The trichoplein–AurA pathway must suppress primary cilia assembly in order for cells to exit G1.
We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1400-x) contains supplementary material, which is available to authorized users.
Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAFspecific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to a-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. Significance: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.
Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut.
SummaryThe keratin cytoskeleton performs several functions in epithelial cells and provides regulated interaction sites for scaffold proteins, including trichoplein. Previously, we found that trichoplein was localized on keratin intermediate filaments and desmosomes in welldifferentiated, non-dividing epithelia. Here, we report that trichoplein is widely expressed and has a major function in the correct localization of the centrosomal protein ninein in epithelial and non-epithelial cells. Immunocytochemical analysis also revealed that this protein is concentrated at the subdistal to medial zone of both mother and daughter centrioles. Trichoplein binds the centrosomal proteins Odf2 and ninein, which are localized at the distal to subdistal ends of the mother centriole. Trichoplein depletion abolished the recruitment of ninein, but not Odf2, specifically at the subdistal end. However, Odf2 depletion inhibited the recruitment of trichoplein to a mother centriole, whereas ninein depletion did not. In addition, the depletion of each molecule impaired MT anchoring at the centrosome. These results suggest that trichoplein has a crucial role in MT-anchoring activity at the centrosome in proliferating cells, probably through its complex formation with Odf2 and ninein.
Wnt proteins direct embryonic patterning, but the regulatory basis of their distribution and signal reception remain unclear. Here, we show that endogenous Wnt8 protein is distributed in a graded manner in Xenopus embryo and accumulated on the cell surface in a punctate manner in association with “N-sulfo-rich heparan sulfate (HS),” not with “N-acetyl-rich HS”. These two types of HS are differentially clustered by attaching to different glypicans as core proteins. N-sulfo-rich HS is frequently internalized and associated with the signaling vesicle, known as the Frizzled/Wnt/LRP6 signalosome, in the presence of Wnt8. Conversely, N-acetyl-rich HS is rarely internalized and accumulates Frzb, a secreted Wnt antagonist. Upon interaction with Frzb, Wnt8 associates with N-acetyl-rich HS, suggesting that N-acetyl-rich HS supports Frzb-mediated antagonism by sequestering Wnt8 from N-sulfo-rich HS. Thus, these two types of HS clusters may constitute a cellular platform for the distribution and signaling of Wnt8.
Sfrp is a secreted Wnt antagonist that directly interacts with Wnt ligand. We show here that inactivation of Sfrp1, Sfrp2, and Sfrp5 leads to fused somites formation in early-somite mouse embryos, simultaneously resulting in defective convergent extension (CE), which causes severe shortening of the anteroposterior axis. These observations indicate the redundant roles of Sfrp1, Sfrp2, and Sfrp5 in early trunk formation. The roles of the Sfrps were genetically distinguished in terms of the regulation of Wnt pathways. Genetic analysis combining Sfrps mutants and Loop-tail mice revealed the involvement of Sfrps in CE through the regulation of the planar cell polarity pathway. Furthermore, Dkk1-deficient embryos carrying Sfrp1 homozygous and Sfrp2 heterozygous mutations display irregular somites and indistinct intersomitic boundaries, which indicates that Sfrps-mediated inhibition of the Wnt/beta-catenin pathway is necessary for somitogenesis. Our results suggest that Sfrps regulation of the canonical and noncanonical pathways is essential for proper trunk formation.
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