Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta

Trophoblast giant cells (TGCs) are the first terminally differentiated subtype to form in the trophoblast cell lineage in rodents. In addition to mediating implantation, they are the main endocrine cells of the placenta, producing several hormones which regulate the maternal endocrine and immune systems and promote maternal blood flow to the implantation site. Generally considered a homogeneous population, TGCs have been identified by their expression of genes encoding placental lactogen 1 or proliferin. In the present study, we have identified a number of TGC subtypes, based on morphology and molecular criteria and demonstrated a previously underappreciated diversity of TGCs. In addition to TGCs that surround the implantation site and form the interface with the maternal deciduas, we demonstrate at least three other unique TGC subtypes: spiral artery-associated TGCs, maternal blood canal-associated TGCs and a TGC within the sinusoidal spaces of the labyrinth layer of the placenta. All four TGC subtypes could be identified based on the expression patterns of four genes: Pl1, Pl2, Plf (encoded by genes of the prolactin/prolactin-like protein/placental lactogen gene locus), and Ctsq (from a placental-specific cathepsin gene locus). Each of these subtypes was detected in differentiated trophoblast stem cell cultures and can be differentially regulated; treatment with retinoic acid induces Pl1/Plf+ TGCs preferentially. Furthermore, cell lineage tracing studies indicated unique origins for different TGC subtypes, in contrast with previous suggestions that secondary TGCs all arise from Tpbpa+ ectoplacental cone precursors.     

Activin promotes differentiation of cultured mouse trophoblast stem cells towards a labyrinth cell fate

Prolonged maintenance of trophoblast stem (TS) cells requires fibroblast growth factor (FGF) 4 and embryonic fibroblast feeder cells or feeder cell-conditioned medium. Previous studies have shown that TGF-β and Activin are sufficient to replace embryonic fibroblast-conditioned medium. Nodal, a member of the TGF-β superfamily, is also known to be important in vivo for the maintenance of TS cells in the developing placenta. Our current studies indicate that TS cells do not express the Nodal co-receptor, Cripto, and do not respond directly to active Nodal in culture. Conversely, Activin subunits and their receptors are expressed in the placenta and TS cell cultures, with Activin predominantly expressed by trophoblast giant cells (TGCs). Differentiation of TS cells in the presence of TGC-conditioned medium or exogenous Activin results in a reduction in the expression of TGC markers. In line with TGC-produced Activin representing the active component in TGC-conditioned medium, this differentiation-inhibiting effect can be reversed by the addition of follistatin. Additional experiments in which TS cells were differentiated in the presence or absence of exogenous Activin or TGF-β show that Activin but not TGF-β results in the maintenance of expression of TS cell markers, prolongs the expression of syncytiotrophoblast markers, and significantly delays the expression of spongiotrophoblast and TGC markers. These results suggest that Activin rather than TGF-β (or Nodal) acts directly on TS cells influencing both TS cell maintenance and cell fate, depending on whether the cells are also exposed to FGF4.     

The roles of ERAS during cell lineage specification of mouse early embryonic development

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.     

Glial cell lineage in vivo in the mouse cerebellum

Glial cells of the cerebellum originate from cells of the ventricular germinative layer, but their lineage has not been fully elucidated. For studying the glial cell lineage in vivo by retrovirus-mediated gene transfer, we introduced a marker retrovirus into the ventricular germinative layer of embryonic day 13 mice. In the resulting adult cerebella, virus-labeled glial cells were grouped in discrete clusters, and statistical analysis showed that these clusters represented clones in high probability. Of 71 of the virus-labeled glial clusters, 33 clusters were composed of astrocytes/Bergmann glia, 10 were composed of only white matter astrocytes, and 24 were composed of only oligodendrocytes. No glial clusters contained virus-labeled neurons. These results suggest that astrocytes/Bergmann glia, white matter astrocytes and oligodendrocytes immediately arise from separate glial precursors: these three glial lineages may diverge in the course of cerebellar development.     

Neurogenin 3 and the enteroendocrine cell lineage in the adult mouse small intestinal epithelium

It is thought that small intestinal epithelial stem cell progeny, via Notch signaling, yield a Hes1-expressing columnar lineage progenitor and an Atoh1 (also known as Math1)-expressing common progenitor for all granulocytic lineages including enteroendocrine cells, one of the body's largest populations of endocrine cells. Because Neurogenin 3 (Neurog3) null mice lack enteroendocrine cells, Neurog3-expressing progenitors derived from the common granulocytic progenitor are thought to produce the enteroendocrine lineage, although more recent work indicates that Neurog3+ progenitors also contribute to non-enteroendocrine lineages. We aimed to test this model and better characterize the progenitors leading from the stem cells to the enteroendocrine lineage. We investigated clones derived from enteroendocrine precursors and found no evidence of a common granulocytic progenitor that routinely yields all granulocytic lineages. Rather, enteroendocrine cells are derived from a short-lived bipotential progenitor whose offspring, probably via Notch signaling, yield a Neurog3+ cell committed to the enteroendocrine lineage and a progenitor committed to the columnar lineage. The Neurog3+ cell population is heterogeneous; only about 1/3 are slowly cycling progenitors, the rest are postmitotic cells in early stages of enteroendocrine differentiation. No evidence was found that Neurog3+ cells contribute to non-enteroendocrine lineages. Revised lineage models for the small intestinal epithelium are introduced.     

Role of cell adhesion in the specification of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

To clarify the role of cell adhesion in the specification of pigment cell lineage in sea urchin embryos, cell contacts were inhibited by Ca²⁺-free artificial seawater (ASW) treatment, and the number of differentiated pigment cells was examined by the method devised for the present study. Obtained results showed that inhibition of cell contacts during mid-to-late blastula stage greatly affects the number of pigment cells. Treatment with Ca²⁺-free ASW during 7.5–10.5h of development drastically decreased the number of pigment cells, indicating that cell adhesion during this period is indispensable for the specification of pigment cell lineage. On the other hand, the number of pigment cells were increased by the treatment during 9.5–12.5 h of development. It was suggested that this increase was caused by excess divisions of the precursor cells, that is, the division schedule of the precursor cells was altered by inhibition of cell contacts at this period. Interestingly, the number of pigment cells was a multiple of four in a majority of embryos in which pigment cells were drastically decreased in number. These findings suggest that the founder blastomeres of the pigment cell lineage are specified during 7–10 h of development, and that these blastomeres divide twice before they differentiate into pigment cells.     

Fluorescent latex microparticles: A non-invasive short-term cell lineage marker suitable for use in the mouse early embryo

Summary We have examined the potential of fluorescent latex microparticles for use as a short term cell lineage marker in the mouse preimplantation embryo. Isolated blastomeres and intact embryos rapidly adsorb and subsequently endocytose the particles (0.2 m diameter) from a monodisperse suspension in normal medium, so that cytoplasmic endocytic organelles, but not the cytosol itself, becomes labelled. Latex fluorescence, either within intact embryos, disaggregated cells or thick resin sections, is stable during UV irradiation. The development of labelled embryos, both in terms of sequential morphological changes and their time of expression, was comparable to controls and resulted in blastocysts with normal cell numbers and capacity for tissue differentiation. Latex fluorescence is preserved within all the progeny of labelled blastomeres over several cell cycles (e.g. from 8-cell stage to 64-cell stage) and is not transmitted to unlabelled cells either by exocytosis or via midbodies. The particles are particularly suitable for labelling exclusively the entire population of outside cells in the intact embryo from the 16-cell stage onwards.     

Environmental control of lineage plasticity and stem cell memory

Tissue-resident stem cells (SCs) are critical players in the maintenance of tissue homeostasis. SCs reside in complex and uniquely anatomically organized microenvironments (SC niches), that carefully control SC lineage outputs depending on localized tissue needs. Upon environmental perturbations and tissue stressors, SCs respond and restore the tissue to homeostasis, as well as protect it from secondary assaults. Critical to this function are two key processes, SC lineage plasticity and SC memory. In this review, we delineate the multifactorial determinants and key principles underlining these two remarkable SC behaviors. Understanding lineage plasticity and SC memory will be critical not only to design new regenerative therapies but also to determine how these processes are altered in a multitude of pathologies such as cancer and chronic tissue damage.     

Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst

In the mouse blastocyst, some cells of the inner cell mass (ICM) develop into primitive endoderm (PE) at the surface, while deeper cells form the epiblast. It remained unclear whether the position of cells determines their fate, such that gene expression is adjusted to cell position, or if cells are pre-specified at random positions and then sort. We have tracked and characterised dynamics of all ICM cells from the early to late blastocyst stage. Time-lapse microscopy in H2B-EGFP embryos shows that a large proportion of ICM cells change position between the surface and deeper compartments. Most of this cell movement depends on actin and is associated with cell protrusions. We also find that while most cells are precursors for only one lineage, some give rise to both, indicating that lineage segregation is not complete in the early ICM. Finally, changing the expression levels of the PE marker Gata6 reveals that it is required in surface cells but not sufficient for the re-positioning of deeper cells. We provide evidence that Wnt9A, known to be expressed in the surface ICM, facilitates re-positioning of Gata6-expressing cells. Combining these experimental results with computer modelling suggests that PE formation involves both cell sorting movements and position-dependent induction.     

Formation and specification of neurons during the development of the leech central nervous system

In the leech embryo, neurogenesis takes place within the context of a stereotyped cell lineage. The prospective germ layers are formed during the early cleavage divisions by the reorganization and segregation of circumscribed domains within the cytoplasm of the fertilized egg. The majority of central neurons arise from the ectoderm, and central neuroblasts are distributed throughout both the length and width of each ectodermal hemisegment. Much of the segmental ganglion arises from medial neuroblasts, but there are also lateral ectodermal neuroblasts and mesodermal neuroblasts that migrate into the nascent ganglion from peripheral sites of origin. Some of these migratory cells are committed to neurogenesis prior to reaching their central destination. In addition, the leech embryo exhibits a secondary phase of neurogenesis that is restricted to the two sex segment ganglia. Secondary neurogenesis requires that a mitogenic or trophic signal be conveyed from the peripherally located male sex organ to a particular set of centrally located neuroblasts, apparently via already differentiated central neurons that innervate the sex organ. The differential specification of neuronal phenotypes within the leech central nervous system occurs in multiple steps. Some aspects of a neuron's identity are already specified at the time of its terminal cell division and would seem to involve the lineal inheritance of developmental commitments made by one of the neuron's progenitors. This lineage-based identity can then be modified by interactions between the postmitotic neuron and other neurons or non-neuronal target cells encountered during its terminal differentiation. © 1995 John Wiley & Sons, Inc.     

The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two‐celled proembryo stage

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo in vitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor in vitro is highly similar to its undisturbed in vivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both in vivo and in vitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two‐celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo in vivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.     

Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification

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Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta

An increasing number of genes known to be critical for cell cycle control, differentiation, and tumor suppression have been found to impact development of the placenta. To elucidate how these genes contribute to development of embryonic and extra-embryonic lineages, we generated a transgenic mouse in which the Cre transgene is driven by placenta-specific regulatory sequences from the human CYP19 gene. Using ROSA26 conditional reporter mice, we could detect expression of the CYP19-Cre transgene throughout the extra-embryonic ectoderm and in the ectoplacental cone at embryonic day 6.5 (E6.5). By E11.5, recombination of LoxP reporter sites was detected in all derivatives of trophoblast stem cells, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. We conclude that the CYP19-Cre transgenic mouse developed here can be used in combination with conditional alleles to distinguish between embryonic and extra-embryonic gene function, and to begin to map the period of time when gene function is critical during development.     

Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta

The placenta is composed of multiple trophoblast cell types that have diverse endocrine, vascular and nutrient transport functions. We have developed a transgenic system to investigate the developmental and functional roles of specific cell types using conditional expression of a cytotoxin to induce cell ablation in transgenic mice. The Tpbpa gene is expressed in ectoplacental cone cells starting between embryonic days (E) 7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. Tpbpa-positive cells are progenitors of many trophoblast subtypes including three subtypes of trophoblast giant cells (TGCs) and glycogen trophoblast cells. We used a Cre recombinase transgene driven by the Tpbpa promoter to irreversibly activate a diphtheria toxin A (DTA) transgene. Cre/DTA double transgenic placentas showed dramatic reduction of Tpbpa-positive spongiotrophoblast cells by E10.5 and conceptuses died by ~ E11.5. The number of cells associated with maternal blood spaces, spiral artery TGCs (SpA-TGCs) and canal TGCs, and glycogen trophoblast cells were reduced. The loss of these specific trophoblast subtypes, especially SpA-TGCs, was correlated with a decrease in maternal spiral artery diameters, indicating a critical role of these cells in modulating the maternal vasculature. In contrast, parietal TGCs were not significantly reduced by progenitor cell ablation, suggesting that there is compensatory growth of this population and indeed a population of Ascl2 (Mash2)-positive/Tpbpa-negative cells was increased in the spongiotrophoblast layer in the Cre/DTA double transgenics. Our work demonstrates that the Tpbpa-positive lineage is essential for placental function and particularly critical for maternal vasculature remodeling.