Germ Layer Induction Research Articles

Zebrafish embryonic explants undergo genetically encoded self-assembly.

Embryonic stem cell cultures are thought to self-organize into embryoid bodies, able to undergo symmetry-breaking, germ layer specification and even morphogenesis. Yet, it is unclear how to reconcile this remarkable self-organization capacity with classical experiments demonstrating key roles for extrinsic biases by maternal factors and/or extraembryonic tissues in embryogenesis. Here, we show that zebrafish embryonic tissue explants, prepared prior to germ layer induction and lacking extraembryonic tissues, can specify all germ layers and form a seemingly complete mesendoderm anlage. Importantly, explant organization requires polarized inheritance of maternal factors from dorsal-marginal regions of the blastoderm. Moreover, induction of endoderm and head-mesoderm, which require peak Nodal-signaling levels, is highly variable in explants, reminiscent of embryos with reduced Nodal signals from the extraembryonic tissues. Together, these data suggest that zebrafish explants do not undergo bona fide self-organization, but rather display features of genetically encoded self-assembly, where intrinsic genetic programs control the emergence of order.

Capacitation of human naïve pluripotent stem cells for multi-lineage differentiation.

ABSTRACTHuman naïve pluripotent stem cells (PSCs) share features with the pre-implantation epiblast. They therefore provide an unmatched opportunity for characterising the developmental programme of pluripotency in Homo sapiens. Here, we confirm that naïve PSCs do not respond directly to germ layer induction, but must first acquire competence. Capacitation for multi-lineage differentiation occurs without exogenous growth factor stimulation and is facilitated by inhibition of Wnt signalling. Whole-transcriptome profiling during this formative transition highlights dynamic changes in gene expression, which affect many cellular properties including metabolism and epithelial features. Notably, naïve pluripotency factors are exchanged for postimplantation factors, but competent cells remain devoid of lineage-specific transcription. The gradual pace of transition for human naïve PSCs is consistent with the timespan of primate development from blastocyst to gastrulation. Transcriptome trajectory during in vitro capacitation of human naïve cells tracks the progression of the epiblast during embryogenesis in Macaca fascicularis, but shows greater divergence from mouse development. Thus, the formative transition of naïve PSCs in a simple culture system may recapitulate essential and specific features of pluripotency dynamics during an inaccessible period of human embryogenesis.

Culture and Host Transfer of Xenopus Oocytes for Maternal mRNA Depletion and Genome Editing Experiments.

The early development of Xenopus critically depends on maternal components stored in the egg. Because important events such as axis formation are triggered immediately after fertilization, it is often desirable to perturb gene function before this occurs. Oocytes can be experimentally manipulated in vitro, prior to maturation, and subsequently fertilized or otherwise activated to develop, and then observed for any embryological defects. Available methods for fertilizing cultured oocytes include in vitro fertilization following oocyte vitelline envelope removal, nuclear transplantation, intracytoplasmic sperm injection, and transferring oocytes to the body cavity of ovulating host females (host transfer). This chapter outlines this host transfer method, which has been used to elucidate basic mechanisms of axis formation, germ-layer induction, and primordial germ cell specification. Methods for obtaining, culturing, transferring, and fertilizing Xenopus oocytes are described. This method has typically been used to alter maternal gene function by antisense oligonucleotide-mediated mRNA knockdown, but is also useful for mRNA or protein overexpression, including the expression of genome-editing reagents prior to fertilization.

The development and metamorphosis of the indirect developing acorn worm Schizocardium californicum (Enteropneusta: Spengelidae)

