Development, Growth & Differentiation

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (~130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

The hallmark of sessile adult ascidians is a vase-like shape with a single oral and atrial siphon. Ciona, however, develops two atrial siphons after metamorphosis, which subsequently fuse into one. The mechanisms underlying this fusion are unknown. This study suggests that Hox2 controls this process. Hox2-knockout animals using Transcription-Activator-Like Effector Nuclease (TALEN) retain two atrial siphons throughout their lives. During normal fusion, epidermal cells between the siphons flatten along the anterior-posterior axis. This cellular flattening does not occur in Hox2-knockout animals, suggesting that the shape change in the epidermal cells produces tension, allowing the atrial siphon openings to converge at the midline for fusion. Hox2-knockout animals lack cupular organs, which are suspected hydrodynamic sensors in the internal epithelium of the fused atrial siphon and on the sperm duct. Among several knockout attempts, atrial siphon fusion was reproduced by only one TALEN pair, suggesting that this phenotype is driven by a mutation having a broader effect than those abolishing protein function. Many ascidians, unlike Ciona, develop a single atrial siphon shortly after metamorphosis. Our findings suggest that a phylogenetically conserved gene, Hox2, establishes this group-specific atrial siphon formation mechanism in Ciona.

Zebrafish (Danio rerio) possess remarkable regenerative capacity, making them an ideal model for studying the molecular mechanisms underlying tissue regeneration. In this article we report the identification of blastema linked exosome like extracellular vesicles (EVs) in zebrafish, that to the vesicles were plausibly being translocated in the proximo-distal axis through filipodia. We further thoroughly examined the exosome like EVs isolated from regenerating tissues of zebrafish caudal fins to characterize their nucleic acid cargo and evaluate their potential regulatory functions in regeneration. Caudal fins were amputated and allowed to regenerate and exosome like EVs isolated from blastema tissues displayed increased abundance compared to non-amputated controls. RNA sequencing identified a dynamic cluster of EV linked microRNAs (miRs). These differentially expressed miRs, including dre-miR-21, dre-miR-200b, dre-miR-218a and dre-let-7e were upregulated and associated with promoting proliferation, migration, differentiation, and tumour suppression pathways. Moreover, dre-miR-100, dre-miR-146a and dre-miR-200c regulated osteogenic differentiation, inflammatory signalling, epithelial-mesenchymal transition, and cell adhesion. Regeneration is generally believed to be coordinated only by local morphogen diffusion. Through this study it is indicative that filipodia bound EVs might have a pivotal role in long-range communication between blastema and the proximal tissues during the regeneration process. A detailed analyses of the miR targets and their validation could potentially lead to novel advancement and solutions in the field of regeneration and regenerative medicine in the near future.

Bergmann glia-like progenitors (BGLPs) are transient astroglial progenitors in the postnatal cerebellum, but how their lineage potential changes during development remains incompletely understood. Our previous electroporation-based study suggested that P0 BGLPs possess broader lineage potential than P6 BGLPs. Here, we performed recombination-based lineage tracing by cerebellar surface application of tamoxifen to Ai9/+; GlastCreERT2/+ mice and temporally analyzed the progeny of BGLPs labeled at P0, P3, P6, and P8. We found that BGLPs undergo progressive lineage restriction during postnatal development. P0 BGLPs gave rise to Bergmann glial cells (BGs), inner granule cell layer astrocytes (IGL astrocytes), white matter astrocytes (WM astrocytes), and molecular layer inhibitory neurons (ML-INs), confirming our previous electroporation-based findings. In contrast, P3 BGLPs generated BGs, IGL astrocytes, and WM astrocytes, whereas P6 BGLPs generated BGs and IGL astrocytes, and P8 BGLPs generated predominantly BGs. Thus, BGLP lineage output was progressively restricted from four progeny categories at P0 to a predominantly BG-restricted output by P8, suggesting that BGLPs dynamically adjust their cellular output during postnatal cerebellar maturation. Additional temporal analyses suggested that ML-INs are unlikely to be generated directly from P0 BGLPs, but may arise indirectly through astrocyte-like progenitors (AsLPs) and inhibitory neuron progenitors (INPs). These findings identify postnatal BGLPs as a useful in vivo model for studying progressive lineage restriction and stage-specific cellular supply during cerebellar development.

