Eve Seuntjens

Summary: Octopus vulgaris and other cephalopods are of increasing interest as neurobiological model organisms. This protocol describes a method to record calcium activity from individual cells in acute brain slices from Octopus vulgaris hatchlings during exogenous application of neurotransmitters. Using this protocol, we characterized single-cell responses to specific neurotransmitters in the optic lobes, which process visual information. The approach is readily adaptable to other cephalopods and small invertebrate species. Graphical

Quantifying animal behavior across species with complex, deformable body plans requires capturing full-body shape rather than sparse skeletal keypoints. Yet accessible, end-to-end tools that take researchers from mask annotation through model training to reliable multi-object tracking are lacking. Here we present OCTRON, a fully trainable pipeline for multi-object segmentation, detection, and tracking that is broadly applicable across experimental setups and organisms. OCTRON leverages large segmentation foundation models to rapidly generate experimenter-supervised annotations from minimal input, overcoming the manual labeling bottleneck of supervised segmentation. These annotations train compact, task-specific models: segmentation networks for extracting pixel-level information such as shape and appearance, or lightweight detection networks for fast centroid tracking. Coupled with multi-object trackers and an interactive track-curation interface in napari, OCTRON enables complete behavioral quantification with minimal manual intervention. We validate OCTRON across species from transparent marine annelids to camouflaging cuttlefish, and in multi-species behavioral assays.

The short-lived and rapidly aging African turquoise killifish, Nothobranchius furzeri GRZ is a unique model to study vertebrate aging. Current genomic and full-length transcriptomic sequencing lacks full gene annotations, resulting in poor mapping in bulk and single-cell transcriptomic studies. In our efforts to reannotate the transcriptome of the killifish telencephalon, we combined long-read (Smrt-Isoseq) and short-read transcriptome sequencing approaches. A total of 17,008 full-length isoforms, including 6763 novel ones were obtained (51 bp to 7,500 bp). The killifish telencephalon comprises 25% multi-exon genes, while over 50% are mono-exon genes. We discovered novel non-coding and coding sequences in both young and aged telencephali. We integrated long-read and RNA-seq data to construct a comprehensive transcriptome and profiled expression dynamics across the aging telencephalon. Our gene models demonstrate greater detail and accuracy than Ensembl, with more precise polyA locations. Alternative splicing analysis revealed 29 events altered with aging, which involved changes in ribosome function, gap junction and mRNA surveillance pathways. These generated resources pave the way for future functional genomic studies in this biogerontology model.

Cell types are fundamental functional units that can be traced across the tree of life. Rapid advances in single-cell technologies, coupled with the phylogenetic expansion in genome sequencing, present opportunities for the molecular characterization of cells across a broad range of organisms. Despite these developments, our understanding of eukaryotic cell diversity remains limited and we are far from decoding this diversity from genome sequences. Here we introduce the Biodiversity Cell Atlas initiative, which aims to create comprehensive single-cell molecular atlases across the eukaryotic tree of life. This community effort will be phylogenetically informed, rely on high-quality genomes and use shared standards to facilitate comparisons across species. The Biodiversity Cell Atlas aspires to deepen our understanding of the evolution and diversity of life at the cellular level, encompassing gene regulatory programs, differentiation trajectories, cell-type-specific molecular profiles and inter-organismal interactions.

Venom plays a central role in the predatory ecology of coleoid cephalopods (octopuses, squids, and cuttlefish), yet the mechanisms governing venom release from the posterior salivary gland (PSG) are unknown. Using a multimodal approach combining X-ray micro-histotomography, histological stainings, in situ hybridization, comparative phylogenetics, and ex vivo imaging across multiple coleoid species, we characterize the structural and neuronal regulatory organization of the PSG. We verified that the gland comprises two distinct tubular systems: secretory tubules specialized for venom production and smooth–striated tubules positioned to facilitate venom transport toward the beak for injection into its prey. Molecular localization of filamentous and α-actin confirms a circular smooth muscle layer surrounding the tubules. Mapping of six neuronal markers, including neurofilament (NF-H), synapsin, and muscle-type nicotinic acetylcholine receptors, reveals dense and stereotyped neural innervation closely associated with the muscular compartments. Comparative phylogenetic analyses of cys-loop ligand-gated ion channel sequences indicate a predominancy of excitatory acetylcholine- and dopamine-gated receptors in coleoid venom glands, implicating potential molecular agents involved in neural control of venom release. Consistent with neural regulation, ex vivo stimulation of the PSG elicits calcium signaling throughout the gland. Together, our results reveal a conserved venom gland structure among octopuses, squids, and cuttlefish, with a spatially distinct neuromuscular tissue organization indicating venom production and release sites modulated by a network of neuronal agents. This work provides a mechanistic framework into venom gland organization and molecular regulation of venom release in one of the oldest venomous lineages.

The African turquoise killifish (Nothobranchius furzeri) is becoming a favorable model for neurobiological research. The combination of a short lifespan and a declining neuroregenerative capacity upon aging makes it ideally suited for research on brain aging and regeneration. A remarkable cellular diversity makes up the young-adult killifish telencephalon, characterized by highly proliferative non-glial progenitors and spatially distinct radial glia subtypes. In contrast to a relatively slow embryonic development, hatching is followed by a period of accelerated growth, in which the brain experiences a period of rapid expansion and maturation. In this study, we quantified the growth progression and maturation of the killifish telencephalon during early post-embryonic development. We discovered that, similar to in zebrafish, neuro-epithelial cells abut the neurogenic niches from early life onwards. Spatial data revealed qualitative and quantitative differences along the anterior-posterior axis and between pallial and subpallial regions in terms of growth pace. We confirmed generation of GABAergic neurons from the subpallial neurogenic niche and glutamatergic neurons from two pallial niches. Our data further showed a more widespread appearance of inhibitory neurons at hatching compared to in zebrafish.

Summary: Octopus vulgaris , the common octopus, has a complex brain that evolved independently from that of vertebrates, raising the possibility that it may be built using novel circuit motifs or non-canonical neurochemistry. Through systematic characterization of octopus ligand-gated ion channel genes, we identified novel ionotropic receptors for two neurotransmitters, dopamine and acetylcholine, that play key roles in visual circuits. One of these, DopC1, encodes a dopamine-gated cation channel expressed in deeper layers of the optic lobe (inner granular layer and medulla). A second, AChRB1, encodes an acetylcholine-gated anion channel expressed in photoreceptors and dopaminergic outer granular layer neurons. Dopamine drives excitation, particularly in deeper, DopC1 enriched layers, and thereby may play an important role in feed-forward excitation, whereas acetylcholine evokes inhibition, possibly mediating negative feedback from deeper layers. The octopus visual system thus shows fundamental differences in both neurochemistry and wiring compared to mammals, implying distinct mechanisms of visual information processing.

There has been a great interest in common octopus Octopus vulgaris aquaculture over recent years and commercial production is coming within reach. In addition, O. vulgaris is a short-lived semelparous species with a long spawning period. Therefore, monitoring ovary maturation in cultured octopuses can be useful in the scheduling of hatchery operations and management of culture resources. The present work aimed to assess the ovary maturity status in cultured and wild common octopus by means of a noninvasive technique, ultrasonography, to anticipate the time of spawning in aquaculture. A total of 27 octopuses (15 cultured and 12 wild ones) in different ovarian maturation stages were used in ultrasonographic procedures. Ultrasound-based ovary volumes were compared with real ovarian volumes and then the ultrasonography-based gonadosomatic index (GSIU) was estimated. Data from cultured females indicate that GSIU was initially below 0.3 cm ³ per 100 g of body weight in immature animals, and attained a fast increase until reaching about 4.0 cm ³ per 100 g of body weight right before spawning. The steep increase in relative volume occurred similarly in females of different body weights. In addition, the specific growth rate of cultured animals decreases with time, and with GSIU. Lastly, a linear mixed model indicated a significantly negative and logarithmic relationship between the time to spawning and GSIU in captive females. That relationship will be useful to predict spawning time in immature and maturing females, helping in the management of octopus broodstocks in research and production facilities.

Conditional deletion of Elp3 in the mouse forebrain leads to microcephaly at birth. In this study, we demonstrate that these mice also develop postnatal hydrocephalus, associated with an enlargement of the brain ventricles. In wild-type mice, ependymal motile cilia are properly aligned to facilitate the circulation of cerebrospinal fluid (CSF) within the ventricles. Our findings reveal that Elp3 loss induces endoplasmic reticulum (ER) stress and upregulation of ATF4 expression in ependymal cell progenitors, which compromises Notch signaling and accelerates their maturation. This is accompanied by a disruption in the establishment of rotational and translational polarities of the motile cilia of maturing ependymal cells, resulting in disorganized cilia bundles. Collectively, these molecular abnormalities lead to the premature and abnormal development of ependymal cells, culminating in cilia beating dysfunction, impaired CSF clearance, and the development of hydrocephalus.