BackgroundEnteropneusts are benthic marine invertebrates that belong to the deuterostome phylum Hemichordata. The two main clades of enteropneusts are defined by differences in early life history strategies. In the Spengelidae and Ptychoderidae, development is indirect via a planktotrophic tornaria larva. In contrast, development in the Harrimanidae is direct without an intervening larval life history stage. Most molecular studies in the development and evolution of the enteropneust adult body plan have been carried out in the harrimanid Saccoglossus kowalevskii. In order to compare these two developmental strategies, we have selected the spengelid enteropneust Schizocardium californicum as a suitable indirect developing species for molecular developmental studies. Here we describe the methods for adult collecting, spawning and larval rearing in Schizocardium californicum, and describe embryogenesis, larval development, and metamorphosis, using light microscopy, immunocytochemistry and confocal microscopy.ResultsAdult reproductive individuals can be collected intertidally and almost year-round. Spawning can be triggered by heat shock and large numbers of larvae can be reared through metamorphosis under laboratory conditions. Gastrulation begins at 17 h post-fertilization (hpf) and embryos hatch at 26 hpf as ciliated gastrulae. At 3 days post-fertilization (dpf), the tornaria has a circumoral ciliary band, mouth, tripartite digestive tract, protocoel, larval muscles and a simple serotonergic nervous system. The telotroch develops at 5 dpf. In the course of 60 days, the serotonergic nervous system becomes more elaborate, the posterior coeloms develop, and the length of the circumoral ciliary band increases. At the end of the larval stage, larval muscles disappear, gill slits form, and adult muscles develop. Metamorphosis occurs spontaneously when the larva reaches its maximal size (ca. 3 mm), and involves loss and reorganization of larval structures (muscles, nervous system, digestive tract), as well as development of adult structures (adult muscles, tripartite body organization).ConclusionsThis study will enable future research in S. californicum to address long standing questions related to the evolution of axial patterning mechanisms, germ layer induction, neurogenesis and neural patterning, the mechanisms of metamorphosis, the relationships between larval and adult body plans, and the evolution of metazoan larval forms.

Cardiogenic programming of human pluripotent stem cells by dose-controlled activation of EOMES

Master cell fate determinants are thought to induce specific cell lineages in gastrulation by orchestrating entire gene programs. The T-box transcription factor EOMES (eomesodermin) is crucially required for the development of the heart—yet it is equally important for endoderm specification suggesting that it may act in a context-dependent manner. Here, we define an unrecognized interplay between EOMES and the WNT signaling pathway in controlling cardiac induction by using loss and gain-of-function approaches in human embryonic stem cells. Dose-dependent EOMES induction alone can fully replace a cocktail of signaling molecules otherwise essential for the specification of cardiogenic mesoderm. Highly efficient cardiomyocyte programming by EOMES mechanistically involves autocrine activation of canonical WNT signaling via the WNT3 ligand, which necessitates a shutdown of this axis at a subsequent stage. Our findings provide insights into human germ layer induction and bear biotechnological potential for the robust production of cardiomyocytes from engineered stem cells.

Germline competency of human embryonic stem cells depends on eomesodermin.

In humans, germline competency and the specification of primordial germ cells (PGCs) are thought to occur in a restricted developmental window during early embryogenesis. Despite the importance of specifying the appropriate number of PGCs for human reproduction, the molecular mechanisms governing PGC formation remain largely unexplored. Here, we compared PGC-like cell (PGCLC) differentiation from 18 independently derived human embryonic stem cell (hESC) lines, and discovered that the expression of primitive streak genes were positively associated with hESC germline competency. Furthermore, we show that chemical inhibition of TGFβ and WNT signaling, which are required for primitive streak formation and CRISPR/Cas9 deletion of Eomesodermin (EOMES), significantly impacts PGCLC differentiation from hESCs. Taken together, our results suggest that human PGC formation involves signaling and transcriptional programs associated with somatic germ layer induction and expression of EOMES.

FGF and canonical Wnt signaling cooperate to induce paraxial mesoderm from tailbud neuromesodermal progenitors through regulation of a two-step epithelial to mesenchymal transition.

Mesoderm induction begins during gastrulation. Recent evidence from several vertebrate species indicates that mesoderm induction continues after gastrulation in neuromesodermal progenitors (NMPs) within the posteriormost embryonic structure, the tailbud. It is unclear to what extent the molecular mechanisms of mesoderm induction are conserved between gastrula and post-gastrula stages of development. Fibroblast growth factor (FGF) signaling is required for mesoderm induction during gastrulation through positive transcriptional regulation of the T-box transcription factor brachyury We find in zebrafish that FGF is continuously required for paraxial mesoderm (PM) induction in post-gastrula NMPs. FGF signaling represses the NMP markers brachyury (ntla) and sox2 through regulation of tbx16 and msgn1, thereby committing cells to a PM fate. FGF-mediated PM induction in NMPs functions in tight coordination with canonical Wnt signaling during the epithelial to mesenchymal transition (EMT) from NMP to mesodermal progenitor. Wnt signaling initiates EMT, whereas FGF signaling terminates this event. Our results indicate that germ layer induction in the zebrafish tailbud is not a simple continuation of gastrulation events.

Detection of Smad Signaling in Zebrafish Embryos.

Nodal and BMPs play critical roles in germ layer induction and patterning in early zebrafish embryos. Smad2/3 and Smad1/5/8 are intracellular effectors of Nodal and BMPs, respectively. These Smads regulate, in cooperation with other factors, transcription of hundreds of target genes in the nucleus. The activity and stability of Smads are regulated by phosphorylation modifications. To better understand the regulatory network of Smads-mediated signaling and its biological implications, it is necessary to monitor the signaling activity in an in vivo model system. In this chapter, we describe the methods used in zebrafish embryos for dissecting Smads signaling, including TGF-β/Nodal- and BMP-responsive luciferase reporter assays, Western blotting for Smads, co-immunoprecipitation for Smads and their interacting proteins, chromatin-immunoprecipitation for identification of Smad2-binding sites, and immunostaining for detection of active Smad1/5/8.

Small C-terminal Domain Phosphatase 3 Dephosphorylates the Linker Sites of Receptor-regulated Smads (R-Smads) to Ensure Transforming Growth Factor β (TGFβ)-mediated Germ Layer Induction in Xenopus Embryos

Germ layer induction is one of the earliest events shortly after fertilization that initiates body formation of vertebrate embryos. In Xenopus, the maternally deposited transcriptional factor VegT promotes the expression of zygotic Nodal/Activin ligands that further form a morphogen gradient along the vegetal-animal axis and trigger the induction of the three germ layers. Here we found that SCP3 (small C-terminal domain phosphatase 3) is maternally expressed and vegetally enriched in Xenopus embryos and is essential for the timely induction of germ layers. SCP3 is required for the full activation of Nodal/Activin and bone morphogenetic protein signals and functions via dephosphorylation in the linker regions of receptor-regulated Smads. Consistently, the linker regions of receptor-regulated Smads are heavily phosphorylated in fertilized eggs, and this phosphorylation is gradually removed when embryos approach the midblastula transition. Knockdown of maternal SCP3 attenuates these dephosphorylation events and the activation of Nodal/Activin and bone morphogenetic protein signals after midblastula transition. This study thus suggested that the maternal SCP3 serves as a vegetally enriched, intrinsic factor to ensure a prepared status of Smads for their activation by the upcoming ligands during germ layer induction of Xenopus embryos.

Maternal Factors Regulate Embryonic Development in Zebrafish

During fertilization in animals, a sperm launches its pronucleus into the egg. The zygotic diploid nucleus is formed by fusion of male and female pronuclei, however, the zygotic genome is transcriptionally inactive during the first few mitotic divisions. A logic speculation is that early embryonic development relies on maternal factors such as RNA and proteins that are supplied by the egg. Forward genetic studies in invertebrates, mainly performed in the 1980s, uncover essential roles of maternal factors in determining the oocyte polarities, the embryonic axes and pattern formation. In the past two decades, analyses of maternal mutants in zebrafish and mice also reveal important functions of maternal factors in embryonic development. This review mainly focuses on identified functions of maternal factors in developmental processes of the zebrafish embryo, including oocyte maturation, egg activation, blastomere cleavage, clearance of maternal mRNAs, zygotic genome activation as well as germ layer induction and patterning and body axis formation. The findings in model animals will provide a base for studying mechanisms of human infertility, miscarriage and birth defects.

