Development (Cambridge, England)
Marczenke, M;Sunaga-Franze, DY;Popp, O;Althaus, IW;Sauer, S;Mertins, P;Christ, A;Allen, BL;Willnow, TE;
PMID: 34698766 | DOI: 10.1242/dev.200080
Growth arrest-specific 1 (GAS1) acts as a co-receptor to Patched 1 promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in iPSC-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating Notch signaling, essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives Notch pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating Notch and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.
J Am Soc Nephrol. 2018 Dec 4.
Mukherjee M, deRiso J, Otterpohl K, Ratnayake I, Kota D, Ahrenkiel P, Chandrasekar I, Surendran K.
PMID: 30514723 | DOI: 10.1681/ASN.2018040440
Abstract Background Notch signaling is required during kidney development for nephron formation and principal cell fate selection within the collecting ducts. Whether Notch signaling is required in the adult kidney to maintain epithelial diversity, or whether its loss can trigger principal cell transdifferentiation (which could explain acquired diabetes insipidus in patients receiving lithium) is unclear. Methods To investigate whether loss of Notch signaling can trigger principal cells to lose their identity, we genetically inactivated Notch1 and Notch2, inactivated the Notch signaling target Hes1, or induced expression of a Notch signaling inhibitor in all of the nephron segments and collecting ducts in mice after kidney development. We examined renal function and cell type composition of control littermates and mice with conditional Notch signaling inactivation in adult renal epithelia. In addition, we traced the fate of genetically labeled adult kidney collecting duct principal cells after Hes1 inactivation or lithium treatment. Results Notch signaling was required for maintenance of Aqp2-expressing cells in distal nephron and collecting duct segments in adult kidneys. Fate tracing revealed mature principal cells in the inner stripe of the outer medulla converted to intercalated cells after genetic inactivation of Hes1 and, to a lesser extent, lithium treatment. Hes1 ensured repression of Foxi1 to prevent the intercalated cell program from turning on in mature Aqp2+ cell types. Conclusions Notch signaling via Hes1 regulates maintenance of mature renal epithelial cell states. Loss of Notch signaling or use of lithium can trigger transdifferentiation of mature principal cells to intercalated cells in adult kidneys.
Saunders-Wood, T;Egawa, N;Zheng, K;Giaretta, A;Griffin, H;Doorbar, J;
PMID: 35019722 | DOI: 10.1128/JVI.01181-21
Papillomaviruses exclusively infect stratified epithelial tissues and cause chronic infections. To achieve this, infected cells must remain in the epithelial basal layer alongside their uninfected neighbours for years or even decades. To examine how papillomaviruses achieve this, we used the in vivo MmuPV1 model of lesion formation and persistence. During early lesion formation, an increased cell density in the basal layer, as well as a delay in the infected cells commitment to differentiation was apparent in cells expressing MmuPV1 E6/E7 RNA. Using cell culture models, keratinocytes exogenously expressing MmuPV1 E6, but not E7, recapitulated this delay in differentiation post-confluence and also grew to a significantly higher density. Cell competition assays further showed that MmuPV1 E6 expression led to a preferential persistence of the cell in the first layer, with control cells accumulating almost exclusively in the second layer. Interestingly, the disruption of MmuPV1 E6 binding to MAML1 protein abrogated these phenotypes. This suggests that the interaction between MAML1 and E6 is necessary for the lower (basal) layer persistence of MmuPV1 E6 expressing cells. Our results indicate a role for E6 in lesion establishment by facilitating the persistence of infected cells in the epithelial basal layer; a mechanism that is most likely shared by other papillomavirus types. Interruption of this interaction is predicted to impede persistent papillomavirus infection and consequently provides a novel treatment target. Importance Persistent infection with high-risk HPV types can lead to development of HPV-associated cancers, and persistent low-risk HPV infection causes problematic diseases, such as recurrent respiratory papillomatosis. The management and treatment of these conditions poses a considerable economic burden. Maintaining a reservoir of infected cells in the basal layer of the epithelium is critical for the persistence of infection in the host, and our studies using the mouse papillomavirus model suggest that E6 gene expression leads to the preferential persistence of epithelial cells in the lower layers during stratification. The E6 interaction with MAML1, a component of the Notch pathway, is required for this phenotype, and is linked to E6 effects on cell density and differentiation. These observations are likely to reflect a common E6 role that is preserved amongst papillomaviruses, and provide us with a novel therapeutic target for the treatment of recalcitrant lesions.
De Schepper, S;Ge, JZ;Crowley, G;Ferreira, LSS;Garceau, D;Toomey, CE;Sokolova, D;Rueda-Carrasco, J;Shin, SH;Kim, JS;Childs, T;Lashley, T;Burden, JJ;Sasner, M;Sala Frigerio, C;Jung, S;Hong, S;
PMID: 36747024 | DOI: 10.1038/s41593-023-01257-z
Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.
