The maternal store of the xlgv7 mRNA in full-grown oocytes is not required for normal development in Xenopus

We have attempted to analyze the function of a maternal mRNA xlgv7 which is distributed as an animal-vegetal gradient in stage 6 oocytes using a combination of antisense oligodeoxynucleotide injection into oocytes followed by in vitro maturation and fertilization. Injection of 20 ng of the antisense oligodeoxynucleotide resulted in the destruction of the xlgv7 mRNA to undetectable levels. Upon maturation and fertilization the resulting embryos develop with no specific defects suggesting that the maternal store of xlgv7 in stage 6 oocytes is not required and that the embryo can develop solely with the maternal store of the xlgv7 protein. Also, these results demonstrate the feasibility of this approach in destroying a specific maternal RNA and assaying its effect on development.     

Stella is a maternal effect gene required for normal early development in mice

stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella null offspring, stella-deficient females displayed severely reduced fertility due to a lack of maternally inherited Stella-protein in their oocytes. Indeed, we demonstrate that embryos without Stella are compromised in preimplantation development and rarely reach the blastocyst stage. stella is thus one of few known mammalian maternal effect genes, as the phenotypic effect on embryonic development is mainly a consequence of the maternal stella mutant genotype. Furthermore, we show that STELLA that is expressed in human oocytes is also expressed in human pluripotent cells and in germ cell tumors. Interestingly, human chromosome 12p, which harbours STELLA, is consistently overrepresented in these tumors. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumors.     

XBtg2 is required for notochord differentiation during early Xenopus development

The notochord is essential for normal vertebrate development, serving as both a structural support for the embryo and a signaling source for the patterning of adjacent tissues. Previous studies on the notochord have mostly focused on its formation and function in early organogenesis but gene regulation in the differentiation of notochord cells itself remains poorly defined. In the course of screening for genes expressed in developing notochord, we have isolated Xenopus homolog of Btg2 (XBtg2). The mammalian Btg2 genes, Btg2/PC3/TIS21, have been reported to have multiple functions in the regulation of cell proliferation and differentiation but their roles in early development are still unclear. Here we characterized XBtg2 in early Xenopus laevis embryogenesis with focus on notochord development. Translational inhibition of XBtg2 resulted in a shortened and bent axis phenotype and the abnormal structures in the notochord tissue, which did not undergo vacuolation. The XBtg2-depleted notochord cells expressed early notochord markers such as chordin and Xnot at the early tailbud stage, but failed to express differentiation markers of notochord such as Tor70 and 5-D-4 antigens in the later stages. These results suggest that XBtg2 is required for the differentiation of notochord cells such as the process of vacuolar formation after determination of notochord cell fate.     

The polycomb-group gene Ezh2 is required for early mouse development

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.     

The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro.

Previous studies have suggested that the vav protooncogene plays an important role in hematopoiesis. To study this further, we have ablated the vav protooncogene by homologous recombination in embryonic stem (ES) cells. Homozygous vav (-/-) ES clones differentiate normally in culture and generate cells of erythroid, myeloid and mast cell lineages. Mice heterozygous for the targeted vav allele do not display any obvious abnormalities. However, homozygous embryos die very early during development. Crosses of vav (+/-) heterozygous mice yield apparently normal vav (-/-) E3.5 embryos but not post-implantation embryos (> or = E7.5). Furthermore, homozygous vav (-/-) blastocysts do not hatch in vitro. These results indicate that vav is essential for an early developmental step(s) that precedes the onset of hematopoiesis. Consistent with the phenotypic analysis of vav (-/-) embryos, we have identified Vav immunoreactivity in the extra-embryonic trophoblastic cell layer but not in the inner embryonic cell mass of E3.5 preimplantation embryos or in the egg cylinder of E6.5 and E7.5 post-implantation embryos. These results suggest that the vav gene is essential for normal trophoblast development and for implantation of the developing embryo.     

The ocular albinism type 1 gene product is an N-glycoprotein but glycosylation is not required for its subcellular distribution.

The ocular albinism type 1 (OA1) gene product is a membrane glycoprotein that may play a role in controlling melanosome growth and maturation. A number of mutations in the OA1 gene lead to ocular albinism due at least in part to retention of the aberrant protein in the endoplasmic reticulum. To examine whether N-glycosylation plays a role in the post-translational trafficking of the Oa1 protein, we constructed a series of mutant mouse Oa1 cDNAs encoding an Oa1-green fluorescent protein fusion in which some or all of the potential glycosylation sites were eliminated by site-directed mutagenesis. Biochemical studies in transfected cells treated with tunicamycin and peptide:N-glycosidase F suggest that asparagine at amino acid 106 is essential for N-glycosylation of the protein. Mutation at amino acid 106 that eliminated glycosylation did not affect the endo/lysosomal distribution of the Oa1 protein in either COS cells or cultured murine melanocytes.     

Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

The art gene product of human immunodeficiency virus is required for replication.

A mutation that alters the initiation codon of the art gene of the human immunodeficiency virus renders the virus replication defective. The replication defect of this mutation can be complemented in trans by the art gene product. These findings indicate that the art protein is essential for replication and, as such, represents a good target for development of new antiviral agents that could be useful in the control of human immunodeficiency virus infection.     

The histidine triad protein Hint is not required for murine development or Cdk7 function

The histidine triad (HIT) protein Hint has been found to associate with mammalian Cdk7, as well as to interact both physically and genetically with the budding yeast Cdk7 homologue Kin28. To study the function of Hint and to explore its possible role in modulating Cdk7 activity in vivo, we have characterized the expression pattern of murine Hint and generated Hint-deficient (Hint(-/-)) mice. Hint was widely expressed during mouse development, with pronounced expression in several neuronal ganglia, epithelia, hearts, and testes from embryonic day 15 onward. Despite this widespread expression, disruption of Hint did not impair murine development. Moreover, Hint-deficient mice had a normal life span and were apparently healthy. Histological examination of tissues with high Hint expression in wild-type animals did not show signs of abnormal pathology in Hint(-/-) mice. Functional redundancy within the HIT family was addressed by crossing Hint(-/-) mice with mice lacking the related HIT protein, Fhit, and by assaying the expression levels of the HIT protein gene family members Hint2 and Hint3 in Hint(+/+) and Hint(-/-) tissues. Finally, Cdk7 kinase activity and cell cycle kinetics were found to be comparable in wild-type and Hint(-/-) mouse embryonic fibroblasts, suggesting that Hint may not be a key regulator of Cdk7 activity.     

The GCR2 gene family is not required for ABA control of seed germination and early seedling development in Arabidopsis

Background

The plant hormone abscisic acid (ABA) regulates diverse processes of plant growth and development. It has recently been proposed that GCR2 functions as a G-protein-coupled receptor (GPCR) for ABA. However, the structural relationships and functionality of GCR2 have been challenged by several independent studies. A central question in this controversy is whether gcr2 mutants are insensitive to ABA, because gcr2 mutants were shown to display reduced sensitivity to ABA under one experimental condition (e.g. 22°C, continuous white light with 150 µmol m^-2 s⁻¹) but were shown to display wild-type sensitivity under another slightly different condition (e.g. 23°C, 14/10 hr photoperiod with 120 µmol m⁻² s⁻¹). It has been hypothesized that gcr2 appears only weakly insensitive to ABA because two other GCR2-like genes in Arabidopsis, GCL1 and GCL2, compensate for the loss of function of GCR2.

Principal Findings

In order to test this hypothesis, we isolated a putative loss-of-function allele of GCL2, and then generated all possible combinations of mutations in each member of the GCR2 gene family. We found that all double mutants, including gcr2 gcl1, gcr2 gcl2, gcl1 gcl2, as well as the gcr2 gcl1 gcl2 triple mutant displayed wild-type sensitivity to ABA in seed germination and early seedling development assays, demonstrating that the GCR2 gene family is not required for ABA responses in these processes.

Conclusion

These results provide compelling genetic evidence that GCR2 is unlikely to act as a receptor for ABA in the context of either seed germination or early seedling development.     