IGF promotes cardiac lineage induction by selective expansion of cardiogenic mesoderm in vitro

Purpose: A thorough understanding of the developmental processes that direct embryonic stem cells (ESCs) towards a cardiac fate is essential for translational application of ESCs in cardiac disease modeling and therapy. Methods: This study set out to identify, by high throughput screening, key signaling pathways that regulate early cardiac lineage formation. Results: We screened a panel of 42 cytokines/growth factors for their ability to induce greater Nkx2.5+ cardiac progenitor cell (CPC) formation during ES cell in vitro differentiation using an Nkx2.5 cardiac enhancer-eGFP transgenic mouse ES cell line. Consistent with previous reports, the treatment of differentiating ESCs with Wnt3a and FGF-2 resulted in increased proportion of Nkx2.5-eGFP+ cells (Wnt3a; 5.3±0.05 and FGF-2; 4.0-fold ± 1.3 over untreated baseline, respectively). Surprisingly, treatment with insulin-like growth factor (IGF) family of ligands (e.g. IGF-1, IGF-2, insulin) resulted in expansion of Nkx2.5+ CPCs by 7.4-fold ± 0.79 (IGF-1; 2.71%±0.27 vs. 0.37%±0.03); 6.3-fold ± 0.46 (IGF-2; 2.96%±0.25 vs. 0.47%±0.07) and 6.3-fold ± 0.35 (insulin; 2.85%±0.01 vs. 0.45%±0.03), respectively. To examine the mechanism of IGF-mediated cardiac lineage expansion, we quantified the abundance of endoderm, ectoderm, and mesoderm populations after IGF treatment and found that IGF selectively increased the number of mesendodermal cells (Brachyury+ cell population: IGF-I treated; 14.3%±1.51, IGF-II treated; 19.3%±0.95, and insulin treated 17.3%±1.51 compared with negative control; 3.95%±0.56), but not of ectodermal (Nestin+) populations. This was corroborated by analysis of lineage marker expression using quantitative PCR. Mechanistically, IGF treatment selectively enhanced the proliferation of Brachyury+ cells (Brachyury+/Ki-67+ cells = 46.2%±7.27, 46.0%±12.7, and 50.3%±8.58 after IGF-I, IGF-II, or insulin supplementation vs. 26.0%±8.34 in untreated control). Furthermore, IGFs appear to directly signal to mesodermal cells through phosphorylation of the Akt/mTOR pathway. Conclusions: Our data uncover a novel role for IGF family ligands to regulate cardiac lineage induction through its ability to expand the mesodermal cell population. These findings contribute to our understanding of early developmental events during germ layer induction and provide new approaches to enhance in vitro cardiogenesis for regenerative medicine applications.

Pluripotent stem cells in development and disease

Learning Outcome 1: The origins and characteristics of ESCs and iPSCs. Learning Outcome 2: How to control pluripotent stem cell differentiation to direct their development along specific lineages. Learning Outcome 3: The power of pluripotent stem cell technology to study development, disease mechanisms and therapy. Abstract: Recent breakthroughs in the stem cell biology field have demonstrated the potential of pluripotent Embryonic Stem Cells (ESCs) to generate any cell type in the body. This technology has been underpinned by advances in elucidating the regulatory networks involved in stemcell self-renewal andpluripotency, but also by insights provided by developmental biology regarding the transcriptional regulators and signaling pathways that set up the body plan and control organogenesis in the embryo. Efficient ESC differentiation protocols have been established, and continue to be modified and improved, that model mammalian development by recapitulating gastrulation in vitro through the induction of ectoderm, mesoderm and endoderm germ layers, and subsequently directing their further differentiation along their respective downstream lineages. The field of pluripotent stem cell biology has also been pushed to the forefront of biomedical researchby the important discovery of reprogramming somatic cells to anESClike state, generating induced Pluripotent Stem cells (iPSCs). The ability to reprogram cells has offered the unique opportunity to generate patient-specific iPSCs, and combine this with lineage-specific differentiation protocols to further model human disease in a dish and understand disease mechanisms. Taken together, pluripotent stem cell technology has provided the unique tools to not only model mammalian development, but also to generate functional cell types and tissues in vitro that can be used for drug discovery, for gaining insights into disease, as well as genetic rescue and potential autologous cell-based regenerative and replacement therapies. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

GFP reporters as tools for investigating bone cell biology and regenerative medicine

