Development, Growth & Differentiation

The neural crest and neurogenic placodes, which arise from the neural plate border, give rise to morphological characteristics that distinguish vertebrates from invertebrates (Gans & Northcutt, 1983). Recent studies have suggested that embryos of ascidians, a group of tunicates that are the closest invertebrate relatives of vertebrates, possess cells that share an evolutionary origin with vertebrate neural crest cells (Abitua et al., 2012; Fatieieva et al., 2025; Ishida & Satou, 2024; Stolfi et al., 2015; Todorov et al., 2024; Waki et al., 2015) and neurogenic placode cells (Abitua et al., 2015; Ikeda et al., 2013; Liu et al., 2023; Liu & Satou, 2019; Manni et al., 2004; Mazet et al., 2005; Papadogiannis et al., 2022; Wagner & Levine, 2012). To dissect the neural plate border of ascidian embryos at the molecular level, and to gain deeper insights into evolutionary origins of the neural crest and neurogenic placodes, we comprehensively analyzed expression patterns of transcription factor genes at single-cell resolution. We demonstrated that the ascidian neural plate border consists of three domains with distinct gene expression profiles: the anterior, inner lateral, and outer lateral domains. A cross-species comparison of transcriptomes from ascidians and zebrafish suggests that the anterior domain is homologous to zebrafish neurogenic placodes, the inner lateral domain to the neural crest and tail bud and the outer lateral domain to the median fin fold ectoderm. We propose that a tripartite neural plate border was present in the last common ancestor of vertebrates and tunicates, providing a blueprint for evolutionary emergence of vertebrate morphological novelties.

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.

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.

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.

Meiosis is a fundamental process in producing both sperm and eggs, yet recombination landscapes often exhibit sexual differences, known as heterochiasmy. Since meiotic proteins are generally expressed in both sexes, the molecular mechanism driving heterochiasmy remains elusive. The -kleisin subunit gene of meiotic cohesin, Rec8, is expressed bisexually in mammals, while its putative teleost ortholog, rec8a, is expressed in a female-biased manner, presumably due to the presence of its paralog originating from the teleost-specific whole-genome duplication (TGD). Here, we elucidated the evolutionary history and expression dynamics of -kleisin genes across teleost lineages. Through comprehensive phylogenetic and synteny analyses, we revealed that major teleost lineages retain two copies of rec8 and rad21, with rec8 loci experiencing drastic chromosomal rearrangements immediately after the TGD. Using in situ hybridization and single-cell transcriptome data in medaka and zebrafish, we demonstrated a conserved sexually biased expression pattern: rec8a is predominantly female-biased, whereas rec8b exhibits male-biased expression during gametogenesis. Furthermore, comparative epigenetic analyses revealed that the conserved sexually biased expression is driven by lineage-specific cis-regulatory elements, rather than conserved ones. Motif analyses imply that regulatory rewiring by transcription factors, including foxl2l in particular, might have played a crucial role in the establishment and maintenance of this paralog divergence. Our findings highlight how whole-genome duplication and subsequent genomic and epigenetic rewiring subdivided the bisexual function of rec8, offering insights into sexually distinct meiotic regulation. HighlightsO_LITeleosts possess a unique -kleisin repertoire originating from the TGD. C_LIO_LITeleost rec8 paralogs exhibit conserved sex-biased expression during meiosis. C_LIO_LIDrastic genomic rearrangements after the duplication rewired the teleost rec8 loci. C_LIO_LIThe conserved expression pattern is governed by lineage-specific CREs. C_LIO_LIThose CREs harbor similar types of TFBSs such as Fox-family TFs. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/731870v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@c8c84dorg.highwire.dtl.DTLVardef@1d65668org.highwire.dtl.DTLVardef@c2d732org.highwire.dtl.DTLVardef@1be54a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Regenerative capacity varies widely across flatworms (Platyhelminthes). Whereas catenulids, microstomids and planarians can regenerate a complete head de novo, other flatworms cannot. This striking diversity raises a longstanding evolutionary question: does whole-body regeneration represent an ancestral trait that was subsequently lost in multiple lineages, or did it evolve convergently? Addressing this question requires comparative analyses of the molecular mechanisms underlying regeneration across phylogenetically diverse flatworms. Here, we focus on Wnt signaling, a deeply conserved regulator of antero-posterior (A-P) patterning and a central determinant of head-versus-tail identity during planarian regeneration, to establish a mechanistic framework for such comparisons. Although Wnt signaling has been studied extensively in planarians and parasitic neodermatans, its evolution and deployment in other flatworm clades remain poorly characterized. To address this gap, we characterized the complement of Wnt signaling components in two early-diverging flatworm clades, Catenulida and Macrostomorpha, with particular emphasis on expression and function in the catenulid Stenostomum brevipharyngium. Phylogenetic analyses reveal the ancient loss of six Wnt families and one secreted Frizzled-related protein (sFRP) family in the last common ancestor of flatworms, followed by additional lineage-specific gene losses and expansions. Moreover, several Wnt pathway components display markedly divergent expression patterns between catenulids and other flatworms, while functional analyses indicate corresponding differences in their regenerative deployment. Together, our findings reveal a dynamic evolutionary history of the flatworm Wnt signaling toolkit and establish a comparative framework for testing whether the molecular circuitry underlying head regeneration is ancestrally conserved or has evolved independently in distinct flatworm lineages.

