Early Embryo Lethality Research Articles

Endocardial HDAC3 is required for myocardial trabeculation

Failure of proper ventricular trabeculation is often associated with congenital heart disease. Support from endocardial cells, including the secretion of extracellular matrix and growth factors is critical for trabeculation. However, it is poorly understood how the secretion of extracellular matrix and growth factors is initiated and regulated by endocardial cells. We find that genetic knockout of histone deacetylase 3 in the endocardium in mice results in early embryo lethality and ventricular hypotrabeculation. Single cell RNA sequencing identifies significant downregulation of extracellular matrix components in histone deacetylase 3 knockout endocardial cells. Secretome from cultured histone deacetylase 3 knockout mouse cardiac endothelial cells lacks transforming growth factor ß3 and shows significantly reduced capacity in stimulating cultured cardiomyocyte proliferation, which is remarkably rescued by transforming growth factor ß3 supplementation. Mechanistically, we identify that histone deacetylase 3 knockout induces transforming growth factor ß3 expression through repressing microRNA-129-5p. Our findings provide insights into the pathogenesis of congenital heart disease and conceptual strategies to promote myocardial regeneration.

A maternal-effect Padi6 variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos.

Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.

Abstract EC.01: Endocardial Hdac3 Promotes Myocardial Trabeculation Through A MicroRNA Pathway

A key process during early heart development is myocardial trabeculation, which is the formation of bundles of myocardium extending into the chamber. The failure of trabeculation often contributes to many forms of congenital heart defects. Proper trabeculation requires coordination among multiple cell types and signaling communication. For instance, growth signals released from the endocardium are required for trabecular myocardial growth and maturation. However, it is poorly understood how these signaling cascades are initiated and regulated in the endocardium. Recently, our lab demonstrated that epicardial histone deacetylase 3 (HDAC3) is required for myocardial compaction. In this current study, we found that the genetic deletion of Histone deacetylase 3 (Hdac3) in the cardiac endothelial cells in mice resulted in early embryo lethality presenting as a hypotrabeculation cardiac phenotype, suggesting that endocardial Hdac3 deletion disrupted critical signals from the endocardium for trabecular myocardium growth. When cultured with supernatant from Hdac3 knockout (KO) mouse cardiac endothelial cells (MCECs), wild-type (WT) mouse embryonic cardiomyocytes also showed decreased proliferation. Single cell RNA-sequencing identified that transforming growth factor ß3 (Tgfß3) along with their extracellular matrix (ECM) downstream targets was significantly downregulated in Hdac3 deficient endocardial cells. Interestingly, chemical inhibition of deacetylase catalytic activity alone resulted in significant downregulation of Tgfß3 in cultured Hdac3 KO MCECs, suggesting that HDAC3 induces Tgfß3 expression in an enzymatic dependent manner. Mechanistically, we identified that miR-129 was significantly upregulated in Hdac3 KO MCECs and Hdac3 endocardial KO hearts. Overexpression of miR-129 repressed Tgfß3 expression in WT MCECs, whereas knockdown of miR-129 restored Tgfß3 expression in Hdac3 KO MCECs. Our findings reveal a critical signaling pathway in which endocardial HDAC3 promotes trabecular myocardium growth by stimulating Tgfß through repressing miR-129, providing novel insights into elucidating the etiology of congenital heart defects and conceptual strategies to promote myocardial regeneration.

TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice.

Members of PARP family are responsible for poly(ADP-ribose) (pADPr) posttranslational modification synthesis. They are intensively studied proteins with more than 20,500 related papers in PubMed database search to date. PARG, the main enzyme that degrades pADPr, is unfairly attracted less attention, and 40 times less papers (a little more than 500) are related to its functioning. The difficulties to work with PARG knockout animals due to its early embryo lethality could be one reason for this huge difference. Mice PARG-specific antibodies are not available from any vendor, which also complicates the research process. There is one available for public PARG knockout mice line generated by KOMP project. It has LacZ cassette, which replaces three critical exons in PARG gene. Here, we present the method to genotype these mice with Taqman qPCR multiplex approach. It allows to work with a small amount of DNA material like early embryo stages and to separate maternal DNA contamination. The modification of this method is also applicable for studying PARG conditional knockouts and identifying the success of floxed PARG gene exon deletion by Cre-driven recombination.

