Comparative motif discovery combined with comparative transcriptomics yields accurate targetome and enhancer predictions

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date. After six months, it is available under a Creative Commons License Attribution-NonCommercial 3.0 Unported License.-- et al.The identification of transcription factor binding sites, enhancers, and transcriptional target genes often relies on the integration of gene expression profiling and computational cis-regulatory sequence analysis. Methods for the prediction of cis-regulatory elements can take advantage of comparative genomics to increase signal-to-noise levels. However, gene expression data are usually derived from only one species. Here we investigate tissue-specific cross-species gene expression profiling by high-throughput sequencing, combined with cross-species motif discovery. First, we compared different methods for expression level quantification and cross-species integration using Tag-seq data. Using the optimal pipeline, we derived a set of genes with conserved expression during retinal determination across Drosophila melanogaster, Drosophila yakuba, and Drosophila virilis. These genes are enriched for binding sites of eye-related transcription factors including the zinc-finger Glass, a master regulator of photoreceptor differentiation. Validation of predicted Glass targets using RNA-seq in homozygous glass mutants confirms that the majority of our predictions are expressed downstream from Glass. Finally, we tested nine candidate enhancers by in vivo reporter assays and found eight of them to drive GFP in the eye disc, of which seven colocalize with the Glass protein, namely, scrt, chp, dpr10, CG6329, retn, Lim3, and dmrt99B. In conclusion, we show for the first time the combined use of cross-species expression profiling with cross-species motif discovery as a method to define a core developmental program, and we augment the candidate Glass targetome from a single known target gene, lozenge, to at least 62 conserved transcriptional targets.This work is funded by research grants from Research Foundation Flanders (FWO, grant G.0704.11N), University of Leuven (CREA/10/014 and PF/10/016), and Human Frontiers Science Program (RGY0070/2011). M.N.S. is funded by a PhD fellowship from FWO.Peer Reviewe

Editorial: Evolution of Postembryonic Development

info:eu-repo/semantics/publishedVersio

Vein or trachea first? A comparative and genetic study to test the hypothesis of the pleural gill origin of the insect wing

Motivation: How insect wings emerged as an evolutionary novelty that permitted the extraordinary radiation and diversification among insects is a long-standing question that remains unanswered. The fossil record suggests that the first wings evolved in aquatic insects from respiratory gills, lateral movable flat pads articulated with the trunk pleural (lateral) region (1). Extant representatives of these flying insect ancestors are the Ephemeropterans, or mayflies. Still today, their aquatic nymphs bear gills along the abdominal segments. Extant wings are formed by a bilayered epithelial wing span. Rigidity is conferred by veins, epithelial thickenings that form hollow tubes that radiate along the proximodistal wing axis. Veins allow the flow of hemolymph and are traversed by trachea (respiratory tubes) and nerves (2) coming from sensory organs distributed along the wing margin or present on the veins. Therefore, the routes taken by trachea and nerves coincide with the veins. In the model organism, veins are genetically specified early, before any trachea invade the wing. Therefore, anatomical and genetic information seem to indicate that veins would be required for trachea and sensory organs to develop (i.e. veins come first). However, mayfly gills are invaded by branching trachea and have a number of sensory (and osmoregulatory) organs along the margin and on its surface, and yet they show no signs of veins. Therefore, is the gills were the ancestors of the wings, either gills from extant species lost the veins, or tracheal invasion of the wing does not depend on veins –although their precise routes might depend on where veins are.Methods: To address this question we made comparative studies between Cloeon dipterum, and two dipteran species, Drosophila melanogaster and Episyrphus balteatus using immunostainings and fly genetics.Results and Conclusions: In this work, I set to first, describe the tracheal invasion of the developing wing in Drosophila and two other species, another fly (Episyrphus balteatus) as well as in the mayfly Cloeon dipterum, to compare it to the development and pattern of tracheal invasion in Cleon’s gills. Second, using a genetic approach, I test the mutual dependencies between trachea, veins and nerves for their establishment. If the wing originated from tracheated movable gills without vein tracks, wing trachea in Drosophila should be able to invade tthe developing wing without genetically defined veins

A novel image segmentation algorithm with applications on confocal microscopy analysis

Motivation: Developing cells change their gene expression profiles dynamically upon induction by proper triggers, typically diffusible morphogens that are spatially distributed (1). These changes impact cell cycle and apoptosis regulators differentially, eventually determining the final structure and size of the mature organs (2). A quantitative model that links gene regulation and tissue growth must be provided with precise experimental data at cell resolution level in order to proceed to its validation, which in some cases is essential for model screening (i.e. reverse ingineering methods). Image analysis from laser confocal microscopy (LCM) has already been used to address modelling problems in developmental tissues such as these (3). However current methods for LCM segmentation rely upon watershed algorithms that show variable efficiency, relatively high parametrization and oversegmentation problems that are critical on very aggregated objects (4). Here we present a different segmentation method based on the maximum complementary n-ball set (MCnB set) concept. The segmentation algorithm takes a full MCnB set as a starting graph representation of the whole stack, which is later contracted using a parallel implementation approach.Results: We assayed the performance by segmenting a randomly generated set of spheres with different resolutions, signal aggregation levels and densities, and compared to the results delivered by a common segmentation free software, (i.e. Vaa3D), which is based on watersheds (5). We also applied this comparison on DAPI stained samples from Drosophila eye-antenna imaginal discs. The results indicate that the mean square displacement of detected spheres centroids is higher in the 3D watershed implementation results than when our method is applied. The same results are obtained when the number of sets or their size are checked instead.Conclusions: The results indicate that our method is adequate enough for image segmentation in three dimensions. It makes no assumptions on what the shape or signal features of the objects are, and does not require any calibration since it can proceed with no specific user parameters. Moreover it beats at least one segmentation method that has already been set up for counting and segmentation. Since the shape of the voxel aggregates is not critical, we sugget that further implementations could be potentially applied in higher dimension samples with interesting applications in developmental biology (i.e. 4D 'movies' segmentation). However one major drawback is that at least one operation runs with a O(n^2) time complexity, which is time (and memory) consuming for very big images

Mechanisms of organ size variation: differential gene expression analysis during the development of two fly eyes of very different sizes

Motivation: Often, organs of related species differ in size in spite of having the same function and general structure. However, the mechanisms that control the variation of organ size, are in most part unknown. This project aims to understand some of those mechanisms that control organ size variation. We will use the eyes of Diptera (flies) as model, because, first, the eyes of flies can be found in a wide range of sizes; and second, eye development in the reference fly model Drosophila melanogaster is simple: the eye primordium is a flat epithelium formed initially by proliferative multipotent progenitors. Differentiation of these progenitors depends on the advance of a signalling wave that induces the differentiation of these progenitors into retinal cells. What are the mechanisms that vary during development of flies that have eyes formed by different cell numbers? We will investigate this problem in Drosophila melanogaster and Episyrphus balteatus, the eyes of which comprise about 20,000 and 100,000 cells.Methods: To identify mechanisms of differential eye size we will compare the transcriptional profiles of eye promordia from Drosophila and Episyrphus at equivalent developmental times. To account for species-specific gene expression differences not related to eye-specific changes, we will compare the transcriptional profiles of wing primordia from both species. Total RNA is isolated from the primordia to generate sequencing libraries. Eye-specific differentially expressed genes will then be identified and characterized globally, using bioinformatic tools and selected genes will be analyzed by in situ hybridization in developing eye primordia using DIG-labelled RNA probes.Prior knowledge of Drosophila eye development suggests a list of interesting candidate genes as potentially involved in eye size control: these are the Wnt-1 wingless (wg) and the BMP2/4 decapentaplegic (dpp). We expect that the patterns of them will vary from Drosophila to Episyrphus. These patterns will be examined through in situ hybridization in eye primordia.In order to structure these data from transcriptomes into a genetic hierarchy (or a gene regulatory network), it would be necessary to establish regulatory relationships between transcription factors and relevant targets. To do so, we are carrying the ChIPmentation-sequencing experiments to identify the binding profile, genome wide, of a set of transcription factors that play essential roles in early eye development

The nucleolar protein Viriato/Nol12 is required for the growth and differentiation progression activities of the Dpp pathway during Drosophila eye development

Drosophila Decapentaplegic (Dpp), a member of the BMP2/4 class of the TGF-βs, is required for organ growth, patterning and differentiation. However, much remains to be understood about the mechanisms acting downstream of these multiple roles. Here we investigate this issue during the development of the Drosophila eye. We have previously identified viriato (vito) as a dMyc-target gene encoding a nucleolar protein that is required for proper tissue growth in the developing eye. By carrying out a targeted in vivo double-RNAi screen to identify genes and pathways functioning with Vito during eye development, we found a strong genetic interaction between vito and members of the Dpp signaling pathway including the TGF-β receptors tkv (type I), put (type II), and the co-Smad medea (med). Analyzing the expression of the Dpp receptor Tkv and the activation pattern of the pathway’s transducer, p-Mad, we found that vito is required for a correct signal transduction in Dpp-receiving cells. Overall, we validate the use of double RNAi to find specific genetic interactions and, in particular, we uncover a link between the Dpp pathway and Vito, a nucleolar component. vito would act genetically downstream of Dpp, playing an important role in maintaining a sufficient level of Dpp activity for the promotion of eye disc growth and regulation of photoreceptor differentiation in eye development

Identification and Analysis of Conserved cis-Regulatory Regions of the MEIS1 Gene

Meis1, a conserved transcription factor of the TALE-homeodomain class, is expressed in a wide variety of tissues during development. Its complex expression pattern is likely to be controlled by an equally complex regulatory landscape. Here we have scanned the Meis1 locus for regulatory elements and found 13 non-coding regions, highly conserved between humans and teleost fishes, that have enhancer activity in stable transgenic zebrafish lines. All these regions are syntenic in most vertebrates. The composite expression of all these enhancer elements recapitulate most of Meis1 expression during early embryogenesis, indicating they comprise a basic set of regulatory elements of the Meis1 gene. Using bioinformatic tools, we identify a number of potential binding sites for transcription factors that are compatible with the regulation of these enhancers. Specifically, HHc2:066650, which is expressed in the developing retina and optic tectum, harbors several predicted Pax6 sites. Biochemical, functional and transgenic assays indicate that pax6 genes directly regulate HHc2:066650 activity.This work was funded through grants BFU2009-07044 (MICINN) and Proyecto de Excelencia CVI 2658 (Junta de Andalucía) to FC and BFU2010-14839 (MICINN), CSD2007-00008 and Proyecto de Excelencia CVI-3488 to JLGS. JLR is a recipient of a JAE-DOC contract from the Spanish National Research Council (CSIC)

dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.MINECO Spain grants: (BFU2012-34324, BFU2015- 66040); Research Foundation—Flanders FWO grants: (G.0640.13, G.0791.14, PhD fellowship); Fundação para a Ciência e Tecnologia grant: (IF/01031/2012)

Thermo-optical performance of a solar funnel cooker

Funnel type solar cookers are simple, effective and have multiple advantages in practical use, but there is limited data available in scientific literature about their thermo-optical performance. This work aims to fill this lack of data. A well known model of solar funnel cooker has been subject to a series of experimental tests, indoor and outdoor, using different working fluids (water and oil). The experimental data has been used to calibrate the parameters of a simple thermal model. The calibrated model predictions show good agreement with experimental data and figures of merit describing the thermo-optical performance of the cooker are provided.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Restless Legs Syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon

This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported).-- et al.Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.The project was supported by Fritz-Thyssen-Stiftung, Cologne, Germany (10.09.2.146; 10.12.2.183), KKF-TUM (8766156), DAAD (0811963), and COST (“HOX and TALE homeoproteins in Development and Disease”). B.S. was partially supported by DFG grants (WI 1820/4-1; WI 1820/5-1) and a TUM-Excellence stipend. The KORA study was financed by the Helmholtz Zentrum München, which is funded by the German Federal Ministry of Education and Research (BMBF) and by the State of Bavaria. KORA research was supported within the Munich Center of Health Sciences (MC Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ. J.L.G.-S. and F.C. acknowledge funding of the Spanish and the Andalusian Governments and the Feder program for grants (BFU2010-14839, BFU2009-07044, CSD2007-00008, and Proyectos de Excelencia CVI-3488 and CVI 2658). This work was funded in part by a grant from the German Federal Ministry of Education and Research (BMBF) to the German Center for Diabetes Research (DZD), to the German Mouse Clinic (Infrafrontier: 01KX1012), to the German Center for Neurodegenerative Diseases (DZNE), Germany; by the Initiative and Networking Fund of the Helmholtz Association in the framework of the Helmholtz Alliance for Mental Research in an Ageing Society (HA-215); and the Munich Cluster for Systems Neurology (EXC 1010 SyNergy) and its Collaborative Research Center (CRC) 870/2 “Assembly and Function of Neuronal Circuits.”Peer Reviewe