iScience

SummaryEstimating deep-time diversification patterns and the establishment of extant biodiversity represent major challenges in macroevolution. Fossil record data provide essential information to address these topics, but their heterogeneous temporal and geographical distributions require using analytical approaches to process these data. Gardiner et al.1 (hereafter GEA) used a deep-learning model2 and a fossil-occurrences dataset3 to estimate neoselachian richness over the last 145 myr. Results and DiscussionGEA1 found that neoselachian diversity increased throughout the Cretaceous, was little impacted by the Cretaceous-Paleogene (K/Pg) mass extinction ([~]10% species loss), and peaked in the mid-Eocene but declined until the Present. While the Cretaceous increase in neoselachian richness is well known4, the other findings of GEA1 are at odds with current knowledge. With the exception of lamniform sharks, the perceived decrease in species richness in the recent past is most likely due to a drop in available fossil record data combined with difficulties in identifying extant species in the fossil record5. Similarly, all previous analyses of the impact of the K/Pg mass extinction on elasmobranch diversification have reported high extinction rates, a marked diversity drop, and delayed recovery6-7, despite heterogeneity across clades, ecology, and geographical distribution7. Taking the K/Pg as an example, we demonstrate that the discrepancies between GEA1s results and current consensus is most likely due to a combination of incomplete, unverified, and incorrect fossil-occurrence data with inappropriate methodology.

In the search for universals shaping acoustic communication across species, we increasingly look for patterns known from human languages and music in non-human animals. These parallels are often explored separately and with limited ecological context. Here, we take a deep dive into the temporal structure of a complex call used by the little auk (Alle alle), a pelagic seabird with elaborate vocal behaviour and socially complex colonial life. Based on syllable durations, intervals and silences, we examine its conformance to linguistic laws, rhythmic structure and information content. This reveals intricate problems of temporal organisation: while the calls conform not only to linguistic laws of brevity but also to the initial and final lengthening known from human prosody, these effects interact with the internal structure of the call and information carried within it. To our knowledge, this is the first time that conformance to multiple linguistic laws, exceeding simple vocal efficiency, has been described for a non-human, non-vocal learning animal. The calls rhythmic structure shows a progressive rallentando -- a systematic slowing driven by changes in syllable and silence durations and the intervals between syllable onsets. The exact patterns of this rallentando are indicative of the callers sex and individually specific. These results reveal how seabird communication is shaped not only by efficiency universals, but also the specific pressures of colonial life. Our work highlights the temporal structure as an important axis of communication evolution, but also serves as a reminder to consider the species ecological reality and the function, not only presence, of temporal organisation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/713940v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13de3a8org.highwire.dtl.DTLVardef@2d64adorg.highwire.dtl.DTLVardef@2ca53aorg.highwire.dtl.DTLVardef@113c38d_HPS_FORMAT_FIGEXP M_FIG C_FIG

The end-Permian mass extinction (EPME) represents the most severe biotic crisis of the Phanerozoic Eon on Earth and has been well documented in marine taxa. However, its impact on terrestrial organisms and ecosystems remains incompletely understood. Here we present a high-resolution reconstruction of terrestrial diversification dynamics and spatial reorganization across the Permo-Triassic boundary (PTB) using comprehensive occurrence data of macroplants, sporomorphs and vertebrates. Terrestrial responses to the EPME show highly temporal, regional and taxonomic heterogeneities. Plants experienced a genus-level diversity loss of [~] 6.7%, across the PTB, whilst vertebrates, a lagged decline from the late Permian, peaking at a diversity loss of [~] 66.7%. Global distributions of plant and vertebrate show converging on similar latitudinal gradients post the PTB. Plant diversity loss is disproportionately high in low-latitude and tropical regions and progressively lower toward mid- and high-latitudes. Our study facilitates a fine-grained understanding to terrestrial macroevolution in geologic history through multi-analysis of a large volume of fossil data. Our findings challenge the long-held notion of global terrestrial collapse and mass extinction in plants during the PTB and offer a deep-time analogue for uneven response of extant terrestrial biodiversity to ongoing climate change.

Sparsely active granule cells in the dentate gyrus (DG) project onto CA3 pyramidal neurons through mossy fibers (MF), forming a feedforward network that ensures even similar inputs activate distinct and minimally overlapping populations of CA3 neurons. This process, known as pattern separation, is a key computational feature at this synapse. However, such sparse activity and connectivity increase the risk of information loss, as multiple presynaptic inputs are typically required to elicit a postsynaptic action potential. Interestingly, MF synapses exhibit robust short-term plasticity (STP), enabling a dynamic increase in release probability during brief bursts of presynaptic activity. This mechanism ensures that a short, high-frequency burst from a single granule cell can reliably generate a spike in its postsynaptic CA3 target. Unlike other hippocampal synapses, MF boutons are large, with multiple active zones, each coupled to a cluster of voltage-dependent calcium channels (VDCCs). MF boutons also possess a large readily releasable pool of vesicles. The functional consequences of this unusual synaptic design remain largely unexplored. In fact, the MF synapse is often depicted as a synapse with multiple sites, each behaving as an independent transmission line, analogous to several CA3 boutons contacting a single CA1 dendrite. We developed a physiologically realistic spatial model of the MF bouton to investigate how its peculiar structural and functional properties affect synaptic signaling and plasticity. Contrary to earlier assumptions, our computational model revealed, release sites are not independent transmission units. Crosstalk between calcium domains is necessary for the observed strong STP and for the timely activation of CA3 neurons. VDCCs in the MF bouton are only loosely coupled to active zones, and the distance between active zones is relatively large. In addition to the synaptic design and the known role of calbindin-D28k and synaptotagmin-7 in STP, we find that loose coupling of VDCCs to active zones and large inter-AZ distances are crucial. These features keep the basal release probability low, and their combined action is required to generate the facilitation that triggers postsynaptic action potentials in response to bursts filtering out non-informative dentate activity, and provides a strong rationale for the mossy fibers synaptic architecture.

Unraveling how adult neurons reshape their architecture is key to understanding post-developmental plasticity. Drosophila clock neurons, which remodel their terminals on a daily basis, offer a unique model to examine the mechanisms underlying structural plasticity. In this study, we examine the impact of the experimental design on the remodeling process. We established a simple fixation protocol that preserves tissue integrity and prevents its deformation while enabling the fixation of a larger number of individuals within the appropriate time window. We show that intrinsic (i.e., targeting fluorescent reporters to the membrane) or extrinsic (i.e., temperature) variables may influence this dynamic process. Examining ex vivo preparations, we found that the s-LNv terminals display numerous thin filopodia extending from their synaptic boutons. However, these fine membrane protrusions are lost upon fixation, as they could only be accurately visualized ex vivo. Finally, we present MorphoScope, a Python-based interface that eliminates observer bias in complexity measurements. Altogether, we present a powerful and robust model to investigate the principles of adult neuronal plasticity, with implications extending beyond circadian biology.

For animals to interact effectively with their environment, the brain must integrate sensory information and generate appropriate motor responses. Multiple neuronal circuits contribute to this process, and identifying their roles remains a central focus in neuroscience. The recently developed photoswitchable compound Carbadiazocine controls neuronal firing across species. It modulates larval zebrafish locomotion and alleviates neuropathic pain in rodents in a reversible, light-induced manner. Given its effects on both motor and somatosensory circuits, we investigated the impact of Carbadiazocine on sensorimotor behaviors. We focused on the optomotor response in zebrafish larvae and assessed its potential as a tool for circuit perturbation and behavioral analysis, for the first time combined with photopharmacology. We performed experiments in head-fixed and free-swimming larvae to assess their capacity to detect and follow the direction of optic flow, as well as to characterize swimming speed patterns and individual tail bout properties following administration of the two Carbadiazocine photoisomers. In both paradigms, treatment with the pre-illuminated compound led to a decrease in accuracy in responding to optic flow (correct turning percentage dropping from [~]95 % to [~]80 % in head-fixed experiments and correctness decreasing from [~]65 % to [~]20 % in free-swimming experiments). Speed analysis revealed an increased number and duration of fast movements with a decrease in number and duration of slow movements, even during periods without visual stimulation. Tail bout analysis further showed an increase in 15-30 Hz bout frequencies, corresponding to incomplete, irregular tail movements. All these effects were absent when the dark-relaxed compound was administered. Together, these findings deepen our understanding of sensorimotor transformations and lay the foundations to probe native neuronal circuits underlying behavior in diverse animal species using a wide dynamic range of photostimulation patterns.

Tumor development and progression involve complex cell-cell interactions and dynamic co-evolution between cancer cells, immune cells and stromal cells in the tumour microenvironment, and this may influence therapeutic resistance. A large proportion of this network relies on direct physical interactions between cells, particularly T-cell mediated interactions. Cell-cell communication inference has now become routine in downstream scRNAseq analysis, but this mostly fails to capture physical cell-cell interactions due to tissue dissociation. Doublets occur naturally in scRNA-seq and are usually excluded from analysis. However, they may represent directly interacting cells that remain undissociated during library preparation. In the present study, we uncover the physical interaction landscape of the ovarian tumour microenvironment using the scRNAseq datasets from 13 treatment-naive ovarian cancer patients. Focusing on T-cell-Macrophage (T-Mac) interaction doublet, we reveal the modulatory effect of macrophages on T cells and the potential influence of this interaction on therapeutic response. Our findings show that T-Macs from resistant patients are functionally polarized to the M2 phenotype and engage T cells to induce T-cell exhaustion. Whereas, T-Macs from sensitive patients are predominantly of the M1 polarized phenotype, physically engaging T cells that lack exhaustion signatures. We also demonstrate that T cells and macrophages in T-Mac doublet are interacting primarily for the purpose of antigen presentation, with the enrichment of several ligand-receptor pairs involved in TCR-MHC interactions and immune synapse formations. We partly validated some of these findings from a spatial transcriptomics dataset of ovarian cancer patients from a separate cohort.

Recent advancements in tomography produce imaging data of geological materials (rocks and fossils) at trillion-voxel scales with multi-channels. Such high-resolution datasets are potentially keys to unveil evolutionary biological information with various shapes and sizes that have not been ever discovered. Volume rendering is an ideal visualization approach for them because it treats all voxels without relying on user-defined surface boundaries. However, these large-scale real-world tomographic data have rarely been volume-rendered at their native resolution, limiting the examination of rich morphological information. Here, we demonstrate a de facto standard volume-rendering pipeline running on a graphical processing unit (GPU)-equipped supercomputing system toward multi-channel, trillion-voxel tomographic data. Our workflow preserves original resolution, capturing detailed morphological information spanning microscopic to macroscopic scales. Systematic comparison of node types shows that GPU memory, rather than host memory, is the primary bottleneck. Our results establish a baseline for large-scale, multi-channel volume rendering of real tomographic data and demonstrate its applicability to geological samples. This work is presented as a practical demonstration of large-scale volume visualization.

Spatiotemporal organisation of biological molecules is a key driver of cellular processes, including many post-transcriptional epigenetic processes. The germline-specific germ granules are biomolecular condensates that act as hubs for mRNA and small RNA processing and are core regulators of germline gene expression programming. Germ granules have been studied extensively in C. elegans, and recent developments have led to many subdivisions of the germ granule into specialised compartments. Rapid advancements in microscopy and protein-protein interaction (PPI) screening techniques have produced a large amount of data towards characterising the localisation of proteins to specific granules. However, common methods used to probe PPIs are limited in their ability to robustly detect valid interactions, especially the multivalent and sometimes transient ones observed in granule environments. Here we perform a meta-analysis of granule protein interaction screens. While these experiments generally enrich for proteins matching the profile of granule-associated proteins, we find that when considering screens individually, reproducibility is surprisingly low, highlighting not only the variability inherent in these methods but also the dynamic nature of the PPI networks present in granules. We developed an algorithm to provide a measure of each proteins association with specific granules across various experiments. By further clustering and investigation of the resulting score matrix, we demonstrate the power of this holistic approach to provide deeper insights into germ granule organisation and highlight novel can provide a resource to better inform future investigations into granules and their constituent proteins.

In the context of multistability-driven diseases, like cancer, spatio-temporal plasticity plays a significant role to achieve a spectrum of phenotypic variations. The interplay between gene regulatory networks and environmental factors, such as resource competition and spatial diffusion, plays a crucial role in determining cellular behaviour and phenotypic heterogeneity. Though reaction-diffusion frameworks have been widely applied in developmental biology, less attention has been paid to the simultaneous effects of resource competition and growth feedback on spatial organization. In this paper, we observed that a bistable genetic circuit under high resource competition due to growth feedback gives rise to multiple emergent phenotypes, as observed in cancer systems. Furthermore, we observed how spatial diffusion coupled with intrinsic nonlinearity can drive the emergence of distinct spatial dynamics over time. The observed spatiotemporal plasticity can also be driven by the comparative stability of the fixed points, diffusivity, and asymmetry of diffusion. Our findings highlight that growth-induced resource competition combined with diffusion can provide deeper insights into metastasis and cancer progression.

A delicate balance between the quiescent and proliferative states of neural stem cells (NSCs) is important for neurogenesis and homeostasis. Histone deacetylase 4 (HDAC4) variants are associated with neurodevelopmental disorders, however, its role in early brain development remains elusive. In this study, we demonstrate that Drosophila HDAC4 plays a crucial role in neural stem cells (NSCs) reactivation and brain development. Depletion of HDAC4 results in notable defects in NSC reactivation, while its overexpression leads to premature reactivation. HDAC4 is SUMOylated at Lys902, which enhances its protein stability by preventing HDAC4 from undergoing ubiquitin-proteasome-mediated degradation. Moreover, phosphorylation of HDAC4 by salt-inducible kinase 3 (SIK3), an AMPK-related kinase, allows cytoplasmic localization of HDAC4 and enhances the association between HDAC4 and Warts, a core kinase of the Hippo pathway. This HDAC4-Wts association inhibits Warts activity, and in turn, the inactivation of the Hippo pathway, triggering NSC reactivation. Finally, genetic epistasis experiments support the SIK3-HDAC4-Warts axis during NSC reactivation. In conclusion, our findings identify HDAC4 as a molecular switch that integrates SUMOylation, ubiquitination, and the Hippo pathway to govern NSC reactivation.

Though whole-genome duplication (WGD) contributes to cancer progression, the mechanism of post-WGD cell proliferation remains unclear. Here, using 6-day live-imaging, we analyzed the proliferation dynamics of more than 150 post-WGD HCT116 cell lineages. A quantitative comparison of mitotic patterns and cell fates between proliferative and non-proliferative lineages revealed that multipolar chromosome segregation in early mitosis is a key factor limiting the proliferative capacity of post-WGD progenies. Multipolar chromosome segregation suppressed post-WGD cell viability, particularly when accompanied by drastic chromosome loss or when it repeatedly occurred. Tracing proliferative lineages elucidated that they proliferated mainly by imposing the risk of multipolar chromosome segregation on one of two sub-lineages that formed after the first bipolar division. Meanwhile, a considerable proportion of proliferative lineages consisted entirely of progeny of early multipolar chromosome segregation events. Our results highlight key cellular events that determine the proliferation dynamics and diversity of post-WGD progenies, providing a fundamental reference for understanding WGD-associated bioprocesses. Summary statementLive image tracing of >150 cell lineages reveals the cross-generation dynamics of multipolar chromosome segregation that determine the fates of post-whole-genome duplication progeny cells.

Anticancer drug resistance is challenging to overcome because it can arise through both intrinsic and acquired mechanisms, each driven by distinct cellular machinery. In particular, there is a sharp need for therapies that target hormone-insensitive prostate tumors due to the growing incidence of castration-resistant prostate cancer. Optimizing the pathways that regulate apoptosis in prostate cancer offers a promising strategy to induce apoptosis and inhibit tumor progression, since these mechanisms do not depend on hormonal signaling. Here, we identified strategies to enhance apoptosis in prostate cancer cells. We used several computational tools (including sensitivity analysis, particle swarm optimization, and ImageJ) to design an ordinary differential equation model of caspase-mediated prostate cancer apoptosis signaling. We apply the model to identify key modalities that increase the propensity toward apoptosis across three separate pro-apoptotic drugs (Tocopheryloxybutyrate, Narciclasine, and Celecoxib). Overall, we demonstrate that apoptosis dynamics can be accurately captured in response to each of the three drugs and identify which features of the model represent viable targets for overcoming intrinsic drug resistance.

Recovering well-preserved vertebrate remains from underwater caves has provided critical insights into archaeological and palaeontological records worldwide. However, understanding how bone assemblages form and are modified in underwater environments remains limited due to stable low energy burial conditions that produce time-averaged deposits, and underwater settings that hinder traditional recording and recovery methods. This study applies an actualistic taphonomic framework to three assemblages of domesticate animal bones (N = 231) from two underwater caves, Green Waterhole and Gouldens Sinkhole, near Mount Gambier, South Australia, encompassing known submerged (wet; N = 134) and dry (N = 97) burial conditions. The assemblages were examined to assess how wet and dry cave environments impact bone distribution, surface and microstructural modification. Radiocarbon dating of 41 specimens indicates that domesticate fauna were deposited over decadal and centennial timescales, allowing taphonomic signatures to be contextualised through time. Statistically significant differences were identified between wet and dry burial contexts. Bones recovered from wet contexts exhibit mostly better preservation, including skeletal elemental completeness, surface, and microstructure, than those from dry caves. However, some of the submerged specimens also have elevated frequencies of bone surface corrosion with macroscopic evidence for heterogenous black biological staining, algal or biofilm attack, and a distinctive form of circular etching. Histotaphonomy further reveals patterns of peripheral cyanobacterial tunnelling across most bones recovered from submerged contexts. Bones from dry environments were dominated by terrestrially linked tunnelling across all regions of the bone cortex. These findings can be explained by variation in light availability across different cave zones which influences biological activity and, in turn, the expression of taphonomic markers on bone externally and at the microstructural level. This is the first study to provide a benchmark bone dataset for reconstructing depositional histories and post-depositional reworking in underwater cave environments under a taphonomic framework.

The morphology of the palate has long constituted the primary basis for differentiating between the two deepest clades of crown group birds, Neognathae and Palaeognathae. However, published literature on the bird palate is dominated by classical anatomical descriptions pre-dating the advent of contemporary three-dimensional imaging techniques, hindering our understanding of bird palate disparity and development. Pterygoid segmentation, the process by which the rostral portion of the pterygoid separates and fuses with the palatine during ontogeny in neognathous birds, remains a poorly understood aspect of avian cranial development despite giving rise to an important component of the cranial kinetic system. Here, we use micro-computed tomography to explore ontogenetic change of the palate during the process of pterygoid segmentation across an unprecedentedly broad taxonomic sample of immature and mature birds. We found that direct evidence of post-hatching pterygoid segmentation was restricted to the major avian subclade Neoaves. Based on morphological and topological similarities, we hypothesise that the rostral projection of the pterygoid observed in Anatidae/Anseres and potentially Anhimidae and Megapodiidae, which we term the hemipterygoid process, is homologous with the hemipterygoid of neoavians, though it does not undergo segmentation. We posit that the origin of a discrete hemipterygoid (as observable in some crownward stem-birds) originated prior to the origin of the process of pterygoid segmentation; however, it remains ambiguous whether pterygoid segmentation is a synapomorphy of Neornithes, Neognathae, or Neoaves. Overall, our study clarifies the process of avian pterygoid segmentation and raises new questions regarding the major morphological modifications that have characterised the evolutionary history of the avian bony palate.

Spatial-numerical associations reflect the tendency to map small numerosities to the left and larger numerosities to the right. Although widely documented in humans and some non-human species, its presence in teleost fish has remained unclear. Here, we investigated whether zebrafish exhibit a spatial-numerical association by presenting fish with a left-right numerical discrimination task in a controlled behavioral assay. Zebrafish showed a reliable leftward preference when selecting smaller numerosities and a rightward preference when selecting larger ones, indicating a systematic coupling between numerical magnitude and spatial direction. These results provide the first evidence of a number-space association in zebrafish and demonstrate that number-space mapping extends to a basal vertebrate lineage. This establishes zebrafish as a tractable model for probing the neurobiological foundations of number-space associations. HighlightsO_LIZebrafish map numerical magnitudes onto left-right spatial positions. C_LIO_LISpatial biases are more robust for small numerosities but still present for larger ones. C_LIO_LIContinuous physical controls disproportionately affect the strength of spatial mapping for large sets. C_LI

The dorsal raphe nucleus (DRN) serotonergic (5-HT) system has been implicated in regulating sleep and motor control; however, its specific role remains controversial. In this study, we found that optogenetic activation of DRN 5-HT neurons in larval zebrafish induced a quiescent state and a reduced response to acoustic stimuli. Unlike sleep, the induced quiescent state was not accompanied by a loss of postural control, and nighttime activation of DRN 5-HT neurons led to subsequent sleep rebound. Whole brain light field imaging combined with demixed principal component analysis (dPCA) revealed distinct neural subspaces related to DRN activation, sound responses, and motor activity. DRN 5-HT activation selectively modulated the motor-related subspace while leaving the sound-evoked subspace unaffected. Unlike DRN activation, sleep induced by mepyramine significantly altered sound-evoked neuronal activity patterns. Further analysis demonstrated that serotonin had a graded effect on the motor subspace, wherein downstream neurons responsible for particular bout types were more significantly influenced. Together, these results elucidate that serotonergic modulation promotes behavioral quiescence through a hierarchical regulation of motor populations.

The blood vasculature has a high capacity for structural regeneration, driven by the blood endothelial cells (BECs) that comprise it. This regenerative process, which involves BEC migration and proliferation to form these complex tissues, is linked to low frequency (< 0.1 Hz) calcium spiking that precedes these activities. However, we need new approaches to stimulating angiogenic responses in tissue engineering applications. By conducting experiments that manipulate local ionic concentrations and developing a simple, yet powerful, computational analysis, we demonstrate that sodium-calcium cross-talk is a crucial component that regulates the calcium signaling and downstream angiogenic responses. Activation and deactivation of the inositol triphosphate 3 receptors (IP3Rs) on the endoplasmic reticulum (ER) and the switch between forward and reverse modes of the sodium-calcium exchanger (NCX) are proposed to be the key mechanisms underlying calcium oscillations when cells are exposed to temporary cationic depletion. The spiking is suggested to be a release of intracellular calcium mediated by IP3R activity, and transport in or out of the cell is driven by NCX for the calcium oscillatory signaling pattern. The NCX and IP3R both contribute to manage intracellular calcium and ionic concentration as initially there is a long ER deactivation period while intracellular sodium slowly increases until a sudden onset of calcium is released by the ER. Other calcium and sodium ion channels can change this resonant coupling of ER and NCX to alter the inter-spike duration. Synchronization of the spiking intervals between cells is triggered by stimulating with vascular endothelial growth factor (VEGF), which induces a propagating wave of intracellular calcium across the 2D tissue culture, prior to coordinated cell migration and proliferation towards the VEGF source. This wave, which can be artificially induced and studied using electrical stimulation, suggests that the underlying sodium-calcium crosstalk mechanism introduces intracellular calcium polarization, whose orientation is transferred across cells through spike synchronization. Thus, control of calcium signaling dynamics through regulation of ionic depletion can serve as useful method for generating angiogenic responses in engineered tissue constructs.