Role of O ‐linked glucose‐fucose disaccharide modification of thrombospondin type I repeats in protein folding and embryo development

We propose that Protein O-fucosyltransferase 2 (POFUT2) and β3-glucosyltransferase (B3GLCT) function to ensure efficient folding of forty-nine proteins containing tandemly repeated Thrombospondin Type 1 Repeats (TSRs). In the endoplasmic reticulum (ER), POFUT2 and B3GLCT recognize properly folded TSRs and act sequentially to add the unusual O-linked Glucoseβ1-3Fucose disaccharide to either a serine or threonine residue located within the POFUT2 consensus sequence (C-X-X-S/T-C). Once all TSRs are modified the target protein exits the ER. In crystal structures the disaccharide forms H-bonds and van der Waals interactions with underlying amino acids in the TSR, raising the possibility that the disaccharide functions as a surrogate amino acid to stabilize the correctly folded TSR. Results from in vitro folding/refolding assays provide evidence that the disaccharide stabilizes the TSR fold and accelerates the overall rate of protein folding. Consistent with this prediction, loss of POFUT2 or B3GLCT in cell culture impairs trafficking of POFUT2/B3GLCT targets. To begin to evaluate the function of the disaccharide in vivo, we generated mouse loss of function mutations in Pofut2 and B3glct. Loss of Pofut2 causes early embryo lethality due to a block in gastrulation, resulting from functional reduction of ADAMTS9 in extraembryonic tissue. In contrast, B3glct animals survive, but have craniofacial and skeletal abnormalities, defects in pigmentation, soft tissue syndactyly, and develop hydrocephalus. The mouse B3glct phenotype shares similarities to Peters Plus syndrome in humans, caused by recessive mutations in B3GLCT. We propose that hydrocephalus in B3glct mutants results from the combined effects of the mutation on ADAMTS9 and ADAMTS20 in ependymal cells and Sco-Spondin (SSPO) in the subcommissural organ. The distinct differences in the effects of these mutations during mouse development support a model whereby the O-linked fucose is essential for folding and secretion of all POFUT2/B3GLCT target proteins and that B3GLCT-mediated extension to the glucose-fucose disaccharide is only required by a subset of sensitive targets.

Rapid Way to Generate Mouse Models for In Vivo Studies of the Endothelium.

A single layer of squamous endothelial cells (ECs), the endothelium, regulates the flow of substance and fluid into and out of a tissue. The endothelium is also involved in vasculogenesis, the formation of new blood vessels, which is a crucial process for organ development in the embryo and fetus. Because most murine mutations of genes involved in EC development cause early embryo lethality, EC-specific conditional knockout (cKO) mouse models are indispensable for in vivo studies. cKO mice including the floxed allele can be generated through advanced approaches including embryonic stem cell-mediated gene targeting or the CRISPR/Cas system. EC-specific mouse models can be generated through further breeding of floxed mice with a Cre driver line, the latest information of which is available in the Jackson Cre Repository or the EUCOMMTOOLS project. Because it takes a long time (generally 1–2 years) to generate EC-specific mouse models, researchers must thoroughly design and plan a breeding strategy before full-scale mouse experiments, which saves time and money for in vivo study. In summary, revolutionary technical advances in embryo manipulation and assisted reproduction technologies have made it easier to generate EC-specific mouse models, which have been used as essential resources for in vivo studies of the endothelium.

Role of O‐linked glucose‐fucose disaccharide modification of thrombospondin type I repeats in protein folding and embryo development

We propose that Protein O‐fucosyltransferase 2 (POFUT2) and β3‐glucosyltransferase (B3GLCT) function to ensure efficient folding of forty‐nine proteins containing tandemly repeated Thrombospondin Type 1 Repeats (TSRs). In the endoplasmic reticulum (ER), POFUT2 and B3GLCT recognize properly folded TSRs and act sequentially to add the unusual O‐linked Glucoseβ1‐3Fucose disaccharide to either a serine or threonine residue located within the POFUT2 consensus sequence (C‐X‐X‐S/T‐C). Once all TSRs are modified the target protein exits the ER. In crystal structures the disaccharide forms H‐bonds and van der Waals interactions with underlying amino acids in the TSR, raising the possibility that the disaccharide functions as a surrogate amino acid to stabilize the correctly folded TSR. Results from in vitro folding/refolding assays provide evidence that the disaccharide stabilizes the TSR fold and accelerates the overall rate of protein folding. Consistent with this prediction, loss of POFUT2 or B3GLCT in cell culture impairs trafficking of POFUT2/B3GLCT targets. To begin to evaluate the function of the disaccharide in vivo, we generated mouse loss of function mutations in Pofut2 and B3glct. Loss of Pofut2 causes early embryo lethality due to a block in gastrulation. In contrast, B3glct animals survive, but have craniofacial and skeletal abnormalities, defects in pigmentation, soft tissue syndactyly, and develop hydrocephalus. The mouse B3glct phenotype shares similarities to Peters Plus syndrome in humans, caused by recessive mutations in B3GLCT. The distinct differences in the effects of these mutations during mouse development support a model whereby the O‐linked fucose is essential for folding and secretion of all POFUT2/B3GLCT target proteins and that B3GLCT‐mediated extension to the glucose‐fucose disaccharide is only required by a subset of sensitive targets.Support or Funding InformationThese studies were supported by funding from NIH R01 HD090156 and NIH R01 HD096030 and 2019 Hydrocephalus Association Innovator Award.Model for how POFUT2/B3GLCT mediated addition of the O‐linked glucose‐fucose disaccharide promotes efficient folding of target proteins containing the Thrombospondin Type 1 Repeats (TSR) fold. Addition of fucose prevents reentry of the TSR to the folding cycle and stabilizes the fold. Extension with glucose provides further stability to the TSR. Once the target protein is fully modified it exits the endoplasmic reticulum (ER).Figure 1

AtBUD13 affects pre-mRNA splicing and is essential for embryo development in Arabidopsis.

Pre-mRNA splicing is an important step for gene expression regulation. Yeast Bud13p (bud-site selection protein 13) regulates the budding pattern and pre-mRNA splicing in yeast cells; however, no Bud13p homologs have been identified in plants. Here, we isolated two mutants that carry T-DNA insertions at the At1g31870 locus and shows early embryo lethality and seed abortion. At1g31870 encodes an Arabidopsis homolog of yeast Bud13p, AtBUD13. Although AtBUD13 homologs are widely distributed in eukaryotic organisms, phylogenetic analysis revealed that their protein domain organization is more complex in multicellular species. AtBUD13 is expressed throughout plant development including embryogenesis and AtBUD13 proteins is localized in the nucleus in Arabidopsis. RNA-seq analysis revealed that AtBUD13 mutation predominantly results in the intron retention, especially for shorter introns (≤100 bases). Within this group of genes, we identified 52 genes involved in embryogenesis, out of which 22 are involved in nucleic acid metabolism. Our results demonstrate that AtBUD13 plays critical roles in early embryo development by effecting pre-mRNA splicing.

Conditional deletion of platelet derived growth factor receptor alpha (Pdgfra) in urorectal mesenchyme causes mesenchyme apoptosis and urorectal developmental anomalies in mice.

In mammals, urorectal development starts at early embryonic stage, defective urorectal development results in anorectal malformations, which are common congenital developmental defects of the anus and the urethra in newborns. The etiology and embryology of the defects are still largely unknown. Platelet-derived growth factor receptor alpha (Pdgfra) is a cell surface receptor tyrosine kinase, upon binding to its ligands (Pdgfa-d), mediates intracellular signaling and regulates embryonic development. The expression of Pdgfra is tightly regulated in the developing urorectal mesenchyme, and its dysregulation is associated with urorectal defects in animals with urorectal defects. Knockout of Pdgfra induces early embryo lethality which precludes investigation of Pdgfra in urorectal development. To address the temporal requirement of Pdgfra in urorectal development, we conditionally deleted Pdgfra in Pdgfra-expressing tissues using a tamoxifen inducible Cre-loxP approach in mice, examined the urorectal development in Pdgfra conditional knockout (Pdgfra-cKO) embryos. We showed that conditional deletion of Pdgfra in Pdgfra-expressing tissues at E10-E11 caused cloaca septation defect, anteriorly displaced anus, defective urogenital folds development and abnormal urethra tubularization in both male and female mice. Furthermore, we showed that Pdgfra was required for the survival of urorectal mesenchyme, deletion of Pdgfra caused apoptosis in the peri-cloacal, the peri-urethra and the urorectal septum mesenchyme of Pdgfra-cKO mutants, associated with an induction of p53, Ndrg1 and activation of caspase-3 in Pdgfra-cKO embryos. In conclusion, Pdgfra is required for the development and survival of the urorectal mesenchyme in embryo, dysregulated Pdgfra signaling induced urorectal defects in mice resembling human congenital diseases of anorectal malformations and hypospadias. Perturbation of PDGFRA signaling may contribute to anorectal malformations and hypospadias in human.

Generation of an induced pluripotent stem cell line from chorionic villi of a Turner syndrome spontaneous abortion

A major cause of spontaneous abortions is chromosomal abnormality of foetal cells. We report the generation of an induced pluripotent stem cell line from the fibroblasts isolated from chorionic villi of an early spontaneously aborted foetus with Turner syndrome. The Turner syndrome villus induced pluripotent stem cell line is transgene free, retains the original XO karyotype, expresses pluripotency markers and undergoes trilineage differentiation. This pluripotent stem cell model of Turner syndrome should serve as a tool to study the developmental abnormalities of foetus and placenta that lead to early embryo lethality and profound symptoms like infertility in 45 XO survivors.

P21-Activated Kinase 2 Regulates Endothelial Development and Function through the Bmk1/Erk5 Pathway.

p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/Erk5 pathway as a critical mediator of endothelial Pak2 signaling.

Cubilin, a High Affinity Receptor for Fibroblast Growth Factor 8, Is Required for Cell Survival in the Developing Vertebrate Head

Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

Targeted disruption of Mcm10 causes defective embryonic cell proliferation and early embryo lethality

Minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein, which plays a key role in the initiation of eukaryotic chromosomal DNA replication and elongation. To elucidate the physiological importance of MCM10 in vivo, we generated conventional knockout mice. No MCM10-null embryos were recovered after E8.5, and the mutation was found to be lethal before the implantation stage. Mutant embryos showed apparently normal growth until the morula stage, but growth defects after this stage. The dramatic reduction of 5-bromo-2-deoxyuridine (BrdU) incorporation in the mutant embryo, followed by cell death, suggests that defective cell proliferation may underlie this developmental failure. Taken together, these findings provide the first unequivocal genetic evidence for an essential and non-redundant physiological role of MCM10 during murine peri-implantation development.

The Phosphoinositide Kinase PIKfyve Is Vital in Early Embryonic Development: PREIMPLANTATION LETHALITY OF PIKfyve−/− EMBRYOS BUT NORMALITY OF PIKfyve+/− MICE

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P(2) and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown. To clarify the importance of PIKfyve and PIKfyve lipid products, in this study, we have characterized the first mouse model with global deletion of the pikfyve gene using the Cre-loxP approach. We report that nearly all PIKfyve(KO/KO) mutant embryos died before the 32-64-cell stage. Cultured fibroblasts derived from PIKfyve(flox/flox) embryos and rendered pikfyve-null by Cre recombinase expression displayed severely reduced DNA synthesis, consistent with impaired cell division causing early embryo lethality. The heterozygous PIKfyve(WT/KO) mice were born at the expected Mendelian ratio and developed into adulthood. PIKfyve(WT/KO) mice were ostensibly normal by several other in vivo, ex vivo, and in vitro criteria despite the fact that their levels of the PIKfyve protein and in vitro enzymatic activity in cells and tissues were 50-55% lower than those of wild-type mice. Consistently, steady-state levels of the PIKfyve products PtdIns(3,5)P(2) and PtdIns5P selectively decreased, but this reduction (35-40%) was 10-15% less than that expected based on PIKfyve protein reduction. The nonlinear decrease of the PIKfyve protein versus PIKfyve lipid products, the potential mechanism(s) discussed herein, may explain how one functional allele in PIKfyve(WT/KO) mice is able to support the demands for PtdIns(3,5)P(2)/PtdIns5P synthesis during life. Our data also shed light on the known human disorder linked to PIKFYVE mutations.

Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5' external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis.

YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis

BackgroundIn flowering plants, gametogenesis generates multicellular male and female gametophytes. In the model system Arabidopsis, the male gametophyte or pollen grain contains two sperm cells and a vegetative cell. The female gametophyte or embryo sac contains seven cells, namely one egg, two synergids, one central cell and three antipodal cells. Double fertilization of the central cell and egg produces respectively a triploid endosperm and a diploid zygote that develops further into an embryo. The genetic control of the early embryo patterning, especially the initiation of the first zygotic division and the positioning of the cell plate, is largely unknown.ResultsHere we report the characterization of a mutation, yaozhe (yao), that causes zygote arrest and misplacement of cell plate of the zygote, leading to early embryo lethality. In addition, gametophyte development is partially impaired. A small portion of the mutant embryo sacs are arrested at four-nucleate stage with aberrant nuclear positioning. Furthermore, the competence of male gametophytes is also compromised. YAO encodes a nucleolar protein with seven WD-repeats. Its homologues in human and yeast have been shown to be components of the U3 snoRNP complex and function in 18S rRNA processing. YAO is expressed ubiquitously, with high level of expression in tissues under active cell divisions, including embryo sacs, pollen, embryos, endosperms and root tips.ConclusionsPhenotypic analysis indicated that YAO is required for the correct positioning of the first zygotic division plane and plays a critical role in gametogenesis in Arabidopsis. Since YAO is a nucleolar protein and its counterparts in yeast and human are components of the U3 snoRNP complex, we therefore postulate that YAO is most likely involved in rRNA processing in plants as well.

