SOX9 Governs Gastric Mucous Neck Cell Identity and Is Required for Injury-Induced Metaplasia.

BACKGROUND & AIMS: Acute and chronic gastric injury induces alterations in differentiation within the corpus of the stomach called pyloric metaplasia. Pyloric metaplasia is characterized by the death of parietal cells and reprogramming of mitotically quiescent zymogenic chief cells into proliferative, mucin-rich spasmolytic polypeptide-expressing metaplasia (SPEM) cells. Overall, pyloric metaplastic units show increased proliferation and specific expansion of mucous lineages, both by proliferation of normal mucous neck cells and recruitment of SPEM cells. Here, we identify Sox9 as a potential gene of interest in the regulation of mucous neck and SPEM cell identity in the stomach. METHODS: We used immunostaining and electron microscopy to characterize the expression pattern of SRY-box transcription factor 9 (SOX9) during murine gastric development, homeostasis, and injury in homeostasis, after genetic deletion of Sox9 and after targeted genetic misexpression of Sox9 in the gastric epithelium and chief cells. RESULTS: SOX9 is expressed in all early gastric progenitors and strongly expressed in mature mucous neck cells with minor expression in the other principal gastric lineages during adult homeostasis. After injury, strong SOX9 expression was induced in the neck and base of corpus units in SPEM cells. Adult corpus units derived from Sox9-deficient gastric progenitors lacked normal mucous neck cells. Misexpression of Sox9 during postnatal development and adult homeostasis expanded mucous gene expression throughout corpus units including within the chief cell zone in the base. Sox9 deletion specifically in chief cells blunts their reprogramming into SPEM. CONCLUSIONS: Sox9 is a master regulator of mucous neck cell differentiation during gastric development. Sox9 also is required for chief cells to fully reprogram into SPEM after injury.

Fat and Dachsous cadherins in mammalian development.

Cell growth and patterning are critical for tissue development. Here we discuss the evolutionarily conserved cadherins, Fat and Dachsous, and the roles they play during mammalian tissue development and disease. In Drosophila, Fat and Dachsous regulate tissue growth via the Hippo pathway and planar cell polarity (PCP). The Drosophila wing has been an ideal tissue to observe how mutations in these cadherins affect tissue development. In mammals, there are multiple Fat and Dachsous cadherins, which are expressed in many tissues, but mutations in these cadherins that affect growth and tissue organization are context dependent. Here we examine how mutations in the Fat and Dachsous mammalian genes affect development in mammals and contribute to human disease.

MnFe2O4@SiO2@CeO2 core-shell nanostructures for applications in water remediation.

Removal of dye pollutants from wastewater is among the most important emerging needs in environmental science and engineering. The main objective of our work is to develop new magnetic core-shell nanostructures and explore their use for potential removal of pollutants from water using an external magnetic field. Herein, we have prepared magnetic core-shell nanoparticles that demonstrated excellent dye pollutant adsorbent properties. These nanoparticles are composed of a manganese ferrite magnetic core coated with silica, to protect the core and enable further functionalisation, then finally coated with ceria, which is shown to be an effective adsorbent. The magnetic core-shell nanostructures have been synthesized by a modification of solvothermal synthesis. The nanoparticles were fully characterised at each stage of the synthesis by powder X-ray diffraction (pXRD), transmission electron microscopy (TEM), vibrating sample magnetometry (VSM) and Fourier transform infrared spectroscopy (FTIR). These particles were found to be effective in removing methylene blue (MB) dye from water, which was validated by UV-visible (UV-vis) spectroscopy. These particles can be quickly removed from solution using a permanent magnet and then can be recycled after being placed in the furnace at 400 °C to burn off any organic residues. The particles were found to retain their ability to adsorb the pollutant after several cycles and TEM images of the particles after several cycles showed no change in the morphology. This research demonstrated the capacity of magnetic core-shell nanostructures to be used for water remediation.

Expanded directly binds conserved regions of Fat to restrain growth via the Hippo pathway.

The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. Here, we show that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. We show that direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, we find Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth.

Transgenic force sensors and software to measure force transmission across the mammalian nuclear envelope in vivo.

