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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.

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.

Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells. The Ras/Raf/MEK/ERK pathway is required for EC function during angiogenesis. Although in vitro studies implicate ERK1 and ERK2 in endothelial cell survival, their precise role in angiogenesis in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in endothelial cells during murine development, resulting in embryonic lethality due to severely reduced angiogenesis. Deletion of Erk1 and Erk2 in primary endothelial cells resulted in decreased cell proliferation and migration, but not in increased apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells, and decreased DNA synthesis could be observed in endothelial cells during embryogenesis. Interestingly, both Paxillin and Focal Adhesion Kinase were expressed at lower levels in endothelial cells lacking Erk1 and Erk2 both in vivo and in vitro, leading to defects in the organization of the cytoskeleton and in cell motility. The regulation of Paxillin and Focal Adhesion Kinase expression occurred post-transcriptionally. These results demonstrate that ERK1 and ERK2 coordinate endothelial cell proliferation and migration during angiogenesis.

The metabolism of poly(ADP-ribose) (PAR) is critical for genomic stability in multicellular eukaryotes. Here, we show that the failure to degrade PAR by means of disruption of the murine poly(ADP-ribose) glycohydrolase (PARG) gene unexpectedly causes early embryonic lethality and enhanced sensitivity to genotoxic stress. This lethality results from the failure to hydrolyze PAR, because PARG null embryonic day (E) 3.5 blastocysts accumulate PAR and concurrently undergo apoptosis. Moreover, embryonic trophoblast stem cell lines established from early PARG null embryos are viable only when cultured in medium containing the poly(ADP-ribose) polymerase inhibitor benzamide. Cells lacking PARG also show reduced growth, accumulation of PAR, and increased sensitivity to cytotoxicity induced by N-methyl-N′-nitro-N-nitrosoguanidine and menadione after benzamide withdrawal. These results provide compelling evidence that the failure to degrade PAR has deleterious consequences. Further, they define a role for PARG in embryonic development and a protective role in the response to genotoxic stress.

Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse.

Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3a G672E/G672E /Polr3b +/R103H double mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease.

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation （XLMR）. However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21^Cipl/WAF and G2/M cell cycle arrest, which could be partially rescued by silencing of p21^Cipl/WAF. Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.

The extracellular sulfatases Sulf1 and Sulf2 remove specific 6‐O‐sulfate groups from heparan sulfate, thereby modulating numerous signalling pathways underlying development and homeostasis. In vitro data have suggested that the two enzymes show functional redundancy. To elucidate their in vivo functions and to further address the question of a putative redundancy, we have generated Sulf1‐ and Sulf2‐deficient mice. Phenotypic analysis of these animals revealed higher embryonic lethality of Sulf2 knockout mice, which can be associated with neuroanatomical malformations during embryogenesis. Sulf1 seems not to be essential for developmental or postnatal viability, as mice deficient in this sulfatase show no overt phenotype. However, neurite outgrowth deficits were observed in hippocampal and cerebellar neurons of both mutant mouse lines, suggesting that not only Sulf2 but also Sulf1 function plays a role in the developing nervous system. Behavioural analysis revealed differential deficits with regard to cage activity and spatial learning for Sulf1‐ and Sulf2‐deficient mouse lines. In addition, Sulf1‐specific deficits were shown for synaptic plasticity in the CA1 region of the hippocampus, associated with a reduced spine density. These results reveal that Sulf1 and Sulf2 fulfil non‐redundant functions in vivo in the development and maintenance of the murine nervous system.

Aurora A (also known as STK15/BTAK in humans), a putative oncoprotein naturally overexpressed in many human cancers, is a member of the conserved Aurora protein serine/threonine kinase family that is implicated in the regulation of G 2 –M phases of the cell cycle. In vitro studies utilizing antibody microinjection, siRNA silencing and small molecule inhibitors have indicated that Aurora A functions in early as well as late stages of mitosis. However, due to limitations in specificity of the techniques, exact functional roles of the kinase remain to be clearly elucidated. In order to identify the physiological functions in vivo , we have generated Aurora A null mouse embryos, which show severe defects at 3.5 d.p.c. (days post-coitus) morula/blastocyst stage and lethality before 8.5 d.p.c. Null embryos at 3.5 d.p.c. reveal growth retardation with cells in mitotic disarray manifesting disorganized spindle, misaligned and lagging chromosomes as well as micronucleated cells. These findings provide the first unequivocal genetic evidence for an essential physiological role of Aurora A in normal mitotic spindle assembly, chromosome alignment segregation and maintenance of viability in mammalian embryos.