Reconstruction and deconstruction of human somitogenesis in vitro.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.

MeCP2 Represses the Rate of Transcriptional Initiation of Highly Methylated Long Genes.

Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.

Activity-induced MeCP2 phosphorylation regulates retinogeniculate synapse refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MECP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here, we show that MeCP2 is phosphorylated at four residues in the mouse brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from the synapse refinement defect previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period.

LPS-mediated release of ATP from urothelial cells occurs by lysosomal exocytosis.

BACKGROUND: While numerous studies have confirmed ATP's importance in bladder physiology/pathophysiology, the literature is still conflicted regarding the mechanism of ATP release from the urothelium. Multiple mechanisms have been identified including non-vesicular release via pannexin channels as well as vesicular release via a mechanism blocked by botulinum toxin. Recently, it has been shown that lysosomes contain significant stores of ATP which can be released extracellularly in response to Toll-like receptor (TLR) stimulation. OBJECTIVE: The goal of the current study was to determine if lysosomal exocytosis occurs in urothelial cells in response to TLR4 stimulation by its agonist, bacterial lipopolysaccharide (LPS). MATERIALS AND METHODS: Human urothelial cells from an immortalized cell line (TRT-HU1) were treated with bacterial LPS (100 µg/ml) or the nicotinic agoinist cytisine (100 µM) and extracellular release of ATP and lysosomal acid phosphatase were measured. Pannexin-mediated ATP release and lysosomal ATP release were differentiated using Brilliant Blue FCF to inhibit pannexin channels and glycyl-l-phenylalanine-ß-naphthylamide (GPN) to destroy lysosomes. The mechanisms controlling lysosomal exocytosis were examined using lysosomal pH measurements using LysoSensor dye and intracellular calcium signaling using Fura-2. RESULTS: Stimulation of TRT-HU1 cells with LPS significantly increased ATP release, which was inhibited by GPN, but not by Brilliant Blue FCF. Conversely, stimulation with cytisine induced ATP release that was sensitive to Brilliant Blue FCF but not GPN. LPS stimulation also induced the release of the lysosomal acid phosphatases. LPS increased lysosomal pH and direct alkalization of lysosomal pH using chloroquine or bafilomycin A1 induced ATP and acid phosphatase release, indicating an important role for pH in lysosomal exocytosis. Additionally, stimulation of lysosomal transient receptor potential mucolipin 1 calcium channels evoked intracellular calcium transients as well as ATP release. CONCLUSION: These data indicate that LPS-induced ATP release from urothelial cells is mediated by lysosomal exocytosis, a vesicular mechanism distinctly separate from non-vesicular release via pannexin channels.

Activity-Induced MeCP2 Phosphorylation Regulates Retinogeniculate Synapse Refinement.

Mutations in MECP2 give rise to Rett syndrome (RTT), an X-linked neurodevelopmental disorder that results in broad cognitive impairments in females. While the exact etiology of RTT symptoms remains unknown, one possible explanation for its clinical presentation is that loss of MeCP2 causes miswiring of neural circuits due to defects in the brain's capacity to respond to changes in neuronal activity and sensory experience. Here we show that MeCP2 is phosphorylated at four residues in the brain (S86, S274, T308, and S421) in response to neuronal activity, and we generate a quadruple knock-in (QKI) mouse line in which all four activity-dependent sites are mutated to alanines to prevent phosphorylation. QKI mice do not display overt RTT phenotypes or detectable gene expression changes in two brain regions. However, electrophysiological recordings from the retinogeniculate synapse of QKI mice reveal that while synapse elimination is initially normal at P14, it is significantly compromised at P20. Notably, this phenotype is distinct from that previously reported for Mecp2 null mice, where synapses initially refine but then regress after the third postnatal week. We thus propose a model in which activity-induced phosphorylation of MeCP2 is critical for the proper timing of retinogeniculate synapse maturation specifically during the early postnatal period. SIGNIFICANCE STATEMENT: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that predominantly affects girls. RTT is caused by loss of function mutations in a single gene MeCP2. Girls with RTT develop normally during their first year of life, but then experience neurological abnormalities including breathing and movement difficulties, loss of speech, and seizures. This study investigates the function of the MeCP2 protein in the brain, and how MeCP2 activity is modulated by sensory experience in early life. Evidence is presented that sensory experience affects MeCP2 function, and that this is required for synaptic pruning in the brain. These findings provide insight into MeCP2 function, and clues as to what goes awry in the brain when the function of MeCP2 is disrupted.

Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing.

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.

Ectodomain-specific E-cadherin antibody suppresses skin SCC growth and reduces tumor grade: a multitargeted therapy modulating RTKs and the PTEN-p53-MDM2 axis.

Tumor cell survival consists of an intricate balance between cell growth and cell death pathways involving receptor tyrosine kinases [RTK; i.e., HER1-4, insulin-like growth factor-1 receptor (IGF-1R), etc.], MDM2, and the tumor suppressor proteins phosphatase and tensin homolog deleted on chromosome ten (PTEN) and p53. We recently demonstrated that shedded E-cadherin extracellular domain fragment (sEcad) is a valid oncogenic target that is significantly increased in human clinical skin squamous cell cancers (SCC) samples, UV-induced mouse tumors, and cells and promotes tumor cell proliferation, migration, and invasion by interacting and activating with the HER-phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) axis. In resected human SCC tumors, we reported enhanced sEcad-HER1, sEcad-HER2, and sEcad-IGF-1R, but not FL-Ecad-RTK interactions. Here, we demonstrate that a sEcad antibody against the ectodomain of E-cadherin suppressed SCC growth and increased tumor differentiation in orthotopic cutaneous SCC xenografts by inhibiting proliferation and inducing apoptosis. A similar anti-sEcad antibody-induced inhibition of proliferation and induction of cell death was evident in PAM212 cells in vitro. Mechanistically, anti-sEcad administration upregulated an array of cell death pathways (i.e., Bad, active caspase-3, and cleaved PARP) and inhibited inhibitors of apoptosis (IAP; survivin, livin, etc.), RTKs (HER1, HER2, p95HER2, and IGF-1R), MAPK and PI3K/mTOR prosurvival signaling. Interestingly, in anti-sEcad mAb-treated tumors and PAM212 cells, this effect was associated with a profound increase in membrane, cytosolic, and nuclear levels of PTEN; enhanced cytosolic p53; and a decrease in MDM2 levels. Overall, our studies suggest that an antibody-based therapy against sEcad may be a novel therapeutic platform for cutaneous SCCs by hampering key proto-oncogenes (RTKs, IAPs, and MDM2) and activating potent tumor suppressor proteins (PTEN and p53) intricately linked to tumor growth and survival.

Monoclonal antibody against the ectodomain of E-cadherin (DECMA-1) suppresses breast carcinogenesis: involvement of the HER/PI3K/Akt/mTOR and IAP pathways.

PURPOSE: Although targeted therapies against HER2 have been one of the most successful therapeutic strategies for breast cancer, patients eventually developed acquired resistance from compensatory upregulation of alternate HERs and mitogen-activated protein kinase-phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling. As we and others have shown that the soluble ectodomain fragment of E-cadherin exerts prooncogenic effects via HER1/2-mediated binding and activation of downstream prosurvival pathways, we explored whether targeting this ectodomain [DECMA-1 monoclonal antibody (mAb)] was effective in the treatment of HER2-positive (HER2(+)) breast cancers. EXPERIMENTAL DESIGN: MMTV-PyMT transgenic mice and HER2(+)/E-cadherin-positive MCF-7 and BT474 trastuzumab-resistant (TtzmR) cells were treated with the DECMA-1 mAb. Antitumor responses were assessed by bromodeoxyuridine incorporation, apoptosis, and necrosis. The underlying intracellular prooncogenic pathways were explored using subcellular fractionation, immunoprecipitation, fluorescence microscopy, and immunoblotting. RESULTS: Treatment with DECMA-1 mAb significantly delayed tumor onset and attenuated tumor burden in MMTV-PyMT mice by reducing tumor cell proliferation and inducing apoptosis without any detectable cytotoxicity to mice or end-organs. In vitro treatment of MCF-7 and BT474 TtzmR cells reduced proliferation and induced cancer cell apoptosis. Importantly, this inhibition of breast tumorigenesis was due to concomitant downregulation, via ubiquitin-mediated degradation through the lysosome and proteasome pathways, of all HER family members, components of downstream PI3K/Akt/mTOR prosurvival signaling and suppression of inhibitor of apoptosis proteins. CONCLUSIONS: Our results establish that the E-cadherin ectodomain-specific mAb DECMA-1 inhibits Ecad(+)/HER2(+) breast cancers by hindering tumor growth and inducing apoptosis via downregulation of key oncogenic pathways involved in trastuzumab resistance, thereby establishing a novel therapeutic platform for the treatment of HER2(+) breast cancers.