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Caspase-8 joins RIPK at the death Caspase-8 mediates apoptosis induced by 'death receptors' on the cell's surface. At the same time, it is able to prevent receptor interacting protein kinase (RIPK)-dependent necrosis. Without caspase-8, mice die during embryonic development, but why this happens is not clear. Two groups show that this lethality is not caused by the absence of apoptosis, but by the RIPK3-dependent necrosis that is unleashed without caspase-8. Mice lacking both caspase-8 and RIP3 develop into viable, immunocompetent adults, but have a progressive lymphoaccumulative disease similar to that in mice that lack the CD95 death receptor. Oberst et al . also show that caspase-8 forms a proteolytically active complex with FLICE-like inhibitory protein long (FLIPL), and that this complex is required for protection against RIP3-dependent necrosis. Caspase-8 mediates apoptosis induced by death receptors. At the same time, this protease is able to prevent RIP-dependent necrosis. Without caspase-8 mice die during their embryonic development. Two papers now show that lethality is not caused by the absence of apoptosis, but by RIP3-dependent necrosis that is unleashed without caspase-8. Mice that lack both caspase-8 and RIP3 develop into viable, immunocompetent, fertile adult mice, but suffer from a progressive lymphoaccumulative disease similar to mice that lack the death receptor CD95. Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns 1 . Casp8 suppresses RIP3–RIP1 (also known as RIPK3–RIPK1) kinase complex-dependent 2 , 3 , 4 necroptosis 5 that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus 6 . Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7 ). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8 −/− Rip3 −/− double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency ( lpr/lpr ; also known as Fas ). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.

Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1E861A, where E861A denotes Glu861→Ala861). Adar1E861A/E861A embryos died at ∼E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)–sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3′ untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1E861A/E861A were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

Transcription of a long non-coding RNA, known as upperhand ( Uph ) located upstream of the HAND2 transcription factor is required to maintain transcription of the Hand2 gene by RNA polymerase, and blockade of Uph expression leads to heart defects and embryonic lethality in mice. upperhand regulates Hand2 expression in early cardiogenesis The expression of the transcription factor HAND2 is controlled by several upstream enhancer elements, confined in a region delimited by the presence of the chromatin mark H3K27Ac. Eric Olson and colleagues have found that the transcription of long non-coding RNA located upstream of HAND2 is required to maintained these chromatin marks and let the RNA polymerase transcribe the Hand2 gene. Preventing the expression of this long non-coding RNA with a termination cassette leads to defects in heart development in mice. HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes 1 , 2 , 3 , 4 . Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality 1 , 5 . Hand2 expression is tightly regulated by upstream enhancers 6 , 7 that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications 8 . Here we show that transcription of a Hand2- associated long non-coding RNA, which we named upperhand ( Uph ), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome 9 , the Uph – Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.

This study establishes an important role for the enzyme Tet1 in erasing genomic imprinting in vivo — mice with a knockout of paternal Tet1 give rise to progeny with imprinting defects and associated growth and development defects, which leads to early embryonic lethality; furthermore, analysis of the DNA methylation dynamics in reprogramming primordial germ cells (PGCs) suggests that Tet1 is required at a late stage of the reprogramming process, in the second wave of DNA demethylation in PGCs. Tet1 removal of genomic imprinting Genomic imprinting mediated by allele-specific DNA methylation must be erased during the genome-wide reprogramming that occurs in primordial germ cells. Here, Yi Zhang and colleagues establish an important role for the methylcytosine dioxygenase Tet1 in erasing genomic imprinting in vivo . Mice with a knockout of paternal Tet1 give rise to progeny with imprinting defects and associated growth and development defects, which leads to early embryonic lethality. Analysis of the DNA methylation dynamics in reprogramming primordial germ cells suggests that Tet1 is required at a late stage of the reprogramming process, in a second wave of DNA demethylation. Genomic imprinting is an allele-specific gene expression system that is important for mammalian development and function 1 . The molecular basis of genomic imprinting is allele-specific DNA methylation 1 , 2 . Although it is well known that the de novo DNA methyltransferases Dnmt3a and Dnmt3b are responsible for the establishment of genomic imprinting 3 , how the methylation mark is erased during primordial germ cell (PGC) reprogramming remains unclear. Tet1 is one of the ten-eleven translocation family proteins, which have the capacity to oxidize 5-methylcytosine (5mC) 4 , 5 , 6 , specifically expressed in reprogramming PGCs 7 . Here we report that Tet1 has a critical role in the erasure of genomic imprinting. We show that despite their identical genotype, progenies derived from mating between Tet1 knockout males and wild- Peg10 and Peg3, which exhibit aberrant hypermethylation in the paternal allele of differential methylated regions (DMRs). RNA-seq reveals extensive dysregulation of imprinted genes in the next generation due to paternal loss of Tet1 function. Genome-wide DNA methylation analysis of embryonic day 13.5 PGCs and sperm of Tet1 knockout mice revealed hypermethylation of DMRs of imprinted genes in sperm, which can be traced back to PGCs. Analysis of the DNA methylation dynamics in reprogramming PGCs indicates that Tet1 functions to wipe out remaining methylation, including imprinted genes, at the late reprogramming stage. Furthermore, we provide evidence supporting the role of Tet1 in the erasure of paternal imprints in the female germ line. Thus, our study establishes a critical function of Tet1 in the erasure of genomic imprinting.

