Resolving Fates and Single-Cell Transcriptomes of Hematopoietic Stem Cell Clones by PolyloxExpress Barcoding.

Lineage tracing reveals hematopoietic stem cell (HSC) fates, while single-cell RNA sequencing identifies snapshots of HSC transcriptomes. To obtain information on fate plus transcriptome in the same cell, we developed the PolyloxExpress allele, enabling Cre-recombinase-dependent RNA barcoding in situ. Linking fates to single HSC transcriptomes provided the information required to identify transcriptional signatures of HSC fates, which were not apparent in single-HSC transcriptomes alone. We find that differentiation-inactive, multilineage, and lineage-restricted HSC clones reside in distinct regions of the transcriptional landscape of hematopoiesis. Differentiation-inactive HSC clones are closer to the origin of the transcriptional trajectory, yet they are not characterized by a quiescent gene signature. Fate-specific gene signatures imply coherence of clonal HSC fates, and HSC output toward short-lived lineage progenitors indicates stability of HSC fates over time. These combined analyses of fate and transcriptome under physiological conditions may pave the way toward identifying molecular determinants of HSC fates.

Using Cre-recombinase-driven Polylox barcoding for in vivo fate mapping in mice.

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox. In Polylox, random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice (Rosa26Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox, including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR-Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2-3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis.

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

LlFH1-mediated interaction between actin fringe and exocytic vesicles is involved in pollen tube tip growth.

Pollen tube tip growth is an extreme form of polarized cell growth, which requires polarized exocytosis based on a dynamic actin cytoskeleton. However, the molecular basis for the connection between actin filaments and exocytic vesicles is unclear. Here, we identified a Lilium longiflorum pollen-specific formin (LlFH1) and found that it localized at the apical vesicles and plasma membrane (PM). Overexpression of LlFH1 induced excessive actin cables in the tube tip region, and downregulation of LlFH1 eliminated the actin fringe. Fluorescence recovery after photobleaching (FRAP) analysis revealed that LlFH1-labeled exocytic vesicles exhibited an initial accumulation at the shoulder of the apex and coincided with the leading edge of the actin fringe. Time-lapse analysis suggested that nascent actin filaments followed the emergence of the apical vesicles, implying that LlFH1 at apical vesicles could initiate actin polymerization. Biochemical assays showed that LlFH1 FH1FH2 could nucleate actin polymerization, but then capped the actin filament at the barbed end and inhibited its elongation. However, in the presence of lily profilins, LlFH1 FH1FH2 could accelerate barbed-end actin elongation. In addition, LlFH1 FH1FH2 was able to bundle actin filaments. Thus, we propose that LlFH1 and profilin coordinate the interaction between the actin fringe and exocytic vesicle trafficking during pollen tube growth of lily.

FIMBRIN1 is involved in lily pollen tube growth by stabilizing the actin fringe.

An actin fringe structure in the subapex plays an important role in pollen tube tip growth. However, the precise mechanism by which the actin fringe is generated and maintained remains largely unknown. Here, we cloned a 2606-bp full-length cDNA encoding a deduced 77-kD fimbrin-like protein from lily (Lilium longiflorum), named FIMBRIN1 (FIM1). Ll-FIM1 was preferentially expressed in pollen and concentrated at actin fringe in the subapical region, as well as in longitudinal actin-filament bundles in the shank of pollen tubes. Microinjection of Ll-FIM1 antibody into lily pollen tubes inhibited tip growth and disrupted the actin fringe. Furthermore, we verified the function of Ll-FIM1 in the fim5 mutant of its closest relative, Arabidopsis thaliana. Pollen tubes of fim5 mutants grew with a larger diameter in early stages but could recover into normal forms in later stages, despite significantly slower growth rates. The actin fringe of the fim5 mutants, however, was impaired during both early and late stages. Impressively, stable expression of fim5pro:GFP:Ll-FIM1 rescued the actin fringe and the growth rate of Arabidopsis fim5 pollen tubes. In vitro biochemical analysis showed that Ll-FIM1 could bundle actin filaments. Thus, our study has identified a fimbrin that may stabilize the actin fringe by cross-linking actin filaments into bundles, which is important for proper tip growth of lily pollen tubes.

Control of the actin cytoskeleton in root hair development.

The development of root hair includes four stages: bulge site selection, bulge formation, tip growth, and maturation. The actin cytoskeleton is involved in all of these stages and is organized into distinct arrangements in the different stages. In addition to the actin configuration, actin isoforms also play distinct roles in the different stages. The actin cytoskeleton is regulated by actin-binding proteins, such as formin, Arp2/3 complex, profilin, actin depolymerizing factor, and villin. Some upstream signals, i.e. calcium, phospholipids, and small GTPase regulate the activity of these actin-binding proteins to produce the proper actin configuration. We constructed a working model on how the actin cytoskeleton is controlled by actin-binding proteins and upstream signaling in root hair development based on the current literature: at the tip of hairs, actin polymerization appears to be facilitated by Arp2/3 complex that is activated by small GTPase, and profilin that is regulated by phosphatidylinositol 4,5-bisphosphate. Meanwhile, actin depolymerization and turnover are likely mediated by villin and actin depolymerizing factor, which are stimulated by calcium. At the shank, actin cables are produced by formin and villin. Under the complicated interaction, the actin cytoskeleton is controlled spatially and temporally during root hair development.