Cells and Development最新文献

Extensive communication at the stem cell-niche interface and asymmetric stem cell division is key for the homeostasis of the Drosophila male germline stem cell system. To improve our understanding of these processes, we analysed the function of the mitotic checkpoint complex (MCC) component Bub3 and the nucleoporin Nup75, a component of the nuclear pore complex realizing the transport of signalling effector molecules to the nucleus, in the Drosophila testis. By lineage-specific interference, we found that the two genes control germline development and maintenance. Bub3 is continuously required in the germline, as its loss results in the beginning in an over-proliferation of early germ cells and later on in loss of the germline. The absence of the germline lineage in such testes has dramatic cell non-autonomous consequences, as cells co-expressing markers of hub and somatic cyst cell fates accumulate and populate in extreme cases the whole testis. Our analysis of Nups showed that some of them are critical for lineage maintenance, as their depletion results in the loss of the affected lineage. In contrast, Nup75 plays a role in controlling proliferation of early germ cells but not differentiating spermatogonia and seems to be involved in keeping hub cells quiescent. In sum, our analysis shows that Bub3 and Nup75 are required for male germline development and maintenance.

Plant growth is driven by apical meristems at the shoot and root growth points, which comprise continuously active stem cell populations. While many of the key factors involved in homeostasis of the shoot apical meristem (SAM) have been extensively studied under artificial constant growth conditions, only little is known how variations in the environment affect the underlying regulatory network. To shed light on the responses of the SAM to ambient temperature, we combined 3D live imaging of fluorescent reporter lines that allowed us to monitor the activity of two key regulators of stem cell homeostasis in the SAM namely CLAVATA3 (CLV3) and WUSCHEL (WUS), with computational image analysis to derive morphological and cellular parameters of the SAM. Whereas CLV3 expression marks the stem cell population, WUS promoter activity is confined to the organizing center (OC), the niche cells adjacent to the stem cells, hence allowing us to record on the two central cell populations of the SAM. Applying an integrated computational analysis of our data we found that variations in ambient temperature not only led to specific changes in spatial expression patterns of key regulators of SAM homeostasis, but also correlated with modifications in overall cellular organization and shoot meristem morphology.

Ceramide induces autophagy upon starvation via downregulation of nutrient transporters. To elucidate the mechanism by which starvation regulates autophagy in mouse embryos, the present study investigated nutrient transporter expression and the effect of C2-ceramide on in vitro embryo development, apoptosis, and autophagy. The transcript levels of the glucose transporters Glut1 and Glut3 were high at the 1- and 2-cell stages, and gradually decreased at the morula and blastocyst (BL) stages. Similarly, expression of the amino acid transporters L-type amino transporter-1 (LAT-1) and 4F2 heavy chain (4F2hc) gradually decreased from the zygote to the BL stage. Upon ceramide treatment, expression of Glut1, Glut3, LAT-1, and 4F2hc was significantly reduced at the BL stage, while expression of the autophagy-related genes Atg5, LC3, and Gabarap and synthesis of LC3 were significantly induced. Ceramide-treated embryos exhibited significantly reduced developmental rates and total cell numbers per blastocyst, and increased levels of apoptosis and expression of Bcl2l1 and Casp3 at the BL stage. Ceramide treatment significantly decreased the average mitochondrial DNA copy number and mitochondrial area at the BL stage. In addition, ceramide treatment significantly decreased mTOR expression. These results suggest that ceramide-induced autophagy promotes apoptosis by following downregulation of nutrient transporters during mouse embryogenesis.

Peroxiredoxins (Prdxs) are thiol-dependent enzymes that scavenge peroxides. Previously, we found that Prdxs were hyperoxidized in a Parkinson's disease model induced by paraquat (PQ), which led to their inactivation, perpetuating reactive oxygen species (ROS) formation. Herein, we evaluated the redox state of the typical 2-Cys-Prx subgroup. We found that PQ induces ROS compartmentalization in different organelles, reflected by the 2-Cys-Prdx hyperoxidation pattern detected by redox eastern blotting. 2-Cys Prdxs are most vulnerable to hyperoxidation, while atypical 2-Cys Peroxiredoxin 5 (Prdx5) is resistant and is expressed in multiple organelles, such as mitochondria, peroxisomes, and cytoplasm. Therefore, we overexpressed human Prdx5 in the dopaminergic SHSY-5Y cell line using the adenoviral vector Ad-hPrdx5. Prdx5 overexpression was confirmed by western blotting and immunofluorescence (IF) and effectively decreased PQ-mediated mitochondrial and cytoplasmic ROS assessed with a mitochondrial superoxide indicator and DHE through IF or flow cytometry. Decreased ROS mediated by Prdx5 in the main subcellular compartments led to overall cell protection against PQ-induced cell death, which was demonstrated by flow cytometry using Annexin V labeling and 7-AAD. Therefore, Prdx5 is an attractive therapeutic target for PD, as its overexpression protects dopaminergic cells from ROS and death, which warrants further experimental animal studies for its subsequent application in clinical trials.

Coil-coiled domain containing 85c (Ccdc85c) is a causative gene for congenital hydrocephalus and subcortical heterotopia with frequent brain hemorrhage. We established Ccdc85c knockout (KO) rats and investigated the roles of CCDC85C and intermediate filament protein expression, including nestin, vimentin, GFAP, and cytokeratin AE1/AE3 during the lateral ventricle development in KO rats to evaluate the role of this gene. We found altered and ectopic expression of nestin and vimentin positive cells in the wall of the dorso-lateral ventricle in the KO rats during development from the age of postnatal day (P) 6, whereas both protein expression became faint in the wild-type rats. In the KO rats, there was a loss of cytokeratin expression on the surface of the dorso-lateral ventricle with ectopic expression and maldevelopment of ependymal cells. Our data also revealed disturbed GFAP expression at postnatal ages. These findings indicate that lack of CCDC85C disrupts the proper expression of intermediate filament proteins (nestin, vimentin, GFAP, and cytokeratin), and CCDC85C is necessary for normal neurogenesis, gliogenesis, and ependymogenesis.

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1.

The pharyngula stage of vertebrate development is characterized by stereotypical arrangement of ectoderm, mesoderm, and neural tissues from the anterior spinal cord to the posterior, yet unformed tail. While early embryologists over-emphasized the similarity between vertebrate embryos at the pharyngula stage, there is clearly a common architecture upon which subsequent developmental programs generate diverse cranial structures and epithelial appendages such as fins, limbs, gills, and tails. The pharyngula stage is preceded by two morphogenetic events: gastrulation and neurulation, which establish common shared structures despite the occurrence of cellular processes that are distinct to each of the species. Even along the body axis of a singular organism, structures with seemingly uniform phenotypic characteristics at the pharyngula stage have been established by different processes. We focus our review on the processes underlying integration of posterior axial tissue formation with the primary axial tissues that creates the structures laid out in the pharyngula. Single cell sequencing and novel gene targeting technologies have provided us with new insights into the differences between the processes that form the anterior and posterior axis, but it is still unclear how these processes are integrated to create a seamless body. We suggest that the primary and posterior axial tissues in vertebrates form through distinct mechanisms and that the transition between these mechanisms occur at different locations along the anterior-posterior axis. Filling gaps that remain in our understanding of this transition could resolve ongoing problems in organoid culture and regeneration.

Just over one decade ago, it was discovered that hematopoietic stem cells (HSCs) could directly respond to inflammatory cytokines by mounting a proliferative response thought to mediate the emergency production of mature blood cells. In the intervening years, we have gained mechanistic insight into this so-called activation process and have started to learn such a response may come at a cost in terms of ultimately resulting in HSC exhaustion and hematologic dysfunction. In this review article, we report the progress we have made in understanding the interplay between infection, inflammation and HSCs during the funding period of the Collaborative Research Center 873 “Maintenance and Differentiation of Stem Cells in Development and Disease”, and place this work within the context of recent output by others working within this field.

Stem cell populations are defined by their capacity to self-renew and to generate differentiated progeny. These unique characteristics largely depend on the stem cell micro-environment, the so-called stem cell niche. Niches were identified for most adult stem cells studied so far, but we know surprisingly little about how somatic stem cells and their niche come together during organ formation. Using the neuromasts of teleost fish, we have previously reported that neural stem cells recruit their niche from neighboring epithelial cells, which go through a morphological and molecular transformation. Here, we tackle quantitative, temporal, and clonal aspects of niche formation in neuromasts by using 4D imaging in transgenic lines, and lineage analysis in mosaic fish. We show that niche recruitment happens in a defined temporal window during the formation of neuromasts in medaka, and after that, the niche is enlarged mainly by the proliferation of niche cells. Niche recruitment is a non-clonal process that feeds from diverse epithelial cells that do not display a preferential position along the circumference of the forming neuromast. Additionally, we cover niche formation and expansion in zebrafish to show that distant species show common features during organogenesis in the lateral line system. Overall, our findings shed light on the process of niche formation, fundamental for the maintenance of stem cells not only in medaka but also in many other multicellular organisms.