Biology subjects

Popsuj, S.

Publications and source records attributed to Popsuj, S..

5 recordsLinked to original sources

bioRxiv2026

Validated CRISPR/Cas9 guide RNAs targeting neurodevelopmental genes in the tunicate Ciona robusta

The development of the central nervous system (CNS) depends on tightly regulated gene expression programs that guide neural progenitor differentiation and neuronal subtype specification. The tunicate Ciona robusta provides a powerful and simplified model for dissecting the genetic control of nervous system development, with a larval CNS composed of just over 200 neurons and sensory cells. Although CRISPR/Cas9-mediated mutagenesis is now routinely used in Ciona, validated single-guide RNAs (sgRNAs) have yet to be validated for key neural genes. Here, we report the design and experimental validation of 25 novel sgRNAs targeting eight conserved genes encoding conserved proteins involved in neurodevelopment and neural function, including six transcription factors (Cdx, Foxb, Sox1/2/3, Dmbx, Engrailed, and Mnx) and two neural effector genes (Tyrosinase and Slc18a3/VAChT). Candidate sgRNAs were selected using CRISPOR and tested for mutagenesis efficiency using Illumina-based target site amplicon sequencing. All sgRNAs induced insertions or deletions at their target loci, with most genes yielding at least one sgRNA with mutagenesis efficacy exceeding 30%, with the exception of Dmbx, for which maximal efficacy reached 25%. We further compared measured mutagenesis rates with predicted Doench 16 and Doench Ruleset 3 (RS3) scores, observing a modest but improved correlation with RS3 predictions. Based on these results, we recommend considering both scoring algorithms, with RS3 potentially offering improved predictive value for Ciona.

developmental biology↗

bioRxiv2025

Regulation of motor neuron differentiation in the Ciona larva

Here we investigate the gene regulatory networks responsible for the differentiation of cholinergic neurons in the Ciona larval motor ganglion, which functions as the organisms central pattern generator for swimming and dispersal. We demonstrate conserved roles motor neuron-enriched transcription factors Neurogenin and Onecut, with Neurogenin likely sitting atop the regulatory cascade. We also identify a key role for the transcription factor Nkx6 in specifying one motor neuron subtype, Motor Neuron 1 (MN1), and show that the secreted Wnt pathway inhibitor Dkk3 is required by MN1 for its unique neuromuscular endplates. We propose that Dkk3 interacts with LRP receptors in target muscle cells, through conserved NxI/V/F motifs shared with another key secreted neuromuscular synapse effector, Agrin. Taken together, our results provide critical insights into the development and evolution of cholinergic neurons involved in chordate locomotion.

developmental biology↗

bioRxiv2025

A protocadherin mediates cell-cell adhesion and integrity of the oral placode in the tunicate Ciona

In chordate embryos, placodes are ectodermal thickenings around the borders of the neural plate that give rise to various sensory organs and cell types. While generally thought to be a vertebrate-specific innovation, homologous placodes are proposed to exist in non-vertebrate chordates as well. In Ciona robusta, a solitary tunicate, the adult mouth (the oral siphon) is derived from one such "cranial-like" placode in the larva, which we term the oral siphon placode (OSP). At embryonic and larval stages, the OSP consists of a small rosette of cells that forms from the neuropore at the anteriormost extent of neural tube closure. While the morphogenesis of the OSP and its physical separation from other surface ectoderm structures have been described in detail, how this is regulated at the molecular level is currently unknown. Here we show the involvement of protocadherin-mediated cell-cell adhesion in the segregation and structural cohesiveness of the OSP. Protocadherin.e (Pcdhe.e) is expressed specifically in the OSP but not in other surface ectoderm cells. CRISPR/Cas9-mediated disruption of Pcdh.e in these cells results in loss of OSP structural integrity and ability to physically separate from other structures derived from the same cell lineage. Overexpression of Pcdh.e throughout the anterior surface ectoderm results in similar loss of a physically separate and distinct OSP territory. Furthermore, we show that Pcdh.e expession in the OSP depends on oral placode-specific transcription factors such as Six1/2 and Pitx. Our results suggest that OSP integrity and morphogenesis require precise regulation of a homotypic cell-cell adhesion molecule, which might reflect a conserved mechanism for placode formation in chordates.

developmental biology↗

bioRxiv2024

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.

developmental biology↗

bioRxiv2023

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.

developmental biology↗