Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis.

Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8-12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal-ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

The autophagy conundrum in cancer: influence of tumorigenic metabolic reprogramming.

Tumorigenesis is often accompanied by metabolic changes that favor rapid energy production and increased biosynthetic capabilities. These metabolic adaptations promote the survival and proliferation of tumor cells, and in conjunction with the hypoxic and metabolically challenged tumor microenvironment, influence autophagic activity. Autophagy is a catabolic process that allows cellular macromolecules to be broken down and re-utilized as metabolic precursors. Stimulation of autophagy promotes the survival of tumor cells under stressful metabolic and environmental conditions, and counters the potentially deleterious effects of mitochondrial dysfunction and the ROS that these organelles generate. However, inhibition of autophagy has also been reported to fuel tumorigenesis. In spite of the advances in our understanding of the relationship between autophagy and tumorigenesis, it remains unclear whether the therapeutic approaches targeting autophagy should aim to increase or decrease autophagic flux in tumor tissues in human patients. Here, we review how metabolic reprogramming influences autophagic activity in tumors, and discuss how inhibition of autophagy might be exploited to target tumor cells that show altered metabolism.

In vivo generation and characterization of a soluble form of the Semliki forest virus fusion protein.

During infection of host cells, a number of enveloped animal viruses are known to produce soluble forms of viral membrane glycoproteins lacking the transmembrane domain. The roles of such soluble glycoproteins in viral life cycles are incompletely understood, but in several cases they are believed to modulate host immune response and viral pathogenesis. Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells through low-pH-dependent fusion and buds from the plasma membrane. Fusion is mediated by the E1 subunit of the SFV spike protein. Previous studies described the in vivo generation of E1s, a truncated soluble form of E1, under conditions in which budding is inhibited in mammalian host cells. We have here examined the properties of E1s generation and the biological activity of E1s. E1s cleavage required spike protein transport out of the endoplasmic reticulum and was independent of virus infection. Cell surface E1 efficiently acted as a precursor for E1s. E1s generation was strongly pH dependent in BHK cells, with optimal cleavage at a pH of < or =7.0, conditions that inhibited the budding of SFV but not the budding of the rhabdovirus vesicular stomatitis virus. The pH dependence of E1s production and SFV budding was unaffected by the stability of the spike protein dimer but was a function of the host cell. Similar to the intact virus and in vitro-generated E1 ectodomain, treatment of E1s at low pH in the presence of target membranes triggered specific acid-dependent conformational changes. Thus, under a variety of conditions, SFV-infected cells can produce a soluble form of E1 that is biologically active.