Segment regeneration in earthworms is a remarkable example of postembryonic morphogenesis, yet its fidelity and cellular mechanisms remain incompletely understood. The present study investigated posterior segment regeneration in adult specimens of the earthworm model Eisenia andrei from wound closure to the 5th postoperative week using anatomical, histological, and ultrastructural approaches. Rapid wound closure occurred through fusion of the cut edges of the body wall and midgut without direct involvement of coelomocytes. The regeneration blastema consisted of dedifferentiated epithelial and muscle cells, innervated by fibers from the last intact ventral nerve cord ganglion. Coelomocytes accumulated in the last intact segments and were primarily involved in debris clearance. Notably, early regenerating tissues lacked collagen fibers, which appeared only after the third postoperative week and remained sparse until the fifth week, whereas original segments exhibited intense, region-specific collagen deposition. Transmission electron microscopy revealed characteristic cytological changes in distinct stages of body wall regeneration, including muscle dedifferentiation and the emergence of collagen-producing fibroblasts. These findings indicate that early cell migration, proliferation, and orientation in the blastema proceed independently of collagen and that collagen functions as a delayed structural scaffold, supporting tissue integrity without impeding regeneration. Importantly, no scar formation was observed between old and new tissues, resembling scarless fetal wound healing. Overall, we clarified previously controversial cellular mechanisms and propose a new, comprehensive model for the early stages of segment regeneration. Our results highlight that coordinated dedifferentiation, spatiotemporal extracellular remodeling, and delayed collagen deposition underlie effective, scar-free regeneration in earthworms, offering insights into conserved mechanisms of regenerative repair across metazoans and potential strategies for enhancing tissue regeneration in mammals.

Evolution of nervous systems is a long debated topic, and similar mechanisms of conditional neural specification linked to dorsal-ventral (D-V) axis formation across some taxa have been used to support homology. We tested for autonomous versus conditional neural specification in two distantly related annelids,Capitella teletaandPlatynereis dumerilii, using blastomere isolations. Our results support previous work inC. teletaand further demonstrate that the autonomous specification of anterior neural tissue and for the first time in trunk neural tissue for both annelids. InC. teleta, we found evidence for conditional pro-neural and anti-neural signals for the VNC. Animal caps lacking vegetal macromeres at the 16-cell stage form a brain and a D-V axis but not a VNC while the addition of any single macromere rescues VNC fate. This suggests that animal micromeres other than 2d produce an anti-neural signal while a pro-neural signal is produced vegetally and that VNC specification is decoupled from D-V axis formation. Taken together, our study suggests possible conservation of autonomous specification of the brain and VNC within Annelida, raising interesting questions of how mechanisms controlling neural specification evolved in Spiralia.

Characterization of a myoinhibitory peptide and its role in neuroimmune regulation in Litopenaeus vannamei during Vibrio harveyi infection.

A neural connectivity atlas for fly flight control.

In Drosophila motoneurons, spatiotemporal dendritic patterns are established in the ventral nerve cord. While many guidance cues have been identified, the mechanisms of temporal regulation remain unknown. Previously, we identified the actin modulator Cdc42 GTPase as a key factor in this process. In this report, we further identify the upstream factors that activate Cdc42. Using single-cell genetics, FRET-based imaging, and biochemical techniques, we demonstrate that the guanine nucleotide exchange factor Vav is anchored to the plasma membrane via the Eph receptor tyrosine kinase, enabling Cdc42 activation. VAMP-associated protein 33 (Vap33), a potential Eph ligand supplied non-cell-autonomously, may induce Eph autophosphorylation, initiating downstream signaling. Traditionally known as an ER-resident protein, Vap33 is secreted extracellularly at the onset of Cdc42 activation, acting as a temporal cue. In humans, VAPB—the ortholog of Vap33—is similarly secreted in the spinal cord, and its dysregulation leads to amyotrophic lateral sclerosis type 8 (ALS8). Our findings may help inform future studies on how VAPB signaling contributes to motor circuit formation in both physiological and disease contexts.

Nuclear hormone receptor regulation of PAL-1/Caudal mediates ventral nerve cord assembly in C. elegans.

Whole-body glucose uptake in crayfish (Procambarus clarkii): A study of sexual dimorphism via [18F]FDG MicroPET imaging.

ABSTRACTTo optimize health, organisms must coordinate energy intake and expenditure and apportion related behaviors to appropriate times of day. In the fruit fly, Drosophila melanogaster, the SIFamide (SIFa) neuropeptide impacts multiple behavioral outputs important for energy regulation, including reproductive activity, sleep, and feeding. SIFa‐expressing neurons receive convergent inputs from circadian and homeostatic brain regions and extend elaborate projections throughout the central nervous system. Consistent with this distribution pattern, the SIFa receptor (SIFaR) is widely expressed in the brain and ventral nerve cord, providing the anatomical substrate for SIFa signaling to influence a broad range of neuronal functions. To further explore the pleiotropic role of SIFa signaling in behavioral control, we have assessed survival, locomotor activity, sleep, and feeding in SIFaR mutant flies, as well as in flies with RNA interference‐induced reduction of SIFaR expression. We find that loss of SIFaR has a complex effect on fly survival that is background‐ and allele‐specific. However, outcrossed SIFaR mutant flies are viable, enabling monitoring of adult behavior. These flies exhibit elevated locomotor activity, reduced sleep, and increased feeding at specific times of day. We also find that SIFaR mutations drastically decrease starvation resistance. These results suggest a prominent role for SIFaR in integrating homeostatic and circadian information to coordinate the magnitude and timing of energy balance‐related behaviors.

Pax6 plays a highly conserved role in the formation of the eye, development, and patterning of the nervous system across bilaterians. Nevertheless, there are no studies focusing on the role of Pax6 during asexual reproduction, a developmental trajectory that is widespread among metazoans. The present study represents the first investigation of Pax6 gene expression during agametic propagation in annelids. We identified in the asexually reproducing annelid Nais communis four homologs of Pax6 and examined their developmental patterns by in situ hybridization. To establish a morphological basis for the expression patterns, we used immunohistochemistry and confocal laser scanning microscopy to describe the nervous system architecture of the growing adults and remodeling of the original ventral nerve cord, development of the new brain, ventral ganglia, peripheral nerves, and sensory organs in asexually reproducing worms. Our results support the hypothesis of an evolutionarily conserved function of Pax6 genes in the development of the eye and other sensory organs, as well as the central nervous system, among bilaterians, regardless of developmental trajectory. On the other hand, identified Pax6 homologs show differential expression within the developing new head and tail ends. Differences in spatiotemporal expression patterns may be evidence of functional diversification of duplicated homologs.

SummarySleep is a continuous behavior across the lifespan, yet its features and functions evolve markedly with development1–4. In Drosophila melanogaster, as in mammals, early life sleep differs from mature sleep5,6, but it is unknown whether disparate sleep regulatory mechanisms underlie these changes. Here, we identify distinct populations of octopaminergic (OA) neurons that promote arousal in larval and adult flies, thus revealing a developmental switch in sleep-wake circuit architecture. Of eight OA neurons present in the sub-esophageal zone (SEZ) of the nervous system at both life stages, dedicated, non-overlapping subsets drive arousal in larvae versus adults. Morphologic and connectomic analyses show that larval OA arousal neurons project primarily to the ventral nerve cord and lack substantial sensory input, suggesting a circuit logic optimized for internally driven arousal during early development. In contrast, adult OA arousal neurons target higher brain regions involved in cognition and receive rich multimodal sensory input, supporting wakefulness in response to environmental cues. These findings highlight a developmental transition in arousal circuitry that mirrors changing ecological demands, with juvenile systems organized to prioritize growth and feeding, insulated from sensory disturbance, and mature systems supporting sensory-guided behavior. Our results support a model of sleep regulation as a developmentally dynamic process, in which shared neuromodulators like OA operate through distinct cellular substrates tailored to life stage–specific behavioral priorities.

Nicotinic acetylcholine receptors function with adhesion molecule SAX-7 to reverse cell orientation during migration.

ABSTRACTTremor is a common movement disorder associated with several neurodegenerative diseases, yet its mechanisms are not well understood. Using a machine-learning method, Feature Learning-based Leg segmentation and Tracking (FLLIT), we previously characterised gait and tremor signatures in a Drosophila model for spinocerebellar ataxia 3 (SCA3) and found them to be analogous to those in human SCA3. Here, we carried out a functional screen for neuronal populations that underlie tremor and found that dysfunction of a specific population of neurons in the ventral nerve cord (VNC) is necessary and sufficient for tremor. Adult-onset expression of mutant ATXN3 in, or genetic hypo-activation of, these neurons led to tremor, indicating their important role in adult motor control. RNA-sequencing and functional experiments showed that dysfunction of GABAergic neurons, and not that of other neurotransmitter populations tested, causes tremor. Finally, we identified a small subset of ∼30 predominantly GABAergic neurons within the adult VNC that are essential for smooth walking. This study demonstrates that tremor in SCA3 flies arises from GABAergic dysfunction, and that FLLIT can be used to dissect motor control mechanisms.

In the Drosophila nervous system, neuroblasts (NBs) divide to produce a clone of neurons that establish distinct fates via precise timing and patterning of transcription factors (TFs). The final step in neurogenesis is a Notch/Numb asymmetric division that produces one daughter neuron that has active Notch signaling (NotchON) and one that does not (NotchOFF). NotchON neurons are well characterized, but the NotchOFF progeny are understudied due to lack of molecular markers. Here, we have identified Fer3 (forty-eight related 3) as a NotchOFF-specific transcription factor expressed in the NB7-1, NB6-1 and NB5-2 lineages. Fer3 is inhibited by Notch signaling in post-mitotic neurons, thereby restricting its expression to NotchOFF neurons. In some contexts, Fer3 is a transcriptional repressor, but we find that Fer3 misexpression generates ectopic Dbx+ neurons, and this is more penetrant when we misexpress a Fer3:activation domain fusion protein. Moreover, Fer3 is sufficient to induce Dbx expression in a neuroblast lineage that does not endogenously express Dbx or Fer3. This work presents the first known NotchOFF exclusive TF in the developing embryonic Drosophila ventral nerve cord.

IPPK-1 and IP6 contribute to ventral nerve cord assembly in C. elegans.

Evolutionary conservation of midline repulsive signaling by Robo family receptors in flies and mice.

Neuropeptide SIFamide (SIFa) neurons in Drosophila melanogaster have been characterized by their exceptionally elaborate arborization patterns, which extend from the brain into the ventral nerve cord (VNC). SIFa neurons are equipped to receive signals that integrate both internal physiological cues and external environmental stimuli. These signals enable the neurons to regulate energy balance, sleep patterns, metabolic status, and circadian timing. These peptidergic neurons are instrumental in orchestrating the animal’s internal states and refining its behavioral responses, yet the precise molecular underpinnings of this process remain elusive. Here, we demonstrate that SIFa neurons coordinate a range of behavioral responses by selectively integrating inputs and outputs in a context-dependent manner. These neurons engage in a feedback loop with sNPF neurons in the VNC, modifying behaviors such as longer mating duration (LMD) and shorter mating duration (SMD). Additionally, SIFa neurons interact with dopamine and glutamate to differentially regulate sleep and mating duration. Activating SIFa neurons leads to reduced mating duration and increased food intake, while deactivating them reduces food intake. Overall, these findings demonstrate the importance of SIFa neurons in absorbing inputs and turning them into behavioral outputs, shedding light on animal’s intricate behavioral orchestration.

Neuropeptide SIFamide (SIFa) neurons in Drosophila melanogaster have been characterized by their exceptionally elaborate arborization patterns, which extend from the brain into the ventral nerve cord (VNC). SIFa neurons are equipped to receive signals that integrate both internal physiological cues and external environmental stimuli. These signals enable the neurons to regulate energy balance, sleep patterns, metabolic status, and circadian timing. These peptidergic neurons are instrumental in orchestrating the animal's internal states and refining its behavioral responses, yet the precise molecular underpinnings of this process remain elusive. Here, we demonstrate that SIFa neurons coordinate a range of behavioral responses by selectively integrating inputs and outputs in a context-dependent manner. These neurons engage in a feedback loop with sNPF neurons in the VNC, modifying behaviors such as longer mating duration (LMD) and shorter mating duration (SMD). Additionally, SIFa neurons interact with dopamine and glutamate to differentially regulate sleep and mating duration. Activating SIFa neurons leads to reduced mating duration and increased food intake, while deactivating them reduces food intake. Overall, these findings demonstrate the importance of SIFa neurons in absorbing inputs and turning them into behavioral outputs, shedding light on animal's intricate behavioral orchestration.

Cell size is strongly correlated with several biological processes, including the cell cycle and growth. Here, we investigated the regulation of stem cell size during Drosophila central nervous system (CNS) development and its association with cell fate. We note that neural stem cells (NSC) in different regions of the ventral nerve cord increase their size at different rates. Thoracic NSCs grow at a faster rate compared with those in the abdominal region during larval development. We show that in addition to the known role in apoptosis and nervous system remodeling, larval expression of abdA is crucial in regulating the rate of postembryonic NSCs size increase, their timely exit from G2 phase and mitotic rate. We demonstrate that when abdA expression is lost in abdominal NSCs, their size increases, they exhibit a shorter G2 phase, enter mitosis earlier, and divide more rapidly. Conversely, the introduction of abdA in thoracic NSCs slows their growth and delays their entry into mitosis. We demonstrate that abdA-mediated NSC size regulation acts downstream of their nutrition-induced activation, thereby fine-tuning the stem cell potential spatiotemporally. This study highlights the instructive role of abdA in regulating various fates of larval NSCs during CNS patterning.

The C. elegans ventral nerve cord (VNC) provides a genetically tractable model for investigating the developmental mechanisms involved in neuronal positioning and organization. The VNC of newly hatched larvae contains a set of 22 motoneurons organized into three distinct classes (DD, DA, and DB) that show consistent positioning and arrangement. This organization arises from the action of multiple convergent genetic pathways, which are poorly understood. To better understand these pathways, accurate and efficient methods for quantifying motoneuron cell body positions within large microscopy datasets are required. Here, we present VNC-Dist (Ventral Nerve Cord Distances), a software toolkit that replaces manual measurements with a faster and more accurate computer-assisted approach, combining machine learning and other tools, to quantify neuron cell body positions in the VNC. The VNC-Dist pipeline integrates several components: manual neuron cell body localization using Fiji’s multipoint tool, deep learning-based worm segmentation with modified Segment Anything Model (SAM), accurate spline-based measurements of neuronal distances along the VNC, and built-in tools for statistical analysis and graphing. To demonstrate the robustness and versatility of VNC-Dist, we applied it to several genetic mutants known to disrupt neuronal positioning in the VNC. This toolbox will enable batch acquisition and analysis of large datasets across genotypes, thereby advancing investigations into the cellular and molecular mechanisms that govern VNC neuronal positioning and arrangement.

Just as genomes revolutionized molecular genetics, connectomes (maps of neurons and synapses) are transforming neuroscience. To date, the only species with complete connectomes are worms1–3 and sea squirts4 (103-104 synapses). By contrast, the fruit fly is more complex (108 synaptic connections), with a brain that supports learning and spatial memory5,6 and an intricate ventral nerve cord analogous to the vertebrate spinal cord7–11. Here we report the first adult fly connectome that unites the brain and ventral nerve cord, and we leverage this resource to investigate principles of neural control. We show that effector cells (motor neurons, endocrine cells and efferent neurons targeting the viscera) are primarily influenced by local sensory cells in the same body part, forming local feedback loops. These local loops are linked by long-range circuits involving ascending and descending neurons organized into behavior-centric modules. Single ascending and descending neurons are often positioned to influence the voluntary movements of multiple body parts, together with endocrine cells or visceral organs that support those movements. Brain regions involved in learning and navigation supervise these circuits. These results reveal an architecture that is distributed, parallelized and embodied (tightly connected to effectors), reminiscent of distributed control architectures in engineered systems12,13.