Nuclear mechanotransduction is a growing field with exciting implications for the regulation of gene expression and cellular function. Mechanical signals may be transduced to the nuclear interior biochemically or physically through connections between the cell surface and chromatin. To define mechanical stresses upon the nucleus in physiological settings, we generated transgenic mouse strains that harbour FRET-based tension sensors or control constructs in the outer and inner aspects of the nuclear envelope. We knocked-in a published esprin-2G sensor to measure tensions across the LINC complex and generated a new sensor that links the inner nuclear membrane to chromatin. To mitigate challenges inherent to fluorescence lifetime analysis in vivo, we developed software (FLIMvivo) that markedly improves the fitting of fluorescence decay curves. In the mouse embryo, the sensors responded to cytoskeletal relaxation and stretch applied by micro-aspiration. They reported organ-specific differences and a spatiotemporal tension gradient along the proximodistal axis of the limb bud, raising the possibility that mechanical mechanisms coregulate pattern formation. These mouse strains and software are potentially valuable tools for testing and refining mechanotransduction hypotheses in vivo.

The inner nuclear membrane protein NEMP1 supports nuclear envelope openings and enucleation of erythroblasts.

Nuclear envelope membrane proteins (NEMPs) are a conserved family of nuclear envelope (NE) proteins that reside within the inner nuclear membrane (INM). Even though Nemp1 knockout (KO) mice are overtly normal, they display a pronounced splenomegaly. This phenotype and recent reports describing a requirement for NE openings during erythroblasts terminal maturation led us to examine a potential role for Nemp1 in erythropoiesis. Here, we report that Nemp1 KO mice show peripheral blood defects, anemia in neonates, ineffective erythropoiesis, splenomegaly, and stress erythropoiesis. The erythroid lineage of Nemp1 KO mice is overrepresented until the pronounced apoptosis of polychromatophilic erythroblasts. We show that NEMP1 localizes to the NE of erythroblasts and their progenitors. Mechanistically, we discovered that NEMP1 accumulates into aggregates that localize near or at the edge of NE openings and Nemp1 deficiency leads to a marked decrease of both NE openings and ensuing enucleation. Together, our results for the first time demonstrate that NEMP1 is essential for NE openings and erythropoietic maturation in vivo and provide the first mouse model of defective erythropoiesis directly linked to the loss of an INM protein.

The Hippo pathway regulates axis formation and morphogenesis in Hydra.

How did cells of early metazoan organisms first organize themselves to form a body axis? The canonical Wnt pathway has been shown to be sufficient for induction of axis in Cnidaria, a sister group to Bilateria, and is important in bilaterian axis formation. Here, we provide experimental evidence that in cnidarian Hydra the Hippo pathway regulates the formation of a new axis during budding upstream of the Wnt pathway. The transcriptional target of the Hippo pathway, the transcriptional coactivator YAP, inhibits the initiation of budding in Hydra and is regulated by Hydra LATS. In addition, we show functions of the Hippo pathway in regulation of actin organization and cell proliferation in Hydra. We hypothesize that the Hippo pathway served as a link between continuous cell division, cell density, and axis formation early in metazoan evolution.

Postnatal expression profiles of atypical cadherin FAT1 suggest its role in autism.

Genetic studies have linked FAT1 (FAT atypical cadherin 1) with autism spectrum disorder (ASD); however, the role that FAT1 plays in ASD remains unknown. In mice, the function of Fat1 has been primarily implicated in embryonic nervous system development with less known about its role in postnatal development. We show for the first time that FAT1 protein is expressed in mouse postnatal brains and is enriched in the cerebellum, where it localizes to granule neurons and Golgi cells in the granule layer, as well as inhibitory neurons in the molecular layer. Furthermore, subcellular characterization revealed FAT1 localization in neurites and soma of granule neurons, as well as being present in the synaptic plasma membrane and postsynaptic densities. Interestingly, FAT1 expression was decreased in induced pluripotent stem cell (iPSC)-derived neural precursor cells (NPCs) from individuals with ASD. These findings suggest a novel role for FAT1 in postnatal development and may be particularly important for cerebellum function. As the cerebellum is one of the vulnerable brain regions in ASD, our study warrants further investigation of FAT1 in the disease etiology.

Correction: The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila.

[This corrects the article DOI: 10.1371/journal.pone.0152259.].

The NEMP family supports metazoan fertility and nuclear envelope stiffness.

Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in NEMP1 (nuclear envelope membrane protein 1) with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in Drosophila, Caenorhabditis elegans, zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line. Biochemical, biophysical, and genetic studies reveal that NEMP proteins support the mechanical stiffness of the germline nuclear envelope via formation of a NEMP-EMERIN complex. These data indicate that the germline nuclear envelope has specialized mechanical properties and that NEMP proteins play essential and conserved roles in fertility.

Ancestral roles of atypical cadherins in planar cell polarity.

