Cis-regulatory control of the initial neurogenic pattern of onecut gene expression in the sea urchin embryo

Specification of the ciliated band (CB) of echinoid embryos executes three spatial functions essential for postgastrular organization. These are establishment of a band about 5 cells wide which delimits and bounds other embryonic territories; definition of a neurogenic domain within this band; and generation within it of arrays of ciliary cells that bear the special long cilia from which the structure derives its name. In S. purpuratus the spatial coordinates of the future ciliated band are initially and exactly determined by the disposition of a ring of cells that transcriptionally activate the onecut homeodomain regulatory gene, beginning in blastula stage, long before the appearance of the CB per se. Thus the cis-regulatory apparatus that governs onecut expression in the blastula directly reveals the genomic sequence code by which these aspects of the spatial organization of the embryo are initially determined. We screened the entire onecut locus and its flanking region for transcriptionally active cis-regulatory elements, and by means of BAC recombineered deletions identified three separated and required cis-regulatory modules that execute different functions. The operating logic of the crucial spatial control module accounting for the spectacularly precise and beautiful early onecut expression domain depends on spatial repression. Previously predicted oral ectoderm and aboral ectoderm repressors were identified by cis-regulatory mutation as the products of goosecoid and irxa genes respectively, while the pan-ectodermal activator SoxB1 supplies a transcriptional driver function

Geometric control of ciliated band regulatory states in the sea urchin embryo

The trapezoidal ciliated band (CB) of the postgastrular sea urchin embryo surrounds the oral ectoderm, separating it from adjacent embryonic territories. Once differentiated, the CB is composed of densely arranged cells bearing long cilia that endow the larva with locomotion and feeding capability. The spatial pattern from which the CB will arise is first evidenced during pregastrular stages by expression of the pioneer gene onecut. Immediately after gastrulation, the CB consists of four separate regulatory state domains, each of which expresses a unique set of transcription factors: (1) the oral apical CB, located within the apical neurogenic field; (2) the animal lateral CB, which bilaterally separates the oral from aboral ectoderm; (3) the vegetal lateral CB, which bilaterally serves as signaling centers; and (4) the vegetal oral CB, which delineates the boundary with the underlying endoderm. Remarkably, almost all of the regulatory genes specifically expressed within these domains are downregulated by interference with SoxB1 expression, implying their common activation by this factor. Here, we show how the boundaries of the CB subdomains are established, and thus ascertain the design principle by which the geometry of this unique and complex regulatory state pattern is genomically controlled. Each of these boundaries, on either side of the CB, is defined by spatially confined transcriptional repressors, the products of regulatory genes operating across the border of each subdomain. In total this requires deployment of about ten different repressors, which we identify in this work, thus exemplifying the complexity of information required for spatial regulatory organization during embryogenesis

General approach for in vivo recovery of cell type-specific effector gene sets

Differentially expressed, cell type-specific effector gene sets hold the key to multiple important problems in biology, from theoretical aspects of developmental gene regulatory networks (GRNs) to various practical applications. Although individual cell types of interest have been recovered by various methods and analyzed, systematic recovery of multiple cell type-specific gene sets from whole developing organisms has remained problematic. Here we describe a general methodology using the sea urchin embryo, a material of choice because of the large-scale GRNs already solved for this model system. This method utilizes the regulatory states expressed by given cells of the embryo to define cell type and includes a fluorescence activated cell sorting (FACS) procedure that results in no perturbation of transcript representation. We have extensively validated the method by spatial and qualitative analyses of the transcriptome expressed in isolated embryonic skeletogenic cells and as a consequence, generated a prototypical cell type-specific transcriptome database

Numerical simulation of 3D free surface flows in time dependent curvilinear coordinates

We propose a three dimensional non-hydrostatic shock-capturing numerical model for the simulation of wave propagation, transformation and breaking, which is based on an original integral formulation of the contravariant Navier-Stokes equations, devoid of Christoffel symbols, in general time-dependent curvilinear coordinates

Genome-wide identification of enhancer elements

We present a prospective genome-wide regulatory element database for the sea urchin embryo and the modified chromosome capture-related methodology used to create it. The method we developed is termed GRIP-seq for genome-wide regulatory element immunoprecipitation and combines features of chromosome conformation capture, chromatin immunoprecipitation, and paired-end next-generation sequencing with molecular steps that enrich for active cis-regulatory elements associated with basal transcriptional machinery. The first GRIP-seq database, available to the community, comes from S. purpuratus 24 hpf embryos and takes advantage of the extremely well-characterized cis-regulatory elements in this system for validation. In addition, using the GRIP-seq database, we identify and experimentally validate a novel, intronic cis-regulatory element at the onecut locus. We find GRIP-seq signal sensitively identifies active cis-regulatory elements with a high signal-to-noise ratio for both distal and intronic elements. This promising GRIP-seq protocol has the potential to address a rate-limiting step in resolving comprehensive, predictive network models in all systems

Feasibility of mini combined cycles for naval applications

The objective of energy production with low environmental impact will have, in the near future, high potential of development also for naval applications. The containment of pollutant emissions can be achieved by the combined use of an innovative mini gas-steam combined cycle with thermal energy cogeneration to feed the ship thermal utilities, in place of the current Diesel engine application, and liquefied natural gas as fuel (LNG). The present work is focused on the definition of the architecture of the plant, by selecting optimal distribution of pressure and temperature and repartition of power between Gas Turbine (GT), Steam Turbine (ST) and thermal utilities, as well as on the choice and sizing of the individual components. The main purpose is the definition of a compact, high efficiency, system. The proposed basic mini-cycle ranges from 2 MW to 10 MW electric power. Thanks to the combined heat and power cogeneration plant adopted, for an overall electrical efficiency of about 30%, a total return (thermal + electricity) of about 75% can be achieved. An example of plant providing large power, in a partially modular arrangement is also proposed

Genome-wide assessment of differential effector gene use in embryogenesis

Six different populations of cells were isolated by FACS from disaggregated late blastula and gastrula stage sea urchin embryos according to the regulatory states expressed in these cells, as reported by recombineered BACs producing fluorochromes. Transcriptomes recovered from these embryonic cell populations revealed striking, early differential expression of large cohorts of effector genes. The six cell populations were presumptive pigment cells, presumptive neurogenic cells, presumptive skeletogenic cells, cells from the stomodeal region of the oral ectoderm, ciliated band cells, and cells from the endoderm/ectoderm boundary that will give rise both to hindgut and to border ectoderm. Transcriptome analysis revealed that each of these domains specifically expressed several hundred effector genes at significant levels. Annotation indicates the qualitative individuality of the functional nature of each cell population, even though they were isolated from embryos only 1 to 2 days old. In no case was more than a tiny fraction of the transcripts enriched in one population also enriched in any other of the six populations studied. As was particularly clear in the cases of the presumptive pigment, neurogenic, and skeletogenic cells, all three of which represent precociously differentiating cell types of this embryo, most specifically expressed genes of given cell types are not significantly expressed at all in the other cell types. Thus at the effector gene level a dramatic, cell type specific pattern of differential gene regulation is established well before any significant embryonic morphogenesis has occurred

Blind and reference-free fluorescence lifetime estimation via consumer time-of-flight sensors

Fluorescence lifetime imaging (FLI) is a popular method for extracting useful information that is otherwise unavailable from a conventional intensity image. Usually, however, it requires expensive equipment, is often limited to either distinctly frequency- or time-domain modalities, and demands calibration measurements and precise knowledge of the illumination signal. Here, we present a generalized time-based, cost-effective method for estimating lifetimes by repurposing a consumer-grade time-of-flight sensor. By developing mathematical theory that unifies time- and frequency-domain approaches, we can interpret a time-based signal as a combination of multiple frequency measurements. We show that we can estimate lifetimes without knowledge of the illumination signal and without any calibration. We experimentally demonstrate this blind, reference-free method using a quantum dot solution and discuss the method’s implementation in FLI applications

Measurement of scaling laws for shock waves in thermal nonlocal media

We are able to detect the details of spatial optical collisionless wave-breaking through the high aperture imaging of a beam suffering shock in a fluorescent nonlinear nonlocal thermal medium. This allows us to directly measure how nonlocality and nonlinearity affect the point of shock formation and compare results with numerical simulations.Comment: 4 pages, 4 figure

TIME-RELATED QUALITY DIMENSIONS OF URBAN REMOTELY SENSED BIG DATA

Abstract. Our rapidly changing world requires new sources of image based information. The quickly changing urban areas, the maintenance and management of smart cities cannot only rely on traditional techniques based on remotely sensed data, but also new and progressive techniques must be involved. Among these technologies the volunteer based solutions are getting higher importance, like crowd-sourced image evaluations, mapping by satellite based positioning techniques or even observations done by unskilled people. Location based intelligence has become an everyday practice of our life. It is quite enough to mention the weather forecast and traffic monitoring applications, where everybody can act as an observer and acquired data – despite their heterogeneity in quality – provide great value. Such value intuitively increases when data are of better quality. In the age of visualization, real-time imaging, big data and crowd-sourced spatial data have revolutionary transformed our general applications. Most important factors of location based decisions are the time-related quality parameters of the used data. In this paper several time-related data quality dimensions and terms are defined. The paper analyses the time sensitive data characteristics of image-based crowd-sourced big data, presents quality challenges and perspectives of the users. The data quality analyses focus not only on the dimensions, but are also extended to quality related elements, metrics. The paper discusses the connection of data acquisition and processing techniques, considering even the big data aspects. The paper contains not only theoretical sections, strong practice-oriented examples on detecting quality problems are also covered. Some illustrative examples are the OpenStreetMap (OSM), where the development of urbanization and the increasing process of involving volunteers can be studied. This framework is continuing the previous activities of the Remote Sensing Data Quality Working Group (ICWGIII/IVb) of the ISPRS in the topic focusing on the temporal variety of our urban environment.</p