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PubMed2025

Foxh1 is a locus-specific PRC2 recruiter governing germ layer silencing.

Polycomb Repressive Complex 2 (PRC2) establishes H3K27me3 marks to shape spatiotemporal gene expression during embryogenesis. While its dysregulation is linked to developmental disorders, cancer, and aging, the mechanisms guiding PRC2 to specific genomic loci remain a subject of ongoing debate. A prevailing model proposes that PRC2 recruitment occurs via its intrinsic affinity for chromatin rather than through sequence-specific transcription factors. Here, we provide evidence that the maternally deposited pioneer transcription factor Foxh1 plays a critical role in directing PRC2 to specific genomic loci during zygotic genome activation in Xenopus. Foxh1 is a critical transcription factor mediating Nodal signaling, but it also plays an earlier role by pre-binding enhancers prior to signaling activation. This pre-binding is essential for forming enhanceosome complexes that trigger mesendodermal gene expression and drive gastrulation, in cooperation with other maternal transcription factors. Using maternal Foxh1-null embryos, we demonstrate that Foxh1 directly recruits Ezh2, the catalytic subunit of PRC2, to Foxh1-bound loci. Loss of Foxh1 impairs Ezh2 recruitment, leading to a global reduction in H3K27me3. These findings support a dual-function model in which Foxh1 not only activates endodermal gene expression in endoderm, but also recruits PRC2 to silence the same genes in ectoderm. This dual activity of Foxh1 allows the spatially coordinated epigenetic states of the endodermal gene regulatory program during early embryogenesis.

CRISPR/Cas9↗
PubMed1988

Beta 3 tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis.

During embryogenesis, the beta 3 tubulin gene of Drosophila is transcribed predominantly in the mesoderm. We have raised antibodies specific to the C-terminal domain of the beta 3 tubulin and analysed by immunostaining the distribution of this tubulin isotype during Drosophila embryogenesis. The protein is first detectable in the cephalic mesoderm at maximal germband extension. Shortly afterwards, beta 3 tubulin is expressed in single cells at identical positions of the thoracic and abdominal segments. We suggest that these cells represent muscle pioneer cells of Drosophila. During later embryonic development the somatic musclature, visceral musculature, dorsal vessel and macrophages contain beta 3 tubulin. In dorsalizing mutants dorsal, snail and twist, which do not form a ventral furrow during gastrulation, beta 3 expression is greatly reduced but not completely abolished. Our analysis shows that beta 3 tubulin immunostaining characterizes the differentiation of mesodermal derivatives during embryogenesis.

Animals↗
PubMed1994

Interacting functions of snail, twist and huckebein during the early development of germ layers in Drosophila.

Two zygotic genes, snail (sna) and twist (twi), are required for mesoderm development, which begins with the formation of the ventral furrow. Both twi and sna are expressed ventrally in the blastoderm, encode transcription factors and promote the invagination of the ventral furrow by activating or repressing appropriate target genes. However, sna and twi alone do not define the position of the ventral furrow, since they are also expressed in ventral cells that do not invaginate. We show that huckebein (hkb) sets the anterior and the posterior borders of the ventral furrow, but acts by different modes of regulation. In the posterior part of the blastoderm, hkb represses the expression of sna in the endodermal primordium (which we suggest to be adjacent to the mesodermal primordium). In the anterior part, hkb antagonizes the activation of target genes by twi and sna. Here, bicoid permits the co-expression of hkb, sna and twi, which are all required for the development of the anterior digestive tract. We suggest that mesodermal fate is determined where sna and twi but not hkb are expressed. Anteriorly hkb together with sna determines endodermal fate, and hkb together with sna and twi are required for foregut development.

Animals↗
PubMed1997

Analysis of factors controlling primary germ layer formation and early hematopoiesis using embryonic stem cell in vitro differentiation.

Differentiation and subsequent development are intricately interwoven processes operating as an integrated whole to form the organism. As an approach to examine these interactions in early mammalian development, we used embryonic stem (ES) cell in vitro differentiation. ES cells can, depending upon the environment differentiated to neuroectoderm, mesoderm and hematopoietic cells. We developed a serum-free, chemically defined medium (CDM) in which ES cells survive and differentiate. In CDM, in the absence of exogenous factors, ES cells form neuroectoderm, upregulating the early neural marker Pax-6. This is consistent with the view that neuroectoderm development can represent a default state, where the absence or sequestration of mesoderm inducing factors permits neuroectoderm formation. In contrast, if CDM is supplemented with bone morphogenetic protein (BMP) 2 or 4 a process resembling primitive streak formation, least at the molecular level occurs, with the formation of mesoderm and subsequently endothelial and hematopoietic cells. If used with care, ES cell in vitro differentiation can act as a guide in understanding the environment which controls early differentiation events in mammals.

Animals↗
PubMed2006

Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation.

BACKGROUND: This study was designed to establish human embryonic stem cell (hESC) lines, to identify the differences when maintained in serum-containing versus serum-free medium and to test their potential of in vitro differentiation. METHODS: Procedures including immunosurgery were performed on 11 donated human blastocysts to establish hESC lines. The cell lines were characterized and maintained using either serum-free or serum-containing media to compare their morphology, Oct-4 expression, apoptosis and growth speed. Differentiation of these lines was evaluated by the morphology and the expression of genes belonging to the three embryonic germ layers and the germ cell lineage. RESULTS: Three hESC lines were established, and they grew at similar speed in both media (serum-containing or serum-free), but hESC cultured in serum-containing medium yielded significantly higher percentages of morphologically good colonies and cells expressing Oct-4. These cell lines differentiated spontaneously in vitro into cells expressing markers belonging to all three embryonic germ layers and germ cell markers, including c-Kit, STELLA, VASA and growth differentiation factor 9 (GDF9), in directly adherent culture. CONCLUSIONS: Three hESC lines with Taiwanese ancestry have been established, and they retain the in vitro differentiation potential with or without embryoid body (EB) formation. The data support that hESC may be capable of differentiation into germ cells although further confirmation is needed. It is also suggested that strategies such as stepwise adaptation will be needed before implementing a serum-free culture condition for hESC lines that have previously been derived in a medium containing serum.

Apoptosis↗
PubMedDate not supplied

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine.

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.

Adult↗
PubMed1978

[Changes in gastrulation processes during phylogenesis in the animal world].

While treating gastrulation only as the entoderm individualization and formation of a double layer germ we do not take into consideration the alterations which gastrulation process has undergone in phylogenesis of different animal types. On the other hand, if entoderm formation (in vertebrates--with discoblastula) is not included in the notion of gastrulation, it will result in a complete incompartibility of gastrulation processes in other groups of animal kingdom. In birds and mammals, gastrulation is a double phasic process: the first phase--entoderm individualization by means of delamination (in combination with immigration), double layer germ formation; the second phase--individualization of mesoderm and chorda from the epiblast composition, a triple layer germ formation, the axial complex germs formation. During phylogenesis of the animal kingdom not only means and mechanisms of gastrulation change but also the contents of the process. For example, in Chordata besides increase in number of germ layers, gastrulation also includes the formation of the axial germ complex. As a result of gastrulational rearrangements of the blastula cellular maternal, the gastrula cell complex (germ layers and germs) come into a new system of interrelationships owing to which architectonic organizational bases of each particular animal type is laid down.

Animals↗
PubMed2004

Adult reserve stem cells and their potential for tissue engineering.

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher orders. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50-70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Animals↗
PubMed2003

The Xenopus LIM-homeodomain protein Xlim5 regulates the differential adhesion properties of early ectoderm cells.

One of the earliest lineage restriction events in embryogenesis is the specification of the primary germ layers: ectoderm, mesoderm and endoderm. In Xenopus, germ layer specification occurs prior to gastrulation and requires the transcription factor VegT both for the cell-autonomous specification of endoderm and the generation of mesoderm-inducing signals. In the absence of VegT, ectoderm is formed in all regions of the embryo. In this work, we show that VegT-depleted vegetal cells (prospective endoderm) behave like animal cells in sorting assays and ectopically express early markers of ectoderm. To gain insight into how ectoderm is specified, we looked for candidate ectoderm-specific genes that are ectopically expressed in VegT-depleted embryos, and examined the role of one of these, the LIM homeobox gene Xlim5, in ectoderm development. We show that overexpression of Xlim5 in prospective endoderm cells is sufficient to impair sorting of animal cells from vegetal cells but is not sufficient (at similar doses) to induce ectoderm-specific genes. In whole embryos, Xlim5 causes vegetal cells to segregate inappropriately to other germ layers and express late differentiation markers of that germ layer. Inhibition of Xlim5 function using an Engrailed repressor construct or a morpholino oligonucleotide causes loss of animal cell adhesion or delay in neural fold morphogenesis, respectively, without significantly affecting early ectoderm gene expression. Taken together, our results provide evidence that a primary role for Xlim5 is to specifically regulate differential cell adhesion behaviour of the ectoderm.

Animals↗
PubMed1988

Communication compartments in the gastrulating mouse embryo.

We characterized the pattern of gap junctional communication in the 7.5-d mouse embryo (at the primitive streak or gastrulation stage). First we examined the pattern of dye coupling by injecting the fluorescent tracers, Lucifer Yellow or carboxyfluorescein, and monitoring the extent of dye spread. These studies revealed that cells within all three germ layers are well coupled, as the injected dye usually spread rapidly from the site of impalement into the neighboring cells. The dye spread, however, appeared to be restricted at specific regions of the embryo. Further thick section histological analysis revealed little or no dye transfer between germ layers, indicating that each is a separate communication compartment. The pattern of dye movement within the embryonic ectoderm and mesoderm further suggested that cells in each of these germ layers may be subdivided into smaller communication compartments, the most striking of which are a number of "box-like" domains. Such compartments, unlike the restrictions observed between germ layers, are consistently only partially restrictive. In light of these results, we further monitored ionic coupling to determine if some coupling might nevertheless persist between germ layers. For these studies, Lucifer Yellow was coinjected while ionic coupling was monitored. The injected Lucifer Yellow facilitated the identification of the impalement sites, both in the live specimen and in thick sections in the subsequent histological analysis. By using this approach, all three germ layers were shown to be ionically coupled, indicating that gap junctional communication is maintained across the otherwise dye-uncoupled "germ layer compartments." Thus our results demonstrate that partially restrictive communication compartments are associated with the delamination of germ layers in the gastrulating mouse embryo. The spatial distribution of these compartments are consistent with a possible role in the underlying development.

Animals↗
PubMed2002

Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm.

The nematode Caenorhabditis elegans is a triploblastic ecdysozoan, which, although it contains too few cells during embryogenesis to create discernible germ "layers," deploys similar programs for germ layer differentiation used in animals with many more cells. The endoderm arises from a single progenitor, the E cell, and is selected from among three possible fates by a three-state combinatorial regulatory system involving intersecting cell-intrinsic and intercellular signals. The core gene regulatory cascade that drives endoderm development, extending from early maternal regulators to terminal differentiation genes, is characterized by activation of successive tiers of transcription factors, including a sequential cascade of redundant GATA transcription factors. Each tier is punctuated by a cell division, raising the possibility that intercession of one cell cycle round, or DNA replication, is required for activation of the next tier. The existence of each tier in the regulatory hierarchy is justified by the assignment of a unique task and each invariably performs at least two functions: to activate the regulators in the next tier and to perform one other activity distinct from that of the next tier. While the regulatory inputs that initiate endoderm development are highly divergent, they mobilize a gene regulatory network for endoderm development that appears to be common to all triploblastic metazoans. Genome-wide functional genomic approaches, including identification of >800 transcripts that exhibit the same regulatory patterns as a number of endoderm-specific genes, are contributing to elucidation of the complete endoderm gene regulatory network in C. elegans. Dissection of the architecture of the C. elegans endoderm network may provide insights into the evolutionary plasticity and origins of this germ layer.

Animals↗
PubMed2003

LvTbx2/3: a T-box family transcription factor involved in formation of the oral/aboral axis of the sea urchin embryo.

T-box family transcription factors have been identified in many organisms and are frequently associated with patterning events during embryonic development. With an interest in the molecular basis of patterning in the sea urchin embryo, we identified several members of the T-box family in Lytechinus variegatus. Here, we report the cloning and characterization of an ortholog of the Tbx2/3 subfamily, LvTbx2/3. To characterize the spatial distribution of LvTbx2/3 protein throughout sea urchin embryogenesis, a polyclonal antiserum was generated. Nuclear localization of LvTbx2/3 initiated at the mesenchyme blastula stage and protein was present into the pluteus stage. Localization was asymmetric throughout this period and costaining with marker genes indicated that asymmetry was about the oral/aboral (O/A) axis. Asymmetric distribution of LvTbx2/3 was observed in the aboral territories of all three germ layers. In the skeletogenic mesoderm lineage, LvTbx2/3 expression was dynamic because expression appeared initially in all skeletogenic mesenchyme cells (PMCs) but, subsequently, became refined solely to the aboral ones during skeletogenesis. To determine if the aboral expression of LvTbx2/3 is linked between germ layers, and to place LvTbx2/3 in the sequence of events that specifies the O/A axis, the effects of a series of perturbations to O/A polarity on LvTbx2/3 expression in each germ layer were examined. Preventing the nuclear localization of beta-catenin, pharmacological disruption of the O/A axis with NiCl(2), overexpression of BMP2/4 and disruption of the extracellular matrix all blocked LvTbx2/3 expression in all germ layers. This indicates that expression of LvTbx2/3 in the aboral territories of each germ layer is a common aspect of O/A specification, downstream of the molecular events that specify the axis. Furthermore, blocking the nuclear localization of beta-catenin, overexpression of BMP2/4 and disruption of the extracellular matrix also prevented the oral (stomodael) expression of LvBrachyury (LvBrac) protein, indicating that the O/A axis is established by a complex series of events. Last, the function of LvTbx2/3 in the formation of the O/A axis was characterized by examining the phenotypic consequences of ectopic expression of LvTbx2/3 mRNA on embryonic development and the expression of marker genes that identify specific germ layers and tissues. Ectopic expression of LvTbx2/3 produced profound morphogenetic defects in derivatives of each germ layer with no apparent loss in specification events in those tissues. This indicates that LvTbx2/3 functions as a regulator of morphogenetic movements in the aboral compartments of the ectoderm, endoderm and mesoderm.

Amino Acid Sequence↗
PubMed2001

[Lineage-switching by pluripotent cells derived from adults].

When proceeding normally, embryonic morphogenesis begins with germ layer formation through the process of gastrulation. Each primordial germ layer gives rise to a particular set of lineages. Until recently, it was considered that fate switches between germ layers were impossible. In the last two or three years however, a fair number of such switches have been described (Table I), the most spectacular of which entails the differentiation of neural stem cells into various derivatives. This unexpected plasticity opens important prospects for cell therapy. Stem cells, which are the cells that display this plasticity, are defined by the two properties of self renewal and pluripotency. They are set apart during ontogeny and are responsible for maintaining the homeostasis of a tissue. This notion, first established in the case of hematopoietic stem cells was later extended to other fast renewing cells, such as those in the intestinal epithelium or epidermis, and more recently to cells reputedly non-renewable, i.e. neurons. A new strategy has been described, which has the interesting feature that it can be applied to the isolation of stem cells from various lineages. It consists in sorting out cells on the basis of the efflux of Hoechst 33342 dye (Goodell et al., 1996). When a cell suspension stained with this dye is examined under two distinct wave lengths, a "side population" (SP), characterized by weak fluorescence, can be identified and sorted out. The dye efflux property of these cells is due to the activity of the mdr (multidrug resistance) gene, which encodes a protein responsible for the building of a canal which serves to extrude toxins from the cells. A means of distinguishing a truly multipotent stem cell from a progenitor committed to a specific lineage has been reported. This consists in the expression of the Pax7 gene. Pax7-/- mouse muscles have no satellite cells, i.e. they miss the cells normally responsible for the regeneration of muscle. In contrast they do have an SP population. These SP cells are incapable of differentiating into muscle, but give rise to 10 times more hematopoietic colonies, when cloned in vitro, than SP cells from wild type muscle do. Thus Pax7 appears to be a commitment gene, in the absence of which stem cells cannot become specified to the muscle lineage. As a conclusion, this review emphasizes various features of the recent findings: 1) the unexpected plasticity uncovered in recent years is restricted to the stem cells of each tissue; 2) the switch in phenotype has to be "forced" on these stem cells by drastic experimental conditions enforced in the host: often sublethal irradiation is superimposed on a genetic deficiency. Progress in this field, concerning both conceptual and applied aspects, will require the identification of the factors characterizing the niches which promote integration and fate switches of stem cells, probably a combination of growth factors and intercellular interactions. Finally a key issue, before any therapeutical applications can be considered, is how to control the proliferation of transplanted stem cells in their new environment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