Organ homeostasis is orchestrated by time- and spatially restricted cell proliferation. Studies identifying cells with superior proliferative capacities often rely on the lineage tracing of a subset ...of cell populations, which introduces a potential selective bias. In this work, we developed a genetic system proliferation tracer (ProTracer) by incorporating dual recombinases to seamlessly record the proliferation events of entire cell populations over time in multiple organs. In the mouse liver, ProTracer revealed more hepatocyte proliferation in distinct zones during liver homeostasis, injury repair, and regrowth. Clonal analysis showed that most of the hepatocytes labeled by ProTracer had undergone cell division. By genetically recording proliferation events of entire cell populations, ProTracer enables the unbiased detection of specific cellular compartments with enhanced regenerative capacities.
Abstract
Cardiac regeneration involves the generation of new cardiomyocytes from cycling cardiomyocytes. Understanding cell-cycle activity of pre-existing cardiomyocytes provides valuable information ...to heart repair and regeneration. However, the anatomical locations and in situ dynamics of cycling cardiomyocytes remain unclear. Here we develop a genetic approach for a temporally seamless recording of cardiomyocyte-specific cell-cycle activity in vivo. We find that the majority of cycling cardiomyocytes are positioned in the subendocardial muscle of the left ventricle, especially in the papillary muscles. Clonal analysis revealed that a subset of cycling cardiomyocytes have undergone cell division. Myocardial infarction and cardiac pressure overload induce regional patterns of cycling cardiomyocytes. Mechanistically, cardiomyocyte cell cycle activity requires the Hippo pathway effector YAP. These genetic fate-mapping studies advance our basic understanding of cardiomyocyte cell cycle activity and generation in cardiac homeostasis, repair, and regeneration.
The Cre-loxP recombination system is the most widely used technology for in vivo tracing of stem or progenitor cell lineages. The precision of this genetic system largely depends on the specificity ...of Cre recombinase expression in targeted stem or progenitor cells. However, Cre expression in nontargeted cell types can complicate the interpretation of lineage-tracing studies and has caused controversy in many previous studies. Here we describe a new genetic lineage tracing system that incorporates the Dre-rox recombination system to enhance the precision of conventional Cre-loxP-mediated lineage tracing. The Dre-rox system permits rigorous control of Cre-loxP recombination in lineage tracing, effectively circumventing potential uncertainty of the cell-type specificity of Cre expression. Using this new system we investigated two topics of recent debates-the contribution of c-Kit
cardiac stem cells to cardiomyocytes in the heart and the contribution of Sox9
hepatic progenitor cells to hepatocytes in the liver. By overcoming the technical hurdle of nonspecific Cre-loxP-mediated recombination, this new technology provides more precise analysis of cell lineage and fate decisions and facilitates the in vivo study of stem and progenitor cell plasticity in disease and regeneration.
Organs of the body require vascular networks to supply oxygen and nutrients and maintain physiological function. The blood vessels of different organs are structurally and functionally heterogeneous ...in nature. To more precisely dissect their distinct in vivo function in individual organs, without potential interference from off-site targets, it is necessary to genetically target them in an organ-specific manner.
The objective of this study was to generate a genetic system that targets vascular endothelial cells in an organ- or tissue-specific manner and to exemplify the potential application of intersectional genetics for precise, target-specific gene manipulation in vivo.
We took advantage of 2 orthogonal recombination systems, Dre-rox and Cre-loxP, to create a genetic targeting system based on intersectional genetics. Using this approach, Cre activity was only detectable in cells that had expressed both Dre and Cre. Applying this new system, we generated a coronary endothelial cell-specific Cre (
) and a brain endothelial cell-specific Cre (
). Through lineage tracing, gene knockout and overexpression experiments, we demonstrated that
and
efficiently and specifically target blood vessels in the heart and brain, respectively. By deletion of vascular endothelial growth factor receptor 2 using
, we showed that vascular endothelial growth factor signaling regulates angiogenesis in the central nervous system and also controls the integrity of the blood-brain barrier.
We provide 2 examples to illustrate the use of intersectional genetics for more precise gene targeting in vivo, namely manipulation of genes in blood vessels of the heart and brain. More broadly, this system provides a valuable strategy for tissue-specific gene manipulation that can be widely applied to other fields of biomedical research.
The liver has remarkable capability to regenerate, employing mechanism to ensure the stable liver-to-bodyweight ratio for body homeostasis. The source of this regenerative capacity has received great ...attention over the past decade yet still remained controversial currently. Deciphering the sources for hepatocytes provides the basis for understanding tissue regeneration and repair, and also illustrates new potential therapeutic targets for treating liver diseases. In this review, we describe recent advances in genetic lineage tracing studies over liver stem cells, hepatocyte proliferation, and cell lineage conversions or cellular reprogramming. This review will also evaluate the technical strengths and limitations of methods used for studies on hepatocyte generation and cell fate plasticity in liver homeostasis, repair and regeneration.
Erasure-code-based storage systems suffer from problems such as long repair time and low I/O performance, resulting in high repair costs. For many years, researchers have focused on reducing the cost ...of repairing erasure-code-based storage systems. In this study, we discuss the demerits of node selecting, data transferring and data repair in erasure-code-based storage systems. Based on the network topology and node structure, we propose SDNC-Repair, a cooperative data repair strategy based on erasure code for SDS (Software Defined Storage), and describe its framework. Then, we propose a data source selection algorithm that senses the available network bandwidth between nodes and a data flow scheduling algorithm in SDNC-Repair. Additionally, we propose a data repair method based on node collaboration and data aggregation. Experiments illustrate that the proposed method has better repair performance under different data granularities. Compared to the conventional repair method, although the SDNC-Repair is more constrained by the cross-rack bandwidth, it improves system throughput effectively and significantly reduces data repair time in scenarios where multiple nodes fail and bandwidth is limited.
The ubiquitination status of RIPK1 is considered to be critical for cell fate determination. However, the in vivo role for RIPK1 ubiquitination remains undefined. Here we show that mice expressing ...RIPK1
which is defective in RIPK1 ubiquitination die during embryogenesis. This lethality is fully rescued by concomitant deletion of Fadd and Ripk3 or Mlkl. Mechanistically, cells expressing RIPK1
are more susceptible to TNF-α induced apoptosis and necroptosis with more complex II formation and increased RIPK1 activation, which is consistent with the observation that Ripk1
lethality is effectively prevented by treatment of RIPK1 kinase inhibitor and is rescued by deletion of Tnfr1. However, Tnfr1
Ripk1
mice display systemic inflammation and die within 2 weeks. Significantly, this lethal inflammation is rescued by deletion of Ripk3. Taken together, these findings reveal a critical role of Lys376-mediated ubiquitination of RIPK1 in suppressing RIPK1 kinase activity-dependent lethal pathways during embryogenesis and RIPK3-dependent inflammation postnatally.
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