Fossilized embryos with extraordinary cellular preservation appear in the Late Neoproterozoic and Cambrian, coincident with the appearance of animal body fossils. It has been hypothesized that microbial processes are responsible for preservation and mineralization of organic tissues. However, the actions of microbes in preservation of embryos have not been demonstrated experimentally. Here, we show that bacterial biofilms assemble rapidly in dead marine embryos and form remarkable pseudomorphs in which the bacterial biofilm replaces and exquisitely models details of cellular organization and structure. The experimental model was the decay of cleavage stage embryos similar in size and morphology to fossil embryos. The data show that embryo preservation takes place in 3 distinct steps: (i) blockage of autolysis by reducing or anaerobic conditions, (ii) rapid formation of microbial biofilms that consume the embryo but form a replica that retains cell organization and morphology, and (iii) bacterially catalyzed mineralization. Major bacterial taxa in embryo decay biofilms were identified by using 16S rDNA sequencing. Decay processes were similar in different taphonomic conditions, but the composition of bacterial populations depended on specific conditions. Experimental taphonomy generates preservation states similar to those in fossil embryos. The data show how fossilization of soft tissues in sediments can be mediated by bacterial replacement and mineralization, providing a foundation for experimentally creating biofilms from defined microbial species to model fossilization as a biological process.bacterial ͉ developmental evolution ͉ metazoan origins ͉ taphonomy ͉ Cambrian

The conserved checkpoint kinases Chk1 and Rad53-Dun1 block the metaphase to anaphase transition by the phosphorylation and stabilization of securin, and block the mitotic exit network regulated by the Bfa1-Bub2 complex. However, both chk1 and rad53 mutants are able to exit from mitosis and initiate a new cell cycle, suggesting that both pathways have supporting functions in restraining anaphase and in blocking the inactivation of mitotic cyclin-Cdk1 complexes. Here we find that the cyclic-AMP-dependent protein kinase (PKA) pathway supports Chk1 in the regulation of mitosis by targeting the mitotic inducer Cdc20. Cdc20 is phosphorylated on PKA consensus sites after DNA damage, and this phosphorylation requires the Atr orthologue Mec1 and the PKA catalytic subunits Tpk1 and Tpk2. We show that the inactivation of PKA or expression of phosphorylation-defective Cdc20 proteins accelerates securin and Clb2 destruction in chk1 mutants and is sufficient to remove most of the DNA damage-induced delay. Mutation of the Cdc20 phosphorylation sites permitted the interaction of Cdc20 with Clb2 under conditions that should halt cell cycle progression. These data show that PKA pathways regulate mitotic progression through Cdc20 and support the DNA damage checkpoint pathways in regulating the destruction of Clb2 and securin.

The production of mature eosinophils is a tightly orchestrated process with the aim to sustain normal eosinophil levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including eosinophil-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with eosinophil maturation (1199 genes) than with eosinophil-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eosinophil progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting eosinophils. Our analyses also delineated a 976-gene eosinophil-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with eosinophils. EoPs and eosinophils, but not granulocyte-monocyte progenitors (GMPs) or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during eosinophil development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and eosinophils with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during eosinophil development.

Eosinophils originate in the bone marrow from an eosinophil lineage–committed, IL-5 receptor alpha (IL-5Rα)–positive, hematopoietic progenitor (eosinophil progenitor). Indeed, IL-5 is recognized as a critical regulator of eosinophilia and has effects on eosinophil progenitors, eosinophil precursors and mature eosinophils. However, substantial levels of eosinophils remain after IL-5 neutralization or genetic deletion, suggesting that there are alternative pathways for promoting eosinophilia. Herein, we investigated the contributory role of IL-5 accessory cytokines on the final stages of eosinophil differentiation. IL-5 stimulation of low-density bone marrow cells resulted in expression of a panel of cytokines and cytokine receptors, including several ligand-receptor pairs. Notably, IL-4 and IL-4Rα were expressed by eosinophil precursors and mature eosinophils. Signaling through IL-4Rα promoted eosinophil maturation when IL-5 was present, but IL-4 stimulation in the absence of IL-5 resulted in impaired eosinophil survival, suggesting that IL-4 cooperates with IL-5 to promote eosinophil differentiation. In contrast, CCL3, an eosinophil precursor–produced chemokine that signals through CCR1, promotes terminal differentiation of CCR1-positive eosinophil precursors in the absence of IL-5, highlighting an autocrine loop capable of sustaining eosinophil differentiation. These findings suggest that brief exposure to IL-5 is sufficient to initiate a cytokine cooperative network that promotes eosinophil differentiation of low-density bone marrow cells independent of further IL-5 stimulation.

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, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1⌬ cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1⌬ dun1⌬ cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

The purpose of this study was to explore the skin transcriptional profile in pediatric localized scleroderma (LS) to provide a better understanding of the altered immune and fibrotic pathways promoting disease. LS is a progressive disease of the skin and underlying tissue that causes significant functional disability and disfigurement, especially in developing children. RNA sequencing (RNAseq) technology allows for improved understanding of relevant cellular expression through transcriptome analysis of phases during LS disease progression (more active/inflammatory vs. inactive/fibrotic) and also permits the use of RNA extracted from existing paraffin-embedded skin tissue, which is important in pediatrics. A strong correlation was observed between the comparison of genes expressed between fresh (RNAlater) and paraffinized skin in healthy and LS subjects, supporting the use of paraffinized tissue. LS gene signatures compared to healthy controls showed a distinct expression of an inflammatory response gene signature (IRGS) composed of IFNγ-, IFNα-, and TNFα-associated genes. GSEA© enrichment analysis showed that the IRGS, including interferon-inducible chemokines such as CXCL9, CXCL10, CXCL11, and IFNγ itself, was more highly expressed in LS patients with more inflammatory lesions. The use of paraffinized skin for sequencing was proven to be an effective substitute for fresh skin by comparing gene expression profiles. The prevalence of the IFNγ signature in the lesion biopsies of active LS patients indicates that these genes reflect clinical activity parameters and may be the promoters of early, inflammatory disease.

Eosinophils are produced in the bone marrow from CD34+ eosinophil lineage–committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. These findings suggest that increased eosinophil lineage–committed progenitor production is an important process in disease-associated eosinophilia. The pathways central to the biology of the eosinophil lineage–committed progenitor remain largely unknown. Thus, developing new methods to investigate the regulators of eosinophil lineage–committed progenitor differentiation is needed to identify potential therapeutic targets to specifically inhibit eosinophil production. We tested cytokine regimens to optimize liquid cultures for the study of eosinophil lineage–committed progenitor and eosinophil precursor differentiation into mature eosinophils. Stem cell factor (but not fms-related tyrosine kinase 3 ligand) was required for optimal yield of eosinophils. Furthermore, we evaluated the effects of cell preservation and scale on the culture, successfully culturing functional eosinophils from fresh and frozen murine bone marrow cells and in a standard-sized and 96-well culture format. In summary, we have developed an adaptable culture system that yields functionally competent eosinophils from murine low-density bone marrow cells and whose cytokine regime includes expansion of progenitors with stem cell factor alone with subsequent differentiation with interleukin 5.