A LINE1-Nucleolin Partnership Regulates Early Development and ESC Identity.

Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.

Inhibition of mTOR induces a paused pluripotent state.

Cultured pluripotent stem cells are a cornerstone of regenerative medicine owing to their ability to give rise to all cell types of the body. Although pluripotent stem cells can be propagated indefinitely in vitro, pluripotency is paradoxically a transient state in vivo, lasting 2-3 days around the time of blastocyst implantation. The exception to this rule is embryonic diapause, a reversible state of suspended development triggered by unfavourable conditions. Diapause is a physiological reproductive strategy widely employed across the animal kingdom, including in mammals, but its regulation remains poorly understood. Here we report that the partial inhibition of mechanistic target of rapamycin (mTOR), a major nutrient sensor and promoter of growth, induces reversible pausing of mouse blastocyst development and allows their prolonged culture ex vivo. Paused blastocysts remain pluripotent and competent-able to give rise to embryonic stem (ES) cells and live, fertile mice. We show that both naturally diapaused blastocysts in vivo and paused blastocysts ex vivo display pronounced reductions in mTOR activity, translation, histone modifications associated with gene activity and transcription. Pausing can be induced directly in cultured ES cells and sustained for weeks without appreciable cell death or deviations from cell cycle distributions. We show that paused ES cells display a remarkable global suppression of transcription, maintain a gene expression signature of diapaused blastocysts and remain pluripotent. These results uncover a new pluripotent stem cell state corresponding to the epiblast of the diapaused blastocyst and indicate that mTOR regulates developmental timing at the peri-implantation stage. Our findings have implications in the fields of assisted reproduction, regenerative medicine, cancer, metabolic disorders and ageing.

Porcupine-dependent Wnt signaling controls stromal proliferation and endometrial gland maintenance through the action of distinct WNTs.

Wnt signaling has been shown to be important in orchestrating proper development of the female reproductive tract. In the uterus, six members of the Wnt family are expressed in the neonatal endometrium and deletion of individual Wnt genes often leads to similar phenotypes, suggesting an interaction of these genes in uterine development and function. Furthermore, Wnts may have complementary functions, which could mask the identification of their individual functional role in single gene deletions. To circumvent this issue, we have generated a deletion of the Porcupine homolog within the female reproductive tract using progesterone receptor-Cre mice (PgrCre/+); preventing Wnt secretion from the producing cells. We show that Porcupine-dependent Wnt signaling, unlike previously reported, is dispensable for postnatal gland formation but is required for post-pubertal gland maintenance as well as for stromal cell proliferation. Furthermore, our results demonstrate that WNT7a is sufficient to restore post-pubertal endometrial gland formation. Although WNT5a did not restore gland formation, it rescued stromal cell proliferation; up-regulating several secreted factors including Fgf10 and Ihh. Our results further elucidate the roles of Wnt signaling in uterine development and function as well as provide an ideal system to address individual Wnt functions in the uterus.

Regulation of porcupine-dependent Wnt signaling is essential for uterine development and function.

Six members of the Wnt family are expressed in the female reproductive tract. Their collective function ensures proper development of the uterus, preparing it for pregnancy during adulthood. Here, we take advantage of the fact that Porcn, a prerequisite for all Wnt secretion, is located on the X chromosome, to generate females that were mosaic for Porcn throughout the reproductive tract. Porcnflox/+ females were mated with progesterone receptor (Pgr)-Cre males (PgrCre/+ ) to generate females that were heterozygous for Porcupine in all tissues of the female reproductive tract, resulting in mosaicism due to random X-inactivation. We demonstrated that Porcn mosaic females are extremely subfertile and exhibit a large spectrum of phenotypes ranging from morphologically normal uteri to uteri with extremely enlarged cystic glands. Decreased fertility in Porcupine mosaic females was not associated with phenotype severity and was observed regardless of whether or not cystic glands were enlarged. By crossing-in a GFP reporter on the wild-type X chromosome, we were able to correlate endometrial gland hyperplasia with a mostly Porcupine mutant stroma, demonstrating the role of stromal Wnts in the regulation of endometrial gland proliferation. Finally, we demonstrated that fertility issues within mosaic females were due to a reduced response to estrogen and to abnormal Tcf/Lef signaling across the mesometrial-anti-mesometrial axis during the window of implantation.

Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.

Wnt/ß-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth cells as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (Porcn(Del)/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating that epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of Porcn(Del) organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable and that stromal production of Wnts can fully support normal murine intestinal homeostasis.

Porcupine-dependent Wnt activity within the uterine epithelium is essential for fertility.

The secretion of mammalian Wnt ligands within the cell is dependent on the activity of Porcupine, a gene located on the X-chromosome that encodes for a membrane-bound O-acyl transferase. Here, we report that postnatal ablation of Porcupine in the uterine luminal epithelium alone results in the decrease in endometrial gland number. Despite having uterine glands, mutant females are completely infertile. Epithelial ablation of Porcupine causes defects in timely apposition of the lumen, along with failure to respond to artificial decidual induction. Interestingly, progesterone supplementation was able to rescue the initiation of decidualization, but the decidua was not maintained and subsequently resorbed. Transcriptome analysis demonstrated that deletion of Porcupine in the epithelium resulted in the stromal dysregulation of members of the Wnt signaling pathway (Lef1, Wnt4, and Wnt16), dysregulation of receptors and ligands in the Notch signaling pathway (Notch1, Notch4, and Dll4) as well as Hoxa10. Our results demonstrate the crucial requirement of Wnt signaling in the epithelium for fertility and demonstrate that epithelial Wnts regulate stromal Wnt gene expression as well as regulating the expression of essential signaling factors and effectors required for successful embryo implantation.

Porcn-dependent Wnt signaling is not required prior to mouse gastrulation.

In mice and humans the X-chromosomal porcupine homolog (Porcn) gene is required for the acylation and secretion of all 19 Wnt ligands and thus represents a bottleneck for all Wnt signaling. We have generated a mouse line carrying a floxed allele for Porcn and used zygotic, oocyte-specific and visceral endoderm-specific deletions to investigate embryonic and extra-embryonic requirements for Wnt ligand secretion. We show that there is no requirement for Porcn-dependent secretion of Wnt ligands during preimplantation development of the mouse embryo. Porcn-dependent Wnts are first required for the initiation of gastrulation, where Porcn function is required in the epiblast but not the visceral endoderm. Heterozygous female embryos, which are mutant in both trophoblast and visceral endoderm due to imprinted X chromosome inactivation, complete gastrulation but display chorio-allantoic fusion defects similar to Wnt7b mutants. Our studies highlight the importance of Wnt3 and Wnt7b for embryonic and placental development but suggest that endogenous Porcn-dependent Wnt secretion does not play an essential role in either implantation or blastocyst lineage specification.

Location of transient ectodermal progenitor potential in mouse development.

Ectoderm is one of the three classic germ layers in the early mouse embryo, with the capacity to develop into both the central nervous system and epidermis. Because it is a transient phase of development with few molecular markers, the early ectoderm is the least understood germ layer in mouse embryonic development. In this work, we studied the differentiation potential of isolated ectoderm tissue in response to BMP signaling at various developmental stages (E6.5, E7.0 and E7.5), and identified a transient region in the anterior-proximal side of the embryo at E7.0 that possesses the ability to become neural or epidermal ectoderm in response to the absence or presence of BMP4, respectively. Furthermore, we demonstrated that inhibition of Nodal signaling could direct the pluripotent E6.5 epiblast cells towards ectoderm lineages during differentiation in explants in vitro. Our work not only improves our understanding of ectodermal layer development in early embryos, but also provides a framework for regenerative differentiation towards ectodermal tissues.

Phenotypic annotation of the mouse X chromosome.

Mutational screens are an effective means used in the functional annotation of a genome. We present a method for a mutational screen of the mouse X chromosome using gene trap technologies. This method has the potential to screen all of the genes on the X chromosome without establishing mutant animals, as all gene-trapped embryonic stem (ES) cell lines are hemizygous null for mutations on the X chromosome. Based on this method, embryonic morphological phenotypes and expression patterns for 58 genes were assessed, approximately 10% of all human and mouse syntenic genes on the X chromosome. Of these, 17 are novel embryonic lethal mutations and nine are mutant mouse models of genes associated with genetic disease in humans, including BCOR and PORCN. The rate of lethal mutations is similar to previous mutagenic screens of the autosomes. Interestingly, some genes associated with X-linked mental retardation (XLMR) in humans show lethal phenotypes in mice, suggesting that null mutations cannot be responsible for all cases of XLMR. The entire data set is available via the publicly accessible website (http://xlinkedgenes.ibme.utoronto.ca/).

Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos.

Wnt signaling plays important roles in development and disease. The X-chromosomal Porcupine homolog gene (Porcn) encodes an evolutionary conserved member of the membrane bound O-acyl transferase (MBOAT) superfamily that has been shown to be required for the palmitoylation and secretion of Wnt3a, a mechanism that has been suggested to be conserved for all mammalian Wnt ligands. PORCN mutations in humans cause Focal Dermal Hypoplasia (FDH), a disorder causing developmental defects in heterozygous females and embryonic lethality in hemizygous males. In this study, Porcn mutant mouse embryonic stem (ES) cells were used to analyze the role of Porcn in mammalian embryonic development. In vitro, we show an exclusive requirement for Porcn in Wnt secreting cells and further, that any of the four Porcn isoforms is sufficient to allow for the secretion of functional Wnt3a. Embryos generated by aggregation of Porcn mutant ES cells with wildtype embryos fail to complete gastrulation in vivo, but remain in an epiblast-like state, similar to Wnt3 and Gpr177/Wls mutants. Consistent with this phenotype, in vitro differentiated mutant ES cells fail to generate endoderm and mesoderm derivatives. Taken together, these data confirm the importance of Porcn for Wnt secretion and gastrulation and suggest that disruption of early development underlies the male lethality of human PORCN mutants.

PhiC31 integrase facilitates genetic approaches combining multiple recombinases.

Homologous and site-specific DNA recombination has revolutionized genetic engineering. The reliability of recombinases such as Cre and FLP has allowed scientists to design complex strategies to study gene function in mammals. However, the retention of recombination sites in the genome limits the use of Cre and FLP recombinases in subsequent modifications. Access to additional recombinases in the ES cell toolbox would enormously widen the number of possibilities to manipulate the genome. In the method presented here, we combine the use of PhiC31, a site-specific integrase, with FLP to obtain site-specific insertion and replacement in pre-inserted docking sites in the genome of mouse ES cells. This method allows for the integration of any sequence of interest in a pre-defined locus, leaving Cre recombinase available for downstream applications. The selection strategy is based on a silent selection marker activated by a plasmid-delivered promoter, making the integration system highly reliable and reducing the need for extensive molecular screens. This article describes how to create "dockable" mouse embryonic stem (ES) cell lines, integrate incoming vectors, and analyze the resulting clones. Current applications of this technology are also discussed.

Unwind and transcribe: chromatin reprogramming in the early mammalian embryo.

Within the first few days of life, the unipotent gametic genomes are rapidly reprogrammed to support emergence of pluripotent cells in the early mammalian embryo. It is now appreciated that this crucial stage of development involves dramatic changes to chromatin at multiple levels, such as DNA methylation, histone modifications, histone mobility, and higher-order chromatin organization. Technological advances are beginning to allow genome-wide views of this chromatin reprogramming, and provide new approaches to functionally dissect its regulation. Here we review recent insights into the dynamic chromatin environment of the early mouse embryo. New data challenge long-held assumptions, for example, with regards to the asymmetry of DNA methylation of the parental genomes or the onset of functional zygotic genome activation. We discuss how impaired chromatin reprogramming can lead to early embryonic lethality, but might also have delayed effects that only manifest later in embryogenesis or postnatally, potentially influencing the propensity for adult-onset diseases.

Sox17-mediated XEN cell conversion identifies dynamic networks controlling cell-fate decisions in embryo-derived stem cells.

Little is known about the gene regulatory networks (GRNs) distinguishing extraembryonic endoderm (ExEn) stem (XEN) cells from those that maintain the extensively characterized embryonic stem cell (ESC). An intriguing network candidate is Sox17, an essential transcription factor for XEN derivation and self-renewal. Here, we show that forced Sox17 expression drives ESCs toward ExEn, generating XEN cells that contribute to ExEn when placed back into early mouse embryos. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. To orchestrate this conversion process, Sox17 acts in autoregulatory and feedforward network motifs, regulating dynamic GRNs directing cell fate. Sox17-mediated XEN conversion helps to explain the regulation of cell-fate changes and reveals GRNs regulating lineage decisions in the mouse embryo.

The Hippo pathway member Nf2 is required for inner cell mass specification.

During mammalian development, the first two lineages to be specified are the trophectoderm (TE) and the inner cell mass (ICM). The Hippo pathway kinases Lats 1 and 2 (Lats1/2) and the transcriptional coactivator Yap play important roles in this specification process [1]. In outside cells of the embryo, Yap is nuclear localized and cooperates with Tead4 to induce the TE-specifying transcription factor Cdx2. In inside cells, Lats1/2 phosphorylate Yap and prevent its nuclear localization. The factors acting upstream of Lats1/2 and Yap in this context have not been identified. Here, we demonstrate that the upstream Hippo pathway member Nf2/Merlin is required for Lats1/2-dependent Yap phosphorylation in the preimplantation embryo. Injection of dominant-negative Nf2 mRNA causes Yap mislocalization and ectopic Cdx2 expression, effects that can be rescued by overexpression of Lats2 kinase. Zygotic Nf2 mutant blastocysts have mild defects in Yap localization and Cdx2 expression, but these become much more severe upon removal of both maternal and zygotic Nf2. The inside cells of maternal-zygotic mutants fail to establish a pluripotent ICM and form excess TE, resulting in peri-implantation lethality. Together, these data establish a clear role for Nf2 upstream of Yap in the preimplantation embryo and demonstrate that Hippo signaling is essential to segregate the ICM from the TE.

Zygotic Porcn paternal allele deletion in mice to model human focal dermal hypoplasia.

In mouse and humans, the X-chromosomal Porcupine homolog (Porcn) gene is required for the acylation and secretion of all 19 Wnt ligands, thus representing a bottleneck in the secretion of Wnt ligands. In humans, mutations in PORCN cause the X-linked dominant syndrome Focal Dermal Hypoplasia (FDH, OMIM#305600). This disorder is characterized by ecto-mesodermal dysplasias and shows a highly variable phenotype, potentially due to individual X chromosome inactivation patterns. To improve the understanding of human FDH, we have established a mouse model by generation of Porcn heterozygous animals carrying a zygotic deletion of the paternal allele. We show that heterozygous female fetuses display variable defects that do not significantly affect survival in the uterus, but lead to perinatal lethality in more than 95% of females. Rare survivors develop to adulthood and display variable skeletal and skin defects, representing an adult zygotic mouse model for human FDH. Although not frequently reported in humans, we also observed bronchopneumonia, rhinitis, and otitis media in these animals, suggesting a potential link between Porcn function and the normal development of ciliated cells in these tissues.

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.