptors.
However, we could not verify a role for Rab26 in the autophagic turnover of synaptic
vesicles in neurons.
Additional assays, such as the RUSH system, can be useful to support functional
analyses. While this acute, synchronous release system could be established for Rabs in
developing photoreceptors and salivary glands, wing imaginal discs proofed to be more
sensitive and no working conditions could be established. Using the RUSH assay, different
release dynamics with ‘fast’ as well as ‘slow’ releasing Rab GTPases could be identified as
shown in Manuscript 3. Further, two nervous system-enriched Rab proteins, namely Rab23
and Rab26, show a clear re-localization from the cell body to the axon terminals, which is
in agreement with their predominant synaptic neuropil localization revealed by expression
profiling.
In conclusion, the findings made during my doctoral work will contribute to a better
understanding of the functional requirements of neurons regarding Rab-mediated membrane trafficking. The complete rab null mutant collection as well as the RUSH and LAMA transgenic toolbox provide a strong basis for further investigations of individual Rab
functions during development and homeostasis
Synaptic circuits for visual navigation in the fly brain
No full textElucidating neural circuit mechanisms that drive complex behavior and cognition requires a multidisciplinary approach combining neuroanatomy with neurophysiology and behavioral science. This integration of different disciplines works particularly well in the model organism Drosophila melanogaster, as it benefits from the rich arsenal of molecular and genetic tools that have been developed over the last century. A focus of research are the circuit elements that drive local computation in the optic lobes. However, considerably less is known about the pathways that leave the optic lobes and converge in the central complex, which is considered the navigation center in Drosophila.
In this thesis I present the complete synaptic pathway connecting the optic lobes to the central complex. Starting from the terminals of inner photoreceptors in the medulla all the way to the dendrites of EPG neurons, the Drosophila counterpart of head-direction cells in mammals. Initially, by studying light microscopically the distribution of putative presynaptic sites of inner photoreceptors I turned to connectomic reconstructions as the first complete EM volume of an adult female brain (full adult fly brain FAFB) became available. First, I manually reconstructed a set of inner photoreceptors and all pre- and postsynaptic partners as well as annotated all synapses among them. In a second step, recent advances in automatic image segmentation of the FAFB volume allowed me to proofread the complete set of projection neurons connecting the photoreceptors to the central complex across multiple neurons and neuropils.
I found that the neurons along the pathway fall into different subchannels that most likely carry information from qualitatively different visual modalities into the central complex. By mapping putative visual fields to the input neurons of EPG neurons I discovered a diverse mapping of visual space in the central complex. As a population the entirety of the fly’s visual field is covered, however the visual fields of individual neurons can differ drastically. Some neurons possess diffuse visual fields while others have discrete spot- or vertical bar-like visual fields supporting the idea of combinatorial integration of different visual modalities in the navigation center of Drosophila.
These findings form the basis for analyzing the integration of visual modalities within the central complex, which could not only accelerate research in this area, but also underscore the crucial role of vision in spatial navigation
Identifizierung von Protogenin als neuartiger Oberflächenmarker für frühe kortikale neurale Stammzellen
No full textDuring mammalian corticogenesis, a wide diversity of neural stem cells (NSCs) orchestrate the development and organization of the cortex. The pool of NSCs initially expands through proliferative symmetric divisions, and sequentially starts dividing asymmetrically to give rise to the diverse cell types residing within the cortical layers. Throughout this process, cortical NSCs undergo extensive modifications in their transcriptomic profile and chromatin landscape contributing to the formation of heterogeneous progenitor populations. Although much progress has been made towards understanding cell-fate specification during human corticogenesis the mechanisms responsible for the temporal lineage specification of NSCs remain largely unknown. Understanding the variability of these distinct NSC populations is key for developing an in vitro system that allows for the homogeneous and unlimited culture of the desired NSC type which is crucial for cell replacement-based therapies. Hence, one of the main aims in our lab is to identify and discern these distinct NSC types which sequentially appear during cortical development with the objective to better understand these cell stages and, eventually, being able to manipulate them in vitro.
In order to address this question, my project is focused on developing a strategy to isolate the early cortical NSC population for its characterization and potential manipulation. The main approach is to identify a cell surface marker to enable the isolation of these cells from our in vitro culture by fluorescence-activated cell sorting. By profiling our hiPSC-derived cortical progenitors at different stages by means of single-cell RNA sequencing, we selected potential candidate markers that were validated using immunofluorescence and sequencing methods.
In this study, we identify Protogenin (PRTG) as a novel surface marker for early human cortical NSCs that can be used to isolate this population in vitro. We provide evidence that early expression of the novel marker correlates with cortical lineage specification. Furthermore, by sorting for such marker at early stages of neural induction we can prospectively isolate three distinct cortical subpopulations, resulting in highly pure subtype-specific NSC cultures.
