Understanding the regulatory interactions that control gene expression during the development of novel tissues is a key goal of evolutionary developmental biology. Here, we show that Mbnl3 has undergone a striking process of evolutionary specialization in eutherian mammals resulting in the emergence of a novel placental function for the gene. Mbnl3 belongs to a family of RNA-binding proteins whose members regulate multiple aspects of RNA metabolism. We find that, in eutherians, while both Mbnl3 and its paralog Mbnl2 are strongly expressed in placenta, Mbnl3 expression has been lost from nonplacental tissues in association with the evolution of a novel promoter. Moreover, Mbnl3 has undergone accelerated protein sequence evolution leading to changes in its RNA-binding specificities and cellular localization. While Mbnl2 and Mbnl3 share partially redundant roles in regulating alternative splicing, polyadenylation site usage and, in turn, placenta maturation, Mbnl3 has also acquired novel biological functions. Specifically, Mbnl3 knockout (M3KO) alone results in increased placental growth associated with higher Myc expression. Furthermore, Mbnl3 loss increases fetal resource allocation during limiting conditions, suggesting that location of Mbnl3 on the X chromosome has led to its role in limiting placental growth, favoring the maternal side of the parental genetic conflict.

Embryonic development yields many different cell types in response to just a few families of inductive signals. The property of signal-receiving cells that determines how they respond to inductive signals is known as competence, and it differs in different cell types. Here, we explore the ways in which maternal factors modify chromatin to specify initial competence in the frog Xenopus tropicalis. We identify early-engaged regulatory DNA sequences, and infer from them critical activators of the zygotic genome. Of these, we show that the pioneering activity of the maternal pluripotency factors Pou5f3 and Sox3 determines competence for germ layer formation by extensively remodelling compacted chromatin before the onset of inductive signalling. This remodelling includes the opening and marking of thousands of regulatory elements, extensive chromatin looping, and the co-recruitment of signal-mediating transcription factors. Our work identifies significant developmental principles that inform our understanding of how pluripotent stem cells interpret inductive signals.

In this issue of Developmental Cell, Paraiso and colleagues report that antisense morpholino oligomers (MOs), frequently used by developmental biologists to inhibit gene function, do not cause immunogenic side effects at gastrula stages in the clawed frog Xenopus. This valuable observation is consistent with our previous work showing that MOs can activate components of the innate immune system, where, although we did not explore the exact stage of onset of the response, we commented that “this cell-intrinsic reaction was first detected during neurulation and intensified during axial elongation” (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Our earlier work was aimed at identifying side effects that might derive from the use of MOs in Xenopus tropicalis (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To this end, we performed a rigorous test in which we compared the transcriptomes of embryos carrying mutations in the Brachyury genes tbxt and tbxt.2 (formerly known as t and t2) with the transcriptomes of embryos in which the functions of the same genes had been inhibited by MOs. We assayed our results during late tailbud stages (7–15 h after gastrulation) and found that a cocktail of tbxt/tbxt.2 MOs (containing one translation- and one splice-blocking MO for each gene) activates a large number of genes associated with the innate immune system. These included complement component 3a receptor 1 (c3ar1), tumor protein 53 (tp53), and tp53 inducible nuclear protein (tp53inp1). This immune response was not restricted to translation-blocking MOs, as Paraiso et al., 2019Paraiso K.D. Blitz I.L. Zhou J.J. Cho K.W.Y. No compelling evidence that morpholinos elicit an innate immune response during early Xenopus embryogenesis.Dev. Cell. 2019; 49 (this issue): 643-650Scopus (7) Google Scholar suggest, because similar observations were made using the tbxt splice-blocking MO as well as the standard control MO (which was originally designed to block aberrant splicing caused by a point mutation in an intron of the human β-globin gene, and which is responsible for β-thalassemia). Similarly, the other side effect we observed, that of off-target mis-splicing, was not restricted to splice-blocking MOs (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Paraiso et al., 2019Paraiso K.D. Blitz I.L. Zhou J.J. Cho K.W.Y. No compelling evidence that morpholinos elicit an innate immune response during early Xenopus embryogenesis.Dev. Cell. 2019; 49 (this issue): 643-650Scopus (7) Google Scholar also suggest that the upregulation of c3ar1 may not be part of an innate immune response but linked to the Brachyury phenotype, in which posterior mesoderm fails to form. This is unlikely because this gene is also upregulated by the standard control MO (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, Paraiso et al., 2019Paraiso K.D. Blitz I.L. Zhou J.J. Cho K.W.Y. No compelling evidence that morpholinos elicit an innate immune response during early Xenopus embryogenesis.Dev. Cell. 2019; 49 (this issue): 643-650Scopus (7) Google Scholar) and because its upregulation, like that of tp53 and tp53inp1, occurs throughout the MO-injected embryo (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). We also know that c3ar1 is not upregulated in tbxt/tbxt.2 mutant embryos (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Analysis of the activation of c3ar1, tp53, and tp53inp1 revealed that expression increases with dose and developmental stage (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). We also observed that MOs with higher guanine-cytosine (GC) contents tend to elicit stronger responses, a result reminiscent of the ability of unmethylated CpG DNA to stimulate the mammalian immune system. Unmethylated CpG DNA is a pathogen-associated molecular pattern detected by immune cell-specific Toll-like receptors (TLRs) (Hemmi et al., 2000Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. A Toll-like receptor recognizes bacterial DNA.Nature. 2000; 408: 740-745Crossref PubMed Scopus (5358) Google Scholar, Krieg et al., 1995Krieg A.M. Yi A.K. Matson S. Waldschmidt T.J. Bishop G.A. Teasdale R. Koretzky G.A. Klinman D.M. CpG motifs in bacterial DNA trigger direct B-cell activation.Nature. 1995; 374: 546-549Crossref PubMed Scopus (3089) Google Scholar). The TLR signal mediator MyD88 induces NF-κB transcription factors and MAP kinases, which in turn trigger the complement component system and release pro-inflammatory cytokines and protective molecules including a truncated form of Tp53, which lacks the pro-apoptotic transactivation domain (Khoury and Bourdon, 2010Khoury M.P. Bourdon J.-C. The isoforms of the p53 protein.Cold Spring Harb. Perspect. Biol. 2010; 2: a000927Crossref PubMed Scopus (139) Google Scholar, Leleux et al., 2017Leleux J.A. Pradhan P. Roy K. Biophysical attributes of CpG presentation control TLR9 signaling to differentially polarize systemic immune responses.Cell Rep. 2017; 18: 700-710Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Work in zebrafish has previously demonstrated that MOs cause the upregulation of truncated Tp53 through the increased use of an internal promoter (Chen et al., 2009Chen J. Ng S.M. Chang C. Zhang Z. Bourdon J.-C. Lane D.P. Peng J. p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish.Genes Dev. 2009; 23: 278-290Crossref PubMed Scopus (127) Google Scholar); we now find the same is true in Xenopus tropicalis (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To ask how general is the ability of MOs to elicit an immune-related response, we analyzed the datasets now used by Paraiso and colleagues (Campbell et al., 2016Campbell E.P. Quigley I.K. Kintner C. Foxn4 promotes gene expression required for the formation of multiple motile cilia.Development. 2016; 143: 4654-4664Crossref PubMed Scopus (28) Google Scholar, Chung et al., 2014Chung M.-I. Kwon T. Tu F. Brooks E.R. Gupta R. Meyer M. Baker J.C. Marcotte E.M. Wallingford J.B. Coordinated genomic control of ciliogenesis and cell movement by RFX2.eLife. 2014; 3: e01439Crossref PubMed Google Scholar, Dichmann et al., 2015Dichmann D.S. Walentek P. Harland R.M. The alternative splicing regulator Tra2b is required for somitogenesis and regulates splicing of an inhibitory Wnt11b isoform.Cell Rep. 2015; 10: 527-536Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, Marlétaz et al., 2015Marlétaz F. Maeso I. Faas L. Isaacs H.V. Holland P.W.H. Cdx ParaHox genes acquired distinct developmental roles after gene duplication in vertebrate evolution.BMC Biol. 2015; 13: 56Crossref PubMed Scopus (10) Google Scholar, Noiret et al., 2016Noiret M. Mottier S. Angrand G. Gautier-Courteille C. Lerivray H. Viet J. Paillard L. Mereau A. Hardy S. Audic Y. Ptbp1 and Exosc9 knockdowns trigger skin stability defects through different pathways.Dev. Biol. 2016; 409: 489-501Crossref PubMed Scopus (10) Google Scholar). We omitted datasets lacking biological replicates and also those sampled at gastrula stages (Chiu et al., 2014Chiu W.T. Charney Le R. Blitz I.L. Fish M.B. Li Y. Biesinger J. Xie X. Cho K.W.Y. Genome-wide view of TGFβ/Foxh1 regulation of the early mesendoderm program.Development. 2014; 141: 4537-4547Crossref PubMed Scopus (51) Google Scholar, Ding et al., 2017Ding Y. Ploper D. Sosa E.A. Colozza G. Moriyama Y. Benitez M.D.J. Zhang K. Merkurjev D. De Robertis E.M. Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis.Proc. Natl. Acad. Sci. USA. 2017; 114: E3081-E3090Crossref Scopus (28) Google Scholar, Nakamura et al., 2016Nakamura Y. de Paiva Alves E. Veenstra G.J.C. Hoppler S. Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules.Development. 2016; 143: 1914-1925Crossref PubMed Scopus (89) Google Scholar, Yasuoka et al., 2014Yasuoka Y. Suzuki Y. Takahashi S. Someya H. Sudou N. Haramoto Y. Cho K.W. Asashima M. Sugano S. Taira M. Occupancy of tissue-specific cis-regulatory modules by Otx2 and TLE/Groucho for embryonic head specification.Nat. Commun. 2014; 5: 4322Crossref PubMed Scopus (37) Google Scholar) because we recognized, based on unpublished in situ hybridization data and as now shown by Paraiso et al., 2019Paraiso K.D. Blitz I.L. Zhou J.J. Cho K.W.Y. No compelling evidence that morpholinos elicit an innate immune response during early Xenopus embryogenesis.Dev. Cell. 2019; 49 (this issue): 643-650Scopus (7) Google Scholar, that this was too early to detect the immune response. Figure 6A of our paper (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), corroborated in Table S2 of Paraiso et al., 2019Paraiso K.D. Blitz I.L. Zhou J.J. Cho K.W.Y. No compelling evidence that morpholinos elicit an innate immune response during early Xenopus embryogenesis.Dev. Cell. 2019; 49 (this issue): 643-650Scopus (7) Google Scholar, is consistent with the idea that MOs used in other studies can induce c3ar1, tp53, and tp53inp1 in a stage-dependent, dose-dependent, and GC-dependent manner. It is important that one considers all three of these variables when analyzing the immune-related response. For example, it is not surprising, bearing in mind the stage dependency, that even MOs with a high GC content do not activate c3ar1 during gastrula stages (Paraiso et al., 2019Paraiso K.D. Blitz I.L. Zhou J.J. Cho K.W.Y. No compelling evidence that morpholinos elicit an innate immune response during early Xenopus embryogenesis.Dev. Cell. 2019; 49 (this issue): 643-650Scopus (7) Google Scholar). As Paraiso et al., 2019Paraiso K.D. Blitz I.L. Zhou J.J. Cho K.W.Y. No compelling evidence that morpholinos elicit an innate immune response during early Xenopus embryogenesis.Dev. Cell. 2019; 49 (this issue): 643-650Scopus (7) Google Scholar note, MOs also stimulate a stage-dependent and dose-dependent immune response in zebrafish embryos (Lai et al., 2018Lai J.K.H. Gagalova K. Stainier D.Y.R. Induction of interferon-stimulated genes and cellular stress pathways by morpholinos.bioRxiv. 2018; https://doi.org/10.1101/479188Crossref Google Scholar). Our analysis of these data suggests that the zebrafish response is also GC-dependent, with the egfl7 MO (52% GC) triggering a ubiquitous and stronger upregulation of immune interferon-stimulated genes than the vegfaa and standard control MOs (28% and 32% GC, respectively). The ongoing work of Paraiso and colleagues on gastrula-stage embryos should not be compromised by our discovery of a morpholino-induced immune response. However, if studying embryos beyond the gastrula stage, we suggest that researchers continue to bear our results in mind and note that “with regard to the immune reaction, we estimate that most of it could be avoided by designing MOs with a GC content of ≤40% and performing MO dosage optimization” (Gentsch et al., 2018Gentsch G.E. Spruce T. Monteiro R.S. Owens N.D.L. Martin S.R. Smith J.C. Innate immune response and off-target mis-splicing are common morpholino-induced side effects in Xenopus.Dev. Cell. 2018; 44: 597-610.e10Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001-157), the UK Medical Research Council (FC001-157), and the Wellcome Trust (FC001-157). Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus EmbryogenesisParaiso et al.Developmental CellMay 20, 2019In BriefGene expression interference by morpholinos has been questioned because of unwanted side effects, including immune response induction by a limited set of morpholinos. By performing a meta-analysis of available transcriptomic datasets, Paraiso et al. show that induction of an immune response is not a general side effect of morpholinos during early embryogenesis. Full-Text PDF Open Archive

Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects.

ABSTRACTEmbryonic development yields many different cell types in response to just a few families of inductive signals. The property of a signal-receiving cell that determines how it responds to such signals, including the activation of cell type-specific genes, is known as its competence. Here, we show how maternal factors modify chromatin to specify initial competence in the frogXenopus tropicalis. We identified the earliest engaged regulatory DNA sequences, and inferred from them critical activators of the zygotic genome. Of these, we showed that the pioneering activity of the maternal pluripotency factors Pou5f3 and Sox3 predefines competence for germ layer formation by extensively remodeling compacted chromatin before the onset of signaling. The remodeling includes the opening and marking of thousands of regulatory elements, extensive chromatin looping, and the co-recruitment of signal-mediating transcription factors. Our work identifies significant developmental principles that inform our understanding of how pluripotent stem cells interpret inductive signals.

Material Supplemental http://genesdev.cshlp.org/content/suppl/2014/09/02/28.17.1873.DC1.html References http://genesdev.cshlp.org/content/28/17/1873.full.html#ref-list-1 This article cites 39 articles, 15 of which can be accessed free at: Open Access Open Access option. Genes & Development Freely available online through the License Commons Creative . http://creativecommons.org/licenses/by/4.0 License (Attribution 4.0 International), as described at , is available under a Creative Commons Genes & Development This article, published in

Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify microRNA (miRNA)-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we found that three miRNA families, miR-20, miR-92, and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the proapoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are primed for not only differentiation but also cell death.

At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

The two first cell fate decisions taken in the mammalian embryo generate three distinct cell lineages: one embryonic, the epiblast, and two extraembryonic, the trophoblast and primitive endoderm. miRNAs are essential for early development, but it is not known if they are utilized in the same way in these three lineages. We find that in the pluripotent epiblast they inhibit apoptosis by blocking the expression of the proapoptotic protein Bcl2l11 (Bim) but play little role in the initiation of gastrulation. In contrast, in the trophectoderm, miRNAs maintain the trophoblast stem cell compartment by directly inhibiting expression of Cdkn1a (p21) and Cdkn1c (p57), and in the primitive endoderm, they prevent differentiation by maintaining ERK1/2 phosphorylation through blocking the expression of Mapk inhibitors. Therefore, we show that there are fundamental differences in how stem cells maintain their developmental potential in embryonic and extraembryonic tissues through miRNAs.

Background: During early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE) and the primitive endoderm (PrE). Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression.Results: In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES), trophectoderm stem (TS) and extra-embryonic endoderm (XEN) stem cell lines that were derived from the inner cell mass (ICM), TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac) and repressive (H3K27me3) chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3). Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation.Conclusions: These data provide evidence that the diversification of defined embryonic and extra-embryonic lineages is accompanied by chromatin remodelling at specific loci. Stem cell lines from the ICM, TE and PrE can each dominantly reprogramme somatic cells but reset gene expression differently, reflecting their separate lineage identities and increasingly restricted developmental potentials.

Background: X chromosome inactivation is the mechanism used in mammals to achieve dosage compensation of X-linked genes in XX females relative to XY males. Chromosome silencing is triggered in cis by expression of the non-coding RNA Xist. As such, correct regulation of the Xist gene promoter is required to establish appropriate X chromosome activity both in males and females. Studies to date have demonstrated co-transcription of an antisense RNA Tsix and low-level sense transcription prior to onset of X inactivation. The balance of sense and antisense RNA is important in determining the probability that a given Xist allele will be expressed, termed the X inactivation choice, when X inactivation commences.Results: Here we investigate further the mechanism of Xist promoter regulation. We demonstrate that both sense and antisense transcription modulate Xist promoter DNA methylation in undifferentiated embryonic stem (ES) cells, suggesting a possible mechanistic basis for influencing X chromosome choice. Given the involvement of sense and antisense RNAs in promoter methylation, we investigate a possible role for the RNA interference (RNAi) pathway. We show that the Xist promoter is hypomethylated in ES cells deficient for the essential RNAi enzyme Dicer, but that this effect is probably a secondary consequence of reduced levels of de novo DNA methyltransferases in these cells. Consistent with this we find that Dicer-deficient XY and XX embryos show appropriate Xist expression patterns, indicating that Xist gene regulation has not been perturbed.Conclusion: We conclude that Xist promoter methylation prior to the onset of random X chromosome inactivation is influenced by relative levels of sense and antisense transcription but that this probably occurs independent of the RNAi pathway. We discuss the implications for this data in terms of understanding Xist gene regulation and X chromosome choice in random X chromosome inactivation.