Effective communication between cells is essential for the typical development and behaviour of an organism. In this context, gap junctions represent the most universally preserved components at cellular membranes of multicellular organisms, facilitating metabolic and electrical connections between cells. Disruptions in these junctions have been linked to various developmental abnormalities and pathological conditions in humans. The invertebrate gap junction proteins, referred to as innexins, exhibit conserved cellular and molecular mechanisms of functioning with their vertebrate counterparts, known as connexins. Consequently, they provide valuable means for studying and understanding the functions of gap junctions in development. In the Drosophila embryo, innexin-2 is expressed in the amnioserosa and ectoderm, where it is required for epithelial morphogenesis. Genetic depletion of innexin-2 results in cuticular defects and embryonic lethality. Pannier, a GATA family transcription factor, is a key regulator of dorsal tissue development in Drosophila and is expressed in the amnioserosa, dorsal ectoderm and the dorsal vessel during embryogenesis. Pannier mutants exhibit defects in dorsal closure, cuticle formation, and cardiac specification. Although substantial evidence from vertebrate systems indicate that connexin expression is regulated by transcription factors such as GATA4, Nkx2.5, Tbx2, Tbx3, and Tbx5, whether a similar regulatory relationship exists between these transcription factors and gap junction proteins in Drosophila remains unknown. In this study, we investigate how innexin mediated intercellular communication impacts pannier dependent morphogenetic processes during Drosophila embryogenesis.

The endoderm is specified at the onset of gastrulation and subsequently undergoes extensive migration before forming an epithelial sheet that gives rise to multiple organs, including the gut and respiratory tracts. Although the gene regulatory network underlying endoderm specification and the later processes that regionalize the gut are increasingly well understood, comparatively little is known about the intervening developmental events. Using single cell transcriptomics, we profiled the zebrafish sox17 lineage, comprising endoderm and dorsal forerunner cells, throughout and immediately after gastrulation. We found that dorsal forerunner cells remain transcriptionally homogeneous while undergoing coordinated temporal changes, associated with ciliogenesis and epithelial organization, during assembly of Kupffers vesicle. In contrast, endoderm cells transition from a migratory to an epithelial transcriptional state while progressively acquiring distinct regional identities. These findings indicate that endoderm regionalization emerges within the context of a broadly shared transcriptional program associated with migration and epithelialization.

Mammal tails have long been recognized for their diversity of morphological form and function, however, there remains a substantial gap between the motivation to understand and emulate the various performance functions of the tail and what is known about tail anatomy. In this study, we were motivated to discover the anatomical foundations of the fast, whipping motions of the tail of the lesser Egyptian jerboa (Jaculus jaculus), which may aid in the quick changes of direction as the animal escapes from predators using ricochetal bipedal hopping. We employed microCT scans, dissections, and museum data to describe the musculoskeletal anatomy of the jerboa in comparison with the laboratory mouse (Mus musculus) and rat (Rattus norvegicus). While many aspects of tail anatomy are conserved across these species, the jerboa does possess unique characteristics such as an extremely long tail arising from caudal vertebral elongation, development of extensive dorsal musculature differentiated into lateral and medial components to increase points of skeletal attachment, and a novel anatomical feature - the bi-lobed cranial transverse process - which serves as a supernumerary dorsal tendon attachment site and possible brace to protect the ventral tendons and intrinsic muscles for a section of caudal vertebrae which likely experiences high mechanical stress.

Conserved dorsoventral patterning systems have been proposed as evidence for a common evolutionary origin of centralized nervous systems in Bilateria, yet functional evidence outside vertebrates and arthropods remains limited. Here, we investigated the role of pax6 in the annelid Platynereis dumerilii using a mutant carrying a 61 bp deletion in the paired-domain coding region. Loss of pax6 disrupted ventral neuroectodermal patterning at 34 hpf, causing a shift in nk2.2 expression, narrowing of the nk6 domain, and downregulation of pax3/7, while msx expression remained largely unaffected. These early patterning defects were followed by selective neuronal abnormalities at 48 hpf, including displacement of TrpH-positive serotonergic cells and loss of posterior hb9-positive motoneuron domains. By 6 dpf, additional defects were observed in TrpH, ChAT, VAChT, and nk2.2 expression, accompanied by severe disruption of ventral nerve cord morphology and loss of the characteristic rope-ladder architecture. Together, these findings identify pax6 as a key regulator linking dorsoventral progenitor patterning, neuronal subtype specification, and nervous system morphogenesis in Platynereis. Our results provide functional evidence that the conserved dorsoventral patterning network plays an essential role in annelid ventral nerve cord development and support the view that important components of bilaterian nervous system patterning predate the divergence of major animal lineages.

This study presents a systematic revision of the suborder Astrophorina for the temperate Pacific coast of the United States and Canada. Major findings include a reduction in the number of species previously thought to range into the region from Japan; validation of most Geodia species erected by Lendenfeld (1910), which were later synonymized by de Laubenfels (1932); the formal description of 10 new species (Poecillastra alaskensis sp. nov., Vulcanella explorata sp. nov., Vulcanella rupta sp. nov., Stelletta cardenasi sp. nov., Stelletta nicolenya sp. nov., Stelletta limuwensis sp. nov., Dercitus (Stoeba) giveni sp. nov., Penares anyapax sp. nov., Penares foxi sp. nov., and Thenea diastra sp. nov.); and one new combination, Penares orientalis comb. nov. Extensive SCUBA-based collection efforts yielded new samples for 11 of the 26 species identified in the region, which enabled an integrative taxonomic approach that combined field photography, fresh material for DNA sequencing, and improved characterization of species ranges and morphological variability in previously described taxa. Illumina sequencing generated complete nuclear ribosomal haplotypes for five species, while Sanger sequencing of the 28S and cox1 loci placed 20 of the 26 species within molecular phylogenies. The use of very short "mini-barcode" amplicons also enabled sequence recovery from historic type specimens up to 137 years old. This study additionally reports the discovery of sponge grounds of abundant, large Geodia at diving depths in Southern California. Together, these results substantially advance our understanding of global astrophorid diversity and systematics, and the biogeography of sponge diversity in the Northeast Pacific. Note about species names: this pre-print is not intended to be a publication of the associated species names for the purposes of zoological nomenclature.

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

In contrast to many other developing systems, in which axon pathfinding and synaptogenesis are separated in time, the pioneering axon of the individually identifiable caudal primary motor neuron in embryonic zebrafish forms en passant synapses during its stereotypical ventral growth. How simultaneous synaptic differentiation and axon pathfinding are coordinated is not fully understood. Here we ask what the role of the tac1 gene, coding for the synaptic tachykinin neuropeptides, is in this unique axon differentiation process. The gene is expressed during axon outgrowth and its disruption results in increased branch length of CaP axons and subtle morphological defects of the pre-synapse. These abnormalities are accompanied by a robust [~]1.5-fold increase in motor neuron activity and in spontaneous early contractions in tac1-deficient embryos. Furthermore, pharmacological inhibition of the tachykinin receptor (Tacr1) leads to altered CaP axonal morphology, mimicking the axonal phenotype observed in tac1-deficient zebrafish. These findings suggest that tachykinin neuropeptides modulate formation and activity of en passant synapses and prevent aberrant axon branching during growth of zebrafish motor axons. HIGHLIGHTS- tac1 refines CaP primary motor axon development in zebrafish - Loss of tac1 disrupts presynaptic maturation at the horizontal myoseptum - tac1 mutants show elevated motor neuron activity and spontaneous contractions

The morphogenesis of the optic cup provides a robust system for studying how two apposed epithelial monolayers with distinct properties fold and stretch in a coordinated manner to form the primordial eye. While much research has been conducted on the temporal dynamics of retinal neuroepithelium invagination, the spatial organization and stretching of the retinal pigment epithelium has received less attention. The fish species Astyanax mexicanus offers a unique model to examine the mechanisms of optic tissue morphogenesis through a comparative lens, as it exhibits natural variation in eye development between its river-dwelling and cave-adapted morphs. Using quantitative 3D imaging of optic cups from both morphs, we found that RPE morphogenesis involves transient, graded, and anisotropic cell stretching that patterns the epithelium during optic cup shaping. Analyses of RPE nuclear spacing and cell morphology showed that tissue stretching gradually increases along the proximo-distal axis, suggesting maximal tension in the elongated distal RPE cells aligned along the optic cup meridians. Furthermore, nuclear volumes and apical surface areas of RPE cells scaled spatially along the same axis, independently of endoreplication. In the cavefish natural mutant, RPE expansion was delayed by over six hours and proximal stretching exhibited altered isotropy, indicative of disrupted temporal coordination and suggesting modified mechanical constraints. These results demonstrate that RPE morphogenesis is a highly heterogeneous process from a spatiotemporal perspective, offering new insights into the study of the biomechanical principles of eye development in vertebrates. Summary statementThis study reveals the emergence of cell morphology gradients within the retinal pigment epithelium during morphogenesis of the eye in two distinct populations of the same species of fish.

