The role of the chorio-allantoic placenta as the critical nutrient- and oxygen-supplying organ to nourish the demands of the fetus has been well recognized. This function relies on the successful establishment of the placental feto-maternal exchange unit, or interhaemal barrier, across which all nutrients as well as waste products must pass to cross from the maternal to the fetal blood circulation, or vice versa, respectively. As a consequence, defects in the establishment of this elaborate interface lead to fetal growth retardation or even embryonic lethality, depending on the severity of the defect. Beyond this essential role, however, it has also emerged that the functionality of the feto-maternal interface dictates the proper development of specific embryonic organs, with tightest links observed to the formation of the heart. In this article, we build on the foundational strength of the mouse as experimental model in which the placental causality of embryonic defects can be genetically proven. We discuss in detail the formation of the interhaemal barrier that makes up the labyrinth layer of the murine placenta, including insights into drivers of its formation and the interdependence of the cell types that make up this essential interface, from in vivo and in vitro data using mouse trophoblast stem cells. We highlight mouse genetic tools that enable the elucidation of cause-effect relationships between defects driven by either the trophoblast cells of the placenta or by embryonic cell types. We specifically emphasize gene knockouts for which a placental causality of embryonic heart defects has been demonstrated. This in-depth perspective provides much-needed insights while highlighting remaining gaps in knowledge that are essential for gaining a better understanding of the multi-facetted roles of the placenta in setting us up for a healthy start in life well beyond nutritional support alone.

Coordination between periodicity of somite formation and somite growth is crucial for regular body pattern formation during somitogenesis. Yet, the specific mechanism that links the two processes remains unclear. Using chick embryos, we demonstrate that both temporal and spatial features can be simultaneously controlled by membrane potential (V) of somite-forming cells. Our findings show that somites hyperpolarize as they mature, displaying step-like changes in V observed between specific groups of somites, reflecting the reported onset of biochemical and structural changes within them. We modify V by changing chemical compositions of the microenvironment of the embryo. Alteration of V sets a new pace of somite formation (cell migration and self-assembly) and its concurrent growth (cell proliferation) without disturbing the somite's regular aspect ratio. Our results therefore suggest that V has the ability to orchestrate cell proliferation, migration and self-assembly - processes that are hallmarks of embryogenesis, tumorigenesis and tissue regeneration.

Heterozygous R391 TUBB4B pathogenic variations are responsible for an association of hearing loss and retinal dystrophy in human. With the goal of understanding the functions of TuBB4b and the pathogenic role of R391 variations, we characterized tubB4B in zebrafish and identified the gene regulatory elements necessary and sufficient for expression of TubB4b as in endogenous tissues. Using knock-out and transgenic approaches, we determined that R391 mutations impair neither localization of TubB4B within sensory hair cells (SHC) nor their structure, but induced to a small decrease in SHC number from anterior crista. Expression of R391 mutations in sensory hair cells has no effect on zebrafish audition, suggesting a different equilibrium between various tubulin isotypes in zebrafish possibly due to compensatory mechanisms. The careful expression analysis and transgenic tools generated in this study could help understand how recently described pathogenic variants lead to more severe clinical forms of TUBB4B-related diseases.

The anther is the developmental housing of pollen and therefore the male gametes of flowering plants. The meiotic cells from which pollen are derived must differentiate de novo from somatic anther cells and synchronously develop with the rest of the anther. Anthropogenic control over another development has become crucial for global agriculture so as to maintain inbred lines and generate hybrid seeds of many crops. Understanding the genes that underlie the proper differentiation, developmental landmarks, and functions of each anther cell type is thus fundamental to both basic and applied plant sciences. We investigated the development of the somatic niche of the maize (Zea mays) anther using single-cell RNA-seq (scRNA-seq). Extensive background knowledge on the birth then pace and pattern of cell division of the maize anther cell types and published examples of cell-type gene expression from in situ hybridization allowed us to identify the primary cell types within the anther lobe, as well as the connective cells between the four lobes. We established the developmental trajectories of somatic cell types from pre-meiosis to post-meiosis, identified putative marker genes for the somatic cell types that previously lacked any known specific functions, and addressed the possibility that tapetal cells sequentially differentiate. This comprehensive scRNA-seq dataset of the somatic niche of the maize anther will serve as a baseline for future analyses investigating male-sterile genotypes and the impact of environmental conditions on male fertility in flowering plants.

Stem cells are subject to continuous regulation to ensure that the correct balance between stem cell differentiation and self-renewal is maintained. The dynamic and ongoing nature of stem cell regulation, as well as the complex signaling microenvironment in which stem cells are typically found, means that studying them in their endogenous environment in real time has multiple advantages over static fixed-sample approaches. We recently described a method for long-term, ex-vivo, live imaging of the blood progenitors in the Drosophila larval hematopoietic organ, the Lymph Gland (LG). This methodology has allowed us to analyze multiple aspects of fly hematopoiesis, in real time, in a manner that could not be carried out previously. Here, we describe novel insights derived from our quantitative live imaging approach. These insights include: the identification of extensive filopodia in the progenitors and description of their morphology and dynamics; visualization and quantitative analysis of JAK/STAT signaling in progenitors by the simultaneous tracking of thousands of vesicles containing internalized Domeless receptors; quantitative analysis of the location, morphology, and dynamics of mitochondria in blood progenitors; long-term tracking of patterns of cell division and migration of mature blood cell in the LG; long-term tracking of multiple cell behaviors in the distal committed progenitors; analysis of Ca signaling of blood progenitors in the secondary lobes of the LG. Together, these observations illustrate the power of imaging fly hematopoiesis in real time and identify many previously undescribed processes and behaviors in the LG that are likely to play important roles in the regulation of progenitor differentiation and self-renewal.

An early step in triploblastic embryo differentiation is the formation of the three germ layers. Maternal pioneer transcription factors (TFs) bind to embryonic enhancers before zygotic genome activation, initiating germ layer specification. While maternal TFs' role in establishing epigenetic marks is known, how early pluripotent cells gain spatially restricted epigenetic identities remains unclear. We show that by the early gastrula stage, H3K4me1-marked regions become distinct in each germ layer, with certain chromatin regions forming high density H3K4me1 marked regions (HDRs). Genes associated with these HDRs are more robustly expressed compared to those associated with low density H3K4me1 marked regions (LDRs) in the genome. This process is driven by the sequential actions of maternal and zygotic factors. Knockdown of key maternal endodermal TFs (Otx1, Vegt and Foxh1) leads to a loss of endodermal H3K4me1 marks in endoderm, with a concurrent emergence of ectodermal and mesodermal marks, indicating a shift in chromatin state. This work highlights the importance of coordinated activities of maternal and zygotic TFs in defining the regionally-resolved and dynamic process of chromatin modification conferred by H3K4me1 in the early Xenopus embryo.

The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.

Heat stress has broad effects on an organism and is an inevitable part of life. Embryos face a particular challenge when faced with heat stress - the intricate molecular processes that pattern the embryo can all be affected by heat, and the embryo lacks some of the strategies that adults can use to manage or avoid heat stress. We use Drosophila melanogaster as a model, as insects are capable of developing normally under a wide range of temperatures and are exposed to daily temperature swings as they develop. Research has focused on the heat shock pathway and the transcription of heat shock proteins as the main response to heat and heat damage. This review explores embryonic heat responses beyond the heat shock pathway. We examine the effects of heat from a biochemical standpoint, as well as highlighting other mechanisms of heat stress regulation, such as miRNA activity or other signaling pathways. We discuss how different elements of the heat stress response must be coordinated across the embryo to enable development under a wide range of temperatures. Studying heat stress in Drosophila melanogaster can be a powerful lens into how developmental systems ensure robustness to environmental factors.

The discovery that homeotic genes in Drosophila are conserved and utilized for embryonic development throughout the animal kingdom, including humans, revolutionized the fields of developmental biology and evolutionary developmental biology (evo-devo). In a pair of back-to-back papers published in Cell in 1984, researchers at the Biozentrum in Basel, Switzerland, showed that the homeobox - previously identified as a sequence shared by homeotic genes in Drosophila - was also present in the genome of diverse animals. The first paper (McGinnis et al., 1984a) showed that genomes of both invertebrates and vertebrates contain sequences that cross-hybridized with Drosophila homeobox probes. The second paper (Carrasco et al., 1984) identified a cross-hybridizing sequence in the model system Xenopus laevis. They then isolated the first vertebrate homeobox-containing gene by cloning and sequencing of the corresponding genomic region. Finally, they showed that this gene (AC1, later renamed HoxC6) was expressed during embryonic development, the first evidence that developmentally expressed Drosophila genes could be used to isolate regulators of vertebrate embryonic development. These findings led to a flurry of activity in the evo-devo field, initially focused on isolating Hox genes across diverse species, and then expanding to isolation of other gene families based on Drosophila orthologs, an approach that continues today. This led to the notion of a conserved genetic toolkit for embryonic development, currently accepted, but unexpected at the time of its discovery. We attempt to provide some context for the sea-change in thinking that these discoveries brought about by referring to Jean Piaget's theories about the sequential acquisition of scientific knowledge.

Vertebrate development is regulated by several complex well-characterized morphogen signaling pathways, transcription factors, and structural proteins, but less is known about the enzymatic pathways that regulate early development. We have identified that factors from the inflammation-mediating cyclooxygenase (COX) signaling pathway are expressed at early stages of development in avian embryos. Using Gallus gallus (chicken) as a research model, we characterized the spatiotemporal expression of a subset of genes and proteins in the COX pathway during early neural development stages. Specifically, here we show expression patterns of COX-1, COX-2, and microsomal prostaglandin E synthase-2 (mPGES-2) as well as the genes encoding these enzymes (PTGS1, PTGS2, and PTGES-2). Unique expression patterns of individual players within the COX pathway suggest that they may play non-canonical/non-traditional roles in the embryo compared to their roles in the adult. Future work should examine the function of the COX pathway in tissue specification and morphogenesis and determine if these expression patterns are conserved across species.

Over the past decades, Developmental Biology has been moving steadily from a rather academic subject to an increasingly practical discipline. It has played a role in the development of contraceptive and conceptive (e.g. in vitro fertilization) technologies. The advent of embryonic (ES) and induced pluripotent stem (iPS) cells and advances in organoids, embryoids, and human-non-human chimeras offer promises and ethical challenges. Courses in developmental biology provide both opportunities and challenges for discussing the societal and ethical implications of new technologies. Here we present outcomes of a Society for Developmental Biology workshop on "teaching ethical issues in developmental biology." We point out important considerations and possible approaches, as well as the need to set boundaries on discussions of the critical issues posed by the new science of embryonic engineering.

We have introduced the floxed allele of Smoothened (Smo) carried by the mouse line Smo into the C57BL/6J strain by serial backcross. Recapitulation of the Smo null phenotype was confirmed by deleting the allele using E2a-cre and intercrossing heterozygous Smo ± mice. No homozygous mutant embryos were identified at E9.5, suggesting the null phenotype is at least as severe as that observed on a mixed genetic background. While healthy and fertile homozygous floxed mice were regularly obtained after intercrosses, their numbers at weaning were reduced relative to Mendelian expectation, suggesting the unrecombined allele is itself hypomorphic. This hypothesis is supported by characterization of transcription of the floxed allele, which revealed that its expression was variably reduced relative to wild-type Smo.

In the mouse, there is preferential inactivation of the paternally-derived X chromosome in extraembryonic tissues of early embryos, including trophectoderm and primitive endoderm or hypoblast. Although derivatives of these tissue have long been considered to be purely extraembryonic in nature, recent studies have shown that hypoblast-derived cells of the 'extraembryonic' visceral endoderm make a substantial cellular contribution to the definitive gut of the fetus. This raises questions about the eventual fate of these cells in the adult and potential disease implications due to the skewed inactivation of the paternally derived X in females heterozygous for X-linked mutations. Similar lineage studies of this tissue have not yet been done in human embryos but differences in the pattern of X chromosome inactivation between mouse and humans indicates that preferential X inactivation will not be an issue in human embryos. Nonetheless, comparisons between mouse and human will be important because of the widespread use of the mouse as a model system for study of genetics, development and disease.

The medial longitudinal fasciculus (MLF) is the first axon tract to develop in the ventral vertebrate brain. It originates in the diencephalon and projects caudally into the spinal cord, pioneering the path for later developing axons. Previous anatomical and expression analyses in the chicken suggested Semaphorin 3 A (Sema3A) as the candidate to repel the amniote MLF from the forebrain. However, studies in the zebrafish implicated a distantly related semaphorin with a role in axon fasciculation, not guidance. Thus, the mechanism accounting for the caudal projection of the MLF remains unclear. Here we show that misexpression of Sema3A or grafting of Sema3A-expressing cells into the path of the MLF diverts the axons or blocks their outgrowth in chicken embryos. In vitro, Sema3A exposure resulted in the collapse of MLF growth cones. A dominant-negative approach or siRNA to interfere with the function of the Sema3A receptor Neuropilin1 allowed MLF axons to project rostrally. Together, this suggests that Sema3a repulsion directs the caudal extension of the MLF to pioneer the ventral longitudinal tract.

The surface of epithelial tissues is covered by an apical extracellular matrix (aECM). The aECMs of different tissues have distinct compositions to serve distinct functions, yet how a particular cell type assembles the proper aECM is not well understood. We used the cell type-specific matrix of the C. elegans vulva to investigate the connection between cell identity and matrix assembly. The vulva is an epithelial tube composed of seven cell types descending from EGFR/Ras-dependent (1°) and Notch-dependent (2°) lineages. Vulva aECM contains multiple Zona Pellucida domain (ZP) proteins, which are a common component of aECMs across life. ZP proteins LET-653 and CUTL-18 assemble on 1° cell surfaces, while NOAH-1 assembles on a subset of 2° surfaces. All three ZP genes are broadly transcribed, indicating that cell type-specific ZP assembly must be determined by features of the destination cell surface. The paired box (Pax) transcription factor EGL-38 promotes assembly of 1° matrix and prevents inappropriate assembly of 2° matrix, suggesting that EGL-38 promotes expression of one or more ZP matrix organizers. Our results connect the known signaling pathways and various downstream effectors to EGL-38/Pax expression and the ZP matrix component of vulva cell fate execution. We propose that dedicated transcriptional networks may contribute to cell-appropriate assembly of aECM in many epithelial organs.

The yolk sac is phylogenetically the oldest of the extra-embryonic membranes and plays important roles in nutrient transfer during early pregnancy in many species. In the human this function is considered largely vestigial, in part because the secondary yolk sac never makes contact with the inner surface of the chorionic sac. Instead, it is separated from the chorion by the fluid-filled extra-embryonic coelom and attached to the developing embryo by a relatively long vitelline duct. The coelomic fluid is, however, rich in nutrients and key co-factors, including folic acid and anti-oxidants, derived from maternal plasma and the endometrial glands. Bulk sequencing has recently revealed the presence of transcripts encoding numerous transporter proteins for these ligands. Mounting evidence suggests the human secondary yolk sac plays a pivotal role in the transfer of histotrophic nutrition during the critical phase of organogenesis but also of chemicals such as medical drugs and cotinine. We therefore propose that the early placental villi, coelomic cavity and yolk sac combine to function physiologically as a choriovitelline placenta during the first weeks of pregnancy. We have derived organoids from the mouse yolk sac as proof-of-principle of a model system that could be used to answer many questions concerning the functional capacity of the human yolk sac as a maternal-fetal exchange interface during the first trimester of pregnancy.

The position of cells during development is constantly subject to noise, i.e. cell-level noise. We do not yet fully understand how the cell-level noise coming from processes such as cell division or movement leads to morphological noise, i.e. morphological differences between genetically identical individuals developing in the same environment. To address this question we constructed a large ensemble of random genetic networks regulating cell behaviors (contraction, adhesion, etc.) and cell signaling. We simulated them with a general computational model of development, EmbryoMaker. We identified and studied the dynamics, under cell-level noise, of those networks that lead to the development of animal-like morphologies from simple blastula-like initial conditions. We found that growth by cell division is a major contributor to morphological noise. Self-activatory gene network loops also amplified cell-level noise into morphological noise while long-range signaling and epithelial stiffness tended to reduce morphological noise.

A profound collaboration between the germline and somatic cells of an organism is the creation of a functional gonad. Here we establish a foundation for studying molecular gonadogenesis in the sea urchin by use of RNA-seq, quantitative mRNA measurements, and in-situ hybridizations throughout the life cycle of the variegated sea urchin, Lytechinus variegatus (Lv). We found through three distinct analyses that the ovary and testis of this echinoderm expresses unique transcripts involved in gametogenesis, and also discovered uncharacterized gene products unique to each gonad. We further developed a pipeline integrating timepoint RNA-seq data throughout development to identify hallmark gene expression in gonads. We found that meiotic and candidate genes involved in sex determination are first expressed surprisingly early during larval growth, and well before metamorphosis. We further discovered that individual larvae express varying amounts of male- or female-hallmarks before metamorphosis, including germline, oocyte, sperm, and meiotic related genes. These distinct male- or female-gonad gene profiles may indicate the onset of, and commitment to, development of a bipotential gonad primordium, and may include metabolic differences, supported by the observation that transcripts involved in glycolysis are highly enriched in the ovary compared to the testis. Together these data support a hypothesis that sex determination is initiated prior to metamorphosis in the sea urchin and that the many uncharacterized genes unique to each gonad type characterized herein may reveal unique pathways and mechanisms in echinoderm reproduction.

The sense of taste is mediated primarily by taste buds on the tongue. These multicellular sensory organs are induced, patterned and become innervated during embryogenesis such that a functional taste system is present at birth when animals begin to feed. While taste buds have been considered ectodermal appendages, this is only partly accurate as only fungiform taste buds in the anterior tongue arise from the ectoderm. Taste buds found in the posterior tongue actually derive from endoderm. Nonetheless, both anterior and posterior buds are functionally similar, despite their disparate embryonic origins. In this review, I compare the development of ectodermal vs endodermal taste buds, highlighting the many differences in the cellular and molecular genetic mechanisms governing their formation.

The trophectoderm (TE) epithelium forms the outer layer of the mammalian blastocyst and generates the blastocoel through vectorial transport. Its differentiation during cleavage, studied mainly in mouse, is integrated with blastocyst morphogenesis with key roles for cell polarisation, asymmetric cell divisions, cell signalling, regulatory transcription factors and cellular inheritance. The TE provides a physical and cellular protection to the emerging lineages of the embryo essential for the integrity of blastocyst development. Here, two examples of TE differentiation are considered in some detail where this immediate protective function for embryo survival is assessed: (i) cellular processes from TE at the polar-mural junctional zone in the early blastocyst that later form filopodia traversing the blastocoel, and (ii) the endocytic system which matures and polarises during differentiation. Understanding the broad role for TE in regulating early morphogenesis and environmental protection of the embryo, including these two examples, have clinical as well as biological relevance.

The epiblast is a pluripotent cell population formed in the late blastocyst stage of preimplantation embryos. During the process of epiblast formation from the inner cell mass (ICM) of the early blastocyst, activation of the Hippo pathway transcription factor TEAD by the nuclear translocation of the coactivator protein YAP is required for the robust expression of pluripotency factors. However, the mechanisms that alter YAP localization during epiblast formation remain unknown. Here, we reveal two such mechanisms. Expansion of the blastocoel promotes nuclear YAP localization by increasing cytoplasmic F-actin and reducing YAP phosphorylation. Additionally, cell differentiation regulates YAP. Expression of the junctional Hippo component, AMOT, gradually decreases during epiblast formation through a tankyrase-mediated degradation. SOX2 expression in the ICM is necessary for the reduction of AMOT and YAP phosphorylation. These two mechanisms function in parallel. Thus, the blastocoel-F-actin and SOX2-AMOT axes cooperatively suppress YAP phosphorylation and promote YAP nuclear localization during epiblast formation. The cooperation of these two distinct mechanisms likely contributes to the robustness of epiblast cell differentiation.