Vienna Biocenter

Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21-25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. We disrupted the dicer-1 (dcr-1) gene in mouse embryonic stem (ES) cells by conditional gene targeting and generated Dicer-null ES cells. These cells were viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells displayed severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs were markedly reduced. Re-expression of Dicer in the knockout cells rescued these phenotypes. Our data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing.

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development.

Tissue-resident memory T (Trm) cells permanently localize to portals of pathogen entry, where they provide immediate protection against reinfection. To enforce tissue retention, Trm cells up-regulate CD69 and down-regulate molecules associated with tissue egress; however, a Trm-specific transcriptional regulator has not been identified. Here, we show that the transcription factor Hobit is specifically up-regulated in Trm cells and, together with related Blimp1, mediates the development of Trm cells in skin, gut, liver, and kidney in mice. The Hobit-Blimp1 transcriptional module is also required for other populations of tissue-resident lymphocytes, including natural killer T (NKT) cells and liver-resident NK cells, all of which share a common transcriptional program. Our results identify Hobit and Blimp1 as central regulators of this universal program that instructs tissue retention in diverse tissue-resident lymphocyte populations.

Changes to the symbiotic microbiota early in life, or the absence of it, can lead to exacerbated type 2 immunity and allergic inflammations. Although it is unclear how the microbiota regulates type 2 immunity, it is a strong inducer of proinflammatory T helper 17 (T(H)17) cells and regulatory T cells (T(regs)) in the intestine. Here, we report that microbiota-induced T(regs) express the nuclear hormone receptor RORγt and differentiate along a pathway that also leads to T(H)17 cells. In the absence of RORγt(+) T(regs), T(H)2-driven defense against helminths is more efficient, whereas T(H)2-associated pathology is exacerbated. Thus, the microbiota regulates type 2 responses through the induction of type 3 RORγt(+) T(regs) and T(H)17 cells and acts as a key factor in balancing immune responses at mucosal surfaces.

Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40-NLS-GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response.

In plants, perception of invading pathogens involves cell-surface immune receptor kinases. Here, we report that the Arabidopsis SITE-1 PROTEASE (S1P) cleaves endogenous RAPID ALKALINIZATION FACTOR (RALF) propeptides to inhibit plant immunity. This inhibition is mediated by the malectin-like receptor kinase FERONIA (FER), which otherwise facilitates the ligand-induced complex formation of the immune receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2) with their co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) to initiate immune signaling. We show that FER acts as a RALF-regulated scaffold that modulates receptor kinase complex assembly. A similar scaffolding mechanism may underlie FER function in other signaling pathways.

Selective autophagy of damaged mitochondria requires autophagy receptors optineurin (OPTN), NDP52 (CALCOCO2), TAX1BP1, and p62 (SQSTM1) linking ubiquitinated cargo to autophagic membranes. By using quantitative proteomics, we show that Tank-binding kinase 1 (TBK1) phosphorylates all four receptors on several autophagy-relevant sites, including the ubiquitin- and LC3-binding domains of OPTN and p62/SQSTM1 as well as the SKICH domains of NDP52 and TAX1BP1. Constitutive interaction of TBK1 with OPTN and the ability of OPTN to bind to ubiquitin chains are essential for TBK1 recruitment and kinase activation on mitochondria. TBK1 in turn phosphorylates OPTN's UBAN domain at S473, thereby expanding the binding capacity of OPTN to diverse Ub chains. In combination with phosphorylation of S177 and S513, this posttranslational modification promotes recruitment and retention of OPTN/TBK1 on ubiquitinated, damaged mitochondria. Moreover, phosphorylation of OPTN on S473 enables binding to pS65 Ub chains and is also implicated in PINK1-driven and Parkin-independent mitophagy. Thus, TBK1-mediated phosphorylation of autophagy receptors creates a signal amplification loop operating in selective autophagy of damaged mitochondria.