Aquatic animals deploy adhesives, in numerous essential functions, and reversibility is a key adaptation. The molecular mechanisms of reversible wet adhesion remain poorly understood. Using a model organism, the freshwater cnidarian Hydra vulgaris, we dissect the mechanism of molecular assembly in a secreted adhesive and uncover a glycan and protein-based architecture organized by a lectin-like protein, Hydra vulgaris adhesive protein 1 (HvAb1). We identify HvAb1 as a nonredundant organizer of the adhesive matrix, being basal-disc specific and secreted. Knockdown of HvAb1 severely impaired attachment and disrupted footprint architecture in a mosaic pattern, with only HvAb1-positive regions of the adhesive footprint retaining their normal structure. The adhesive is wheat germ agglutinin (WGA)-reactive and contains a fibrillar chitin-based sub-network, synthesized by a basal-disc-specific chitin synthase. Applying exogeneous chitinase abolished both WGA staining and Hydra attachment, indicating that WGA-positive components perform essential roles in adhesion. Our results therefore describe a glycan-dominated matrix, organized via a lectin-like protein (HvAb1), which is reinforced by chitin and enables reversible adhesion underwater. This establishes Hydra as a tractable model to better understand the principles of reversible adhesion underwater and, potentially, inform future bioinspired, sustainable adhesives.

Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. Key PointsO_LIzebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages C_LIO_LIzebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis C_LI

RNA interference (RNAi) is a widely exploited reverse-genetics tool with potential uses for disease control. Successful RNAi has been reported in trematode-vectoring snails, but the composition of RNAi effector-encoding gene complements, a key driver for RNAi efficiency, remain unstudied in these species. Using bioinformatics and comparative genomics, we searched for orthologues of 115 RNAi effector sequences in genomes or transcriptomes of four snail vectors: Biomphalaria glabrata, B. pfeifferi, Bulinus truncatus, and Lymnaea staginalis. Gene expression patterns of selected RNAi effectors were then examined across developmental stages and tissues of the model B. glabrata snail. At least 74 RNAi-related proteins were conserved across all four species, including core components known to be essential for gene silencing. Classical systemic RNAi-deficient (SID) genes that facilitate systemic RNAi in other systems were absent, suggesting that alternative pathways may compensate for dsRNA uptake and transport. Core effectors of secondary RNAi amplification and heritable RNAi were not detected. Expressions of Dicer-1, Argonaute-2, and the exonuclease Eri-1 did not vary significantly with snail size. A putative RNAi-inhibiting Staufen orthologue showed elevated expression in the ovotestis, while another putative cholesterol-interacting gene was overexpressed in the trunk tissue and may partly contribute to RNAi import. Altogether, our results present the most comprehensive overview of RNAi pathway effectors in major intermediate snail hosts for trematodes. The findings underscore the likely broad potential for RNAi use in trematode intermediate hosts as an experimental tool and potential control method.

Large-scale brain imaging has relied heavily on fluorescent reporters; however, photobleaching and signal variability limit quantitative analysis in intact tissues. Here, we intro-duce MelaCAST (melanin-based scattering CAST imaging), a genetically encoded scattering strategy for whole-brain imaging. AAV-mediated delivery of tyrosinase enables cell-type-specific melanin production, generating stable intracellular scattering contrast throughout the mouse brain. By integrating tissue clearing with scattering tomography, MelaCAST enables non-photobleaching, high-throughput volumetric imaging of genetically defined cell populations in in-tact brains. This approach establishes melanin as a genetically encoded scattering reporter and expands whole-organ imaging beyond fluorescence-based modalities.

