Cells must employ a diversity of strategies to regulate the quantity and functionality of different proteins during development and adult homeostasis. Post-transcriptional regulation of gene transcripts by microRNAs (miRNAs) is recognized as an important mechanism by which the dosage of proteins is regulated. Despite this, the physiological relevance of direct regulation of an endogenous gene transcript by miRNAs in vivo is rarely investigated. PAX6 is a useful model gene for studying miRNA regulation directly. PAX6 is highly dosage-sensitive transcription factor that is dynamically expressed during development of the eye, nose, central nervous system, gut and endocrine pancreas, and is mutated in the haploinsufficiency disease aniridia. Several miRNAs have been implicated in regulating PAX6 in different developmental contexts. Notably, miR-7 appears to regulate Pax6 during specification of olfactory bulb interneurons in the ventricular-subventricular zone (V-SVZ) of the brain and during development of the endocrine pancreas. Here, we produced a bioinformatics tool to enable selective mutation of candidate microRNA recognition elements (MREs) for specific miRNAs while ensuring that new MREs are not inadvertently generated in the process. We then performed the first comprehensive analysis of the mouse Pax6 3’ untranslated region (3’UTR) to identify MREs that may mediate miRNA regulation of Pax6 and to identify miRNAs capable of interacting with the 3’UTR of Pax6. Using Pax6 3’UTR genetic reporter assay, we confirmed that two MREs for miR-7-5 located at 3’UTR positions 517 and 655 function together to regulate PAX6. We generated mice harbouring mutations in the Pax6 3’UTR that disrupt these miR-7-5p MREs, individually or in combination, to explore the biological relevance of miRNA regulation directly. PAX6 protein abundance was elevated in double miR-7-5p MRE mutants relative to wild type and single mutants in the ventral V-SVZ. However, this increase in PAX6 was not associated with an altered dopaminergic periglomerular neuron phenotype in the olfactory bulb. Our findings suggest that, in vivo, microRNA regulation can be mediated through redundant MRE interactions. This work also reveals that directly mutating predicted MREs at the genomic level is necessary to fully characterize the specific phenotypic consequences of miRNA-target regulation. / Graduate

Sturgeons are commonly known as living fossils or ancient giants that diverged from ancient pre-Jurassic teleost lineage approximately ~300 million years ago (Mya). Sturgeons' 85% species are listed as critically endangered in the International Union for Conservation of Nature (IUCN). Sturgeons' reproductive traits such as delay in sexual maturation and periodic interrupted spawning cycles make their rehabilitation more difficult. However, among sturgeon species, the sterlet (Acipenser ruthenus) has shortest sexual maturation period. Therefore, it can be used as a host in surrogate production in sturgeons. Dnd1 was discovered as germ-plasm specific maternal RNA that exclusively expresses in vertebrate germ-line. Various studies have confirmed that dnd1 protein is essential for Primordial Germ Cells (PGCs) migration; and disruption of the PGCs migration affects fish fertility. Dnd1 deficient PGCs in zebrafish transdifferentiate into somatic cells. Previously our colleagues used morpholino oligonucleotide to knock down dnd1 in sterlet to produce germ cell free host for surrogate production. CRISPR/Cas9, a cutting-edge genome editing technology is being used in different research fields; here we thus aimed to harness the power of aforementioned technology to knock out dnd1 in sterlet. No or less number of PGCs were observed in CRISPR/Cas9 injected embryos as compared to control group injected with FITC-dextran only in order to label PGCs. Furthermore, we compared three different sterilization techniques viz., CRISPR/Cas9 and morpholino oligonucleotide (MO) targeting dnd1 and ultraviolet irradiation to eliminate PGCs in sterlet. Our data showed higher hatching and survival rates in CRISPR/Cas9, UV irradiation, and MO knockdown groups, respectively. Interestingly, some embryos treated with CRISPR/Cas9 displayed malformations. We presume that malformations were due to off-target effects and/or due to double injections i.e., injection of CRISPR/Cas9 at animal pole to knock-out the dnd1 and FITC-dextran at vegetal pole. Taking advantages of Iron Oxide nanoparticles (IONs) applications in various burgeoning research fields, we opted to use them to label PGCs in sturgeons. We injected IONs combined with FITC-dextran into vegetal pole of sturgeon embryos, and have successfully labelled the PGCs. Injection of IONs in sturgeons did not affect hatching and survival rates of embryos. Interestingly at 5 dpf, significantly less number of FITC-dextran labelled PGCs in FITC-dextran/IONs injected group were observed when compared with PGCs that were labelled with FITC-dextran only. Less number of PGCs in IONs injected group presumably could be because of interference posed by IONs to PGCs during the course of their migration. This is first study of its kind where germ cells of any species have been labelled by using nanoparticles. In conclusions, this thesis provides information regarding role of Dnd1 protein as potential germ-cell molecular marker in various fish species, and its use for conservation of fish species. Dnd1 knockout sterlet can be potentially used as sterile host for surrogate production in sturgeons. Moreover, labelling of PGCs in sturgeons by using IONs can thus open new avenues to study interactions of nanoparticles with cells that will ultimately help in hyperthermia where cells/tissues are exposed to electromagnetic field increasing temperatures to activate their death. After insertion of IONs to PGC in sturgeon embryo, it could be possible to isolate PGC using a magnetic field or to apply hyperthermia for host sterilization purpose.

IRAK2 besitzt eine Schlüsselrolle im Signalweg des TLR4. Fehlregulationen dieses Signalwegs führen zu fehlgeleiteten Immunreaktionen, die auch die Entstehung und Progression von Krebserkrankungen fördern. Bevor IRAK2 als therapeutisches Ziel in Frage kommen kann, muss erst noch weitere Klarheit über die grundsätzliche Funktionsweise dieses Proteins bestehen. So ist für IRAK2 aufgrund der Substitution einer Aminosäure in der Kinase-Domäne im Vergleich zu IRAK1 noch nicht abschließend geklärt, ob es sich um eine aktive Kinase oder eine Pseudokinase handelt und ob diese Veränderung eine Erhöhung oder eine Erniedrigung der Funktion im TLR4-Signalweg nach sich zieht. Um diese Fragen anzugehen, wurde in dieser Arbeit Asparagin im vermeintlich aktiven Zentrum (Aminosäure 333) wieder zur Asparaginsäure [N333D] revertiert und damit versucht die Phosphorylierungsaktivität zu steigern bzw. vergleichbar zu IRAK1 wiederherzustellen. Das Einbringen der Mutation in IRAK2 erfolgte mittels ortsspezifischer Mutagenese. Mit dieser und anderen Mutanten und mit wildtypischem IRAK2 wurden durch die CRISPR/Cas9-Methode generierte IRAK2-defiziente 264.7 Makrophagen rekonstituiert und damit ein System etabliert, mit dem der Einfluss der Mutation auf den Signalweg des TLR4 nach Stimulation mit LPS quantitativ analysiert werden konnte. Sowohl die indirekte NF-κB-Messung über CD40-Expression als auch die direkte NF-κB-Messung über die NF-κB-getriebene Expression eines Reportergens (cyan fluorescent protein) ergab, dass IRAK2[N333D] die LPS-abhängige NF-κB-Aktivierung über den TLR4 Signalweg schlechter ermöglicht als IRAK2. Insgesamt deuten die Ergebnisse darauf hin, dass die in der Entwicklungsgeschichte aufgetretene Veränderung des aktiven Zentrums von IRAK2 im Vergleich zu IRAK1 zu einer besseren Aktivierung der MyD88-abhängigen NF-κB-Aktivität führte und somit eine erhöhte und länger anhaltende Signalleitung ermöglichte. Diese Erkenntnis kann als weiterer Schritt hin zu einem besseren Verständnis der Funktion des IRAK2-Proteins und zu einer möglichen zukünftigen Verwendung von IRAK2 als Ziel therapeutischer Behandlungen gesehen werden. / Toll-like receptors are fundamental for many immune cells. The protein IRAK2 plays a key role in the signaling pathway downstream of TLR4 and other TLRs. Due to a change at amino acid position 333 of the protein from aspartate (D) to asparagine (N) in the presumed active center of the kinase it is unclear if IRAK2 is an active kinase. In this research the mutation IRAK2-N333D was investigated thereby trying to reconstitute the evolutionarily original version of the protein. Interestingly, overexpression of IRAK2-N333D in IRAK2-deficient RAW 264.7 cells obtained by using the CRISPR/Cas9-system was less effective in activating LPS-induced CD40 expression and NF-κB activity as compared to wild type IRAK2. Our results suggest that the evolutionary alteration in the "catalytic center" of IRAK2 led to improved signal transduction thereby allowing longer-lasting activation of the cells. This knowledge might help to better target IRAK2 in therapies.

The human microbiome has important roles in maintaining health, but dysbiosis of the microbiota can lead to disease. Polymicrobial interactions can result in synergy, producing disease that is worse than the sum of the respective single species infections. Despite this significant impact, synergy is understudied due to the complexity of polymicrobial interactions. Periodontitis is a microbiome-associated disease, and is one of the most common infectious diseases worldwide. Therefore, we have used periodontal disease as a model to study polymicrobial synergy. I have used two complementary approaches to study polymicrobial infections. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans exhibits synergy with streptococci in model murine infections. Because polymicrobial interactions are dependent on organisms’ abilities to sense their environments, I have examined the genetic regulatory mechanisms used by A. actinomycetemcomitans to interact with its environment. Through Northern blot analyses and biochemical approaches, I show that A. actinomycetemcomitans uses non-coding RNAs to regulate amino acid transport. Taking a comparative genomics approach, I demonstrate that A. actinomycetemcomitans DNA uptake systems are evolutionarily linked to genome defense. To describe host-influenced changes in gene expression, I develop a new technique to transcriptionally profile A. actinomycetemcomitans in a murine abscess infection, thereby revealing the importance of specific fermentative and anaerobic respiratory genes for in vivo survival. The long-term goal is to use these studies as a basis to characterize genetic regulatory mechanisms mediating synergy in polymicrobial A. actinomycetemcomitans infections with streptococci and other oral microbes. As a second approach to study polymicrobial infections, I analyze gene expression of healthy and diseased human plaque communities from aggressive periodontitis patients. Profiling ribosome content of healthy and diseased communities, I show that disease communities adopt similar less diverse population structures distinct from healthy populations. In addition to changes in population composition, using community transcriptional profiling I show that a keystone species within diseased communities up-regulates expression of genes involved in making the oral inflammatory molecule butyrate. These studies demonstrate for the first time that microbiome based diseases are marked by gene expression changes in addition to compositional changes. / text

The ability to introduce targeted modifications into genomes and engineer model organisms holds enormous promise for biomedical and technological applications, and has driven the development of tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). To facilitate genome engineering in mammalian cells, we have engineered the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 programmable nuclease systems from Streptococcus pyogenes SF370 (SpCas9) and S. thermophilus LMD-9 (St1Cas9) for mouse and human cell gene editing through heterologous expression of the minimal protein and RNA components. We have demonstrated that Cas9 nucleases can be guided by several short RNAs (sgRNAs) to introduce double stranded breaks (DSB) in the mammalian genome and induce efficient, multiplexed gene modification through non-homologous end-joining-mediated indels or homology-directed repair. Furthermore, we have engineered SpCas9 into a nicking enzyme (SpCas9n) to facilitate recombination while minimizing mutagenic DNA repair processes, and show that SpCas9n can be guided by pairs of appropriately offset sgRNAs to induce DSBs with high efficiency and specificity. In collaboration with Drs. Osamu Nureki and Hiroshi Nishimasu at the University of Tokyo, we further report the crystal structure of SpCas9 in complex with the sgRNA and target DNA, and elucidate the structure-function relationship of the ribonucleoprotein complex. Finally, through a metagenomic screen of orthologs, we have identified an additional small Cas9 from Staphylococcus aureus subsp. aureus (SaCas9) that cleaves mammalian endogenous DNA with high efficiency. SaCas9 can be packaged into adeno-associated virus for effective gene modification in vivo. Together, these technologies open up exciting possibilities for applications across basic science, biotechnology, and medicine.

<p>Synthetic biology facilitates both the design and fabrication of biological components and systems that do not already exist in the natural world. From an engineering point of view, synthetic biology is akin to building a complex machine by assembling simpler parts. Complex genetic machines can also be built by a modular and rational assembly of simpler biological parts. These biological machines can profoundly affect various cellular processes including the transcriptional machinery. In this thesis I demonstrate the utilization of biological parts according to synthetic biology principles to solve three distinct transcription-level problems: 1) How to efficiently select for transgene excision in induced pluripotent stem cells (iPSCs)? 2) How to eliminate transposase expression following piggyBac-mediated transgenesis? 3) How to reprogram cell lineage specification by the dCas9/gRNA transactivator-induced expression of endogenous transcription factors? </p><p>Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells (iPSCs). For translation, it is important to silence or remove the reprogramming factors after induction of pluripotency. In the first study, we design an excisable loxP-flanked lentiviral construct that a) includes all the reprogramming elements in a single lentiviral vector expressed by a strong EF-1&#945; promoter; b) enables easy determination of lentiviral titer; c) enables transgene removal and cell enrichment using LoxP-site-specific Cre-recombinase excision and Herpes Simplex Virus-thymidine kinase/ganciclovir (HSV-tk/gan) negative selection; and d) allows for transgene excision in a colony format. With our design, a reprogramming efficiency comparable to that reported in the literature without boosting molecules can be consistently obtained. To further demonstrate the utility of this Cre-loxP/HSV-tk/gan strategy, we incorporate a non-viral therapeutic transgene (human blood coagulation Factor IX) in the iPSCs, whose expression can be controlled by a temporal pulse of Cre recombinase. The robustness of this platform enables the implementation of an efficacious and cost-effective protocol for iPSC generation and their subsequent transgenesis for downstream studies.</p><p>Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In the second study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the transposase element is deactivated after transposition. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.</p><p>Genome editing by the efficient CRISPR/Cas9 system shows tremendous promise with ease of customization and the capability to multiplex distinguishing it from other such technologies. Endogenous gene activation is another aspect of CRISPR/Cas9 technology particularly attractive for biotechnology and medicine. However, the CRISPR/Cas9 technology for gene activation leaves much room for improvement. In the final study of this thesis we show that the fusion of two transactivation (VP64) domains to Cas9 dramatically enhances gene activation to a level that is sufficient to achieve direct cell reprogramming. Targeted activation of the endogenous Myod1 gene locus with this system leads to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. </p><p>In conclusion, this dissertation demonstrates the power of utilizing biological parts in a rational and systematic way to rectify problems associated with cell fate reprogramming and transposon-based gene delivery. Through design of genetic constructs aided by synthetic biology principles, I aspire to make contributions to the related fields of cellular reprogramming, stem cell differentiation, genomics, epigenetics, cell-based disease models, gene therapy, and regenerative medicine.</p> / Dissertation

Large animal species, such as cattle, sheep and pigs, have great potential value to scientific research. This is due to their physiological similarity to humans, meaning they make excellent disease models in addition to their inherent agricultural value. However, the efficiency with which such animals can be created has been a critical barrier to their use in bioscience. Research into creating genetically modified large animals has not progressed as rapidly as research on smaller mammals, such as mice, for two main reasons. Firstly, technologies such as pluripotent stem cells, which are well established in rodents, are lacking for large animals. Secondly, large animals cannot produce as many offspring within a given time frame as mice or rats. This, combined with the low efficiencies and lack of precision of current transgenic methods, severely reduces the likelihood of obtaining an animal with a desired genotype within a viable amount of time. Recently, new tools known as ’genome editors’ have been developed to facilitate genetic modification of animals. The vastly enhanced efficiency of these editors in comparison to previous gene targeting methods, combined with the fact that genome editors do not require marker genes to be used, mean that creating genetically modified livestock is now far more feasible. This thesis investigates whether two types of genome editor, TALENs and CRISPR/Cas9, can be used to produce genetically modified large animals for a range of applications. Genome editors were combined with interspecific blastocyst complementation techniques to produce chimeric rodents where the haematopoietic system is partially or fully derived from the donor cells. This work was carried out with a long-term aim of producing chimeric animals which could produce human organs suitable for transplantation. Initial blastocyst complementation experiments were carried out by injecting murine ESCs into wildtype rat blastocysts. One animal resulting from these injections showed chimerism in several tissues. Further experiments were carried out using rat ESCs and mouse blastocysts which were either Runx1-/- or Rag1-/-, however no additional chimeras were identified. In addition to these experiments, TALENs and sgRNAs were designed against Runx1 and Rag1 in sheep and pigs in order to create a large animal model for future blastocyst complementation experiments. Increasing animal productivity is a key step in meeting the demands of an increasing global population and tackling future food insecurities. TALENs and sgRNAs for use in the CRISPR/ Cas9 system were created to target the myostatin gene in sheep. Myostatin is a negative regulator of muscle growth and animals which acquire natural inactivating mutations in both myostatin alleles exhibit a well-characterised double-muscled phenotype, where total muscle mass is about 20% greater than that of a wildtype animal. Embryo microinjections were carried out using both types of genome editor and two edited lambs were produced, one from each editor. The TALEN-edited lamb was mosaic for a deletion of arginine 283 which, upon further analysis of the muscle, did not appear to cause a significant phenotype. The CRISPR-edited lamb was heterozygous for a 20bp deletion, causing the formation of a premature stop codon and severe truncation of the mature myostatin protein. Based on data from other myostatin-knockout animals, including the Belgian Blue cattle breed, this truncated protein is not thought to be functional. To determine if this is indeed the case, the CRISPR-edited lamb is now part of a breeding programme to amplify the edited allele. To discover if genome editors could be applied to create disease-resistant animals, the project focused on foot and mouth disease. Through a literature search and bioinformatic analysis of the bovine and porcine proteomes, three host genes which are cleaved by the virus were identified; eIF4A1, eIF4G1 and IKBKG. TALENs were designed to bind and cut at the FMDV protease cleavage sites in all three genes in order to disrupt protease cleavage and reduce viral replication by slowing viral disruption of the host translation and innate immune response pathways. Although none of the TALENs showed any signs of activity, this thesis sets out some potential directions for future work. In conclusion, this thesis shows that, despite some technical issues, genome editors are a promising technology for the creation of genetically modified livestock.

Master of Science / Genetics Interdepartmental Program / Eduard D. Akhunov / The CRISPR/Cas9-based genome editing system holds a great promise to accelerate wheat improvement by helping us to understand the molecular basis of agronomic traits and providing means to modify genes controlling these traits. CRISPR/Cas9 is based on a synthetic guide-RNA (gRNA) that can guide Cas9 nuclease to specific targets in the genome and create double strand breaks (DSB). The DSB are repaired through the error-prone non-homologous end joining process causing insertions or deletions that may result in loss-of-function mutations. Here, we have developed an effective wheat genome editing pipeline. We used next-generation sequencing (NGS) data to estimate genome editing efficiency of many gRNAs using the wheat protoplast assay and choose the most efficient gRNAs for plant transformation. We successfully applied this pipeline to five wheat orthologs of the rice yield component genes that have been identified previously. We obtained edited plants for all these genes and validated the effect of the mutations in TaGW7 on wheat traits, which showed trends similar to those in rice. These results suggest that transferring discoveries made in rice to improve wheat is a feasible and effective strategy to accelerate breeding.

Human pluripotent stem cell (hPSC)-derived neuronal and glial material presents a relatively new opportunity to model human neurophysiology in both health, and disease. Validation of regionally-defined hPSC-derived neurones and glia cultures thus represents the founding blocks of technology that aims to complement existing models. Principally, the relevance of in vitro hPSC-derived material is determined by how representative it is of native material, yet at present the physiology of these cells remains underexplored. Here, electrophysiology and pharmacology are used to functionally assess hPSC-derived excitatory cortical neurones (hECNs), motorneurones (MNs) and oligodendrocyte-lineage cells in the context of regional-specific properties and maturation. These properties are then examined in material derived from hPSCs generated from patients with neurological disorders. This thesis examines of the properties of GABAARs and strychnine-sensitive glycine receptors (GlyRs) in hECNs by assessing their subunit composition, and compares these with studies which have made comparable investigations of rodent tissue where maturation is associated with a shift in GABAA and GlyR compositions. Using pharmacology and RNAseq analysis, GABAAR and GlyRs in hECNs were found to possess receptor populations typical of those reported in the immature cortex. hECNs generated from patients harbouring a mutation to the Disrupted-in-schizophrenia-gene 1 (DISC1), a candidate schizophrenia gene, were then examined. Imbalances in the excitation/inhibition balance are suspected in schizophrenia and, in this regard, the intrinsic excitability properties alongside expression and composition of major neurotransmitter receptors and intracellular chloride concentration were assessed. No obvious differences in excitability or functional expression of AMPARs, GABAARs or NMDARs were observed between case and control derived neurones. Receptor composition and intracellular chloride concentrations were found to be predominantly immature-like, however, AMPAR composition and intracellular chloride concentration were found to be like that of adult cortical neurones. These data are discussed in the context of modelling DISC1-associated pathologies. Thirdly, MNs from hPSCs generated from ALS patients harbouring mutations on the C9ORF72 gene were examined. The hypothesis that increased glutamate-mediated excitoxicity could, in part, be explained by increased expression of Ca2+- permeable AMPARs was examined. The estimated mean single-channel conductance of AMPARs was found to be high in MNs derived from ALS patients, reminiscent of Ca2+-permeable AMPARs and was reversed by gene-editing of the C9ORF72 mutation. Finally, oligodendrocytes generated from ALS patients harbouring TARDBP mutations were examined. Distinctive electrophysiological shifts in oligodendrocytes-lineage cell development are reported. A similar AMPAR phenotype of elevated Ca2+-permeable AMPAR expression was observed in oligodendrocytes derived from two patient hPSC lines and was rescued in an isogenic, gene-edited line, raising the intriguing possibility of convergence in pathophysiologies in the nature of the overlap between cell-type, AMPAR pathology and excitotoxicity in ALS disease progression mechanisms.

Primary liver cancer (PLC) is the second most common cause of cancer death worldwide, preceded only by lung cancer. Current models for PLC either fail to fully recapitulate tumour histology and architecture or are expensive, time consuming and do not allow for personalised drug testing. During the first part of my PhD, I have collaborated with Dr. Laura Broutier in order to established a new 3D in vitro model system for liver cancer. Based on the current knowledge on organoid cultures, we have managed to establish a system to grow primary human liver cancer cells long-term (Broutier et al., in press). Interestingly, the tumour-derived organoids (tumoroids) recapitulate the original tumour histology and genetic alterations and are also able to generate tumours in an in vivo xenograft mouse model after long-term expansion. Furthermore, we have shown that tumoroids can also be successfully used for drug testing, suggesting their use to devise new targeted therapy as well as personalised treatment strategies. Current models to investigate the role of genes in cancer rely mostly on animal studies, which can be very time consuming and cost demanding, especially if resulting in negative outcomes. To overcome this issue, I have set up a protocol for introducing mutations in healthy human liver organoids using the CRISPR-Cas9 technology. Interestingly, after mutating TP53, RNF43 and ZNRF3 either alone or in combination, human organoids undergo genetic alterations and phenotypic changes that partially resemble the ones observed in tumoroids. This data suggests that this system could be used as a screening platform to study gene function before using animal models. In the last part, I have further explored the role of RNF43 and ZNRF3 (R&Z) - two newly identified WNT pathway negative regulators mutated in many cancer types - in the liver using an in vivo mouse model. Interestingly, conditional deletion of R&Z specifically in adult mouse hepatocytes results metabolic changes that eventually lead to extensive liver damage. However, when the liver is challenged to regenerate in a chronic damage model, R&Z mutated livers fail to fully repair and show presence of multiple regenerative nodules. Later, livers develop either focal nodular hyperplasia and/or early hepatocellular carcinoma. These data suggest that R&Z have an important role in both liver metabolic homeostasis and liver regeneration and that their alteration can eventually lead to cancer formation.