Development, Growth & Differentiation

CRISPR-Cas genome editing toolkits have expanded the scope of genetic studies in various emerging model organisms. However, their applications are limited mainly to knockout experiments due to technical difficulties in establishing knock-in strains, which enable in vivo molecular tagging-based experiments. Here, we investigated knock-in strategies in the harlequin ladybug Harmonia axyridis, a model insect for evolutionary developmental biology, which shows more than 200 color pattern variations within a species. We tested several knock-in strategies using synthetic DNA templates. We found that ssDNA templates generated founder knock-in strains efficiently (2.5-11%), whereas the 5 regions of ssDNA templates were frequently deleted when the insert length exceeded [~]40 bases. To overcome this limitation, we designed several 3 extended DNA templates. Fast-annealed 3-extended double-stranded DNA templates, which were designed for tagging endogenous proteins with epitope tags, showed high founder generation efficiency (9.9-20.9%) and accuracy (30.8-85.7%). This strategy is also applicable to the two-spotted cricket Gryllus bimaculatus, suggesting that the fast-annealed 3-extended dsDNA template is a versatile DNA template for generating knock-in strains in emerging model insects for developmental genetic studies. Summary statementFast-annealed 3-extended dsDNA templates facilitate efficient CRISPR-Cas9-mediated knock-in in emerging model insects.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

The brain is one of the most complex animal organs but the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is known to be involved in brain patterning but their specific contributions remain to be elucidated in most cases, including foxQ2II. This transcription factor is known to be conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. However, the contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use an enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

In the multitude of suitable experimental systems used for functional studies in the field of developmental biology, the sea urchin plays a central role due to its amenability to various methods, including both transient and stable transgenesis. Among others, transposable elements represent powerful tools for generating stable transgenic specimens, and Minos transposon turned out to be an excellent genetic tool in marine organisms, despite its efficiency being host-dependent. This study provides new evidence for the activity of Minos transposable elements and their stable integration into the genome of the Mediterranean sea urchin Paracentrotus lividus. Using the Minos-based technology coupled with a fully-automated system used for the qPCR screening of the Minos transposon integration, we devised a new pipeline for performing transgenesis-based functional studies in P. lividus.

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.

Glucose has important roles in the development of the hematopoietic stem cells and the brain in vertebrate embryos; however, in most oviparous animals, the amount of glucose in the yolk is scarce. In zebrafish, gluconeogenesis takes place in the yolk syncytial layer (YSL), an extraembryonic tissue that surrounds the yolk. Gluconeogenic activity have also been observed in extraembryonic YSL-like tissue or endoderm-derived tissues in cloudy catshark, sterlet, and western clawed frog during development. However, it remains unclear when such ability was acquired or how it changed over the evolution of vertebrates. In this study, we used the Arctic lamprey, a cyclostome sister group of jawed vertebrates, to compare changes in metabolite levels and gluconeogenic gene expression patterns during development. Also, gluconeogenic activity was assessed using 13C-labeled substrates. Our metabolite analysis revealed that glucose levels increased during development and that glycerol was actively metabolized to produce glucose. In addition, many gluconeogenic genes were expressed in the muscle, notochord, and epithelium, making a striking contrast to previous observations in the above-mentioned vertebrates. Genomic DNA sequence motif analysis using HOMER and MEME identified common transcription factors binding motifs in the upstream regions of g6pc1/2 and fbp1 across vertebrate lineages. Among them, interestingly, the binding motif for HNF4A was not detected in g6pc1/2 and fbp1 genes of cyclostomes, suggesting distinct transcriptional regulation of gluconeogenesis in cyclostomes. These results indicate that gluconeogenesis is an essential process during development across vertebrate lineages, including cyclostomes, although the tissues and regulatory mechanisms for this function vary among lineages.

The axon initial segment (AIS) undergoes structural plasticity and refines neuronal excitability, yet the underlying mechanisms remain unclear. We here developed an in vivo CRISPR/Cas9 knockout platform using an all-in-one triple-guide RNA vector introduced via electroporation and employed this approach to seek molecules that regulate the developmental shortening of AIS in the chicken nucleus magnocellularis. We have targeted fourteen molecules associated with microtubules and found that knockouts of glycogen synthase kinase 3{beta} (GSK3{beta}) and Tau disabled the AIS shortening. Conversely, overexpression of constitutively active form of GSK3{beta} facilitated the AIS shortening in vivo. This extensive shortening was replicated in slice cultures, which was occluded by stabilization of microtubules. These results suggested that microtubule remodeling by GSK3{beta} activity contributed to the AIS shortening. This study thus provides a genetic approach suitable for genetic screening that allows identifying regulators of the AIS plasticity in the chicken brain.