Oxidative and replication stress underlie genomic instability of cancer cells. Amplifying genomic instability through radiotherapy and chemotherapy has been a powerful but nonselective means of killing cancer cells. Precision medicine has revolutionized cancer therapy by putting forth the concept of selective targeting of cancer cells. Poly(ADP-ribose) polymerase (PARP) inhibitors represent a successful example of precision medicine as the first drugs targeting DNA damage response to have entered the clinic. PARP inhibitors act through synthetic lethality with mutations in DNA repair genes and were approved for the treatment of BRCA mutated ovarian and breast cancer. PARP inhibitors destabilize replication forks through PARP DNA entrapment and induce cell death through replication stress-induced mitotic catastrophe. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) exploit and exacerbate replication deficiencies of cancer cells and may complement PARP inhibitors in targeting a broad range of cancer types with different sources of genomic instability. Here I provide an overview of the molecular mechanisms and cellular consequences of PARP and PARG inhibition. I highlight clinical performance of four PARP inhibitors used in cancer therapy (olaparib, rucaparib, niraparib, and talazoparib) and discuss the predictive biomarkers of inhibitor sensitivity, mechanisms of resistance as well as the means of overcoming them through combination therapy.

Histone N-termini (tails) undergo diverse post-translational modifications,including acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation (van Holde,1988; Wolffe,1998). The discoveries of enzymes that perform these modifications and of chromatin-associated proteins that selectively bind to position-specific histone modifications(Strahl and Allis, 2000; Jenuwein and Allis, 2001)reveals that modified histone N-termini can significantly extend the information potential of the genetic code. Moreover, they appear to index chromatin regions, facilitating epigenetic control, lineage commitment and the overall functional organisation of chromosomes.FIG1Acetylation (Roth et al.,2001) and arginine methylation(Stallcup, 2001) have been linked mainly with transcriptional stimulation. Phosphorylation(Cheung et al., 2000a) instead is a marker for activation of immediate early genes and a signal for mitotic chromatin condensation. Here, we focus on histone lysine methylation. The roles of acetylation, phosphorylation and methylation are summarized in Table 1, and discussion of the interplay between these distinct modifications can be found elsewhere(Zhang and Reinberg, 2001; Berger, 2002; Kouzarides, 2002).At least five methylatable lysine positions exist in the N-termini of histones H3 (K4, K9, K27, K36) and H4 (K20); another occurs in the histone-fold domain of histone H3 (K79)(Feng et al., 2002; Lacoste et al., 2002; Ng et al., 2002; van Leeuwen et al., 2002). For clarity, we focus on H3-K4, H3-K9 and H3-K27 methylation to illustrate the general principles and complexities involved.The mammalian Suv39h enzymes and their S. pombe homologue, Clr4,were the first histone lysine methyltransferases (HMTases) identified(Rea et al., 2000). The conserved SET-domain of the Su(var)3-9-related HMTases catalyzes the methylation of H3-K9, creating a high-affinity binding site for the chromodomain of heterochromatin protein 1 (HP1) proteins(Lachner and Jenuwein, 2002). Other methylatable lysine positions might also be marked by position-specific SET-domain HMTases for methyl-binding chromodomain proteins. The human and mouse genomes each encode ≥50 predicted SET-domain proteins(Kouzarides, 2002) and ≥30 chromodomain-containing sequences (A. Schleiffer and F. Eisenhaber, personal communication). By contrast, S. pombe has only ∼10 putative SET domain HMTases, and S. cerevisiae has not more than seven(Briggs et al., 2001). Lysine residues are mono-, di- and tri-methylated in vivo(Paik and Kim, 1971; van Holde, 1988; Waterborg, 1993). A progressive conversion towards tri-methylation could contribute to the apparent stability of histone lysine methylation and is ideally suited to imparting additional layers of combinatorial control, which might allow both short-term and long-term chromatin imprints.The poster shows the dynamic cycle of histone lysine methylation in transcriptional stimulation or repression. `Exit routes' from this cycle reveal more extended reprogramming of the chromatin structure – for example, during cellular senescence, Polycomb-mediated transcriptional memory,X chromosome inactivation and constitutive heterochromatin formation. In this`road map', the various destinations for a chromatin region are indicated by road signs that reflect distinct methylation positions and states.In euchromatic regions, binding of transcription factors to specific promoter/enhancer sequences is the initiating step in altering a naive chromatin template. If positively acting complexes prevail, promoter-proximal nucleosomes sequentially adopt an activation-specific modification profile(Urnov and Wolffe, 2001; Zhang and Reinberg, 2001; Berger, 2002; Daujat et al., 2002). Fully activated promoters appear to be enriched in