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.

Tunicates are a sister lineage to vertebrates, with compact, relatively simple nervous systems featuring a single central ganglion, reflecting a minimal complement of chordate functional architecture. Although glutamatergic neurons are the most abundant population in vertebrates, their ancestry remains unclear. Here, we used glutamate immunohistochemical labeling (Glutamate IR) to identify glutamatergic elements in the neural system of the pelagic tunicate Doliolum sp. (Thaliacea). Glutamate IR was observed in all major nerves of the central ganglion, including motor-like terminals on the circular bundles of swim muscles, which were themselves labeled. However, the neuronal somata in the central ganglion were not labeled, suggesting glutamate accumulation in axonal processes and terminals. In contrast, we did not identify GABA-containing neural elements. This study suggests that glutamatergic systems were elaborated in the common ancestor of tunicates and vertebrates, although the functional role of glutamate and its role in muscular control need further investigation in these pelagic tunicates.

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.

PAX9 is an evolutionarily conserved paired-box transcription factor that is critical for embryonic development and human diseases. The mouse model has been predominantly used to investigate Pax9 functions. Zebrafish has emerged as a complementary vertebrate model for various human diseases, including cancers. Until recently, the functions of the zebrafish pax9 gene in jaw and hematopoiesis have started to be uncovered. However, detailed pax9 spatiotemporal expression, molecular mechanisms, and potential functions in other zebrafish organs remain largely unknown. With the technical advances in CRISPR-Cas9, non-homologous end joining (NHEJ) has made knockin and knockout a convenient way to examine endogenous gene expression in vivo and to generate a loss-of-function allele simultaneously. Here, we first generated pax9 knockin fish lines by inserting fluorescent proteins at the start of the endogenous pax9 coding region. Then, we examined pax9 expression in real time from early embryonic stages through adulthood. Except for previously reported expression domains, we were able to identify pax9 expression in high resolution in the paired and median fins, where pax9 marks anterior fin rays. Moreover, our knockin and knockout mutants showed increased fin ray number in median fins, but no evident effect on paired fins in pax9 null mutants. Thus, PAX9 is critical for median fin patterning in zebrafish.

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.

Primordial germ cells (PGCs) are the stem cell lineage responsible for transmitting genetic information across generations and are therefore central to studies of germline development, reproduction, and evolution. However, a lack of molecular tools in jawless vertebrates such as lampreys and hagfish has limited comparative insights into the evolutionary origins of vertebrate germline cells. Here, we report the development of the first germline-specific reporter (GSR) in the sea lamprey (Petromyzon marinus), Tg(piwil1: egfp-UTRnanos1), a piwil1-based germline reporter that is driven by the sea lamprey piwil1 regulatory region that directs Green Fluorescent Protein (GFP) expression in early PGCs. Promoter activity was validated by robust GFP expression in PGCs of zebrafish embryos co-injected with Tol2 transposase mRNA. Injection of the construct into sea lamprey embryos resulted in early, persistent, and gonad-localized GFP-positive cells. These results demonstrate stable germline-specific transgene expression in a jawless vertebrate and suggest deep evolutionary conservation of germline regulatory mechanisms between jawless and jawed vertebrates. Furthermore, the piwil1-based germline reporter line enabled lifetime visualization of zebrafish germline cells, revealing that increased PGC abundance during embryogenesis biases toward female sexual differentiation. Together, this study establishes the first GSR system in a jawless vertebrate and provides a useful platform for investigating germline biology and vertebrate germline evolution.

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

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.

Genome-edited mice are widely used to elucidate molecular mechanisms in vivo and are an indispensable tool, particularly for studies aimed at clarifying gene function at the organismal level. Currently, there is an increasing demand for mice in which multiple genes are simultaneously modified in order to investigate interactions among multiple genes. Notably, generating conditional multiple-gene knockout mice with temporal and spatial specificity requires extensive crossing between multiple Cre-driver mice and floxed mice, resulting in a prolonged time frame for line establishment. To address this limitation, we developed Transposon-Enhanced Multi-Plex Orchestration Editing (TEMPO-editing), a single-step strategy for generating multiple-gene-edited mice. TEMPO-editing enables simultaneous modification of multiple genes through the integration of transposon, Cre-loxP, and CRISPR/Cas9 systems. Using the DNA transposon piggyBac, we constructed a single cassette harboring gRNAs targeting genes of interest together with a conditionally expressed Cas9 (lsl-Cas9). By injecting this cassette into fertilized eggs of Cre mice, we enabled the generation of temporally and spatially specific genome edited mice in the F0 generation. In this study, we generated double-gene-edited mice targeting Hoxa13 and Hoxd13, which are key regulators of embryonic body patterning and are essential for autopod development. The phenotype observed in these mice was consistent with the incomplete autopod phenotype previously reported in mice generated by crossing Hoxa13 knockout and Hoxd13 knockout mice. These results demonstrate the utility of TEMPO-editing for the generation of multiple-gene-edited mice in the F0 generation. Notably, this study establishes a simplified strategy for producing conditional multi-gene-edited mice, a process that has traditionally required substantial time and labor using conventional methods.

Mucus-secreting cells and ionocytes play critical roles in many organs. Both cell types are usually distributed as scattered, solitary cells within an epithelium, a pattern presumably optimal for their function. To investigate how they attain their dispersed distributions, we imaged mucus cell and ionocyte precursors in the epidermis of developing zebrafish. Previous reports found that precursors of both cell types are first detected in the ventral epidermis (covering the yolk) before spreading dorsally, but the mechanism driving this progression was unknown. Photoconverting basal epidermal cells in the ventral embryo revealed that some cells actively migrate away from this area to populate the rest of the epidermis. These cells lose their basal cell identity when they start migrating and begin expressing markers of mature mucus cells or ionocytes during migration. These cells travel entirely between the two epithelial layers of the epidermis, occasionally pause migration to divide, and repel one another through contact inhibition of locomotion, behaviors that likely aid in their dispersal. After migrating for about a day, mucus cell and ionocyte precursors intercalate into the superficial epithelial layer of the epidermis, where they complete differentiation. These observations reveal how mucus cells and ionocytes achieve their scattered distributions in the zebrafish epidermis, suggesting that similar processes promote their distribution in other mucosal organs.

MyoD plays a central role in determining the skeletal muscle lineage in vertebrate embryos. The core enhancer (CE) and distal regulatory region (DRR) are the only known MyoD enhancers, and together, they recapitulate all major aspects of MyoD expression in the embryo. However, knocking out each enhancer individually has only modest effects on MyoD expression. Here, we show that embryos lacking both enhancers maintain muscle-specific MyoD expression, indicating the existence of unknown MyoD regulatory elements. Precision run-on sequencing together with available ChIP-seq and DNase I hypersensitivity datasets identified three new candidate enhancer regions within 96 kb of MyoD 5 flanking sequences. Transgenic analysis revealed that DNA elements at -36 and -60 kb are active in all muscle-forming regions, each recapitulating aspects of endogenous MyoD expression. Enhancer activities in muscle regulatory factor-deficient mice suggest that they are components of the auto- and cross-regulatory circuitry that maintains MyoD expression. Analysis of Hi-C data showed that the entire -96 kb region constitutes a loop domain, within which multiple interactions between elements and with the MyoD gene were detected. A larger loop domain delimited by CTCF sites was also identified from -96 kb to +220 kb relative to the MyoD transcriptional start site. These data indicate that the newly identified enhancers are key components of a cis regulatory network that controls the activation and maintenance of MyoD expression in the embryo. One sentence summaryCis regulation of MyoD transcription during development

Koalas (Phascolarctos cinereus) are marsupials highly specialized for arboreal life and are considered a representative example of convergent evolution with primates, which are also adapted to arboreal habitats. The forelimb digital flexors play a crucial role in arboreal locomotion by flexing the digits and enabling the claws or fingers to engage with tree trunks and branches. Although the morphology and function of the digital flexors have been extensively examined in primates, anatomical information on koala digital flexors has not been sufficiently updated in modern anatomical studies. In this study, we examined the digital flexor muscles and their innervation in four forelimbs from three koalas to clarify both shared and distinctive structural features among arboreal mammals. The flexor digitorum profundus (FDP) was markedly developed, formed a U-shaped muscle belly, and received dual innervation from the median and ulnar nerves. In contrast, the flexor digitorum superficialis (FDS) was small, arose from the superficial surface of the FDP, was partially enclosed by the FDP muscle belly, and gave rise to extremely slender tendons inserting into digits II-V. The branch supplying the FDS arose from the main trunk of the median nerve near the carpal region, ascended proximally in a recurrent course, and terminated within the FDS. This unusual course may be consistent with a morphogenetic process underlying FDS formation. These findings indicate that the koala digital flexors are characterized by a markedly developed FDP, a relatively reduced FDS, and a distinctive innervation pattern of the FDS. Thus, even among mammals adapted to arboreal life, koalas and primates appear to have achieved arboreal adaptation through different morphological specializations within the forearm digital flexor system.

The teleost whole-genome duplication (TGD) contributed to functional diversification of opsins. Some TGD paralogs, including those of parapinopsin (PP), Vertebrate Ancient (VA) opsin, and long wavelength-sensitive (LWS) opsin, show different absorption spectra and/or expression patterns. However, our knowledge of detailed evolutionary processes and mechanisms by which TGD contributed to opsin diversification is still limited. Here, we report that TGD paralogs of parietopsin (PT1 and PT2) are retained in extant species. While most species, including the zebrafish, have only PT1, several characins, including the red piranha (Pygocentrus nattereri), retain both PT1 and PT2. The Mexican tetra (Astyanax mexicanus) and catfishes have only PT2. To assess the degree of functional diversification between PT1 and PT2, we characterized spectral properties and expression patterns. Maximum absorption spectra differ slightly among PTs. Those of red piranha PT1, PT2, Mexican tetra PT2, and Japanese catfish (Silurus asotus) PT2 were located at 517 nm, 528 nm, 517 nm, and ~535 nm, respectively. Fluorescence in situ hybridization showed that (1) piranha PT1 and PT2 are expressed in the same pineal cells, and (2) they are also co-expressed with PP1. Based on these findings, functional diversification of opsins after gene duplication is discussed.

During vertebrate limb development, muscles, blood vessels, and nerves undergo coordinated morphogenesis. Limb muscles form through progressive splitting of the dorsal and ventral muscle masses, but the three-dimensional relationships between muscle splitting and surrounding tissues remain incompletely understood. Although blood vessels have been implicated in muscle splitting, whether this association is shared among different muscle masses is unclear. Here, we established a three-dimensional imaging approach for chicken embryonic forelimbs by combining fluorescent vascular labeling, whole-mount immunostaining, tissue clearing, and confocal microscopy. We compared three hydrophilic clearing methods, ScaleS, CUBIC, and SeeDB2, for compatibility with fluorescent ink, LCA-lectin, and PKH26 vascular labeling. SeeDB2 preserved all three vascular signals. We then developed a modified SeeDB2 protocol incorporating blood decolorization and used it to examine muscle, vessel, and nerve relationships from E5.0 to E6.0. In the ventral muscle mass, multiple vascular extensions were observed around the splitting region at E5.5. In contrast, no prominent vascular extension was detected around the splitting region of the dorsal muscle mass despite ongoing splitting at the same stage. Nerve projections also showed distinct spatial patterns between the dorsal and ventral muscle masses. These findings indicate that vascular extension is not a universal feature of limb muscle splitting and suggest that individual splitting events occur within distinct local tissue environments. Our three-dimensional approach provides new morphological insight into the coordinated development of muscle, vasculature, and peripheral nerves in the embryonic limb.

Fibroblasts are readily obtained and expanded and may provide an alternative cell source for cultivated seafood if their latent myogenic potential can be accessed. We examined whether transient exposure to 5-azacytidine (AZA), a cytidine analogue known to convert mouse embryonic fibroblasts into myoblasts, elicits a persistent muscle gene program in embryo-derived zebrafish (Danio rerio) Z3 fibroblasts. Cells were exposed to 0, 2.5, 5 or 10 M AZA for 48 h and maintained in drug-free medium for 3 weeks. RNA-seq identified 744 genes that increased and 670 that decreased consistently across the concentration series. Increased genes encompassed myogenic regulators, fusion factors, sarcomeric components and excitation-contraction machinery, whereas decreased genes were enriched for extracellular-matrix and adhesion functions. In a separate six-dose RT-qPCR experiment (0-20 M AZA) with three biological replicates per concentration, all 19 selected muscle genes were significantly increased at 1.25 M, the lowest concentration tested, whereas 9 of 12 extracellular-matrix/fibroblast genes were significantly decreased. Several muscle genes plateaued or declined at higher concentrations, and multinucleated myotubes were not observed. A separate 5-aza-2'-deoxycytidine (decitabine) series showed a comparable muscle-associated response at 0.25 M. These findings indicate that zebrafish fibroblasts retain access to a myogenic-like transcriptional program, and they provide a model for identifying food-compatible approaches to control fish-cell lineage plasticity for cultivated seafood.

Hox genes play central roles in specifying positional identities along the vertebrate anterior-posterior axis. In mice, genetic analyses have demonstrated that Hox genes distributed among the four Hox clusters contribute to vertebral patterning, with extensive functional redundancy among paralogous genes. Our previous genetic analysis in zebrafish identified important roles for HoxB- and HoxC-related genes in specifying anterior vertebral identities, whereas the contributions of HoxA- and HoxD-related genes remained unresolved. Here, we examined adult zebrafish carrying extensive combinations of hoxaa, hoxab, and hoxda cluster deletions and generated five-gene homozygous mutants carrying frameshift mutations in hoxa3a, hoxa4a, hoxa5a, hoxd3a, and hoxd4a. X-ray micro-CT analysis revealed no obvious alterations in anterior vertebral morphology in either the compound cluster mutants or the five-gene mutants. These results indicate that HoxA/D genes make only a limited detectable contribution to anterior vertebral patterning in zebrafish. Together with our previous findings, they suggest that vertebral patterning functions are distributed unevenly among zebrafish Hox clusters, with a predominant contribution from HoxB/C-related genes.

One of the first genes identified to regulate the spacing of nuclei in the multinucleated myofiber was Kinesin-1. However, the mechanism by which Kinesin-1 or other kinesins regulate myonuclear spacing is not known. Critically, the myofiber lacks centrosomes, and the many myonuclei act as the primary microtubule organizing centers of the cell. Because of this unique re-structuring, we hypothesized that the kinesins that drive centrosomes apart during mitotic spindle elongation may play a similar role in spacing myonuclei. We found that the bipolar Kinesin-5 (Klp61f) and the (-)-end directed Kinesin-14 (ncd) were both necessary for myonuclear spacing at different times, with both being necessary during embryogenesis, but only ncd being necessary in the fully differentiated myofiber. To investigate the shared mechanisms during embryogenesis, we used live-imaging and found that, similar to the mitotic spindle, Klp61f acts as an accelerator for myonuclear movement, whereas ncd acts as a brake contrary to this movement. To investigate these mechanisms and test the hypothesis that this is dependent on microtubule-sliding, we used super-resolution microscopy to visualize and quantify the microtubule network in animals with disrupted Klp61f or ncd. We found that in both cases, there was a decrease in the amount of microtubule overlap between neighboring myonuclei. Furthermore, we found that disrupting ncd led to severe changes in microtubule network organization, supporting our hypotheses that microtubule-sliding is necessary to space myonuclei, and that ncd likely functions through a unique mechanism in the differentiated myofiber to maintain myonuclear spacing. Together, our data supports a model where myonuclear spacing is regulated by a counteracting force generated by different kinesins during embryonic development. Furthermore, one kinesin, ncd, is repurposed in the differentiated myofiber to dynamically crosslink microtubules, a function necessary to anchor nuclei in place. Thus, kinesin motors regulate myonuclear spacing across developmental time by leveraging opposing forces through diverse mechanisms.