Although transcranial magnetic stimulation (TMS) is widely used for brain stimulation, fundamental issues about its underlying mechanisms remain unresolved. We investigated some of these issues experimentally using an intact isolated turtle cerebellum in vitro, employing a novel chamber designed to deliver precisely calibrated induced electric fields along cortical depth. Our results show that single-pulse TMS can directly activate Purkinje cells and climbing fibers, and synaptically activate Purkinje cells via climbing fibers - all within the first 1.2 ms. Specifically, current source density analysis showed that TMS directly (non-synaptically) activated (1) climbing fibers near the bend with intracellular current directed toward the axonal terminals and (2) Purkinje cells directly near the axon initial segment with intracellular current directed toward the distal dendrites. The thresholds for direct activation of climbing fibers and Purkinje cells were found to be very similar, 25 {+/-} 1 V/m. The climbing fibers synaptically activated Purkinje cells, as expected, with intracellular current originating in the proximal dendritic trunk and directed toward the distal dendrites. At higher electric fields (> 58 {+/-} 17 V/m), TMS synaptically activated dendritic currents in Purkinje cells. These results provide new insight into how TMS may activate afferent fibers and cell bodies of cortical neurons.

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.

The mesencephalic trigeminal nucleus (MTN) contains the proprioceptive sensory neurons that innervate mechanoreceptors in the jaw closing muscles. In the chick embryo, MTN neurons are the first neurons generated in the mesencephalon. They arise bilaterally adjacent to the roof plate and then extend their axons ventrally before projecting caudally towards the rhombencephalon. MTN axons remain in a mid - dorsoventral position and pioneer the lateral longitudinal fasciculus. Notably, MTN axons never cross the roof plate, raising the question of which mechanisms underlie this restriction. Here, we investigated the effects of tissue transplants on the guidance of MTN axons. We found that both the diencephalon and the notochord exert repulsive effects on MTN axons, which could partially explain their early trajectory. We have also analysed the potential roles of the guidance cues BMP2/4, GDF7, SLIT and NETRIN in MTN axon navigation, both in vivo and in vitro. We found no evidence for a role of BMP2/4 or GDF7 in directing MTN axons. However, SLIT-ROBO signaling was found to play a significant role. SLIT proteins are repulsive guidance cues expressed by roof and floor plate. Loss or reduced expression of ROBO2 led to aberrant axon meandering within the dorsal midbrain. Most axons eventually reoriented posteriorly, and only a small fraction crossed the roof plate. Unexpectedly, in the absence of ROBO2, MTN somata migrated into the roof plate, resulting in the loss of a defined roof plate region. Taken together, these results suggest that SLIT2-ROBO2 signaling not only prevents MTN axons from crossing the roof plate but also maintains MTN cell bodies adjacent to the roof plate. With regards to MTN neuron guidance, we conclude that additional roof plate - derived factors are likely to co-operate with SLIT proteins to prevent crossing of the roof plate. Another possibility could be that SLIT might signal through additional receptors.

BackgroundDuring embryonic development and regeneration, the growth of any organ must be tightly regulated in order to achieve their optimal final size and become fully functional. Freshwater planarians, with their remarkable plasticity and ability to regenerate any part of their body based upon the presence of adult pluripotent stem cells, provide an ideal model to study how the final organ size is regulated during this process. Also, the fact that planarians are constantly growing and degrowing depending on culture conditions, allows us to study how the size of the different organs is determined under homeostatic cell renewal. ResultsHere, we investigate the role of Smed-pctk-1, a cyclin dependent kinase that belongs to the PCTAIRE subfamily of CDKs, which remains largely understudied. Functional analyses show that Smed-pctk-1 silencing disrupts the normal size of the cephalic ganglia and results in an overgrowth of the eyes both in homeostatic and regenerating planarians. The increase in eye size correlates to an increase in the number of both progenitor and differentiated eye cell types. Phototaxis behavioral assays reveal that Smed-pctk-1 RNAi planarians exhibit a precocious sensitivity to light. ConclusionsOverall, our findings identify Smed-pctk-1 as a key regulator of eye and neural size in planarians, highlighting its contribution to the mechanisms that control organ growth during both regeneration and homeostasis.

In this study, we investigate alliance formation and complexity in male Indo-Pacific bottlenose dolphins around Mikura Island using five years of data collected through underwater observations. Focusing on 18 mature males, we examined affiliative behaviors (proximity and rubbing), consortships, and associations. To determine male relationships, we evaluated a simple model (small units) and a complex model (large units). In both models, units were identified by association, and models were evaluated by the extent to which affiliative behaviors and consortship were concentrated within units. All types of behaviors were more concentrated within units determined by the complex model than the simple model, and thus the former was further investigated. The unit sizes determined by the complex model were three, seven, and eight, and variation in association frequency was observed within units. Within units, two to four males engaged in a single consortship irrespective of association frequency. Given that units are mediated by affiliative and cooperative relationships, it is reasonable to interpret units as alliances. Considering the variation in both alliance size and within-alliance relationships, as well as the fact that only a few males cooperate in a single consortship, we suggest that a multi-level structure is plausible in the Mikura community.

Xenopus laevis has recently emerged as a vital model for studying functional eye regrowth in pre-metamorphic tadpoles. Following eye removal surgery, tailbud embryos have been shown to regenerate a functionally complete eye within a 3-5 day period. While current studies have primarily focused on the signaling mechanisms required for this rapid regeneration, less is known about the specific stem cell populations and modes of regeneration employed by the embryo. In both the adult and tadpole, eye tissue regeneration can be facilitated through a combination of a pre-existing stem cell niche and the transdifferentiation of cells surrounding retinal or lens injuries, depending on the extent of the tissue removal. Notably, in the Xenopus eye regrowth assay, surgeries typically leave behind approximately 15% of the ocular tissue, indicating a post-surgical stem cell niche with potential for regeneration. In this study, we explored the hypothesis that a residual retinal progenitor cell (RPC) niche is critical for the rapid eye regrowth observed in Xenopus tadpoles. By utilizing a photoconvertible protein, EosFP, which changes permanently from green to red fluorescence, we selectively marked retinal progenitor cells (RPCs) in the presumptive eye area with red fluorescence. We then carefully preserved a small population of these red-labeled RPCs within the post-surgical wound. This progenitor cell niche, comprising not only the red-labeled RPCs but also the surrounding cells, creates a unique signaling environment. This specialized microenvironment is crucial, as it may provide specific signals that dictate the developmental outcomes of the RPCs, effectively controlling their fate. Observations made throughout the regrowth process revealed that the eye predominantly regrew from this red-labeled RPC niche within three days, with all retinal layers comprising red-labeled cells. The regrown lens was observed to be composed of a mix of both cells outside the RPC lineage and RPC progeny. Of interest, we observed cells of the closing optic fissure and ventral retina incorporate progeny from cells outside the labeled RPC lineage. These findings support the notion that the primary mode of regeneration in pre-metamorphic Xenopus eye regrowth involves the use of a pre-existing stem cell niche, and may also involve transdifferentiation, thus providing new insights into the mechanisms of embryonic eye regrowth in Xenopus laevis.

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.

Voltage-gated potassium channels (Kv) are a large family of potassium channels composed of 40 members across 12 subtypes. The KCNH genes encode 3 subfamilies of voltage-gated potassium channels: Kv10 (EAG, ether a go go), Kv11 (ERG, EAG-related gene), and Kv12 (ELK, EAG-like K). Kv channels play prominent roles in the neuronal and cardiovascular systems. Mutations in Kv channels have been linked to many human diseases, such as epilepsy, heart arrhythmias, and cancers. Significant progress has been made in understanding protein structures, physiological functions, and the pharmacological modifiers. However, the evolutionary history and gene expression of vertebrate KCNH genes during embryonic development remain largely unknown. We systematically identified and cloned 14 kcnh genes in zebrafish. Then, we examined vertebrate KCNH channel evolution by phylogenetic and syntenic analyses. Our data revealed that the three subtypes of the KCNH gene family have already evolved in invertebrates, long before the emergence of vertebrates. The number of vertebrate KCNH genes increased, most likely due to whole-genome duplications (WGDs). In addition, we examined zebrafish kcnh gene expression during early embryogenesis by in situ hybridization. Each subgroups genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues. Notably, kcnh6a showed robust expression in the developing heart, consistent with its conserved role in cardiac repolarization. Additionally, a few kcnh genes were transiently expressed in nonneural tissues, such as somites and the notochord, suggesting they may have a unique role in embryonic development. Our phylogenetic and developmental analyses of KCNH channels shed light on their evolutionary history and potential roles during embryogenesis, in line with their physiological functions and human channelopathies.

For over four decades, the bivalve Anomalocardia flexuosa has been recorded in Hong Kong coastal waters. However, the known native distribution of this heavily exploited commercial species is restricted to the Atlantic coast of South America, raising questions about the biogeographical validity of the Hong Kong populations. By employing an integrative taxonomic approach combining morphological re-evaluations and molecular phylogenetic analysis of the COI gene, we confirm that the species in Shui Hau, Hong Kong, China, has been historically misidentified. The population belongs to Cryptonema producta (syn. Anomalocardia producta).

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.

We sequenced the nearly complete mitochondrial genome of the hammerhead flatworm Bipalium nobile Kawakatsu and Makino, 1982 using short-read sequencing technology, yielding a 16,018 bp genome comprising 12 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. The composition and order of genes were consistent with those observed in the closely related species Bipalium kewense and Diversibipalium multilineatum, except for the position of tRNA-Glu. Phylogenetic analysis based on all mitochondrial proteins from species within the family Geoplanidae supports the monophyly of a clade comprising B. nobile, B. kewense, and D. multilineatum. The mitochondrial genome sequence obtained in this study provides a valuable resource for investigating the genetic diversity and population structure of B. nobile, a soil-dwelling predator with the potential for global spread as an invasive organism.

Adeno-associated viral (AAV) vectors are foundational tools for dissecting brain structure-function relationships, but AAV serotype tropism varies across brain regions and species, requiring empirical validation to inform experimental design. This need is especially important in non-model organisms, where molecular neuroscience tools remain underdeveloped and access to research subjects is often limited. The Arctic ground squirrel (AGS, Urocitellus parryii) is a valuable model for studying extreme physiology, including metabolic suppression during hibernation and resistance to cerebral ischemia/reperfusion, yet no studies have evaluated AAV performance in the AGS brain. Here, we investigated the ability of AAV serotypes 1, 8, 9, and DJ to transduce the AGS hypothalamus using the human synapsin (hSyn) promoter and directly compared cellular transduction rates in a region implicated in thermoregulation and hibernation. To maximize data collection from a limited experimental population, we used a within-animal, contralateral stereotaxic injection design. Recombinant AAV vectors expressing enhanced green fluorescent protein or mCherry were delivered bilaterally, and reporter expression was analyzed four weeks later. All tested serotypes produced clear and reproducible reporter expression, establishing AAV as a viable molecular tool in the AGS hypothalamus. AAV1 produced significantly greater cellular transduction rates than AAV-DJ (17.2% {+/-} 3.5% vs 8.4% {+/-} 2.9%, paired t-test, p = 0.032). AAV8 and AAV9 showed transduction rates of 22.8% {+/-} 0.6% and 20.1% {+/-} 1.5%, respectively; however, with only two biological replicates per serotype, formal statistical comparison was not performed. These findings provide the first direct characterization of AAV-mediated gene delivery in the AGS brain and establish a foundation for future molecular interrogation of hypothalamic circuits in this extreme mammalian hibernator.

Coleoid cephalopods, squids, cuttlefish, and octopuses, have emerged as powerful model organisms for studying neurobiology, development, and behavior, however, cellular tools for investigating their specialized tissues remain limited. In particular, their venom producing gland, the posterior salivary gland (PSG), has been extensively described anatomically and histologically, yet remains largely inaccessible to experimental investigation at the cellular level. Here, we report the first establishment of primary cell cultures derived from both the optical lobe and PSG tissues of Octopus bimaculoides. Building on recent advances in cephalopod brain cultures, we adapted and optimized dissociation and culture conditions to support short-term survival and attachment of cells in vitro. We show that passive cell release during tissue handling, rather than enzymatic treatment, yields viable cultures from both tissues, and poly-D-lysine markedly improves the adherence of PSG-derived cells. Morphological analyses and fluorescent staining confirm the presence and viability of distinct cell populations, while cell cycle analysis indicates that the majority of cells reside in G0/G1 phase. Notably, O. bimaculoides brain cultures exhibit features comparable to those previously described in squid, suggesting conserved aspects of coleoid cellular physiology. Together, our findings establish a foundational in vitro platform for studying octopus PSG and neural cell biology, providing a tractable system for probing venom biosynthesis, secretion, and neural regulation in coleoid cephalopods.