Muscular hydrostats, muscular structures with no rigid skeleton, are ubiquitous within the animal kingdom, from vertebrate tongues to cephalopod arms1,2, but how they perform complex actions remains poorly understood. One model hydrostat studied for its neural control3-7 and biomechanics8-17 is the feeding system (buccal mass) of the sea hare Aplysia (Fig. 1). The buccal mass (Fig. 1b) performs multiple feeding behaviors by coordinating intrinsic muscles to move a grasper (odontophore)18,19. In this paper, we investigated how mechanical reconfiguration from interacting shape-changing elements facilitates large odontophore protractions. During rejection behaviors, mechanical reconfiguration of the odontophore (elongating its shape to a higher aspect ratio) stretches a protractor muscle (I2), allowing I2 to generate stronger protractions12. In biting behaviors, the odontophore has a similar range of motion. However, during biting, the odontophore has a lower aspect ratio throughout protraction, meaning the I2 muscle alone is insufficient to reach observed protractions due to its length/tension property and reduced mechanical advantage9,10,12,18. By combining new analysis of MRI movies of Aplysia feeding12,18 (Fig. 1) with a new biomechanical model for biting and rejection (Fig. 2), we demonstrate two context-dependent mechanical reconfiguration mechanisms that explain the different ways large protractions are produced in biting and rejection (Fig. 3). The mechanisms integrate shape changes, bending and conforming of muscle structures, and shifts in contact interactions. We propose two mechanical subclasses of muscular hydrostats, "constrained" or "unconstrained" (Fig. 4), that may be morphologically similar but employ different control strategies depending on whether mechanical constraints are reliably present. O_FIG O_LINKSMALLFIG WIDTH=150 HEIGHT=200 SRC="FIGDIR/small/715937v1_fig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1c60cbeorg.highwire.dtl.DTLVardef@16ebd04org.highwire.dtl.DTLVardef@13b65d5org.highwire.dtl.DTLVardef@9aafb0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO Anatomy and kinematics of the Aplysia feeding system (a1) Adult Aplysia californica searching for food and (a2) feeding on Gracilaria macroalgae ((a1) photo credit: Dr. Jeffrey P. Gill, (a2) modified with permission from Bennington et al. 202514). Gray highlight shows the location of the feeding structure, the buccal mass (b). (b) An anatomical diagram of a midline sagittal view of a buccal mass. During feeding, the odontophore (the internal grasper of the buccal mass) protracts through the tubelike I3 muscle. In the midsagittal plane, the I3 is visible as two longitudinal elements, but is one continuous structure that runs circumferentially around the buccal mass. The inner wall of the distal I3 is shown in dark blue. The dashed white line shows the jaw line, which is used as the reference for both the translation and rotation measurements. (c) Configuration of the buccal mass (left: anatomical diagram; middle: MRI frames) showing (c1) peak retraction and (c2) peak protraction. (right) A diagram of the buccal mass was created to highlight key anatomical landmarks for each frame of the MRI video showing a complete biting sequence (d-e). The same diagrammatic representations of the landmarks are shown in (d) and (e) for the protraction and retraction portions of the biting sequence, respectively (See STAR Methods). The frames shown in (c1) and (c2) correspond to the 0 ms and 3410 ms frames, respectively, and are the same between the middle and right portions of the figure. Key frames referred to in the text: t0: start of the behavioral cycle, t1: peak rotation reached, t2: peak translation reached, t3: rotation plateau ended, t4: end of behavioral cycle. (f) Kinematic measurements were taken using the drawn diagrams for each frame in the sequence. See main text for definitions of variables. All scale bars correspond to 10 mm. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/715937v1_fig2.gif" ALT="Figure 2"> View larger version (34K): org.highwire.dtl.DTLVardef@1848bb9org.highwire.dtl.DTLVardef@f126a4org.highwire.dtl.DTLVardef@1ffd5forg.highwire.dtl.DTLVardef@336910_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 2.C_FLOATNO Kinetic/Kinematic biomechanical model of the buccal mass (a) Rest geometry of the biomechanical model. The grasper (odontophore) is modeled as a rigid ellipse (magenta with yellow radula). It is connected to the I1/I3 lumen (blue trapezoid) by the hinge muscle (green). The I2 protractor muscle (red) wraps conformally around the odontophore and attaches at the lateral groove. The net force and torque from the I2 on the odontophore are found by performing an instantaneous force balance on a small arc of the ellipse and integrating across the full region of contact between the I2 and the odontophore. The hinge muscle is modeled as a linearly elastic, geometrically exact beam. At each position along the beams midline, a quasistatic force balance is performed (see STAR Methods). (b1) The tension in the I2 is modeled using the length-tension relationship reported in Yu et al. 1999 scaled by a normalized activation level. (b2) The axial and bending stiffness of the beam hinge were calibrated to ex vivo animal data reported in Sutton et al. 2004. Gray region indicates odontophore displacements observed during biting behaviors (Sutton et al. 2004). (c1-c2) To investigate the effects of mechanical reconfiguration on odontophore position at peak protraction, (c1) the aspect ratio of the odontophore ellipse and (c2) the stretch of the lateral groove were added as additional kinematic constraints. (c1) and (c2) show results from the model but do not correspond to any particular behavior or configuration observed in the animal. These constraints impact the biomechanical model via contact forces from the I1/I3 (see STAR Methods). The lateral groove stretch is converted to a depression angle of the dorsal I1/I3 muscle as a proxy for the wrapping of the dorsal I3 around the odontophore observed during in vivo feeding behaviors (Fig 1). (d-e) MRI frames at peak protraction in (d1, with and without overlay) biting (t2) and (e1, with and without overlay) rejection ({tau}2) compared to corresponding frames from the biomechanical model (d2 and e2, respectively). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/715937v1_fig3.gif" ALT="Figure 3"> View larger version (56K): org.highwire.dtl.DTLVardef@1369a90org.highwire.dtl.DTLVardef@1dda429org.highwire.dtl.DTLVardef@4485d5org.highwire.dtl.DTLVardef@ae6523_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 3.C_FLOATNO Mechanical reconfiguration of the buccal mass (a) Midsagittal kinematics of the buccal mass during a (left) biting and (right) rejection behavior (see also Figs. S1 and S2). Colored circles (diamonds) show data for an individual frame, and the black line shows the two-point moving average of the signal. Vertical dashed lines show concurrent time points in the different kinematic signals (biting: t0: cycle starts, t1: peak rotation, t2: peak translation, t3: rotation plateau ended, t4: cycle ends. Rejection: {tau}0: cycle starts, {tau}1: rotation plateau ends, {tau}2: peak translation, {tau}3: peak rotation, {tau}4: cycle ends). (b) Model configurations for nine different pairs of aspect ratios ({Phi}) and lateral groove stretches ({lambda}LG ) (numbers correspond to the labeled points in (Fig. S6c)). Note that these simulated results from the model do not necessarily correspond to configurations observed in the animal but rather show changes in the systems configuration due to changes in the kinematic parameters. All configurations here were achieved with an I2 activation of AI2 = 65%. (c-d) Sensitivity of the model translation and rotation at peak protraction to lateral groove shortening ({lambda}LG, top row) and aspect ratio change ({Phi}, bottom row) for biting (c) and rejection (d). The y-axis for all panels reports the difference between the model prediction and observed animal value at peak protraction (for translation or rotation) normalized by the range of motion (ROM) for each behavior. For each panel, one kinematic parameter is held fixed (top:{Phi} fixed; bottom:{lambda} LG fixed) at the value observed in the animal at peak protraction, and the other is varied to determine the effect of changing this parameter on the translation and rotation of the odontophore. Vertical dashed lines show the observed value of the varied parameter in the animal at peak protraction. The horizontal dashed line shows 0 difference for reference. The steepness of the difference curve in the vicinity of the vertical dashed line indicates how sensitive the system is to changes in each kinematic parameter near peak protraction. Here, a steeper curve (with a positive or negative slope) indicates greater sensitivity. For biting simulations, AI2 = 15%, and for rejection, AI2 = 90% based on the results of the model validation. Each curve in (c) and (d) is a 1D cross-section of the 2D contour plots shown in Figs. S6-S7. For a complete view of the sensitivity of translation and rotation to lateral groove stretch and aspect ratio across the kinematic configuration space at different I2 activations, see Figs. S6-S7. Note that (c) and (d) use different vertical scales. The smaller scale for the rejection plots was chosen to better show the difference curves for rejection, and it reflects the overall decreased sensitivity to both lateral groove stretch and aspect ratio changes for the rejection behaviors. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/715937v1_fig4.gif" ALT="Figure 4"> View larger version (36K): org.highwire.dtl.DTLVardef@171f4c6org.highwire.dtl.DTLVardef@7d11a7org.highwire.dtl.DTLVardef@11206e3org.highwire.dtl.DTLVardef@82489c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 4.C_FLOATNO Mechanical reconfiguration facilitates behaviors in a variety of constrained hydrostat systems Combinations of the active shape change of internal structures (cyan), changes to the movement constraints and contact interaction (blue), and bending and conforming of structures (magenta) allow constrained hydrostats to mechanically reconfigure their neuromusculature (purple) to perform various behaviors. This can be seen in various systems across various species. As discussed here, the Aplysia buccal mass uses combinations of these mechanisms in (a) biting and (b) rejection behaviors to protract the buccal mass. (c) The pond snail, Lymnaea, has a morphologically similar buccal mass to Aplysia, but its I1/I3 homolog, the anterior jugalis, sits further posterior to the odontophore35, meaning it may more readily rely on the bending of the anterior jugalis and contact interactions during protraction. (d) The octopus and, more broadly, cephalopod buccal masses contain a beak that lacks a fixed articulation. Instead, by activating the lateral mandibular muscle (LMM), the buccal mass can create a stiff rotation point and may shift the function of the posterior mandibular muscle (PMM) from compressing the buccal mass to opening the beak36,37. (e) The human tongue (and other Type I tongues38) sits within the skull and makes use of contact with the hard palate to push food from the oral cavity into the pharynx27,48. (f) Additionally, by changing how the tongue interacts with the palate and teeth, while maintaining the same internal shape, humans can produce various vowel and consonant sounds39,49,50. This use of contact with the palate and teeth is known in the phonetics community as "bracing." Here, by creating a groove in the middle of the tongue, the phonemes /{varepsilon}/ and /ae/ can be produced. By raising the tongue and creating palatal contact while maintaining that groove, these vowels shift to the fricative consonants /s/ and /{theta}/49. Small insets show which of the mechanical configurations are used in each behavior. C_FIG

Heart development is supported by diverse cell types originating from the lateral plate mesoderm (LPM), including the first heart field (FHF), the second heart field (SHF), which is a part of pharyngeal mesoderm, and the newly described juxta-cardiac field (JCF) that harbors progenitor cells of the epicardium. While FGF-MEK-ERK signalling has been implicated in various developmental mechanisms, its role in cardiac specification remains elusive. This signalling pathway involves autoinhibitory loops, acting at translational and post-translational levels, resulting in pulses of ERK activity. We hypothesized that this alternating ERK activity could direct binary cell fate choices during mesodermal specification. Using an in vitro system, we found that inhibition of ERK activity in the LPM, before cardiac commitment, resulted in enriched gene expression of JCF and pharyngeal mesoderm/SHF markers, with high proepicardial marker levels, at the expense of FHF-like cardiomyocyte markers. Our findings reveal a novel contribution of ERK signalling in cell differentiation within the cardiac lineages.