While the transcriptional network of human embryonic stem cells (hESCs) has been extensively studied, relatively little is known about how post-transcriptional modulations determine hESC function. RNA-binding proteins play central roles in RNA regulation, including translation and turnover. Here we show that the RNA-binding protein CSDE1 (cold shock domain containing E1) is highly expressed in hESCs to maintain their undifferentiated state and prevent default neural fate. Notably, loss of CSDE1 accelerates neural differentiation and potentiates neurogenesis. Conversely, ectopic expression of CSDE1 impairs neural differentiation. We find that CSDE1 post-transcriptionally modulates core components of multiple regulatory nodes of hESC identity, neuroectoderm commitment and neurogenesis. Among these key pro-neural/neuronal factors, CSDE1 binds fatty acid binding protein 7 (FABP7) and vimentin (VIM) mRNAs, as well as transcripts involved in neuron projection development regulating their stability and translation. Thus, our results uncover CSDE1 as a central post-transcriptional regulator of hESC identity and neurogenesis.
Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo.
Because tumor cell motility is a requirement for metastasis, we hypothesized that lung tissue harbors substances that induce tumor cell migration. MCF‐7 breast carcinoma cells exposed to small airway epithelial cells and conditioned medium exhibited dose‐dependent tumor cell migration. Among the extracellular matrix proteins in the conditioned medium identified by mass spectrometry, laminin 332 (LM332) had the greatest contribution to the migration of MCF‐7 cells. Immunoblotting and immunohistochemistry for LM332‐specific chains identified LM332 in the lung and in pulmonary epithelial cells. Antibodies to either LM332 or its integrin receptor inhibited MCF‐7 motility, and knockdown of LM332 chains also reduced its migration‐inducing activity. Taken together, these findings implicate LM332 as a component of lung tissue that can induce motility in breast carcinoma cells that have been transported to lung during metastasis. Earlier studies on LM332 in tumor progression have examined LM332 expression in tumor cells. This investigation, in comparison, provides evidence that the tumor promoting potential of LM332 may originate in the lung microenvironment rather than in tumor cells alone. Furthermore, this study provides evidence that the motility‐inducing properties of the microenvironment can reside in epithelial cells. The findings raise the possibility that LM332 plays a role in the pulmonary metastases of breast carcinoma and may provide a target for antimetastasis therapy.
The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.
Poised enhancers (PEs) represent a limited and genetically distinct set of distal regulatory elements that control the induction of developmental genes in a hierarchical and non-redundant manner. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally identified and subsequently characterized using embryonic stem cells (ESC) as an in vitro differentiation system, it is currently unknown whether PEs are functionally conserved in vivo. Here, we generate and mine various types of genomic data to show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that, in mouse pluripotent cells, the interactions between PEs and their bivalent target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. proximity to CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation, preferentially within specific vertebrate clades, with only a small subset being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting evolutionary conserved PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential and non-redundant roles during the induction of major developmental genes in vivo.
Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas-4 null mice leads to embryonic arrest around E9. Centriole loss in Sas-4−/− embryos causes prolonged mitosis and p53-dependent cell death. Studies in vitro discovered a similar USP28-, 53BP1-, and p53-dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas-4−/− embryos. The data indicates that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.
In order to determine whether lung tissue secretes a motility factor for breast cancer, minced fresh human pulmonary tissue was co‐cultured with MCF‐7 breast carcinoma cells. Dose‐dependent MCF‐7 migration was observed, consistent with a motility response to a factor secreted by the lung tissue. Pulmonary epithelial cells were isolated and cultured in defined medium. Concentrated conditioned lung culture medium induced motility in the breast carcinoma cell lines MDA‐MB‐231, SKBR3, and BT‐20 using a Boyden chamber assay. For MCF‐7, the motility was dose dependent by a scattering assay, when the concentrate was either in a soluble form or after deposition on tissue culture plastic. The deposited material was analyzed by mass spectrometry, revealing the α3 and β3 chains of the motility factor laminin 332 (LN332). Blocking antibodies to LN332 and to the LN332 receptor for motility, α3β1 integrin, inhibited motility to the same extent as the inhibition of motility that occurred when these antibodies were applied to MCF‐7 cells incubated with purified LN332. Finally, LN332 in normal lung was confirmed by immunohistochemistry. These experiments together showed that pulmonary LN332 induced motility in MCF‐7. These findings provide a mechanism for the migration of metastatic tumor cells from their arrest in the pulmonary vasculature into the surrounding tissue, allowing their establishment as a metastatic colony.
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