ABSTRACT: Developmental signaling inputs are fundamental for shaping cell fates and behavior. However, traditional fluorescent-based signaling reporters have limitations in scalability and molecular resolution of cell types. We present SABER-seq, a CRISPR-Cas molecular recorder that stores transient developmental signaling cues as permanent mutations in cellular genomes for deconstruction at later stages via single-cell transcriptomics. We applied SABER-seq to record Notch signaling in developing zebrafish brains. SABER-seq has two components: a signaling sensor and a barcode recorder. The sensor activates Cas9 in a Notch-dependent manner with inducible control while the recorder obtains mutations in ancestral cells where Notch is active. We combine SABER-seq with an expanded juvenile brain atlas to identify cell types derived from Notch-active founders. Our data reveals rare examples where differential Notch activity in ancestral progenitors is detected in terminally differentiated neuronal subtypes. SABER-seq is a novel platform for rapid, scalable and high-resolution mapping of signaling activity during development.
Project description:Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, such calcium transients trigger pre-synaptic vesicle release and mediate post-synaptic plasticity. It is challenging to capture the molecular events associated with these localized and ephemeral calcium signals, however. Here we report the development of an engineered biotin ligase that combines the power of genetically encoded calcium indicators with protein proximity labeling. The enzyme, Cal-ID, biotinylates nearby proteins in response to elevated local calcium levels. The biotinylated proteins can be visualized by microscopy and identified via mass spectrometry. Cal-ID mass spectrometry applied to HEK293T cells identified cell cycle-dependent calcium signaling microdomains at centrosomes. Our results from mouse primary neurons indicated active calcium signaling near calcium extrusion sites on the plasma membrane. Therefore, we propose Cal-ID as a biochemical recorder of calcium signaling in living cells.
Project description:We have sequenced messenger RNA isolated from seedling tissue for 19 accessions of Arabidopsis thaliana (with biological replication). The 19 accessions for which RNA-Seq reads were collected have served as the founders for the MAGIC lines, a high-resolution recombinant inbred line mapping resource. RNA sequencing data was used to examine differential gene expression among the accessions.
Project description:We have sequenced messenger RNA isolated from seedling tissue for 19 accessions of Arabidopsis thaliana (with biological replication). The 19 accessions for which RNA-Seq reads were collected have served as the founders for the MAGIC lines, a high-resolution recombinant inbred line mapping resource. RNA sequencing data was used to examine differential gene expression among the accessions. Examination of RNA expression in seedling tissue carried out using biological replicates.
Project description:The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ~380,000 CRISPR target sites and reconstruct dissemination of ~28,000 single cells across multiple metastatic sites. We find cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for in vivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.
Project description:Summary
Extracellular vesicles (EVs) facilitate intercellular communication by transferring cargo between cells in a variety of tissues. However, how EVs achieve cell type-specific intercellular communication is still largely unknown. We found that Notch1 and Notch2 proteins are expressed on the surface of neuronal EVs that have been generated in response to neuronal excitatory synaptic activity. Notch ligands bind these EVs on the neuronal plasma membrane, trigger their internalization, activate the Notch signaling pathway, and drive the expression of Notch target genes. The generation of these neuronal EVs requires the ESCRT-associated protein Alix. Adult Alix conditional knockout mice have reduced hippocampal Notch signaling activation and glutamatergic synaptic protein expression. Thus, EVs facilitate neuron to neuron communication via the Notch receptor-ligand system in the brain.
Project description:Loss or damage to the mandible due to trauma, treatment of oral malignancies, and other diseases is currently treated using bone grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to their mandibular bone, and thus offer an opportunity to understand how to boost intrinsic healing ability. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes. We also show that modulation of Notch signaling during the initial postoperative period results in lasting changes to regenerate bone quantity one month later. Notch signaling is required for mandibular bone healing, as pharmacological inhibition of Notch signaling blocks cartilage callus formation and results in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates regenerative ossification. Mechanistically, we report that postoperative Notch signaling regulates multiple phases of chondroid regeneration and patterns callus metabolic landscape. Given conserved functions of Notch signaling in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing may have therapeutic value.