tri-methylated H3-K4(Santos-Rosa et al., 2002);basal transcription correlates with H3-K4 dimethylation, although the methylation potential of the HMTases involved needs to be defined(Briggs et al., 2001; Nishioka et al., 2002a; Wang et al., 2001a; Santos-Rosa et al., 2002).H3-K9 methylation, by contrast, is present mainly in silenced chromatin domains (Noma et al., 2001; Litt et al., 2001), and the`activated genome' of S. cerevisiae exhibits abundant H3-K4 methylation but lacks apparent H3-K9 di-methylation(Briggs et al., 2001). Recruitment of several H3-K9-specific HMTases induces gene repression within euchromatin (Tachibana et al.,2001; Nielsen et al.,2001; Vandel et al.,2001; Ogawa et al.,2002; Schultz et al.,2002; Tachibana et al.,2002; Yang et al.,2002). G9a and a closely related enzyme appear to be euchromatic HMTases that form complexes with HP1γ and a subset of E2F transcription factors (Ogawa et al., 2002). These enzymes might, by default, repress target promoters that fail to recruit additional activating complexes.In proliferating cells and for G9a-mediated in vivo methylation, the repressive signal appears to be primarily H3-K9 di-methylation(Tachibana et al., 2002) (A. H. Peters, S. Kubicek, L. Perez-Burgos et al., unpublished), although in vitro G9a methylates both H3-K9 and H3-K27. Differences between H3-K9 di- and tri-methylation patterns could underpin the more robust association of inhibitory complexes with the promoters of several cell cycle genes, as cells enter senescence (S. Lowe, personal communication) or have their growth potential restricted by the tumor suppressor Rb, which could recruit additional repressive HMTases (Nielsen et al., 2001).For histone lysine methylation, no `direct' demethylase has been described. Although intermediary enzymes could destabilise the amino-methyl bond by oxidation or radical attack (Chinenov,2002; Falnes et al.,2002; Trewick et al.,2002), reversion of an engaged chromatin region to a more naive state might instead be triggered by transcription-coupled histone replacement,in which the histone H3.3 variant is deposited in place of modified histone H3(Ahmad and Henikoff, 2002a). This mechanism does not operate in transcriptionally silent domains, which might explain turnover of methylated histones in euchromatic regions while allowing persistence of histone methylation in constitutive heterochromatin(Ahmad and Henikoff,2002b).During differentiation, `transcriptional memory' maintains the expression status of certain key regulatory genes over many cell division cycles. This depends on the antagonistic function of polycomb (Pc-G) and trithorax (trx-G)group proteins (Orlando and Paro,1995; Pirrotta,1998). The Pc-G protein enhancer of zeste [E(z)] contains a SET domain and becomes an HMTase when complexed with another early-acting Pc-G protein, extra sex combs (Esc). The Drosophila E(z)-Esc complex(Czermin et al., 2002; Müller et al., 2002) and its mammalian Ezh-Eed counterpart (Cao et al., 2002; Kuzmichev et al.,2002) have an apparent preference for H3-K27 but might also target H3-K9. Ezh/Eed-mediated nucleosome methylation increases in vitro binding of the chromodomain protein polycomb (PC)(Czermin et al., 2002; Kuzmichev et al., 2002). In E(z) mutants, methylation of H3-K27, and probably also H3-K9, is impaired– in a manner suggesting that extended H3-K27 di- and tri-methylation across several nucleosomes (Cao et al.,2002) or dual tri-methylation of H3-K27 and H3-K9[(Czermin et al., 2002) R. Paro, personal communication] might induce stable recruitment of Pc-G complexes. The E(z) HMTase complex could be developmentally regulated such that a di-methylating activity prepares histones for a tri-methylating activity, which propagates transcriptional memory. Fully defining the in vivo methyl mark(s) involved, however, requires the development of highly specific H3-K27 and H3-K9 antibodies.Long-term maintenance of active transcriptional states is regulated by trx-G proteins. The trx-G proteins Trx/MLL(Milne et al., 2002; Nakamura et al., 2002) and Ash-1 each contain a SET domain and display HMTase activity. Whereas a Trx complex performs H3-K4 di-methylation(Czermin et al., 2002; Milne et al., 2002; Nakamura et al., 2002), Ash-1 can methylate H3-K4, H3-K9 and probably also H4-K20(Beisel et al., 2002). Ash-1-mediated methylation apparently prevents binding of the repressive PC and HP1 proteins but facilitates association of the Brahma coactivator(Beisel et al., 2002) –another trx-G protein and a component of nucleosome-mobilising machines. Indeed, H3-K4 methylation can trigger recruitment of the Brahma-related ISWI ATPase (T. Kouzarides, personal communication). Thus, trx-G HMTases may allow propagation of an activated chromatin state by `neutralising' repressive marks(e.g. H3-K9 and H4-K20 methylation) (Fang et al., 2002; Nishioka et al.,2002b), while simultaneously coupling a positive signal (H3-K4 methylation) with chromatin remodelling.Dosage compensation in female mammals involves chromosome-wide inactivation of one X-chromosome (Avner and Heard,2001). H3-K9 methylation is associated with the inactive X chromosome (Xi) (Boggs et al.,2002; Peters et al.,2002; Heard et al.,2001; Mermoud et al.,2002), but H3-K27 tri-methylation might also be a prominent, if not the major, mark (Silva et al.,2003; Plath et al.,2003) (A. H. Peters, S. Kubicek, L. Perez-Burgos et al.,unpublished). Pronounced H3-K27 tri-methylation at the Xi would be consistent with the finding that X-inactivation is independent of Suv39h HMTases and does not require HP1 proteins (Peters et al.,2002). The HMTases that target the Xi, particularly for random X-inactivation, are unidentified. A likely candidate for initiating early methylation imprints is the Ezh-Eed complex, because both Ezh2(Mak et al., 2002) and Eed(Wang et al., 2001c)accumulate at the Xi during imprinted X-inactivation. However, in contrast to Pc-G-mediated gene silencing, there is no evidence for stable association of PC or other Pc-G complexes at the Xi(Silva et al., 2003). Differences in H3-K27 and H3-K9 methylation could discriminate between Pc-G-dependent repression (extended H3-K27 di- and tri-methylation or a combination of H3-K9 tri- and H3-K27 tri-methylation?) and X-inactivation (a combination of H3-K9 di- and H3-K27 tri-methylation?). Alternatively, the Xist RNA could provide an additional signal for recruitment of other,Xi-restricted HMTases and associated silencing complexes. This would be similar to Xist-dependent accumulation of BRCA1(Ganesan et al., 2002) and preclude occupancy by the PC system and HP1 proteins. Subtle differences in the methylation state of lysine positions might also be associated with allele-specific imprinting (Xin et al.,2001; Fournier et al.,2002; Xin et al.,2003).Unlike euchromatin, constitutive heterochromatin lacks apparent transcription units, and instead contains arrays of satellite repeats(Karpen and Allshire, 1997; Csink and Henikoff, 1998). Such repeats appear to give rise – through the RNAi machinery – to small heterochromatic RNAs (shRNAs)(Volpe et al., 2002; Hall et al., 2002; Partridge et al., 2002; Mochizuki et al., 2002; Taverna et al., 2002). These or other RNAs (Maison et al.,2002) might pair with the underlying DNA sequences and bind to chromodomain-like adaptor proteins (Akhtar et al., 2000a) that could recruit Su(var)3-9-related HMTases(Jenuwein, 2002). The H3-K9 methylation signal would then be stabilised and propagated by `interlocking'HP1 molecules to form an extended heterochromatic domain(Nakayama et al., 2001; Hall et al., 2002). Furthermore, H3-K9 methylation can trigger DNA methylation in Neurospora crassa (Tamaru and Selker,2001) and Arabidopsis thaliana(Jackson et al., 2002), and a similar pathway directs DNA methylation at pericentric satellite repeats in mammals (B. Lehnertz, Y. Ueda, A. A. Derijck et al., unpublished). The combination of histone- and DNA-methylation systems(Fahrner et al., 2002; Nguyen et al., 2002; Fuks et al., 2003) probably stabilises silent chromatin domains, safe-guarding gene expression programmes and protecting genome integrity.Pericentric heterochromatin is enriched in tri-methylated H3-K9. This profile is selectively abolished upon disruption of Suv39h HMTases, whereas centromeric regions display Suv39h-independent H3-K9 di-methylation (A. H. Peters, S. Kubicek, L. Perez-Burgos et al., unpublished). Interestingly, in Suv39h dn cells, pericentric heterochromatin exhibits significant H3-K9 mono-methylation (A. H. Peters, S. Kubicek, L. Perez-Burgos et al.,unpublished). Suv39h HMTases are thus tri-methylating enzymes that can convert intermediary methylation states (mono- or di-methylation) into the apparently more stable tri-methylation end state. Regional H3-K9 tri-methylation at transcriptionally inert chromatin domains therefore appears to be a robust hallmark of constitutive heterochromatin.The above examples highlight the exquisite complexity and coding potential of histone lysine methylation in epigenetic control. Position- and state-specific methylation antibodies(Santos-Rosa et al., 2002) (A. H. Peters, S. Kubicek, L. Perez-Burgos et al., unpublished) and the solved 3D-structures of several SET domain enzymes(Trievel et al., 2002; Wilson et al., 2002; Zhang et al., 2002; Jacobs et al., 2002; Min et al., 2002) have started to reveal the functions of mono- (SET7/9; Xiao et al., 2003), di- [G9a(Tachibana et al., 2002) (A. H. Peters, S. Kubicek, L. Perez-Burgos et al., unpublished)] and tri-methylating HMTases [Suv39h (A. H. Peters, S. Kubicek, L. Perez-Burgos et al., unpublished)]. Although the `rules of the road' highlighted in this poster focused on basic mechanisms of transcriptional regulation and chromosome organisation, histone lysine methylation probably affects most chromatin-templated processes – from cell proliferation and tumorigenesis (Varambally et al.,2002) to imprinting, X-inactivation, lineage commitment(Su et al., 2003), aging,stem cell plasticity and the epigenetic reprogramming of the genome.We thank David Allis, Renato Paro, Tony Kouzarides, Neil Brockdorff, Steven Gamblin and Scott Lowe for helpful discussions and for allowing us to cite work prior to its publication. Research in T.J.'s laboratory is supported by the IMP through Boehringer Ingelheim and by funds from the Vienna Economy Promotion Fund (WWFF), an EU-network grant and the Austrian GEN-AU initiative.

Selective autophagy contributes to intracellular homeostasis by mediating the degradation of cytoplasmic material such as aggregated proteins, damaged or over-abundant organelles, and invading pathogens. The molecular machinery for selective autophagy must ensure efficient recognition and sequestration of the cargo within autophagosomes. Cargo specificity can be mediated by autophagic cargo receptors that specifically bind the cargo material and the autophagosomal membrane. Here we review the recent insights into the mechanisms that enable cargo receptors to confer selectivity and exclusivity to the autophagic process. We also discuss their different roles during starvation-induced and selective autophagy. We propose to classify autophagic events into cargo-independent and cargo-induced autophagosome formation events.

Abstract Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl ( Ambystoma mexicanum ) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3 . However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3 −/− and Pax7 −/− mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.

Epigenome modulation potentially provides a mechanism for organisms to adapt, within and between generations. However, neither the extent to which this occurs, nor the mechanisms involved are known. Here we investigate DNA methylation variation in Swedish Arabidopsis thaliana accessions grown at two different temperatures. Environmental effects were limited to transposons, where CHH methylation was found to increase with temperature. Genome-wide association studies (GWAS) revealed that the extensive CHH methylation variation was strongly associated with genetic variants in both cis and trans, including a major trans-association close to the DNA methyltransferase CMT2. Unlike CHH methylation, CpG gene body methylation (GBM) was not affected by growth temperature, but was instead correlated with the latitude of origin. Accessions from colder regions had higher levels of GBM for a significant fraction of the genome, and this was associated with increased transcription for the genes affected. GWAS revealed that this effect was largely due to trans-acting loci, many of which showed evidence of local adaptation.

The mammalian kidney develops in three successive steps from the initial pronephros via the mesonephros to the adult metanephros. Although the nephric lineage is specified during pronephros induction, no single regulator, including the transcription factor Pax2 or Pax8, has yet been identified to control this initial phase of kidney development. In this paper, we demonstrate that mouse embryos lacking both Pax2 and Pax8 are unable to form the pronephros or any later nephric structures. In these double-mutant embryos, the intermediate mesoderm does not undergo the mesenchymal-epithelial transitions required for nephric duct formation, fails to initiate the kidney-specific expression of Lim1 and c-Ret, and is lost by apoptosis 1 d after failed pronephric induction. Conversely, retroviral misexpression of Pax2 was sufficient to induce ectopic nephric structures in the intermediate mesoderm and genital ridge of chick embryos. Together, these data identify Pax2 and Pax8 as critical regulators that specify the nephric lineage.

Since the first reports of a novel severe acute respiratory syndrome (SARS)-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the human population. Recent debate has coalesced around two competing ideas: a "laboratory escape" scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.

The generation of B-lymphocytes from hematopoietic stem cells is controlled by multiple transcription factors regulating distinct developmental aspects. Ikaros and PU.1 act in parallel pathways to control the development of lymphoid progenitors in part by regulating the expression of essential signaling receptors (Flt3, c-Kit, and IL-7R alpha). The generation of the earliest B cell progenitors depends on E2A and EBF, which coordinately activate the B cell gene expression program and immunoglobulin heavy-chain gene rearrangements at the onset of B-lymphopoiesis. Pax5 restricts the developmental options of lymphoid progenitors to the B cell lineage by repressing the transcription of lineage-inappropriate genes and simultaneously activating the expression of B-lymphoid signaling molecules. LEF1 and Sox4 contribute to the survival and proliferation of pro-B cells in response to extracellular signals. Finally, IRF4 and IRF8 together control the termination of pre-B cell receptor signaling and thus promote differentiation to small pre-B cells undergoing light-chain gene rearrangements.

The detailed cellular and molecular mechanisms leading to joint destruction in rheumatoid arthritis, a disease driven by proinflammatory cytokines, are still unknown. To address the question of whether osteoclasts play a pivotal role in this process, transgenic mice that express human TNF (hTNFtg) and that develop a severe and destructive arthritis were crossed with osteopetrotic, c-fos–deficient mice (c-fos–/–) completely lacking osteoclasts. The resulting mutant mice (c-fos–/–hTNFtg) developed a TNF-dependent arthritis in the absence of osteoclasts. All clinical features of arthritis, such as paw swelling and reduction of grip strength, progressed equally in both groups. Histological evaluation of joint sections revealed no difference in the extent of synovial inflammation, its cellular composition (except for the lack of osteoclasts), and the expression of matrix metalloprotein-ase-3 (MMP-3) and MMP-13. In addition, cartilage damage, proteoglycan loss, and MMP-3, -9, and -13 expression in chondrocytes were similar in hTNFtg and c-fos–/–hTNFtg mice. However, despite the presence of severe inflammatory changes, c-fos–/–hTNFtg mice were fully protected against bone destruction. These data reveal that TNF-dependent bone erosion is mediated by osteoclasts and that the absence of osteoclasts alters TNF-mediated arthritis from a destructive to a nondestructive arthritis. Therefore, in addition to the use of anti-inflammatory therapies, osteoclast inhibition could be beneficial for the treatment of rheumatoid arthritis.

Abstract One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid 1 . Here we show that naive human pluripotent stem cells cultured in PXGL medium 2 and triply inhibited for the Hippo, TGF-β and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development 3,4 .