RNA sequencing reveals a diverse and dynamic repertoire of the Xenopus tropicalis transcriptome over development

Xenopus is one of the major model systems for the study of vertebrate embryogenesis and basic cell biological processes. There are multiple advantages to the use of Xenopus as an experimental system, such as the availability of large abundant eggs that are easily manipulated, ready accessibility to any developmental stage, and conservation of cellular pathways between Xenopus and mammals. In the past 50 years, landmark studies on Xenopus have been critical toward our understanding of nuclear reprogramming (Gurdon et al. 1958), embryonic patterning (Harland and Gerhart 1997; De Robertis 2006), membrane channels and receptors (Kusano et al. 1977), and cell cycle control (Murray and Kirschner 1989; Murray et al. 1989; Glotzer et al. 1991). Genomics resources for Xenopus research have emerged in the past 10–15 years. During the early days of the genomics era, several cDNA sequencing efforts, such as EST (expressed sequence tag) projects, have allowed the construction of full length cDNA clones and identification of Xenopus open reading frames (ORFs) (Gilchrist et al. 2004; Morin et al. 2006; Fierro et al. 2007). Microarrays have also been used to investigate the expression levels of annotated genes and gave some insights into transcriptome changes over development as well as expression differences between two closely related frog species, Xenopus laevis and Xenopus tropicalis (Yanai et al. 2011). In addition, forward and reverse genetic screens have uncovered mutations that affect a myriad of organogenesis and differentiation processes in Xenopus (Goda et al. 2006), while a genetic map based on simple sequence length polymorphism (SSLP) markers, which can be used to clone genes identified by mutation, has recently been generated (Wells et al. 2011). Notably, while early developmental and molecular studies have been performed on Xenopus laevis, its closely related cousin Xenopus tropicalis has proven to be more widely used for genetic and genomic research. This is mainly because Xenopus laevis has a more complex pseudotetraploid genome, while Xenopus tropicalis has a smaller and more amenable diploid genome. Hence, the initial genome sequencing effort has been directed mostly at Xenopus tropicalis, whose genome has recently been published (Hellsten et al. 2010). Strikingly, the frog genome is highly syntenic with the human genome, with regions of synteny frequently spanning more than a hundred genes. Nevertheless, although it is largely assembled into multiple scaffolds, the Xenopus tropicalis genome is yet to be sequenced at the same depth and annotated at the same level of details and accuracy as the genomes of human and mouse. Importantly, annotations of protein-coding and noncoding genes are strikingly incomplete, including the widely used RefSeq and Ensembl annotations. The advent of high-throughput sequencing technologies has had an enormous impact on genomics. In particular, such technologies have revolutionized studies of the transcriptome in many species from yeast to humans and have revealed tremendous amounts of complexities and gaps in our understanding of any transcriptome (Wang et al. 2009). Not only does RNA sequencing (RNA-seq) provide a more accurate measurement of expression levels, it provides single nucleotide resolution and has the ability to reveal novel splice junctions, unannotated transcripts, and allele-specific expression. Here, we present the first comprehensive study of the transcriptome of Xenopus tropicalis using RNA-seq over development from a two-cell fertilized embryo to a feeding tadpole. We report evidence for transcription of more than a hundred genes prior to the midblastula transition, when the embryonic genome is generally believed to be transcriptionally silent. We also discovered thousands of novel splicing events, including exon skipping in annotated genes, as well as thousands of unannotated, potentially noncoding transcripts. Hence, our data serve as a valuable resource for developmental biologists and the general genomics community. Furthermore, to extend the reach of our work, we have created an interactive website (http://hci.stanford.edu/∼jcchuang/frog-genes/latest/) that allows users to not only browse the heatmaps in this manuscript but to also query the expression profile of any RefSeq or Ensembl annotated gene with ease.