Modelling TGFβR and Hh pathway regulation of prognostic matrisome molecules in ovarian cancer
Delaine-Smith, R;Maniati, E;Malacrida, B;Nichols, S;Roozitalab, R;Jones, R;Lecker, L;Pearce, O;Knight, M;Balkwill, F;
| DOI: 10.1016/j.isci.2021.102674
In a multi-level ‘deconstruction’ of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix, ECM, molecules, COL11A1, COMP, FN1, VCAN, CTSB and COL1A1, are up-regulated in cancer. Using biopsies, we identified significant associations between TGFβR activity, Hedgehog signalling and these ECM molecules and studied the associations in mono-, co- and tri-culture. Activated omental fibroblasts produced more matrix than malignant cells, directed by TGFβR and Hedgehog signalling crosstalk. We ‘reconstructed’ omental metastases in tri-cultures of HGSOC cells, omental fibroblasts and adipocytes. This combination was sufficient to generate all six ECM proteins and the matrisome expression signature. TGFβR and Hedgehog inhibitor combinations attenuated fibroblast activation, gel and ECM remodelling in these models. The tri-culture model reproduces key features of omental metastases and allows study of diseased-associated ECM.
Li L, Grausam KB, Wang J, Lun MP, Ohli J, Lidov HG, Calicchio ML, Zeng E, Salisbury JL, Wechsler-Reya RJ, Lehtinen MK, Schüller U, Zhao H.
PMID: 26999738 | DOI: 10.1038/ncb3327
Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.
Lawsonia intracellularis infected enterocytes lack sucrase-isomaltase which contributes to reduced pig digestive capacity
Helm, ET;Burrough, ER;Leite, FL;Gabler, NK;
PMID: 34147126 | DOI: 10.1186/s13567-021-00958-2
Lawsonia intracellularis is endemic to swine herds worldwide, however much is still unknown regarding its impact on intestinal function. Thus, this study aimed to characterize the impact of L. intracellularis on digestive function, and how vaccination mitigates these impacts. Thirty-six L. intracellularis negative barrows were assigned to treatment groups (n = 12/trt): (1) nonvaccinated, L. intracellularis negative (NC); (2) nonvaccinated, L intracellularis challenged (PC); and (3) L. intracellularis challenged, vaccinated (Enterisol Ileitis, Boehringer Ingelheim) 7 weeks pre-challenge (VAC). On days post-inoculation (dpi) 0 PC and VAC pigs were inoculated with L. intracellularis. From dpi 19-21 fecal samples were collected for apparent total tract digestibility (ATTD) and at dpi 21, pigs were euthanized for sample collection. Post-inoculation, ADG was reduced in PC pigs compared with NC (41%, P < 0.001) and VAC (25%, P < 0.001) pigs. Ileal gross lesion severity was greater in PC pigs compared with NC (P = 0.003) and VAC (P = 0.018) pigs. Dry matter, organic matter, nitrogen, and energy ATTD were reduced in PC pigs compared with NC pigs (P ≤ 0.001 for all). RNAscope in situ hybridization revealed abolition of sucrase-isomaltase transcript in the ileum of PC pigs compared with NC and VAC pigs (P < 0.01). Conversely, abundance of stem cell signaling markers Wnt3, Hes1, and p27Kip1 were increased in PC pigs compared with NC pigs (P ≤ 0.085). Taken together, these data demonstrate that reduced digestibility during L. intracellularis challenge is partially driven by abolition of digestive machinery in lesioned tissue. Further, vaccination mitigated several of these effects, likely from lower bacterial burden and reduced disease severity.
Activation of notch signaling in dorsal root ganglia innervating knee joints in experimental osteoarthritis
Osteoarthritis and Cartilage
Wang, L;Miller, R;Malfait, A;
| DOI: 10.1016/j.joca.2021.02.480
Purpose: Surgical destabilization of the medial meniscus (DMM) is a widely used mouse model of knee osteoarthritis (OA). The cell bodies of primary sensory neurons innervating the knee joints are located in the lumbar dorsal root ganglia (L3-L5 DRG). Analysis of the gene expression profile of L3-L5 DRG after DMM or sham surgery revealed that innate neuro-immune pathways were strongly regulated, especially in the later stages of the model, 8-16 weeks after DMM, when persistent pain is associated with severe joint damage. In depth analysis of the microarray data further showed that a number of genes encoding molecules in the Notch signaling pathway were regulated, mostly in late-stage disease, along with the upregulation of the gene encoding monocyte chemoattractant protein (MCP)-1/C-C motif chemokine ligand 2 (CCL2). CCL2 is a proalgesic mediator that is released upon tolllike receptor (TLR) 2/4 activation, and plays a key role in initiating and maintaining pain in this model. The aim of this study was to investigate Notch signaling in the knee-innervating DRG of mice with experimental knee OA, and determine the effect of Notch signaling activation on TLR2/4-mediated CCL2 synthesis in cultured DRG cells. Methods: DMM or sham surgery was performed in the right knee of 10- week old male C57BL/6 mice. Ipsilateral L4 DRG from mice 26 weeks after DMM or sham surgery were collected and cryosectioned. Expression of the Notch downstream target gene, Hes1, was detected using RNA in situ hybridization (ISH) (RNAscope, Advanced Cell Diagnostics). Quantification of mRNA expression was performed as calculating H-score of each sample according to the 0-4 five-bin scoring system recommended by the manufacturer, based on the number of cells with the same range of number of dots per cell. Active Notch protein was detected via immunofluorescence (IF) staining using an antibody against Notch intracellular domain (NICD), which is only present after g-secretase cleavage of Notch at S3. For in vitro cultures of DRG cells, bilateral L3-L5 DRG were collected from 10-week old male naïve C57BL/6 mice. Following enzymatic digestion, DRG cells were plated on poly-L-lysine and laminin coated glass coverslips, and cultured in F12 medium supplemented with 1x N2 and 0.5% fetal bovine serum. Inhibition of Notch signaling was achieved by (1) g-secretase inhibitor, DAPT; (2) ADAM-17 inhibitor, TAPI-1; or (3) soluble form of the Jag1 peptide (sJag1). On day 4, cells were pre-treated with DAPT (25 mM), TAPI-1 (20 mM), or sJag1 (40 mM) for 1 hour, followed by addition of the TLR2 agonist, Pam3CSK4 (1 mg/ml), or the TLR4 agonist, LPS (1 mg/ ml). Then, RNA was collected 3 hours later for qRT-PCR to quantify Ccl2 mRNA expression, or culture supernatants were collected 24 hours later to measure the CCL2 protein level using Quantikine Mouse CCL2/JE/ MCP-1 Immunoassay kit from R&D Systems, Inc.
Pepe-Mooney BJ, Dill MT, Alemany A, Ordovas-Montanes J, Matsushita Y, Rao A, Sen A, Miyazaki M, Anakk S, Dawson PA, Ono N, Shalek AK, van Oudenaarden A, Camargo FD.
PMID: 31080134 | DOI: 10.1016/j.stem.2019.04.004
The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasisand after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.
Li, L;Sun, Y;Davis, AE;Shah, SH;Hamed, LK;Wu, MR;Lin, CH;Ding, JB;Wang, S;
PMID: 37269288 | DOI: 10.1016/j.celrep.2023.112596
Neural progenitor cells lengthen their cell cycle to prime themselves for differentiation as development proceeds. It is currently not clear how they counter this lengthening and avoid being halted in the cell cycle. We show that N6-methyladenosine (m6A) methylation of cell-cycle-related mRNAs ensures the proper cell-cycle progression of late-born retinal progenitor cells (RPCs), which are born toward the end of retinogenesis and have long cell-cycle length. Conditional deletion of Mettl14, which is required for depositing m6A, led to delayed cell-cycle exit of late-born RPCs but has no effect on retinal development prior to birth. m6A sequencing and single-cell transcriptomics revealed that mRNAs involved in elongating the cell cycle were highly enriched for m6A, which could target them for degradation and guarantee proper cell-cycle progression. In addition, we identified Zfp292 as a target of m6A and potent inhibitor of RPC cell-cycle progression.
Coppo, R;Kondo, J;Iida, K;Okada, M;Onuma, K;Tanaka, Y;Kamada, M;Ohue, M;Kawada, K;Obama, K;Inoue, M;
PMID: 36718360 | DOI: 10.1016/j.isci.2023.105962
Dynamic changes in cell properties lead to intratumor heterogeneity; however, the mechanisms of nongenetic cellular plasticity remain elusive. When the fate of each cell from colorectal cancer organoids was tracked through a clonogenic growth assay, the cells showed a wide range of growth ability even within the clonal organoids, consisting of distinct subpopulations; the cells generating large spheroids and the cells generating small spheroids. The cells from the small spheroids generated only small spheroids (S-pattern), while the cells from the large spheroids generated both small and large spheroids (D-pattern), both of which were tumorigenic. Transition from the S-pattern to the D-pattern occurred by various extrinsic triggers, in which Notch signaling and Musashi-1 played a key role. The S-pattern spheroids were resistant to chemotherapy and transited to the D-pattern upon drug treatment through Notch signaling. As the transition is linked to the drug resistance, it can be a therapeutic target.
Hurley, EM;Mozolewski, P;Dobrowolski, R;Hsieh, J;
PMID: 37352850 | DOI: 10.1016/j.stemcr.2023.05.018
Alzheimer's disease (AD) is the most common neurodegenerative disorder, but its root cause may lie in neurodevelopment. PSEN1 mutations cause the majority of familial AD, potentially by disrupting proper Notch signaling, causing early unnoticed cellular changes that affect later AD progression. While rodent models are useful for modeling later stages of AD, human induced pluripotent stem cell-derived cortical spheroids (hCSs) allow access to studying the human cortex at the cellular level over the course of development. Here, we show that the PSEN1 L435F heterozygous mutation affects hCS development, increasing size, increasing progenitors, and decreasing post-mitotic neurons as a result of increased Notch target gene expression during early hCS development. We also show altered Aβ expression and neuronal activity at later hCS stages. These results contrast previous findings, showing how individual PSEN1 mutations may differentially affect neurodevelopment and may give insight into fAD progression to provide earlier time points for more effective treatments.
