Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.     

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.     

Experimental phenocopy of a minute maternal-effect mutation alters blastoderm determination in embryos of Drosophila melanogaster

Maternal haploinsufficiency for a third chromosome Minute, M(3)i55, lowers rates of protein synthesis by approximately 30% during the syncytial nuclear cycles of early embryogenesis. The maternal effect of Mi55 also produces segmentation defects (denticle belt fusions) in the posterior abdomen of larvae. Furthermore, embryos from Minute mothers show abnormal expression patterns of the segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage of embryogenesis. We developed a computer-aided analysis to describe the deviations in ftz expression which demonstrates that abnormally narrow ftz stripes occur in segment primordia that become fused in the larva. Unexpectedly, an abnormally wide ftz stripe occurs in segment primordia which do not develop abnormally. In addition, Mi55 produces a general narrowing of all ftz- interstripes. We phenocopied the Minute mutation by injecting wild-type embryos with cycloheximide concentrations which decreased protein synthesis rates to levels comparable with those of Minute embryos. Thus, a general decrease in protein synthesis during early embryogenesis leads to abnormal determination of posterior abdominal segment primordia.     

Determinants of Drosophila fushi tarazu mRNA instability.

The fushi tarazu gene is essential for the establishment of the Drosophila embryonic body plan. When first expressed in early embryogenesis, fushi tarazu mRNA is uniformly distributed over most of the embryo. Subsequently, fushi tarazu mRNA expression rapidly evolves into a pattern of seven stripes that encircle the embryo. The instability of fushi tarazu mRNA is probably crucial for attaining this localized pattern of expression. mRNA stability in transgenic embryos was measured by a new method that does not use drugs or external interference. Experiments using hybrid genes that fuse fushi tarazu sequences to those of the stable ribosomal protein A1 mRNA provide evidence for at least two destabilizing elements in the fushi tarazu mRNA, one located within the 5' one-third of the mRNA and the other near the 3' end (termed FIE3 for ftz instability element 3'). The FIE3 lies within a 201-nucleotide sequence just upstream of the polyadenylation signal and can act autonomously to destabilize a heterologous mRNA. Further deletion constructs identified an essential 68-nucleotide element within the FIE3. Lack of homology between this element and other previously identified destabilization sequences suggests that FIE3 contains a novel RNA destabilization element.     

Time-dependent effects of cycloheximide and alpha-amanitin on meiotic resumption and progression in bovine follicular oocytes

To identify the stage during maturation at which new protein and RNA are synthesized for meiotic resumption, follicular oocytes were cultured in TCM-199 with the protein synthesis inhibitor cycloheximide or the hnRNA synthesis inhibitor alpha-amanitin. Although the meiotic resumption of cumulus-enclosed oocytes was completely blocked by the addition of 25 microg/ml cycloheximide at 4 h after the onset of culture, 23% of oocytes cultured from 5 h post cultivation in the medium with cycloheximide underwent germinal vesicle breakdown (GVBD). By further delaying the addition of cycloheximide, the proportion of oocytes which underwent GVBD increased. Addition of the inhibitor at 8 h or more post cultivation resulted in GVBD occurring in more than 87% of oocytes, though none of them were able to proceed beyond the metaphase I stage. In contrast, the addition of 50 microg/ml alpha-amanitin from the onset of culture significantly reduced the proportion of GVBD to 75% in cumulus-enclosed oocytes, while no significant reduction in the proportions of GVBD was noted in the case of its addition from 1 h of culture onward. However, denuded oocytes were almost insensitive to any treatments with alpha-amanitin. These results indicate that protein synthesis in the oocytes and RNA synthesis in the cumulus cells soon after the onset of culture are necessary for GVBD and that continuous protein synthesis following GVBD is indispensable for progression of the meiotic division in bovine oocytes.     

