Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Understanding how sets of genes are coordinately regulated in space and time to generate the diversity of cell types that characterise complex metazoans is a major challenge in modern biology. The use of high-throughput approaches, such as large-scale in situ hybridisation and genome-wide expression profiling via DNA microarrays, is beginning to provide insights into the complexities of development. However, in many organisms the collection and annotation of comprehensive in situ localisation data is a difficult and time-consuming task. Here, we present a widely applicable computational approach, integrating developmental time-course microarray data with annotated in situ hybridisation studies, that facilitates the de novo prediction of tissue-specific expression for genes that have no in vivo gene expression localisation data available. Using a classification approach, trained with data from microarray and in situ hybridisation studies of gene expression during Drosophila embryonic development, we made a set of predictions on the tissue-specific expression of Drosophila genes that have not been systematically characterised by in situ hybridisation experiments. The reliability of our predictions is confirmed by literature-derived annotations in FlyBase, by overrepresentation of Gene Ontology biological process annotations, and, in a selected set, by detailed gene-specific studies from the literature. Our novel organism-independent method will be of considerable utility in enriching the annotation of gene function and expression in complex multicellular organisms.

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.

The product of the flightless I gene is predicted to provide a link between molecules of an as yet unidentified signal transduction pathway and the actin cytoskeleton. Previous work has shown that weak and severe mutations of the flightless I locus in Drosophila melanogaster cause disruption in the indirect flight muscles and in embryonic cellularization events, respectively, indicative of a regulatory role for the flightless I protein in cytoskeletal rearrangements. A C-terminal domain within flightless I with significant homology to the gelsolin-like family of actin-binding proteins has been identified, but evidence of a direct interaction between endogenous flightless I and actin remains to be shown. In the present study, chick, mouse and Drosophila melanogaster embryos have been examined and the localization of flightless I investigated in relation to the actin cytoskeleton. It is shown that flightless I localization is coincident with actin-rich regions in parasympathetic neurons harvested from chicks, in mouse blastocysts and in structures associated with cellularization in Drosophila melanogaster.

Recent advances in synthetic embryology have opened new avenues for understanding the complex events controlling mammalian peri-implantation development. Here, we show that mouse embryonic stem cells (ESCs) solely exposed to chemical inhibition of SUMOylation generate embryo-like structures comprising anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs give rise to spheroids that self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in a droplet microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell transcriptomics reveals various cellular lineages, including properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures. Transient SUMOylation suppression gradually increases DNA methylation genome wide and repressive mark deposition at Nanog. Interestingly, cell-to-cell variations in SUMOylation levels occur during early embryogenesis. Our approach provides a proof of principle for potentially powerful strategies to explore early embryogenesis by targeting chromatin roadblocks of cell fate change.

Bioengineered cardiac tissues represent a promising strategy for regenerative medicine. However, methods of vascularization and suitable cell sources for tissue engineering and regenerative medicine have not yet been established. In this study, we developed methods for the induction of vascular endothelial cells from mouse embryonic stem (ES) cells using three-dimensional (3D) suspension culture, and fabricated cardiac cell sheets with a pre-vascularized structure by co-culture of mouse ES cell-derived endothelial cells. After induction, isolated CD31+ cells expressed several endothelial cell marker genes and exhibited the ability to form vascular network structures similar to CD31+ cells from neonatal mouse heart. Co-culture of ES cell-derived CD31+ cells with ES cell-derived cardiomyocytes and dermal fibroblasts resulted in the formation of cardiac cell sheets with microvascular network formation. In contrast, microvascular network formation was reduced in co-cultures without cardiomyocytes, suggesting that cardiomyocytes within the cell sheet might enhance vascular endothelial cell sprouting. Polymerase chain reaction array analysis revealed that the expression levels of several angiogenesis-related genes, including fibroblast growth factor 1 (FGF1), were up-regulated in co-culture with cardiomyocytes compared with cultures without cardiomyocytes. The microvascular network in the cardiac sheets was attenuated by treatment with anti-FGF1 antibody. These results indicate that 3D suspension culture methods may be used to prepare functional vascular endothelial cells from mouse ES cells, and that cardiomyocyte-mediated paracrine effects might be important for fabricating pre-vascularized cardiac cell sheets.

Somatic embryogenesis is commonly used for clonal propagation of a wide variety of plant species. Induction of protocorm-like-bodies (PLBs), which are capable of developing into individual plants, is a routine tissue culture-based practice for micropropagation of orchid plants. Even though PLBs are often regarded as somatic embryos, our recent study provides molecular evidence to argue that PLBs are not derived from somatic embryogenesis. Here, we report and characterize the somatic embryonic tissues induced by Phalaenopsis aphrodite LEAFY COTYLEDON1 (PaLEC1) in Phalaenopsis equestris. We found that PaLEC1-induced somatic tissues are morphologically different from PLBs, supporting our molecular study that PLBs are not of somatic embryonic origin. The embryonic identity of PaLEC1-induced embryonic tissues was confirmed by expression of the embryonic-specific transcription factors FUSCA3 (FUS3) and ABSCISIC ACID INSENSITIVE3 (ABI3), and seed storage proteins 7S GLOBULIN and OLEOSIN. Moreover, PaLEC1-GFP protein was found to be associated with the Pa7S-1 and PaFUS3 promoters containing the CCAAT element, supporting that PaLEC1 directly regulates embryo-specific processes to activate the somatic embryonic program in P. equestris. Despite diverse embryonic structures, PaLEC1-GFP-induced embryonic structures are pluripotent and capable of generating new shoots. Our study resolves the long-term debate on the developmental identity of PLB and suggests that somatic embryogenesis may be a useful approach to clonally propagate orchid seedlings.

Human embryonic stem cells (hESCs) differentiated into cardiomyocytes (CM) often develop into complex 3D structures that are composed of various cardiac cell types. Conventional methods to study the electrophysiology of cardiac cells are patch clamp and microelectrode array (MEAs) analyses. However, these methods are not suitable to investigate the contractile features of 3D cardiac clusters that detach from the surface of the culture dishes during differentiation. To overcome this problem, we developed a video-based motion detection software relying on the optical flow by Farnebäck that we call cBRA (cardiac beat rate analyzer). The beating characteristics of the differentiated cardiac clusters were calculated based on the local displacement between two subsequent images. Two differentiation protocols, which profoundly differ in the morphology of cardiac clusters generated and in the expression of cardiac markers, were used and the resulting CM were characterized. Despite these differences, beat rates and beating variabilities could be reliably determined using cBRA. Likewise, stimulation of β-adrenoreceptors by isoproterenol could easily be identified in the hESC-derived CM. Since even subtle changes in the beating features are detectable, this method is suitable for high throughput cardiotoxicity screenings.

The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different "nuclear landscape" in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell-type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML-defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display approximately 1-3 large PML structures of two morphological types: long linear "rods" or elaborate "rosettes", which lack substantial SUMO-1, Daxx, and Sp100. These occur primarily between Day 0-2 of differentiation and become rare thereafter. PML rods may be "taut" between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a "gap" in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML-defined structures.

Human embryonic stem cells (ESCs) can differentiate into endothelial cells in response to stimuli from extracellular cytokines. Transforming growth factor (TGF)-β1 signaling is involved in stem cell renewal and vascular development. Previously, human ESCs were isolated from inner cell mass and a stable ESC line was developed. In the present study, the effects of extracellular TGF-β1 were investigated on human ESC-derived embryoid bodies (EB) in suspension. The structures of the EBs were analyzed with light and electron microscopy, while the cellular composition of the EBs was examined via the expression levels of specific markers. Vascular-like tubular structures and cardiomyocyte-like beating cells were observed in the EBs at day 3 and 8, respectively. The frequencies of vascular-like structures and beating cells in the TGF-β1 treated group were significantly higher compared with the control group (84.31 vs. 12.77%; P<0.001; 37.25 vs. 8.51%; P<0.001, respectively). Electron microscopy revealed the presence of lumens and gap junctions in the sections of the tubular structures. Semiquantitative polymerase chain reaction revealed elevated expression levels of CD31 and fetal liver kinase-1 in EBs cultured with TGF-β1. In addition, extensive staining of von Willebrand factor was observed in the vascular-like structures of TGF-β1-treated EBs. Therefore, the results of the present study may aid the understanding of the underlying mechanisms of human ESC differentiation and improve the methods of propagating specific cell types for the clinical therapy of cardiovascular diseases.

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

The field of stem cell-based embryo-like models is rapidly evolving, providing in vitro models of in utero stages of mammalian development. Here, we detail steps to first establish adherent spheroids composed of three cell types from mouse embryonic stem cells solely treated with a chemical inhibitor of SUMOylation. We then describe procedures for generating highly reproducible gastruloids from these dissociated spheroid cells, as well as embryo-like structures comprising anterior neural and trunk somite-like regions using an optimized microfluidics platform. For complete details on the use and execution of this protocol, please refer to Cossec et al. (2023).1.

DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.

We and others have previously demonstrated that retinal cells can be derived from human embryonic stem cells (hESCs) and induced pluripotent stem cells under defined culture conditions. While both cell types can give rise to retinal derivatives in the absence of inductive cues, this requires extended culture periods and gives lower overall yield. Further understanding of this innate differentiation ability, the identification of key factors that drive the differentiation process, and the development of clinically compatible culture conditions to reproducibly generate functional neural retina is an important goal for clinical cell based therapies. We now report that insulin-like growth factor 1 (IGF-1) can orchestrate the formation of three-dimensional ocular-like structures from hESCs which, in addition to retinal pigmented epithelium and neural retina, also contain primitive lens and corneal-like structures. Inhibition of IGF-1 receptor signaling significantly reduces the formation of optic vesicle and optic cups, while exogenous IGF-1 treatment enhances the formation of correctly laminated retinal tissue composed of multiple retinal phenotypes that is reminiscent of the developing vertebrate retina. Most importantly, hESC-derived photoreceptors exhibit advanced maturation features such as the presence of primitive rod- and cone-like photoreceptor inner and outer segments and phototransduction-related functional responses as early as 6.5 weeks of differentiation, making these derivatives promising candidates for cell replacement studies and in vitro disease modeling.

Somatic embryogenesis (SE) is the most promising method for the quick propagation of desirable plant genotypes. However, application of SE to conifers remains challenging due to our limited knowledge about the genes involved in embryogenesis and the processes that lead to somatic embryo formation. Douglas-fir, an economically important lumber species, possesses a homolog of the angiosperm embryo-regulatory LEC1 gene. In the present study, we analyzed the potential of Douglas-fir PmLEC1 to induce embryonic programs in the vegetative cells of a heterologous host, Arabidopsis thaliana. PmLEC1 complemented the Arabidopsis lec1-1 null mutant and led to a variety of phenotypes ranging from normal morphology to developmental arrest at various stages in the T1 generation. PmLEC1 did not affect the morphology of wild type Arabidopsis T1 plants. More profound results occurred in T2 generations. PmLEC1 expression induced formation of recurrent somatic embryo-like structures in vegetative tissues of the rescued lec1-1 mutant but loss of apical dominance (bushy phenotype) in wild type plants. The activation of embryonic programs in the lec1-1PmLEC1 T2 plants was confirmed by the presence of the embryo-specific transcripts, OLEOSIN and CRUCIFERIN. In contrast, no embryo-like structures, and no OLEOSIN or CRUCIFERIN were observed in PmLEC1-expressing bushy wild type T2 plants.

The H2A/UBIQUITIN-binding proteins AtZRF1a/b have been reported as key regulators involved in multiple processes of Arabidopsis plant growth and development. Yet, the cellular and molecular mechanisms underlying the mutant phenotype remain largely elusive. Here we show that loss-of-function of AtZRF1a/b causes defective root elongation and deformed root apical meristem organization in seedlings. The premature termination of the primary root in the atzrf1a;atzrf1b double mutant is associated with an advanced onset of endoreduplication and subsequent consumption of reservoir stem cells. Cytological analyses using cell type-specific markers and florescent dyes indicate that AtZRF1a/b are involved in maintenance of proper cell layer organization, determinacy of cell identity, and establishment of auxin gradient and maximum at the root tip. During embryogenesis AtZRF1a/b act dominantly in regulating the maintenance of ground tissue initial cells and production of lateral root cap. Lastly, quantitative real-time polymerase chain reaction analysis shows mis-expression of some key genes involved in regulating cell patterning, cell proliferation and/or hormone pathways. Our results provide important insight into AtZRF1a/b function in cell fate determinacy and in establishment and maintenance of proper stem cell reservoir during embryonic and post-embryonic root development.

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

We identified the mouse homolog of human DRAPC1 (APCDD1) gene, shown to be a target of Wnt/beta-catenin signaling pathway in cancer cell lines. Analysis of its spatiotemporal expression in mouse embryos from E7.5 to E14 showed that Drapc1 is expressed during development of the extraembryonic structures, nervous system, vascular system and inner ear. In addition, Drapc1 is expressed in the mesenchyme of several developing organs at sites of epithelio-mesenchymal interactions. Drapc1 expression was also found in the hair follicles of the adult mouse skin. Similarity of Drapc1 expression pattern to location of active beta-catenin in developing mouse embryo further suggests that mouse Drapc1 is a novel in vivo target gene of Wnt/beta-catenin signaling pathway.

The size and growth patterns of the components of the human embryonic heart have remained largely undefined. To provide these data, three-dimensional heart models were generated from immunohistochemically stained sections of ten human embryonic hearts ranging from Carnegie stage 10 to 23. Fifty-eight key structures were annotated and volumetrically assessed. Sizes of the septal foramina and atrioventricular canal opening were also measured. The heart grows exponentially throughout embryonic development. There was consistently less left than right atrial myocardium, and less right than left ventricular myocardium. We observed a later onset of trabeculation in the left atrium compared to the right. Morphometry showed that the rightward expansion of the atrioventricular canal starts in week 5. The septal foramina are less than 0.1 mm2 and are, therefore, much smaller than postnatal septal defects. This chronological, graphical atlas of the growth patterns of cardiac components in the human embryo provides quantified references for normal heart development. Thereby, this atlas may support early detection of cardiac malformations in the foetus.This article has an associated First Person interview with the first author of the paper.

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence.