Progression of the pluripotent epiblast depends upon the NMD factor UPF2

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that degrades RNAs harboring in-frame stop codons in specific contexts. Loss of NMD factors leads to embryonic lethality in organisms spanning the phylogenetic scale, but the mechanism remains unknown. Here, we report that the core NMD factor, UPF2, is required for expansion of epiblast cells within the inner cell mass of mice in vivo. We identify NMD target mRNAs in mouse blastocysts – both canonical and alternatively processed mRNAs – including those encoding cell cycle arrest and apoptosis factors, raising the possibility that NMD is essential for embryonic cell proliferation and survival. In support, the inner cell mass of Upf2-null blastocysts rapidly regresses with outgrowth and is incompetent for embryonic stem cell derivation in vitro. In addition, we uncovered concordant temporal- and lineage-specific regulation of NMD factors and mRNA targets, indicative of a shift in NMD magnitude during peri-implantation development. Together, our results reveal developmental and molecular functions of the NMD pathway in the early embryo.</p

Dynamic Switching of Active Promoter and Enhancer Domains Regulates Tet1 and Tet2 Expression during Cell State Transitions between Pluripotency and Differentiation

The Tet 5-methylcytosine dioxygenases catalyze DNA demethylation by producing 5-hydroxymethylcytosine and further oxidized products. Tet1 and Tet2 are highly expressed in mouse pluripotent cells and downregulated to different extents in somatic cells, but the transcriptional mechanisms are unclear. Here we defined the promoter and enhancer domains in Tet1 and Tet2. Within a 15-kb "superenhancer" of Tet1, there are two transcription start sites (TSSs) with different activation patterns during development. A 6-kb promoter region upstream of the distal TSS is highly active in naive pluripotent cells, autonomously reports Tet1 expression in a transgenic system, and rapidly undergoes DNA methylation and silencing upon differentiation in cultured cells and native epiblast. A second TSS downstream, associated with a constitutively weak CpG-rich promoter, is activated by a neighboring enhancer in naive embryonic stem cells (ESCs) and primed epiblast-like cells (EpiLCs). Tet2 has a CpG island promoter with pluripotency-independent activity and an ESC-specific distal intragenic enhancer; the latter is rapidly downregulated in EpiLCs. Our study reveals distinct modes of transcriptional regulation at Tet1 and Tet2 during cell state transitions of early development. New transgenic reporters using Tet1 and Tet2 cis-regulatory domains may serve to distinguish nuanced changes in pluripotent states and the underlying epigenetic variations.status: publishe

Lineage-specific functions of TET1 in the postimplantation mouse embryo

The mammalian TET enzymes catalyze DNA demethylation. While they have been intensely studied as major epigenetic regulators, little is known about their physiological roles and the extent of functional redundancy following embryo implantation. Here we define non-redundant roles for TET1 at an early postimplantation stage of the mouse embryo, when its paralogs Tet2 and Tet3 are not detectably expressed. TET1 regulates numerous genes defining differentiation programs in the epiblast and extraembryonic ectoderm. In epiblast cells, TET1 demethylates gene promoters via hydroxymethylation and maintains telomere stability. Surprisingly, TET1 represses a majority of epiblast target genes independently of methylation changes, in part through regulation of the gene encoding the transcriptional repressor JMJD8. Dysregulated gene expression in the absence of TET1 causes embryonic defects, which are partially penetrant in an inbred strain but fully lethal in non-inbred mice. Collectively, our study highlights an interplay between the catalytic and non-catalytic activities of TET1 that is essential for normal development.status: publishe

The Neonatal and Adult Human Testis Defined at the Single-Cell Level.

Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are&nbsp;candidates to be used for SSC therapy to treat infertility

TGFβ1-Induced <em>Baf60c</em> Regulates both Smooth Muscle Cell Commitment and Quiescence

<div><p>Smooth muscle cells (SMCs) play critical roles in a number of diseases; however, the molecular mechanism underlying their development is unclear. Although the role of TGFβ1 signaling in SMC development is well established, the downstream molecular signals are not fully understood. We used several rat multipotent adult progenitor cell ((r)MAPC) lines that express levels of <em>Oct4</em> mRNA similar to hypoblast stem cells (HypoSC), and can differentiate robustly to mesodermal and endodermal cell types. TGFβ1 alone, or with PDGF-BB, induces differentiation of rMAPCs to SMCs, which expressed structural SMC proteins, including α-smooth muscle actin (αSMA), and contribute to the SMC coat of blood vessels in vivo. A genome-wide time-course transcriptome analysis revealed that transcripts of <em>Baf60c</em>, part of the SWI/SNF actin binding chromatin remodeling complex D-3 (SMARCD3/BAF60c), were significantly induced during MAPC-SMC differentiation. We demonstrated that BAF60c is a necessary co-regulator of TGFβ1 mediated induction of SMC genes. Knock-down of <em>Baf60c</em> decreased SMC gene expression in rMAPCs whereas ectopic expression of <em>Baf60c</em> was sufficient to commit rMAPCs to SMCs in the absence of exogenous cytokines. TGFβ1 activates <em>Baf60c</em> via the direct binding of SMAD2/3 complexes to the <em>Baf60c</em> promoter region. Chromatin- and co-immunoprecipitation studies demonstrated that regulation of SMC genes by BAF60c is mediated via interaction with SRF binding CArG box-containing promoter elements in SMC genes. We noted that compared with TGFβ1, Baf60c overexpression in rMAPC yielded SMC with a more immature phenotype. Similarly, <em>Baf60c</em> induced an immature phenotype in rat aortic SMCs marked by increased cell proliferation and decreased contractile marker expression. Thus, Baf60c is important for TGFβ-mediated commitment of primitive stem cells (rMAPCs) to SMCs and is associated with induction of a proliferative state of quiescent SMCs. The MAPC-SMC differentiation system may be useful for identification of additional critical (co-)regulators of SMC development.</p> </div

Genome wide transcriptome analysis of rMAPC-SMC differentiation.

<p>rMAPCs (Cl19 and 3c3) were cultured for 6 days with TGFβ1 and PDGF-BB in serum free medium, and RNA harvested from triplicate samples on days 0, 2, 4 and 6. In addition, RNA from cultured RAOSMCs was obtained in duplicate. <b>A</b>. Ingenuity pathway analysis (IPA) of the differentially expressed genes in both clones of rMAPCs identified predominantly developmental and cardiaovascular system associated genes (table represents data obtained as output from IPA). <b>B</b>. Pathways relating to cardiogenesis were among the top canonical pathways (table represents data obtained as output from IPA). <b>C</b>. Validation by PCR of the target TF genes identified by microarray analysis.</p

The Neonatal and Adult Human Testis Defined at the Single-Cell Level.

Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are&nbsp;candidates to be used for SSC therapy to treat infertility

<i>Baf60c</i> is important for the synthetic/proliferative phase of primary smooth muscle cells.

<p>RAOSMCs were either transfected with an on-target anti-<i>Baf60c</i> siRNA pool vs non-targeting pool (NTP) of siRNAs; or transduced with a lentiviral vector encoding for BAF60c or GFP <b>A</b>. qRT-PCR for the expression of <i>Baf60c, Sm-mhc</i>, and α<i>Sma</i> in cells transfected with on-target anti-<i>Baf60c</i> siRNA pool vs non-targeting pool (NTP) of siRNAs (Mean ±s.e.m of n = 4; Student's t-test p<0.05). <b>B</b>. qRT-PCR for the expression of <i>Baf60c, Sm-mhc</i>, and α<i>Sma</i> in cells transduced with either GFP or BAF60c (Mean±s.e.m of n = 3–5). <b>C</b>. Cell cycle analysis using fluorescence activated cell sorting in cells transfected with on-target anti-<i>Baf60c</i> siRNA pool vs non-targeting pool (NTP) of siRNAs (Mean±s.e.m of n = 3; Student's t-test p<0.05).</p