Large-scale phenotyping efforts have demonstrated that approximately 25–30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5–14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation. Analysis of embryonic lethal and sub-viable mouse knockout lines reveals that ablation of many genes affects placental development, and that the occurrence of placental defects is co-associated with abnormal brain, heart and vascular system development. Womb woes increased in mutant mice Large-scale analysis of mouse gene knockouts have demonstrated the prevalence of deleterious defects that arise in the mutant's offspring during development in the womb, thought to reflect defects during the development of the embryo itself. By analysing mouse knockouts from the Deciphering the Mechanisms of Developmental Disorders program, Myriam Hemberger and colleagues have found that knockout of many genes affects placental development and subsequently the development of the brain, heart and vasculature in the embryo. The studies highlight the importance of considering the role of the placenta when analysing developmental disorders.

Inflammatory autoimmune diseases such as systemic lupus erythematosus (SLE) and polyarthritis are characterized by chronic cytokine overproduction, suggesting that the stimulation of host innate immune responses, speculatively by persistent infection or self nucleic acids, plays a role in the manifestation of these disorders. Mice lacking DNase II die during embryonic development through comparable inflammatory disease because phagocytosed DNA from apoptotic cells cannot be adequately digested and intracellular host DNA sensor pathways are engaged, resulting in the production of a variety of cytokines including type I IFN. The cellular sensor pathway(s) responsible for triggering DNA-mediated inflammation aggravated autoimmune disease remains to be determined. However, we report here that Stimulator of IFN Genes (STING) is responsible for inflammation-related embryonic death in DNase II defective mice initiated by self DNA. DNase II-dependent embryonic lethality was rescued by loss of STING function, and polyarthritis completely prevented because cytosolic DNA failed to robustly trigger cytokine production through STING-controlled signaling pathways. Our data provides significant molecular insight into the causes of DNA-mediated inflammatory disorders and affords a target that could plausibly be therapeutically controlled to help prevent such diseases.

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease 1 , 2 and embryonic lethality during mouse development 3 . Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice 4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon. OTULIN, which removes ubiquitin chains deposited by LUBAC, promotes LUBAC activity by preventing its auto-ubiquitination, thereby supporting normal mouse embryo development and preventing pro-inflammatory cell death in adult mice.

Implantation is a key stage during pregnancy, as the fate of the embryo is often decided upon its first contact with the maternal endometrium. Around this time, DCs accumulate in the uterus; however, their role in pregnancy and, more specifically, implantation, remains unknown. We investigated the function of uterine DCs (uDCs) during implantation using a transgenic mouse model that allows conditional ablation of uDCs in a spatially and temporally regulated manner. Depletion of uDCs resulted in a severe impairment of the implantation process, leading to embryo resorption. Depletion of uDCs also caused embryo resorption in syngeneic and T cell-deficient pregnancies, which argues against a failure to establish immunological tolerance during implantation. Moreover, even in the absence of embryos, experimentally induced deciduae failed to adequately form. Implantation failure was associated with impaired decidual proliferation and differentiation. Dynamic contrast-enhanced MRI revealed perturbed angiogenesis characterized by reduced vascular expansion and attenuated maturation. We suggest therefore that uDCs directly fine-tune decidual angiogenesis by providing two critical factors, sFlt1 and TGF-beta1, that promote coordinated blood vessel maturation. Collectively, uDCs appear to govern uterine receptivity, independent of their predicted role in immunological tolerance, by regulating tissue remodeling and angiogenesis. Importantly, our results may aid in understanding the limited implantation success of embryos transferred following in vitro fertilization.

Genomic instability can trigger cellular responses that include checkpoint activation, senescence and inflammation 1 , 2 . Although genomic instability has been extensively studied in cell culture and cancer paradigms, little is known about its effect during embryonic development, a period of rapid cellular proliferation. Here we report that mutations in the heterohexameric minichromosome maintenance complex—the DNA replicative helicase comprising MCM2 to MCM7 3 , 4 —that cause genomic instability render female mouse embryos markedly more susceptible than males to embryonic lethality. This bias was not attributable to X chromosome-inactivation defects, differential replication licensing or X versus Y chromosome size, but rather to ‘maleness’—XX embryos could be rescued by transgene-mediated sex reversal or testosterone administration. The ability of exogenous or endogenous testosterone to protect embryos was related to its anti-inflammatory properties 5 . Ibuprofen, a non-steroidal anti-inflammatory drug, rescued female embryos that contained mutations in not only the Mcm genes but also the Fancm  gene; similar to MCM mutants, Fancm mutant embryos have increased levels of genomic instability (measured as the number of cells with micronuclei) from compromised replication fork repair 6 . In addition, deficiency in the anti-inflammatory IL10 receptor was synthetically lethal with the Mcm4 Chaos3 helicase mutant. Our experiments indicate that, during development, DNA damage associated with DNA replication induces inflammation that is preferentially lethal to female embryos, because male embryos are protected by high levels of intrinsic testosterone. Genomic instability, caused by MCM mutations, results in embryonic lethality that disproportionally affects female mouse embryos and is rescued by testosterone or ibuprofen treatment, both of which ameliorate inflammatory effects.