Visceral Endoderm Expression of Yin-Yang1 (YY1) Is Required for VEGFA Maintenance and Yolk Sac Development

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.     

Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

Cryptococcus neoformans PKH2-01 and PKH2-02 are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied in S. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show that PKH2-02 but not PKH2-01 is required for C. neoformans to tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion of PKH2-02 leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress. PKH2-02 function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using a Galleria mellonella model of low-temperature cryptococcosis, we found that PKH2-02 is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of the pkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion of PKH2-02 affects the interaction between C. neoformans and phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling in C. neoformans is crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence.     

Edd, the murine hyperplastic disc gene, is essential for yolk sac vascularization and chorioallantoic fusion

EDD is the mammalian ortholog of the Drosophila melanogaster hyperplastic disc gene (hyd), which is critical for cell proliferation and differentiation in flies through regulation of hedgehog and decapentaplegic signaling. Amplification and overexpression of EDD occurs frequently in several cancers, including those of the breast and ovary, and truncating mutations of EDD are also observed in gastric and colon cancer with microsatellite instability. EDD has E3 ubiquitin ligase activity, is involved in regulation of the DNA damage response, and may control hedgehog signaling, but a definitive biological role has yet to be established. To investigate the role of Edd in vivo, gene targeting was used to generate Edd knockout (Edd(Delta/Delta)) mice. While heterozygous mice had normal development and fertility, no viable Edd-deficient embryos were observed beyond E10.5, with delayed growth and development evident from E8.5 onward. Failed yolk sac and allantoic vascular development, along with defective chorioallantoic fusion, were the primary effects of Edd deficiency. These extraembryonic defects presumably compromised fetal-maternal circulation and hence efficient exchange of nutrients and oxygen between the embryo and maternal environment, leading to a general failure of embryonic cell proliferation and widespread apoptosis. Hence, Edd has an essential role in extraembryonic development.     

OsPIE1, the Rice Ortholog of Arabidopsis PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1, Is Essential for Embryo Development

Background

The SWR1 complex is important for the deposition of histone variant H2A.Z into chromatin necessary to robustly regulate gene expression during growth and development. In Arabidopsis thaliana, the catalytic subunit of the SWR1-like complex, encoded by PIE1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1), has been shown to function in multiple developmental processes including flowering time pathways and petal number regulation. However, the function of the PIE1 orthologs in monocots remains unknown.

Methodology/Findings

We report the identification of the rice (Oryza sativa) ortholog, OsPIE1. Although OsPIE1 does not exhibit a conserved exon/intron structure as Arabidopsis PIE1, its encoded protein is highly similar to PIE1, sharing 53.9% amino acid sequence identity. OsPIE1 also has a very similar expression pattern as PIE1. Furthermore, transgenic expression of OsPIE1 completely rescued both early flowering and extra petal number phenotypes of the Arabidopsis pie1-2 mutant. However, homozygous T-DNA insertional mutants of OsPIE1 in rice were embryonically lethal, in contrast to the viable mutants in the orthologous genes for yeast, Drosophila and Arabidopsis (Swr1, DOMINO and PIE1, respectively).

Conclusions/Significance

Taken together, our results suggest that OsPIE1 is the rice ortholog of Arabidopsis PIE1 and plays an essential role in rice embryo development.     

Endothelium Expression of Bcl-2 Is Essential for Normal and Pathological Ocular Vascularization

Bcl–2 is an anti-apoptotic protein with important roles in vascular homeostasis and angiogenesis. Mice globally lacking Bcl–2 (Bcl–2 -/-) are small in stature and succumb to renal failure shortly after weaning as a result of renal hypoplasia/cystic dysplasia. We have shown that Bcl–2 -/- mice displayed attenuated retinal vascular development and neovascularization. In vitro studies indicated that in addition to modulating apoptosis, Bcl–2 expression also impacts endothelial and epithelial cell adhesion, migration and extracellular matrix production. However, studies delineating the cell autonomous role Bcl–2 expression plays in the endothelium during vascular development, pruning and remodeling, and neovascularization are lacking. Here we generated mice carrying a conditional Bcl–2 allele (Bcl-2^Flox/Flox) and VE-cadherin-cre (Bcl-2^EC mice). Bcl-2^EC mice were of normal stature and lifespan and displayed some but not all of the retinal vascular defects previously observed in global Bcl–2 deficient mice. Bcl-2^EC mice had decreased numbers of endothelial cells, decreased retinal arteries and premature primary branching of the retinal vasculature, but unlike the global knockout mice, spreading of the retinal superficial vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl-2^EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, Bcl–2 expression in the endothelium plays a significant role during postnatal retinal vascularization, and pathological choroidal but not retinal neovascularization, suggesting vascular bed specific Bcl–2 function in the endothelium.     

Isotopic Labeling of Embryo, Yolk Sac, and Intracellular Parasites of the Embryonated Hen''s Egg by Yolk Replacement

A technique is described which allows the replacement of 50% of the yolk of the embryonated hen's egg with large volumes of diverse but chemically defined solutions. By using an electrosurgical unit and a polyethylene tunnel, the procedure was performed on eggs from days 3 through 7 with greater than 90% surgical success and viability for the short term. More than 50% of the eggs replaced showed viability for 2 weeks, and a significant proportion went full term. ³²PO₄ and amino acids (³H and ¹⁴C) added to the replaced eggs were incorporated into the macromolecules of the embryo and yolk sac as well as into parasitic rickettsiae cultivated in the replaced eggs. The incorporated ³²PO₄ was shown to be assimilated into a variety of biochemical species.     

Isotopic Labeling of Embryo,Yolk Sac,and Intracellular Parasites of the Embryonated Hen's Egg by Yolk Replacement

A technique is described which allows the replacement of 50% of the yolk of the embryonated hen''s egg with large volumes of diverse but chemically defined solutions. By using an electrosurgical unit and a polyethylene tunnel, the procedure was performed on eggs from days 3 through 7 with greater than 90% surgical success and viability for the short term. More than 50% of the eggs replaced showed viability for 2 weeks, and a significant proportion went full term. ³²PO₄ and amino acids (³H and ¹⁴C) added to the replaced eggs were incorporated into the macromolecules of the embryo and yolk sac as well as into parasitic rickettsiae cultivated in the replaced eggs. The incorporated ³²PO₄ was shown to be assimilated into a variety of biochemical species.     

Direct Effects of Microalgae and Protists on Herring (Clupea harengus) Yolk Sac Larvae

This study investigated effects of microalgae (Rhodomonas baltica) and heterotrophic protists (Oxyrrhis marina) on the daily growth, activity, condition and feeding success of Atlantic herring (Clupea harengus) larvae from hatch, through the end of the endogenous (yolk sac) period. Yolk sac larvae were reared in the presence and absence of microplankton and, each day, groups of larvae were provided access to copepods. Larvae reared with microalgae and protists exhibited precocious (2 days earlier) and ≥ 60% increased feeding incidence on copepods compared to larvae reared in only seawater (SW). In the absence and presence of microalgae and protists, life span and growth trajectories of yolk sac larvae were similar and digestive enzyme activity (trypsin) and nutritional condition (RNA-DNA ratio) markedly declined in all larvae directly after yolk sac depletion. Thus, microplankton promoted early feeding but was not sufficient to alter life span and growth during the yolk sac phase. Given the importance of early feeding, field programs should place greater emphasis on the protozooplankton-ichthyoplankton link to better understand match-mismatch dynamics and bottom-up drivers of year class success in marine fish.     

The Type IV Fimbrial Subunit Gene (fimA) of Dichelobacter nodosus Is Essential for Virulence, Protease Secretion, and Natural Competence

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.     

Spink13, an Epididymis-specific Gene of the Kazal-type Serine Protease Inhibitor (SPINK) Family,Is Essential for the Acrosomal Integrity and Male Fertility

Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives.     

The htrA (degP) Gene of Listeria monocytogenes 10403S Is Essential for Optimal Growth under Stress Conditions

This report describes a mutant of Listeria monocytogenes strain 10403S (serotype 1/2a) with a defective response to conditions of high osmolarity, an environment that L. monocytogenes encounters in some ready-to-eat foods. A library of L. monocytogenes clones mutagenized with Tn917 was generated and scored for sensitivity to 4% NaCl in order to identify genes responsible for growth or survival in elevated-NaCl environments. One of the L. monocytogenes Tn917 mutants, designated strain OSM1, was selected, and the gene interrupted by the transposon was sequenced. A BLAST search with the putative translated amino acid sequence indicated that the interrupted gene product was a homolog of htrA (degP), a gene coding for a serine protease identified as a stress response protein in several gram-positive and gram-negative bacteria. An htrA deletion strain, strain LDW1, was constructed, and the salt-sensitive phenotype of this strain was complemented by introduction of a plasmid carrying the wild-type htrA gene, demonstrating that htrA is necessary for optimal growth under conditions of osmotic stress. Additionally, strain LDW1 was tested for its response to temperature and H₂O₂ stresses. The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44°C but at a rate similar to that of the wild-type strain when incubated at 4°C. In addition, strain LDW1 was significantly more sensitive to a 52°C heat shock than the wild-type strain. Strain LDW1 was also defective in its response to H₂O₂ challenge at 37°C, since 100 or 150 μg of H₂O₂ was more inhibitory for the growth of strain LDW1 than for that of the parent strain. The stress response phenotype observed for strain LDW1 is similar to that observed for other HtrA⁻ organisms, which suggests that L. monocytogenes HtrA may play a role in degrading misfolded proteins that accumulate under stress conditions.     

Desmin Is Essential for the Tensile Strength and Integrity of Myofibrils but Not for Myogenic Commitment, Differentiation, and Fusion of Skeletal Muscle

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des −/−) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362–366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255– 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des −/− mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.     

Candida albicans AGE3, the Ortholog of the S. cerevisiae ARF-GAP-Encoding Gene GCS1, Is Required for Hyphal Growth and Drug Resistance

Background

Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus.

Methodology/Principal Findings

Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Δ mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Δ cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Δ cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Δ mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p.

Conclusions/Significance

The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Δ cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Δ cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Δ cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it represents a promising antifungal drug target.     

The Quiescin Q6 Gene (QSCN6) Is a Fusion of Two Ancient Gene Families: Thioredoxin and ERV1

Cell and tissue growth is a dynamic process determined by the fraction of cells in the proliferative cycle, the fraction of cells in quiescence, and the rate of cell death. Genes whose expression is induced at the beginning of the transition from the proliferative cell cycle to quiescence may play an important role in this process. We have identified a gene, Quiescin Q6 (QSCN6), whose expression is induced just as fibroblasts begin to leave the proliferative cycle and enter quiescence. QSCN6 is located on human chromosome 1q24, near the putative hereditary prostate cancer locus (HPC1). A triplet repeat (CTG)_nencodes a putative signal sequence. The gene encodes a 582-amino-acid open reading frame that has domains that are members of two ancient gene families. These domains apparently underwent a gene fusion event during metazoan evolution to create QSCN6. QSCN6 is most closely related to three genes of unknown function fromCaenorhabditis elegansas well as a gene from guinea pig. Analysis of this relationship showed nine Quiescin homology zones (QHZ). QHZ 0 is the putative signal sequence, QHZ 1 is homologous to a thioredoxin domain, and QHZ 2, 3, 4, and 8 are homologous only to themselves, while QHZ 5, 6, and 7 are homologous to the ERV1 gene ofSaccharomyces cerevisiae.In both thioredoxin and ERV1 gene superfamilies, QSCN6 sequences appear to be on distinct branches of their respective phylogenetic trees, consistent with an ancient origin of the QSCN6 gene. We present a model of the origin of QSCN6 and discuss its potential role in growth regulation.