Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.

In C. elegans, hydrogen sulfide (H2S) exposure results in Hif-1 stabilization. hif-1 is required for survival in H2S and constitutive HIF-1 stabilization confers resistance to H2S. H2S-induced HIF-1 reporter activity appears to be independent of VHL-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity.

Hydrogen sulfide (H2S), an endogenously produced small molecule, protects animals from various stresses. Recent studies demonstrate that animals exposed to H2S are long lived, resistant to hypoxia, and resistant to ischemia–reperfusion injury. We performed a forward genetic screen to gain insights into the molecular mechanisms Caenorhabditis elegans uses to appropriately respond to H2S. At least two distinct pathways appear to be important for this response, including the H2S-oxidation pathway and the hydrogen cyanide (HCN)-assimilation pathway. The H2S-oxidation pathway requires two distinct enzymes important for the oxidation of H2S: the sulfide:quinone reductase sqrd-1 and the dioxygenase ethe-1. The HCN-assimilation pathway requires the cysteine synthase homologs cysl-1 and cysl-2. A low dose of either H2S or HCN can activate hypoxia-inducible factor 1 (HIF-1), which is required for C. elegans to respond to either gas. sqrd-1 and cysl-2 represent the entry points in the H2S-oxidation and HCN-assimilation pathways, respectively, and expression of both of these enzymes is highly induced by HIF-1 in response to both H2S and HCN. In addition to their role in appropriately responding to H2S and HCN, we found that cysl-1 and cysl-2 are both essential mediators of innate immunity against fast paralytic killing by Pseudomonas. Furthermore, in agreement with these data, we showed that growing worms in the presence of H2S is sufficient to confer resistance to Pseudomonas fast paralytic killing. Our results suggest the hypoxia-independent hif-1 response in C. elegans evolved to respond to the naturally occurring small molecules H2S and HCN.