Mapping of susceptibility loci for Ebola virus pathogenesis in mice.

Ebola virus (EBOV), a major global health concern, causes severe, often fatal EBOV disease (EVD) in humans. Host genetic variation plays a critical role, yet the identity of host susceptibility loci in mammals remains unknown. Using genetic reference populations, we generate an F2 mapping cohort to identify host susceptibility loci that regulate EVD. While disease-resistant mice display minimal pathogenesis, susceptible mice display severe liver pathology consistent with EVD-like disease and transcriptional signatures associated with inflammatory and liver metabolic processes. A significant quantitative trait locus (QTL) for virus RNA load in blood is identified in chromosome (chr)8, and a severe clinical disease and mortality QTL is mapped to chr7, which includes the Trim5 locus. Using knockout mice, we validate the Trim5 locus as one potential driver of liver failure and mortality after infection. The identification of susceptibility loci provides insight into molecular genetic mechanisms regulating EVD progression and severity, potentially informing therapeutics and vaccination strategies.

Ectopically expressed Airn lncRNA deposits Polycomb with a potency that rivals Xist.

We report that when expressed at similar levels from an isogenic locus, the Airn lncRNA induces Polycomb deposition with a potency that rivals Xist . However, when subject to the same degree of promoter activation, Xist is more abundant and more potent than Airn . Our data definitively demonstrate that the Airn lncRNA is functional and suggest that Xist achieved extreme potency in part by evolving mechanisms to promote its own abundance.

A precisely humanized FCRN transgenic mouse for preclinical pharmacokinetics studies.

Monoclonal antibodies (mAbs) are one of the fastest-growing classes of drugs and have been approved to treat several diseases, including cancers and autoimmune disorders. Preclinical pharmacokinetics studies are performed to determine the therapeutically meaningful dosages and efficacy of candidate drugs. These studies are typically performed in non-human primates; however, using primates is costly and raises ethical considerations. As a result, rodent models that better mimic human-like pharmacokinetics have been generated and remain an area of active investigation. Pharmacokinetic characteristics of a candidate drug, such as half-life, are partly controlled by antibody binding to the human neonatal receptor hFCRN. Due to the abnormally high binding of human antibodies to mouse FCRN, traditional laboratory rodents do not accurately model the pharmacokinetics of human mAbs. In response, humanized rodents expressing hFCRN have been generated. However, these models generally use large inserts randomly integrated into the mouse genome. Here, we report the production and characterization of a CRISPR/Cas9-mediated hFCRN transgenic mouse termed SYNB-hFCRN. Using CRISPR/Cas9-assisted gene targeting, we prepared a strain with a simultaneous knockout of mFcrn and insertion of a hFCRN mini-gene under the control of the endogenous mouse promoter. These mice are healthy and express hFCRN in the appropriate tissues and immune cell subtypes. Pharmacokinetic evaluation of human IgG and adalimumab (Humira®) demonstrate hFCRN-mediated protection. These newly generated SYNB-hFCRN mice provide another valuable animal model for use in preclinical pharmacokinetics studies during early drug development.

Viral protease cleavage of MAVS in genetically modified mice with hepatitis A virus infection.

BACKGROUND & AIMS: Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/- mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range. METHODS: Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS ('mMAVS-VS'), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/- mice. RESULTS: The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/- mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/- mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/- mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/- mice. CONCLUSIONS: MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice. IMPACT AND IMPLICATIONS: The limited host range of human hepatitis viruses could be explained by species-specific viral strategies that disrupt innate immune responses. Both hepatitis A virus (HAV) and hepatitis C virus express viral proteases that cleave the innate immune adaptor protein MAVS, in human but not mouse cells. However, the impact of this immune evasion strategy has never been assessed in vivo. Here we show that HAV 3ABC protease cleavage of MAVS enhances viral replication and lessens liver inflammation in mice lacking interferon receptors, but that it is insufficient by itself to overcome the cross-species barrier to infection in mice. These results enhance our understanding of how hepatitis viruses interact with the host and their impact on innate immune responses.

Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4.

