Weiyang Shi

A distinct class of small RNAs arises from pre-miRNA–proximal regions in a simple chordate

MicroRNAs (miRNAs) have been implicated in various cellular processes. They are thought to function primarily as inhibitors of gene activity by attenuating translation or promoting mRNA degradation. A typical miRNA gene produces a predominant ∼21-nucleotide (nt) RNA (the miRNA) along with a less abundant miRNA* product. We sought to identify miRNAs from the simple chordate Ciona intestinalis through comprehensive sequencing of small RNA libraries created from different developmental stages. Unexpectedly, half of the identified miRNA loci encode up to four distinct, stable small RNAs. The additional RNAs, miRNA-offset RNAs (moRs), are generated from sequences immediately adjacent to the predicted ∼60-nt pre-miRNA. moRs seem to be produced by RNAse III–like processing, are ∼20 nt long and, like miRNAs, are observed at specific developmental stages. We present evidence suggesting that the biogenesis of moRs results from an intrinsic property of the miRNA processing machinery in C. intestinalis.

Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis

The bHLH transcription factor Mesp has an essential but ambiguous role in early chordate heart development. Here, we employ the genetic and morphological simplicity of the basal chordate Ciona intestinalis to elucidate Mesp regulation and function. Characterization of a minimal cardiac enhancer for the Ciona Mesp gene demonstrated direct activation by the T-box transcription factor Tbx6c. The Mesp enhancer was fused to GFP, permitting high-resolution visualization of heart cells as they migrate and divide. The enhancer was also used to drive targeted expression of an activator form of Mesp, which induces heart formation without migration. We discuss the implications of Tbx6-Mesp interactions for the evolution of cardiac mesoderm in invertebrates and vertebrates.

miRTRAP, a computational method for the systematic identification of miRNAs from high throughput sequencing data

MicroRNAs (miRs) have been broadly implicated in animal development and disease. We developed a novel computational strategy for the systematic, whole-genome identification of miRs from high throughput sequencing information. This method, miRTRAP, incorporates the mechanisms of miR biogenesis and includes additional criteria regarding the prevalence and quality of small RNAs arising from the antisense strand and neighboring loci. This program was applied to the simple chordate Ciona intestinalis and identified nearly 400 putative miR loci.

FoxF is essential for FGF-induced migration of heart progenitor cells in the ascidian Ciona intestinalis

Heart development requires precise coordination of morphogenetic movements with progressive cell fate specification and differentiation. In ascidian embryos, FGF/MAPK-mediated activation of the transcription factor Ets1/2 is required for heart tissue specification and cell migration. We found that FoxF is one of the first genes to be activated in heart precursors in response to FGF signaling. We identified the FoxF minimal heart enhancer and used a cis-trans complementation test to show that Ets1/2 can interact with the FoxF enhancer in vivo. Next, we found that FoxF function is required downstream and in parallel to the FGF/MAPK/Ets cascade for cell migration. In addition, we demonstrated that targeted expression of a dominant-negative form of FoxF inhibits cell migration but not heart differentiation, resulting in a striking phenotype: a beating heart at an ectopic location within the body cavity of juveniles. Taken together, our results indicate that FoxF is a direct target of FGF signaling and is predominantly involved in the regulation of heart cell migration.

Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo

Mesodermal tissues arise from diverse cell lineages and molecular strategies in the Ciona embryo. For example, the notochord and mesenchyme are induced by FGF/MAPK signaling, whereas the tail muscles are specified autonomously by the localized determinant, Macho-1. A unique mesoderm lineage, the trunk lateral cells, develop from a single pair of endomesoderm cells, the A6.3 blastomeres, which form part of the anterior endoderm, hematopoietic mesoderm and muscle derivatives. MAPK signaling is active in the endoderm descendants of A6.3, but is absent from the mesoderm lineage. Inhibition of MAPK signaling results in expanded expression of mesoderm marker genes and loss of endoderm markers, whereas ectopic MAPK activation produces the opposite phenotype: the transformation of mesoderm into endoderm. Evidence is presented that a specific Ephrin signaling molecule, Ci-ephrin-Ad, is required to establish asymmetric MAPK signaling in the endomesoderm. Reducing Ci-ephrin-Ad activity via morpholino injection results in ectopic MAPK signaling and conversion of the mesoderm lineage into endoderm. Conversely, misexpression of Ci-ephrin-Ad in the endoderm induces ectopic activation of mesodermal marker genes. These results extend recent observations regarding the role of Ephrin signaling in the establishment of asymmetric cell fates in the Ciona notochord and neural tube.

FGF3 in the floor plate directs notochord convergent extension in the Ciona tadpole

Convergent extension (CE) is the narrowing and lengthening of an embryonic field along a defined axis. It underlies a variety of complex morphogenetic movements, such as mesoderm elongation and neural tube closure in vertebrate embryos. Convergent extension relies on the same intracellular molecular machinery that directs planar cell polarity (PCP) in epithelial tissues, including non-canonical Wnt signaling components. However, it is not known what signals coordinate CE movements across cell fields. In the simple chordate Ciona intestinalis, the notochord plate consists of just 40 cells, which undergo mediolateral convergence (intercalation) to form a single cell row. Here we present evidence that a localized source of FGF3 in the developing nerve cord directs notochord intercalation through non-MAPK signaling. A dominant-negative form of the Ciona FGF receptor suppresses the formation of polarized actin-rich protrusions in notochord cells, resulting in defective notochord intercalation. Inhibition of Ciona FGF3 activity results in similar defects, even though it is expressed in an adjacent tissue: the floor plate of the nerve cord. In Xenopus mesoderm explants, inhibiting FGF signaling perturbs CE and disrupts membrane localization of Dishevelled (Dsh), a key regulator of PCP and CE. We propose that FGF signaling coordinates CE movements by regulating PCP pathway components such as Dsh.

Microinjection of Morpholino Oligos and RNAs in Sea Squirt (Ciona) Embryos

This protocol describes microinjection of morpholino oligos (MOs) and RNAs into sea squirt (Ciona) embryos. This is the method of choice for gene disruption assays. MOs that target the initiating ATG can be used, in addition to those that target splice donor and acceptor sites. The latter method permits the selective inhibition of zygotic mRNAs in cases in which the gene in question is expressed in both the egg and embryo. Although injection is usually performed at the one-cell stage, it is possible to target individual blastomeres, up to the eight-cell stage, thereby permitting lineage-specific knockdown of pleiotropic genes. Injection can also be performed in unfertilized eggs to inhibit maternal genes. Microinjection also permits DiI labeling and lineage tracing.

The Sea Squirt Ciona intestinalis

Sea squirts (Ciona intestinalis) are tunicates (or urochordates), the closest living relatives of the vertebrates. Although the adults are simple, sessile filter feeders, the embryos and larvae possess clear chordate features including a prominent notochord and dorsal, hollow neural tube. Tail-bud-stage embryos and mature swimming tadpoles are composed of approximately 1000 and 2600 cells, respectively, with complete lineage information. This cellular simplicity is coupled with a streamlined genome that has not undergone the duplications seen in vertebrates. A variety of molecular tools have been applied to understanding Ciona embryogenesis. Comparisons of the C. intestinalis genome and the related but divergent Ciona savignyi genome have facilitated the identification of conserved noncoding DNAs, including regulatory DNAs such as tissue-specific enhancers. Systematic in situ hybridization assays and gene-disruption experiments using specific morpholino antisense oligonucleotides have led to the elaboration of provisional gene regulatory networks underlying the specification of key chordate tissues, including the notochord, neural tube, and beating heart. These networks provide a foundation for understanding the mechanistic basis of more complex cell-specification processes in vertebrates, and for understanding the evolutionary origins of distinctive vertebrate characteristics such as the neural crest. Because tunicates and vertebrates are sister groups, there is every indication that the developmental mechanisms revealed in the simple Ciona model will be applicable to comparable processes in vertebrates.

X-gal Staining of Electroporated Sea Squirt (Ciona) Embryos

This protocol describes the fixation, staining, and mounting of sea squirt (Ciona intestinalis) embryos and larvae that have been electroporated with a plasmid DNA containing a cis-regulatory DNA (e.g., tissue-specific enhancer) fused to the lacZ reporter gene. Green fluorescent protein (GFP) reporter genes can be directly visualized in living embryos and larvae, although fixed preparations can use aspects of this protocol.

8 Unraveling Genomic Regulatory Networks in the Simple Chordate, Ciona intestinalis

The ancestral chordate gave rise to three groups, i.e., the vertebrates, cephalochordates, and tunicates (which include the ascidians). While the vertebrates diversified into many familiar aquatic and terrestrial species, the cephalochordates and tunicates remained in the ocean and evolved into highly specialized filter feeders. In the tunicates, acquisition of an endoglucanase gene and cellulose tunic (possibly derived by lateral gene transfer from bacteria) (see Dehal et al. 2002; Matthysse et al. 2004; Nakashima et al. 2004) led to the evolution of a highly divergent adult body plan lacking almost all coelomic cavities. However, the morphogenesis of chordate structures (e.g., notochord and dorsal nerve cord) in tunicate tadpoles has been well conserved despite the “retrograde” evolution of adult anatomy. The recent sequencing of two closely related ascidian species, Ciona intestinalis and Ciona savignyi , has revived the status of the ascidians as a major developmental model system (Dehal et al. 2002; Satoh 2003; Satoh et al. 2003). Research has confirmed that many aspects of ascidian and vertebrate embryogenesis rely on a conserved set of orthologous genes and cellular processes (Satoh 2003; Passamaneck and Di Gregorio 2005). This is clearly the case for development of the nervous system (Meinertzhagen et al. 2004), notochord (Passamaneck and Di Gregorio 2005), and heart (Satou et al. 2004) and may also be true for other tissues and organs, including the blood cells, pharyngeal gill slits, endostyle, and neural crest (Jeffery et al. 2004). However, ascidian embryos are extraordinarily simple, with low cell numbers, rapid development, and well-defined...