Mesenchyme Cells Research Articles

MicroRNA-31 regulates skeletogenesis by direct suppression of Eve and Wnt1

microRNAs (miRNAs) play a critical role in a variety of biological processes, including embryogenesis and the physiological functions of cells. Evolutionarily conserved microRNA-31 (miR-31) has been found to be involved in cancer, bone formation, and lymphatic development. We previously discovered that, in the sea urchin, miR-31 knockdown (KD) embryos have shortened dorsoventral connecting rods, mispatterned skeletogenic primary mesenchyme cells (PMCs) and shifted and expanded Vegf3 expression domain. Vegf3 itself does not contain miR-31 binding sites; however, we identified its upstream regulators Eve and Wnt1 to be directly suppressed by miR-31. Removal of miR-31’s suppression of Eve and Wnt1 resulted in skeletal and PMC patterning defects, similar to miR-31 KD phenotypes. Additionally, removal of miR-31’s suppression of Eve and Wnt1 results in an expansion and anterior shift in expression of Veg1 ectodermal genes, including Vegf3 in the blastulae. This indicates that miR-31 indirectly regulates Vegf3 expression through directly suppressing Eve and Wnt1. Furthermore, removing miR-31 suppression of Eve is sufficient to cause skeletogenic defects, revealing a novel regulatory role of Eve in skeletogenesis and PMC patterning. Overall, this study provides a proposed molecular mechanism of miR-31’s regulation of skeletogenesis and PMC patterning through its cross-regulation of a Wnt signaling ligand and a transcription factor of the endodermal and ectodermal gene regulatory network.

SPECC1L-deficient primary mouse embryonic palatal mesenchyme cells show speed and directionality defects

Cleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.

Prostate Cancer Differentiation - Stromal Cell PENK and Stem Cell Transcription Factors

Prostate cancer cells from luminal-like (differentiated) adenocarcinoma to less luminal-like and more stem-like (undifferentiated) small cell carcinoma retain responsiveness to signaling by stromal cell factor proenkephalin (PENK). Stem-like cancer cells, like stem cells, express stem cell transcription factor (scTF) LIN28A, NANOG, POU5F1, SOX2 as well as a 10-fold lower level of β2-microglobulin (B2M). Forced expression of this scTF quartet can reprogram adenocarcinoma cells, as represented by LuCaP xenograft lines, to stem-, small cell carcinoma-like derivatives with the phenotypic change from scTF−B2Mhi to scTF+B2Mlo. Prostate stromal mesenchyme cells control organ development through secreted hormone factors and heterotypic cell contact. The highly expressed prostate stromal-specific PENK, when transfected into scTF+B2Mlo small cell carcinoma LuCaP 145.1, caused down-regulation of scTF and up-regulation of B2M to indicate differentiation. When transfected into scTF-reprogrammed adenocarcinoma-like LNCaP cells, PENK reversed the reprogramming through down-regulation of scTF with attendant changes in cell appearance and colony morphology. When transfected into adenocarcinoma LuCaP 70CR, PENK up-regulated the expression of anterior gradient 2 (AGR2), a marker associated with prostate cancer differentiation and better patient survival. Thus, PENK can induce differentiation to counter cancer de-differentiation due to activation of scTF. Loss of cancer differentiation leads to an untreatable disease. Patient survival would benefit if this can be reversed or prevented. Prostate cancer de-differentiation is likened to reprogramming. Cancer cells containing multiple mutations and being aneuploid can, like normal cells, still undergo differentiation.

Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.