These findings illustrate the utility of PRTG cell-surface sorting for enriching early cortical NSCs in culture and, thus, aiding to develop a more robust and homogenous differentiation protocol. Ultimately, such knowledge should facilitate the generation of highly pure stage- and region-specific NSC populations from patient-derived samples which would provide a reliable source for cell replacement and regenerative therapies
Modality-specific circuits for skylight orientation in the fly visual system
No full textThe fly eye contains different subtypes of unit eyes (ommatidia) with molecularly and morphologically specialized photoreceptors for comparing either between different wavelengths (color vision) or between different angles of the linearly polarized skylight (polarization vision). However, microcircuit differences between those parts of the columnar medulla neuropil computing color versus polarization remain largely unknown. There is virtually nothing known about the circuit elements immediately downstream of polarization-sensitive photoreceptors in the ‘dorsal rim area’ (DRA). In this work, I described the cellular and synaptic architecture of medulla columns that receive skylight polarization input from DRA photoreceptors. I showed that only in the DRA region, R7 and R8 photoreceptors resemble each other by targeting their axons to the same medulla layer. However, within this layer DRA R7 and R8 connect to morphologically distinct Dm target cells (called Dm-DRA1 and Dm-DRA2, respectively). Both Dm-DRA cell types are modality-specific by avoiding contact with color-sensitive photoreceptors. Using the genetic toolbox of Drosophila such as activity-dependent GFP-reconstitution across synaptic partners (GRASP) and the genetically inducible trans-synaptic tracer ‘trans-Tango’, I confirmed that Dm-DRA1 and Dm-DRA2 are the specific post-synaptic targets of DRA.R7 or DRA.R8, respectively. Neither Dm-DRAs overlap with the main synaptic targets of color-sensitive R7 cells (called Dm8 cells), revealing for the first time that skylight polarization is processed by separate modality-specific circuits in the early visual system. These modality-specific differences are not limited only Dm-DRA cells. I described modality-specific cellular and synaptic specializations in other optic lobe cell types in the DRA region of the medulla: the dendritic arbors of certain cell types (neuromodulatory cells and visual projection neurons) specifically avoid the DRA region. Furthermore, Transmedullary (Tm) cells that are post-synaptic to color-sensitive photoreceptors showed modality-specific differences in connectivity or were absent from the DRA. Finally, I contributed a study describing the cellular organization of the ‘anterior visual pathway’ that carries skylight information from the eye to the central brain. In this study, I showed that an optic glomerulus called the anterior optic tubercle (AOTU) receives direct information via different classes of medulla-to-tubercle (MeTu) neurons, terminating in different subdomains of the AOTU. Finally, we hypothesize that different classes of MeTu cells carry different types of skylight information to the central brain via parallel pathways
The development and maintenance of synaptic specificity in the fly visual system
No full textThe daunting complexity of the brain emerges from the large number of neurons it contains and their compartmentalized synaptic interactions at axon terminals and dendrites. Generation of functional neuronal networks requires robust, unambiguous developmental processes to ensure synapse-specific neuronal partner choice and subsequent maintenance mechanisms to keep neurons and particularly synapses healthy and functional over a long time. Defects in wiring and maintenance mechanisms are associated with neuropsychiatric and neurodegenerative disorders.
Having regard to the importance of protein quality control mechanism both during development and function of the nervous system, in this doctoral work, I investigated possible local roles of lysosomal degradation pathways including ubiquitous and neuron-specific endolysosomal degradation and autophagy at axon terminals. Using live imaging in intact Drosophila brains and novel acidification-sensing degradation probes, first, we reported a direct live observation of local protein degradation at axon terminals in large, acidified compartments. These acidic, degradative endocytic compartments undergo continuous flux of fusion and fission of smaller compartments that is reflected by their molecular composition at a given time. Therefore, we named these compartments ‘local hubs’ as they behave as sort-and-degrade stations for local protein turnover at axon terminals. Secondly, we reported differential, cargo-specific sorting of plasma and synaptic vesicle membrane proteins into distinct hubs via two molecularly distinct pathways. Although plasma membrane protein sorting and degradation depends on ubiquitous Rab GTPase, Rab7, synaptic vesicle membrane protein sorting and degradation is Rab7-independent and operated by previously characterized synaptic vesicle proteins V100 and n-Syb. V100, as a subunit of a proton pump, particularly affects acidification of synaptic vesicles hubs, whereas n-Syb is required for the delivery of golgi-derived microvesicles containing acidic hydrolases into synaptic vesicle hubs. Interestingly, autophagy does not overlap with any of these local degradation pathways. Following their formation at axon terminals, they enter in axons without engaging in any fusion/fission events, hence morphologically and dynamically distinct from local hub compartments.
