Biology subjects

Stolfi, A.

Publications and source records attributed to Stolfi, A..

18 recordsLinked to original sources

bioRxiv2026

Fine-tuning neuron number: Lzts as a key regulator of neurogenesis and cell proliferation in Ciona

How progenitor mitotic timing is coupled to the number of neurons in a nervous system remains poorly understood, in part because most model systems have large, variable progenitor pools that obscure this relationship. The non-vertebrate chordate Ciona robusta offers a tractable alternative: its larval nervous system arises from an invariant lineage, including four Bipolar Tail Neurons (BTNs) that are reproducibly generated from the caudal neural plate border. Here we investigate the developmental function of the single Ciona ortholog belonging to the leucine zipper tumor suppressor (Lzts) gene family, whose vertebrate members are implicated in cell-cycle regulation and tumor suppression. In Ciona, Lzts is expressed in several embryonic tissues, including differentiating BTNs. Tissue-specific CRISPR/Cas9 knockout of Lzts in the BTN lineage produced supernumerary BTNs, without expanding the embryonic Neurog+ domain, indicating that Lzts acts downstream of BTN specification to limit neuron number. Lzts1 promotes Cdk1 activity via stabilization of Cdc25C, co-expression of Ciona Cdc25C or a non-phosphorylatable Cdk1/2/3 mutant rescued normal BTN number in Lzts crispants, proving the existence of an ancestral Lzts-Cdc25-Cdk1 circuit controlling neural cell cycle. These findings establish Ciona Lzts as a regulator of mitotic tempo determining the BTN number and suggest that this cell-cycle regulatory axis links proliferation to neurogenesis across chordates in vivo.

developmental biology↗

bioRxiv2026

Using CRISPR/Cas9 to investigate the role of candidate human disease gene orthologs in Ciona

The tunicate Ciona robusta offers a tractable non-vertebrate chordate model for probing gene function via tissue-specific, CRISPR/Cas9-mediated mutagenesis in F0. Building on Arcadia Sciences Zoogle platform, which identifies and ranks orthologs of human genes from various non-traditional model organisms, we carried out a pilot project to probe the developmental roles of three notochord- and endoderm-expressed candidate orthologs of human disease genes (Fcho, Pgm3, and Nckap1) alongside a fourth gene (Plastin) implicated in papilla cell elongation. This preprint compiles and updates a series of research project milestones previously posted episodically on Zenodo. Here we summarize the full results and our conclusion about this pilot project. Using CRISPR/Cas9, we found that tissue-specific knockout of Pgm3 and, to a lesser extent, Fcho caused significant defects in larval tail elongation. Separately, CRISPR knockout of Plastin, an actin-bundling gene expressed throughout the sensory-adhesive papillae of the larva, caused a subtle reduction in papilla cell elongation when combined as a duoble knockout with another actin-bundling protein-encoding gene, Villin. These results identify Pgm3 as the most promising candidate for further development as a Ciona-based model of human disease and demonstrate the utility of tissue-specific CRISPR screening for prioritizing candidate disease gene orthologs identified through comparative genomics platforms like Zoogle.

developmental biology↗

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

An NKX2-5 homolog is required downstream of BMP signaling to pattern the sensory-adhesive organ of a tunicate larva

Tunicates are the sister group to vertebrates within the chordate phylum, yet unlike other chordate groups, they evolved a biphasic lifecycle alternating between motile larvae and sessile adults. The papillae of most tunicate larvae are the key sensory-adhesive organ regulating their settlement and metamorphosis. The papillae are nearly always arranged as a group of three morphologically identical organs that arise from an anterior neural plate border region nested between ventral epidermis and more dorsal/posterior neural tube progenitors. Due to their embryonic origin and molecular signatures, this anterior border has been evolutionarily linked to vertebrate placode regions. It was previously shown that the specification, patterning, and morphogenesis of the embryonic papilla region all depend on BMP signaling, though downstream mechanisms remain poorly understood. Here we show that the NKX2-3/5/6 ortholog NK4 is a key transcription factor that acts downstream of BMP signaling to pattern the papillae in the Ciona embryo. We present evidence that NK4 is activated by BMP signaling and encodes a transcriptional repressor that is required to restrict the expression of the papilla regulatory gene Foxg to three cell clusters that give rise to the three papillae. Loss of NK4 function results in the formation of a single large papilla. In contrast, overexpression of NK4 represses Foxg, eliminating the papillae. We also show that the expression of NK4 is restricted dorsally by the BMP antagonist Chordin, while the ventrally-expressed transcription factor Msx alleviates the repressive effect of NK4, potentially allowing for the characteristic tripartite patterning of the papillae.

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↗

bioRxiv2024

A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is only expressed in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. While in vertebrates Myogenic Regulatory Factors (MRFs) like MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF/MyoD and Early B-Cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

evolutionary biology↗

bioRxiv2024

A gene regulatory network for specification and morphogenesis of a Mauthner Cell homolog in non-vertebrate chordates

Transcriptional regulation of gene expression is an indispensable process in multicellular development, yet we still do not fully understand how the complex networks of transcription factors operating in neuronal precursors coordinately control the expression of effector genes that shape morphogenesis and terminal differentiation. Here we break down in greater detail a provisional regulatory circuit downstream of the transcription factor Pax3/7 operating in the descending decussating neurons (ddNs) of the tunicate Ciona robusta. The ddNs are a pair of hindbrain neurons proposed to be homologous to the Mauthner cells of anamniotes, and Pax3/7 is sufficient and necessary for their specification. We show that different transcription factors downstream of Pax3/7, namely Pou4, Lhx1/5, and Dmbx, regulate distinct "branches" of this ddN network that appear to be dedicated to different developmental tasks. Some of these network branches are shared with other neurons throughout the larva, reinforcing the idea that modularity is likely a key feature of such networks. We discuss these ideas and their evolutionary implications here, including the observation that homologs of all four transcription factors (Pax3/7, Lhx5, Pou4f3, and Dmbx1) are key for the specification of cranial neural crest in vertebrates.

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↗

bioRxiv2023

Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function

Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larvas ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae of transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate "capstone" research course. This small screen revealed at least one gene, Vamp1/2/3, that appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.

developmental biology↗

bioRxiv2023

Specification of distinct cell types in a sensory-adhesive organ for metamorphosis in the Ciona larva

The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least five distinct cell types in the papillae of the model tunicate Ciona, allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult of Ciona.

developmental biology↗

bioRxiv2021

Evolution of a chordate-specific mechanism for myoblast fusion

The size of an animal is determined by the size of its musculoskeletal system. Myoblast fusion is an innovative mechanism that allows for multinucleated muscle fibers to compound the size and strength of individual mononucleated cells. However, the evolutionary history of the control mechanism underlying this important process is currently unknown. The phylum Chordata hosts closely related groups that span distinct myoblast fusion states: no fusion in cephalochordates, restricted fusion and multinucleation in tunicates, and extensive, obligatory fusion in vertebrates. To elucidate how these differences may have evolved, we studied the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed an unexpectedly complex evolutionary history of myoblast fusion in chordates. A pre-vertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Thus, our findings reveal the evolutionary origins of chordate-specific fusogens and illustrate how new genes can shape the emergence of novel morphogenetic traits and mechanisms.

developmental biology↗

bioRxiv2019

Neurogenin regulates effectors of migratory neuron cell behaviors in Ciona

The bipolar tail neurons (BTNs) of Ciona develop according to a highly dynamic, yet highly stereotyped developmental program and thus could serve as an accessible model system for neuronal delamination, migration, and polarized axon outgrowth. Here we used FACS/RNAseq to profile the transcriptional output of Neurogenin in the BTNs, searching for candidate effectors of BTN cell behaviors. We identified several candidate genes that might play conserved roles in similar cell behaviors in other animals, including mammals. Among the more interesting candidates were several microtubule-binding proteins and TGF{beta} pathway antagonists. A small Gi subunit was also found to be upregulated in migrating BTNs, and interfering with its function through expression of a dominant negative inhibited delamination and a complete epithelial-to-mesenchymal transition. We propose models for the regulation of BTN behaviors by the identified candidate effectors, establishing a foundation for testing effector gene functions that might be conserved in chordate neurodevelopment.

developmental biology↗

bioRxiv2019

Neuron subtype-specific effector gene expression in the Motor Ganglion of Ciona

The central nervous system of the Ciona larva contains only 177 neurons. The precise regulation of neuron subtype-specific morphogenesis and differentiation observed in during the formation of this minimal connectome offers a unique opportunity to dissect gene regulatory networks underlying chordate neurodevelopment. Here we compare the transcriptomes of two very distinct neuron types in the hindbrain/spinal cord homolog of Ciona, the Motor Ganglion (MG): the Descending decussating neuron (ddN, proposed homolog of Mauthner Cells in vertebrates) and the MG Interneuron 2 (MGIN2). Both types are invariantly represented by a single bilaterally symmetric left/right pair of cells in every larva. Supernumerary ddNs and MGIN2s were generated in synchronized embryos and isolated by fluorescence-activated cell sorting for transcriptome profiling. Differential gene expression analysis revealed ddN- and MGIN2-specific enrichment of a wide range of genes, including many encoding potential \"effectors\" of subtype-specific morphological and functional traits. More specifically, we identified the upregulation of centrosome-associated, microtubule-stabilizing/bundling proteins and extracellular matrix proteins and axon guidance cues as part of a single intrinsic regulatory program that might underlie the unique polarization of the ddNs, the only descending MG neurons that cross the midline.

