The Drosophila cystoblast differentiation factor, benign gonial cell neoplasm, is related to DExH-box proteins and interacts genetically with bag-of-marbles.

Selection of asymmetric cell fates can involve both intrinsic and extrinsic factors. Previously we have identified the bag-of-marbles (bam) gene as an intrinsic factor for cystoblast fate in Drosophila germline cells and shown that it requires active product from the benign gonial cell neoplasm (bgcn) gene. Here we present the cloning and characterization of bgcn. The predicted Bgcn protein is related to the DExH-box family of RNA-dependent helicases but lacks critical residues for ATPase and helicase functions. Expression of the bgcn gene is extremely limited in ovaries but, significantly, bgcn mRNA is expressed in a very limited number of germline cells, including the stem cells. Also, mutations in bgcn dominantly enhance a bam mutant phenotype, further corroborating the interdependence of these two genes' functions. On the basis of known functions of DExH-box proteins, we propose that Bgcn and Bam may be involved in regulating translational events that are necessary for activation of the cystoblast differentiation program.

Localization and function of Bam protein require the benign gonial cell neoplasm gene product.

Division of a female Drosophila stem cell produces a daughter stem cell and a cystoblast. The cystoblast produces a syncytial cluster of 16 cells by precisely four mitotic divisions and incomplete cytokinesis. Mutations in genes required for cystoblast differentiation, such as bag-of-marbles, block syncytial cluster formation and produce a distinctive "tumorous" or hyperplastic germ cell phenotype. In this paper, we compare the oogenic phenotype of benign gonial cell neoplasm mutations to that of mutations in bam. The data indicate that, like bam, bgcn is required for cystoblast development and that germ cells lacking bgcn become trapped in a stem cell-like state. One indication that germ cells lacking bgcn cannot form cystoblasts is that bgcn stem cells resist genetic ablation by Bam misexpression. Misexpression of Bam eliminates wild-type stem cells, apparently by inducing them to divide as cystoblasts. bgcn stem cells remain active when Bam is misexpressed, probably because they cannot adopt the cystoblast fate. Bgcn activity is not required for Bam protein expression but is essential for the localization of Bam protein to the fusome. Together, the results suggest that Bam and Bgcn cooperatively regulate cystoblast differentiation by controlling localization of Bam protein to the fusome.

The Drosophila stonewall gene encodes a putative transcription factor essential for germ cell development.

The differentiation of Drosophila germ cells is a useful model for studying mechanisms of cell specification. We report the identification of a gene, stonewall, that is required for germ cell development. Mutations in stonewall block proper oocyte differentiation and frequently cause the presumptive oocyte to develop as a nurse cell. Eventually, germ cells degenerate apoptotically. Stonewall is a germ cell nuclear protein; Stonewall has a DNA binding domain that shows similarities to the Myb and Adf-1 transcription factors and has other features that suggest that it is a transcription activating factor. We suggest that Stonewall transcriptional regulation is essential in cystocytes for maturation into specialized nurse cells and oocyte.

orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis.

We describe mutations in the orb gene, identified previously as an ovarian-specific member of a large family of RNA-binding proteins. Strong orb alleles arrest oogenesis prior to egg chamber formation, an early step of oogenesis, whereas females mutant for a maternal-effect lethal orb allele lay eggs with ventralized eggshell structures. Embryos that develop within these mutant eggs display posterior patterning defects and abnormal dorsoventral axis formation. Consistent with such embryonic phenotypes, orb is required for the asymmetric distribution of oskar and gurken mRNAs within the oocyte during the later stages of oogenesis. In addition, double heterozygous combinations of orb and grk or orb and top/DER alleles reveal that mutations in these genes interact genetically, suggesting that they participate in a common pathway. Orb protein, which is localized within the oocyte in wild-type females, is distributed ubiquitously in stage 8-10 orb mutant oocytes. These data will be discussed in the context of a model proposing that Orb is a component of the cellular machinery that delivers mRNA molecules to specific locations within the oocyte and that this function contributes to both D/V and A/P axis specification during oogenesis.

bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis.

In Drosophila, male and female gametes begin development when a stem cell divides to produce a cyst precursor. Subsequently, four special divisions give rise to a cluster of 16 interconnected cystocytes that develop into a single egg or 64 sperm. We identified and characterized a gene, bag-of-marbles (bam), that disrupts cyst formation in both sexes. An apparent null mutation causes abnormal cysts to form containing an excess number of cells that cannot differentiate into gametes. bam function resides within a simple 2.2-kb transcription unit encoding a single 442-amino-acid protein that shows similarity to the product of the ovarian tumor gene. The specific expression of bam RNA within female cystoblasts suggested that it might be involved in the specific cell-cycle alterations that occur during cystocyte divisions.

Persistent estrogen induction of hepatic Xenopus laevis serum retinol binding protein mRNA.

Administration of estradiol-17 beta to male Xenopus laevis induces the hepatic mRNA coding for the serum retinol binding protein (RBP) approximately 10-fold, both in vivo and in primary liver cultures. Estrogen induction of RBP mRNA is completely blocked by the anti-estrogen, hydroxytamoxifen. Testosterone administration reduces the elevated level of RBP mRNA observed in livers of female X. laevis to the constitutive level seen in livers of control male animals, and partially blocks the estrogen induction of RBP mRNA. Intracellular RBP mRNA levels therefore represent a balance between the opposing effects of estradiol-17 beta and testosterone. In marked contrast to the estrogen induction of vitellogenin mRNA, which requires the continuous presence of exogenous estrogen, induction of RBP mRNA persists for at least 4 months after a single injection of estrogen. Runoff transcription measurements demonstrate that persistent induction of RBP mRNA is due to an increased rate of RBP gene transcription. Administration of hydroxytamoxifen abolishes persistent induction of RBP mRNA, suggesting that residual hormone receptor complex plays a role in the persistent induction of RBP gene transcription. The persistent estrogen induction of RBP mRNA provides the first demonstration of long-term activation of the transcription of a hormone-responsive gene in response to a transient dose of a steroid hormone.

Estrogen induces transcription of the Xenopus laevis serum retinol-binding protein gene.

We have isolated and sequenced a full-length cDNA cloned tentatively identified as encoding Xenopus laevis serum retinol-binding protein (RBP) mRNA. The derived amino acid sequence of the Xenopus protein is 63% homologous to the sequence of the human RBP. 17 of 19 amino acids identified as critical in the retinol-binding pocket of human RBP are identical or conservative replacements in the Xenopus protein. The RBP cDNA clone has been used as a hybridization probe to demonstrate that administration of estradiol-17 beta to male X. laevis induces hepatic RBP mRNA 10-fold from its constitutive in vivo level of 1,800 molecules/cell to approximately 18,000 molecules/cell. Using a simplified method for determining relative rates of gene transcription, we demonstrate an estrogen-mediated increase in the rate of RBP gene transcription. These quantitative data provide the first demonstration that a steroid hormone regulates the levels of vertebrate retinol-binding protein mRNA.