Epithelial cells in Hydra perform an unusual combination of functions: they divide continuously like adult stem cells while simultaneously executing the complex physiological tasks of differentiated epithelia. This challenges the traditional distinction between proliferative stem cells and terminally differentiated tissue, raising the question of how a single cell type integrates these opposing roles. Using electron microscopy, we examined morphological characteristics that define the stem-like and differentiated states of Hydras ectodermal and endodermal epithelial cells. Stemness is reflected by nuclear characteristics of active proliferation, including extensive euchromatin, large nucleoli, and the presence of nuage. However, differentiated epithelial cells exhibit strong apical-basal polarity, various endomembrane compartments for endocytosis and transport, specialized secretion mechanisms, and basal muscle processes with dense-core vesicles implicated in hormonal communication. Cryofixation improved ultrastructure preservation, elucidating the pleiomorphic configurations of complex intracellular channel systems traditionally presenting as singular vacuoles. This may shed new light on possible functions of this compartment. Taken together, Hydra epithelial cells combine ancient stem cell traits with highly specialized differentiated functions. This multifunctionality provides insight into the cellular organization of early-branching animals and suggests that multifunctional epithelia may represent an ancestral condition preceding the strict segregation of stem and differentiated cell lineages in bilaterians.

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.

In the zebrafish visual system, accurate retinotopic mapping occurs throughout life as new neurons are generated and integrated into existing circuitry in both the retina and optic tectum (OT). To explore how OT development changes relative to innervation from the retina, we examined cell death and proliferation in genetic and surgical models that disrupt retinal innervation of the OT. Specifically, we compared lakritz (lak) mutants, which have no optic nerves due to a lesion in the atoh7 gene, with either wild-type or one-eyed fish generated through surgical eye removal. We observed elevated cell death, fewer proliferating progenitors, and fewer sox2+ OT neuroepithelial stem cells in lak mutant and denervated OT lobes. To examine whether light-mediated vision contributes to proliferation and survival in the optic tectum, we reared fish in constant darkness and then compared survival and proliferation of OT cells in innervated and non-innervated tecta. We found that OT cells were still more likely to survive and proliferate in the presence of optic nerve innervation even when fish were reared in the dark. To identify molecular pathways that could regulate OT growth, we examined the expression of known mitogens in the zebrafish optic tectum and found evidence that Wnt/{beta}-catenin pathway activity could promote innervation-dependent proliferation in lak mutant tecta. Expression of both wnt3a and the Wnt/{beta}-catenin target gene axin2, as detected by in situ hybridization and RT-qPCR, is decreased in non-innervated tectal lobes. Further supporting an innervation-dependent role for Wnt/{beta}-catenin pathway activation in the zebrafish OT, we found that lak mutants treated with a Wnt-pathway agonist, BIO, exhibited levels of OT cell proliferation that were indistinguishable from wild-type. Together these findings suggest that progenitor cells in the optic tectum produce Wnt3a in response to innervation by the optic nerve, providing new insight into how a vertebrate visual system coordinates growth across its sensory and recipient tissues.

