The positional identity of mouse ES cell-generated neurons is affected by BMP signaling

We investigated the effects of bone morphogenetic proteins (BMPs) in determining the positional identity of neurons generated in vitro from mouse embryonic stem cells (ESCs), an aspect that has been neglected thus far. Classical embryological studies in lower vertebrates indicate that BMPs inhibit the default fate of pluripotent embryonic cells, which is both neural and anterior. Moreover, mammalian ESCs generate neurons more efficiently when cultured in a minimal medium containing BMP inhibitors. In this paper, we show that mouse ESCs produce, secrete, and respond to BMPs during in vitro neural differentiation. After neuralization in a minimal medium, differentiated ESCs show a gene expression profile consistent with a midbrain identity, as evaluated by the analysis of a number of markers of anterior–posterior and dorsoventral identity. We found that BMPs endogenously produced during neural differentiation mainly act by inhibiting the expression of a telencephalic gene profile, which was revealed by the treatment with Noggin or with other BMP inhibitors. To better characterize the effect of BMPs on positional fate, we compared the global gene expression profiles of differentiated ESCs with those of embryonic forebrain, midbrain, and hindbrain. Both Noggin and retinoic acid (RA) support neuronal differentiation of ESCs, but they show different effects on their positional identity: whereas RA supports the typical gene expression profile of hindbrain neurons, Noggin induces a profile characteristic of dorsal telencephalic neurons. Our findings show that endogenously produced BMPs affect the positional identity of the neurons that ESCs spontaneously generate when differentiating in vitro in a minimal medium. The data also support the existence of an intrinsic program of neuronal differentiation with dorsal telencephalic identity. Our method of ESC neuralization allows for fast differentiation of neural cells via the same signals found during in vivo embryonic development and for the acquisition of cortical identity by the inhibition of BMP alone.     

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNA^Met. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNA^Met methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.     

DNA repair mechanisms in embryonic stem cells

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body. Therefore, as a renewable source of various functional cells in the human body, ESCs hold great promise for human cell therapy. During the rapid proliferation of ESCs in culture, DNA damage, such as DNA double-stranded breaks, will occur in ESCs. Therefore, to realize the potential of ESCs in human cell therapy, it is critical to understand the mechanisms how ESCs activate DNA damage response and DNA repair to maintain genomic stability, which is a prerequisite for their use in human therapy. In this context, it has been shown that ESCs harbor much fewer spontaneous mutations than somatic cells. Consistent with the finding that ESCs are genetically more stable than somatic cells, recent studies have indicated that ESCs can mount more robust DNA damage responses and DNA repair than somatic cells to ensure their genomic integrity.     

A close look at the mammalian blastocyst: epiblast and primitive endoderm formation

During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues—the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.     

Gene-manipulated embryonic stem cells for rat transgenesis

Embryonic stem cells (ESCs) are derived from blastocysts and are capable of differentiating into whole tissues and organs. Transplantation of ESCs into recipient blastocysts leads to the generation of germline-competent chimeras in mice. Transgenic, knockin, and knockout gene manipulations are available in mouse ESCs, enabling the production of genetically modified animals. Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. However, in contrast to mouse ESCs, rat ESCs were not established until 2008 because of the difficulty of maintaining pluripotency. Although the use of signaling inhibitors has allowed the generation of rat ESCs, the production of genetically modified rats has been difficult due to problems in rat ESCs after gene introduction. In this review, we will focus on some well-documented examples of gene manipulation in rat ESCs.     

Mesodermal fate decisions of a stem cell: the Wnt switch

Stem cells are a powerful resource for cell-based transplantation therapies in osteodegenerative disorders, but before some kinds of stem cells can be applied clinically, several aspects of their expansion and differentiation need to be better controlled. Wnt molecules and members of the Wnt signaling cascade have been ascribed a role in both these processes in vitro as well as normal development in vivo. However some results are controversial. In this review we will present the hypothesis that both canonical and non-canonical signaling are involved in mesenchymal cell fate regulation, such as adipogenesis, chondrogenesis and osteogenesis, and that in vitro it is a timely switch between the two that specifies the identity of the differentiating cell. We will specifically focus on the in vitro differentiation of adipocytes, chondrocytes and osteoblasts contrasting embryonic and mesenchymal stem cells as well as the role of Wnts in mesenchymal fate specification during embryogenesis.     

Metabolic circuits in neural stem cells

Metabolic activity indicative of cellular demand is emerging as a key player in cell fate decision. Numerous studies have demonstrated that diverse metabolic pathways have a critical role in the control of the proliferation, differentiation and quiescence of stem cells. The identification of neural stem/progenitor cells (NSPCs) and the characterization of their development and fate decision process have provided insight into the regenerative potential of the adult brain. As a result, the potential of NSPCs in cell replacement therapies for neurological diseases is rapidly growing. The aim of this review is to discuss the recent findings on the crosstalk among key regulators of NSPC development and the metabolic regulation crucial for the function and cell fate decisions of NSPCs. Fundamental understanding of the metabolic circuits in NSPCs may help to provide novel approaches for reactivating neurogenesis to treat degenerative brain conditions and cognitive decline.     

Developmental actions of natriuretic peptides in the brain and skeleton

The concept that atrial natriuretic peptide (ANP) and the closely related peptides BNP and CNP might be involved in the ontogeny of several organ systems emerged in the late 1980s. While many of the reported in vitro actions have not been examined in the context of organ development in vivo, recent studies demonstrate that mice which lack or overexpress natriuretic peptides or receptors exhibit pronounced skeletal growth defects. This article discusses how natriuretic peptides and other factors appear to regulate bone growth as an example of how natriuretic peptides might participate in the ontogeny of other organ systems. Evidence indicating that natriuretic peptides regulate neural development is then reviewed. Natriuretic peptides and receptors exhibit complex expression patterns in the developing nervous system, where they have been shown to act on neural cells as early as at the embryonic neural tube stage. Interestingly, both bone and brain growth appear to utilize primarily CNP and the CNP-specific type B receptor, and perhaps the type C receptor. In vitro data indicate that CNP may act on developing neurons, astrocytes and Schwann cells like a classical growth factor, regulating proliferation, patterning, phenotypic specification, survival and axonal pathfinding. Natriuretic peptides might also have roles in the vascularization of the embryonic brain, establishment of the blood-brain and blood-nerve barriers, and perhaps in nerve regeneration.Received 13 April 2004; received after revision 20 May 2004; accepted 27 May 2004     

The specification of neuronal identity in the mammalian cerebral cortex

The determination of neuronal fate in the developing cerebral cortex has been studied by tracking normal cell lineages in the cortex, and by testing the commitment of young cortical neurons to their normal fates. These studies together suggest that neuronal progenitors are multipotent during development and have the potential to produce neurons destined for many or all of the cortical layers. However, the laminar identity of an individual neuron appears to be specified through environmental interactions at the time of the cell's terminal mitotic division, prior to its migration into the cortical plate.     

The specification of neuronal identity in the mammalian cerebral cortex

Summary The determination of neuronal fate in the developing cerebral cortex has been studied by tracking normal cell lineages in the cortex, and by testing the commitment of young cortical neurons to their normal fates. These studies together suggest that neuronal progenitors are multipotent during development and have the potential to produce neurons destined for many or all of the cortical layers. However, the laminar identity of an individual neuron appears to be specified through environmental interactions at the time of the cell's temrinal mitotic division, prior to its migration into the cortical plate.     

(In)discrete charm of the polyembryony: Evolution of embryo cloning

Polyembryonic development, where multiple embryos are formed from a single zygote, evolved at least 15 times in six different phyla in animals. The mechanisms leading to polyembryony and the forces that shaped the evolution of the polyembryonic developmental program have remained poorly understood. Recent studies of the polyembryonic development in the endoparasitic wasp Copidosoma floridanum have revealed that the evolution of polyembryony is associated with the evolution of developmental novelties such as total cleavage, early specification of embryonic and extra-embryonic fates, and a specific cell proliferation phase. These changes cumulatively result in the formation of thousands of embryos from a single egg. Laser ablation studies and analysis of early cell fate specification have revealed that a single blastomere representing the progenitor of the primordial germ cell regulates the proliferation of the embryos. We propose that evolutionary changes in cell cleavage, cell interactions, and the cell-differentiation program, reminiscent of interactions between the germinal stem cell and stem cell niche in fly ovaries, underlies the evolution of polyembryony. Received 30 January 2007; received after revision 21 June 2007; accepted 11 July 2007     

The intestinal epithelium tuft cells: specification and function

The intestinal epithelium, composed of at least seven differentiated cell types, represents an extraordinary model to understand the details of multi-lineage differentiation, a question that is highly relevant in developmental biology as well as for clinical applications. This review focuses on intestinal epithelial tuft cells that have been acknowledged as a separate entity for more than 60?years but whose function remains a mystery. We discuss what is currently known about the molecular basis of tuft cell fate and differentiation and why elucidating tuft cell function has been so difficult. Finally, we summarize the current hypotheses on their potential involvement in diseases of the gastro-intestinal tract.     

Nodal signals pattern vertebrate embryos

Vertebrate embryonic patterning requires several conserved inductive signals–including Nodal, Bmp, Wnt and Fgf signals. Nodal, which is a member of the transforming growth factor β (TGFβ) superfamily, activates a signal transduction pathway that is similar to that of other TGFβ members. Nodal genes, which have been identified in numerous vertebrate species, are expressed in specific cell types and tissues during embryonic development. Nodal signal transduction has been shown to play a pivotal role in inducing and patterning mesoderm and endoderm, and in regulating neurogenesis and left-right axis asymmetry. Antagonists, which act at different steps in the Nodal signal transduction pathway, have been shown to tightly modulate the inductive activity of Nodal. Received 20 October 2005; received after revision 15 November 2005; accepted 25 November 2005     

Control of signaling molecule range during developmental patterning

Tissue patterning, through the concerted activity of a small number of signaling pathways, is critical to embryonic development. While patterning can involve signaling between neighbouring cells, in other contexts signals act over greater distances by traversing complex cellular landscapes to instruct the fate of distant cells. In this review, we explore different strategies adopted by cells to modulate signaling molecule range to allow correct patterning. We describe mechanisms for restricting signaling range and highlight how such short-range signaling can be exploited to not only control the fate of adjacent cells, but also to generate graded signaling within a field of cells. Other strategies include modulation of signaling molecule action by tissue architectural properties and the use of cellular membranous structures, such as signaling filopodia and exosomes, to actively deliver signaling ligands to target cells. Signaling filopodia can also be deployed to reach out and collect particular signals, thereby precisely controlling their site of action.     

Erythropoietin but not VEGF has a protective effect on auditory hair cells in the inner ear

It has recently been shown that the oxygen-regulated factors erythropoietin (Epo) and vascular endothelial growth factor (VEGF) confer protection on different cells, including neuronal-derived ones. The receptors for Epo and VEGF are widely expressed in different organs. Since mammalian auditory hair cells can irreversibly be damaged by different agents, we aimed to identify otoprotective compounds. We focused on the role of Epo and VEGF in the inner ear and review the recent studies. Epo and its receptor are expressed in the inner ear. In vitro experiments on auditory hair cells showed a protective effect of Epo in ischemia- and gentamicin-induced hair cell damage. In contrast, an in vivo study using an animal model of noise-induced hearing loss showed a negative effect of Epo. Also VEGF and its receptors are expressed in the inner ear. Changes in the expression of VEGF or its receptors have been found in the cochlea after noise exposure, transcranial vibration and diabetic or aged animals. Until now, there are no studies about a direct effect of VEGF on auditory hair cells in vitro or in vivo. We could exclude a protective effect of VEGF on gentamicin-induced auditory hair cell damage in vitro. Thus, we conclude that Epo but not VEGF has a protective effect on auditory hair cell damage at least in vitro. (Part of a multi-author review.)