Local and global interneuron function in the retina

Brain regions consist of intricate neuronal circuits with diverse interneuron types. In order to gain mechanistic insights into brain function, it is essential to understand the computational purpose of the different types of interneurons. How does a single interneuron type shape the input-output transformation of a given brain region? Here I investigated how different interneuron types of the retina contribute to retinal computations. I developed approaches to systematically and quantitatively investigate the function of retinal interneurons by combining precise circuit perturbations with a system-wide read-out of activity. I studied the functional roles of a locally acting interneuron type, starburst amacrine cells, and of a globally acting type, horizontal cells. In Chapter 1, I show how a defined genetic perturbation in starburst amacrine cells, the mutation of the FRMD7 gene, leads to specific effects in the direction-selective output channels of the retina. Our findings provide a link between a specific neuronal computation and a human disease, and present an entry point for understanding the molecular pathways responsible for generating neuronal circuit asymmetries. Chapter 2 addresses how mutated FRMD7 in starburst cells and the genetic ablation of starburst cells affect the computation of visual motion in the retina and in primary visual cortex. Chapter 3 addresses how horizontal cells mediate rod depolarization under bright daylight conditions. In Chapter 4, I combined the precise, yet retina-wide, perturbation of horizontal cells with a system-level readout of the retinal output. I uncovered that horizontal cells can differentially shape the response dynamics of individual retinal output channels. Our combined experimental and theoretical work shows how the inhibitory feedback at the first visual synapse shapes functional diversity in the retina

Revitalisierung der Ulster am „Ulstersack“ – Konzept zur Entwicklung eines Uferrandstreifens und Förderung der Eigendynamik

Für einen begradigten und ausgebauten Abschnitt der Ulster am „Ulstersack“ im Grenzgebiet Hessen/Thüringen sind im Rahmen des Projekts „Rhön im Fluss“ Maßnahmen zur Revitalisierung des Gewässers vorgesehen. Neben der Bestandsvermessung und einer Gewässerschau wurde nach dem LAWA-Vor-Ort-Verfahren eine Strukturgüterhebung durchgeführt. Durch ausgesuchte Maßnahmen zur Förderung der Eigenentwicklung soll das in der EU-Wasserrahmenrichtlinie postulierte Umweltqualitätsziel des guten ökologischen Zustands bis zum Jahr 2015 erreicht werden. Mit Blick auf das fließgewässertypische Leitbild werden Einzelmaßnahmen entwickelt und als Maßnahmenkombinationen in drei Varianten vorgestellt. Gleichzeitig wird zur Quantifizierung des Erfolgs die erwartete Strukturgüteverbesserung dargelegt. Neben der Berechnung der Wasserspiegellagen wurde eine Kosten-Wirksamkeitsanalyse zur Bewertung der Kosteneffizienz vorgenommen. Die Ausweisung eines ausreichend breiten Uferrandstreifens als Entwicklungs- und Sukzessionsfläche ist wesentliche Voraussetzung für den Umsetzungserfolg. Die für die Planungsphase gewählte Projektabwicklung ist auf ähnlich gelagerte Projekte übertragbar

RNAi in Budding Yeast

RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y′ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y′ messenger RNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a previously unknown class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi

Discovery of an unconventional centromere in budding yeast redefines evolution of point centromeres

Centromeres are the chromosomal regions promoting kinetochore assembly for chromosome segregation. In many eukaryotes, the centromere consists of up to mega base pairs of DNA. On such "regional centromeres," kinetochore assembly is mainly defined by epigenetic regulation [1]. By contrast, a clade of budding yeasts (Saccharomycetaceae) has a "point centromere" of 120-200 base pairs of DNA, on which kinetochore assembly is defined by the consensus DNA sequence [2, 3]. During evolution, budding yeasts acquired point centromeres, which replaced ancestral, regional centromeres [4]. All known point centromeres among different yeast species share common consensus DNA elements (CDEs) [5, 6], implying that they evolved only once and stayed essentially unchanged throughout evolution. Here, we identify a yeast centromere that challenges this view: that of the budding yeast Naumovozyma castellii is the first unconventional point centromere with unique CDEs. The N. castellii centromere CDEs are essential for centromere function but have different DNA sequences from CDEs in other point centromeres. Gene order analyses around N. castellii centromeres indicate their unique, and separate, evolutionary origin. Nevertheless, they are still bound by the ortholog of the CBF3 complex, which recognizes CDEs in other point centromeres. The new type of point centromere originated prior to the divergence between N. castellii and its close relative Naumovozyma dairenensis and disseminated to all N. castellii chromosomes through extensive genome rearrangement. Thus, contrary to the conventional view, point centromeres can undergo rapid evolutionary changes. These findings give new insights into the evolution of point centromeres