developmental biology↗

bioRxiv2019

A cis-regulatory change underlying the motor neuron-specific loss of terminal selector gene expression in immotile tunicate larvae

The evolutionary history of animal body plans cannot be fully reconstructed without considering the roles of both novelties and losses. Some of the more remarkable examples of massively parallel evolutionary losses in animals comes from many species in the tunicate genus Molgula that have independently lost the swimming larva and instead develop as tail-less, immotile larvae that bypass the period of swimming and dispersal observed in other tunicates, marine invertebrate chordates that alternate between motile larval and sessile adult life cycle stages. The larvae of Molgula occulta and other tail-less species do not fully develop structures that are essential for swimming behavior, including notochord, tail muscles, and otolith, and loss-of-function mutations have been identified in various genes required for the differentiation of these tissues. However, little is known about the extent of development of the larval nervous system in M. occulta. While differentiated neurons might in principle be entirely dispensable to the non-swimming larva, the adult has a fully functional nervous system like any other tunicate. To further investigate this conundrum, we studied the specification and patterning of the M. occulta Motor Ganglion, which is the key central nervous system compartment that drives the motor movements of swimming tunicate larvae. We found that the expression patterns of important regulators of MG neuron subtype specification are highly conserved during the development of the non-swimming larvae of M. occulta, suggesting that the gene networks regulating their expression are largely intact in this species, despite the loss of swimming ability. However, we identified a M. occulta-specific reduction in expression of the important motor neuron terminal selector gene Ebf (Collier/Olf/EBF or COE) in the Motor Ganglion. Although M. occulta Ebf is predicted to encode a fully functional protein, its expression was reduced in developing motor neurons when compared to species with swimming larvae, which was corroborated by measuring allele-specific expression of Ebf in interspecific hybrid embryos produced by crossing M. occulta with the closely related swimming species M. oculata. Comparative reporter construct experiments also revealed a specific cis-regulatory sequence change that underlies the reduced expression of M. occulta Ebf in motor neurons, but not in other tissues and cell types. This points to a potential mechanism for arresting larval motor neuron differentiation in the non-swimming larvae of this species.

evolutionary biology↗

bioRxiv2018

Developmental system drift in motor ganglion patterning between distantly related tunicates

The larval nervous system of the solitary tunicate Ciona is a simple model for the study of chordate neurodevelopment. The development and connectivity of the Ciona Motor Ganglion (MG) has been studied in fine detail, but how this important structure develops in other tunicates is not well known. By comparing gene expression patterns in the developing MG of the distantly related tunicate Molgula occidentalis, we found that its patterning is highly conserved compared to the Ciona MG. MG neuronal subtypes in Molgula were specified in the exact same positions as in Ciona, though the timing of subtype-specific gene expression onset was slightly shifted to begin earlier, relative to mitotic exit and differentiation. In transgenic Molgula embryos electroporated with Dmbx reporter plasmids, we were also able to characterize the morphology of the lone pair of descending decussating neurons (ddNs) in Molgula, revealing the same unique contralateral projection seen in Ciona ddNs and their putative vertebrate homologs the Mauthner cells. Although Dmbx expression labels the ddNs in both species, cross-species transgenic assays revealed significant changes to the cis-regulatory logic underlying Dmbx transcription. We found that Dmbx cis-regulatory DNAs from Ciona can drive highly specific reporter gene expression in Molgula ddNs, but Molgula sequences are not active in Ciona ddNs. This acute divergence in the molecular mechanisms that underlie otherwise functionally conserved cis-regulatory DNAs supports the recently proposed idea that the extreme genetic plasticity observed in tunicates may be attributed to the extreme rigidity of the spatial organization of their embryonic cell lineages.

evolutionary biology↗

bioRxiv2018

Single-cell transcriptome profiling of the Ciona larval brain

The tadpole-type larva of Ciona has emerged as an intriguing model system for the study of neurodevelopment. The Ciona intestinalis connectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, the Ciona tadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of the Ciona larval brain.

developmental biology↗