Despite several reports on formation of autophagosomes at axon terminals, potential physiological roles it may exert still remain largely unknown, especially during neural circuit assembly. Live imaging of developing Drosophila photoreceptor axon terminals with autophagosome markers revealed their formation at the tip of synaptogenic filopodia followed by destabilization of these structures. Consistent with this observation, loss of function analyses of autophagy in developing Drosophila photoreceptors revealed increased stability of synaptogenic filopodia and subsequent increase in synapse numbers. More importantly, autophagy-deficient neurons connect to several aberrant synaptic partners causing neuronal miswiring. Finally, adult flies with miswired brains due to loss of autophagy show distinct and predictable behavioral phenotypes such as prolonged, repetitive visual attention to objects. Interestingly, development at colder temperatures exerts similar effect on filopodial stability as in loss of autophagy where axonal filopodia slow down and stabilize more synaptogenic filopodia. This effect on filopodia stability further leads to increased synapse formation and recruitment of aberrant synaptic partners changing brain wiring pattern. Collectively, these results demonstrate that filopodia kinetics play an important role to restrict or facilitate synaptic partnerships between neurons in close proximity during brain wiring.
In conclusion, my doctoral work contributed to better understanding of local functions of protein degradation machineries and developmental temperature during brain wiring and maintenance. Unexpected roles of such cellular mechanisms and external factors in establishing proper neuronal circuits point to the fact that combinatorial action of several factors in time and space during brain development contribute to the final outcome, a functional brain
Physiological Characterization of the Neural Circuits Mediating Polarization Vision in Drosophila melanogaster
No full textThe ability of animals to navigate their environment, locate food sources, and find mating partners hinges on their capacity to process and integrate information provided by the visual system. At the heart of this complex task lies the intricate web of thousands of individual neurons, each playing a crucial role in the orchestration of sensory information. Understanding the rules and mechanisms guiding this neural computation is a profound pursuit central to the fields of neuroscience and ethology.
My doctoral research advances our understanding of navigation by delving into neural circuitry and information processing mechanisms, particularly emphasizing polarized skylight detection in insects. Focused on Drosophila melanogaster, a powerful model organism, the study explores the intricate visual system comprised of optically isolated unit eyes called ommatidia. Approximately 800 of these units populate the adult retina, facilitating precise spatial sampling. Within the Drosophila retina, different ommatidial subtypes house specialized inner photoreceptors for color perception in the central retina or the detection of skylight polarization in the dorsal rim area (DRA). Visual information undergoes complex processing in the optic lobes before being relayed to higher brain structures, such as the anterior optic tubercle (AOTU) within the visual glomeruli. My thesis contributes to understanding the less well-known ventral polarization vision, exploring local circuitries in the optic lobes, and shedding light on the less-understood aspect of this visual modality. The literature study identifies functionally specialized non-DRA detectors by examining non-celestial polarization vision across diverse insect species, including dragonflies, butterflies, beetles, bugs, and flies. Although the ventral polarization vision in Drosophila melanogaster presents a fascinating modality, the unknown location of the specific circuitry stays hidden. Therefore, I turned my attention to the better-known specific circuitry of skylight polarization vision in the DRA and unveiled modality-specific connectivities of local medulla neurons in the DRA. Including Mt11-like medulla tangential cells that avoid the DRA region. Despite gathering comprehensive information from the entire medulla, these cells lack inputs related to polarized light from the DRA, indicating separate processing of distinct visual attributes within the central brain. Finally, I characterized the anatomical and physiological properties of MeTu-types, modality-specific to the DRA, called MeTu-DRA1 and MeTu-DRA2. Using the genetic toolkit of Drosophila melanogaster, the study showed for the first time that both populations are modality-specific postsynaptic to DRA.R7 photoreceptors only, project to the same subunit of the AOTU and show differences in their morphology as well as connectivity. Although the morphology showed significant differences, single-cell clones revealed a topographic projection of both MeTu-DRA sub-populations from the medulla to the AOTU. Based on these findings, we hypothesized that the anatomical and connectivity differences might result in different physiological response patterns of MeTu-DRA1 and MeTu-DRA2. In order to test this theory, I implemented calcium imaging (using GCaMP) under a 2-photon microscope. I recorded the physiological response properties of Dm-DRA1 (in the medulla) and MeTu-DRA1 and MeTu-DRA2 responses in the AOTU. Interestingly, I could show for the first time that MeTu-DRA1 shows a detailed representation of different ’Angle of Polarization’ (AoP) in the AOTU, and MeTu-DRA2 responses, however, split the AOTU in a dorsal or ventral half pattern. With EM reconstruction, we could identify a more detailed circuitry of the MeTu-DRAs and a new DRA-specific interhemispheric cell type called MeMe-DRAs. Additionally, I could show that only MeTu-DRA2 responds to unpolarized UV flashes presented contralaterally, which is most likely mediated by MeMe-DRAs and presents an early binocular integration of polarized skylight information.
In conclusion, the discoveries made during my doctoral research significantly contribute to our comprehension of the functional characteristics and circuitry of MeTu-DRA neurons in . This comprehensive understanding enhances our knowledge of how binocular integration plays a crucial role in the neural mechanisms guiding polarization vision and navigation
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