Endocardial cells lining the heart lumen are coronary vessel progenitors during embryogenesis. Re-igniting this developmental process in adults could regenerate blood vessels lost during cardiac injury, but this requires additional knowledge of molecular mechanisms. Here, we use mouse genetics and scRNA-seq to identify regulators of endocardial angiogenesis and precisely assess the role of CXCL12/CXCR4 signaling. Time-specific lineage tracing demonstrated that endocardial cells differentiated into coronary endothelial cells primarily at mid-gestation. A new mouse line reporting CXCR4 activity-along with cell-specific gene deletions-demonstrated it was specifically required for artery morphogenesis rather than angiogenesis. Integrating scRNA-seq data of endocardial-derived coronary vessels from mid- and late-gestation identified a Bmp2-expressing transitioning population specific to mid-gestation. Bmp2 stimulated endocardial angiogenesis in vitro and in injured neonatal mouse hearts. Our data demonstrate how understanding the molecular mechanisms underlying endocardial angiogenesis can identify new potential therapeutic targets promoting revascularization of the injured heart.

Genetic deletion of platelet PAR4 results in reduced thrombosis and impaired hemostatic plug stability.

BACKGROUND: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis. OBJECTIVE: We aimed to generate a mouse line with a megakaryocyte/platelet-specific deletion of PAR4 (PAR4fl/fl ;PF4Cre+ ) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice. METHODS: Platelets from PAR4fl/fl ;PF4Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet-specific deletion of PAR4 or wild-type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors. RESULTS: PAR4fl/fl ;PF4Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4-/- and PAR4fl/fl ;PF4Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4-/- mice and PAR4fl/fl ;PF4Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild-type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability. CONCLUSION: We generated a novel PAR4fl/fl ;PF4Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability.

Generation of a Matrix Gla (Mgp) floxed mouse, followed by conditional knockout, uncovers a new Mgp function in the eye.

The ability to ablate a gene in a given tissue by generating a conditional knockout (cKO) is crucial for determining its function in the targeted tissue. Such tissue-specific ablation is even more critical when the gene's conventional knockout (KO) is lethal, which precludes studying the consequences of its deletion in other tissues. Therefore, here we describe a successful strategy that generated a Matrix Gla floxed mouse (Mgp.floxed) by the CRISPR/Cas9 system, that subsequently allowed the generation of cKOs by local viral delivery of the Cre-recombinase enzyme. MGP is a well-established inhibitor of calcification gene, highly expressed in arteries' smooth muscle cells and chondrocytes. MGP is also one of the most abundant genes in the trabecular meshwork, the eye tissue responsible for maintenance of intraocular pressure (IOP) and development of Glaucoma. Our strategy entailed one-step injection of two gRNAs, Cas9 protein and a long-single-stranded-circular DNA donor vector (lsscDNA, 6.7 kb) containing two loxP sites in cis and 900-700 bp 5'/3' homology arms. Ocular intracameral injection of Mgp.floxed mice with a Cre-adenovirus, led to an Mgp.TMcKO mouse which developed elevated IOP. Our study discovered a new role for the Mgp gene as a keeper of physiological IOP in the eye.

Elements at the 5' end of Xist harbor SPEN-independent transcriptional antiterminator activity.

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5' end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

Baseline and innate immune response characterization of a Zfp30 knockout mouse strain.

Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.

Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.

Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFPloxP/stop/loxP knock-in cassette that enables the specific labeling of circulating CD63+ vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-CreERT2 mice to generate CD63emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP+ vesicles that are derived from the endothelium, co-express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63+ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.

Pancreatic cancer-associated inflammation drives dynamic regulation of p35 and Ebi3.