Learning Outcome 1: The origins and characteristics of ESCs and iPSCs. Learning Outcome 2: How to control pluripotent stem cell differentiation to direct their development along specific lineages. Learning Outcome 3: The power of pluripotent stem cell technology to study development, disease mechanisms and therapy. Abstract: Recent breakthroughs in the stem cell biology field have demonstrated the potential of pluripotent Embryonic Stem Cells (ESCs) to generate any cell type in the body. This technology has been underpinned by advances in elucidating the regulatory networks involved in stemcell self-renewal andpluripotency, but also by insights provided by developmental biology regarding the transcriptional regulators and signaling pathways that set up the body plan and control organogenesis in the embryo. Efficient ESC differentiation protocols have been established, and continue to be modified and improved, that model mammalian development by recapitulating gastrulation in vitro through the induction of ectoderm, mesoderm and endoderm germ layers, and subsequently directing their further differentiation along their respective downstream lineages. The field of pluripotent stem cell biology has also been pushed to the forefront of biomedical researchby the important discovery of reprogramming somatic cells to anESClike state, generating induced Pluripotent Stem cells (iPSCs). The ability to reprogram cells has offered the unique opportunity to generate patient-specific iPSCs, and combine this with lineage-specific differentiation protocols to further model human disease in a dish and understand disease mechanisms. Taken together, pluripotent stem cell technology has provided the unique tools to not only model mammalian development, but also to generate functional cell types and tissues in vitro that can be used for drug discovery, for gaining insights into disease, as well as genetic rescue and potential autologous cell-based regenerative and replacement therapies. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

Pivotal roles for eomesodermin during axis formation,epithelium-to-mesenchyme transition and endoderm specification in the mouse

The T-box transcription factor eomesodermin (Eomes) has been implicated as an important component in germ layer induction and patterning in vertebrate embryos. In the mouse, Eomes is essential for development of the trophectoderm lineage and Eomes loss-of-function mutants arrest at implantation. Here, we have used a novel Eomes conditional allele to test Eomes functions in the embryo proper. Eomes-deficient embryos express both Fgf8 and its downstream target Snail at normal levels but surprisingly fail to downregulate E-cadherin. Eomes functional loss thus efficiently and profoundly blocks EMT and concomitant mesoderm delamination. Marker analysis as well as fate-mapping and chimera studies demonstrate for the first time that Eomes is required for specification of the definitive endoderm lineage. We also describe developmental abnormalities in Eomes/Nodal double heterozygotes, and demonstrate that these phenotypes reflect Eomes and Nodal interactions in different tissue sites. Collectively, our experiments establish that Eomes is a key regulator of anteroposterior axis formation, EMT and definitive endoderm specification in the mouse.

Germ Layer Induction in ESC—Following the Vertebrate Roadmap

Controlled differentiation of pluripotential cells takes place routinely and with great success in developing vertebrate embryos. It therefore makes sense to take note of how this is achieved and use this knowledge to control the differentiation of embryonic stem cells (ESCs). An added advantage is that the differentiated cells resulting from this process in embryos have proven functionality and longevity. This unit reviews what is known about the embryonic signals that drive differentiation in one of the most informative of the vertebrate animal models of development, the amphibian Xenopus laevis. It summarizes their identities and the extent to which their activities are dose-dependent. The unit details what is known about the transcription factor responses to these signals, describing the networks of interactions that they generate. It then discusses the target genes of these transcription factors, the effectors of the differentiated state. Finally, how these same developmental programs operate during germ layer formation in the context of ESC differentiation is summarized.

Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells.

The establishment of the primitive streak and its derivative germ layers, mesoderm and endoderm, are prerequisite steps in the formation of many tissues. To model these developmental stages in vitro, an ES cell line was established that expresses CD4 from the foxa2 locus in addition to GFP from the brachyury locus. A GFP-Bry(+) population expressing variable levels of CD4-Foxa2 developed upon differentiation of this ES cell line. Analysis of gene-expression patterns and developmental potential revealed that the CD4-Foxa2(hi)GFP-Bry(+) population displays characteristics of the anterior primitive streak, whereas the CD4-Foxa2(lo)GFP-Bry(+) cells resemble the posterior streak. Using this model, we were able to demonstrate that Wnt and TGF-beta/nodal/activin signaling simultaneously were required for the generation of the CD4-Foxa2(+)GFP-Bry(+) population. Wnt or low levels of activin-induced a posterior primitive streak population, whereas high levels of activin resulted in an anterior streak fate. Finally, sustained activin signaling was found to stimulate endoderm commitment from the CD4-Foxa2(+)GFP-Bry(+) ES cell population. These findings demonstrate that the early developmental events involved in germ-layer induction in the embryo are recapitulated in the ES cell model and uncover insights into the signaling pathways involved in the establishment of mesoderm and endoderm.

Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm

Formation of mesoderm from the pluripotent epiblast depends upon canonical Wnt/beta-catenin signaling, although a precise molecular basis for this requirement has not been established. To develop a robust model of this developmental transition, we examined the role of Wnt signaling during the analogous stage of embryonic stem cell differentiation. We show that the kinetics of Wnt ligand expression and pathway activity in vitro mirror those found in vivo. Furthermore, inhibition of this endogenous Wnt signaling abrogates the functional competence of differentiating ES cells, reflected by their failure to generate Flk1(+) mesodermal precursors and subsequent mature mesodermal lineages. Microarray analysis at various times during early differentiation reveal that mesoderm- and endoderm-associated genes fail to be induced in the absence of Wnt signaling, indicating a lack of germ layer induction that normally occurs during gastrulation in vivo. The earliest genes displaying Wnt-dependent expression, however, were those expressed in vivo in the primitive streak. Using an inducible form of stabilized beta-catenin, we find that Wnt activity, although required, does not autonomously promote primitive streak-associated gene expression in vitro. Our results suggest that Wnt signaling functions in this model system to regulate the thresholds or stability of responses to other effector pathways and demonstrate that differentiating ES cells represent a useful model system for defining complex regulatory interactions underlying primary germ layer induction.

Germ layer induction from embryonic stem cells

Embryonic stem (ES) cells have the potential to develop into all cell types of the adult body. This capability provides the basis for considering the ES cell system as a novel and unlimited source of cells for replacement therapies for the treatment of a wide range of diseases. Before the cell-based therapy potential of ES cells can be realized, a better understanding of the pathways regulating lineage-specific differentiation is required. Current studies suggest that the bone morphogenic protein, transforming growth factor-beta, Wnt, and fibroblast growth factor pathways that are required for gastrulation and germ layer induction in the embryo are also essential for differentiation of ES cells in culture. The current understanding of how these factors influence germ layer induction in both the embryo and in the ES cell differentiation system is addressed in this review.

A molecular basis for Smad specificity.

Bone morphogenetic proteins (BMPs) and activins are members of the TGFbeta superfamily of growth factors, a crucial group of regulators of induction and patterning of embryonic germ layers in metazoa. In early Xenopus embryos, activin, Vgl, and nodal are potent inducers of dorsal mesoderm, whereas BMPs can ventralize mesoderm, repress neural fate, and induce blood differentiation. These characteristic responses rely on ligand-specific signaling pathways, encompassing transmembrane kinase receptors and signal transducers belonging to the Smad family. The overexpression in Xenopus embryos of BMP-activated Smad1 and of activin/Vg1/ nodal-activated Smad2 is sufficient to specifically recapitulate ligand responses. In a search for determinants of a Smad specificity code, we have identified two small regions within the conserved carboxyl-domain that are necessary and sufficient for specific Smad action. Swapping both residue clusters (C1 and C2) between Smadl and Smad2 completely switches Smad effects in vivo. Thus, Smadl with swapped Smad2 clusters responds specifically to BMP but elicits an activin response, while a Smad2 protein containing the Smadl clusters is activated by activin and elicits a BMP response. Furthermore, association between Smads and FAST-1, a mediator of mesoderm induction by activin, is dependent upon the presence of the Smad2 C1 sequence. Finally, the Smadl-specific antagonist Smad6 can inhibit a Smad2 molecule harboring Smadl C1 and C2 sequences. Thus, the C1 and C2 regions of Smads specify the association between Smads and pathway-specific partners, such as FAST-1 and Smad6, and account for activin- and BMP- specific responses in vertebrate embryogenesis.