Biology subjects

Pavel Tomancak

Publications and source records attributed to Pavel Tomancak.

4 recordsLinked to original sources

bioRxiv2016

Light sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development

BACKGROUNDSelective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratorys own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal.\n\nRESULTSOur OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm Maritigrella crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of Maritigrella with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope.\n\nCONCLUSIONSWe conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our gained experience during this project will help future OpenSPIM users with similar ambitions.

Developmental Biology↗

bioRxiv2015

A genome-wide resource for the analysis of protein localisation in Drosophila

The Drosophila genome contains >13,000 protein coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here we present a genome-wide fosmid library of {approx}10,000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins we have created transgenic lines and for a total of 207 lines we have assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we can visualise many proteins at endogenous expression levels and find a large fraction of them localising to subcellular compartments. Using complementation tests we demonstrate that two-thirds of the tagged proteins are fully functional. Moreover, our clones also enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will enable systematic analysis of protein expression and localisation in various cellular and developmental contexts.

Genomics↗

bioRxiv2014

Systematic Imaging Reveals Features of Localized mRNAs and Their Changing Subcellular Destinations in Development

mRNA localization is critical for eukaryotic cells and affects numerous transcripts, yet how cells regulate distribution of many mRNAs to their subcellular destinations is still unknown. We combined transcriptomics and systematic imaging to determine tissue-specific expression and subcellular distribution of 5862 mRNAs during Drosophila oogenesis. mRNA localization is widespread in the ovary, depends on the microtubule cytoskeleton and the mRNAs enriched at the posterior cortex share common localization machinery. Localized mRNAs, in particular the posterior class, have distinct gene features and differ in expression level, 3UTR length and sequence conservation from unlocalized mRNAs. Using cross-tissue comparison we revealed that the localization status of mRNAs differs between epithelial, germline and embryonic cell types and also changes within one cell, the oocyte, over time. This dataset enables the transition from deep mechanistic dissection of singular mRNA localization events towards global understanding of how mRNAs transcribed in the nucleus distribute in cells.

Developmental Biology↗

bioRxiv2013

An Adaptive Threshold in Mammalian Neocortical Evolution

Expansion of the neocortex is a hallmark of human evolution. However, it remains an open question what adaptive mechanisms facilitated its expansion. Here we show, using gyrencephaly index (GI) and other physiological and life-history data for 102 mammalian species, that gyrencephaly is an ancestral mammalian trait. We provide evidence that the evolution of a highly folded neocortex, as observed in humans, requires the traversal of a threshold of [~]109 neurons, and that species above and below the threshold exhibit a bimodal distribution of physiological and life-history traits, establishing two phenotypic groups. We identify, using discrete mathematical models, proliferative divisions of pro-genitors in the basal compartment of the developing neocortex as evolutionarily necessary and sufficient for generating a fourteen-fold increase in daily prenatal neuron production and thus traversal of the neuronal threshold. Finally, using RNA-seq data from fetal human neocortical germinal zones, we show a genomic correlate to the neuron threshold in the differential conservation of long intergenic non-coding RNA. (see arXiv:1304.5412)

Evolutionary Biology↗