Eileen Wagner

Identification of a rudimentary neural crest in a non-vertebrate chordate

Neural crest arises at the neural plate border, expresses a core set of regulatory genes and produces a diverse array of cell types, including ectomesenchyme derivatives that elaborate the vertebrate head. The evolution of neural crest has been proposed to be a key event leading to the appearance of new cell types that fostered the transition from filter feeding to active predation in ancestral vertebrates. However, the origin of neural crest remains controversial, as homologous cell types have not been unambiguously identified in non-vertebrate chordates. Here we show that the tunicate Ciona intestinalis possesses a cephalic melanocyte lineage (a9.49) similar to neural crest that can be reprogrammed into migrating ‘ectomesenchyme’ by the targeted misexpression of Twist (also known as twist-like 2). Our results suggest that the neural crest melanocyte regulatory network pre-dated the divergence of tunicates and vertebrates. We propose that the co-option of mesenchyme determinants, such as Twist, into the neural plate ectoderm was crucial to the emergence of the vertebrate ‘new head’.

FGF signaling establishes the anterior border of the Ciona neural tube

The Ciona tadpole is constructed from simple, well-defined cell lineages governed by provisional gene networks that have been defined via extensive gene disruption assays. Here, we examine the patterning of the anterior neural plate, which produces placodal derivatives such as the adhesive palps and stomodeum, as well as the sensory vesicle (simple brain) of the Ciona tadpole. Evidence is presented that the doublesex-related gene DMRT is expressed throughout the anterior neural plate of neurulating embryos. It leads to the activation of FoxC and ZicL in the palp placode and anterior neural tube, respectively. This differential expression depends on FGF signaling, which inhibits FoxC expression in the anterior neural tube. Inhibition of FGF signaling leads to expanded expression of FoxC, the loss of ZicL, and truncation of the anterior neural tube.

Neural tube patterning by Ephrin, FGF and Notch signaling relays

The motor ganglion (MG) controls the rhythmic swimming behavior of the Ciona intestinalis tadpole. Despite its cellular simplicity (five pairs of neurons), the MG exhibits conservation of transcription factor expression with the spinal cord of vertebrates. Evidence is presented that the developing MG is patterned by sequential Ephrin/FGF/MAPK and Delta/Notch signaling events. FGF/MAPK attenuation by a localized EphrinAb signal specifies posterior neuronal subtypes, which in turn relay a Delta2/Notch signal that specifies anterior fates. This short-range relay is distinct from the patterning of the vertebrate spinal cord, which is a result of opposing BMP and Shh morphogen gradients. Nonetheless, both mechanisms lead to localized expression of related homeodomain codes for the specification of distinct neuronal subtypes. This MG regulatory network provides a foundation for elucidating the genetic and cellular basis of a model chordate central pattern generator.

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.

Diverse ETS transcription factors mediate FGF signaling in the Ciona anterior neural plate

The ascidian Ciona intestinalis is a marine invertebrate belonging to the sister group of the vertebrates, the tunicates. Its compact genome and simple, experimentally tractable embryos make Ciona well-suited for the study of cell-fate specification in chordates. Tunicate larvae possess a characteristic chordate body plan, and many developmental pathways are conserved between tunicates and vertebrates. Previous studies have shown that FGF signals are essential for neural induction and patterning at sequential steps of Ciona embryogenesis. Here we show that two different ETS family transcription factors, Ets1/2 and Elk1/3/4, have partially redundant activities in the anterior neural plate of gastrulating embryos. Whereas Ets1/2 promotes pigment cell formation in lateral lineages, both Ets1/2 and Elk1/3/4 are involved in the activation of Myt1L in medial lineages and the restriction of Six3/6 expression to the anterior-most regions of the neural tube. We also provide evidence that photoreceptor cells arise from posterior regions of the presumptive sensory vesicle, and do not depend on FGF signaling. Cells previously identified as photoreceptor progenitors instead form ependymal cells and neurons of the larval brain. Our results extend recent findings on FGF-dependent patterning of anterior-posterior compartments in the Ciona central nervous system.

Islet is a key determinant of ascidian palp morphogenesis

The anterior-most ectoderm of ascidian larvae contains the adhesive papillae, or palps, which play an important role in triggering the metamorphosis of swimming tadpoles. In Ciona intestinalis, the palps consist of three conical protrusions within a field of thickened epithelium that form late in embryogenesis, as tailbuds mature into larvae. The palp protrusions express the LIM-homeodomain transcription factor Islet. Protrusion occurs through differential cell elongation, probably mediated by Islet, as we find that ectopic expression of Islet is sufficient to promote cell lengthening. FGF signaling is required for both Islet expression and palp morphogenesis. Importantly, we show that Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling. We conclude that Islet is a key regulatory factor governing morphogenesis of the palps. It is conceivable that Islet is also essential for the cellular morphogenesis of placode-derived sensory neurons 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.