Fat, Fat-like, and Dachsous family cadherins are giant proteins that regulate planar cell polarity (PCP) and cell adhesion in bilaterians. Their evolutionary origin can be traced back to prebilaterian species, but their ancestral function(s) are unknown. We identified Fat-like and Dachsous cadherins in Hydra, a member of phylum Cnidaria a sister group of bilaterian. We found Hydra does not possess a true Fat homolog, but has homologs of Fat-like (HyFatl) and Dachsous (HyDs) that localize at the apical membrane of ectodermal epithelial cells and are planar polarized perpendicular to the oral-aboral axis of the animal. Using a knockdown approach we found that HyFatl is involved in local cell alignment and cell-cell adhesion, and that reduction of HyFatl leads to defects in tissue organization in the body column. Overexpression and knockdown experiments indicate that the intracellular domain (ICD) of HyFatl affects actin organization through proline-rich repeats. Thus, planar polarization of Fat-like and Dachsous cadherins has ancient, prebilaterian origins, and Fat-like cadherins have ancient roles in cell adhesion, spindle orientation, and tissue organization.

Hedgehog-Activated Fat4 and PCP Pathways Mediate Mesenchymal Cell Clustering and Villus Formation in Gut Development.

During development, intestinal epithelia undergo dramatic morphogenesis mediated by mesenchymal signaling to form villi, which are required for efficient nutrient absorption and host defense. Although both smooth-muscle-induced physical forces and mesenchymal cell clustering beneath emerging villi are implicated in epithelial folding, the underlying cellular mechanisms are unclear. Hedgehog (Hh) signaling can mediate both processes. We therefore analyzed its direct targetome and revealed GLI2 transcriptional activation of atypical cadherin and planar cell polarity (PCP) genes. By examining Fat4 and Dchs1 knockout mice, we demonstrate their critical roles in villus formation. Analyses of PCP-mutant mice and genetic interaction studies show that the Fat4-Dchs1 axis acts in parallel to the core-Vangl2 PCP axis to control mesenchymal cell clustering. Moreover, live light-sheet fluorescence microscopy and cultured PDGFRα+ cells reveal a requirement for PCP in their oriented cell migration guided by WNT5A. Therefore, mesenchymal PCP induced by Hh signaling drives cell clustering and subsequent epithelial remodeling.

Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow.

The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1) and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease, respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA-binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell-autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome.

Fat/Dachsous family cadherins in cell and tissue organisation.

Precisely controlled organisation at the cellular and tissue level is crucial to establish and maintain complex organisms. The atypical cadherins Fat (Ft), Fat2 and Dachsous (Ds) contribute to this organisation by regulating growth and planar cell polarity. Here we describe the recent advances in understanding how these large cadherins coordinate these processes, and discuss additional progress extending their function in regulation of microtubules, migration and disease.

Localization of YAP activity in developing skeletal rudiments is responsive to mechanical stimulation.

BACKGROUND: Normal skeletal development, in particular ossification, joint formation and shape features of condyles, depends on appropriate mechanical input from embryonic movement but it is unknown how such physical stimuli are transduced to alter gene regulation. Hippo/Yes-Associated Protein (YAP) signalling has been shown to respond to the physical environment of the cell and here we specifically investigate the YAP effector of the pathway as a potential mechanoresponsive mediator in the developing limb skeleton. RESULTS: We show spatial localization of YAP protein and of pathway target gene expression within developing skeletal rudiments where predicted biophysical stimuli patterns and shape are affected in immobilization models, coincident with the period of sensitivity to movement, but not coincident with the expression of the Hippo receptor Fat4. Furthermore, we show that under reduced mechanical stimulation, in immobile, muscle-less mouse embryos, this spatial localization is lost. In culture blocking YAP reduces chondrogenesis but the effect differs depending on the timing and/or level of YAP reduction. CONCLUSIONS: These findings implicate YAP signalling, independent of Fat4, in the transduction of mechanical signals during key stages of skeletal patterning in the developing limb, in particular endochondral ossification and shape emergence, as well as patterning of tissues at the developing synovial joint.

Position effects on promoter activity in Escherichia coli and their consequences for antibiotic-resistance determinants.

The activity of any bacterial promoter is generally supposed to be set by its base sequence and the different transcription factors that bind in the local vicinity. Here, we review recent data indicating that the activity of the Escherichia coli lac operon promoter also depends upon its chromosomal location. Factors that affect promoter activity include the binding of nucleoid-associated proteins to neighbouring sequences, supercoiling and the activity of neighbouring promoters. We suggest that many bacterial promoters might be susceptible to similar position-dependent effects and we review recent data showing that the expression of mobile genes encoding antibiotic-resistance determinants is also location-dependent, both when carried on a bacterial chromosome or a conjugative plasmid.

