ISTA Thesis

During navigation, animals can infer the structure of the environment by computing the optic flow cues elicited by their own movements, and subsequently use this information to instruct proper locomotor actions. These computations require a panoramic assessment of the visual environment in order to disambiguate similar sensory experiences that may require distinct behavioral responses. The estimation of the global motion patterns is therefore essential for successful navigation. Yet, our understanding of the algorithms and implementations that enable coherent panoramic visual perception remains scarce. Here I pursue this problem by dissecting the functional aspects of interneuronal communication in the lobula plate tangential cell network in Drosophila melanogaster. The results presented in the thesis demonstrate that the basis for effective interpretation of the optic flow in this circuit are stereotyped synaptic connections that mediate the formation of distinct subnetworks, each extracting a particular pattern of global motion. Firstly, I show that gap junctions are essential for a correct interpretation of binocular motion cues by horizontal motion-sensitive cells. HS cells form electrical synapses with contralateral H2 neurons that are involved in detecting yaw rotation and translation. I developed an FlpStop-mediated mutant of a gap junction protein ShakB that disrupts these electrical synapses. While the loss of electrical synapses does not affect the tuning of the direction selectivity in HS neurons, it severely alters their sensitivity to horizontal motion in the contralateral side. These physiological changes result in an inappropriate integration of binocular motion cues in walking animals. While wild-type flies form a binocular perception of visual motion by non-linear integration of monocular optic flow cues, the mutant flies sum the monocular inputs linearly. These results indicate that rather than averaging signals in neighboring neurons, gap-junctions operate in conjunction with chemical synapses to mediate complex non-linear optic flow computations. Secondly, I show that stochastic manipulation of neuronal activity in the lobula plate tangential cell network is a powerful approach to study the neuronal implementation of optic flow-based navigation in flies. Tangential neurons form multiple subnetworks, each mediating course-stabilizing response to a particular global pattern of visual motion. Application of genetic mosaic techniques can provide sparse optogenetic activation of HS cells in numerous combinations. These distinct combinations of activated neurons drive an array of distinct behavioral responses, providing important insights into how visuomotor transformation is performed in the lobula plate tangential cell network. This approach can be complemented by stochastic silencing of tangential neurons, enabling direct assessment of the functional role of individual tangential neurons in the processing of specific visual motion patterns. Taken together, the findings presented in this thesis suggest that establishing specific activity patterns of tangential cells via stereotyped synaptic connectivity is a key to efficient optic flow-based navigation in Drosophila melanogaster

Strand asymmetry influences mismatch resolution during a single-strand annealing

Background: Biases of DNA repair can shape the nucleotide landscape of genomes at evolutionary timescales. The molecular mechanisms of those biases are still poorly understood because it is difficult to isolate the contributions of DNA repair from those of DNA damage. Results: Here, we develop a genome-wide assay whereby the same DNA lesion is repaired in different genomic contexts. We insert thousands of barcoded transposons carrying a reporter of DNA mismatch repair in the genome of mouse embryonic stem cells. Upon inducing a double-strand break between tandem repeats, a mismatch is generated if the break is repaired through single-strand annealing. The resolution of the mismatch showed a 60-80% bias in favor of the strand with the longest 3' flap. The location of the lesion in the genome and the type of mismatch had little influence on the bias. Instead, we observe a complete reversal of the bias when the longest 3' flap is moved to the opposite strand by changing the position of the double-strand break in the reporter. Conclusions: These results suggest that the processing of the double-strand break has a major influence on the repair of mismatches during a single-strand annealing.We acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN-2020-06377), the Spanish Ministry of Economy, Industry and Competitiveness (“Centro de Excelencia Severo Ochoa 2013-2017”, Plan Estatal PGC2018-099807-B-I00), of the CERCA Programme/Generalitat de Catalunya, and of the European Research Council (Synergy Grant 609989). VOP was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie programme (665385). We also acknowledge the support of the Spanish Ministry of Economy and Competitiveness (MEIC) to the EMBL partnershi

An experimental assay of the interactions of amino acids from orthologous sequences shaping a complex fitness landscape

Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible.The work was supported by HHMI International Early Career Scientist Program (55007424), the MINECO (BFU2012-31329, BFU2012-37168, BFU2015-68351-P and BFU2015-68723-P), Spanish Ministry of Economy and Competitiveness Centro de Excelencia Severo Ochoa 2013-2017 grant (SEV-2012-0208), the Unidad de Excelencia María de Maeztu funded by the MINECO (MDM-2014-0370), Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat AGAUR program (2014 SGR 0974), the CERCA Programme of the Generalitat de Catalunya, Russian Foundation for Basic Research grant (18-04-01173), the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie programme (665385) and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013, ERC grant agreement 335980_EinME and Synergy Grant 609989). KSS was supported by EMBO long-term fellowship (ALTF 107-2016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript