Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

Collimated Microbeam Reveals that the Proportion of Non-Damaged Cells in Irradiated Blastoderm Determines the Success of Development in Medaka (Oryzias latipes) Embryos

It has been widely accepted that prenatal exposure to ionizing radiation (IR) can affect embryonic and fetal development in mammals, depending on dose and gestational age of the exposure, however, the precise machinery underlying the IR-induced disturbance of embryonic development is still remained elusive. In this study, we examined the effects of gamma-ray irradiation on blastula embryos of medaka and found transient delay of brain development even when they hatched normally with low dose irradiation (2 and 5 Gy). In contrast, irradiation of higher dose of gamma-rays (10 Gy) killed the embryos with malformations before hatching. We then conducted targeted irradiation of blastoderm with a collimated carbon-ion microbeam. When a part (about 4, 10 and 25%) of blastoderm cells were injured by lethal dose (50 Gy) of carbon-ion microbeam irradiation, loss of about 10% or less of blastoderm cells induced only the transient delay of brain development and the embryos hatched normally, whereas embryos with about 25% of their blastoderm cells were irradiated stopped development at neurula stage and died. These findings strongly suggest that the developmental disturbance in the IR irradiated embryos is determined by the proportion of severely injured cells in the blastoderm.

Divergent Expression Patterns and Function of Two cxcr4 Paralogs in Hermaphroditic Epinephelus coioides

Chemokine receptor Cxcr4 evolved two paralogs in the teleost lineage. However, cxcr4a and cxcr4b have been characterized only in a few species. In this study, we identified two cxcr4 paralogs from the orange-spotted grouper, Epinephelus coioides. The phylogenetic relationship and gene structure and synteny suggest that the duplicated cxcr4a/b should result from the teleost-specific genome duplication (Ts3R). The teleost cxcr4 gene clusters in two paralogous chromosomes exhibit a complementary gene loss/retention pattern. Ec_cxcr4a and Ec_cxcr4b show differential and biased expression patterns in grouper adult tissue, gonads, and embryos at different stages. During embryogenesis, Ec_cxcr4a/b are abundantly transcribed from the neurula stage and mainly expressed in the neural plate and sensory organs, indicating their roles in neurogenesis. Ec_Cxcr4a and Ec_Cxcr4b possess different chemotactic migratory abilities from the human SDF-1α, Ec_Cxcl12a, and Ec_Cxcl12b. Moreover, we uncovered the N-terminus and TM5 domain as the key elements for specific ligand–receptor recognition of Ec_Cxcr4a-Ec_Cxcl12b and Ec_Cxcr4b-Ec_Cxcl12a. Based on the biased and divergent expression patterns of Eccxcr4a/b, and specific ligand–receptor recognition of Ec_Cxcl12a/b–Ec_Cxcr4b/a, the current study provides a paradigm of sub-functionalization of two teleost paralogs after Ts3R.

Embryonic transcription is controlled by maternally defined chromatin state

Histone-modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3- and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300 recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences.

Developmentally regulated activity of CRM1/XPO1 during early Xenopus embryogenesis

In this work, we have investigated the role of CRM1/XPO1, a protein involved in specific export of proteins and RNA from the nucleus, in early Xenopus embryogenesis. The cloning of the Xenopus laevis CRM1, XCRM1, revealed remarkable conservation of the protein during evolution (96.7% amino acid identity between Xenopus and human). The protein and mRNA are maternally expressed and are present during early embryogenesis. However, our data show that the activity of the protein is developmentally regulated. Embryonic development is insensitive to leptomycin B, a specific inhibitor of CRM1, until the neurula stage. Moreover, the nuclear localization of CRM1 changes concomitantly with the appearance of the leptomycin B sensitivity. These data suggest that CRM1, present initially in an inactive form, becomes functional before the initiation of the neurula stage during gastrula-neurula transition, a period known to correspond to a critical transition in the pattern of gene expression. Finally, we confirmed the gastrula-neurula transition-dependent activation of CRM1 by pull-down experiments as well as by the study of the intracellular localization of a green fluorescent protein tagged with a nuclear export signal motif during early development. This work showed that the regulated activity of CRM1 controls specific transitions during normal development and thus might be a key regulator of early embryogenesis.

The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. Here we report the isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene. We have monitored the effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos. We found that the ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers were observed. In contrast, expressions of markers of the diencephalon were strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, were ectopically expressed in this region. Therefore, we propose that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. Our data demonstrate that this change can be mimicked by ectopic engrailed expression alone.

Reconstitution of the organizer is both sufficient and required to re-establish a fully patterned body plan in avian embryos

Lateral blastoderm isolates (LBIs) at the late gastrula/early neurula stage (i.e., stage 3d/4) that lack Hensen's node (organizer) and primitive streak can reconstitute a functional organizer and primitive streak within 10–12 hours in culture. We used LBIs to study the initiation and regionalization of the body plan. A complete body plan forms in each LBI by 36 hours in culture, and normal craniocaudal, dorsoventral, and mediolateral axes are re-established. Thus, reconstitution of the organizer is sufficient to re-establish a fully patterned body plan. LBIs can be modified so that reconstitution of the organizer does not occur. In such modified LBIs, tissue-type specific differentiation (with the exception of heart differentiation) and reconstitution of the body plan fail to occur. Thus, the reconstitution of the organizer is not only sufficient to re-establish a fully patterned body plan, it is also required. Finally, our results show that formation and patterning of the heart is under the control of the organizer, and that such control is exerted during the early to mid-gastrula stages (i.e., stages 2–3a), prior to formation of the fully elongated primitive streak.

De novo induction of the organizer and formation of the primitive streak in an experimental model of notochord reconstitution in avian embryos

We have developed a model system for analyzing reconstitution of the notochord using cultured blastoderm isolates lacking Hensen's node and the primitive streak. Despite lacking normal notochordal precursor cells, the notochord still forms in these isolates during the 36 hours in culture. Reconstitution of the notochord involves an inducer, which acts upon a responder, thereby inducing a reconstituted notochord. To better understand the mechanism of notochord reconstitution, we asked whether formation of the notochord in the model system was preceded by reconstitution of Hensen's node, the organizer of the avian neuraxis. Our results show not only that a functional organizer is reconstituted, but that this organizer is induced from the responder. First, fate mapping reveals that the responder forms a density, morphologically similar to Hensen's node, during the first 10–12 hours in culture, and that this density expresses typical markers of Hensen's node. Second, the density, when fate mapped or when labeled and transplanted in place of Hensen's node, forms typical derivatives of Hensen's node such as endoderm, notochord and the floor plate of the neural tube. Third, the density, when transplanted to an ectopic site, induces a secondary neuraxis, identical to that induced by Hensen's node. And fourth, the density acts as a suppressor of notochord reconstitution, as does Hensen's node, when transplanted to other blastoderm isolates. Our results also reveal that the medial edge of the isolate forms a reconstituted primitive streak, which gives rise to the normal derivatives of the definitive primitive streak along its rostrocaudal extent and which expresses typical streak markers. Finally, our results demonstrate that the notochordal inducer also induces the reconstituted Hensen's node and, therefore, acts like a Nieuwkoop Center. These findings increase our understanding of the mechanism of notochord reconstitution, provide new information and a novel model system for studying the induction of the organizer and reveal the potential of the epiblast to regulate its cell fate and patterns of gene expression during late gastrula/early neurula stage in higher vertebrates.

Enzymatischer Abbau der Embryonalhüllen des Lanzettfischchens, Branchiostoma lanceolatum, während des Schlüpfens / Hatching through Enzymatic Breakdown of the Embryonic Coats in the Lancelet, Branchiostoma lanceolatum (Cephalochordata)

A study was made of the hatching process of the lancelet, Branchiostoma lanceolatum (Amphioxus), under laboratory conditions. Embryos in the early neurula stage (5 to 6 pairs of somites) escape through a hole made in the fertilization envelope, following intensive rotatory movements and partial digestion of the envelope. Using the chromogenic substrate Hide Powder Azure (HPA) at pH 8.0, proteolytic activity was detected in the hatching medium. The effect of various protease inhibitors was investigated. Whereas the activity was only slightly affected by inhibitors of trypsin, chymotrypsin or thiol proteases, chelating agents such as EDTA and 1,10-phenanthroline prevented the degradation of HPA, as well as the breakdown of the vitelline membrane of alcohol fixed oocytes by the protease from hatching fluid. These data suggest the presence of a metalloprotease in the hatching medium. How far this enzyme can be compared with hatching enzymes found in echinoderms and fishes (where they have been identified as matrix degrading metalloproteases), awaits further study of the lancelet enzyme, including its isolation and purification.

The Xenopus laevis tail-forming region

A fate map is produced for the Xenopus tail-forming region at the neurula stage by orthotopic grafting of tissue labelled with fluorescein-dextran amine. It is shown that the axial tissues of the tail are derived from a rectangle 700 micrometre wide by 600 micrometre long, while the epidermis of the tail is drawn from a much larger area. The fate map shows that much of the final tail is not formed from the tail bud itself, but by a displacement of trunk axial tissue relative to the proctodaeum. A specification map is also produced by culturing parts of the tail-forming region in vitro or as grafts on a neutral site on host embryos. For the axial tissues this map is identical to the fate map, showing that the tail-forming region is embryologically mosaic. The prospective tail epidermis can, however, regulate defects. It is shown that previous claims of regeneration of the Xenopus tail bud are misleading. Removal of the tail-forming region totally prevents tail development. Removal of the tail bud leads to a partial tail, formed by the normal process of displacement of trunk tissue relative to the proctodaeum. Even when only part of the tail bud is removed the tail is still truncated. This shows that there is no terminal regeneration of the tail at embryonic stages.