A conserved RNA switch for acetylcholine receptor clustering at neuromuscular junctions in chordates.

In mammals, neuromuscular synapses rely on clustering of acetylcholine receptors (AChRs) in the muscle plasma membrane, ensuring optimal stimulation by motor neuron-released acetylcholine neurotransmitter. This clustering depends on a complex pathway based on alternative splicing of Agrin mRNAs by the RNA-binding proteins Nova1/2. Neuron-specific expression of Nova1/2 ensures the inclusion of small "Z" exons in Agrin, resulting in a neural-specific form of this extracellular proteoglycan carrying a short peptide motif that is required for binding to Lrp4 receptors on the muscle side, which in turn stimulate AChR clustering. Here we show that this intricate pathway is remarkably conserved in Ciona robusta, a non-vertebrate chordate in the tunicate subphylum. We use in vivo tissue-specific CRISPR/Cas9-mediated mutagenesis and heterologous "mini-gene" alternative splicing assays in cultured mammalian cells to show that Ciona Nova is also necessary and sufficient for Agrin Z exon inclusion and downstream AChR clustering. We present evidence that, although the overall pathway is well conserved, there are some surprising differences in Nova structure-function between Ciona and mammals. We further show that, in Ciona motor neurons, the transcription factor Ebf is a key activator of Nova expression, thus ultimately linking this RNA switch to a conserved, motor neuron-specific transcriptional regulatory network.

CRISPR/Cas9 protocols for disrupting gene function in the non-vertebrate chordate Ciona.

The evolutionary origins of chordates and their diversification into the three major subphyla of tunicates, vertebrates, and cephalochordates pose myriad questions about the genetic and developmental mechanisms underlying this radiation. Studies in non-vertebrate chordates have refined our model of what the ancestral chordate may have looked like, and have revealed the pre-vertebrate origins of key cellular and developmental traits. Work in the major tunicate laboratory model Ciona has benefitted greatly from the emergence of CRISPR/Cas9 techniques for targeted gene disruption. Here we review some of the important findings made possible by CRISPR in Ciona, and present our latest protocols and recommended practices for plasmid-based, tissue-specific CRISPR/Cas9-mediated mutagenesis.

Specification and survival of post-metamorphic branchiomeric neurons in a non-vertebrate chordate.

Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are replaced by adult-specific ones. The regulatory mechanisms underlying this replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the 'neck', a cellular compartment set aside in the larva to give rise to cranial motor neuron-like neurons post-metamorphosis. Using bulk and single-cell RNA-sequencing analyses, we characterize the transcriptome of the neck downstream of Pax2/5/8. We present evidence that neck-derived adult ciliomotor neurons begin to differentiate in the larva and persist through metamorphosis, contrary to the assumption that the adult nervous system is formed after settlement and the death of larval neurons during metamorphosis. Finally, we show that FGF signaling during the larval phase alters the patterning of the neck and its derivatives. Suppression of FGF converts neck cells into larval neurons that fail to survive metamorphosis, whereas prolonged FGF signaling promotes an adult neural stem cell-like fate.

Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona.

Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.

Specification and survival of post-metamorphic branchiomeric neurons in the hindbrain of a non-vertebrate chordate.

Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are largely replaced by adult-specific ones. Yet the regulatory mechanisms underlying this neural replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the "Neck", a compartment of cells set aside in the larva to give rise to cranial motor neuron-like neurons in the adult. Using bulk and single-cell RNAseq analyses, we also characterize the transcriptome of the Neck downstream of Pax2/5/8. Surprisingly, we find that Neck-derived adult ciliomotor neurons begin to differentiate in the larva, contrary to the long-held assumption that the adult nervous system is formed only after settlement and the death of larval neurons during metamorphosis. Finally, we show that manipulating FGF signaling during the larval phase alters the patterning of the Neck and its derivatives. Suppression of FGF converts Neck cells into larval neurons that fail to survive metamorphosis, while prolonged FGF signaling promotes an adult neural stem cell-like fate instead.

Loss of collagen gene expression in the notochord of the tailless tunicate Molgula occulta.

In tunicates, several species in the Molgulidae family have convergently lost the tailed, swimming larval body plan, including the morphogenesis of the notochord, a major chordate-defining trait. Through the comparison of tailless M. occulta and a close relative, the tailed species M. oculata, we show that notochord-specific expression of the Collagen Type I/II Alpha (Col1/2a) gene appears to have been lost specifically in the tailless species. Using CRISPR/Cas9-mediated mutagenesis in the tailed laboratory model tunicate Ciona robusta, we demonstrate that Col1/2a plays a crucial role in the convergent extension of notochord cells during tail elongation. Our results suggest that the expression of Col1/2a in the notochord, although necessary for its morphogenesis in tailed species, is dispensable for tailless species. This loss is likely a result of the accumulation of cis-regulatory mutations in the absence of purifying selective pressure. More importantly, the gene itself is not lost, likely due to its roles in other developmental processes, including during the adult stage. Our study further confirms the Molgulidae as an interesting family in which to study the evolutionary loss of tissue-specific expression of indispensable genes.

Ebf Activates Expression of a Cholinergic Locus in a Multipolar Motor Ganglion Interneuron Subtype in Ciona.

Conserved transcription factors termed "terminal selectors" regulate neuronal sub-type specification and differentiation through combinatorial transcriptional regulation of terminal differentiation genes. The unique combinations of terminal differentiation gene products in turn contribute to the functional identities of each neuron. One well-characterized terminal selector is COE (Collier/Olf/Ebf), which has been shown to activate cholinergic gene batteries in C. elegans motor neurons. However, its functions in other metazoans, particularly chordates, is less clear. Here we show that the sole COE ortholog in the non-vertebrate chordate Ciona robusta, Ebf, controls the expression of the cholinergic locus VAChT/ChAT in a single dorsal interneuron of the larval Motor Ganglion, which is presumed to be homologous to the vertebrate spinal cord. We propose that, while the function of Ebf as a regulator of cholinergic neuron identity conserved across bilaterians, its exact role may have diverged in different cholinergic neuron subtypes (e.g., interneurons vs. motor neurons) in chordate-specific motor circuits.

Neurobiology: Swimming at the Intersection of Light and Gravity.

Many animals use gravity as a spatial reference to help navigate their surroundings, but how they do so is not well understood. A new study reveals that a representative of our closest invertebrate relatives, the tunicate Ciona, processes light and gravity cues through a simple neural circuit to decide when and how to swim.