Transient plasma membrane domains called lipid rafts have emerged as important regulators of signal transduction. These territories are formed by lipid-lipid and lipid-protein interactions, and these local interactions can be scaffolded by resident lipid raft organizing protein family members. While roles for lipid rafts have been described for multiple signaling pathways in many contexts, their in vivo prevalence and role during embryonic development remains incompletely understood. Here we examined gene expression for the Raftlin family of lipid raft organizing proteins, Raftlin (RFTN1) and Raftlin-2 (RFTN2), over the course of early vertebrate development, with a focus on neural crest cell dynamics. By analyzing transcriptomic data across vertebrate species, we identified conserved patterns of RFTN1 and RFTN2 expression across species, where RFTN1 is broadly expressed at low levels, while RFTN2 is distinctly enriched in neural crest cells. We used fluorescent in situ hybridization to spatially define Raftlin gene expression patterns in the early avian embryo. Our results show that RFTN1 is broadly expressed with periods of enrichment in the developing paraxial mesoderm. In contrast, RFTN2 expression is strongly enriched in neural crest cells, beginning during specification and persisting through migration, with additional expression in both the cranial and intermediate mesoderm. Together, these patterns suggest that Raftlins may play important roles in regulating signaling during development with specific roles in somitogenesis and in neural crest and mesodermal cell migrations.

The red palm weevil, Rhynchophorus ferrugineus, is the most economically destructive palm pest worldwide, threatening livelihoods, food security, and ecosystems across 49 countries. Weevil management currently relies predominantly on chemical insecticides, raising significant environmental and public health concerns. Despite its global agricultural importance, genetic approaches to pest management and the mechanistic basis of genome-editing strategies in Rhynchophorus remain largely unexplored. Here, we employed CRISPR/Cas9 genome editing to disrupt the R. ferrugineus ommochrome biosynthetic pathway -- a multi-enzymatic metabolic cascade that converts tryptophan into ommochrome pigments, including brown, yellow, and red pigments. We targeted two key pathway components: the ATP-binding cassette transporter white and the heme peroxidase cardinal. Both genes were ubiquitously expressed, with peak expression levels in the gut, fat body, and head. Elevated transcript levels were observed across early, mid, and late pupal stages and in 0-, 1-, and 2-day-old adult males and females, consistent with the progression of eye pigmentation throughout the R. ferrugineus life cycle. Embryonic microinjection of a single guide RNA (sgRNA)-Cas9 ribonucleoprotein complex targeting white produced in the Generation-0 (G0) adults with a distinct, white-eyed phenotype with a brownish outer margin, in contrast to the black eyes of wild-type adults. Genome-edited cardinal mutant adults displayed a translucent, brownish-white-eyed phenotype, with white streaks that gradually transitioned to a persistent translucent reddish-brown eye coloration. Mutations in both genes were confirmed in G0 adults by genomic DNA sequencing. Mutant adults were crossed to generate heterozygous G1 (+/-), G2 (-/-, -/+, and +/+), and G3 lines (-/-) with genotypes verified as carrying 2-, 3-, 9-, and 13-nucleotide deletions. A stable, heritable eye-color phenotype was established in homozygous knockout (-/-) G3 lines for both white and cardinal, confirmed by unambiguous indel (insertions/deletions) genotyping. Inheritance analysis revealed that both genes are X-linked, following a classical Mendelian sex-linked pattern: paternal alleles are transmitted exclusively to daughters, while maternal alleles are inherited equally by both daughters and sons. This study establishes the first fully homozygous knockout strain in R. ferrugineus and, by characterizing sex-linked inheritance in a coleopteran system, advances our understanding of how CRISPR/Cas9 can be efficiently applied to destructive palm weevil species. The present study represents the first report of CRISPR/Cas9 genome editing in any weevil (Curculionidae), using white and cardinal as marker genes. These findings provide a valuable platform for functional genomics and genome engineering in R. ferrugineus and offer a translational framework for genome editing in the invasive South American palm weevil, R. palmarum, laying a solid foundation for the development of gene-drive strategies aimed at sustainable palm weevil population control.

The sea star Patiria miniata is a widely used and powerful model organism for cell, developmental, and reproductive biology, but it has lacked genetic tools for expressing transgenes or endogenously tagging proteins. We developed a protocol to endogenously tag a broadly expressed actin gene and to express additional fluorescent markers from the same locus, using CRISPR/Cas9 genome editing. We also identified and isolated a promoter sequence of this actin gene which drives expression of transgenes. This promoter and transgene cassette can be introduced via a plasmid into the genome and persist through metamorphosis into the juvenile stage. Robust methods to induce metamorphosis that result in healthy juveniles are essential for developing stable transgenic lines and have been lacking in the field. Here we report a fast and efficient approach to induce the metamorphosis of larvae into healthy juveniles by introducing surf clam shells. Thus, we present a reliable method to generate both CRISPR/Cas9 knock-in and plasmid-integrated transgenic juvenile P. miniata, enabling future research on their fascinating biology, including regeneration, oogonial stem cells, metamorphosis, and more.

