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Full Paper Proc. of Int. Conf. on Advances in Communication and Information Technology 2011

3D Visual Integration of Spatio-Temporal Gene Expression Patterns on Digital Atlas of Zebrafish Embryo using Web Service D. Potikanond, F. J. Verbeek Section Imaging and Bioinformatics, Leiden Institute of Advanced Computer Science Leiden University, Leiden, The Netherlands Email: {dpotikan, fverbeek}@liacs.nl Abstract—Gene expression patterns analysis with microarray provides quantitative information that shows how a gene is expressed under a particular condition. Whole mount in situ hybridization, on the other hand, can be used to capture the spatio-temporal characteristics of the gene expression pattern. Therefore, visual integration of gene expression data from both techniques with a digital atlas data of a model-organism can help identifying not only spatial and temporal but also quantitative aspects of gene expression in different stages of development. In this paper, we present an approach using web services to provide an integrative online visualization of gene expression patterns in within a digital atlas of zebrafish in different stages of development. We developed SOAP web services that provide programmatic access to the 3D data and spatial-temporal whole mount gene expression data to our readily developed information systems; the 3D digital atlas of zebrafish development and the Gene Expression Management System (GEMS). We also created web applications that exploit the newly developed web services to retrieve data from our repositories. The web applications also uses the web services to retrieve relevant quantitative microarray analysis gene expression data from community resources; i.e. the ArrayExpress Atlas. All the gene expression patterns data and the 3D atlas data are subsequently integrated using ontology based mapping. In order to deliver the integrated visualization to end users, we developed a Java based 3D-viewer client that can be integrated in a web interface allowing users to visualize the information over Internet.

temporal and spatial aspects of gene expression in developmental is a crucial step for additional functional analysis of genes. The microarray technique [4] is one of the major experimental breakthroughs enabling high throughput measurement and analysis of the expression patterns of (tens of) thousands of genes simultaneously [5]. However, in multicellular organism such as zebrafish, gene expression influences the development of a cell or group of cells. Therefore whole-specimen microarray analysis cannot fully document the spatio-temporal relations. Whole mount in situ hybridization, on the other hand, can be used to obtain such information. To this end, we built the Gene Expression Management System (GEMS) [6] as an information system for 3D spatio-temporal gene expression patterns which are generated through Fluorescent In Situ Hybridization (zebraFISH) [7] protocol. There are a number of information systems providing information on zebrafish anatomy and/or gene expression data such as the Zebrafish Information Network (ZFIN) [8], ArrayExpress [9], Entrez Gene [10] and Ensembl [11]. However, the anatomical data and gene expression data are typically not integrated nor represented in such a way that they can be visualized jointly in a 3D context. To help understanding the spatio-temporal context of genes expression and the involvement in changing anatomical structures, it is important to have a visualization system that integrates data from these different domains. For example, in the mouse (Mus musculus), there are the e-Mouse Atlas Project (EMAP) [12], the e-Mouse Atlas of Gene Expression (EMAGE) [13] and the Digital Atlasing and Standardization in the Mouse Brain [14]. The Berkeley Drosophila Transcription Network Project (BDTNP) provides resources and visualization tools for viewing 3D gene expression patterns in early Drosophila embryo at cellular resolution [15, 16]. However, there is no such thing available for zebrafish. In this paper, we describe an approach to provide web services that helps visualizing gene expression information within 3D graphical models of zebrafish atlas. This way, detailed information about gene expression in zebrafish becomes available embedded into their 3D spatial context. To provide programming interfaces to access our 3D reconstruction data and gene expression patterns data, we have created the 3D reconstruction (TDR) and the GEMS web services. Even though GEMS provides semantic information on spatio-temporal gene expression, it is yet to provide quantitative gene expression data. Hence we need

Index Terms—Visualization, Web services, Zebrafish Atlas, 3D reconstruction, Gene expression patterns

I. INTRODUCTION 3D imaging and graphical models have been used effectively as a common technical framework for representing spatial information in biomedical research. Among the wellknown techniques for capturing 3D data are the serial sectioning methods [1, 2]. These methods are used to produce 3D contour information from multiple regions of interest (ROIs) in 3D data and thereby allowing reconstructing 3D surface models. Earlier, we created the 3D digital atlas of zebrafish development [3] which provides an online 3D visualization of the anatomy in the zebrafish embryo. It serves as a framework of reference for researchers. Therefore in the context of the atlas, ROIs are anatomical domains in zebrafish embryo. One of the crucial challenges in developmental biology and molecular genetics is to determine how genes interact to control biological processes. Identifying both © 2011 ACEEE DOI: 02.CIT.2011.01. 28

Full Paper Proc. of Int. Conf. on Advances in Communication and Information Technology 2011 to retrieve this related information from a microarray gene expression resource, the ArrayExpress Atlas [17, 18]. The ArrayExpress Atlas of Gene Expression contains a subset of curated and re-annotated archive data from the ArrayExpress Repository which is one of the recommended international repositories to archive publication related functional genomics data [19]. It can be queried for individual gene expression under different biological conditions across experiments. In order to integrate these data correctly we need to provide a basis for cross-domain communication. In this work, we used controlled vocabularies from standard ontologies to annotate the anatomical domains and genes domains on both atlas and gene expression patterns data. This allows our data to be linked together and also enables interoperability and communication with external community resources as well. To this end, we have developed the Bio-Visualization web service as an intermediate component that is responsible for retrieving related information from the underlying web services, including the ArrayExpress Atlas web service. The Bio-Visualization filters and integrates all related gene expression data from external information source(s) onto the existing reconstruction model in order to generate a new visualization model. The web service is designed to be extensible to support more external information source in the future. In support of this work, we developed web applications based on the web services that provide the underlying data required. The web applications provide an overview and allow users to query on our 3D digital atlas along with related gene expression data. In order to deliver the data to end users, we integrate our Java based 3D-viewer (TDRViewer) with the web applications allowing users to visualize the integrated visualization over Internet.

The image datasets for spatial gene expression patterns, on the other hand, are produced using the zebraFISH protocol. The patterns are acquired with the confocal laser scanner microscope as multi-channel 3D images containing the outline of the embryo and the spatial patterns of gene expression in separated channels. The next step is to create 3D reconstruction models from both atlas and gene expression image datasets using our reconstruction software, TDR3Dbased [2] (Fig. 1). The reconstruction software is basically a tool for 3D annotation and surface reconstruction. We used a graphical annotation to specify domains of interest which, in this context, are the boundaries of anatomical domains and/or patterns of gene expression. Textual annotation is accomplished by attaching a term to each graphical annotation. Anatomical domains are annotated with anatomical terms from the Developmental Anatomy Ontology (DAOZ) [21] and gene expression patterns data are annotated with proper gene terms from GEMS. In fact, all controlled vocabularies in both DAOZ and GEMS are extracted from the standard ontologies, i.e.,

II. CONSTRUCTING INFORMATON MODEL In this section the acquisition of the raw data for creating both reconstruction models and patterns of gene expression will be discussed. The 3D reconstruction models and 3D gene expression patterns data in GEMS are created from 3D-image dataset, however, from different modalities. The input data in both cases need to be annotated using terms from standard ontologies. The only difference is that the annotation for reconstruction models is done before submitting the models to the TDR repository whereas annotation for the gene expression data in GEMS has to be done as part of the submission process. A. The 3D Reconstruction Models In the past few years, we have produced a number of 3Dmodels of for the zebrafish atlas as well as spatial patterns of gene expression, in a range of developmental stages; 24, 36, 48, 72 hours post-fertilisation (hpf ) [20]. The first step is to acquire raw data. The 3D image datasets for atlas were acquired in both a normal and high resolution from histological section images of a zebrafish embryo using our dedicated acquisition station [2]. ÂŠ 2011 ACEEE DOI: 02.CIT.2011.01.28

Figure 1. The TDR-3Dbase reconstruction software. This figure shows how to create a 3D reconstruction model of 24 hpf zebrafish embryo. The 3D reconstruction dataset consists of a model description (TDRML) file, section images, contour information and 3D surface information.

ZFIN Anatomical Ontology and the Gene Ontology (GO) [22] respectively. A reconstruction model also contains metadata that need to be annotated correctly with terms from our ontologies, e.g., the stage of development. Annotation for reconstruction models has to be done in the reconstruction 57

Full Paper Proc. of Int. Conf. on Advances in Communication and Information Technology 2011 software and therefore completes before submitting the model to the atlas repository. Each 3D reconstruction model is considered as a single instance of data and is described by a model description, 3D Reconstruction Markup Language (TDRML), which provides scalability and extensibility, both of which are very important for a project that is subject to updates in order to improve quality of the data. Moreover, TDRML facilitates easy exchange between different platforms. Each model description contains information about metadata, section images and annotated domains. Each domain is attached with its contour information and 3D surface data. All of the information described in the model description file will be extracted and subsequently aggregated into a relational database management system, i.e., MySQL. This process is realized by submitting the reconstruction instance to the TDR data repository through a web application.

from standard ontologies provides us the capability to integrate our data with a broad range of external bioinformatics resources, i.e., ZFIN, Ensembl, ArrayExpress Atlas. Therefore, mapping the gene expression data from GEMS and ArrayExpress Atlas onto the 3D reconstruction models is relatively straightforward. This mapping helps answering the question in which anatomical structures in zebrafish a gene of interest is expressed at a particular developmental stage of the embryo. For example, “Which anatomical structures of zebrafish that the gene fgf8a is expressed at the developmental stage of High-pec?” The result from mapping is the list of structures where the gene of interest is expressed, along with other related quantitative experimental data such as P-value and the significant of gene expression. This result will be used later on by the web services to generate proper 3D visualization. III. VISUALIZATION OF GENE EXPRESSION IN 3D GRAPHICAL MODEL

B. The Gene Expression Patterns Data Other than providing controlled vocabulary for textual annotation, GEMS aims to be an integrative information system and repository for 3D spatio-temporal patterns of gene expression. It provides links to related gene expression data on other external gene expression resources [6]. GEMS is capable of organizing and comparing multiple spatial patterns of gene expression at tissue level. GEMS uses the same 3D gene expression patterns image datasets as those for creating reconstruction model for input data. For each 3D image dataset, we used the DAOZ to provide common terms to describe anatomical features and the developmental stages, e.g., list of anatomical structures and developmental stage where a particular gene is expressed. We used terms from GO to describe the expressed gene in the image datasets. In addition, the input image datasets are annotated with imaging conditions and preparation protocol as well. All data annotations have to be done during data submission process. Due to the lacking of array-based functional genomics data in our local resources, we retrieve this information from an external microarray analysis gene expression resource, the ArrayExpress Atlas [18]. ArrayExpress Atlas is a curated set of gene expression datasets that are publicly available through a web services. The query results from the web services are the corresponding experiments and p-values for the differentially expressed genes. WikiPathways Atlas Mapper [23] is an example of online biological pathway resource that provides visualization of an integrative pathway interactions data and gene expression data from ArrayExpress Atlas.

A. Mapping Gene Expression Data to Geometry In this context, the gene expression data can be classified into geometric and non-geometric data. Geometric gene expression data refers to the 3D graphical representation of the locations where a gene is expressed, which, in our case, are the surface data of 3D gene expression patterns derived from 3D image datasets. This type of gene expression data can be mapped directly into the 3D visualization scene together with other 3D anatomical structures data from the zebrafish atlas. Non-geometric gene expression data, i.e., the semantic and quantitative analysis microarray gene expression data, is represented by 3D annotations which can be visualized by using 2D/3D texts and symbols and are integrated into the 3D scene. Typically, there is a lot of quantitative and semantic gene expression information compare to the limited area in the visualization scene, therefore pop-up table and dialog box containing links to further information on external information resources will be used. B. Emphasized Visualization One approach for visualization gene expression data is to hide and emphasize the geometric data of 3D gene expression and 3D anatomical structures. Important objects, or even just a certain object of interest, are highlighted whereas less important objects are hidden, removed or reduced in perceptibility. Apart from the removal case, this technique can be accomplished using only color, transparency and outlines for the visualization.

C. Ontology Based Data Mapping For the visualization of the gene expression data within 3D reconstruction model, both data models have to be integrated. In the 3D reconstruction models, we annotate 3D anatomical domains with anatomical terms from DAOZ, such as YOLK, DIENCEPHALON and ECTODERM, while we annotate 3D gene expression domains with standard gene symbols derived from GO, such as fgf8a and hoxa9a. We annotate all of the 3D models together with the stage of developmental. Annotating the datasets with terms derived © 2011 ACEEE DOI: 02.CIT.2011.01.28

IV. VISUALIZATION SERVICE ARCHITECTURE In this section we will discuss the service architecture of our visualization service (Fig. 2). Various information sources are accessed to retrieve the required data. The reconstruction data repository of 3D atlas data and the 3D patterns of gene expression are stored in a MySQL database server and the server file system. In addition, in to facilitate access to our repositories, TDR and GEMS web services have been implemented. These web services can be used to develop 58

