A new human embryonic cell type associated with activity of young transposable elements allows definition of the inner cell mass.

There remains much that we do not understand about the earliest stages of human development. On a gross level, there is evidence for apoptosis, but the nature of the affected cell types is unknown. Perhaps most importantly, the inner cell mass (ICM), from which the foetus is derived and hence of interest in reproductive health and regenerative medicine, has proven hard to define. Here, we provide a multi-method analysis of the early human embryo to resolve these issues. Single-cell analysis (on multiple independent datasets), supported by embryo visualisation, uncovers a common previously uncharacterised class of cells lacking commitment markers that segregates after embryonic gene activation (EGA) and shortly after undergo apoptosis. The discovery of this cell type allows us to clearly define their viable ontogenetic sisters, these being the cells of the ICM. While ICM is characterised by the activity of an Old non-transposing endogenous retrovirus (HERVH) that acts to suppress Young transposable elements, the new cell type, by contrast, expresses transpositionally competent Young elements and DNA-damage response genes. As the Young elements are RetroElements and the cells are excluded from the developmental process, we dub these REject cells. With these and ICM being characterised by differential mobile element activities, the human embryo may be a "selection arena" in which one group of cells selectively die, while other less damaged cells persist.

Engineered LINE-1 retrotransposition in nondividing human neurons.

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.

The impact of transposable elements on mammalian development.

Despite often being classified as selfish or junk DNA, transposable elements (TEs) are a group of abundant genetic sequences that have a significant impact on mammalian development and genome regulation. In recent years, our understanding of how pre-existing TEs affect genome architecture, gene regulatory networks and protein function during mammalian embryogenesis has dramatically expanded. In addition, the mobilization of active TEs in selected cell types has been shown to generate genetic variation during development and in fully differentiated tissues. Importantly, the ongoing domestication and evolution of TEs appears to provide a rich source of regulatory elements, functional modules and genetic variation that fuels the evolution of mammalian developmental processes. Here, we review the functional impact that TEs exert on mammalian developmental processes and discuss how the somatic activity of TEs can influence gene regulatory networks.

Ratiometric Two-Photon Near-Infrared Probe to Detect DPP IV in Human Plasma, Living Cells, Human Tissues, and Whole Organisms Using Zebrafish.

DPP IV, otherwise known as CD26 lymphocyte T surface antigen, is a transmembrane glycoprotein also found in circulation in the blood. It plays an important role in several processes like glucose metabolism and T-cell stimulation. Moreover, it is overexpressed in renal, colon, prostate, and thyroid human carcinoma tissues. It can also serve as a diagnostic in patients with lysosomal storage diseases. The biological and clinical importance of having readouts for the activity of this enzyme, in physiological and disease conditions, has led us to design a near-infrared (NIR) fluorimetric probe that also has the characteristics of being ratiometric and excitable by two simultaneous NIR photons. The probe consists of assembling an enzyme recognition group (Gly-Pro) (Mentlein, 1999; Klemann et al., 2016) on the two-photon (TP) fluorophore (derivative of dicyanomethylene-4H-pyran, DCM-NH2) disturbing its NIR characteristic internal charge transfer (ICT) emission spectrum. When the dipeptide group is released by the DPP IV-specific enzymatic action, the donor-acceptor DCM-NH2 is restored, forming a system that shows high ratiometric fluorescence output. With this new probe, we have been able to detect, quickly and efficiently, the enzymatic activity of DPP IV in living cells, human tissues, and whole organisms, using zebrafish. In addition, due to the possibility of being excited by two photons, we can avoid the autofluorescence and subsequent photobleaching that the raw plasma has when it is excited by visible light, achieving detection of the activity of DPP IV in that medium without interference.

Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis.

Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. In this work, we have used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. We show that reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, our results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, we show that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, our results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells.

Wingless signaling and the control of cell shape in Drosophila wing imaginal discs.

The control of cell morphology is important for shaping animals during development. Here we address the role of the Wnt/Wingless signal transduction pathway and two of its target genes, vestigial and shotgun (encoding E-cadherin), in controlling the columnar shape of Drosophila wing disc cells. We show that clones of cells mutant for arrow (encoding an essential component of the Wingless signal transduction pathway), vestigial or shotgun undergo profound cell shape changes and are extruded towards the basal side of the epithelium. Compartment-wide expression of a dominant-negative form of the Wingless transducer T-cell factor (TCF/Pangolin), or double-stranded RNA targeting vestigial or shotgun, leads to abnormally short cells throughout this region, indicating that these genes act cell autonomously to maintain normal columnar cell shape. Conversely, overexpression of Wingless, a constitutively-active form of the Wingless transducer beta-catenin/Armadillo, or Vestigial, results in precocious cell elongation. Co-expression of Vestigial partially suppresses the abnormal cell shape induced by dominant-negative TCF. We conclude that Wingless signal transduction plays a cell-autonomous role in promoting and maintaining the columnar shape of wing disc cells. Furthermore, our data suggest that Wingless controls cell shape, in part, through maintaining vestigial expression.

Physical mechanisms shaping the Drosophila dorsoventral compartment boundary.

BACKGROUND: Separating cells with distinct identities and fates by straight and sharp compartment boundaries is important for growth and pattern formation during animal development. The physical mechanisms shaping compartment boundaries, however, are not fully understood. RESULTS: We combine theory and quantitative experiments to investigate the roles of different mechanisms to shape compartment boundaries. Our theoretical work shows that cell elongation created by anisotropic stress, cell proliferation rate, orientation of cell division, and cell bond tension all have distinct effects on the morphology of compartment boundaries during tissue growth. Our experiments using the developing Drosophila wing reveal that the roughness of the dorsoventral compartment boundary is dynamic and that it decreases during development. By measuring tissue relaxation in response to laser ablation of cell bonds at different developmental times, we demonstrate that decreased boundary roughness correlates with increased cell bond tension along the compartment boundary. Finally, by using experimentally determined values for cell bond tension, cell elongation and bias in orientation of cell division in simulations of tissue growth, we can reproduce the main features of the time evolution of the dorsoventral compartment boundary shape. CONCLUSIONS: Local increase of cell bond tension along the boundary as well as global anisotropies in the tissue contribute to shaping boundaries in cell networks. We propose a simple scenario that combines time-dependent cell bond tension at the boundary, oriented cell division, and cell elongation in the tissue that can account for the main features of the dynamics of the shape of the dorsoventral compartment boundary.

Dpp signaling promotes the cuboidal-to-columnar shape transition of Drosophila wing disc epithelia by regulating Rho1.

Morphogenesis is largely driven by changes in the shape of individual cells. However, how cell shape is regulated in developing animals is not well understood. Here, we show that the onset of TGFbeta/Dpp signaling activity correlates with the transition from cuboidal to columnar cell shape in developing Drosophila melanogaster wing disc epithelia. Dpp signaling is necessary for maintaining this elongated columnar cell shape and overactivation of the Dpp signaling pathway results in precocious cell elongation. Moreover, we provide evidence that Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, we demonstrate that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. Our results identify a cell-autonomous role for Dpp signaling in promoting and maintaining the elongated columnar shape of wing disc cells and suggest that Dpp signaling acts by regulating Rho1 and MRLC.

Increased cell bond tension governs cell sorting at the Drosophila anteroposterior compartment boundary.

Subdividing proliferating tissues into compartments is an evolutionarily conserved strategy of animal development [1-6]. Signals across boundaries between compartments can result in local expression of secreted proteins organizing growth and patterning of tissues [1-6]. Sharp and straight interfaces between compartments are crucial for stabilizing the position of such organizers and therefore for precise implementation of body plans. Maintaining boundaries in proliferating tissues requires mechanisms to counteract cell rearrangements caused by cell division; however, the nature of such mechanisms remains unclear. Here we quantitatively analyzed cell morphology and the response to the laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings. We found that mechanical tension is approximately 2.5-fold increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632 [7], an inhibitor of Rho-kinase whose main effector is Myosin II [8]. Simulations using a vertex model [9] demonstrate that a 2.5-fold increase in local cell bond tension suffices to guide the rearrangement of cells after cell division to maintain compartment boundaries. Our results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries.