Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor.

The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.

A repressible female-specific lethal genetic system for making transgenic insect strains suitable for a sterile-release program

We have developed a tetracycline-repressible female-specific lethal genetic system in the vinegar fly Drosophila melanogaster. One component of the system is the tetracycline-controlled transactivator gene under the control of the fat body and female-specific transcription enhancer from the yolk protein 1 gene. The other component consists of the proapoptotic gene hid under the control of a tetracycline-responsive element. Males and females of a strain carrying both components are viable on medium supplemented with tetracycline, but only males survive on normal medium. A strain with such properties would be ideal for a sterile-insect release program, which is most effective when only males are released in the field.

Polysomal ribonuclease 1 exists in a latent form on polysomes prior to estrogen activation of mRNA decay

Estrogen induces a global change in the translation profile of Xenopus hepatocytes, replacing serum protein synthesis with production of the yolk protein precursor vitellogenin. This is accomplished by the coordinate destabilization of serum protein mRNAs and the transcriptional induction and subsequent stabilization of vitellogenin mRNA. Previous work identified an endonuclease activity whose appearance on polysomes correlated with the disappearance of serum protein mRNAs. This enzyme, polysomal ribonuclease 1 (PMR1), is a novel member of the peroxidase gene family. The current study examined the association of PMR1 with its mRNA targets on polysomes and mRNPs. The highest amount of polysome-bound PMR1 was observed prior to estrogen induction of mRNA decay. Its distribution on sucrose density gradients matched the absorbance profile of polysome-bound mRNA, suggesting that PMR1 forms a latent complex with mRNA. Following dissociation with EDTA the 62 kDa PMR1 sedimented with a larger complex of >670 kDa. Estrogen induces a 22-fold increase in unit enzymatic activity of polysome-bound PMR1, and a time-dependent loss of PMR1 from polysomes in a manner that mirrors the disappearance of albumin mRNA. These data suggest that the key step in the extensive estrogen-induced change in mRNA decay in Xenopus liver is activation of a latent mRNA endonuclease associated with its target mRNA.

Purification and physical properties of the male and female double sex proteins of Drosophila.

The double sex gene (dsx) encodes two proteins, DSX(M) and DSX(F), that regulate sex-specific transcription in Drosophila. These proteins bind target sites in DNA from which the male-specific DSX(M) represses and the female-specific DSX(F) activates transcription of yolk protein (Yp) genes. We investigated the physical properties of these DSX proteins, which are identical in their amino-terminal 397 residues but are entirely different in their carboxyl-terminal sequences (DSX(F), 30 amino acids; DSX(M), 152 amino acids). DSX(M) and DSX(F) were overexpressed in cultured insect cells and purified to near homogeneity. Gel filtration chromatography and glycerol gradient sedimentation showed that at low concentrations both proteins are dimers of highly asymmetrical shape. The axial ratios are approximately 18:1 (DSX(M), 860 X 48 angstroms; DSX(F), 735 X 43 angstroms). At higher concentrations, the proteins form tetramers. Through use of a novel, double crosslinking assay (protein-DNA plus protein-protein), we demonstrated that a DNA regulatory site binds to both monomers of the DSX dimer and to only two monomers of the tetramer. Furthermore, binding another DNA molecule to what we presume is the second and identical site in the tetramer dramatically shifts the equilibrium from tetramers to dimers. These oligomerization and DNA binding properties are indistinguishable between the male and female proteins.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

Essential role for p38α mitogen-activated protein kinase in placental angiogenesis

The p38 family of mitogen-activated protein kinases (MAPKs) mediates signaling in response to environmental stresses and inflammatory cytokines, but the requirements for the p38 MAPK pathway in normal mammalian development have not been elucidated. Here, we show that targeted disruption of the p38α MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. Although chorioallantoic placentation is initiated appropriately in p38α null homozygotes, placental defects are manifest at 10.5 days postcoitum as nearly complete loss of the labyrinth layer and significant reduction of the spongiotrophoblast. In particular, p38α mutant placentas display lack of vascularization of the labyrinth layer as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis. Furthermore, p38α mutants display abnormal angiogenesis in the embryo proper as well as in the visceral yolk sac. Thus, our results indicate a requirement for p38α MAPK in diploid trophoblast development and placental vascularization and suggest a more general role for p38 MAPK signaling in embryonic angiogenesis.