ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis.

During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.

Epithelial dynamics during early mouse development.

The first epithelia to arise in an organism face the challenge of maintaining the integrity of the newly formed tissue, while exhibiting the behavioral flexibility required for morphogenetic processes to occur effectively. Epithelial cells integrate biochemical and biomechanical cues, both intrinsic and extrinsic, in order to bring about the molecular changes which determine their morphology, behavior and fate. In this review we highlight recent advances in our understanding of the various dynamic processes that the emergent epithelial cells undergo during the first seven days of mouse development and speculate what the future holds in understanding the mechanistic bases for these processes through integrative approaches.

Cell competition acts as a purifying selection to eliminate cells with mitochondrial defects during early mouse development.

Cell competition is emerging as a quality-control mechanism that eliminates unfit cells in a wide range of settings from development to the adult. However, the nature of the cells normally eliminated by cell competition and what triggers their elimination remains poorly understood. In mice, 35% of epiblast cells are eliminated before gastrulation. Here we show that cells with mitochondrial defects are eliminated by cell competition during early mouse development. Using single-cell transcriptional profiling of eliminated mouse epiblast cells, we identify hallmarks of cell competition and mitochondrial defects. We demonstrate that mitochondrial defects are common to a range of different loser cell types and that manipulating mitochondrial function triggers cell competition. Moreover, we show that in the mouse embryo, cell competition eliminates cells with sequence changes in mt-Rnr1 and mt-Rnr2, and that even non-pathological changes in mitochondrial DNA sequences can induce cell competition. Our results suggest that cell competition is a purifying selection that optimizes mitochondrial performance before gastrulation.

Pituitary hyperplasia with Sertoli cell-only and 47,XYY syndromes: an uncommon triad.

We report the case history of a 32-year-old man with no phenotypical abnormalities who presented with infertility. Semen analysis revealed azoospermia and testicular biopsy confirmed Sertoli cell-only (SCO) syndrome. Karyotyping revealed 47,XYY and pituitary hyperplasia was found on MRI pituitary. In our patient, 47,XYY karyotype is likely to have given rise to SCO syndrome that in turn resulted in pituitary hyperplasia. The patient was evaluated by various members of the multidisciplinary team including the pituitary surgeon, endocrinologist and andrologist. The patient's partner successfully delivered a healthy baby via in vitro fertilisation with donor sperm. This triad of diagnoses (SCO syndrome, 47,XYY karyotype and pituitary hyperplasia) has not been reported previously. SCO syndrome should be considered in the presence of azoospermia, elevated follicle-stimulating hormone, low inhibin-B and normal testosterone levels. Our case report also highlights the importance of excluding genetic causes of infertility even when the patient has no phenotypical abnormalities.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay.

Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.

Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development.

Oncogenic PIK3CA induces centrosome amplification and tolerance to genome doubling.

Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca H1047R hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA H1047R mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution.

Vps34 PI 3-kinase inactivation enhances insulin sensitivity through reprogramming of mitochondrial metabolism.

Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.

ASPP2 links the apical lateral polarity complex to the regulation of YAP activity in epithelial cells.

The Hippo pathway, by tightly controlling the phosphorylation state and activity of the transcription cofactors YAP and TAZ is essential during development and tissue homeostasis whereas its deregulation may lead to cancer. Recent studies have linked the apicobasal polarity machinery in epithelial cells to components of the Hippo pathway and YAP and TAZ themselves. However the molecular mechanism by which the junctional pool of YAP proteins is released and activated in epithelial cells remains unknown. Here we report that the tumour suppressor ASPP2 forms an apical-lateral polarity complex at the level of tight junctions in polarised epithelial cells, acting as a scaffold for protein phosphatase 1 (PP1) and junctional YAP via dedicated binding domains. ASPP2 thereby directly induces the dephosphorylation and activation of junctional YAP. Collectively, this study unearths a novel mechanistic paradigm revealing the critical role of the apical-lateral polarity complex in activating this localised pool of YAP in vitro, in epithelial cells, and in vivo, in the murine colonic epithelium. We propose that this mechanism may commonly control YAP functions in epithelial tissues.

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells.

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.

Do the effects of testosterone on muscle strength, physical function, body composition, and quality of life persist six months after treatment in intermediate-frail and frail elderly men?

CONTEXT: Short-term testosterone (T) treatment in frail elderly men improves muscle mass and strength. It is unclear whether these effects can be maintained post treatment. OBJECTIVE: To assess the durability of androgen effects in frail men. DESIGN AND SETTING: Single center, randomized, double-blind, placebo-controlled trial to investigate the effects of 6 months T (25-75 mg daily) on muscle strength, body composition, physical function, and quality of life (QoL). Participants were assessed at the end of treatment (6 months) and 6 months after treatment cessation (12 months). PARTICIPANTS: 274 intermediate-frail and frail elderly men aged 65-90 years with low T levels. RESULTS: Mean T increased from 11.1 (3.1) nmol/liter at baseline to 18.4 (3.5) nmol/liter at 6 months, then declined to 10.5 (3.7) nmol/L at 12 months, in the T-treated group. Isometric knee extension peak torque increased in the T-treated group compared with placebo to give an adjusted mean difference (95% CI) between groups of 8.1 (-0.2 to 16.5) Nm at 6 months. Lean mass increased in the T-treated group giving a difference between groups of 1.2 (0.8 to 1.7) kg at 6 months. Somatic and sexual symptoms improved during treatment. None of these differences between groups remained at 12 months. Prostate specific antigen (PSA) levels and haematocrit increased slightly during treatment but returned to baseline by 12 months. CONCLUSION: The effects of 6-month T treatment on muscle strength, lean mass, and QoL in frail men are not maintained at 6 months post treatment.

Management of adrenal incidentaloma: size still matters.

A 56-year-old man was found to have an adrenal incidentaloma on a CT scan of the abdomen. Clinically and biochemically, the mass was not functional. MRI scan revealed a heterogeneously enhancing, T2-hyperintense, right-sided adrenal mass (4.5×6.5 cm). Meta-iodo-benzylguanidine scan was normal, making a diagnosis of pheochromocytoma unlikely. As the mass was larger that 4 cm, it was excised and histopathological examination revealed a rare, composite tumour: benign adrenal adenoma with haemangiomatous and myelolipomatous components. This case highlights the difficulties encountered by a clinician faced with investigating a potentially malignant adrenal mass (based on size) and correlates radiological findings with a rare histopathological specimen.

Use of the viral 2A peptide for bicistronic expression in transgenic mice.

BACKGROUND: Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner. RESULTS: To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line. CONCLUSION: The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site.