ATR3 encodes a diflavin reductase essential for Arabidopsis embryo development

*The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable. *Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the N-terminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2. *Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein. *This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

The plant adherin AtSCC2 is required for embryogenesis and sister‐chromatid cohesion during meiosis in Arabidopsis

Adherin plays an important role in loading the cohesin complex onto chromosomes, and is essential for the establishment of sister-chromatid cohesion. We have identified and analyzed the Arabidopsis adherin homolog AtSCC2. Interestingly, the sequence analysis of AtSCC2 and of other putative plant adherin homologs revealed the presence of a PHD finger, which is not found in their fungal and animal counterparts. AtSCC2 is identical to EMB2773, and mutants show early embryo lethality and formation of giant endosperm nuclei. A role for AtSCC2 in sister-chromatid cohesion was established by using conditional RNAi and examining meiotic chromosome organization. AtSCC2-RNAi lines showed sterility, arising from the following defects in meiotic chromosome organization: failure of homologous pairing, loss of sister-chromatid cohesion, mixed segregation of chromosomes and chromosome fragmentation. The mutant phenotype, which included defects in chromosome organization and cohesion in prophase I, is distinct from that of the Arabidopsis cohesin mutant Atrec8, which retains centromere cohesion up to anaphase I. Immunostaining experiments revealed the aberrant distribution of the cohesin subunit AtSCC3 on chromosomes, and defects in chromosomal axis formation, in the meiocytes of AtSCC2-RNAi lines. These results demonstrate a role for AtSCC2 in sister-chromatid cohesion and centromere organization, and show that the machinery responsible for the establishment of cohesion is conserved in plants.

Vascular defects and liver damage by the acute inactivation of the VHL gene during mouse embryogenesis

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene leads to the development of central nervous system hemangioblastomas, pheochromocytomas and renal cell carcinomas. The biological role of the VHL gene during development is poorly understood because of early lethality of VHL-null embryos. To overcome early embryo lethality observed in the conventional knockout mouse, we introduced a tamoxifen-inducible Cre (CreERTM) transgene for the stage specific inactivation of the VHL gene. Acute tamoxifen-induced inactivation of the VHL gene at E10.5 resulted in embryonic lethality between E14.5 and E15.0 with extensive hemorrhage and necrosis, while littermate controls showed normal development. Examination of the VHL-inactivated embryos between E10.5 and E14.5 revealed dilated blood vessels, hemorrhage and necrotizing liver damage. Concomitant with severe hemorrhage and abnormal vasculature at E15.0, blood circulation in the yolk sac was impaired in the VHL-inactivated embryos, which may be the cause of embryo death. Placental development looked normal before embryo death (E14.5); however, at E16.5 following embryo death, we observed reduced growth of the placental labyrinthine layer. Inactivation of the VHL gene resulted in hypoxia-inducible factor (HIF)-1α stabilization and induction of its target genes, VEGF and CAIX, in mouse embryonic fibroblasts (MEFs). In addition, we observed lactate overproduction and acidification of culture media by the inactivation of the VHL gene. Thus, by using a novel conditional VHL knockout mouse model, we could show that the VHL gene plays an important role in the developing vasculature and liver during embryogenesis through regulation of HIF-1α and its target genes. This mouse model will be useful for the screening of anti-HIF or anti-VEGF drugs in vivo. Additionally, this acute VHL inactivation system may provide a useful tool for the in vivo study of genes that cause early embryonic lethality.

AtDEK1 is essential for specification of embryonic epidermal cell fate

The specification of epidermal (L1) identity occurs early during plant embryogenesis. Here we show that, in Arabidopsis, AtDEK1 encodes a key component of the embryonic L1 cell-layer specification pathway. Loss of AtDEK1 function leads to early embryo lethality characterized by a severe loss of cell organization in the embryo proper and abnormal cell divisions within the suspensor. Markers for L1 identity, ACR4 and ATML1, are not expressed in homozygous mutant embryos. In order to clarify the function of AtDEK1 further, an RNAi knockdown approach was used. This allowed embryos to partially complete embryogenesis before losing AtDEK1 activity. Resulting seedlings showed a specific loss of epidermal cell identity within large portions of the cotyledons. In addition, meristem structure and function was systematically either reduced or entirely lost. AtDEK1 expression is not restricted to the L1 epidermal cell layer at any stage in development. This is consistent with AtDEK1 playing an upstream role in the continuous generation or interpretation of positional information required for epidermal specification. Our results not only identify a specific role for AtDEK1 during embryogenesis, but underline the potential key importance of L1 specification at the globular stage for subsequent progression through embryogenesis.