Early Postimplantation Mouse Development Research Articles

Single-Cell Rna-Seq Reveals Cellular Heterogeneity of Pluripotency Transition and X-Chromosome Dynamics During Early Postimplantation Mouse Development

Following implantation, the epiblast (EPI) cells transit from the naive to the primed pluripotency state in preparation for gastrulation, a momentous developmental milestone of the formation of the three primary germ layers. To investigate the molecular attributes of this critical developmental process of lineage specification and the dynamic changes in X-chromosome activity, we performed Smart-seq2 single-cell RNA-seq analysis of 1724 mouse E5.25 to E6.5 embryonic cells. We identified three distinct cellular states in the EPI cells that mirrors the transition of postimplantation EPI along the continuum of naive to primed pluripotency and the acquisition of primitive streak propensity over time. The transition of the EPI cellular state was accompanied by enhanced interaction with the visceral endoderm mediated by inductive signalling activity. By tracing X-chromosome activity at the allelic resolution, we discovered that X-chromosome reactivation (XCR) was initiated prior to the complete silencing of imprinted X-chromosome in the EPI, and followed by asynchronous random X-chromosome inactivation (XCI). Moreover, imprinted X-chromosome inactivation proceeded with a faster kinetics in the visceral endoderm compared to the extraembryonic ectoderm. Our study has outlined in detail a molecular roadmap of the emergent process of lineage specification and X-chromosome reprogramming during mouse pregastrulation development.

Sfrp5 is not essential for axis formation in the mouse

Secreted frizzled related protein (Sfrp) genes encode extracellular factors that can modulate Wnt signaling. During early post-implantation mouse development Sfrp5 is expressed in the anterior visceral endoderm (AVE) and the ventral foregut endoderm. The AVE is important in anterior-posterior axis formation and the ventral foregut endoderm contributes to multiple gut tissues. Here to determine the essential role of Sfrp5 in early mouse development we generated Sfrp5-deficient mice by gene targeting. We report that Sfrp5-deficient mice are viable and fertile. To determine whether the absence of an axis phenotype might be due to genetic redundancy with Dkk1 in the AVE we generated Sfrp5;Dkk1 double mutant mice. AVE development and primitive streak formation appeared normal in Sfrp5(-/-);Dkk1(-/-) embryos. These results indicate that Sfrp5 is not essential for axis formation or foregut morphogenesis in the mouse and also imply that Sfrp5 and Dkk1 together are not essential for AVE development.

Structure of the Predominant Protein Arginine Methyltransferase PRMT1 and Analysis of Its Binding to Substrate Peptides

PRMT1 is the predominant type I protein arginine methyltransferase in mammals and highly conserved among all eukaryotes. It is essential for early postimplantation development in mouse. Here we describe the crystal structure of rat PRMT1 in complex with the reaction product AdoHcy and a 19 residue substrate peptide containing three arginines. The results reveal a two-domain structure—an AdoMet binding domain and a barrel-like domain—with the active site pocket located between the two domains. Mutagenesis studies confirmed that two active site glutamates are essential for enzymatic activity, and that dimerization of PRMT1 is essential for AdoMet binding. Three peptide binding channels are identified: two are between the two domains, and the third is on the surface perpendicular to the strands forming the β barrel.

Arginine N-methyltransferase 1 is required for early postimplantation mouse development, but cells deficient in the enzyme are viable.

Protein arginine N-methyltransferases have been implicated in a variety of processes, including cell proliferation, signal transduction, and protein trafficking. In this study, we have characterized essentially a null mutation induced by insertion of the U3betaGeo gene trap retrovirus into the second intron of the mouse protein arginine N-methyltransferase 1 gene (Prmt1). cDNAs encoding two forms of Prmt1 were characterized, and the predicted protein sequences were found to be highly conserved among vertebrates. Expression of the Prmt1-betageo fusion gene was greatest along the midline of the neural plate and in the forming head fold from embryonic day 7.5 (E7.5) to E8.5 and in the developing central nervous system from E8.5 to E13.5. Homozygous mutant embryos failed to develop beyond E6.5, a phenotype consistent with a fundamental role in cellular metabolism. However, Prmt1 was not required for cell viability, as the protein was not detected in embryonic stem (ES) cell lines established from mutant blastocysts. Low levels of Prmt1 transcripts (approximately 1% of the wild-type level) were detected as assessed by a quantitative reverse transcription-PCR assay. Total levels of arginine N-methyltransferase activity and asymmetric N(G), N(G)-dimethylarginine were reduced by 85 and 54%, respectively, while levels of hypomethylated substrates were increased 15-fold. Prmt1 appears to be a major type I enzyme in ES cells, and in wild-type cells, most substrates of the enzyme appear to be maintained in a fully methylated state.

Identification of Endogenous Retinoids, Enzymes, Binding Proteins, and Receptors during Early Postimplantation Development in Mouse: Important Role of Retinal Dehydrogenase Type 2 in Synthesis of All-trans-Retinoic Acid

Specific combinations of nuclear retinoid receptors acting as ligand-inducible transcription factors mediate the essential role of retinoids in embryonic development. Whereas some data exist on the expression of these receptors during early postimplantation development in mouse, little is known about the enzymes controlling the production of active ligands for the retinoid receptors. Furthermore, at early stages of mouse development virtually no data are available on the presence of endogenous retinoids. In the present study we have used a recently developed high-performance liquid chromatographic (HPLC) technique to identify endogenous retinoids in mouse embryos down to the egg cylinder stage. All-trans-retinoic acid, a ligand for the retinoic acid receptors, was detected in embryos dissected as early as 7.5 dpc (i.e., a combination of midstreak until late allantoic bud stage embryos). At these stages, we detected mRNA coding for all the retinoid receptors, retinoid binding proteins, and two enzymes able to convert retinol to retinal (retinol dehydrogenase 5 (RDH5) and alcohol dehydrogenase 4 (ADH4)). We also detected retinal dehydrogenase type 2 (RALDH2), an enzyme capable of oxidising the final step in the all-trans-retinoic acid synthesis. In egg cylinder stage mouse embryos no all-trans-retinoic acid was detected. However, at this stage its precursor all-trans-retinal was present. In accordance with these HPLC observations, RDH5 and ADH4 were expressed, but no transcripts coding for enzymes that oxidise retinal to retinoic acid. Therefore, our results suggest that RALDH2 is a key regulator in initiating retinoic acid synthesis sometime between the mid-primitive streak stage and the late allantoic bud stage in mouse embryos.

Requirement for theXrcc1DNA Base Excision Repair Gene during Early Mouse Development

Surveillance and repair of DNA damage are essential for maintaining the integrity of the genetic information that is needed for normal development. Several multienzyme pathways, including the excision repair of damaged or missing bases, carry out DNA repair in mammals. We determined the developmental role of theX-ray cross-complementing (Xrcc)-1gene, which is central to base excision repair, by generating a targeted mutation in mice. Heterozygous matings producedXrcc1−/−embryos at early developmental stages, but notXrcc1−/−late-stage fetuses or pups. Histology showed that mutant (Xrcc1−/−) embryos arrested at embryonic day (E) 6.5 and by E7.5 were morphologically abnormal. The most severe abnormalities observed in mutant embryos were in embryonic tissues, which showed increased cell death in the epiblast and an altered morphology in the visceral embryonic endoderm. Extraembryonic tissues appeared relatively normal at E6.5–7.5. Even without exposure to DNA-damaging agents, mutant embryos showed increased levels of unrepaired DNA strand breaks in the egg cylinder compared with normal embryos.Xrcc1−/−cell lines derived from mutant embryos were hypersensitive to mutagen-induced DNA damage.Xrcc1mutant embryos that were also made homozygous for a null mutation inTrp53underwent developmental arrest after only slightly further development, thus revealing aTrp53-independent mechanism of embryo lethality. These results show that an intact base excision repair pathway is essential for normal early postimplantation mouse development and implicate an endogenous source of DNA damage in the lethal phenotype of embryos lacking this repair capacity.

Analysis of the role of dystroglycan in early postimplantation mouse development.

Analysis of the role of dystroglycan in early postimplantation mouse development.

Thymosin β10 mRNA expression during early postimplantation mouse development

The β-thymosins are a family of monomeric actin sequestering peptides that regulate actin dynamics within the cells. During embryogenesis the control of actin polymerization is essential in processes such as cell migration, angiogenesis and neurogenesis. Here we report that the levels of thymosin β 10 (T β 10) mRNA strongly increase during early postimplantation mouse embryogenesis as well as during in vitro P19 cell differentiation, indicating that this peptide plays an important role in early development. Moreover, analysis of the spatial distribution of T β 10 mRNA in 9.5–12.5 days postcoitum mouse embryos showed a remarkable presence of this transcript in mesenchymal structures as well as in the mantle layer of spinal cord. Interestingly, we observed differences in the distribution of the mRNAs encoding T β 10 and T β 4, another member of the β-thymosin family, suggesting different roles for these peptides during mouse embryogenesis.

The thymosin β4 gene is strongly activated in neural tissues during early postimplantation mouse development

We studied the temporal and spatial distribution of the mRNA encoding for thymosin β 4 (Tβ 4), a small acidic actin-sequestering peptide, during the early postimplantation mouse development. Analysis of total embryo RNA demonstrated a strong activation of Tβ 4 gene after gastrulation and coincident with neurulation. In situ hybridization showed that Tβ 4 mRNA was strongly expressed in the central nervous system and peripheral ganglia, paralleling the gradient of neuronal differentiation. An intense signal was also observed in intraventricular macrophages and blood vessels. The role of Tβ 4 in mammalian neuroembryogenesis is discussed.

The yeast homolog of H < beta > 58, a mouse gene essential for embryogenesis, performs a role in the delivery of proteins to the vacuole.

The highly conserved and ubiquitously expressed mouse gene H < beta > 58, identified through insertional mutagenesis, has been shown to be essential for early postimplantation development in mouse, but the mechanism by which it acts is unknown (Radice et al. 1991; Lee et al. 1992). We report here the identification of a yeast gene related to the H < beta > 58 gene and provide biochemical and genetic evidence for its function within the cell. The gene, PEP8, plays a role in the delivery of proteins to the vacuole. Disruption of the gene did not affect cell viability. However, the disruptants were shown to have a defect in the processing of the soluble vacuolar proteases but not the membrane vacuolar hydrolases. The processing defect appeared to be a consequence of the inability of the soluble vacuolar hydrolase to reach the vacuole. Although a small amount of the vacuolar precursors was mis-sorted to the extracellular medium, mis-sorting did not appear to be the primary defect in these cells. Pep8p was identified by epitope tagging of the protein. Biochemical fractionation indicated that the protein was peripherally bound to membranes. Immuno-gold electron microscopy indicated that the Pep8p localized to vacuolar membranes. Complementation experiments with the mouse H < beta > 58 cDNA revealed that a Pep8p-H < beta > 58 fusion protein in which the carboxy-terminal 85 amino acids of Pep8p were replaced by the carboxy-terminal 115 amino acids of H < beta > 58 was functional.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles.

The homeoproteins HNF1 (LFB1/HNF1-A) and vHNF1 (LFB3/HNF1 beta) interact with an essential control element of a group of liver-specific genes. During development, these putative target genes are initially expressed in the visceral endoderm of the yolk sac and subsequently in fetal liver. To assess the possible involvement of HNF1 and/or vHNF1 as transcriptional regulators in the early steps of visceral endoderm differentiation, we have analyzed the expression pattern of both factors both in vitro during differentiation of murine F9 embryonal carcinoma cells and in vivo during early postimplantation mouse development. We show here that differentiation of F9 cells into either visceral or parietal endoderm is accompanied by a sharp induction in vHNF1 mRNA and protein. By contrast, only low levels of aberrantly sized HNF1 transcripts, but not DNA-binding protein, are found in F9 cells and its differentiated derivatives. At 6-7.5 days of gestation, high levels of vHNF1 mRNA are present in the visceral extraembryonic endoderm, which co-localize with transcripts of the transthyretin gene. HNF1 transcripts are first detected in the yolk sac roughly two embryonic days later, after the developmental onset of transcription of target genes. As development proceeds, discrepancies are observed between the level of transcripts of both vHNF1 and HNF1 and their respective nuclear binding proteins, notably in the yolk sac and embryonic kidney. In addition, we show that two alternative spliced isoforms of vHNF1 mRNA, vHNF-A and vHNF1-B, are expressed in both embryonic and adult tissues. Taken together, these data suggest that vHNF1 participates as a regulatory factor in the initial transcriptional activation of the target genes in the visceral endoderm of the yolk sac, whereas the later appearance of HNF1 could be required for maintenance of their expression. Our results also provide evidence of a posttranscriptional level of control of vHNF1 and HNF1 gene expression during development, in addition to the spatial restriction in transcription.

Expression pattern of Motch, a mouse homolog of Drosophila Notch, suggests an important role in early postimplantation mouse development.

The Notch gene of Drosophila encodes a large transmembrane protein involved in cell-cell interactions and cell fate decisions in the Drosophila embryo. To determine if a gene homologous to Drosophila Notch plays a role in early mouse development, we screened a mouse embryo cDNA library with probes from the Xenopus Notch homolog, Xotch. A partial cDNA clone encoding the mouse Notch homolog, which we have termed Motch, was used to analyze expression of the Motch gene. Motch transcripts were detected in a wide variety of adult tissues, which included derivatives of all three germ layers. Differentiation of P19 embryonal carcinoma cells into neuronal cell types resulted in increased expression of Motch RNA. In the postimplantation mouse embryo Motch transcripts were first detected in mesoderm at 7.5 days post coitum (dpc). By 8.5 dpc, transcript levels were highest in presomitic mesoderm, mesenchyme and endothelial cells, while much lower levels were detected in neuroepithelium. In contrast, at 9.5 dpc, neuroepithelium was a major site of Motch expression. Transcripts were also abundant in cell types derived from neural crest. These data suggest that the Motch gene plays multiple roles in patterning and differentiation of the early postimplantation mouse embryo.

Permissiveness to murine leukemia virus expression during preimplantation and early postimplantation mouse development

Permissiveness to Moloney Murine Leukemia Virus (MoMuLV) expression was examined during preimplantation and early postimplantation development of the mouse embryo. Blastocysts and 8th, 9th and 10th day postimplantation embryos were infected in vitro with a MoMuLV-based retroviral vector expressing the lacZ gene driven off an internal rat beta-actin promoter. Beta-galactosidase-positive cells were identified in all embryonic tissues including inner cell mass, epiblast, mesoderm, endoderm and definitive ectoderm. In contrast, embryos infected with a MoMuLV-based vector expressing the lacZ gene driven off the viral LTR showed beta-galactosidase-positive cells only in mesoderm and definitive ectoderm. We conclude that permissiveness to transcriptional activity of the LTR is acquired immediately upon differentiation of epiblast during gastrulation of the mouse embryo.

The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells

In early postimplantation mouse development, transferrin synthesis appears to be a marker of visceral endoderm cell types. Transferrin was identified using immunoperoxidase staining, in the proximal (visceral) endoderm of the sixth-day egg cylinder, in some tissues at later stages, and in the visceral yolk sac (VYS) at all stages examined. Since the location of a plasma protein does not necessarily indicate its site of synthesis, the incorporation of labeled amino acids into transferrin was studied. Synthesis could be detected in egg cylinders on the seventh day of gestation onwards and in the VYS at all stages. However, although endoderm was the likely tissue source, its ability to synthesize transferrin after its isolation from the embryo was either much reduced or absent. The data are suggestive of a modulating influence by mesoderm and other cell types on transferrin synthesis in visceral endoderm cells. Three types of endoderm-like cells which are produced by teratocarcinoma embryonal carcinoma (EC) cells were analyzed for transferrin synthesis to assess possible parallels with the embryo. Embryoid bodies from PSA1 EC cells contained some outer endoderm cells which stained for transferrin and others which did not. The endoderm line PSA5E but not PYS-2 synthesized transferrin. The third type of endoderm-like cell (END cells) synthesized very little (OC15S1) or no (PC13 clone 5) transferrin. The conclusion that PSA5E, OC15 END, and some differentiated PSA1 cells have visceral endoderm-like character while PYS-2 reflects parietal endoderm phenotype is in agreement with published data.