Fungal regulatory evolution: cis and trans in the balance

available in PMC 2010 June 17.Regulatory divergence is likely a major driving force in evolution. Comparative genomics is being increasingly used to infer the evolution of gene regulation. Ascomycota fungi are uniquely suited among eukaryotes for regulatory evolution studies, due to broad phylogenetic scope, many sequenced genomes, and tractability of genomic analysis. Here we review recent advances in the identification of the contribution of cis- and trans-factors to expression divergence. Whereas current strategies have led to the discovery of surprising signatures and mechanisms, we still understand very little about the adaptive role of regulatory evolution. Empirical studies including experimental evolution, comparative functional genomics and hybrid and engineered strains are showing early promise toward deciphering the contribution of regulatory divergence to adaptation.Human Frontier Science Program (Strasbourg, France)Howard Hughes Medical InstituteBurroughs Wellcome Fund (Career Award)Alfred P. Sloan Foundatio

EvoChromo: towards a synthesis of chromatin biology and evolution

Over the past few years, interest in chromatin and its evolution has grown. To further advance these interests, we organized a workshop with the support of The Company of Biologists to debate the current state of knowledge regarding the origin and evolution of chromatin. This workshop led to prospective views on the development of a new field of research that we term ‘EvoChromo’. In this short Spotlight article, we define the breadth and expected impact of this new area of scientific inquiry on our understanding of both chromatin and evolution

Author response

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly

Comparative Genomics Reveals Two Novel RNAi Factors in Trypanosoma brucei and Provides Insight into the Core Machinery

The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3′-5′ exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites

Genome and Transcriptome Analysis of the Food-Yeast Candida utilis

The industrially important food-yeast Candida utilis is a Crabtree effect-negative yeast used to produce valuable chemicals and recombinant proteins. In the present study, we conducted whole genome sequencing and phylogenetic analysis of C. utilis, which showed that this yeast diverged long before the formation of the CUG and Saccharomyces/Kluyveromyces clades. In addition, we performed comparative genome and transcriptome analyses using next-generation sequencing, which resulted in the identification of genes important for characteristic phenotypes of C. utilis such as those involved in nitrate assimilation, in addition to the gene encoding the functional hexose transporter. We also found that an antisense transcript of the alcohol dehydrogenase gene, which in silico analysis did not predict to be a functional gene, was transcribed in the stationary-phase, suggesting a novel system of repression of ethanol production. These findings should facilitate the development of more sophisticated systems for the production of useful reagents using C. utilis

Tinkering Evolution of Post-Transcriptional RNA Regulons: Puf3p in Fungi as an Example

Genome-wide studies of post-transcriptional mRNA regulation in model organisms indicate a “post-transcriptional RNA regulon” model, in which a set of functionally related genes is regulated by mRNA–binding RNAs or proteins. One well-studied post-transcriptional regulon by Puf3p functions in mitochondrial biogenesis in budding yeast. The evolution of the Puf3p regulon remains unclear because previous studies have shown functional divergence of Puf3p regulon targets among yeast, fruit fly, and humans. By analyzing evolutionary patterns of Puf3p and its targeted genes in forty-two sequenced fungi, we demonstrated that, although the Puf3p regulon is conserved among all of the studied fungi, the dedicated regulation of mitochondrial biogenesis by Puf3p emerged only in the Saccharomycotina clade. Moreover, the evolution of the Puf3p regulon was coupled with evolution of codon usage bias in down-regulating expression of genes that function in mitochondria in yeast species after genome duplication. Our results provide a scenario for how evolution like a tinker exploits pre-existing materials of a conserved post-transcriptional regulon to regulate gene expression for novel functional roles