Autocatalytic ftz activation and metameric instability induced by ectopic ftz expression

Inappropriate expression of the Drosophila pair-rule gene, fushi tarazu (ftz), causes cuticular pattern deletions apparently complementary to those in ftz larvae. We show that the two patterns actually originate similarly, in both cases affecting the even-numbered parasegmental boundaries. The reciprocal cuticular patterns derive from differing patterns of selector gene expression (homoeotic transformations). The primary effect of ectopic ftz activity is to broaden ftz domains by autocatalytic activation of endogenous ftz expression in an additional anterior cell. This activates engrailed (en) and represses wingless (wg) expression, consistent with their proposed combinatorial control by ftz (and other pair-rule genes) to define parasegmental primordia. We propose that the anterior margin of each ftz stripe is normally defined by the posterior even-skipped (eve) boundary.     

Regulation and function of the Drosophila segmentation gene fushi tarazu

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a pattern of seven stripes at the blastoderm stage. Two cis-acting control elements are required for this expression: the zebra element, which confers the striped pattern by mediating the effects of a subset of segmentation genes; and the upstream element, an enhancer element requiring ftz+ activity for its action. Fusion of the upstream element to a basal promoter results in activation of the heterologous promoter in a ftz-dependent striped pattern, supporting the idea that ftz regulates itself by acting through its enhancer. The upstream element can also confer expression patterns similar to that of the homeotic gene Antennapedia, suggesting that a similar element may play a role in the activation of Antennapedia.     

Detection of inhibitors of protein and nucleic acid synthesis using oocytes of Xenopus laevis microinjected with the herpes thymidine kinase gene

We have developed an assay that measures the inhibition of protein synthesis and can be used in conjunction with a whole embryo bioassay that detects the ability of a chemical to cause fetotoxicity, malformation and abnormal growth. The assay involves microinjecting the herpes thymidine kinase gene into stage 6 oocytes of Xenopus laevis then exposing the oocytes to a test compound for 18-24 h. The inhibition of thymidine kinase (TK) expression caused by an inhibitor is then measured by simple enzyme assay. Protein synthesis inhibitors such as cycloheximide, puromycin and emetine all inhibited TK synthesis. Concentrations of cycloheximide (1.4 X 10(-4) mg/ml) and puromycin (0.04 mg/ml) near the 96 h embryo LC50 inhibited thymidine kinase expression by 78% and 97%, respectively but emetine (0.01 mg/ml) had no effect. However, 0.1 mg/ml emetine inhibited TK synthesis by almost 50%. The RNA synthesis inhibitor, actinomycin D (0.013 mg/ml) inhibited TK expression by 61%. DNA synthesis inhibitors hydroxyurea (2.0 mg/ml), cytosine arabinoside (2.0 mg/ml) and ethidium bromide (0.02 mg/ml) failed to inhibit the expression of the TK gene even though these concentrations were near the 96 h embryo LC50. The whole embryo bioassay cannot differentiate the DNA synthesis inhibitors from the RNA and protein synthesis inhibitors but the oocyte assay can. This type of molecular test data can help separate classes of teratogens such as DNA synthesis inhibitors from nonteratogenic compounds such as protein synthesis inhibitors and allow the extrapolation of test data to other species.     

Zygotically active genes that affect the spatial expression of the fushi tarazu segmentation gene during early Drosophila embryogenesis

The establishment of the segmental body pattern of Drosophila requires the coordinated functions of three classes of zygotically active genes early in development. We have examined the effects of mutations in these genes on the spatial expression of the fushi tarazu (ftz) pair-rule segmentation gene. Mutations in four gap loci and in three pair-rule loci dramatically affect the initial pattern of transverse stripes of ftz-containing nuclei. Five other pair-rule genes and several other loci that affect the larval cuticular pattern do not detectably affect ftz expression. No simple regulatory relationships can be deduced. Rather, expression of the ftz gene depends upon the interactions among the different segmentation genes active at each position along the anterior-posterior axis of the early embryo.     

Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA.

Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.