Mast cell lineage diversion of T lineage precursors by the essential T cell transcription factor GATA-3

GATA-3 is essential for T cell development from the earliest stages. However, abundant GATA-3 can drive T lineage precursors to a non–T cell fate, depending on Notch signaling and developmental stage. Here, overexpression of GATA-3 blocked the survival of pro–T cells when Notch-Delta signals were present but enhanced viability in their absence. In fetal thymocytes at the double-negative 1 (DN1) stage and DN2 stage but not those at the DN3 stage, overexpression of GATA-3 rapidly induced respecification to the mast cell lineage with high frequency by direct transcriptional 'reprogramming'. Normal DN2 thymocytes also showed mast cell potential when interleukin 3 and stem cell factor were added in the absence of Notch signaling. Our results suggest a close relationship between the pro–T cell and mast cell programs and a previously unknown function for Notch in T lineage fidelity

Stochastic Cytokine Expression Induces Mixed T Helper Cell States

During eukaryotic development, the induction of a lineage-specific transcription factor typically drives differentiation of multipotent progenitor cells, while repressing that of alternative lineages. This process is often mediated by some extracellular signaling molecules, such as cytokines that can bind to cell surface receptors, leading to activation and/or repression of transcription factors. We explored the early differentiation of naive CD4 T helper (Th) cells into Th1 versus Th2 states by counting single transcripts and quantifying immunofluorescence in individual cells. Contrary to mutually exclusive expression of antagonistic transcription factors, we observed their ubiquitous co-expression in individual cells at high levels that are distinct from basal-level co-expression during lineage priming. We observed that cytokines are expressed only in a small subpopulation of cells, independent from the expression of transcription factors in these single cells. This cell-to-cell variation in the cytokine expression during the early phase of T helper cell differentiation is significantly larger than in the fully differentiated state. Upon inhibition of cytokine signaling, we observed the classic mutual exclusion of antagonistic transcription factors, thus revealing a weak intracellular network otherwise overruled by the strong signals that emanate from extracellular cytokines. These results suggest that during the early differentiation process CD4 T cells acquire a mixed Th1/Th2 state, instructed by extracellular cytokines. The interplay between extracellular and intracellular signaling components unveiled in Th1/Th2 differentiation may be a common strategy for mammalian cells to buffer against noisy cytokine expression.National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874)National Institutes of Health (U.S.) (Pioneer Award)National Institutes of Health (U.S.) (Grant R01-GM068957

Bottom-Water Conditions in a Marine Basin after the Cretaceous–Paleogene Impact Event: Timing the Recovery of Oxygen Levels and Productivity

An ultra-high-resolution analysis of major and trace element contents from the Cretaceous–Paleogene boundary interval in the Caravaca section, southeast Spain, reveals a quick recovery of depositional conditions after the impact event. Enrichment/depletion profiles of redox sensitive elements indicate significant geochemical anomalies just within the boundary ejecta layer, supporting an instantaneous recovery –some 102 years– of pre-impact conditions in terms of oxygenation. Geochemical redox proxies point to oxygen levels comparable to those at the end of the Cretaceous shortly after impact, which is further evidenced by the contemporary macrobenthic colonization of opportunistic tracemakers. Recovery of the oxygen conditions was therefore several orders shorter than traditional proposals (104–105 years), suggesting a probable rapid recovery of deep-sea ecosystems at bottom and in intermediate waters.This research was supported by Projects CGL2009-07603, CGL2008-03007, CGL2012-33281 and CGL2012-32659 (Secretaría de Estado de I+D+I, Spain), Projects RNM-3715 and RNM 05212, and Research Groups RNM-178 and 0179 (Junta de Andalucía)

Human cord blood derived immature basophils show dual characteristics, expressing both basophil and eosinophil associated proteins

Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FceRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cellspecific transcripts, i.e. b-tryptase, serglycin and FceRI a-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development. The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo