Modelling the development of Barrett’s Oesophagus: Towards better treatment of Oesophageal Adenocarcinoma

© 2020 Elhadi IichBarrett’s oesophagus (BO) is a metaplastic condition in which the normal squamous epithelium of the oesophagus is replaced by a columnar gastro-intestinal like epithelium due to repeated gastro-intestinal reflux. BO is generally accepted to be a precursor condition with the potential to develop into oesophageal adenocarcinoma. Consensus for the cell of origin for Barrett’s oesophagus is still lacking. Different sources for the cell of origin have been proposed, one of which is the submucosal gland (SMG) and duct cells of the oesophagus. This hypothesis, however, has not been properly studied due to the lack of proper model systems. In this thesis, pig and human SMGs and ducts were characterised and compared to each other to assess the suitability of pig SMGs as a substitute for human SMGs using immunohistology and multiplex immunofluorescence staining. Pig epithelial SMG cells were also further characterised using single cell RNA sequencing. Furthermore, organoid culture systems were developed to functionally assess the potential of the progenitor cells found using histologic and transcriptomic characterisation. Pig and human SMGs show different distributions at the distal end of their respective oesophagi but are largely similar in cell type and progenitor cell marker expression as demonstrated by histologic characterisation. Both pig and human SMGs showed progenitor cell marker expression in their respective basal duct cells (CD49f and p75) and the myoepithelial cells (CD49f), suggesting that both the ductal and glandular compartments to contain their own respective pool of progenitor cells. The transcriptomic analysis of pig SMGs single cell RNA sequencing largely supports their role in maintaining oesophageal homeostasis, as previously is known for human. Furthermore, the pseudotime trajectory inference data support the notion of the basal duct cells to be progenitor which give rise luminal duct cells. Interestingly, the data suggest the myoepithelial cells to be progenitor cells that could give rise to both basal duct cells and the gland compartment cells. Sorting and organoid culturing of basal duct cells demonstrated their capacity to grow into squamous spheroids, supporting their role in contributing to normal oesophageal repair. This process could be inhibited by treatment with retinoic acid. Similarly, culture of gland compartment cells gave rise to spheroids with two distinct morphologies. Dense type spheroids showed a squamous morphology but also mucin production found in BO. The second type of spheroid showed a cystic morphology and similarly produces BO type mucin. Finally, viral induction of intestinalisation in submucosal basal duct cells, in the current culture system, did not show metaplastic changes similar to BO. In summary, pig SMGs show great similarity to human SMGs. The pig SMGs contain progenitor cells in their ductal and gland compartments. These progenitors can be purified and cultured in vitro. The current developed protocols could be used to test the hypothesis that the SMGs contain the cell of origin of BO

FGFR-mediated ERK1/2 signaling contributes to mesendoderm and definitive endoderm formation in vitro

Summary: The differentiation of human pluripotent stem cells into the SOX17+ definitive endoderm (DE) germ layer is important for generating tissues for regenerative medicine. Multiple developmental and stem cell studies have demonstrated that Activin/Nodal signaling is the primary driver of definitive endoderm formation. Here, we uncover that the FGF2-FGFR-ERK1/2 signaling contributes to mesendoderm and SOX17+ DE formation. Without ERK1/2 signaling, the Activin/Nodal signaling is insufficient to drive mesendoderm and DE formation. Besides FGF2-FGFR-mediated signaling, IGF1R signaling possibly contributes to the ERK1/2 signaling for DE formation. We identified a temporal relationship between Activin/Nodal-SMAD2 and FGF2-FGFR-ERK1/2 signaling in which Activin/Nodal-SMAD2 participates in the initiation of mesendoderm and DE specification that is followed by increasing activity of FGF2-FGFR-ERK1/2 to facilitate and permit the successful generation of SOX17+ DE. Overall, besides the role of Activin/Nodal signaling for DE formation, our findings shed light on the contribution of ERK1/2 signaling for mesendoderm and DE formation

Release of Notch activity coordinated by IL-1β signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration

AbstractWhile the acquisition of cellular plasticity in adult stem cells is essential for rapid regeneration after tissue injury, little is known about the underlying molecular mechanisms governing this process. Our data reveal the coordination of airway progenitor differentiation plasticity by inflammatory signals during alveolar regeneration. Upon damage, IL-1β signalling-dependent modulation of Jag1/2 expression in ciliated cells results in the inhibition of Notch signalling in secretory cells, which drives reprogramming and acquisition of differentiation plasticity. We identify a core role for the transcription factor Fosl2/Fra2 in secretory cell fate conversion to alveolar type 2 (AT2) cells retaining the distinct genetic and epigenetic signatures of secretory lineages. We furthermore reveal that KDR/FLK-1+ human secretory cells display a conserved capacity to generate AT2 cells via Notch inhibition. Our results demonstrate the functional role of a IL-1β-Notch-Fosl2 axis for the fate decision of secretory cells during injury repair, proposing a new potential therapeutic target for human lung alveolar regeneration.</jats:p

Genome-wide analysis reveals NRP1 as a direct HIF1α-E2F7 target in the regulation of motorneuron guidance in vivo

In this study, we explored the existence of a transcriptional network co-regulated by E2F7 and HIF1α, as we show that expression of E2F7, like HIF1α, is induced in hypoxia, and because of the previously reported ability of E2F7 to interact with HIF1α. Our genome-wide analysis uncovers a transcriptional network that is directly controlled by HIF1α and E2F7, and demonstrates both stimulatory and repressive functions of the HIF1α-E2F7 complex. Among this network we reveal Neuropilin 1 (NRP1) as a HIF1α-E2F7 repressed gene. By performing in vitro and in vivo reporter assays we demonstrate that the HIF1α-E2F7 mediated NRP1 repression depends on a 41 base pairs 'E2F-binding site hub', providing a molecular mechanism for a previously unanticipated role for HIF1α in transcriptional repression. To explore the biological significance of this regulation we performed in situ hybridizations and observed enhanced nrp1a expression in spinal motorneurons (MN) of zebrafish embryos, upon morpholino-inhibition of e2f7/8 or hif1α. Consistent with the chemo-repellent role of nrp1a, morpholino-inhibition of e2f7/8 or hif1α caused MN truncations, which was rescued in TALEN-induced nrp1ahu10012 mutants, and phenocopied in e2f7/8 mutant zebrafish. Therefore, we conclude that repression of NRP1 by the HIF1α-E2F7 complex regulates MN axon guidance in vivo

Genome-wide analysis reveals NRP1 as a direct HIF1α-E2F7 target in the regulation of motorneuron guidance in vivo

In this study, we explored the existence of a transcriptional network co-regulated by E2F7 and HIF1α, as we show that expression of E2F7, like HIF1α, is induced in hypoxia, and because of the previously reported ability of E2F7 to interact with HIF1α. Our genome-wide analysis uncovers a transcriptional network that is directly controlled by HIF1α and E2F7, and demonstrates both stimulatory and repressive functions of the HIF1α -E2F7 complex. Among this network we reveal Neuropilin 1 (NRP1) as a HIF1α-E2F7 repressed gene. By performing in vitro and in vivo reporter assays we demonstrate that the HIF1α-E2F7 mediated NRP1 repression depends on a 41 base pairs 'E2F-binding site hub', providing a molecular mechanism for a previously unanticipated role for HIF1α in transcriptional repression. To explore the biological significance of this regulation we performed in situ hybridizations and observed enhanced nrp1a expression in spinal motorneurons (MN) of zebrafish embryos, upon morpholino-inhibition of e2f7/8 or hif1α. Consistent with the chemo-repellent role of nrp1a, morpholino-inhibition of e2f7/8 or hif1α caused MN truncations, which was rescued in TALEN-induced nrp1a(hu10012) mutants, and phenocopied in e2f7/8 mutant zebrafish. Therefore, we conclude that repression of NRP1 by the HIF1α-E2F7 complex regulates MN axon guidance in vivo