From Pioneer to Repressor: Bimodal foxd3 Activity Dynamically Remodels Neural Crest Regulatory Landscape In Vivo

The neural crest (NC) is a transient embryonic stem cell-like population characterized by its multipotency and broad developmental potential. Here, we perform NC-specific transcriptional and epigenomic profiling of foxd3-mutant cells in vivo to define the gene regulatory circuits controlling NC specification. Together with global binding analysis obtained by foxd3 biotin-ChIP and single cell profiles of foxd3-expressing premigratory NC, our analysis shows that, during early steps of NC formation, foxd3 acts globally as a pioneer factor to prime the onset of genes regulating NC specification and migration by re-arranging the chromatin landscape, opening cis-regulatory elements and reshuffling nucleosomes. Strikingly, foxd3 then gradually switches from an activator to its well-described role as a transcriptional repressor and potentially uses differential partners for each role. Taken together, these results demonstrate that foxd3 acts bimodally in the neural crest as a switch from "permissive" to "repressive" nucleosome and chromatin organization to maintain multipotency and define cell fates

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19

Tracking chromatin dynamics and bimodal foxd3 mechanism on neural crest gene regulation in vivo

The neural crest (NC) is a transient embryonic stem cell-like population characterised by its multipotency and broad developmental potential. The core NC transcription factor foxd3, known to mediate late NC lineage decisions via transcriptional repression, is also pervasively expressed in the pre-migratory NC progenitors, where its putative role remains elusive. To elucidate the mechanistic and temporal mode of foxd3 action, we analysed its genomewide binding profiles across multiple stages of zebrafish development. To this end, we have developed and used our novel biotin ChIP-seq approach allowing to specifically biotinylate factors of interest, such as foxd3 protein, in vivo. Foxd3 binding maps interpreted within the context of NC-specific transcriptional and epigenomic profiles of foxd3-mutant and control cells reveal that foxd3 acts bimodally throughout the NC ontogeny. We showed that first foxd3 functions globally as a pioneer factor to prime the onset of NC specification and migration genes by re-arranging their chromatin landscape. Strikingly, foxd3 then gradually switches from an activator to its well-described role as a transcriptional repressor and potentially uses different partners for each role. By employing two enhancer reporter zebrafish lines, imaging and -omics tools, we have also shown that auto-regulation of foxd3 gene locus is involved during early cell-fate decisions and embryo patterning. In the cranial embryo regions, positive auto-regulatory foxd3 feedback is driving foxd3 expression that is needed for the NC and nervous system development. This auto-regulatory loop is ensured by a set of foxd3 cis-regulatory elements that exhibit NC-like regulatory signatures. Contrarily, foxd3 is auto-regulated by a different set of enhancers and, as a result, is expressed to lower levels during the apparent neuromesodermal progenitor specification in the caudal embryo regions. Taken together, this thesis demonstrates that foxd3 needs to be tightly auto-regulated during early cell lineage decisions. Also, later in the NC development, foxd3 switches from a chromatin activator to a repressor in order to maintain NC multipotency and define its cell fates.</p

Single-cell atlas of early chick development reveals gradual segregation of neural crest lineage from the neural plate border during neurulation

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harboring neural crest and cranial placode progenitors. Here, we a generate single-cell atlas of the developing chick epiblast from late gastrulation through early neurulation stages to define transcriptional changes in the emerging ‘neural plate border’ as well as other regions of the epiblast. Focusing on the border territory, the results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridization. Developmental trajectory analysis infers that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined neural crest and placode lineages. The data also can be mined to reveal changes throughout the developing epiblast

Nanoscale dynamics of cholesterol in the cell membrane

Cholesterol constitutes approximately 30%-40% of the mammalian plasma membrane-a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analog, whereas the other one was significantly faster. When a cholesterol analog was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane

Nanoscale dynamics of cholesterol in the cell membrane

Cholesterol constitutes approximately 30%-40% of the mammalian plasma membrane-a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analog, whereas the other one was significantly faster. When a cholesterol analog was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane