A one-dimensional model of PCP signaling: polarized cell behavior in the notochord of the ascidian Ciona.

Despite its importance in development and physiology the planar cell polarity (PCP) pathway remains one of the most enigmatic signaling mechanisms. The notochord of the ascidian Ciona provides a unique model for investigating the PCP pathway. Interestingly, the notochord appears to be the only embryonic structure in Ciona activating the PCP pathway. Moreover, the Ciona notochord as a single-file array of forty polarized cells is a uniquely tractable system for the study of polarization dynamics and the transmission of the PCP pathway. Here, we test models for propagation of a polarizing signal, interrogating temporal, spatial and signaling requirements. A simple cell-cell relay cascading through the entire length of the notochord is not supported; instead a more complex mechanism is revealed, with interactions influencing polarity between neighboring cells, but not distant ones. Mechanisms coordinating notochord-wide polarity remain elusive, but appear to entrain general (i.e., global) polarity even while local interactions remain important. However, this global polarizer does not appear to act as a localized, spatially-restricted determinant. Coordination of polarity along the long axis of the notochord requires the PCP pathway, a role we demonstrate is temporally distinct from this pathway's earlier role in convergent extension and intercalation. We also reveal polarity in the notochord to be dynamic: a cell's polarity state can be changed and then restored, underscoring the Ciona notochord's amenability for in vivo studies of PCP.

Trophoblast syncytialisation necessitates mitochondrial function through estrogen-related receptor-γ activation.

Human pregnancy needs a correct placentation which depends on adequate cytotrophoblast proliferation, differentiation and invasion. In this study, using specific mitochondrial respiratory chain inhibitors, we observed a decrease of hormone production (hCG and leptin) and cell fusion of human primary villous cytotrophoblasts (CT). These results demonstrated that mitochondria are involved in the control of CT differentiation process. Moreover, we also observed a decrease of mitochondrial mass associated with an increase of mitochondrial DNA during CT differentiation. Furthermore, lactate production increased during CT differentiation suggesting that anaerobic metabolism was enhanced in differentiated CTs, and that the role of mitochondria in CT fusion is not only related to its energetic function. Otherwise, the orphan nuclear receptor, estrogen-related receptor γ (ERRγ) is known to orchestrate transcriptional control of energy metabolism genes. In this study, using RNA knockdown and transcriptional activation with DY131 (an ERRγ agonist), we clearly demonstrated that ERRγ promotes hormone production and cell fusion indicating that ERRγ is a key positive transcriptional factor involved in CT differentiation. Finally, we showed that ERRγ promotes mitochondrial biogenesis and function during CT differentiation, and that the role of ERRγ during trophoblast differentiation is mainly mediated by the control of mitochondrial functions.

[Would D'Arcy Thompson have predicted a topological control of apoptosis?]. / D'Arcy Thompson aurait-il prédit un contrôle topologique de l'apoptose ?

The laws that drive morphogenesis remain a major biological question. Today's views emphasize molecular autonomous processes rather than physical and mechanical constraints proposed by d'Arcy Thompson earlier on. In Ciona intestinalis oocyte, follicular cells formed by two distinct sets of geometrically-ordered epithelial monolayers positioned over the egg control apoptosis, implying that physically-predetermined shapes play a role in the control of cell determinism. In follicular cells ideally positioned over the spherical geometry of the egg, a drastic, optimized and polarized inward apoptosis sequence directly results from this positioning, suggesting the existence of some apoptotic master cells which control the destiny of neighboring cells. This concept could shed a new light on the origin of massive apoptosis phases that take place during embryogenesis in vertebrates (e.g., cavitation, inter-digitation). It could also be applied to specific therapeutic strategies to fight cancer.

Adiponectin regulates glycogen metabolism at the human fetal­maternal interface

Throughout the entire first trimester of pregnancy, fetal growth is sustained by endometrial secretions, i.e. histiotrophic nutrition. Endometrial stromal cells (EnSCs) accumulate and secrete a variety of nutritive molecules that are absorbed by trophoblastic cells and transmitted to the fetus. Glycogen appears to have a critical role in the early stages of fetal development, since infertile women have low endometrial glycogen levels. However, the molecular mechanisms underlying glycogen metabolism and trafficking at the fetal­maternal interface have not yet been characterized. Among the various factors acting at the fetal­maternal interface, we focused on adiponectin ­ an adipocyte-secreted cytokine involved in the control of carbohydrate and lipid homeostasis. Our results clearly demonstrated that adiponectin controls glycogen metabolism in EnSCs by (i) increasing glucose transporter 1 expression, (ii) inhibiting glucose catabolism via a decrease in lactate and ATP productions, (iii) increasing glycogen synthesis, (iv) promoting glycogen accumulation via phosphoinositide-3 kinase activation and (v) enhancing glycogen secretion. Furthermore, our results revealed that adiponectin significantly limits glycogen endocytosis by human villous trophoblasts. Lastly, we demonstrated that once glycogen has been endocytosed into placental cells, it is degraded into glucose molecules in lysosomes. Taken as a whole, the present results demonstrate that adiponectin exerts a dual role at the fetal­maternal interface by promoting glycogen synthesis in the endometrium and conversely reducing trophoblastic glycogen uptake. We conclude that adiponectin may be involved in feeding the conceptus during the first trimester of pregnancy by controlling glycogen metabolism in both the uterus and the placenta.

