The developmental potential of the C. elegans RFX transcription factor DAF-19

Die Wahrnehmung und korrekte Interpretation von Signalen aus der Umwelt ist entscheidend für das Überleben jedes Organismus. Es ist eine grosse Herausforderung die Aufnahme und Weiterleitung von sensorischer Information zu studieren, speziell in den komplexen Nervensystemen höherer Organismen. Der Rundwurm C. elegans besitzt ein relativ simples Nervensystem aus 302 Neuronen. 60 davon haben Cilien (haar-ähnliche, sensorische Fortsätze) und sind somit die wichtigste Aufnahmequelle externer, sensorischer Information. Die Verhaltensmuster von C. elegans sind zahlreich, was den Rundwurm zu einem idealen Modellorganismus macht, in dem man die Funktion von sensorischen Neuronen studieren kann. RFX Transkriptionsfaktoren sind essentiell für die Bildung von Cilien in vielen Organismen, unter anderem in Mäusen und Menschen. Das Fehlen von DAF-19, dem einzigen RFX Protein in C. elegans führt zum Fehlen aller Cilien und somit zur Unfähigkeit sensorische Information aufnehmen zu können. In Paper I beschreiben und charakterisieren wir drei Isoformen von DAF-19. Isoform DAF-19C ist spezifisch für Neuronen mit Cilien. Der Zusatz von DAF-19C genügt alle Cilien-bedingten Phänotypen in daf-19 Mutanten wiederherzustellen. DAF-19A/B kommen in allen nicht-ciliierten Neuronen vor und regulieren dort synaptische Funktionen. Unsere Arbeit beschreibt zum ersten Mal dass RFX Proteine nicht nur für die Aufnahme, sondern auch für die Weiterleitung von Signalen notwendig sind. In Paper II untersuchen wir mögliche zell-autonome Funktionen von DAF-19 in ciliierten Neuronen. Wir etablieren und testen ein genetisches „Reparatur“ Verfahren, welches in vivo die Analyse einzelner ciliierter Neuronen sowohl auf dem Zell- als auch auf dem Organismus-Niveau erlaubt. Durch Wiederherstellung der Funktion von DAF-19C nur in bestimmten, einzelnen sensorischen Neuronen (in daf-19 genetischen Mutanten) kreieren wir Tiere mit nur einem einzelnen, isolierten, jedoch funktionsfähigen ciliierten Neuron, wohingegen jedwede anderweitige sensorische Signale von diesen Tieren nicht aufgenommen werden können. Dieses experimentelle System kann dazu benutzt werden spezifische sensorische Fragen in Bezug auf ciliierte Neuronen oder Schritte der frühen Cilienentwicklung zu studieren. In Paper III untersuchen wir die mögliche übergeordnete Rolle von DAF-19 in der Entwicklung von Cilien. Wir exprimieren daf-19C in verschiedenen Zelltypen und versuchen so die Bildung von Cilien in nicht-ciliierten Zellen zu induzieren. Dabei entdecken wir ein wahrscheinlich regulatorisches Netzwerk, welches festlegt, in welchen Zellen Cilien gebildet werden können und in welchen nicht. Wir nehmen an, dass isoform-spezifische Suppressoren von DAF-19 das für jeweils verschiede Zelltypen spezifische Aktivierungspotential regulieren.The detection and correct interpretation of environmental signals is crucial for the survival of every organism. Studying mechanisms of sensory perception and signal transmission is a challenging task, especially in organisms with complex neuronal networks. The nematode C. elegans possesses a rather simple neuronal network of 302 neurons. 60 of them have cilia (hair-like surface structures), which are the main source of external sensory input. C. elegans executes a large number of different behaviors and is therefore an excellent model organism in which to study sensory neuron function. RFX transcription factors are essential for cilia formation in many organisms including mice and humans. Lack of the C. elegans RFX transcription factor DAF-19 leads to the complete absence of cilia and consequently of sensory input. In Paper I we describe and functionally characterize three different isoforms of DAF-19. We find that the short isoform DAF-19C is specifically expressed in ciliated sensory neurons and sufficient to rescue all cilia-related phenotypes of daf-19 mutants. The long isoforms DAF-19A/B function in all non-ciliated neurons, where they are required to maintain synaptic functions. Thus, we show for the first time that an RFX protein is not only required for signal detection, but also for signal transmission. In Paper II we explore cell-autonomous functions of DAF-19 in ciliated sensory neurons (CSNs). We establish and test a genetic rescue system that allows the in vivo analysis of isolated CSNs at both cellular and systemic levels. Using daf-19 mutants and cell-specific rescue of DAF-19 function we generate animals with single, functional CSNs, which otherwise are completely devoid of any environmental input through cilia. This system can be used to study specific sensory issues concerning CSNs or early steps of ciliogenesis. Finally, in Paper III we explore a potential master regulatory role of DAF-19. We attempt to induce ectopic cilia in C. elegans by expressing DAF-19C in various non-ciliated cell types and discover a likely regulatory network that governs in which cell types cilia can be made. We hypothesize that isoform-specific suppressors of DAF-19 regulate this cell-type-specific ciliogenic potential

