How an organism detects organ injury to initiate regeneration remains one of the central unresolved questions in regenerative biology. The liver is the only solid organ in mammals capable of complete regeneration, a process initiated by hepatocyte growth factor ( Hgf ) upregulation, yet the mechanism linking liver injury to Hgf induction has been unknown. Here we identify an albumin-based sensing mechanism that continuously reports the liver's functional status to hepatic stellate cells to gate the regenerative program. Using single-molecule RNA FISH, parabiosis, and an ex vivo plasma assay, we show that stellate cells are the predominant source of Hgf and that a circulating, protein-dependent signal suppresses Hgf expression when the liver is functional. Biochemical fractionation, immunodepletion, and albumin knockout mice together demonstrate that this suppressive signal is a molecule carried by albumin rather than albumin itself. Untargeted metabolomics identified retinol as the albumin-associated suppressive molecule, which we confirm is sufficient to restore Hgf suppression in injured liver plasma. Conversely, long-chain fatty acids that rise after hepatectomy, specifically palmitate and linoleate, which compete for the same albumin binding site, are sufficient to derepress Hgf in healthy plasma. These data support a dual-input model in which albumin functions as an AND gate, integrating retinol loss and fatty acid gain as coincident indicators of liver injury before permitting Hgf derepression. Consistent with this model, acute in vivo knockdown of albumin is sufficient to derepress Hgf in stellate cells and drive liver overgrowth. These findings establish albumin as an active integrator of physiological signals that governs tissue-scale regenerative decisions and reveal that the liver is constitutively poised to regenerate, gatekept by signals that directly reflect its own functional status.

The inability of most human organs to regenerate themselves after injury underlies the lifelong morbidity of numerous diseases. As we continue to seek solutions for these intractable conditions, the liver emerges as an inspiring and informative exception. The liver is the only solid organ that can completely regenerate itself. At the core of this extraordinary feat of organ physiology lie two equally exceptional features of cell biology. First, liver regeneration is driven not by stem cells, but rather by the proliferation of the liver's differentiated cells. Second, many of these liver cells are polyploid, yet still able to execute proper cell division. Understanding how liver cells maintain proliferative capacity as differentiated cells and how they execute mitosis faithfully in a polyploid state could offer powerful insights toward engineering regenerative capacity in other organs. The liver thus offers not only proof that mammalian organ regeneration is possible, but also a blueprint for achieving this long-standing goal of regenerative medicine.

A complete understanding of the genetic determinants underlying mammalian physiology and disease is limited by the capacity for high-throughput genetic dissection in the living organism. Genome-wide CRISPR screening is a powerful method for uncovering the genetic regulation of cellular processes, but the need to stably deliver single guide RNAs to millions of cells has largely restricted its implementation to ex vivo systems. There thus remains a need for accessible high-throughput functional genomics in vivo. Here, we establish genome-wide screening in the liver of a single mouse and use this approach to uncover regulation of hepatocyte fitness. We uncover pathways not identified in cell culture screens, underscoring the power of genetic dissection in the organism. The approach we developed is accessible, scalable, and adaptable to diverse phenotypes and applications. We have hereby established a foundation for high -throughput functional genomics in a living mammal, enabling comprehensive investigation of physiology and disease.

Much of our understanding of chromosome segregation is based on cell culture systems. Here, we examine the importance of the tissue environment for chromosome segregation by comparing chromosome segregation fidelity across several primary cell types in native and nonnative contexts. We discover that epithelial cells have increased chromosome missegregation outside of their native tissues. Using organoid culture systems, we show that tissue architecture, specifically integrin function, is required for accurate chromosome segregation. We find that tissue architecture enhances the correction of merotelic microtubule-kinetochore attachments, and this is especially important for maintaining chromosome stability in the polyploid liver. We propose that disruption of tissue architecture could underlie the widespread chromosome instability across epithelial cancers. Moreover, our findings highlight the extent to which extracellular context can influence intrinsic cellular processes and the limitations of cell culture systems for studying cells that naturally function within a tissue.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Aneuploidy, a state of karyotype imbalance, is a hallmark of cancer. Changes in chromosome copy number have been proposed to drive disease by modulating the dosage of cancer driver genes and by promoting cancer genome evolution. Given the potential of cells with abnormal karyotypes to become cancerous, do pathways that limit the prevalence of such cells exist? By investigating the immediate consequences of aneuploidy on cell physiology, we identified mechanisms that eliminate aneuploid cells. We find that chromosome missegregation leads to further genomic instability that ultimately causes cell-cycle arrest. We further show that cells with complex karyotypes exhibit features of senescence and produce pro-inflammatory signals that promote their clearance by the immune system. We propose that cells with abnormal karyotypes generate a signal for their own elimination that may serve as a means for cancer cell immunosurveillance.

Control of both tissue architecture and scale is a fundamental translational roadblock in tissue engineering. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. We fabricated a structurally organized engineered tissue unit that expanded in response to regenerative cues after implantation into mice with liver injury. Specifically, we found that tissues containing patterned human primary hepatocytes, endothelial cells, and stromal cells in a degradable hydrogel expanded more than 50-fold over the course of 11 weeks in mice with injured livers. There was a concomitant increase in graft function as indicated by the production of multiple human liver proteins. Histologically, we observed the emergence of characteristic liver stereotypical microstructures mediated by coordinated growth of hepatocytes in close juxtaposition with a perfused vasculature. We demonstrated the utility of this system for probing the impact of multicellular geometric architecture on tissue expansion in response to liver injury. This approach is a hybrid strategy that harnesses both biology and engineering to more efficiently deploy a limited cell mass after implantation.

Aneuploidy, the state of having gained or lost chromosomes, is a hallmark of cancer. Approximately 90% of tumors have gained or lost at least one chromosome. In spite of aneuploidy occurring as frequently as, if not more often than, disruption of the p53 pathway, whether and how aneuploidy influences tumorigenesis is still poorly understood. Here, we take advantage of large-scale tumor sequencing efforts to assess karyotypic alterations across many cancer types and review recent sequencing studies that show how karyotypes change in space and time. We further summarize findings that describe the effects of aneuploidy on untransformed cells, the mechanisms by which aneuploidy could drive tumorigenesis, and the potential to target aneuploidy for cancer therapy.

Aneuploidy, an imbalanced karyotype, is a widely observed feature of cancer cells that has long been hypothesized to promote tumorigenesis. Here we evaluate the fitness of cells with constitutional trisomy or chromosomal instability (CIN) in vivo using hematopoietic reconstitution experiments. We did not observe cancer but instead found that aneuploid hematopoietic stem cells (HSCs) exhibit decreased fitness. This reduced fitness is due at least in part to the decreased proliferative potential of aneuploid hematopoietic cells. Analyses of mice with CIN caused by a hypomorphic mutation in the gene Bub1b further support the finding that aneuploidy impairs cell proliferation in vivo. Whereas nonregenerating adult tissues are highly aneuploid in these mice, HSCs and other regenerative adult tissues are largely euploid. These findings indicate that, in vivo, mechanisms exist to select against aneuploid cells.

Megabase-scale copy number variants (CNVs) can have profound phenotypic consequences. Germline CNVs of this magnitude are associated with disease and experience negative selection. However, it is unknown whether organismal function requires that every cell maintain a balanced genome. It is possible that large somatic CNVs are tolerated or even positively selected. Single-cell sequencing is a useful tool for assessing somatic genomic heterogeneity, but its performance in CNV detection has not been rigorously tested. Here, we develop an approach that allows for reliable detection of megabase-scale CNVs in single somatic cells. We discover large CNVs in 8%-9% of cells across tissues and identify two recurrent CNVs. We conclude that large CNVs can be tolerated in subpopulations of cells, and particular CNVs are relatively prevalent within and across individuals.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Extensive chromosomal rearrangement – chromothripsis – is seen in several cancers. Imaging and sequencing of single cells shows that this phenomenon can occur inside cellular anomalies known as micronuclei. See Article p.179

Abstract Aneuploidy, the state in which cells have an abnormal number of chromosomes, is a hallmark of most human cancers. Despite aneuploidy's near-ubiquitous presence in tumors, it is unknown whether aneuploidy is a driver or passenger in cancer progression. Aneuploidy is normally prevented by the spindle assembly checkpoint (SAC), a critical cellular mechanism which blocks chromosomal missegregation by inhibiting premature sister chromatid separation during mitosis. Although transgenic mouse models with defective SAC proteins exist, the dysfunctional proteins are expressed constitutively from birth in all tissues, a milieu that differs from that of an in vivo human tumor in which cancer cells develop aneuploidy de novo in the context of a euploid organism. In order to generate a mouse model that more closely approximates this tumor environment, we will insert single copies of shRNA hairpins that target SAC proteins BubR1 or Mad2 into the mouse genome at the Col1A1 locus using recombination mediated cassette exchange. shRNA expression will be under the control of a tetracycline responsive element in tissues that co-express rtTA, leading to temporally and spatially inducible disruption of the SAC, and thus aneuploidy. In vitro shRNA construct validation experiments indicate our shRNAs successfully knockdown target RNA and protein levels, as well as functionally promote mitotic slippage after nocodazole arrest, which is indicative of an impaired SAC. We will use this mouse model to investigate the role of increased chromosomal missegregation on tumor initiation and evolution, as well as the impact of increased aneuploidy on normal organ function. [Daniel Eichberg is a Howard Hughes Medical Institute Medical Research Fellow.] Citation Format: Daniel Eichberg, Kristin Knouse, Angelika Amon. Temporally and spatially inducible mouse model of aneuploidy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2429. doi:10.1158/1538-7445.AM2014-2429

Whole-chromosome copy number alterations, also known as aneuploidy, are associated with adverse consequences in most cells and organisms. However, high frequencies of aneuploidy have been reported to occur naturally in the mammalian liver and brain, fueling speculation that aneuploidy provides a selective advantage in these organs. To explore this paradox, we used single cell sequencing to obtain a genome-wide, high-resolution assessment of chromosome copy number alterations in mouse and human tissues. We find that aneuploidy occurs much less frequently in the liver and brain than previously reported and is no more prevalent in these tissues than in skin. Our results highlight the rarity of chromosome copy number alterations across mammalian tissues and argue against a positive role for aneuploidy in organ function. Cancer is therefore the only known example, in mammals, of altering karyotype for functional adaptation.

The studies that implicated CIN and aneuploidy in ageing.

During the course of breast cancer progression, normally dormant tumour-promoting effects of transforming growth factor beta (TGFbeta), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro-migratory TGFbeta 'switch' in mammary epithelial cells in vitro, we found that TGFbeta stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFbeta receptor, ALK5, as evidenced by studies using short hairpin RNA-resistant ALK5 mutants in ALK5-depleted cells and in vitro kinase assays. Functionally, Smad1/5 co-depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFbeta-stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFbeta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGFbeta switch in mammary epithelial cells.