A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage.

The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology.

Influence of pDNA availability on transfection efficiency of polyplexes in non-proliferative cells.

We succeeded in visualizing plasmid DNA (pDNA) in the nucleus and cytosol of non-proliferative cells after transfection with linear polyethylenemine (lPEI) and histidinylated lPEI (His16-lPEI). This was possible with confocal microscope by using pDNA labelled with quantum dots. Indeed pDNA labelled with Cy3 leads to false positive nuclear localization because the saturation of the fluorescence signal overestimated the volume occupied by Cy3-pDNA. Moreover, Cy3 brightness was too weak to detect low amount of pDNA. About 20 to 40 pDNA copies were detected in the nucleus after the transfection of pDNA labelled with quantum dots. Transfection efficiency and cellular imaging data suggested that the cytosolic availability of pDNA, including endosome escape and/or polyplexes dissociation, is crucial for its nuclear delivery. In vitro transcription assay and transfection of cells allowing cytosolic gene expression concluded to better cytosolic availability of pDNA within His16-lPEI polyplexes. Cryo-TEM analyses revealed that His16-lPEI polyplexes exhibited a spherical shape and an amorphous internal structure which differed from the high degree of order of lPEI polyplexes. Altogether, this comparative study indicated that the high transfection efficiency of non-proliferative cells with His16-lPEI polyplexes was related to the amorphous structure and the facilitated dissociation of the assemblies.

Methods toward in vivo measurement of zebrafish epithelial and deep cell proliferation.

We present a strategy for automatic classification and density estimation of epithelial enveloping layer (EVL) and deep layer (DEL) cells, throughout zebrafish early embryonic stages. Automatic cells classification provides the bases to measure the variability of relevant parameters, such as cells density, in different classes of cells and is finalized to the estimation of effectiveness and selectivity of anticancer drug in vivo. We aim at approaching these measurements through epithelial/deep cells classification, epithelial area and thickness measurement, and density estimation from scattered points. Our procedure is based on Minimal Surfaces, Otsu clustering, Delaunay Triangulation, and Within-R cloud of points density estimation approaches. In this paper, we investigated whether the distance between nuclei and epithelial surface is sufficient to discriminate epithelial cells from deep cells. Comparisons of different density estimators, experimental results, and extensively accuracy measurements are included.

Topological control of life and death in non-proliferative epithelia.

Programmed cell death is one of the most fascinating demonstrations of the plasticity of biological systems. It is classically described to act upstream of and govern major developmental patterning processes (e.g. inter-digitations in vertebrates, ommatidia in Drosophila). We show here the first evidence that massive apoptosis can also be controlled and coordinated by a pre-established pattern of a specific 'master cell' population. This new concept is supported by the development and validation of an original model of cell patterning. Ciona intestinalis eggs are surrounded by a three-layered follicular organization composed of 60 elongated floating extensions made of as many outer and inner cells, and indirectly spread through an extracellular matrix over 1200 test cells. Experimental and selective ablation of outer and inner cells results in the abrogation of apoptosis in respective remaining neighbouring test cells. In addition incubation of outer/inner follicular cell-depleted eggs with a soluble extract of apoptotic outer/inner cells partially restores apoptosis to apoptotic-defective test cells. The 60 inner follicular cells were thus identified as 'apoptotic master' cells which collectively are induction sites for programmed cell death of the underlying test cells. The position of apoptotic master cells is controlled by topological constraints exhibiting a tetrahedral symmetry, and each cell spreads over and can control the destiny of 20 smaller test cells, which leads to optimized apoptosis signalling.

Fertilization regulates apoptosis of Ciona intestinalis extra-embryonic cells through thyroxine (T4)-dependent NF-kappaB pathway activation during early embryonic development.

In Ciona intestinalis, the elimination of extra-embryonic test cells during early stage of development is delayed by a fertilization signal. Test cells undergo a caspase-dependent apoptosis event repressed by thyroxine (T4)-activated NF-kappaB. When apoptosis was experimentally blocked, the hatching stage was delayed. The incubation of unfertilized eggs with a 1-h-fertilized egg extract or purified T4 restored apoptosis in test cells at a similar timing than found in fertilized eggs. Ciona expresses specific genes forming a functional IkappaB/NF-kappaB pathway. One, Ci-p65, was transiently induced upon fertilization via T4 and found to exert its anti-apoptotic role in test cells nuclei as well as in a reconstituted cell system. Blocking NF-kappaB activity by dexamethasone-induced overexpression of Ci-IkappaB abrogated the repression of apoptosis in test cells. Overall, the data are consistent for defining a central coupling role of both T4 and NF-kappaB during early embryo development.