Genetic Tools for Cell Lineage Tracing and Profiling Developmental Trajectories in the Skin

Abstract

The epidermis is the body's first line of protection against dehydration and pathogens, continually regenerating the outermost protective skin layers throughout life. During both embryonic development and wound healing, epidermal stem and progenitor cells must respond to external stimuli and insults to build, maintain, and repair the cutaneous barrier. Recent advances in CRISPR-based methods for cell lineage tracing have remarkably expanded the potential for experiments that track stem and progenitor cell proliferation and differentiation over the course of tissue and even organismal development. Additional tools for DNA-based recording of cellular signaling cues promise to deepen our understanding of the mechanisms driving normal skin morphogenesis and response to stressors as well as the dysregulation of cell proliferation and differentiation in skin diseases and cancer. In this review, we highlight cutting-edge methods for cell lineage tracing, including in organoids and model organisms, and explore how cutaneous biology researchers might leverage these techniques to elucidate the developmental programs that support the regenerative capacity and plasticity of the skin.

Keywords: CRISPR; DNA barcoding; DNA typewriter; Genome editing; Prime editor.

Figure 1:. Dynamic DNA barcoding enhances lineage tracing capacity and can be multiplexed with single-cell transcriptomics.

(a) Static barcoding introduces a unique barcode per cell in a population during a single labeling event (e.g., lentiviral transduction); the founder cell’s barcode is then stably passed on to all its progeny. (b) Dynamic barcoding continually diversifies a DNA barcode over multiple generations of cells to permit reconstruction of a more complex lineage tree. (c) Dynamic lineage tracing in tissues like the epidermis requires introduction of a genome editor (e.g., Cas9), an array of DNA targets that become the edited barcode, and guide RNA(s) to direct the genome editor to the targets; the lineage of all cells within the tissue can be deciphered by the similarity of their final edited barcode. (d) By embedding a DNA barcode (BC) under the control of a promoter that recruits RNA polymerase-II (e.g., placing the barcode within the 5’ or 3’ UTR of a fluorescent protein or other selection marker driven by a constitutive promoter), the barcode itself is transcribed into mRNA including a poly-A tail; thus, when capturing the entire transcriptome with a poly-dT primer in scRNAseq, the barcode is read along with all other mRNAs so that a cell’s transcriptional state can be captured along with its lineage.

Figure 2:. Prime editing-based lineage tracing records the order of DNA edits and events linked to edits.

(a) Prime editing-based lineage tracing can record the order in which edits were introduced by using a synthetic DNA target introduced into the cell’s genome. DNA Typewriter uses a tandem array of identical prime editing target sites in which all but the first in the series are truncated to prevent editing. The prime editing guide RNA (pegRNA) edits the first target site with high specificity; the resulting edit disrupts the first target site (preventing subsequent editing) and completes (unlocks) the second target site so that it is now recognized by the pegRNA; similar editing and unlocking of the subsequent target sites occurs such that cells accumulate defined DNA edits in a sequentially ordered fashion within the barcode. (b) Prime editing-based recording methods chronicle the temporal order of biological events by linking production of a pegRNA to a specific signal or event (e.g., Wnt/β-catenin signal activity, phage infection, light exposure, or other signals of interest); pegRNAs are placed under transcriptional control of a signal/event-responsive promoter and encode a specific edit to the barcode when a biological event of interest is experienced by the cell. Using DNA Typewriter, biological events are thus sequentially recorded into the editing target, allowing for reconstruction of event order based on the final edited sequence of the barcode.

Figure 3:. Cas9-based lineage tracing can map the fate of progenitor cells during the development of organoids, organs, and whole organisms.

Cas9-based methods dynamically generate diverse barcodes over many cellular generations and have been applied to map the development of whole zebrafish and its organs, mouse embryogenesis and hematopoiesis, and human induced pluripotent stem cell (iPSC)-derived cerebroids. An array of Cas9 targets (often linked to a transcribed fluorescent protein [FP]) and gRNAs are delivered into stem or progenitor cells; subsequent editing generates the barcode diversity needed for complex lineage tracing; importantly, the Cas9 editor, itself, can either be constitutively or inducibly expressed (e.g., under tetracycline- or heat shock-inducible promoters) to allow temporal control over when editing occurs during development. Such technology could be applied to animal or organoid models of skin to comprehensively map the developmental plan of the epidermis and its appendages.

Figure 4:. Lineage tracing in cancer models can reveal key oncogenic steps and drivers of metastatic behavior.

(a) Lineage tracing allows investigators to track a cancer progenitor cell through its clonal expansion, epithelial-to-mesenchymal transition (EMT), and leaving the site of origin through the vasculature to metastasize, seed, and grow in distant sites. The resultant barcode sequence at secondary metastatic sites can identify the clone of origin and can be coupled with transcriptomic analysis to understand the critical drivers of its metastatic behavior, which could dictate the therapeutic strategy. (b) Cancer cells can be engineered to express the machinery for DNA editing-based lineage tracing studies; this approach has revealed the origins and drivers of metastases in mouse xenograft models of lung and pancreatic carcinomas and could be adapted to study metastatic behavior in melanoma or other invasive skin cancers.