Cnidarians possess remarkable powers of regeneration, but the cellular and molecular mechanisms underlying this capability are unclear. Studying the hydrozoan Hydractinia echinata we show that a burst of stem cell proliferation occurs following decapitation, forming a blastema at the oral pole within 24 hr. This process is necessary for head regeneration. Knocking down Piwi1, Vasa, Pl10 or Ncol1 expressed by blastema cells inhibited regeneration but not blastema formation. EdU pulse-chase experiments and in vivo tracking of individual transgenic Piwi1+ stem cells showed that the cellular source for blastema formation is migration of stem cells from a remote area. Surprisingly, no blastema developed at the aboral pole after stolon removal. Instead, polyps transformed into stolons and then budded polyps. Hence, distinct mechanisms act to regenerate different body parts in Hydractinia. This model, where stem cell behavior can be monitored in vivo at single cell resolution, offers new insights for regenerative biology.DOI: http://dx.doi.org/10.7554/eLife.05506.001
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma-extracellular matrix-tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.
BackgroundCellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined.MethodsHuman iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment.ResultsWe demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation.ConclusionsHigh levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0425-x) contains supplementary material, which is available to authorized users.
Ultrastructural analysis, that is, electron microscopy, particularly immunogold labeling, will enable direct visualization of subcellular compartments. This in conjunction with the analysis of tissues lacking specific Rho GTPases, and Nox components will facilitate a detailed examination of the interactions of these structures with the actin cytoskeleton. In combination with the analysis of ROS production, including its subcellular location, these data will contribute significantly to our understanding of this intricate network under physiological conditions. Based on this, in vivo and in vitro studies can then be combined to elucidate the signaling pathways involved and their targets.
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