The dorsal protein is distributed in a gradient in early Drosophila embryos

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is closely related to the vertebrate proto-oncogene c-rel. Genetic experiments suggest that dorsal represents one of the last (if not the last) steps in the maternal pathway involved in establishing dorsal-ventral polarity in the early embryo. Even though the dorsal RNA is uniformly distributed in the embryo, we have found that the dorsal protein is specifically localized in peripheral nuclei of syncytial and cellular blastoderm stage embryos, and it is distributed in a ventral-to-dorsal gradient. These findings suggest possible mechanisms for how the dorsal protein may communicate maternal positional information to the zygotic genome.     

The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila

The maternal morphogen dorsal (dl) plays a key role in the establishment of dorsal-ventral polarity in Drosophila. We present evidence that the graded distribution of dl protein is initiated by selective nuclear transport. The dl protein is uniformly distributed throughout the cytoplasm of early embryos, but approximately 90 min after fertilization, dl protein present in ventral but not dorsal regions is selectively transported to the nucleus. Mutations in maternally active genes that regulate dl disrupt this transport process, resulting in an inactive, cytoplasmically localized form of the dl protein. Selective nuclear transport of dl protein was reproduced in tissue culture cells. The wild-type dl protein is largely restricted to the cytoplasm, while truncated proteins are predominantly localized within the nucleus. Transient cotransfection assays suggest that dl activates expression from several promoters in an apparently sequence-independent manner. We discuss the role of nuclear transport as a regulated process in gene expression and development.     

Multiple extracellular activities in Drosophila egg perivitelline fluid are required for establishment of embryonic dorsal-ventral polarity.

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment of the embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity is defined within the perivitelline compartment surrounding the embryo by the ventral formation of a ligand for the Toll receptor. Here, by transplantation of perivitelline fluid we demonstrate the presence of three separate activities present in the perivitelline fluid that can restore dorsal-ventral polarity to mutant easter, snake, and sp?tzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos.     

Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product

Drosophila females that lack Toll gene activity produce dorsalized embryos, in which all embryonic cells behave like the dorsal cells of the wild-type embryo. Injection of wild-type cytoplasm into young Toll- embryos restores their ability to produce a normal dorsal-ventral pattern in a position-dependent manner. No matter where the cytoplasm is injected relative to the dorsal-ventral axis of the egg shell, the position of the injected cytoplasm defines the ventralmost part of the rescued pattern. Although injection of wild-type cytoplasm into mutants at six other dorsal-group loci also restores the ability to produce lateral and ventral structures, only Toll- embryos lack any residual dorsal-ventral polarity. Experiments suggest that the activity of the Toll product is normally regulated by other dorsal-group genes and that the function of the Toll product is to provide the source for a morphogen gradient in the dorsal-ventral axis of the wild-type embryo.     

Dorsal ventral polarity and pattern formation in the Drosophila embryo

The establishment of polarity along the dorsal-ventral axis of the Drosophila embryo requires the graded distribution of the dorsal morphogen. Several maternal genes are responsible for the formation of the gradient and their products act in an ordered series of events that begins during oogenesis and involves two different cell types, the oocyte and the follicle cells. The last step in the series results in selective nuclear localization of dorsal proteins, dorsal is thought to regulate the expression of zygotic genes in a concentration dependent way. The zygotic genes determine cell fates in specific regions of the embryo and direct other genes involved in the processes of differentiation.     

Dissociation of the dorsal-cactus complex and phosphorylation of the dorsal protein correlate with the nuclear localization of dorsal

The formation of dorsal-ventral polarity in Drosophila requires the asymmetric nuclear localization of the dorsal protein along the D/V axis. This process is regulated by the action of the dorsal group genes and cactus. We show that dorsal and cactus are both phosphoproteins that form a stable cytoplasmic complex, and that the cactus protein is stabilized by its interaction with dorsal. The dorsal-cactus complex dissociates when dorsal is targeted to the nucleus. While the phosphorylation of cactus remains apparently unchanged during early embryogenesis, the phosphorylation state of dorsal correlates with its release from cactus and with its nuclear localization. This differential phosphorylation event is regulated by the dorsal group pathway.     

cactus, a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos.

