Methods for lineage tracing on the organism-wide level

Determining the lineage origin of cell types is a major goal in developmental biology. Furthermore, lineage tracing is a powerful approach for understanding the origin of developmental defects as well as the origin of diseases such as cancer. There is now a variety of complementary approaches for identifying lineage relationships, ranging from direct observation of cell divisions by light microscopy to genetic labeling of cells using inducible recombinases and fluorescent reporters. A recent development, and the main topic of this review article, is the use of high-throughput sequencing data for lineage analysis. This emerging approach holds the promise of increased multiplexing capacity, allowing lineage analysis of large cell numbers up to the organism-wide level combined with simultaneous transcription profiling by single cell RNA sequencing

Massively parallel single cell lineage tracing using CRISPR/Cas9 induced genetic scars

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many different cell types. Single-cell RNAsequencing (scRNA-seq) has become a widely-used method due to its ability to identify all cell types in a tissue or organ in a systematic manner(1-3). However, a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here, we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNAseq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses, LINNAEUS (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences) can be used as a systematic approach for identifying the lineage origin of novel cell types, or of known cell types under different conditions

Mit Einzelzell-Genomik die Entscheidungen von Zellen verfolgen [Deciphering cell fate decisions using single cell genomics]

During embryonic development, cells need to take a series of successive fate decisions in order to reach their final differentiated stage. Understanding the processes that give rise to the multitude of different cell types in an organism is a major question in developmental biology. New methods in single cell genomics enable researchers to decipher the transcriptional programs and gene regulatory mechanisms that underlie cell fate decisions during embryonic development

Patched receptors sense, interpret and establish an epidermal Hedgehog signalling gradient

By employing the sensitivity of single molecule fluorescent in situ hybridisation (smFISH) we have precisely quantified the levels and defined the temporal and spatial distribution of Hedgehog signalling activity during embryonic skin development, and uncovered that there is a Hedgehog signalling gradient along the proximal-distal axis of developing hair follicles. In order to explore the contribution of Hedgehog receptors Ptch1 and Ptch2 in establishing the epidermal signalling gradient, we quantitated the level of pathway activity generated in Ptch1 and Ptch1;Ptch2-deficient skin and defined the contribution of each receptor to regulation of the levels of Hedgehog signalling identified in wild-type skin. Moreover, we show that both the cellular phenotype and level of pathway activity featured in Ptch1;Ptch2-deficient cells faithfully recapitulates the Peak level of endogenous Hedgehog signalling detected at the base of developing follicles, where the concentration of endogenous Shh is predicted to be highest. Taken together, these data demonstrate that both Ptch1 and Ptch2 play a crucial role in sensing the concentration of Hedgehog ligand and regulating the appropriate dose-dependent response

Simultaneous lineage tracing and cell-type identification using CRISPR-Cas9-induced genetic scars

A key goal of developmental biology is to understand how a single cell is transformed into a full-grown organism comprising many different cell types. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ. However, organizing the resulting taxonomy of cell types into lineage trees to understand the developmental origin of cells remains challenging. Here we present LINNAEUS (lineage tracing by nuclease-activated editing of ubiquitous sequences)-a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas, and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions

Licensing of primordial germ cells for gametogenesis depends on genital ridge signaling

In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge

Massively parallel clonal analysis using CRISPR/Cas9 induced genetic scars

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many cells. Although impressive progress has been made in lineage tracing using imaging approaches, analysis of vertebrate lineage trees has mostly been limited to relatively small subsets of cells. Here we present scartrace, a strategy for massively parallel clonal analysis based on Cas9 induced genetic scars in the zebrafish

Variability of an early developmental cell population underlies stochastic laterality defects

Embryonic development seemingly proceeds with almost perfect precision. However, it is largely unknown how much microscopic variability is hidden beneath this macroscopic accuracy. Here, we quantified embryo-to-embryo variability in vertebrate development, by studying cell number variation in the zebrafish endoderm. We noticed that the size of a subpopulation of the endoderm, the dorsal forerunner cells (which later form the left-right organizer), is highly variable between individual embryos. We found that the frequency of left-right laterality defects is increased drastically in embryos with a low number of dorsal forerunner cells, and we observed that these fluctuations are largely nonhereditary. Hence, a stochastic variation in early development leads to a remarkably strong macroscopic phenotype. These fluctuations appear to be caused by variable deposition of maternal factors involved in specification of the dorsal forerunner cells. In summary, we here dissect cause and consequence of embryo-to-embryo variability in a vertebrate model

Long-term imaging reveals behavioral plasticity during C. elegans dauer exit

During their lifetime, animals must adapt their behavior to survive in changing environments. This ability requires the nervous system to adjust through dynamic expression of neurotransmitters and receptors but also through growth, spatial reorganization and connectivity while integrating external stimuli. For instance, despite having a fixed neuronal cell lineage, the nematode Caenorhabditis elegans’ nervous system remains plastic throughout its development. Here, we focus on a specific example of nervous system plasticity, the C. elegans dauer exit decision. Under unfavorable conditions, larvae will enter the non-feeding and non-reproductive dauer stage and adapt their behavior to cope with a new environment. Upon improved conditions, this stress resistant developmental stage is actively reversed to resume reproductive development. However, how different environmental stimuli regulate the exit decision mechanism and thereby drive the larva’s behavioral change is unknown. To fill this gap, we developed a new open hardware method for long-term imaging (12h) of C. elegans larvae. We identified dauer-specific behavioral motifs and characterized the behavioral trajectory of dauer exit in different environments to identify key decision points. Combining long-term behavioral imaging with transcriptomics, we find that bacterial ingestion triggers a change in neuropeptide gene expression to establish post-dauer behavior. Taken together, we show how a developing nervous system can robustly integrate environmental changes, activate a developmental switch and adapt the organism’s behavior to a new environment

Variability of an early developmental cell population underlies stochastic laterality defects

Embryonic development seemingly proceeds with almost perfect precision. However, it is largely unknown how much underlying microscopic variability is compatible with normal development. Here, we quantify embryo-to-embryo variability in vertebrate development by studying cell number variation in the zebrafish endoderm. We notice that the size of a sub-population of the endoderm, the dorsal forerunner cells (DFCs, which later form the left-right organizer), exhibits significantly more embryo-to-embryo variation than the rest of the endoderm. We find that, with incubation of the embryos at elevated temperature, the frequency of left-right laterality defects is increased drastically in embryos with a low number of DFCs. Furthermore, we observe that these fluctuations have a large stochastic component among fish of the same genetic background. Hence, a stochastic variation in early development leads to a remarkably strong macroscopic phenotype. These fluctuations appear to be associated with maternal effects in the specification of the DFCs