Project description:Incisor enamel organ epithelial cells were isolated and enzymatically processed from postnatal day 4 mice transgenic for Amelx-promoter driven tdTomato. Single cell suspensions were subjected to fluorescence-activated cell sorting (FACS) to isolate tdTomato positive ameloblasts. tdTomato-positive cells were isolated from enamel organ epithelial from the incisors of three mouse lines, which were Mmp20+/+ -AT4 (WT), Mmp20-/--AT4 (KO), Mmp20+/+ Tg-AT4 (Tg, overexpress MMP20). We used Fukuoka Dental College mouse ameloblast PCR array panel to quantitate gene expression of genes associated with enamel formation, cell migration and cell adhesion from ameloblasts.
Project description:Nephridial (excretory organ) symbionts are widespread in lumbricid earthworms and the complexity of the nephridial symbiont communities varies greatly between earthworm species. The two most common symbionts are the well-described Verminephrobacter and less well-known Flexibacter-like bacteria. Verminephrobacter are present in almost all lumbricid earthworms, they are species-specific, vertically transmitted, and have presumably been associated with their hosts since the origin of lumbricids. Flexibacter-like symbionts have been reported from about half the investigated earthworms; they are also vertically transmitted. To investigate the evolution of this tri-partite symbiosis, phylogenies for 18 lumbricid earthworm species were constructed based on two mitochondrial genes, NADH dehydrogenase subunit 2 (ND2) and cytochrome c oxidase subunit I (COI), and compared to their symbiont phylogenies based on RNA polymerase subunit B (rpoB) and 16S rRNA genes. The two nephridial symbionts showed markedly different evolutionary histories with their hosts. For Verminephrobacter, clear signs of long-term host-symbiont co-evolution with rare host switching events confirmed its ancient association with lumbricid earthworms, likely dating back to their last common ancestor about 100 million years (MY) ago. In contrast, phylogenies for the Flexibacter-like symbionts suggested an ability to switch to new hosts, to which they adapted and subsequently became species-specific. Putative co-speciation events were only observed with closely related host species; on that basis, this secondary symbiosis was estimated to be minimum 45 MY old. Based on the monophyletic clustering of the Flexibacter-like symbionts, the low 16S rRNA gene sequence similarity to the nearest described species (<92%) and environmental sequences (<94.2%), and the specific habitat in the earthworm nephridia, we propose a new candidate genus for this group, Candidatus Nephrothrix.
Project description:Almost all lumbricid earthworms (oligochaetaLumbricidae) harbor extracellular species-specific bacterial symbionts of the genus Verminephrobacter (Betaproteobacteria) in their nephridia. The symbionts have a beneficial effect on host reproduction and likely live on their host's waste products. They are vertically transmitted and presumably associated with earthworms already at the origin of Lumbricidae 62-136 million years ago. The Verminephrobacter genomes carry signs of bottleneck-induced genetic drift, such as accelerated evolutionary rates, low codon usage bias, and extensive genome shuffling, which are characteristic of vertically transmitted intracellular symbionts. However, the Verminephrobacter genomes lack AT bias, size reduction, and pseudogenization, which are also common genomic hallmarks of vertically transmitted, intracellular symbionts. We propose that the opportunity for genetic mixing during part of the host-symbiont life cycle is the key to evade drift-induced genome erosion. Furthermore, we suggest the earthworm-Verminephrobacter association as a new experimental system for investigating host-microbe interactions, and especially for understanding genome evolution of vertically transmitted symbionts in the presence of genetic mixing.
Project description:Background: Ependymomas encompass multiple, clinically relevant tumor types based on localization and molecular profiles. Although tumors of the methylation class “spinal ependymoma” (SP-EPN) represent the most common intramedullary neoplasms in children and adults, their developmental origin is ill-defined, molecular data are scarce, and the potential heterogeneity within SP-EPN remains unexplored. The only known recurrent genetic events in SP-EPN are loss of chromosome 22q and NF2 mutations, but neither types and frequency of these alterations nor their clinical meaning have been described in a large, epigenetically defined series. Methods: We mapped SP-EPN transcriptomes (n=76) to developmental atlases of the developing and adult spinal cord to uncover potential developmental origins of these tumors. In addition, transcriptomic, epigenetic (n=234), genetic (n=140), and clinical analyses (n=115) were integrated for a detailed overview on this entity. Results: Integration of transcriptomic ependymoma data with single-cell atlases of the spinal cord identified mature adult ependymal cells to display highest similarities to SP-EPN. Unsupervised hierarchical clustering of tumor data together with integrated analysis of methylation profiles identified two molecular SP-EPN subtypes. Subtype 1 predominantly contained NF2 wild type sequences with regular NF2 expression but revealed more extensive copy number alterations. Subtype 2 harbored previously known germline or sporadic NF2 mutations and was NF2-deficient in most cases, more often showed multilocular disease, and demonstrated a significantly reduced progression-free survival. Conclusion: Based on integrated molecular profiling of a large tumor series we identify two distinct SP-EPN subtypes with important implications for genetic counseling, patient surveillance, and drug development priorities.