The tnpR gene product of TnA is required for transposition immunity

Summary A mutant of TnA no longer recognizing immune plasmids has been isolated. The mutation is complemented in trans by a functional tnpR gene. The requirement for wild type tnpR gene product for the establishment of transposition immunity was confirmed by the use of a derivative of transposon Tn3 in which both the tnpA and the tnpR genes are partly deleted. This deleted Tn3 was shown to transpose onto an immune plasmid in the presence of a wild type tnpA gene but not in the presence of both tnpA and tnpR genes.     

Tomato hydroxymethylglutaryl-CoA reductase is required early in fruit development but not during ripening.

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and the level of its mRNA have been determined at various stages of tomato fruit development. The HMGR reaction makes mevalonate, a necessary component in the synthesis of all isoprene containing compounds, such as sterols and carotenoids. A cDNA clone encoding the active site region of HMGR has been isolated from a tomato library derived from young-fruit mRNA. The clone hybridizes to a one- or two-copy fragment in high-stringency DNA gel blot analyses and detects an mRNA of approximately 3.0 kb. Both HMGR activity and mRNA levels are high in early stages of tomato fruit development, when rapid cell division occurs, as well as in the subsequent early stages of cellular expansion. In contrast, ripening fruit have very low levels of reductase activity and mRNA, even though large amounts of the carotenoid lycopene are synthesized during this period. Furthermore, in vivo inhibition of HMGR during early fruit stages disrupts subsequent development, whereas inhibition during later stages of fruit expansion has no apparent effect on ripening. We conclude that the pool of mevalonate responsible for the synthesis of phytosterols is synthesized primarily during the first half of tomato fruit development. In addition, the final period of fruit expansion and ripening is not dependent upon HMGR activity, but instead utilizes a preexisting pool of pathway intermediates or requires the use of salvage pathways in the cell.     

The F-box protein Fbw7 is required for cerebellar development

The F-box protein Fbw7 (also known as Fbxw7, hCdc4 and Sel-10) functions as a substrate recognition component of a SCF-type E3 ubiquitin ligase. SCF(Fbw7) facilitates polyubiquitination and subsequent degradation of various proteins such as Notch, cyclin E, c-Myc and c-Jun. Fbw7 is highly expressed in the nervous system and controls neural stem cell differentiation and apoptosis via Notch and c-Jun during embryonic development (Hoeck et al., 2010). Fbw7 deletion in the neural lineage is perinatal lethal and thus prohibits studying the role of Fbw7 in the adult nervous system. fbw7 mRNA is highly expressed in the postnatal brain and to gain insights into the function of Fbw7 in postnatal neurogenesis we analysed Fbw7 function in the cerebellum. We generated conditional Fbw7-knockout mice (fbw7^?Cb) by inactivating Fbw7 specifically in the cerebellar anlage. This resulted in decreased cerebellar size, reduced Purkinje cell number and defects in axonal arborisation. Moreover, Fbw7-deficient cerebella showed supranumeral fissures and aberrant progenitor cell migration. Protein levels of the Fbw7 substrates Notch1 and N-terminally phosphorylated c-Jun were upregulated in fbw7^?Cb mice. Concomitant deletion of c-Jun, and also the junAA knock-in mutation which specifically abrogates c-Jun N-terminal phosphorylation, rescued Purkinje cell numbers and arborisation in the fbw7^?Cb background. Taken together these data demonstrate that Fbw7 is essential during cerebellar development, and identify N-terminally phosphorylated c-Jun as an important substrate of SCF(Fbw7) during neurogenesis.     

The Klebsiella pneumoniae PII protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL-mediated nif gene regulation

Summary The role of theKlebsiella pneumoniae P_II protein (encoded byglnB) in nitrogen regulation has been studied using two classes ofglnB mutants. In Class I mutants P_II appears not to be uridylylated in nitrogen-limiting conditions and in Class II mutants P_II is not synthesised. The effects of these mutations on expression from nitrogen-regulated promoters indicate that P_II is not absolutely required for nitrogen control. Furthermore the uridylylated form of P_II(P_II-UMP) plays a significant role in the response to changes in nitrogen status by counteracting the effect of P_II on NtrB-mediated dephosphorylation of NtrC. P_II is not involved in thenif-specific response to changes in nitrogen status mediated by NifL.