PPP3CA silence regulates MET process, cell apoptosis, proliferation and migration in metanephric mesenchyme cells

Kidney is one of the most important organs of the body and the mammalian kidney development is essential for kidney unit formation. The key process of kidney development is metanephric development, where mesenchymal-epithelial transition (MET) plays a crucial role. Here we investigated the biological function of PPP3CA in metanephric mesenchyme (MM) cells. qRT-PCR and Western blotting were used to detect PPP3CA and MET makers expression in mK3, mK4 cells respectively at mRNA and protein level. Subsequently, PPP3CA was stably knocked down via lentivirus infection in mK4 cells. Flow cytometry, EdU/CCK-8 assay, wound healing assay were conducted to clarify the regulation of PPP3CA on cell apoptosis, proliferation and migration respectively. PPP3CA was expressed higher in epithelial-like mK4 cells than mesenchyme-like mK3 cells. Thus, PPP3CA was silenced in mK4 cells and PPP3CA deficiency promoted E-cadherin expression, cell apoptosis. Moreover, PPP3CA knock down attenuated cell proliferation and cell migration in mK4 cell. The underlying mechanism was associated with the dephosphorylation of PPP3CA on ERK1/2. Taken together, our results indicated that PPP3CA mediated MET process and cell behaviors of MM cells, providing new foundation for analyzing potential regulator in kidney development process.

Lrig1 Regulates Epithelial to Mesenchyme Transition (EMT) in Pancreatic Duct Glands (PDG), an Epithelial Stem Cell Compartment Important in Regeneration and Cancer

Lrig1, an epithelial stem cell marker and tumor suppressor, is uniquely expressed within PDGs in pancreas. PDGs are an epithelial progenitor compartment and likely compartment-of-origin for PDAC. Here, in vivo GFP-lineage tagging of Lrig1 expressing PDG cells identify GFP tagged cells in mesenchyme but not in the main duct epithelium, identifying a cell within a PDG epithelial compartment capable of EMT to regenerate its own micro-environment. Mechanisms regulating this EMT switch results from a loss of Lrig1. Characterization of the GFP-positive mesenchyme and PDG cells confirms EMT. ScRNASeq of epithelial spheroids revealed enhanced mesenchymal features in Lrig1 deficient cells, while Lrig1 overexpression abrogates mesenchymal differentiation. In vivo, in vitro and ex vivo studies reveal the Lrig1 loss within this stem cell compartment results in enhanced EGF and TGF-β signaling, expansion of stem cell, proliferative and mesenchymal markers in addition to the up-regulation of a master regulator of stemness and EMT, PSPC1. In vivo murine models of PDAC (KPC) which tag and lose Lrig1 in the PDG compartment results in the expansion of the PDG stem cell compartment which now resembles low-grade PanINs in addition to the formation of an extensive GFP-positive desmoplasia. In vitro, Lrig1 regulates proliferation and EMT in cancer cell lines. These studies identify Lrig1 as a regulator of stemness and EMT in regeneration and cancer.

Heterogeneity and hierarchy of the tissue stem cells in the human newborn vocal fold mucosa.

ObjectivesThere is growing evidence that the cells in the maculae flavae (MFe) are tissue stem cells and the MFe are a stem cell niche of the human vocal fold mucosa. Heterogeneity and hierarchy of tissue stem cells in the MFe of newborn vocal fold were investigated in vivo.Study designHistologic analysis of the human vocal folds.MethodsFive normal human newborn vocal folds were investigated under transmission electron microscopy and light microscopy.ResultsCobblestone‐like polygonal cells, vocal fold stellate cell‐like cells, and fibroblast‐like spindle cells were intermingled in the newborn MFe in vivo, indicating that the cells in the MFe had heterogeneity. However, cobblestone‐like polygonal cells were predominant. Free ribosomes were well developed in the cytoplasm. The cells in some cases formed gap junctions with each other. The cells in some cases were attached to other cells and formed cell junctions with each other. These findings indicated cells in the newborn maculae flavae possessed features of mesenchymal cells (cells in mesenchyme). Colony‐forming‐unit‐like cell aggregate was observed, indicating the cells in the newborn MFe had stemness. The cobblestone‐like polygonal cells expressed SSEA‐3 (a human pluripotent stem cell marker), indicating they were at the top of a cellular hierarchy in the stem cell system.ConclusionsThe cells in the MFe of the human newborn vocal fold mucosa had heterogeneity and hierarchy in the stem cell system in vivo. At birth, newborn maculae flavae are ready to start the growth of the vocal fold mucosa as a vibrating tissue.

Cellular bicarbonate accumulation and vesicular proton transport promote calcification in the sea urchin larva.

The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires [Formula: see text] concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [Formula: see text] mediated by the [Formula: see text] cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.

Association Study of Genetic Variants in Autophagy Pathway and Risk of Non-syndromic Cleft Lip With or Without Cleft Palate.

Although genetic variants in autophagy pathway genes were associated with the risk of oral cancers and early development in embryos, their associations with non-syndromic cleft lip with or without cleft palate (NSCL/P) risk remained unclear. A two-stage case-control study (2,027 NSCL/P cases and 1,843 controls) was performed to investigate the associations between single nucleotide polymorphisms (SNPs) in 23 autophagy pathway genes and NSCL/P susceptibility. The logistic regression model was used to calculate effects of SNPs on NSCL/P susceptibility. Gene-based analysis was performed via the sequence kernel association test (SKAT) and multi-marker analysis of genomic annotation (MAGMA) methods. Expression quantitative trait loci (eQTL) analysis was conducted using NSCL/P lip tissue samples. Gene expression during embryonic development was evaluated using RNA-Seq. Functional roles were explored by luciferase activity assay, cell apoptosis, proliferation, and cycle in vitro. Rs2301104 in HIF1A was significantly associated with NSCL/P susceptibility in the combined analysis (OR: 1.29, 95% CI: 1.09–1.29, P = 3.39 × 10–03), and showed strong evidence of association heterogeneity (P = 9.06 × 10–03) with obvious association in the female (OR: 1.80; 95% CI: 1.32–2.45; P = 1.79 × 10–04). The G allele of rs2301104 was associated with enhanced transcription activity and high expression of HIF1A compared with that of C allele. Moreover, rs2301104 exhibited an eQTL effect for HIF1A with its GC/CC genotypes associated with decreased HIF1A expression compared with those with GG genotypes (P = 3.1 × 10–2). Knockdown of HIF1A induced cell apoptosis and inhibited cell proliferation in human embryonic palate mesenchyme (HEPM) and human oral epithelium cells (HOEC). This study demonstrated that rs2301104 in autophagy pathway gene HIF1A was associated with susceptibility of NSCL/P.

Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

ABSTRACTOsteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.

A sticky problem for digits

Development Cell death during development is important for finger and toe separation, but additional developmental programs are also required for proper digitation. Kashgari et al. show that, after the death of the mesenchyme cells from which digits form, a structure called the interdigital epithelial tongue invaginates into the mesenchyme, allowing the digits to bifurcate. In normal digit formation, the outermost layer of the embryonic epidermis, the periderm, prevents digit adhesion. However, after the digits separate, they sometimes fuse back together, causing a condition known as syndactyly. This occurs when the transcription factor grainyhead-like 3 (GRHL3) is lacking, which results in the usually nonsticky periderm becoming sticky. Dev. Cell. 52 , 764 (2020).

Regulation of TAZ Expression by Cyclic Stretch

It is increasingly recognized that fibrosis‐related phenotype transitions (e.g. EMT and fibrobrolast‐myofibroblast transition) are brought about by the interplay between chemical (e.g. TGFβ) and mechanical (e.g. stretch) stimuli, which, in part, act through cytoskeleton‐regulated transcription factors. Among these, the Hippo effector TAZ and the Rho/actin‐regulated myocardin‐related transcription factor (MRTF) are TGFβ‐ and mechano‐sensitive transcriptional co‐activators, which play key roles in the pathogenesis of organ fibrosis. Previously we have shown a multilevel crosstalk between TAZ and MRTF. Moreover, we found that TGFβ is a strong inducer of TAZ protein and mRNA expression, an effect mediated by p38‐dependent MRTF activation, independent of a net nuclear translocation of MRTF or TAZ. These results revealed that, in addition to TAZ nuclear translocation, the regulation of TAZ expression (a hitherto poorly characterized process) is also a critical feature of fibrogenesis. Since mechanical stimuli are prominent inducers of fibrosis, we sought to determine if cyclic stretch impacts TAZ expression and to investigate the potential role of the RhoGTPase/MRTF signaling pathway in this process. Cyclic stretch (10%, 1 Hz) increased MRTF‐transcriptional activity, measured by a CArG box‐ (cis‐element for MRTF) containing luciferase reporter construct, between 3 and 6 hr in C3H10T1/2 cells. Overexpression of Rho family small GTPases (RhoA, Rac1 and Cdc42) known to be activated by stretch and to induce MRTF translocation, were sufficient to enhance the activity of a TAZ promoter luciferase reporter (TAZ‐Luc). Consistently, cyclic stretch increased TAZ protein and mRNA expression, and activated TAZ‐Luc. TAZ‐Luc activation was abolished by MRTF inhibition or the mutation of the CArG box within the reporter (mtTAZ‐Luc). The stretch‐induced activation of TAZ expression was a universal phenomenon, since similar effects were observed in Interstitial Rat Mesenchyme Cells (IRMC) and Human Pulmonary Microvascular Endothelial Cells (HPMEC) as well. Finally, since the redox‐regulator pirin was recently suggested to be an upstream regulator of MRTF, we checked its potential involvement in the regulation of TAZ expression. While CCT‐251236, a pirin‐binding compound abolished the TGFβ‐induced TAZ expression, pirin silencing failed to recapitulate this effect. This suggests that the drug has alternative targets and MRTF can be activated in a pirin‐independent manner. Taken together, our studies show that critical chemical and mechanical fibrogenic stimuli increase TAZ expression and MRTF is a key mediator of this effect, both via classic (Rho‐mediated, translocation‐dependent) and by “non‐canonical” (phosphorylation‐induced activation) mechanisms. We propose that the ensuing rise in TAZ expression potently accelerates and exacerbates fibrogenic phenotype transitions.Support or Funding InformationKidney Foundation of Canada and Natural Sciences and Engineering Research Council of Canada (AK)

Pou3f4-expressing otic mesenchyme cells promote spiral ganglion neuron survival in the postnatal mouse cochlea.

During inner ear development, primary auditory neurons named spiral ganglion neurons (SGNs) are surrounded by otic mesenchyme cells, which express the transcription factor Pou3f4. Mutations in Pou3f4 are associated with DFNX2, the most common form of X-linked deafness and typically include developmental malformations of the middle ear and inner ear. It is known that interactions between Pou3f4-expressing mesenchyme cells and SGNs are important for proper axon bundling during development. However, Pou3f4 continues to be expressed through later phases of development, and potential interactions between Pou3f4 and SGNs during this period had not been explored. To address this, we documented Pou3f4 protein expression in the early postnatal mouse cochlea and compared SGNs in Pou3f4 knockout mice and littermate controls. In Pou3f4y/- mice, SGN density begins to decline by the end of the first postnatal week, with approximately 25% of SGNs ultimately lost. This period of SGN loss in Pou3f4y/- cochleae coincides with significant elevations in SGN apoptosis. Interestingly, this period also coincides with the presence of a transient population of Pou3f4-expressing cells around and within the spiral ganglion. To determine if Pou3f4 is normally required for SGN peripheral axon extension into the sensory domain, we used a genetic sparse labeling approach to track SGNs and found no differences compared with controls. We also found that Pou3f4 loss did not lead to changes in the proportions of Type I SGN subtypes. Overall, these data suggest that otic mesenchyme cells may play a role in maintaining SGN populations during the early postnatal period.

Putative stem cells in the hemolymph and in the intestinal submucosa of the solitary ascidian Styela plicata

BackgroundIn various ascidian species, circulating stem cells have been documented to be involved in asexual reproduction and whole-body regeneration. Studies of these cell population(s) are mainly restricted to colonial species. Here, we investigate the occurrence of circulating stem cells in the solitary Styela plicata, a member of the Styelidae, a family with at least two independent origins of coloniality.ResultsUsing flow cytometry, we characterized a population of circulating putative stem cells (CPSCs) in S. plicata and determined two gates likely enriched with CPSCs based on morphology and aldehyde dehydrogenase (ALDH) activity. We found an ALDH + cell population with low granularity, suggesting a stem-like state. In an attempt to uncover putative CPSCs niches in S. plicata, we performed a histological survey for hemoblast-like cells, followed by immunohistochemistry with stem cell and proliferation markers. The intestinal submucosa (IS) showed high cellular proliferation levels and high frequency of undifferentiated cells and histological and ultrastructural analyses revealed the presence of hemoblast aggregations in the IS suggesting a possible niche. Finally, we document the first ontogenetic appearance of distinct metamorphic circulatory mesenchyme cells, which precedes the emergence of juvenile hemocytes.ConclusionsWe find CPSCs in the hemolymph of the solitary ascidian Styela plicata, presumably involved in the regenerative capacity of this species. The presence of proliferating and undifferentiated mesenchymal cells suggests IS as a possible niche.

Nubp2 is required for cranial neural crest survival in the mouse

The N-ethyl-N-nitrosourea (ENU) ←forward genetic screen is a useful tool for the unbiased discovery of novel mechanisms regulating developmental processes. We recovered the dorothy mutation in such a screen designed to recover recessive mutations affecting craniofacial development in the mouse. Dorothy embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies, including a severe midfacial clefting phenotype. We used exome sequencing to discover a missense mutation in nucleotide binding protein 2 (Nubp2) to be causative. This finding was confirmed by a complementation assay with the dorothy allele and an independent Nubp2 null allele (Nubp2null). We demonstrated that Nubp2 is indispensable for embryogenesis. NUBP2 is implicated in both the cytosolic iron/sulfur cluster assembly pathway and negative regulation of ciliogenesis. Conditional ablation of Nubp2 in the neural crest lineage with Wnt1-cre recapitulates the dorothy craniofacial phenotype. Using this model, we found that the proportion of ciliated cells in the craniofacial mesenchyme was unchanged, and that markers of the SHH, FGF, and BMP signaling pathways are unaltered. Finally, we show evidence that the phenotype results from a marked increase in apoptosis within the craniofacial mesenchyme.

Effects of circular RNA circ-SOD2 on intestinal epithelial barrier and ulcerative colitis

OBJECTIVE To explore the expression profiling of circRNAs in ulcerative colitis(UC) and then determine the significantly changed circRNA and its influences on intestinal epithelial barrier. METHODS In this study, we selected 5 pairs of inflamed and normal colorectal mucosa tissues from UC patients to perform circRNAs microarray and identified the differentially expressed circRNAs in the UC inflamed colorectal mucosa tissues, and quantitative real-time PCR was used to identify the expression change of circ-SOD2 in 30 UC patients' inflamed and normal colorectal mucosa tissues. We detected the expression of circ-SOD2 in Caco2 and NCM460 cells after being treated with inflammatory factors (LPS, TNF-α, IL1-β). Fluorescence in situ hybridization (FISH) was used to determine the cellular location of circ-SOD2 in the UC colorectal mucosal tissues. The circ-SOD2 overexpression vector was constructed and produced and then transfected into Caco2 cells to examine the cells' trans-epithelial electrical resistance (TEER), permeability of FITC-dextran and the alterations of epithelial barrier related molecules. RESULTS We found 264 circRNAs (111 increased and 153 decreased) differentially expressed in the inflamed colon mucosa compared with normal colon mucosa using a P-value 1.5-fold change cutoff. To validate the circRNA microarray results, we selected some circRNAs to perform qRT-PCR based on the following criteria: (1) circRNAs raw data >100 in each sample, (2) fold-change >2, (3) P<0.05. We identified 10 dysregulated circRNA, among them, circ-SOD2 was upregulated with maximum fold-change in the UC inflamed colorectal mucosa tissues. Then we identified circ-SOD2 was upregulated significantly through quantitative real-time PCR (qRT-PCR) in expanded 30 paired colorectal mucosa tissues(P<0.001). After treatments with LPS, TNF-α and IL1-β, circ-SOD2 was upregulated in Caco2 and NCM460 cells at different points from 1 to 7 h. Fluorescence in situ hybridization (FISH) indicated that circ-SOD2 located in intestinal epithelium mostly and few in mesenchyme and inflammatory cells. The overexpression of circ-SOD2 in Caco2 cells resulted in a decrease of transepithelial electrical resistance (TEER), an increase of the FITC-dextran permeability and the downregulation of epithelial barrier related molecule CLDN-8 (P<0.05). CONCLUSION The dysregulation of circRNAs existed in UC inflamed colorectal mucosa, among which, the upregulated circ-SOD2 weakened the intestinal epithelial barrier and thus might promote the occurrence of ulcerative colitis.

Gastrulation in the sea urchin.

Gastrulation is arguably the most important evolutionary innovation in the animal kingdom. This process provides the basic embryonic architecture, an inner layer separated from an outer layer, from which all animal forms arise. An extraordinarily simple and elegant process of gastrulation is observed in the sea urchin embryo. The cells participating in sea urchin gastrulation are specified early during cleavage. One outcome of that specification is the expression of transcription factors that control each of the many subsequent morphogenetic changes. The first of these movements is an epithelial-mesenchymal transition (EMT) of skeletogenic mesenchyme cells, then EMT of pigment cell progenitors. Shortly thereafter, invagination of the archenteron occurs. At the end of archenteron extension, a second wave of EMT occurs to release immune cells into the blastocoel and primordial germ cells that will home to the coelomic pouches. The archenteron then remodels to establish the three parts of the gut, and at the anterior end, the gut fuses with the stomodaeum to form the through-gut. As part of the anterior remodeling, mesodermal coelomic pouches bud off the lateral sides of the archenteron tip. Multiple cell biological processes conduct each of these movements and in some cases the upstream transcription factors controlling this process have been identified. Remarkably, each event seamlessly occurs at the right time to orchestrate formation of the primitive body plan. This review covers progress toward understanding many of the molecular mechanisms underlying this sequence of morphogenetic events.

The evolution of a new cell type was associated with competition for a signaling ligand.

There is presently a very limited understanding of the mechanisms that underlie the evolution of new cell types. The skeleton-forming primary mesenchyme cells (PMCs) of euechinoid sea urchins, derived from the micromeres of the 16-cell embryo, are an example of a recently evolved cell type. All adult echinoderms have a calcite-based endoskeleton, a synapomorphy of the Ambulacraria. Only euechinoids have a micromere-PMC lineage, however, which evolved through the co-option of the adult skeletogenic program into the embryo. During normal development, PMCs alone secrete the embryonic skeleton. Other mesoderm cells, known as blastocoelar cells (BCs), have the potential to produce a skeleton, but a PMC-derived signal ordinarily prevents these cells from expressing a skeletogenic fate and directs them into an alternative developmental pathway. Recently, it was shown that vascular endothelial growth factor (VEGF) signaling plays an important role in PMC differentiation and is part of a conserved program of skeletogenesis among echinoderms. Here, we report that VEGF signaling, acting through ectoderm-derived VEGF3 and its cognate receptor, VEGF receptor (VEGFR)-10-Ig, is also essential for the deployment of the skeletogenic program in BCs. This VEGF-dependent program includes the activation of aristaless-like homeobox 1 (alx1), a conserved transcriptional regulator of skeletogenic specification across echinoderms and an example of a "terminal selector" gene that controls cell identity. We show that PMCs control BC fate by sequestering VEGF3, thereby preventing activation of alx1 and the downstream skeletogenic network in BCs. Our findings provide an example of the regulation of early embryonic cell fates by direct competition for a secreted signaling ligand, a developmental mechanism that has not been widely recognized. Moreover, they reveal that a novel cell type evolved by outcompeting other embryonic cell lineages for an essential signaling ligand that regulates the expression of a gene controlling cell identity.

Requirement of Hyaluronan Synthase-2 in Craniofacial and Palate Development

Cleft palate is a common major birth defect resulting from disruption of palatal shelf growth, elevation, or fusion during fetal palatogenesis. Whereas the molecular mechanism controlling palatal shelf elevation is not well understood, a prevailing hypothesis is that region-specific accumulation of hyaluronan, a predominant extracellular glycosaminoglycan in developing palatal mesenchyme, plays a major role in palatal shelf elevation. However, direct genetic evidence for a requirement of hyaluronan in palate development is still lacking. In this study, we show that Has2, 1 of 3 hyaluronan synthases in mammals, plays a major role in hyaluronan synthesis in the neural crest–derived craniofacial mesenchyme during palatogenesis in mice. We analyzed developmental defects caused by tissue-specific inactivation of Has2 throughout the cranial neural crest lineage or specifically in developing palatal or mandibular mesenchyme, respectively, using Wnt1-Cre, Osr2-Cre, and Hand2-Cre transgenic mice. Inactivation of Has2 either throughout the neural crest lineage or specifically in the developing palatal mesenchyme caused reduced palatal shelf size and increased palatal mesenchyme cell density prior to the time of normal palatal shelf elevation. Whereas both Has2f/f;Wnt1-Cre and Has2f/f;Osr2-Cre mutant mice exhibit cleft palate at complete penetrance, the Has2f/f; Wnt1-Cre fetuses showed dramatically reduced mandible size and complete failure of palatal shelf elevation, whereas Has2f/f;Osr2-Cre fetuses had normal mandibles and delayed palatal shelf elevation. All Has2f/f;Hand2-Cre pups showed reduced mandible size and about 50% of them had cleft palate with disruption of palatal shelf elevation. Results from explant culture assays indicate that disruption of palatal shelf elevation in Has2f/f;Hand2-Cre mutant fetuses resulted from physical obstruction by the malformed mandible and tongue. Together, these data indicate that hyaluronan plays a crucial intrinsic role in palatal shelf expansion and timely reorientation to the horizontal position above the tongue as well as an important role in mandibular morphogenesis that secondarily affects palatal shelf elevation.

Mouse embryonic palatal mesenchymal cells maintain stemness through the PTEN-Akt-mTOR autophagic pathway

BackgroundBoth genetic and environmental factors are implicated in the pathogenesis of cleft palate. However, the molecular and cellular mechanisms that regulate the development of palatal shelves, which are composed of mesenchymal cells, have not yet been fully elucidated. This study aimed to determine the stemness and multilineage differentiation potential of mouse embryonic palatal mesenchyme (MEPM) cells in palatal shelves and to explore the underlying regulatory mechanism associated with cleft palate formation.MethodsPalatal shelves excised from mice models were cultured in vitro to ascertain whether MEPM are stem cells through immunofluorescence and flow cytometry. The osteogenic, adipogenic, and chondrogenic differentiation potential of MEPM cells were also determined to characterize MEPM stemness. In addition, the role of the PTEN-Akt-mTOR autophagic pathway was investigated using quantitative RT-PCR, Western blotting, and transmission electron microscopy.ResultsMEPM cells in culture exhibited cell surface marker expression profiles similar to that of mouse bone marrow stem cells and exhibited positive staining for vimentin (mesodermal marker), nestin (ectodermal marker), PDGFRα, Efnb1, Osr2, and Meox2 (MEPM cells markers). In addition, exposure to PDGFA stimulated chemotaxis of MEPM cells. MEPM cells exhibited stronger potential for osteogenic differentiation as compared to that for adipogenic and chondrogenic differentiation. Undifferentiated MEPM cells displayed a high concentration of autophagosomes, which disappeared after differentiation (at passage four), indicating the involvement of PTEN-Akt-mTOR signaling.ConclusionsOur findings suggest that MEPM cells are ectomesenchymal stem cells with a strong osteogenic differentiation potential and that maintenance of their stemness via PTEN/AKT/mTOR autophagic signaling prevents cleft palate development.