Steroid hormone regulation of C. elegans and Drosophila aging and life history

In the last two decades it has become clear that hormones and gene mutations in endocrine signaling pathways can exert major effects on lifespan and related life history traits in worms, flies, mice, and other organisms. While most of this research has focused on insulin/insulin-like growth factor-1 signaling, a peptide hormone pathway, recent work has shown that also lipophilic hormones play an important role in modulating lifespan and other life history traits. Here we review how steroid hormones, a particular group of lipophilic hormones, affect life history traits in the nematode worm (Caenorhabditis elegans) and the fruit fly (Drosophila melanogaster), with a particular focus on longevity. Interestingly, a comparison suggests that parallel endocrine principles might be at work in worms and flies in these species and that steroid hormones interact with the gonad to affect lifespan

Systematic Identification of Genes that Regulate Neuronal Wiring in the Drosophila Visual System

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring

Worms With a Single Functional Sensory Cilium Generate Proper Neuron-Specific Behavioral Output

Studying the development and mechanisms of sensory perception is challenging in organisms with complex neuronal networks. The worm Caenorhabditis elegans possesses a simple neuronal network of 302 neurons that includes 60 ciliated sensory neurons (CSNs) for detecting external sensory input. C. elegans is thus an excellent model in which to study sensory neuron development, function, and behavior. We have generated a genetic rescue system that allows in vivo analyses of isolated CSNs at both cellular and systemic levels. We used the RFX transcription factor DAF-19, a key regulator of ciliogenesis. Mutations in daf-19 result in the complete absence of all sensory cilia and thus of external sensory input. In daf-19 mutants, we used cell-specific rescue of DAF-19 function in selected neurons, thereby generating animals with single, fully functional CSNs. Otherwise and elsewhere these animals are completely devoid of any environmental input through cilia. We demonstrated the rescue of fully functional, single cilia using fluorescent markers, sensory behavioral assays, and calcium imaging. Our technique, functional rescue in single sensory cilia (FRISSC), can thus cell-autonomously and cell-specifically restore the function of single sensory neurons and their ability to respond to sensory input. FRISSC can be adapted to many different CSNs and thus constitutes an excellent tool for studying sensory behaviors, both in single animals and in populations of worms. FRISSC will be very useful for the molecular dissection of sensory perception in CSNs and for the analysis of the developmental aspects of ciliogenesis

Distinct Isoforms of the RFX Transcription Factor DAF-19 Regulate Ciliogenesis and Maintenance of Synaptic Activity

Neurons form elaborate subcellular structures such as dendrites, axons, cilia, and synapses to receive signals from their environment and to transmit them to the respective target cells. In the worm Caenorhabditis elegans, lack of the RFX transcription factor DAF-19 leads to the absence of cilia normally found on 60 sensory neurons. We now describe and functionally characterize three different isoforms of DAF-19. The short isoform DAF-19C is specifically expressed in ciliated sensory neurons and sufficient to rescue all cilia-related phenotypes of daf-19 mutants. In contrast, the long isoforms DAF-19A/B function in basically all nonciliated neurons. We discovered behavioral and cellular phenotypes in daf-19 mutants that depend on the isoforms daf-19a/b. These novel synaptic maintenance phenotypes are reminiscent of synaptic decline seen in many human neurodegenerative disorders. The C. elegans daf-19 mutant worms can thus serve as a molecular model for the mechanisms of functional neuronal decline