B cells are important modulators of immune responses both in autoimmunity and cancer. We have previously shown that B regulatory (Breg) cells promote pancreatic cancer via production of IL35, a heterodimeric cytokine comprised of the subunits p35 (Il12a) and Ebi3. However, it is not known how production of IL35 is regulated in vivo in the context of cancer-associated inflammation. To begin addressing this question, we have generated a knock-in mouse model, Il12aGFP, where an IRES-emGFP gene was inserted within the 3' UTR of the Il12a locus. EmGFP signal in B cells from the Il12aGFP mice correlated with expression of p35 mRNA and protein. Using this model, we observed that in addition to Bregs, expression of GFP (p35) is upregulated in several other B cell subtypes in response to cancer. We assessed the expression of the other IL35 subunit, Ebi3, using a published tdTomato reporter model. We determined that Ebi3 expression was more tightly regulated in vivo and in vitro, suggesting that stimuli affecting Ebi3 upregulation are more likely to result in production of full IL35 heterodimer. We were also able to detect GFP and Tomato signal in myeloid & T cell lineages suggesting that these reporter models could also be used for tracking IL12-, IL27- and IL35-producing cells. Furthermore, using primary B cells isolated from reporter mice, we identified BCR, CD40 and TLR pathways as potential drivers of IL35 expression. These findings highlight the importance of pancreatic cancer-associated inflammatory processes as drivers of cytokine expression and provide a tool to dissect both disease-associated regulation of IL12- and IL35-competent lineage cells as well as establish assays for pharmacological targeting of individual subunits of heterodimeric IL12 family cytokines.

lncRNA-Induced Spread of Polycomb Controlled by Genome Architecture, RNA Abundance, and CpG Island DNA.

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.

RETRACTED: A 5' fragment of Xist can sequester RNA produced from adjacent genes on chromatin.

Xist requires Repeat-A, a protein-binding module in its first two kilobases (2kb), to repress transcription. We report that when expressed as a standalone transcript in mouse embryonic stem cells (ESCs), the first 2kb of Xist (Xist-2kb) does not induce transcriptional silencing. Instead, Xist-2kb sequesters RNA produced from adjacent genes on chromatin. Sequestration does not spread beyond adjacent genes, requires the same sequence elements in Repeat-A that full-length Xist requires to repress transcription and can be induced by lncRNAs with similar sequence composition to Xist-2kb. We did not detect sequestration by full-length Xist, but we did detect it by mutant forms of Xist with attenuated transcriptional silencing capability. Xist-2kb associated with SPEN, a Repeat-A binding protein required for Xist-induced transcriptional silencing, but SPEN was not necessary for sequestration. Thus, when expressed in mouse ESCs, a 5' fragment of Xist that contains Repeat-A sequesters RNA from adjacent genes on chromatin and associates with the silencing factor SPEN, but it does not induce transcriptional silencing. Instead, Xist-induced transcriptional silencing requires synergy between Repeat-A and additional sequence elements in Xist. We propose that sequestration is mechanistically related to the Repeat-A dependent stabilization and tethering of Xist near actively transcribed regions of chromatin.

Mice Expressing Low Levels of CalDAG-GEFI Exhibit Markedly Impaired Platelet Activation With Minor Impact on Hemostasis.

OBJECTIVE: The tight regulation of platelet adhesiveness, mediated by the αIIbß3 integrin, is critical for hemostasis and prevention of thrombosis. We recently demonstrated that integrin affinity in platelets is controlled by the guanine nucleotide exchange factor, CalDAG-GEFI (CD-GEFI), and its target, RAP1. In this study, we investigated whether low-level expression of CD-GEFI leads to protection from thrombosis without pathological bleeding in mice. APPROACH AND RESULTS: Cdg1(low) mice were generated by knockin of human CD-GEFI cDNA into the mouse Cdg1 locus. CD-GEFI expression in platelets from Cdg1(low) mice was reduced by ≈90% when compared with controls. Activation of RAP1 and αIIbß3 was abolished at low agonist concentrations and partially inhibited at high agonist concentrations in Cdg1(low) platelets. Consistently, the aggregation response of Cdg1(low) platelets was weaker than that of wild-type platelets, but more efficient than that observed in Cdg1(-/-) platelets. Importantly, Cdg1(low) mice were strongly protected from arterial and immune complex-mediated thrombosis, with only minimal impact on primary hemostasis. CONCLUSIONS: Together, our studies suggest the partial inhibition of CD-GEFI function as a powerful new approach to safely prevent thrombotic complications.

Mucosal pemphigus vulgaris anti-Dsg3 IgG is pathogenic to the oral mucosa of humanized Dsg3 mice.

There are two major clinical subsets of pemphigus vulgaris (PV)-mucosal PV (mPV) and mucocutaneous PV (mcPV). The mPV subset exhibits anti-human desmoglein (Dsg) 3 autoantibodies that fail to recognize murine Dsg3 (mDsg3); thus, passive transfer experiments of mPV IgG into wild-type (WT) mice have been unsuccessful at inducing disease. We therefore generated a fully humanized Dsg3 (hDSG3) murine model utilizing a hDsg3 transgenic animal crossed to the mDsg3 knockout line. Expression of hDsg3 in the mucosa rescues the mDsg3 knockout phenotype. Well-characterized mPV sera bind mucosal epithelia from the hDsg3 mice, but not mucosal tissues from WT mice, as detected by indirect immunofluorescence (IF). The majority of mPV sera preferentially recognize hDsg3 compared with mDsg3 by immunoprecipitation as well. Passive transfer of mPV IgG into adult hDsg3 mice, but not WT mice, induces suprabasilar acantholysis in mucosal tissues, thus confirming the pathogenicity of mPV anti-hDsg3 IgG in vivo. Human anti-hDsg3 antibodies are detected in perilesional mucosa as well as in sera of recipient mice by IF. These findings suggest that the Dsg3 epitopes targeted by pathogenic mPV IgG are human specific. This hDsg3 mouse model will be invaluable in studying the clinical transition from mPV to mcPV.

Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology.

Humanized mouse models have become increasingly important and widely used in modeling human diseases in biomedical research. Immunodeficient mice such as NOD-Rag1-/-IL2RgammaC-null (NRG) or NOD-SCID-IL2RgammaC-null (NSG) mice are critical for efficient engraftment of human cells or tissues. However, their genetic modification remains challenging due to a lack of embryonic stem cells and difficulty in the collection of timed embryos after superovulation. Here, we report the generation of gene knockout NRG mice by combining in vitro fertilization (IVF) and CRISPR/Cas9 technology. Sufficient numbers of fertilized embryos were produced through IVF, and a high rate of Fah gene targeting was achieved with microinjection of Cas9 mRNA, gRNA and single strand oligonucleotide DNA (ssDNA) into the embryos. The technology paves the way to construct NRG or NSG mutant mice to facilitate new humanized mouse models. The technology can also be readily adapted to introduce mutations in other species such as swine and non-human primates.

Requirements for Aurora-A in tissue regeneration and tumor development in adult mammals.

Aurora-A is a kinase involved in the formation and maturation of the mitotic spindle and chromosome segregation. This kinase is frequently overexpressed in human cancer, and its activity may confer resistance to antitumoral drugs such as Taxol. Inhibition of Aurora-A results in mitotic defects, and this kinase is considered as an attractive therapeutic target for cancer. Nevertheless, the specific requirements for this kinase in adult mammalian tissues remain unclear. Conditional genetic ablation of Aurora-A in adult tissues results in polyploid cells that display a DNA-damage-like response characterized by the upregulation of p53 and the cell-cycle inhibitor p21(Cip1). This is accompanied by apoptotic, differentiation, or senescence markers in a tissue-specific manner. Therapeutic elimination of Aurora-A prevents the progression of skin and mammary gland tumors. However, this is not due to significant levels of apoptosis or senescence, but because Aurora-A-deficient tumors accumulate polyploid cells with limited proliferative potential. Thus, Aurora-A is required for tumor formation in vivo, and the differential response observed in various tissues might have relevant implications in current therapeutic strategies aimed at inhibiting this kinase in the treatment of human cancer.

Efficient division and sampling of cell colonies using microcup arrays.

A microengineered array to sample clonal colonies is described. The cells were cultured on an array of individually releasable elements until the colonies expanded to cover multiple elements. Single elements were released using a laser-based system and collected to sample cells from individual colonies. A greater than an 85% rate in splitting and collecting colonies was achieved using a 3-dimensional cup-like design or "microcup". Surface modification using patterned titanium deposition of the glass substrate improved the stability of microcup adhesion to the glass while enabling minimization of the laser energy for splitting the colonies. Smaller microcup dimensions and slotting the microcup walls reduced the time needed for colonies to expand into multiple microcups. The stem cell colony retained on the array and the collected fraction within released microcups remained undifferentiated and viable. The colony samples were characterized by both reporter gene expression and a destructive assay (PCR) to identify target colonies. The platform is envisioned as a means to rapidly establish cell lines using a destructive assay to identify desired clones.

Conditional Aurora A deficiency differentially affects early mouse embryo patterning.

Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.