Four-jointed knock-out delays renal failure in an ADPKD model with kidney injury.

Autosomal Dominant Polycystic Kidney Disease is characterised by the development of fluid-filled cysts in the kidneys which lead to end-stage renal disease (ESRD). In the majority of cases, the disease is caused by a mutation in the Pkd1 gene. In a previous study, we demonstrated that renal injury can accelerate cyst formation in Pkd1 knock-out (KO) mice. In that study, we found that after injury four-jointed (Fjx1), an upstream regulator of planar cell polarity and the Hippo pathway, was aberrantly expressed in Pkd1 KO mice compared to WT. Therefore, we hypothesised a role for Fjx1 in injury/repair and cyst formation. We generated single and double deletion mice for Pkd1 and Fjx1, and we induced toxic renal injury using the nephrotoxic compound 1,2-dichlorovinyl-cysteine. We confirmed that nephrotoxic injury can accelerate cyst formation in Pkd1 mutant mice. This caused Pkd1 KO mice to reach ESRD significantly faster; unexpectedly, double KO mice survived significantly longer. Cyst formation was comparable in both models, but we found significantly less fibrosis and macrophage infiltration in double KO mice. Taken together, these data suggest that Fjx1 disruption protects the cystic kidneys against kidney failure by reducing inflammation and fibrosis. Moreover, we describe, for the first time, an interesting (yet unidentified) mechanism that partially discriminates cyst growth from fibrogenesis. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

FAT4 Fine-Tunes Kidney Development by Regulating RET Signaling.

FAT4 mutations lead to several human diseases that disrupt the normal development of the kidney. However, the underlying mechanism remains elusive. In studying the duplex kidney phenotypes observed upon deletion of Fat4 in mice, we have uncovered an interaction between the atypical cadherin FAT4 and RET, a tyrosine kinase receptor essential for kidney development. Analysis of kidney development in Fat4-/- kidneys revealed abnormal ureteric budding and excessive RET signaling. Removal of one copy of the RET ligand Gdnf rescues Fat4-/- kidney development, supporting the proposal that loss of Fat4 hyperactivates RET signaling. Conditional knockout analyses revealed a non-autonomous role for Fat4 in regulating RET signaling. Mechanistically, we found that FAT4 interacts with RET through extracellular cadherin repeats. Importantly, expression of FAT4 perturbs the assembly of the RET-GFRA1-GDNF complex, reducing RET signaling. Thus, FAT4 interacts with RET to fine-tune RET signaling, establishing a juxtacrine mechanism controlling kidney development.

Fat regulates expression of four-jointed reporters in vivo through a 20 bp element independently of the Hippo pathway.

Development of an organism requires accurate coordination between the growth of a tissue and orientation of cells within the tissue. The large cadherin Fat has been shown to play a role in both of these processes. Fat is involved in the establishment of planar cell polarity and regulates growth through the Hippo pathway, a developmental cascade that controls proliferation and apoptosis. Both Fat and the Hippo pathway are known to regulate transcription of four-jointed, although the nature of this regulation is unknown. In this study, we test whether Fat affects four-jointed transcription via or independently of Hippo pathway. Our analysis of the four-jointed regulatory region reveals a 1.2 kb element that functions as an enhancer for graded expression of Four-jointed in the eye imaginal disc. Within this enhancer element, we identify a 20 bp fragment that is critical for regulation by Fat but not by Hippo. Our findings suggest that Fat and the Hippo pathway control four-jointed expression independently of each other and none of the transcription factors known to function downstream of the Hippo pathway are required to regulate four-jointed expression through the 1.2 kb element.

Homozygous frameshift mutations in FAT1 cause a syndrome characterized by colobomatous-microphthalmia, ptosis, nephropathy and syndactyly.

A failure in optic fissure fusion during development can lead to blinding malformations of the eye. Here, we report a syndrome characterized by facial dysmorphism, colobomatous microphthalmia, ptosis and syndactyly with or without nephropathy, associated with homozygous frameshift mutations in FAT1. We show that Fat1 knockout mice and zebrafish embryos homozygous for truncating fat1a mutations exhibit completely penetrant coloboma, recapitulating the most consistent developmental defect observed in affected individuals. In human retinal pigment epithelium (RPE) cells, the primary site for the fusion of optic fissure margins, FAT1 is localized at earliest cell-cell junctions, consistent with a role in facilitating optic fissure fusion during vertebrate eye development. Our findings establish FAT1 as a gene with pleiotropic effects in human, in that frameshift mutations cause a severe multi-system disorder whereas recessive missense mutations had been previously associated with isolated glomerulotubular nephropathy.