Developmental Biology

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

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.

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.

Wnt/{beta}-catenin signaling is a critical pathway that regulates nephron progenitor renewal versus differentiation as well as nephron patterning. In addition to its role as a transcription co-factor, {beta}-catenin also functions as a structural component of adherens junctions, where it interacts with cadherins to link cell-cell contacts to the cytoskeleton. However, the relationship between the nuclear and junctional localization of {beta}-catenin during vertebrate nephron development remains poorly understood. To define how endogenous {beta}-catenin localization changes during nephrogenesis, we optimized an accelerated-turnover {beta}-catenin chromobody for live imaging in Xenopus embryos. Using in vivo imaging of Xenopus pronephric development, we visualized endogenous {beta}-catenin within the nuclear, cytoplasmic, and junctional compartments. Across successive developmental stages, {beta}-catenin became progressively enriched at epithelial junctions during nephron maturation while remaining abundant within nuclear and cytoplasmic compartments. Quantitative analyses indicate that epithelial maturation is accompanied by coordinated expansion and partitioning of multiple intracellular {beta}-catenin pools rather than a simple redistribution from nuclear to junctional compartments.

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.

Eph and ephrin genes encode receptor-ligand pairs that mediate contact-dependent cell signaling and are essential for nervous system development. However, less is known about the role of Ephrin signaling during adult tissue homeostasis and regeneration. Here, we investigated the role of Ephrin signaling in neural patterning in the planarian Schmidtea mediterranea. We discovered that RNAi against the Eph receptor EphR1 led to striking ectopic expression of the mechanosensory neuron markers pkd1L-2 and hmcn-1-L, without obvious disruption of the overall architecture of the central nervous system. To investigate the basis of this phenotype, we identified additional Eph receptor homologs and four putative ephrin ligands and assessed their function. An RNAi screen revealed that ephrin-1 phenocopies the defects of EphR1 RNAi. Temporal analyses of EphR1 and ephrin-1 inhibition revealed a progressive increase in pkd1L-2+ and hmcn-1-L+ cells, indicating an unappreciated role for Ephrin signaling in regulating neural patterning and cell number during adult tissue homeostasis. Together, these findings provide a framework for dissecting Ephrin-dependent mechanisms in adult tissue maintenance and regeneration.

The nervous system consists of the brain and associated structures that are replete with highly specialised neurons and glia. Central to the development of the brain are Neural Stem Cells, which self-renew to maintain their number and also give rise to differentiated progeny. To identify genes involved in the maintenance of Drosophila neural stem cells, neuroblasts, we performed a protein expression screen followed by a protein knockdown screen using deGradFP system. Through this, we identified CSN7, a COP9 signalosome (CSN) subunit, which is enriched in neuroblasts and essential for neural development. CSN is a highly conserved multi-protein complex that regulates proteasome-mediated protein degradation via modulation of Cullin-RING E3 ligases. We found that loss of CSN7 and CSN1b lead to a decrease in neuroblast size and a reduced mitotic index. Our results show that CSN7/CSN1b regulates Akt-TOR signalling in the developing larval brain. Furthermore, we found that this regulation is mediated by Cul1. Overall, our work describes a hitherto undescribed role for the components of the CSN complex in neural development.

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.

Amphibians develop both external and internal gills during ontogeny, offering an opportunity to investigate the developmental relationship between these positionally distinct respiratory organs. Although internal gills of vertebrates are widely accepted to arise from pharyngeal endoderm, external gills have long been regarded as purely ectodermal outgrowths, obscuring their relationship to other vertebrate gills. Here, we combine histological analysis with direct lineage tracing in the Mexican axolotl (Ambystoma mexicanum) and the African clawed frog (Xenopus laevis) to resolve the embryonic origin of amphibian gills. We show that the external gill develops as a continuous epithelial extension of the pharyngeal endoderm, which forms its basal epithelium and reaches the distal gill tip. In the frog, this extension remains continuous with the epithelium giving rise to the internal gills. Rather than representing separate epithelial structures, external and internal gills therefore arise from a shared epithelial domain of the pharyngeal endoderm. These findings resolve a longstanding question concerning the embryonic origin of amphibian gills and provide a developmental viewpoint for understanding how spatially diverse vertebrate gills can evolve through repeated modification of a conserved endodermal tissue.

Epithelial tube morphogenesis is critical for the function of many organs. Basement membranes underlie epithelia and their remodeling is a key step to reach the correct size and shape. Key regulators that mediate basement membrane remodeling for tube elongation and branching remain largely unknown. We analyze the expression and function of AdamTS-B, a matrix metalloprotease, in the respiratory system of Drosophila. Here we show, that AdamTS-B is expressed early in tracheal development during placode formation. We generated a mutant line of AdamTS-B, which is lethal. Analysis of trachea morphogenesis in this AdamTS-B mutant reveal a function in tube elongation and cell migration. Our results suggest that AdamTS-B control BM remodeling required for organ shape.

The laminar architecture of the mammalian neocortex depends on precise radial migration of newborn neurons to the appropriate cortical layers. This process is governed by the integration of extracellular signals with cell-intrinsic transcriptional programs. BMP signaling has been previously demonstrated to be essential for radial migration of late-born (E15.5) upper-layer cortical neurons. However, the gene expression programs downstream of BMP signaling that regulate this process remained unknown. To address this, we combined temporally targeted in utero electroporation with GeoMx Digital Spatial Profiling (DSP) to map BMP-responsive transcriptional programs in E15.5-born layer II/III neurons at two defined developmental timepoints: E17.5, when neurons actively migrate through the intermediate zone, and postnatal day 0 (P0), when they have completed migration and have attained their laminar position. BMP inhibition produced largely non-overlapping transcriptional changes at these two stages. At E17.5, chromatin-regulatory programs and ribosomal protein gene expression were collectively upregulated upon BMP inhibition. However, by P0, the same cohort of ribosomal genes exhibited downregulation while membrane lipid biosynthesis and synaptic specialization pathways became dominant, revealing a stage-dependent transcriptional switch. A subset of shared BMP-responsive genes was regulated in opposite directions at these two stages, which lent further support to the hypothesis that there is a temporal reorganization of BMP-dependent transcriptional outputs. We selected four candidates from among the BMP-responsive genes for functional studies, namely Mfap4, Olfm2, Adora1, and Arpp21, which belong to diverse functional categories, including extracellular matrix proteins, G protein-coupled receptors, secreted glycoproteins, and RNA-binding proteins. RNAi-mediated knockdown of all four candidates resulted in radial migration defects that closely phenocopied inhibition of BMP signaling, establishing these genes as functional effectors of the BMP signaling pathway regulating neuronal migration.

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

NANOG is a conserved regulator of epiblast identity and is essential for hypoblast development and embryo survival after the blastocyst stage in mice. However, its role during post-blastocyst development remains poorly understood in non-rodent mammals. Here, we investigated the function of NANOG during ovine embryogenesis using CRISPR/Cas9-mediated gene ablation. NANOG ablation did not affect blastocyst formation or the initial development of the epiblast. However, NANOG-deficient blastocysts contained significantly fewer SOX17+ hypoblast cells by day (D) 8, followed by reduced hypoblast migration during early post-hatching stages in D12 in vitro embryos. These defects were transient, as hypoblast development was comparable between NANOG-deficient and wild-type conceptuses during subsequent in vivo conceptus elongation from embryonic day (E) 11 onwards. In contrast, although extraembryonic membranes development and conceptus elongation proceeded normally, NANOG-deficient conceptuses lacked SOX2+ epiblast cells and failed to form an embryonic disc (ED) from E11 onwards. These findings demonstrate that NANOG is dispensable for hypoblast development but is required for epiblast maintenance and ED formation during conceptus elongation in sheep.

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.

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 specificity protein 7 (SP7), also known as Osterix (OSX), is a zinc-finger transcription factor essential for osteoblast differentiation and skeletal development. Hereafter, the protein is referred to as OSX/SP7 throughout the manuscript. Although OSX/SP7 has been extensively studied in mammals and teleost fish, its developmental expression pattern in chicken (Gallus gallus) has not been described. Using an experimentally isolated chicken OSX/SP7 sequence, we examined OSX/SP7 mRNA expression during embryogenesis by in situ hybridization. As expected, OSX/SP7 expression was detected in developing skeletal elements undergoing ossification. Unexpectedly, transcripts were also observed in the neuroepithelium, retina, central nervous system, embryonic muscles and integument. Notably, OSX/SP7 expression was present in the neural tube from Hamburger and Hamilton stage 9 and persisted in the developing central nervous system until at least HH40, suggesting that OSX/SP7 functions during avian development may extend beyond osteogenesis. In addition, we reconstructed the chicken OSX/SP7 coding sequence and inferred its associated untranslated regions. The reconstructed ORF was independently supported by maternal and paternal haplotype-resolved chicken genome assemblies and retained the characteristic domain architecture of vertebrate OSX/SP7 proteins despite substantial divergence outside the DNA-binding domain. Taken together, these findings broaden the developmental landscape of OSX/SP7 expression in birds and provide new molecular resources for future studies of OSX/SP7 regulation and function during vertebrate development.

Temporal patterning is essential for generation of neuronal diversity in the developing vertebrate nervous system. A shared sequence of temporal transcription factors (tTFs) partitions neurons by birth date across much of the central nervous system. The molecular mechanisms that control the timing of expression of these tTFs are incompletely understood. Here, we provide evidence that this process is under the control of microRNAs (miRNAs). Perturbing miRNA biogenesis by conditional deletion of Dgcr8 from the developing mouse nervous system extends the window during which neurons with early identities are generated, but abolishes late identities in the spinal cord, hindbrain and midbrain, and holds progenitors in an early transcriptional state. We further identify miRNAs of the let-7 and miR-9 families, which target transcriptional regulators of early progenitor and neuronal identities, as candidates for mediating this effect. Throughout the nervous system, levels of these miRNAs rise during the neurogenic period in progenitors, while levels in neurons correlate with their birth-date. Moreover, the sensitivity of tTF-encoding transcripts to miR-9 inversely correlates with their expression sequence, suggesting a model in which miR-9 controls the chronology of neuronal cell fate transitions by targeting specifiers of early progenitor and neuronal temporal identity in a level-dependent manner. Consistently, perturbations of miR-9 expression dynamics by gain and loss-of-function approaches perturb temporal patterning. Together, our results demonstrate a critical, evolutionary conserved role for miRNAs in temporal patterning and suggest that a temporal miR-9 expression gradient controls the timing of tTF expression in large parts of the developing vertebrate nervous system.

Scoliosis is a three-dimensional deformity of the vertebral column that commonly emerges during growth, yet the biological mechanisms that initiate and drive curve progression remain poorly understood. Serotonin (5-hydroxytryptamine [5-HT]) has long been linked to scoliosis through its relationship with pineal melatonin signaling and through genetic associations with human disease, but its role in spinal morphogenesis has remained unclear. Here, using zebrafish, we show that serotonergic signaling is required for normal spine morphology. Transient exposure to 5-HT during development induces later spinal abnormalities, while loss of Tph2, the principal enzyme required for neuronal 5-HT synthesis, causes progressive three-dimensional spinal curvature during juvenile growth, mimicking aspects of adolescent idiopathic scoliosis (AIS). tph2 mutants undergo initially normal vertebral patterning while targeted ablation of the pineal gland does not induce scoliosis, arguing against a pineal or early skeletal origin for the 5-HT-associated spinal curves. The Reissner fiber, which has been linked to spinal curvature in zebrafish, also formed normally in tph2 mutants. Instead, mutants showed a marked reduction in Fev-expressing intraspinal serotonergic neurons together with increased locomotor activity. Mutants exhibited longer swim bouts and shorter periods of inactivity between bouts. Importantly, these motor defects were evident shortly before the onset of overt spinal curvature. Together, these findings identify neuronal serotonin as a critical regulator of spinal stability and support a model in which altered serotonergic control of motor function changes the mechanical environment experienced by the growing spine, increasing its susceptibility to progressive curvature.

Early mammalian development progresses through a small number of cleavage divisions with extensive transcriptional and epigenetic reprogramming that result in the very first lineage decisions, leading to blastocyst formation. Although classical embryological studies suggested that developmental progression can occur independently of normal cell division, the extent to which DNA replication contributes to these processes remains unclear. In particular, whether replication is required simply for proliferation or also for establishing the cellular states that regulate stage-specific gene expression is unknown. Here we show that mouse preimplantation development can proceed without DNA replication, but it is required to maintain proper heterochromatin organization at the 2-cell stage, the time of zygotic genome activation. The inhibition of DNA replication with aphidicolin blocked cleavage divisions without preventing zygotic genome activation, embryo morphogenesis, activation of lineage-specification factors, or the establishment of stage-specific transcriptional programs. However, replication arrest selectively altered the expression of developmental genes associated with late-replicating, Polycomb-enriched chromatin domains, and also reduced global levels of H3K27me3 and H3K9me3. These findings indicate that developmental timing in the early embryo is largely uncoupled from DNA replication and cell-cycle progression. Instead, DNA replication contributes to transcriptional fidelity by establishing or maintaining repressive chromatin states during the extensive genome reprogramming that accompanies zygotic genome activation.

Renal cysts are categorized as non-pathogenic simple cysts and pathogenic malignant cysts based on their pathophysiological status. Cyst formation is divided by cyst initiation and cyst progression/promotion. Pathogenic cysts are thought to be developed through continuous initiation followed by progression until pathogenic status is achieved. Although many genetic and environmental factors are identified to cause pathogenic cyst formation, the mechanisms that discriminate cyst initiation and progression are poorly understood. Using genetic mutation models of ETV transcription factors, ETV1, ETV4, and ETV5, and a pharmacological inhibitor of hedgehog signaling, cyclopamine, we identified one of the mechanisms regulating cyst initiation and progression. Nephron specific deletion of ETV4 and ETV5 initiated cyst formation. However, cyst initiation did not continue as animals grow, and a limited number of the initial cysts underwent further growth. Additional deletion of ETV1 was required for continuous initiation in addition to promotion of cyst growth. Furthermore, administration of cyclopamine attenuated promotion of cyst progression but had little effect on cyst initiation. Therefore, we provide evidence that cyst initiation and progression is genetically and molecularly distinct and can be modulated. This information provides new insight into how to control renal cyst initiation and progression and can be used to suppress pathogenic cyst growth.