Deconvolving organogenesis in space and time via spatial transcriptomics in thick tissues.

Organ development is guided by a space-time landscape that constraints cell behavior. This landscape is challenging to characterize for the hair follicle - the most abundant mini organ - due to its complex microscopic structure and asynchronous development. We developed 3DEEP, a tissue clearing and spatial transcriptomic strategy for characterizing tissue blocks up to 400 µm in thickness. We captured 371 hair follicles at different stages of organogenesis in 1 mm3 of skin of a 12-hour-old mouse with 6 million transcripts from 81 genes. From this single time point, we deconvoluted follicles by age based on whole-organ molecular pseudotimes to animate a stop-motion 3D atlas of follicle development along its trajectory. We defined molecular stages for hair follicle organogenesis and characterized the order of emergence for its structures, differential signaling dynamics at its top and bottom, morphogen shifts preceding and accompanying structural changes, and series of structural changes leading to the formation of its canal and opening. We further found that hair follicle stem cells and their niche are established and stratified early in organogenesis, before the formation of the hair bulb. Overall, this work demonstrates the power of increased depth of spatial transcriptomics to provide a four-dimensional analysis of organogenesis.

Temporal recording of mammalian development and precancer.

Temporal ordering of cellular events offers fundamental insights into biological phenomena. Although this is traditionally achieved through continuous direct observations1,2, an alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible marks that enables retrospective temporal ordering3-5. Using a multipurpose, single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, with incorporation of cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during mouse embryonic development, unconventional developmental relationships between cell types and new epithelial progenitor states by their unique genetic histories. Analysis of mouse adenomas, coupled to multiomic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyclonal initiation in 15-30% of colonic precancers, showing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses, to explore the origins and timing of development and tumorigenesis in mammalian systems.

The use of ectopic volar fibroblasts to modify skin identity.

Skin identity is controlled by intrinsic features of the epidermis and dermis and their interactions. Modifying skin identity has clinical potential, such as the conversion of residual limb and stump (nonvolar) skin of amputees to pressure-responsive palmoplantar (volar) skin to enhance prosthesis use and minimize skin breakdown. Greater keratin 9 (KRT9) expression, higher epidermal thickness, keratinocyte cytoplasmic size, collagen length, and elastin are markers of volar skin and likely contribute to volar skin resiliency. Given fibroblasts' capacity to modify keratinocyte differentiation, we hypothesized that volar fibroblasts influence these features. Bioprinted skin constructs confirmed the capacity of volar fibroblasts to induce volar keratinocyte features. A clinical trial of healthy volunteers demonstrated that injecting volar fibroblasts into nonvolar skin increased volar features that lasted up to 5 months, highlighting a potential cellular therapy.

Identifying a gene signature of metastatic potential by linking pre-metastatic state to ultimate metastatic fate.

Identifying the key molecular pathways that enable metastasis by analyzing the eventual metastatic tumor is challenging because the state of the founder subclone likely changes following metastatic colonization. To address this challenge, we labeled primary mouse pancreatic ductal adenocarcinoma (PDAC) subclones with DNA barcodes to characterize their pre-metastatic state using ATAC-seq and RNA-seq and determine their relative in vivo metastatic potential prospectively. We identified a gene signature separating metastasis-high and metastasis-low subclones orthogonal to the normal-to-PDAC and classical-to-basal axes. The metastasis-high subclones feature activation of IL-1 pathway genes and high NF-κB and Zeb/Snail family activity and the metastasis-low subclones feature activation of neuroendocrine, motility, and Wnt pathway genes and high CDX2 and HOXA13 activity. In a functional screen, we validated novel mediators of PDAC metastasis in the IL-1 pathway, including the NF-κB targets Fos and Il23a, and beyond the IL-1 pathway including Myo1b and Tmem40. We scored human PDAC tumors for our signature of metastatic potential from mouse and found that metastases have higher scores than primary tumors. Moreover, primary tumors with higher scores are associated with worse prognosis. We also found that our metastatic potential signature is enriched in other human carcinomas, suggesting that it is conserved across epithelial malignancies. This work establishes a strategy for linking cancer cell state to future behavior, reveals novel functional regulators of PDAC metastasis, and establishes a method for scoring human carcinomas based on metastatic potential.

A Rigorous Framework to Classify the Postduplication Fate of Paralogous Genes.

Gene duplication has a central role in evolution; still, little is known on the fates of the duplicated copies, their relative frequency, and on how environmental conditions affect them. Moreover, the lack of rigorous definitions concerning the fate of duplicated genes hinders the development of a global vision of this process. In this paper, we present a new framework aiming at characterizing and formally differentiating the fate of duplicated genes. Our framework has been tested via simulations, where the evolution of populations has been simulated using aevol, an in silico experimental evolution platform. Our results show several patterns that confirm some of the conclusions from previous studies, while also exhibiting new tendencies; this may open up new avenues to better understand the role of duplications as a driver of evolution.

Post-translation digital data encoding into the genomes of mammalian cell populations.

High resolution cellular signal encoding is critical for better understanding of complex biological phenomena. DNA-based biosignal encoders alter genomic or plasmid DNA in a signal dependent manner. Current approaches involve the signal of interest affecting a DNA edit by interacting with a signal specific promoter which then results in expression of the effector molecule (DNA altering enzyme). Here, we present the proof of concept of a biosignal encoding system where the enzyme terminal deoxynucleotidyl transferase (TdT) acts as the effector molecule upon directly interacting with the signal of interest. A template independent DNA polymerase (DNAp), TdT incorporates nucleotides at the 3' OH ends of DNA substrate in a signal dependent manner. By employing CRISPR-Cas9 to create double stranded breaks in genomic DNA, we make 3'OH ends available to act as substrate for TdT. We show that this system can successfully resolve and encode different concentrations of various biosignals into the genomic DNA of HEK-293T cells. Finally, we develop a simple encoding scheme associated with the tested biosignals and encode the message "HELLO WORLD" into the genomic DNA of HEK-293T cells at a population level with 91% accuracy. This work demonstrates a simple and engineerable system that can reliably store local biosignal information into the genomes of mammalian cell populations.

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Temporal recording of mammalian development and precancer.

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo , while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding.

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.

Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites.

Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

Recording Temporal Signals with Minutes Resolution Using Enzymatic DNA Synthesis.

Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with subhour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3'-ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals such as Co2+, Ca2+, and Zn2+ concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co2+ concentration to within 1 min over a 60 min period. Finally, we engineer TdT to allosterically turn off in the presence of a physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single-step change in the Ca2+ signal to within 1 min over a 60 min period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with a resolution of minutes.

Lineage barcoding in mice with homing CRISPR.

Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.

Terminator-free template-independent enzymatic DNA synthesis for digital information storage.

DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA.

Developmental barcoding of whole mouse via homing CRISPR.

In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation resulted in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We used these recordings for reliable post hoc reconstruction of the earliest lineages and investigation of axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.

Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae.

Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.

Rapidly evolving homing CRISPR barcodes.

We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9-hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization.

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Highly multiplexed subcellular RNA sequencing in situ.

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

Physical principles for scalable neural recording.

Simultaneously measuring the activities of all neurons in a mammalian brain at millisecond resolution is a challenge beyond the limits of existing techniques in neuroscience. Entirely new approaches may be required, motivating an analysis of the fundamental physical constraints on the problem. We outline the physical principles governing brain activity mapping using optical, electrical, magnetic resonance, and molecular modalities of neural recording. Focusing on the mouse brain, we analyze the scalability of each method, concentrating on the limitations imposed by spatiotemporal resolution, energy dissipation, and volume displacement. Based on this analysis, all existing approaches require orders of magnitude improvement in key parameters. Electrical recording is limited by the low multiplexing capacity of electrodes and their lack of intrinsic spatial resolution, optical methods are constrained by the scattering of visible light in brain tissue, magnetic resonance is hindered by the diffusion and relaxation timescales of water protons, and the implementation of molecular recording is complicated by the stochastic kinetics of enzymes. Understanding the physical limits of brain activity mapping may provide insight into opportunities for novel solutions. For example, unconventional methods for delivering electrodes may enable unprecedented numbers of recording sites, embedded optical devices could allow optical detectors to be placed within a few scattering lengths of the measured neurons, and new classes of molecularly engineered sensors might obviate cumbersome hardware architectures. We also study the physics of powering and communicating with microscale devices embedded in brain tissue and find that, while radio-frequency electromagnetic data transmission suffers from a severe power-bandwidth tradeoff, communication via infrared light or ultrasound may allow high data rates due to the possibility of spatial multiplexing. The use of embedded local recording and wireless data transmission would only be viable, however, given major improvements to the power efficiency of microelectronic devices.