Biology subjects

Christiaen, L.

Publications and source records attributed to Christiaen, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Evolution of CTCF underlies the establishment of loop extrusion as a predominant chromatin folding mechanism in vertebrates

CTCF is a DNA-binding protein that emerged in the last bilaterian ancestor and that plays a central role in 3D genome organization. Yet, despite its relevance, genomic studies have revealed notable differences in CTCF function across animal clades. In vertebrates, CTCF is essential for TAD organization through cohesin-mediated loop extrusion, whereas its contribution to chromatin organization outside this clade appears to be more limited. Here we investigate the evolution of CTCF as an architectural factor through a cross-species functional replacement approach, in which orthologs from divergent bilaterian lineages are expressed in either mouse embryonic stem cells or Drosophila embryos. Chromosome conformation analyses demonstrated that the ability of CTCF to mediate loop extrusion-dependent TAD formation is encoded within the vertebrate protein. Yet, this property alone is insufficient to establish vertebrate-like genome architecture in an invertebrate background, suggesting the existence of additional lineage-specific factors. ChIP-seq analyses further revealed that CTCF DNA-binding specificity has been progressively refined during bilaterian evolution, with the vertebrate lineage exhibiting a preferential shift towards distal genomic regions that is consistent with an expansion of regulatory domains. Notably, despite failing to form TADs, the ortholog from a non-vertebrate chordate partially rescued the lethality associated with the loss of the vertebrate CTCF protein, indicating that its architectural and regulatory functions are evolutionary independent. Further Hi-C analyses in tunicate and cyclostome species suggest that the ability of CTCF to act as a loop extrusion barrier was already present in the last common ancestor of all vertebrates. Altogether, our findings establish the diversification of CTCF regulation and function as key drivers for the specialization of bilaterian 3D genome organization.

genomics↗

bioRxiv2025

Epithelial cell extrusion underlies starvation-induced cell loss in a sea anemone

Epithelia likely predate the last common animal ancestor, yet the evolutionary origin and environmental regulation of epithelial remodelling remain poorly understood. Here, we show that extensive, starvation-induced cell loss in the sea anemone Nematostella vectensis is associated with epidermal cell extrusion. This process involves formation of a rosette-like arrangement in which an apoptotic, extruding cell is surrounded by a phospho-ERK1/2-positive ring, accompanied by basal translocation of adherens junction components. Combining chemical perturbations with computational quantification of extrusion and cell density, we show that apoptosis is necessary but not sufficient for rosette formation, and that ERK1/2 signalling limits epidermal extrusion density. We further find that extrusion activity increases during starvation, and that extruded cells may be recycled by efferocytosis. Together, our findings indicate that epithelial cell extrusion has physiological roles in sea anemones and that its key hallmarks are evolutionarily ancient, predating the last common ancestor of sea anemones, flies and vertebrates.

cell biology↗

bioRxiv2025

Genome-wide expression gradient estimation based on local pseudotime in single cell RNA sequencing

Single cell genomics measures the internal state of individual cells and makes it possible to describe biological phenomena such as cell type heterogeneity, developmental progression, and dynamic differentiation using computational methods. A prominent approach to facilitate downstream analysis, for large collection of cells, is to connect individual cells by a k-Nearest Neighbor Graph (kNN-G). The kNN-G is among other things used by methods that derive pseudotime, a temporal ordering of cells. However, pseudotime methods require knowledge of initial states of the trajectories created. Alternatives have been invented that estimate gene rate of transcription e.g. by RNA velocity but these methods dont generalize to all individual genes for estimating transcriptional rate of change along pseudotime. While these methods can derive pseudo progression de novo by extrapolating transcription velocity vectors they are limited to subsets of genes where intronic reads are captured with sufficient magnitude. Here we present pseudovelocity, an alternative method for calculating a local rate of change in transcription based on the kNN-G and diffusion-based pseudotime, to derive RNA velocity estimates for individual genes, and demonstrate how it improves the gene-based estimation of RNA velocity for downstream analysis. This package can be accessed here: https://gitlab.com/Xparx/pseudovelocity.

bioinformatics↗

bioRxiv2024

A simple inland culture system provides insights into ascidian post-embryonic developmental physiology

Maintenance and breeding of experimental organisms are fundamental to life sciences, but both initial and running costs, and hands-on zootechnical demands can be challenging for many laboratories. Here, we aimed to further develop a simple protocol for reliable inland culture of tunicate model species of the Ciona genus. We cultured both Ciona robusta and Ciona intestinalis in controlled experimental conditions, with a focus on dietary variables, and quantified growth and maturation parameters. From statistical analysis of these standardized datasets, we gained insights into the post-embryonic developmental physiology of Ciona, and inferred an improved diet and culturing conditions for sexual maturation. We showed that body length is a critical determinant of both somatic and sexual maturation, which suggests the existence of systemic control mechanisms of resource allocation toward somatic growth or maturation, and supports applying size selection as a predictor of reproductive fitness in our inland culture, to keep the healthiest animals at low density in the system. In the end, we successfully established a new protocol, including size selection, to promote both sperm and eggs production. Our protocol using small tanks will empower researchers to initiate inland Ciona cultures with low costs and reduced space constraints.

developmental biology↗

bioRxiv2024

Cell cycle-driven transcriptome maturation confers multilineage competence to cardiopharyngeal progenitors

During development, stem and progenitor cells divide and transition through germ layer- and lineage-specific multipotent states to generate the diverse cell types that compose an animal. Defined changes in biomolecular composition underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, multipotent cardiopharyngeal progenitors display multilineage transcriptional priming, whereby both the cardiac and pharyngeal muscle programs are partially active and coexist in the same progenitor cells, while their daughter cells engage in a cardiac or pharyngeal muscle differentiation path only after cell division. Here, using the tunicate Ciona, we studied the acquisition of multilineage competence and the coupling between fate decisions and cell cycle progression. We showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single cell RNA-seq (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S and G2 phases. We termed this process "transcriptome maturation", and identified candidate "mature genes", including the Rho GAP-coding gene Depdc1, which peak in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and proper oriented and asymmetric division that influences subsequent fate decisions, illustrating the concept of "behavioral competence". Both classic feedforward circuits and coupling with cell cycle progression drive transcriptome maturation, uncovering distinct levels of coupling between cell cycle progression and fateful molecular transitions. We propose that coupling competence and fate decision with the G2 and G1 phases, respectively, ensures the timely deployment of lineage-specific programs.

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

The Chordate Origins of Heart Regeneration

The human heart is infamous for not healing after infarction in adults, prompting biomedical interest in species that can regenerate damaged hearts. In such animals as zebrafish and neonatal mice, cardiac repair relies on remaining heart tissue supporting cardiomyocyte proliferation. Natural de novo cardiogenesis in post-embryonic stages thus remains elusive. Here we show that the tunicate Ciona, an ascidian among the closest living relatives to the vertebrates, can survive complete chemogenetic ablation of the heart and loss of cardiac function, and recover both cardiac tissue and contractility. As in vertebrates, Ciona heart regeneration relies on Bone Morphogenetic Protein (BMP) signaling-dependent proliferation of cardiomyocytes, providing insights into the evolutionary origins of regenerative cardiogenesis in chordates. Remarkably, prospective lineage tracing by photoconversion of the fluorescent protein Kaede suggested that new cardiomyocytes can emerge from endodermal lineages in post-metamorphic animals, providing an unprecedented case of regenerative de novo cardiogenesis. Finally, while embryos cannot compensate for early losses of the cardiogenic lineage, forming heartless juveniles, developing animals gain their regenerative ability during metamorphosis, uncovering a fundamental transition between deterministic embryogenesis and regulative post-embryonic development.

developmental biology↗

bioRxiv2023

Structure primed embedding on the transcription factor manifold enables transparent model architectures for gene regulatory network and latent activity inference

The modeling of gene regulatory networks (GRNs) is limited due to a lack of direct measurements of regulatory features in genome-wide screens. Most GRN inference methods are therefore forced to model relationships between regulatory genes and their targets with expression as a proxy for the upstream independent features, complicating validation and predictions produced by modeling frameworks. Separating covariance and regulatory influence requires aggregation of independent and complementary sets of evidence, such as transcription factor (TF) binding and target gene expression. However, the complete regulatory state of the system, e.g. TF activity (TFA) is unknown due to a lack of experimental feasibility, making regulatory relations difficult to infer. Some methods attempt to account for this by modeling TFA as a latent feature, but these models often use linear frameworks that are unable to account for non-linearities such as saturation, TF-TF interactions, and other higher order features. Deep learning frameworks may offer a solution, as they are capable of modeling complex interactions and capturing higher-order latent features. However, these methods often discard central concepts in biological systems modeling, such as sparsity and latent feature interpretability, in favor of increased model complexity. We propose a novel deep learning autoencoder-based framework, StrUcture Primed Inference of Regulation using latent Factor ACTivity (SupirFactor), that scales to single cell genomic data and maintains interpretability to perform GRN inference and estimate TFA as a latent feature. We demonstrate that SupirFactor outperforms current leading GRN inference methods, predicts biologically relevant TFA and elucidates functional regulatory pathways through aggregation of TFs.

systems biology↗

bioRxiv2022

Rnf149-related is an FGF/MAPK-independent regulator of pharyngeal muscle fate specification

During embryonic development, cell fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors to both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study the cardiopharyngeal fate specification with cellular resolution, as only 2 pairs of cardiopharyngeal multipotent progenitors give rise to the heart and to pharyngeal muscles (aka atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented asymmetric divisions. Here, we identify the primed gene Rnf149-related (Rnf149-r), which becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss knock-out of Rnf149-r function impairs atrial siphon muscle morphogenesis, and down-regulates Tbx1/10 and Ebf, two key determinants of the pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of loss of FGF-MAPK signaling in the cardiopharyngeal lineage, and integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations identified a significant overlap between FGF-MAPK and Rnf149-r targets. However, functional interaction assays suggested the Rnf149-r does not directly modulate the activity of the FGF-MAPK-Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF-MAPK signaling on shared targets, as well as on FGF-MAPK-independent targets through (a) separate pathway(s).

developmental biology↗

bioRxiv2021

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline

During development, remodeling of the cellular transcriptome and proteome underlies cell fate decisions and, in somatic lineages, transcription control is a major determinant of fateful biomolecular transitions. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focused on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic mRNAs, including Pem-1, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II (RNAPII), and nascent transcripts from the Mef2 locus. Using PGC-specific Mef2 transcription as a read-out, we uncovered a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline, which relies on successive cellular remodeling and JAK inhibition, and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

developmental biology↗

bioRxiv2021

Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that these multicellular arrangements confer biochemical and biomechanical properties that contribute to tissue scale organization. The cardiopharyngeal progenitors of the tunicate Ciona provide the simplest possible model of collective cell migration. They form cohesive bilateral cell pairs, leader-trailer polarized along the migration path as they migrate between the ventral epidermis and trunk endoderm. Here, circumventing difficulties in quantifying cellular mechanics in live embryos, we use the Cellular Potts Model to computationally probe the distributions of forces consistent with the shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we first determine that cardiopharyngeal progenitors display hallmarks of supracellular organization, with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. Combined 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy that contributes to aligning collective polarity with the direction of migration, as observed with three or more cells both in silico and in vivo. Our approach reveals emerging properties of the migrating collective. Specifically, cell pairs are more persistent, thus migrating over longer distances, and presumably with higher accuracy. Finally, simulations suggest that polarized cell pairs literally join forces to deform the trunk endoderm, as they migrate through the extracellular space. We thus propose that the polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.

developmental biology↗

bioRxiv2020

Single cell multi-omic analysis identifies a Tbx1-dependent multilineage primed population in the murine cardiopharyngeal mesoderm

The poles of the heart and branchiomeric muscles of the face and neck are formed from the cardiopharyngeal mesoderm (CPM) within the pharyngeal apparatus. The formation of the cardiac outflow tract and branchiomeric muscles are disrupted in patients with 22q11.2 deletion syndrome (22q11.2DS), due to haploinsufficiency of TBX1, encoding a T-box transcription factor. Here, using single cell RNA-sequencing, we identified a multilineage primed population (MLP) within the CPM, marked by the Tbx1 lineage, which has bipotent properties to form cardiac and skeletal muscle cells. The MLPs are localized within the nascent mesoderm of the caudal lateral pharyngeal apparatus and provide a continuous source of progenitors that undergo TBX1-dependent progression towards maturation. Tbx1 also regulates the balance between MLP maintenance and maturation while restricting ectopic non-mesodermal gene expression. We further show that TBX1 confers this balance by direct regulation of MLP enriched genes and downstream pathways, partly through altering chromatin accessibility. Our study thus uncovers a new cell population and reveals novel mechanisms by which Tbx1 directs the development of the pharyngeal apparatus, which is profoundly altered in 22q11.2DS.

developmental biology↗

bioRxiv2020

GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate

GATA4/5/6 transcription factors play essential, conserved roles in heart development. How these factors mediate the transition from multipotent mesoderm progenitors to a committed cardiac fate is unclear. To understand how GATA4/5/6 modulate cell fate decisions we labelled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at pre-cardiac time points spanning gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was dramatically altered. In addition to the expected absence of cardiac mesoderm, we observed a concomitant expansion of cranial-pharyngeal mesoderm. Functional genetic analyses in zebrafish and the invertebrate chordate Ciona, which possess a single GATA4/5/6 homolog, revealed an essential and cell-autonomous role for GATA4/5/6 in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, the maintenance and repression of GATA4/5/6 activity plays a critical, evolutionarily conserved role in early development.

developmental biology↗

bioRxiv2020

Asymmetric second generation genomic incompatibility in interspecific crosses between Ciona robusta and Ciona intestinalis

Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species in the tunicate genus Ciona, Ciona robusta and Ciona intestinalis can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed inland culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids indicated that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. Asymmetrical second generation inviability and infertility suggested that interspecific genomic incompatibilities involved interactions between the maternal, zygotic and mitochondrial genomes during development. This study paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model.

evolutionary biology↗

bioRxiv2020

Optimal tuning of weighted kNN- and diffusion-based methods for denoising single cell genomics data

The analysis of single-cell genomics data presents several statistical challenges, and extensive efforts have been made to produce methods for the analysis of this data that impute missing values, address sampling issues and quantify and correct for noise. In spite of such efforts, no consensus on best practices has been established and all current approaches vary substantially based on the available data and empirical tests. The k-Nearest Neighbor Graph (kNN-G) is often used to infer the identities of, and relationships between, cells and is the basis of many widely used dimensionality-reduction and projection methods. The kNN-G has also been the basis for imputation methods using, e.g., neighbor averaging and graph diffusion. However, due to the lack of an agreed-upon optimal objective function for choosing hyperparameters, these methods tend to oversmooth data, thereby resulting in a loss of information with regard to cell identity and the specific gene-to-gene patterns underlying regulatory mechanisms. In this paper, we investigate the tuning of kNN- and diffusion-based denoising methods with a novel non-stochastic method for optimally preserving biologically relevant informative variance in single-cell data. The framework, Denoising Expression data with a Weighted Affinity Kernel and Self-Supervision (DEWAKSS), uses a self-supervised technique to tune its parameters. We demonstrate that denoising with optimal parameters selected by our objective function (i) is robust to preprocessing methods using data from established benchmarks, (ii) disentangles cellular identity and maintains robust clusters over dimension-reduction methods, (iii) maintains variance along several expression dimensions, unlike previous heuristic-based methods that tend to oversmooth data variance, and (iv) rarely involves diffusion but rather uses a fixed weighted kNN graph for denoising. Together, these findings provide a new understanding of kNN- and diffusion-based denoising methods and serve as a foundation for future research. Code and example data for DEWAKSS is available at https://gitlab.com/Xparx/dewakss/-/tree/Tjarnberg2020branch.

bioinformatics↗

bioRxiv2019

A simple method to quantify proteins from one thousand cells

The rise of single-cell transcriptomics has created an urgent need for similar approaches that use a minimal number of cells to quantify expression levels of proteins. We integrated and optimized multiple recent developments to establish a proteomics workflow to quantify proteins from as few as 1,000 mammalian stem cells. The method uses chemical peptide labeling, does not require specific equipment other than cell lysis tools, and quantifies >2,500 proteins with high reproducibility. We validated the method by comparing mouse embryonic stem cells and in vitro differentiated motor neurons. We identify differentially expressed proteins with small fold-changes, and a dynamic range in abundance similar to that of standard methods. Protein abundance measurements obtained with our protocol compare well to corresponding transcript abundance and to measurements using standard inputs. The protocol is also applicable to other systems, such as FACS-purified cells from the tunicate Ciona. Therefore, we offer a straightforward and accurate method to acquire proteomics data from minimal input samples.

systems biology↗

bioRxiv2019

Comparative analysis of diverse cell states establishes an epigenetic basis for inferring regulatory genes governing cell identity

Determining genes orchestrating cell differentiation in development and disease remains a fundamental goal of cell biology. This study establishes a genome-wide metric based on the gene-repressive tri-methylation of histone 3 lysine 27 (H3K27me3) across hundreds of diverse cell types to identify genetic regulators of cell differentiation. We introduce a computational method, TRIAGE, that uses discordance between gene-repressive tendency and expression to identify genetic drivers of cell identity. We apply TRIAGE to millions of genome-wide single-cell transcriptomes, diverse omics platforms, and eukaryotic cells and tissue types. Using a wide range of data, we validate TRIAGEs performance for identifying cell-type specific regulatory factors across diverse species including human, mouse, boar, bird, fish, and tunicate. Using CRISPR gene editing, we use TRIAGE to experimentally validate RNF220 as a regulator of Ciona cardiopharyngeal development and SIX3 as required for differentiation of endoderm in human pluripotent stem cells. A record of this papers Transparent Peer Review process is included in the Supplemental Information.

cell 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↗