Full Paper Proc. of Int. Conf. on Advances in Communication and Information Technology 2011 client applications providing users a functionality to retrieve and modify the reconstruction and gene expression patterns data in the repositories. The Bio-Visualization web service is an intermediate component that provides standard interfaces for retrieving data from local and external web services. In this work, we developed web applications that allow users to browse and query 3D models in the zebrafish atlas and related patterns of gene expression. The web application uses the Bio-Visualization web service to get related microarray data from external information sources, i.e. the ArrayExpress Atlas, and deliver an online visualization of gene expression data within 3D reconstruction models to end users using Java applets. A. Web Services The TDR web service is implemented to enable query access to the 3D reconstruction data in the zebrafish atlas repository. In similar fashion, the GEMS web service is implemented to provide access to data in GEMS. Both web services can be accessed through the Simple Object Access protocol (SOAP), and the data structure and available functions are described in Web Service Description Language (WSDL). Both SOAP and WSDL are commonly supported standards [24]. With TDR web service, a complete or partial reconstruction model description can be downloaded in TDRML format (Fig. 3). It provides also interfaces to retrieve binary data of a particular reconstruction model, for instance, section images, contour and surface reconstruction information. Together, a client obtains all necessary data to create a 3D visualization of a reconstruction model. GEMS web service provides a query interface for the client to retrieve gene expression data based on annotated information, for instance, gene of interest, stage of development and location where the gene is expressed. All the text-based results are returned in XML format. Both web services also allow the client software to publish information to their underlying data repository as well. The Bio-Visualization web service is implemented as the intermediate component for a client. The web service uses TDR and GEMS web services to get access to data in local repositories. In addition, Bio-Visualization web service also uses the ArrayExpress Atlas web service to retrieve related experimental array-based gene expression data from the ArrayExpress Repository. The web service allows the user to query for condition-specific based on set of genes by name, organism, and developmental stage. What is returned from ArrayExpress Atlas web service is an XML containing the list of corresponding experimental data related to the gene of interest, each with P-values and an up/down characterizing the significance and direction of differentially expressed genes [17]. The result is in Microarray Gene Expression Markup Language (MAGE-ML) [25] (Fig. 4). The XML-based format has been developed by The Functional Genomics Data (FGED) society [26] and Object Management Group (OMG) [27].

ÂŠ 2011 ACEEE DOI: 02.CIT.2011.01. 28

Figure 2. System architecture of visualization service.

After receiving XML results from all of the underlying web services, the Bio-Visualization web service filters out the unnecessary information received from the ArrayExpress Atlas such as the data that is related to the anatomical parts which do not exist in the 3D reconstruction model of interest. The filtered microarray data will be mapped onto the 3D reconstruction data received from TDR web service and the extended version of TDRML will be generated. This version of TDRML contains not only the original 3D reconstruction data but also contains the quantitative microarray data related each anatomical structure existing in the 3D model of interest. In the end, the output TDRML will be delivered to the visualization client, the TDRViewer, over Internet along with the related binary data, i.e., section images, 3D contour and surface information. The Bio-Visualization web service is designed to be extensible in order to support more external information resources in the future. From the client point-of-view, the Bio-Visualization web service provides a consistent programming interface for client to retrieve data from heterogeneous sources. B. Web Applications The web applications provide query web interface allowing users to search for the reconstruction model of interest based on anatomical structures, developmental

Full Paper Proc. of Int. Conf. on Advances in Communication and Information Technology 2011

Figure 3. An example of TDRML resulted from TDR web service: a complete model description for 3D reconstruction model of spatial gene expression patterns: 14-3-3 in 48 hpf zebrafish embryo. The geometrical gene expression data is outlined with red box.

Figure 5. The first page of the web application shows the list of available reconstruction models of atlas and 3D gene expression.

Figure 6. The model information page shows links to the related 3D gene expression model and the related whole mount in situ hybridization data in GEMS. More information about each anatomical structure can also be found by following the link to external resource, ZFIN

Figure 4. An example of XML result from ArrayExpress Atlas web service. The first part of the result contains gene information such as GO and Ensembl identifiers, organism and gene name. The second part contains a list of microarray gene expression data from different experiments.

C. The TDRViewer In order to provide 3D interactive visualization over the Internet, we have been developing and improving a highly portable 3D reconstruction model viewer, TDRViewer (Fig. 7). This viewer is an improved version of the atlas viewer we developed earlier for the digital atlas of zebrafish development. TDRViewer is implemented using Java technology and can be used as a stand-alone application or can be integrated with a web interface as a Java applet allowing online interactive visualization. The TDRViewer allows users to visualize our datasets in both 2D and 3D views. The 2D view shows a particular section image together with its 2D graphical annotations of the domains of interest; anatomical structures for atlas dataset and the areas where a gene is expressed for spatial gene expression data. The user has options to change the zooming level and the section image. The 3D view provides 3D

stages (Fig. 5). For each reconstruction model, the web applications also provide the links, based on the developmental stage, to the related 3D gene expression patterns models and the related whole mount in situ hybridization experimental data from GEMS (Fig. 6). The data access layer of the web applications was implemented to adopt the newly introduced Bio-Visualization web service. The query performed by user is subsequently executed using the underlying web services. The web applications allow users to publish new 3D data of atlas and gene expression to the corresponding repository as well. As the web applications receive all required 3D visualization data from the Bio-Visualization web service, they pass the data to the client, a Java-based 3D viewer applet to deliver the visualization to users. ÂŠ 2011 ACEEE DOI: 02.CIT.2011.01.28

Full Paper Proc. of Int. Conf. on Advances in Communication and Information Technology 2011 visualization in one of the three view modes: contour view, solid view, and surface view. In the 3D view, user has options to visualize section plane and section images in 3D scene as well. In this paper, we integrate the TDRViewer with our web application. After receiving the (extended version of) TDRML from the server, the TDRViewer parses all the data and requests for additional binary data described in TDRML; section images, 3D contour and surface information. Aside from the TDRML file, all binary data are compressed on the server before sending and decompressed after receiving at the viewer.

alized directly into the 3D scene while the non-geometric data can be visualized as 3D annotations using texts and symbols. More information on each microarray experiment and results can be found by following the available link which redirects user to the ArrayExpress Repository web site. V. CONCLUSIONS We have developed a visualization system that provides online visualization of gene expression information within 3D reconstruction model for the early developmental stages of zebrafish; i.e., 24, 36, 48 and 72 hpf. To support this, we have implemented TDR and GEMS web services that provide interfaces for a client to access our 3D reconstruction and 3D gene expression patterns data in the repositories. We also implemented an intermediate web service, the BioVisualization, as a client for retrieving data from local and external web services, i.e., TDR, GEMS and ArrayExpress Atlas. The Bio-Visualization is responsible for filtering unrelated experimental data received from the ArrayExpress Atlas and mapping the result onto the 3D reconstruction model. Mapping all aspects of related gene expression patterns data is accomplished by using an ontology based mapping; using annotated ontology terms to query related gene expression data from local and external resources. The Bio-Visualization web service generates an extended model description, TDRML, which contains not only the original reconstruction data but also the related gene expression data. The web service is designed to be extensible to support more information resources in the future. It also provides a standard data interface to retrieve data from underlying web services. In order to deliver the visualization to end users, a web application is developed. The web application provides a query web interface allowing users to search for the reconstruction model of interest based on anatomical structures and developmental stages. The web application also incorporates the TDRViewer applet allowing users to visualize the graphically combined data interactively over the Internet. The geometric representation of the gene expression data such as the area where the gene is expressed can be directly integrated into a 3D scene with 3D anatomical domains but other gene expression data that do not have a geometric representation (i.e. microarray data) can be visualized as 3D annotations. To limit the amount of annotated information in the 3D scene, a pop-up menu or dialog box containing links to further information on external information resources will be used. In this way, users are able to derive relations between the spatial information of 3D reconstruction models and patterns of gene expression in a 3D context.

Figure 7. TDRViewer in the digital atlas of zebrafish: a surface view of 3D digital atlas of a 48 hpf zebrafish embryo.

Figure 8. A surface visualization with a 3D section image of gene expression patterns: 14-3-3 gamma2 in a 48 hpf embryo; the gene expression is annotated in white together with some reference anatomical structures. Related microarray gene expression data on the gene 14-3-3 from ArrayExpress Atlas are annotated in the lower left corner of the 3D scene. This information indicates the anatomical structures that this gene is expressed and how much it is expressed. The annotation also provides links to all related experimental data in the ArrayExpress Atlas.

ACKNOWLEDGMENTS The authors wish to express their gratitude to Gerda Lamers, Esther Dondorp, Rebecca Schoon, Laura Bertens, Monique Welten, Willemijn Spoor and Aimy Sels for providing the experimental data and creating 3D reconstruction models from atlas and 3D gene expression patterns datasets.

The viewer uses the available geometric data to construct 3D scene and overlaying the gene expression data onto the 3D graphical model of the reconstruction data. As previously mentioned, the geometric gene expression data can be visu ÂŠ 2011 ACEEE DOI: 02.CIT.2011.01.28

Full Paper Proc. of Int. Conf. on Advances in Communication and Information Technology 2011 This work is partially supported by Netherlands’ council for Scientific Research (NWO) and a personal grant from the Ministry of Science and Technology, Thai Government.

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