Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish.

Primordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this post-migratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same time point, PGC nuclei become extremely gyrated, displaying general broad opening of chromatin and high levels of intergenic transcription. This is accompanied by changes in nuage morphology, expression of large loci (PGC-expressed non-coding RNA loci, PERLs) that are enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any time point, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.

Signalling codes for the maintenance and lineage commitment of embryonic gastric epithelial progenitors.

The identity of embryonic gastric epithelial progenitors is unknown. We used single-cell RNA-sequencing, genetic lineage tracing and organoid assays to assess whether Axin2- and Lgr5-expressing cells are gastric progenitors in the developing mouse stomach. We show that Axin2+ cells represent a transient population of embryonic epithelial cells in the forestomach. Lgr5+ cells generate both glandular corpus and squamous forestomach organoids ex vivo Only Lgr5+ progenitors give rise to zymogenic cells in culture. Modulating the activity of the WNT, BMP and Notch pathways in vivo and ex vivo, we found that WNTs are essential for the maintenance of Lgr5+ epithelial cells. Notch prevents differentiation of the embryonic epithelial cells along all secretory lineages and hence ensures their maintenance. Whereas WNTs promote differentiation of the embryonic progenitors along the zymogenic cell lineage, BMPs enhance their differentiation along the parietal lineage. In contrast, WNTs and BMPs are required to suppress differentiation of embryonic gastric epithelium along the pit cell lineage. Thus, coordinated action of the WNT, BMP and Notch pathways controls cell fate determination in the embryonic gastric epithelium.

Genetic requirements for repair of lesions caused by single genomic ribonucleotides in S phase.

Single ribonucleoside monophosphates (rNMPs) are transiently present in eukaryotic genomes. The RNase H2-dependent ribonucleotide excision repair (RER) pathway ensures error-free rNMP removal. In some pathological conditions, rNMP removal is impaired. If these rNMPs hydrolyze during, or prior to, S phase, toxic single-ended double-strand breaks (seDSBs) can occur upon an encounter with replication forks. How such rNMP-derived seDSB lesions are repaired is unclear. We expressed a cell cycle phase restricted allele of RNase H2 to nick at rNMPs in S phase and study their repair. Although Top1 is dispensable, the RAD52 epistasis group and Rtt101Mms1-Mms22 dependent ubiquitylation of histone H3 become essential for rNMP-derived lesion tolerance. Consistently, loss of Rtt101Mms1-Mms22 combined with RNase H2 dysfunction leads to compromised cellular fitness. We refer to this repair pathway as nick lesion repair (NLR). The NLR genetic network may have important implications in the context of human pathologies.

ID2 controls differentiation of enteroendocrine cells in mouse small intestine.

AIMS: The mammalian gut is the largest endocrine organ. Dozens of hormones secreted by enteroendocrine cells regulate a variety of physiological functions of the gut but also of the pancreas and brain. Here, we examined the role of the helix-loop-helix transcription factor ID2 during the differentiation of intestinal stem cells along the enteroendocrine lineage. METHODS: To assess the functions of ID2 in the adult mouse small intestine, we used single-cell RNA sequencing, genetically modified mice, and organoid assays. RESULTS: We found that in the adult intestinal epithelium Id2 is predominantly expressed in enterochromaffin and peptidergic enteroendocrine cells. Consistently, the loss of Id2 leads to the reduction of Chromogranin A-positive enteroendocrine cells. In contrast, the numbers of tuft cells are increased in Id2 mutant small intestine. Moreover, ablation of Id2 elevates the numbers of Serotonin+ enterochromaffin cells and Ghrelin+ X-cells in the posterior part of the small intestine. Finally, ID2 acts downstream of BMP signalling during the differentiation of Glucagon-like peptide-1+ L-cells and Cholecystokinin+ I-cells towards Neurotensin+ PYY+ N-cells. CONCLUSION: ID2 plays an important role in cell fate decisions in the adult small intestine. First, ID2 is essential for establishing a differentiation gradient for enterochromaffin and X-cells along the anterior-posterior axis of the gut. Next, ID2 is necessary for the differentiation of N-cells thus ensuring a differentiation gradient along the crypt-villi axis. Finally, ID2 suppresses the commitment of secretory intestinal epithelial progenitors towards tuft cell lineage and thus controls host immune response to commensal and parasitic microbiota.

IgE Epitope Profiling for Allergy Diagnosis and Therapy - Parallel Analysis of a Multitude of Potential Linear Epitopes Using a High Throughput Screening Platform.

Immunoglobulin E (IgE) is pivotal for manifestation and persistence of most immediate-type allergies and some asthma phenotypes. Consequently, IgE represents a crucial target for both, diagnostic purposes as well as therapeutic approaches. In fact, allergen-specific immunotherapy - aiming to re-route an IgE-based inflammatory response into an innocuous immune reaction against the allergen - is the only curative approach for IgE-mediated allergic diseases known so far. However, this requires the cognate allergen to be known. Unfortunately, even in well-characterized allergics or asthmatics, often just a small fraction of total IgE can be assigned to specific target allergens. To overcome this knowledge gap, we have devised an analytical platform for unbiased IgE target epitope detection. The system relies on chemically produced random peptide libraries immobilized on polystyrene beads ("one-bead-one-compound (OBOC) libraries") capable to present millions of different peptide motifs simultaneously to immunoglobulins from biological samples. Beads binding IgE are highlighted with a fluorophore-labeled anti-IgE antibody allowing fluorescence-based detection and isolation of positives, which then can be characterized by peptide sequencing. Setting-up this platform required an elaborate optimization process including proper choice of background suppressants, secondary antibody and fluorophore label as well as incubation conditions. For optimal performance our procedure involves a sophisticated pre-adsorption step to eliminate beads that react nonspecifically with anti-IgE secondary antibodies. This step turned out to be important for minimizing detection of "false positive" motifs that otherwise would erroneously be classified as IgE epitopes. In validation studies we were able to retrieve artificial test-peptide beads spiked into our library by using IgE directed against those test-peptides at physiological concentrations (≤20 IU/ml of specific IgE), and disease-relevant bead-bound epitopes of the major peanut allergen Ara h 2 by screening with sera from peanut allergics. Thus, we established a platform with which one can find and validate new immunoglobulin targets using patient material which displays a largely unknown immunoglobulin repertoire.