In ascidian embryos, gene expression from the zygotic genome begins between the 8- and 16-cell stages. While most zygotic genes are expressed in specific cell lineages at these stages, transcription factors that provide spatial cues for establishing specific expression patterns are insufficient to activate target genes at normal physiological levels. Gata.a is a transcription factor that provides spatial cues for targets expressed specifically in the animal hemisphere. Intriguingly, it is also required for physiological-level expression of many zygotic genes expressed in the vegetal hemisphere. In the present study, we found that Tbx21 and Klf6/7 augment the latter function of Gata.a. To determine the global extent of genes under control of these factors, we identified genes zygotically activated in early embryos using RNA-sequencing of BrU-labelled zygotic mRNAs. Our results revealed that approximately 80% of all zygotically activated genes were under control of these three factors. That is, together, Gata.a, Tbx21, and Klf6/7 are necessary to regulate target gene expression at physiological levels. This requirement for a specific set of broadly distributed factors resembles those of pioneer transcription factors that trigger zygotic genome activation (ZGA) in other animals, including flies and vertebrates. Regulatory factors involved in ZGA vary among animals, and our results indicate that ascidians use a distinct set of transcription factors for ZGA.

Neural crest (NC) multipotent stem cells give rise to many tissues including most of the peripheral nervous system, pigment cells and the craniofacial mesenchyme and skeleton. During gastrulation and early neurulation, cranial NC cells are specified in the ectoderm territory located between the anterior neural plate ectoderm and the future pre-placodal and lateral non-neural ectoderm. At the end of neurulation, NC cells undergo an epithelial-to-mesenchymal transition and migrate to various locations in the developing embryo where they differentiate. While the fine-tuning of NC specification is increasingly being elucidated, many questions remain, including how microRNAs may govern expression of gene programs during these processes. MicroRNAs are short non-coding 20-22 nucleotides-long RNAs which regulate gene expression through post-transcriptional repression. We have identified miR-219 as a candidate regulator of Xenopus NC development. Here, miR-219-dependent molecular pathways were investigated by morpholino knock-down and reveal NC phenotypes. The development of the NC and adjacent ectoderm was evaluated using whole mount in situ hybridization of key markers (pax3, zic1, xhe2, sox10, snai2, sox2), alcian blue cartilage staining, phenotype analysis, RNA sequencing of microdissected dorsal ectoderm and microRNA rescue experiments. While neural induction is mainly unaffected, miR-219 depletion alters gene expression programs associated with neural border development, resulting in loss of NC specification. HighlightsO_LImiR-219 depletion expands the neural border territory and disrupts neural crest specification. C_LIO_LImiR-219 depletion phenotypes are rescued with miRNA mimics. C_LIO_LImiR-219 morphant neural border expansion is rescued by pax3 depletion. C_LIO_LIRNA-seq reveals specific gene program modulation in miR-219 morphant neural crest. C_LIO_LImiR-219 is predicted to directly downregulate the neural gene Hes5.3. C_LI

Multiple hypotheses have been formulated to explain differences in tissue repair ability across vertebrates. One hypothesis posits that the accessibility of actively cycling stromal cells within uninjured tissue confers access to a proliferative population in response to tissue damage. This hypothesis further suggests that animals with an indeterminate growth mode possess an actively cycling cell population necessary for growth that can be readily accessed for tissue regeneration. Moreover, the absence of an actively cycling population in connective tissue provides a mechanism that restricts regeneration in animals with determinate growth whose cells are refractory to cell cycle progression and proliferation to produce new tissue for morphogenesis. Here, we explore this paradigm using an EdU-BrdU pulse chase strategy in four different vertebrate species: two with determinate (Acomys dimidiatus and Mus musculus) and two with indeterminate modes of growth (Danio rerio and Ambystoma mexicanum). We find that although indeterminate growers do possess a small population of actively cycling cells, this population does not contribute to regeneration. Moreover, we found that while Acomys does not possess a population of actively cycling stromal cells, cells re-enter the cell cycle de novo in these animals to contribute to regeneration. Furthermore, testing this hypothesis allowed us to ask whether tissue injury could stimulate cell cycle re-entry - a so-called primed state - in cells at distance from the injury site in these four species and we did not find evidence of such priming in stromal or epidermal tissue. HighlightsO_LICell cycle re-entry is a common response to injury in regenerative and non-regenerative vertebrates that is independent of actively cycling stromal cells in uninjured connective tissue C_LIO_LIActively cycling cells do not contribute to regenerative healing in spiny mice, axolotls or zebrafish. C_LIO_LIOur data do not support systemic cell cycle activation in response to injury. C_LI

The vertebrate neural retina is composed of several neuronal types that precisely organize into layers, with photoreceptors facing the outer surface and the projection neurons, retinal ganglion cells, at the innermost layer. This organization, essential for its function, is established during early development through a complex process involving cell-cell interactions such as adhesion. In the case of photoreceptors, two adhesion complexes, based on the adhesive proteins Cadherin2 and Crumbs, appear essential for their correct localization at the outer nuclear layer (ONL). We here aimed at better characterizing the role of the scaffolding protein PALS1, a central component of the Crumbs complex. Through a validated pals1a/nok morpholino knockdown strategy in zebrafish embryos, we demonstrate that its reduced expression causes photoreceptor progenitors to initially disperse as actively migrating cells, to then coalesce into cell groups around the central retina. They eventually start polarizing, forming rosette-like structures with the apical border towards the inside. Conversely, in organoids derived from uncommitted neuroepithelial retinal progenitors, PALS1 deficiency causes an inversion of their localization from internal rosette-like structures to an organized superficial layer. In both conditions, photoreceptors show signs of polarization, with apical borders towards the inside of rosettes in wild-type organoids, to surface-directed apical borders in morphants. Altogether, our results support previous observations of the pivotal function of the Crumbs complex in ONL formation, but also indicate that either the Crumbs complex, or PALS1 itself, are central for the delicate balance in differential cell adhesion partly responsible for retinal lamination.

Insects show extreme diversity and have long intrigued biologists. Recent technological advancements, such as gene editing and transgenesis, should in principle enable the use of almost any insect species for biological research. However, in practice, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug Oncopeltus fasciatus, we first generate a useful eye- and body-color mutant strain by using CRISPR/Cas9-mediated genome editing. Then we use this strain to develop an efficient piggyBac-mediated transgenesis system using a nuclear targeting approach. We show that incorporating 1x and 3x nuclear localization signals (NLS) into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus. Taking advantage of both the useful mutant strain and the efficient transgenesis system, we successfully integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated site-specific integration and histone-labelled strains for live fluorescence imaging. We further provide evidence that the Q system, a binary expression system, is functional in this species, paving the way for future sophisticated genetic manipulations including functional assays for cis-regulatory elements. Together, our results not only expand the genetic toolkit of O. fasciatus as a comparative model insect, but also provide a practical framework for developing efficient transgenesis in other non-traditional model organisms.

Uveal Melanoma (UM) is the most common eye cancer, with a metastatic mortality rate of 80%. Only 1-3% of patients have detectable UM at metastasis, and UM exhibits punctuated early growth. Doxycycline has recently been shown to inhibit metabolic processes exploited by cancer cells and reduce cancer cell growth in models of liver cancer. We hypothesized doxycycline may also be effective in UM and therefore tested doxycycline treatment in an eye organoid model of uveal melanoma. Using a stem cell line whereby BAP1 can be knocked down with a tetracycline-inducible system, we differentiated this line into a whole eye organoid model termed self-formed ectodermal autonomous multi-zone of ocular cells (SEAM). We found an enhanced proliferation in neural crest cells within the SEAM colonies. To identify the neural crest cells, we conducted single-cell RNA sequencing (scRNA-seq) analysis utilizing the Seurat R toolkit to pinpoint genes within neural crest clusters. To confirm the results of the in silico scRNA-seq analysis, genes with notable functions and differential expression in the neural crest cluster in relation to UM proliferation, angiogenesis, and oxidative phosphorylation were analyzed through immunofluorescence and RT-qPCR. Based on the scRNA-seq analysis, immunofluorescence, and RT-qPCR, the novel BAP1 KD (UM phenotype) model was found to replicate UM-relevant gene and protein expressions effectively, so the BAP1 KD (UM phenotype) was then treated with doxycycline to evaluate its effect on UM metastasis. Subsequent analysis found that doxycycline significantly inhibited UM growth, angiogenesis, and oxidative phosphorylation in the BAP1 KD (UM phenotype) model more than that of the control model, perhaps due to doxycycline targeting higher regions with more mitochondrial activity, indicating doxycyclines therapeutic potential in treating UM.

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.