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Gametophytes and embryo ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae)

Among the flowering plants, the gametophyte development and reproductive biology of orchids is particularly poorly understood. Cypripedium japonicum is a perennial herb, native to East Asia. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. Light microscopy and SEM methods were used to study the development of the gametes and embryo. The complete reproductive cycle was developed based on our observations. Anther development begins under the soil and meiosis of pollen cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid-late June. The pattern of embryo sac formation is bisporic and there are six nuclei. Triple fertilization results in the endosperm nucleus. A globular embryo is formed after multiple cell division and 9 weeks after pollination the entire embryo sac is occupied by embryo. Overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient to characterize the entire genus. Based on the available information a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.\n\nHighlightManual pollination, reproductive biology and seed development process in Cypripedium japonicum Thunb., a ladys slipper orchid endemic to East Asia

developmental biology↗

Generation of Human Taste Bud Organoids as a Human-Mimetic Platform for Modeling Taste Perception

We established a human taste bud organoid system derived from circumvallate papillae. This model has been highly anticipated in the field of taste research, where feasible approaches for validating taste biology discovered in rodent models have been limited. Through a stepwise exploratory strategy, we systematically identified and optimized the niche factors required to maintain taste bud organoids and promote their differentiation. This human taste bud organoid system comprises Type I-IV taste receptor cells (TRCs) as well as stem/progenitor cells, and its sensory receptor cells exhibit calcium responses to taste stimuli. Using this system, we identified robust Wnt signaling as a requirement for optimal TRC fate progression, uncovered a human-specific transcriptional program in LGR5 cells, and identified previously unrecognized molecular markers for Type I TRCs. By recapitulating native human taste bud cell diversity and function, this organoid provides a tractable platform for studying human taste biology and dysfunction.

developmental biology↗

Establishment of a novel non-integrated human pluripotent stem cell-based gastruloid model

Embryo loss and pregnancy disorders are prevalent worldwide, with both conditions critically associated with dysfunctioning gastrulation processes. Gastrulation and post-gastrulation organogenesis are crucial stages of embryonic development that establish the blueprint for body part formation. These processes involve the sequential generation of three germ layer cells and primordial germ cells, as well as the assembly of the precursor tissues for body parts. However, due to ethical limitations associated with studying human embryogenesis, a more detailed understanding of gastrulation and post-gastrulation organogenesis remains elusive. To ensure that the knowledge obtained from gastruloids is biologically meaningful and clinically relevant, it is critical to create high-fidelity human embryo models that closely mimic embryogenesis in vivo. Here, we developed a two-stage derivation gastruloids in vitro based on human pluripotent stem cells. Morphological tracking mimicks the developmental processes of models from Carnegie Stage 4 (CS4) to early CS7. Our gastruloids exhibit key structures characteristic of human embryos, including amniotic cavity, embryonic disc, primitive streak, primary yolk sac, secondary yolk sac, and blood islets. Comparison of our cell lineage development maps showed that gastruloids closely resembled human natural CS7 gastrula. Our gastruloids exhibited transcriptional characteristics that mimicked the molecular pathways observed in natural embryos development. Importantly, we found that in our model, extraembryonic mesoderm originates from the yolk sac and primordial germ cells originate from the posterior epiblast of the embryonic disc. Moreover, we found that thalidomide affects the differentiation of three germ layer cells, resulting in the arrest of human gastruloid development. In conclusion, by establishing a human gastruloid, we were able to gain valuable insights into the mechanisms responsible for human gastrulation and shed light on the causes of early embryo loss and pregnancy disorders.

developmental biology↗

How Dickkopf molecules and Wnt/beta-catenin interplay to self-organise the Hydra body axis

The antagonistic interplay between canonical Wnt signalling and Dickkopf (Dkk) proteins is fundamental to tissue organisation, including stem cell differentiation and body-axis formation. Disruptions in this interaction are linked to various human diseases, yet the mechanisms enabling robust body-axis formation through {beta}-catenin/Wnt-Dkk interactions remain unclear. A key model system for Wnt-driven pattern formation is the pre-bilaterian organism Hydra, where two ancestral Dkk proteins interact with Wnt signalling to self-organise the body axis. While Hydra patterning has been extensively studied using the activator-inhibitor framework, a model integrating experimentally identified molecules has been lacking. Here, we introduce a mathematical model that incorporates both Dkks and their experimentally observed interactions with Wnt signalling. Numerical and analytical studies show that this network alone is sufficient to drive de novo body-axis formation across a broad parameter range. Our mutual inhibition model provides a biologically grounded realization of the general local-activation/long-range-inhibition (LALI) principle of de novo pattern formation, offering a mechanistic explanation for the observed Dkk and Wnt expression patterns under various conditions. Unlike previous models, it is directly grounded in experimental data, links injury response to pattern formation, and remains robust against perturbations. Author SummaryHow organisms form and regenerate complex body structures is a fundamental question in biology. In the freshwater animal Hydra, which can regenerate its entire body from a small tissue fragment, a molecular signalling system involving Wnt proteins and their inhibitors, the Dickkopf (Dkk) family, plays a central role in organising the body axis. While these molecules are known to interact, their exact roles and how they collectively shape large-scale patterns have remained unclear--especially since their activity does not fully align with established pattern formation models. In this study, we present a new mathematical model that captures the observed interactions between Wnt and two Dkk molecules in Hydra. We show that a mechanism based on mutual inhibition--rather than the traditional interplay between activator and inhibitor molecules--can explain the emergence of a stable body axis and the results of various perturbation experiments. Our work offers new insights into the design principles of biological pattern formation and emphasizes the importance of exploring alternative mechanisms beyond classical theories.

developmental biology↗

Temporal specificity and heterogeneity of the fly immune cells' transcriptional landscape

Immune cells provide defense against the non-self, however recent data suggest roles well beyond innate immunity, in processes as diverse as development, metabolism and tumor progression. Nevertheless, the heterogeneity of these cells remains an open question. Using bulk RNA sequencing we find that the Drosophila immune cells (hemocytes) display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single cell RNA sequencing we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct cellular processes e.g. proliferation, phagocytosis, metabolic homeostasis and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation that triggers the differentiation of a novel cell type, the lamellocyte. This first molecular atlas provides precious insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.

developmental biology↗

Effect of changing the direction of the force vector of the mandible in relation to the skull base: study in rabbits

The temporomandibular joint has a great capacity for functional adaptation. The aim of this study was to evaluate, bilaterally, the influence of unilateral modification of the direction of the mandibular force vector in relation to the skull base in rabbits. Thirty New Zealand rabbits (Oryctolagus cuniculus L.) were randomly divided into two groups (n = 15/group): test (modification of the mandibular force vector) and control (no modification of the mandibular force vector). The animals were killed at 20, 40, and 60 days postoperatively. Histomorphometric evaluation of the temporal and condylar joint structures bilaterally always showed significant differences (P < 0.05) between the test and control groups on both sides of the TMJ. The results demonstrate that the rabbit temporal bone and mandibular condyle showed significant adaptive capacity as a biological response to mechanical forces on both the operated and opposite sides.

developmental biology↗

Acto3D: user- and budget-friendly software for multichannel high-resolution three-dimensional imaging

Advances in fluorescence microscopy and tissue-clearing technology have revolutionized three-dimensional (3D) imaging of fluorescently labeled tissues, organs, and embryos. However, the complexity and high cost of existing software and computer solutions for such imaging limit its widespread adoption by researchers with limited resources. We here introduce Acto3D as a user- and budget-friendly, open-source computer software application designed to streamline the generation and observation of high-resolution 3D images of targets labeled with multiple fluorescent probes. Acto3D features an intuitive interface that simplifies the importation, visualization, and analysis of data sets, has an associated tool for annotation of vascular lumens, and incorporates multiple fluorescence channels for comprehensive imaging. Underpinned by an integrated graphics processing unit, Acto3D allows accurate image reconstruction and efficient data processing without the need for expensive high-performance computers. We validated the software by imaging mouse embryonic structures. Acto3D thus constitutes a cost-effective and efficient platform to support biological research.

developmental biology↗

Live imaging human embryos reveals mitotic errors and lineage specification prior to implantation

Meiotic and mitotic chromosome segregation errors in human development have been studied mostly prior to and at the time of fertilisation. Despite chromosomal errors being a leading cause of miscarriage and infertility, chromosome missegregation has not been extensively studied at later stages of human development. Here we optimised labelling, light-sheet live imaging and semi-quantitative analysis of human embryos and reveal chromosome segregation errors just prior to implantation. We found that human embryos exhibited a number of chromosome missegregation events including multipolar spindle formation, lagging chromosomes, misalignment and chromosome slippage. We found that the majority of lagging chromosomes were passively inherited by one of the daughter cells, instead of reincorporating into the nuclei, suggesting a distinct pattern of micronuclei inheritance. By semi-automated segmentation, we tracked the position of labelled cells in human embryos and observed that while most labelled cells remained segregated to the outside, and therefore restricted to a placental-progenitor fate, there was evidence of a rare cell migration to cells positioned on the inside, which suggests that there may be plasticity. Altogether, we found that mitotic chromosome segregation errors arise just prior to implantation, which has implications for our understanding of biological events that contribute to aneuploidy mosaicism.

developmental biology↗

Charting Postnatal Heart Development Using In Vivo Single-Cell Functional Genomics

The transition at birth, marked by increased circulatory demands and rapid growth, necessitates extensive remodeling of the hearts structure, function, and metabolism. This transformation requires precise spatial and temporal coordination among diverse cardiac cell types; central to this process is cardiomyocyte maturation, yet the regulatory mechanisms driving these changes remain poorly understood. Here, we present a temporal and spatial atlas of postnatal hearts by integrating single-nucleus transcriptomics with image-based spatial transcriptomics, which uncovers the dynamic regulatory networks of cardiomyocyte maturation. To functionally interrogate candidate regulators in vivo, we developed Probe-based Indel-detectable Perturb-seq (PIP-seq), a high-throughput platform that uses probe-based chemistry to directly capture sgRNA expression, perturbation status, and transcriptomic profiles at single-nucleus resolution. Applying PIP-seq to postnatal cardiac development identified 21 novel regulators of cardiomyocyte maturation, highlighting critical nodal points in this process. Our study establishes a high-resolution framework for dissecting postnatal heart development, underscoring the integrative and highly ordered roles of microenvironment and intercellular communication in cardiomyocyte maturation. Importantly, PIP-seq enables systematic, high-throughput exploration of gene function and networks underlying complex biological processes in their native in vivo context.

developmental biology↗

SpinePy enables automated 3D spatiotemporal quantification of multicellular in vitro systems

Organoids and stem-cell-based embryo models such as gastruloids are powerful systems to quantitatively study morphogenesis and patterning. This requires 3D analysis in reference frames that emerge dynamically over development, but are less stereotypical than in vivo. Meaningful statistical comparison and interpretation of biological and physical quantities in space and time -- such as signaling activity, gene expression or cell flows -- depend on proper quantification in these internal and dynamic coordinate systems, especially in in vitro systems that naturally exhibit larger variation. Here, we present a computational framework, packaged as a modular Python toolkit termed SpinePy, that identifies the emergent primary body axis ("spine") of individual gastruloids and constructs a local, dynamic coordinate system aligned to their morphology. SpinePy enables 3D quantification relative to evolving axes and statistical comparisons of morphodynamics, patterning, and densities across structures with varying geometries. We validate and benchmark SpinePy using both synthetic and experimental gastruloid data, providing practical insights into method performance. Using this framework, we generate 3D patterning maps from gastruloids formed with different initial cell numbers (N0). This reveals distinct patterning classes that are better explained by gastruloid volumes than by N0. While demonstrated in gastruloids, SpinePy is broadly applicable to any multicellular system where analysis relative to evolving internal axes is needed, advancing quantitative and comparative spatial biology.

developmental biology↗

Mining functional genes and characterizing cellular transcriptomic profiles in the single-cell atlas of adult Spodoptera litura ovary

Understanding the reproductive biology of non-model organisms remains challenging due to the limited availability of high-resolution molecular resources. Here, we present a comprehensive single-cell transcriptomic atlas of the adult ovary of Spodoptera litura (S. litura), a highly polyphagous agricultural pest with a polytrophic meroistuc ovary. By integrating single-cell RNA sequencing with cross-species comparison to Drosophila melanogaster (D. melanogaster), we define major germline and somatic cell populations and delineate conserved and species-specific features of ovarian cell composition. To enhance the interpretability and reuse of this dataset, we combine transcriptomic profiling with in situ hybridization to validate cluster-specific molecular markers across ovarian cell types. We further apply RNA interference (RNAi) to assess the contributions of germline-enriched genes (Hsc70-4, Wech, Polo, Path) to ovarian development and fecundity. Trajectory inference, together with SCENIC and CellChat analyses, provides a system-level view of transcriptional regulatory programs and predicted intercellular communication pathways during oogenesis in S. litura. Collectively, this work establishes a valuable resource for studying lepidopteran insect oogenesis, offering a comparative framework for reproductive biology in non-model insects and highlighting potential targets for RNAi-based pest control strategies.

developmental biology↗

Interactive Effects of Biological Maturation and Relative Age Effect on Talent Identification for U16 Elite Soccer Players

This retrospective study aims to explore the interactive effects of biological maturation and relative age effect (RAE) on talent identification. 56 male elite soccer players matched for chronological age (15.08{+/-}0.41 years) were studied. Test items included anthropometry (height, body mass, sitting height, leg length, BMI and Quetelet index), physiology (power, speed, agility, speed endurance and aerobic performance), soccer-specific skills (passing, shooting and dribbling), psychology (achievement motivation, orientation and resilience) and biological maturation (APHV) tests. The test results were analyzed independent sample t-test, Pearson correlation analysis, and stratified regression. Conclusion: Biological maturation significantly influences anthropometry (height, weight and Quetelet index), lower limb explosive, and speed (single-leg jump, standing triple jump, and 30-m sprint) in U16 male elite soccer players in Shanghai. The relative age effect shows no significant impact on talent selection indicators, which is attributed to the accumulated training load effect. The mechanisms of biological maturation and RAE in youth soccer talent selection are distinct and operate independently.

developmental biology↗

The comprehensive ontology of the anatomy and development of the solitary ascidian Ciona: the swimming larva and its metamorphosis.

Ciona robusta (Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona, a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 hour post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource "TunicAnatO", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

developmental biology↗

Conserved and unique transcriptional features of pharyngeal arches in the skate (Leucoraja erinacea) and evolution of the jaw

The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers bapx1 and gdf5 and pro-chondrogenic transcription factors barx1 and gsc, and the dorsalising transcription factor pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

developmental biology↗

A landscape of differentiated biological processes involved in the initiation of sex differentiation in zebrafish

Zebrafish (Danio rerio) has been used as a promising animal model to study gonadal development and gametogenesis. Although previous studies have identified critical molecules participating in zebrafish gonad differentiation, a landscape view of the biological processes involved in this process is still lacking. Here we isolated intact zebrafish differentiating gonads, at 25 days post-fertilization (dpf) and 30 dpf, and conducted RNA-seq analysis between the juvenile gonads that tended to develop into ovaries or testes. Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the level of biological process. During ovary differentiation, the biological processes related to metabolic activities in production of energy and maternal substances, RNA degradation, and DNA repair were enriched. During testis differentiation, the biological processes related to cell proliferation, differentiation, and morphogenesis were enriched, with a total of 15 signaling pathways. Notably, we reveal that the immune-related processes are extensively involved in the regulation of testis development. Overall, this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.

developmental biology↗

Dermal appendage-dependent patterning of zebrafish atoh1a+ Merkel cells

Touch system function requires precise interactions between specialized skin cells and somatosensory axons, as exemplified by the vertebrate mechanosensory Merkel cell-neurite complex. Development and patterning of Merkel cells and associated neurites during skin organogenesis remains poorly understood, partly due to the in utero development of mammalian embryos. Here, we discover Merkel cells in the zebrafish epidermis and identify Atonal homolog 1a (Atoh1a) as a marker of zebrafish Merkel cells. We show that zebrafish Merkel cells derive from basal keratinocytes, express neurosecretory and mechanosensory machinery, extend actin-rich microvilli, and complex with somatosensory axons, all hallmarks of mammalian Merkel cells. Merkel cells populate all major adult skin compartments, with region-specific densities and distribution patterns. In vivo photoconversion reveals that Merkel cells undergo steady loss and replenishment during skin homeostasis. Merkel cells develop concomitant with dermal appendages along the trunk, and preventing dermal appendage formation reduces Merkel cell density by affecting both cell differentiation and maintenance. By contrast, altering dermal appendage morphology changes the distribution, but not density, of Merkel cells. Overall, our studies provide insights into touch system maturation during skin organogenesis and establish zebrafish as an experimentally accessible in vivo model for the study of Merkel cell biology.

developmental biology↗

Essential Role for Trf2 in Cardiac Development and Function

Telomere Repeat-binding Factor 2 (Trf2) is essential for protecting our telomeres. While Trf2 global deletion is lethal, its role in organ-specific development, particularly in the heart, remains less understood. In this study, we investigated the role of Trf2 in cardiac development and function. Our studies reveal that cardiomyocyte (CM)-specific loss of Trf2 leads to profound defects in heart morphology, including impaired ventricular wall formation and compromised CM proliferation, concurrent with no CM telomere length attrition. Further, in vivo functional assessment and molecular analyses of CM-Trf2 deficient ventricles revealed severe cardiac dysfunction and, interestingly, altered nuclear envelope gene expression, respectively. Our work provides new insights into the essential role of Trf2 in heart development and function, and potential avenues for therapeutic intervention targeting telomere biology.

developmental biology↗

Effects of Social Status on Reproductive Success, Physiology and Maternal Care in Socially housed Female Rhesus Macaques

This study examined the long-term effects of low social status on reproductive success and seasonal changes in reproductive and stress hormones of adult female rhesus macaques. Rhesus are a matriarchal, matrilineal species that form hierarchical social hierarchies maintained by aggression. Thus, animals in the bottom of the hierarchy (low social status: subordinate) experience high levels of aggression from those in higher social ranks and therefore must remain vigilant. Social subordination generates chronic psychosocial stress and could negatively impact reproductive function. Twenty-seven adult female rhesus monkeys (13 dominant -DOM-, 14 subordinate -SUB-; 8-11 years old) were studied for stress neuroendocrine function -measuring basal plasma levels of cortisol-, as well as for reproductive endocrine function -measuring basal levels of estradiol (E2). In addition, we examined the reproductive success rates of these females (defined as number of live births/years with male access), their success rates raising infants that survived to 1 year, and the characteristics and effectiveness of their maternal care. Our findings show significantly lower reproductive success rates in SUB than DOM females, blunted E2 seasonal changes, driven by higher E2 levels in the anovulatory season and more attentive maternal care of offspring early in life than dominant females. Age at first birth was negatively associated with infant survival rate across ranks. Interestingly, higher levels of reproductive hormones (E2) during the anovulatory season predicted lower reproductive success, although the effect was driven by animals lactating at that point. Overall, these findings suggest that subordinate female macaques show a phenotype consistent with impaired reproductive function. Simple SummaryThis study examined the long-term effects of low social status on reproductive success and seasonality of reproductive and stress physiology of female rhesus monkeys. The despotic rhesus social hierarchy is matrilineal and maintained by aggression. Males leave their natal group in bachelor groups around puberty, but females remain, so social status of group members is passed down the female line. Rhesus with low social status (subordinate) experience significantly more aggression and harassment than their higher-ranking counter parts (dominant). This makes rhesus macaques an ideal model for studying chronic psychosocial stress and its long-term effects. Our findings show significantly lower reproductive success rates (number of live births based on access to males; lower infant survival rates) in subordinate than dominant females. Subordinate females also show blunted seasonal changes of the gonadal hormone estradiol (E2), driven by higher E2 levels in the anovulatory season than dominant animals, as well as more attentive maternal care of offspring early in life than dominant animals. Age at first birth was negatively associated with infant survival rate across ranks. Interestingly, higher levels of reproductive hormones (E2) during the anovulatory season were associated with lower reproductive success, although the effect was driven by animals lactating at that point. Overall, these findings suggest that female macaques with lower social status show a phenotype consistent with impaired reproductive function, though the biological mechanisms involved need to be further explored.

developmental biology↗