Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K‐M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K‐M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice.     

rRNA accumulation and protein synthetic patterns in growing mouse oocytes

The rRNA contents of mouse primordial oocytes, three stages of growing oocytes, full-grown oocytes, and ovulated ova have been measured by hybridization of RNA samples to excess 3H-DNA complementary to rRNA. Since it was known from previous work that rRNA is stable, the results when plotted against days of oocyte growth indicated that rRNA was synthesized at a constant rate over the first 9 days of growth and about 1.5 times faster in the last 5 days. The maximum value of 0.3 ng per oocyte was attained by about 14 days of growth in oocytes 59 micrometers in diameter, well below the maximum diameter of 77 micrometers for full-grown oocytes. The stability of proteins synthesized in mid-growth phase oocytes was measured by labeling for 5 h with 35S-methionine and then following the decline of incorporated label during a 48h chase; 40% of the label decayed with a half-life of 11 h. and 60% was apparently stable. The two-dimensional electrophoretic patterns of labeled proteins synthesized by growing and full-grown oocytes were compared. The principal change was the appearance or great increase in intensity of several spots in full-grown oocytes as compared to growing oocytes. Egg proteins separated on a two-dimensional gel were visualized by silver staining. The cytoskeletal proteins actin, tubulin, and putative intermediate filament protein, as well as putative lactate dehydrogenase, were synthesized in growing and full-grown oocytes, and accumulated to form a significant portion of bulk egg protein.     

Aortic carboxypeptidase-like protein is expressed in collagen-rich tissues during mouse embryonic development

Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis.     

TTP, a C3H zinc finger protein gene, is expressed in mouse ovarian oocytes

The gene TTP, encoding a C3H zinc finger protein of the TIS11 family, is expressed in growing mouse oocytes. The gene is downregulated in Graafian follicles shortly before ovulation. This corresponds to a possible function in regulation of maternal mRNA translation, a function attributed to related C3H class genes in Caenorhabditis elegans, zebrafish, and Xenopus.     

Staufen1 is expressed in preimplantation mouse embryos and is required for embryonic stem cell differentiation

Pluripotent mouse embryonic stem (mES) cells derived from the blastocyst of the preimplantation embryo can be induced to differentiate in vitro along different cell lineages. However the molecular and cellular factors that signal and/or determine the expression of key genes, and the localisation of the encoded proteins, during the differentiation events are poorly understood. One common mechanism by which proteins can be targeted to specific regions of the cell is through the asymmetric localisation of mRNAs and Staufen, a double-stranded RNA binding protein, is known to play a direct role in mRNA transport and localisation. The aims of the present study were to describe the expression of Staufen in preimplantation embryos and mES cells and to use RNA interference (RNAi) to investigate the roles of Staufen1 in mES cell lineage differentiation. Western blotting and immunocytochemistry demonstrated that Staufen is present in the preimplantation mouse embryo, pluripotent mES cells and mES cells stimulated to differentiate into embryoid bodies, but the Staufen staining patterns did not support asymmetric distribution of the protein. Knockdown of Staufen1 gene expression in differentiating mES cells reduced the synthesis of lineage-specific markers including Brachyury, alpha-fetoprotein (AFP), PAX-6, and Vasa. There was however no significant change in either the gene expression of Nanog and Oct4, or in the synthesis of SSEA-1, all of which are key markers of pluripotency. These data indicate that inhibition of Staufen1 gene expression by RNAi affects an early step in mES cell differentiation and suggest a key role for Staufen in the cell lineage differentiation of mES cells.     

Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse

Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.     

The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development

Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2α and/or CK2α′) and two CK2β regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2β gene is required for early embryonic development, while the CK2α′ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2α gene in the mouse germ line. Embryos lacking CK2α have a marked reduction in CK2 activity in spite of the presence of the CK2α′ subunit. CK2α^−/− embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2α appears to play an essential and uncompensated role in mammalian development.     

Stem-loop binding protein is required for retinal cell proliferation,neurogenesis, and intraretinal axon pathfinding in zebrafish

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3′-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5 dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.     

Inter-alpha-inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse oocytes.

This report characterizes the effects of excess hyaluronan (HA) upon the expansion of the cumulus oocyte complex (COC) within intact follicles and upon ovulation and oocyte viability in mice. Covalent linkage between heavy chains of the inter-alpha-inhibitor (IalphaI) family of serum glycoproteins and HA is necessary for optimal cumulus extracellular matrix (cECM) stabilization and cumulus expansion. Intravenous administration of HA oligosaccharides inhibited the binding of IalphaI to endogenous HA, disrupting the process of expansion and resulting in a reduction in the size of the cumulus mass. Western blot and immunocytochemical analyses of COCs from HA-treated animals demonstrated a reduction of IalphaI heavy chains within the cECM. Additionally, HA-treated immature animals ovulated 56.3% fewer COCs compared to control animals. The developmental potential of COCs in HA-treated animals was also tested. Extended periods of oviductal storage of COCs ovulated by HA-injected adult mice resulted in a reduction of normal embryos and a significant increase in the proportion of fragmented oocytes/embryos. These observations support the view that covalent binding of IalphaI heavy chains to HA is required for optimal cumulus expansion, extrusion of the COCs from the follicle at ovulation, and maintenance of oocyte viability within the oviduct.     

The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse

Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.     

A transmembrane protein EIG121L is required for epidermal differentiation during early embryonic development

Epidermal differentiation in the ventral ectoderm of Xenopus embryos is regulated by the bone morphogenetic protein (BMP) pathway. However, it remains unclear how the BMP pathway is activated and induces the epidermal fate in the ventral ectoderm. Here, we identify a novel player in the BMP pathway that is required for epidermal differentiation during Xenopus early embryonic development. We show that Xenopus EIG121L (xEIG121L) protein, an evolutionarily conserved transmembrane protein, is expressed in the ventral ectoderm at the gastrula and neurula stages. Almost complete knockdown of xEIG121L protein with antisense morpholino oligonucleotides in early Xenopus embryos results in severe developmental defects, including the inhibition of epidermal differentiation and the induction of neural genes. Remarkably, our analysis shows that BMP/Smad1 signaling is severely suppressed in the xEIG121L knockdown ectoderm. Moreover, immunoprecipitation and immunostaining experiments suggest that xEIG121L protein physically interacts, and co-localizes, with BMP receptors. Thus, our results identify a novel regulator of the BMP pathway that has a positive role in BMP signaling and plays an essential role in epidermal differentiation during early embryonic development.     

G2E3 is a dual function ubiquitin ligase required for early embryonic development

G2E3 is a putative ubiquitin ligase (E3) identified in a microarray screen for mitotic regulatory proteins. It shuttles between the cytoplasm and nucleus, concentrating in nucleoli and relocalizing to the nucleoplasm in response to DNA damage. In this study, we demonstrate that G2E3 is an unusual ubiquitin ligase that is essential in early embryonic development to prevent apoptotic death. This protein has a catalytically inactive HECT domain and two distinct RING-like ubiquitin ligase domains that catalyze lysine 48-linked polyubiquitination. To address in vivo function, we generated a knock-out mouse model of G2E3 deficiency that incorporates a beta-galactosidase reporter gene under control of the endogenous promoter. Animals heterozygous for G2E3 inactivation are phenotypically normal with no overt change in development, growth, longevity, or fertility, whereas G2E3 null embryos die prior to implantation. Although normal numbers of G2E3(-/-) blastocysts are present at embryonic day 3.5, these blastocysts involute in culture as a result of massive apoptosis. Using beta-galactosidase staining as a marker for protein expression, we demonstrate that G2E3 is predominantly expressed within the central nervous system and the early stages of limb bud formation of the developing embryo. In adult animals, the most intense staining is found in Purkinje cell bodies and cells lining the ductus deferens. In summary, G2E3 is a dual function ubiquitin ligase essential for prevention of apoptosis in early embryogenesis.     

Soluble acidic complexes containing histones H3 and H4 in nuclei of Xenopus laevis oocytes

Oocyte nuclei of Xenopus laevis contain nucleosomal-core histones in large amounts and in a soluble, non-chromatin-bound form. Supernatant fractions (100,000 X g) from isolated nuclei are enriched in complexes containing histones H3 and H4, which are of distinct size (5.6S by sucrose gradient centrifugation, approximate molecular weight of 270,000 by gel filtration) and negatively charged (isoelectric at pH 4.4). These complexes bind to DEAE-Sephacel and can be separated from nucleoplasmin. In diverse fractionation experiments, histones H3 and H4 have been found to comigrate with a pair of polypeptides of molecular weight 110,000 that represent the most acidic major protein present in these nuclei. After enrichment by gel filtration, ion exchange chromatography and electrophoresis, this pair of acidic polypeptides has been the only nonhistone protein detected in the histone-complex fraction. We suggest that in the oocyte nucleus, large proportions of the soluble histones H3 and H4 are not contained in complexes of all four nucleosomal-core histones but are differentially associated with specific, very acidic proteins into distinct 5.6S complexes.     

Borealin is differentially expressed in ES cells and is essential for the early development of embryonic cells

Maintaining undifferentiated state and self-renewal ability of embryonic stem cells is a process that many genes and factors participate in. Using bioinformatics analyses and suppression subtractive hybridization we cloned a novel human gene related to the proliferation of human embryonic stem (hES) cells and its mouse homologue and identified them as being borealin. Our data demonstrated that borealin was highly expressed in undifferentiated ES cells, mouse pre-implantation embryos and the brain of 8.5–9.5 day post-coitum mouse embryos. Furthermore, following Borealin depletion by microinjecting anti-Borealin antibody into the zygotes the mouse embryos were arrested at the 2 or 4-cell stage and chromosomes could not correctly localize at the equator plane of the mitotic spindle and most cells had two or more nuclei. Taken together, these results indicate that Borealin plays a crucial role in the early mouse embryonic development.