Memorizing a new phone number or address is hard at first, but becomes easier with repetition, as information shifts from working memory to long-term memory. Here we investigated how repetition affects the storage and transition of different aspects of mnemonic information by comparing univariate neural markers of active object storage with multivariate decoding of memory content. Thirty participants encoded lateralized stimuli from a continuous shape space into memory. Memory items were repeated six times in a row to induce learning. In line with earlier work, EEG recordings revealed that repetition led to a reduction in contralateral delay activity (CDA), a measure of active storage that has been taken to reflect a pointer-like representation of the individual object or its original source. In contrast, shape decoding during the retention and also after an impulse perturbation remained constant across repetitions. These results suggest that learning over repetitions reflects the abolishment of active and individuated object memory representations while passive, source-independent memory representations are retained.

Intelligent behavior depends on the brain's ability to represent multiple features of the environment simultaneously while keeping those representations independent [1,2]. Patients with Alzheimer's disease often mix up objects, people, and events [3-7], raising the possibility that disease mixes up the way that information is represented in the brain. Here we show that the independence of visual representations progressively breaks down during early stages of disease progression in a rhesus macaque model of Alzheimer's disease and related dementias [8-10]. In visual area V4, representations of different visual features become progressively less independent, such that the representation of one feature is increasingly influenced by the value of another. We term this loss of independence neuronal feature confusion. This neuronal change predicts a specific behavioral consequence: because feature representations become less independent, preferences associated with one visual feature increasingly influence visually guided choices associated with other, independent features. Using an analogous image-selection task, we found the same behavioral signature in people with mild cognitive impairment, distinguishing them from age-matched controls. These results identify a specific and measurable alteration in neuronal population representations that predicts a behavioral change observed across species. More broadly, these findings demonstrate that neuronal population representations can guide the development of sensitive, non-invasive behavioral methods for early detection of functional changes associated with Alzheimer's disease.

Ethological tasks promise to engage the integrated perception, memory, and decision-making that define natural behavior, yet laboratory implementations are often so sparsified that they may fail to recruit the very cognitive processes of interest. We tested whether increasing environmental complexity in a standard task could expose this hidden cognition. Mice that were already expert hunters in a bare arena were challenged to capture live insect prey in arenas filled with objects that obstruct movement, occlude vision, and offer the prey places to hide. Despite their prior mastery, the added complexity revealed an entire layer of learning that the simple task failed to engage: rather than refining the sensorimotor details of pursuit, mice reorganized how they searched the environment. Across trajectory, kinematic, and object-referenced analyses, learning was expressed predominantly within the search state. To analyze behavior in explicit relation to environmental structure, we developed an open-source framework-a compact ethogram with hierarchical, pose- and object-based classification-that links each action to its environmental context. Unsupervised analyses revealed structured search dynamics across multiple timescales, and a minimal, interpretable agent-based model showed that short-term spatial memory and object-specific value are together sufficient to reproduce the non-random structure of search, including a learned, non-backtracking bias that emerged within the first days of object exposure. Classifiers further showed that mice selectively acquired the object interactions most likely to expose hidden prey. Reproducible with inexpensive materials, the paradigm and its analysis tools offer a sensitive behavioral readout of search, memory, and strategy for studies that conventional low-dimensional assays leave unresolved.

Light is a fundamental regulator of human physiology and behaviour. Whether prior light exposure shapes subsequent higher-order cognition and mood beyond the period of exposure remains unknown. We tested this in a within-subject, randomised crossover experiment in which 24 healthy young adult males completed a multimodal cognitive battery following 2 x15 min of full-spectrum light (FL; median 1,029 melanopic equivalent daylight illuminance [mEDI]) or standard indoor light (SL; median 234 mEDI), with all testing conducted under identical dim illumination. FL improved Digit-Symbol Substitution Test accuracy and promoted digit-directed gaze reallocation, consistent with more efficient associative encoding. On the Balloon Analogue Risk Task, FL reduced reward-seeking behaviour and suppressed backward-referencing gaze transitions linking current and prior-trial reward information. Mood declined following SL but remained stable after FL. Sustained attention, vigilance, and subjective sleepiness were unaffected. Our findings identify pre-task FL exposure as a selective primer of higher-order cognition and mood, independent of alertness.