The dorsoventral pattern of the Drosophila embryo is mediated by a gradient of nuclear localization of the dorsal protein which acts as a morphogen. Establishment of the nuclear concentration gradient of dorsal protein requires the activities of the 10 maternal 'dorsal group' genes whose function results in the positive regulation of the nuclear uptake of the dorsal protein. Here we show that in contrast to the dorsal group genes, the maternal gene cactus acts as a negative regulator of the nuclear localization of the dorsal protein. While loss of function mutations of any of the dorsal group genes lead to dorsalized embryos, loss of cactus function results in a ventralization of the body pattern. Progressive loss of maternal cactus activity causes progressive loss of dorsal pattern elements accompanied by the expansion of ventrolateral and ventral anlagen. However, embryos still retain dorsoventral polarity, even if derived from germline clones using the strongest available, zygotic lethal cactus alleles. In contrast to the loss-of-function alleles, gain-of-function alleles of cactus cause a dorsalization of the embryonic pattern. Genetic studies indicate that they are not overproducers of normal activity, but rather synthesize products with altered function. Epistatic relationships of cactus with dorsal group genes were investigated by double mutant analysis. The dorsalized phenotype of the dorsal mutation is unchanged upon loss of cactus activity. This result implies that cactus acts via dorsal and has no independent morphogen function. In all other dorsal group mutant backgrounds, reduction of cactus function leads to embryos that express ventrolateral pattern elements and have increased nuclear uptake of the dorsal protein at all positions along the dorsoventral axis. Thus, the cactus gene product can prevent nuclear transport of dorsal protein in the absence of function of the dorsal group genes. Genetic and cytoplasmic transplantation studies suggest that the cactus product is evenly distributed along the dorsoventral axis. Thus the inhibitory function that cactus product exerts on the nuclear transport of the dorsal protein appears to be antagonized on the ventral side. We discuss models of how the action of the dorsal group genes might counteract the cactus function ventrally.     

Windbeutel is required for function and correct subcellular localization of the Drosophila patterning protein Pipe

Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restriction of pipe gene expression to a ventral subpopulation of follicle cells. Pipe, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of an extracellular serine proteolytic cascade which defines the ventral side of the embryo. When pipe is expressed uniformly in the follicle cell layer, a strong ventralization of the resulting embryos is observed. Here, we show that this ventralization is dependent on the other members of the dorsal group of genes controlling dorsal-ventral polarity, but not on the state of the Epidermal Growth Factor Receptor signal transduction pathway which defines egg chamber polarity. Pipe protein expressed in vertebrate tissue culture cells localizes to the endoplasmic reticulum. Strikingly, coexpression of the dorsal group gene windbeutel in those cells directs Pipe to the Golgi. Similarly, Pipe protein exhibits an altered subcellular localization in the follicle cells of females mutant for windbeutel. Thus, Windbeutel protein enables the correct subcellular distribution of Pipe to facilitate its pattern-forming activity.     

A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is determined by a morphogen gradient established by the action of 12 maternal-effect genes: the dorsal group genes and cactus. One of the dorsal group genes, dorsal (dl), encodes the putative morphogen. Although no overall asymmetry in the distribution of dorsal protein is observed, a gradient of nuclear concentration of dl protein is established during cleavage stages, with a maximum at the ventral side of the egg. At the dorsal side of the egg, the protein remains in the cytoplasm. Nuclear localization of the dl protein, and hence gradient formation, is blocked in dorsalizing alleles of all of the other dorsal group genes, while in ventralizing mutants nuclear localization extends to the dorsal side of the egg. A correlation between dl protein distribution and embryonic pattern in mutant embryos indicates that the nuclear concentration of the dl protein determines pattern along the dorsoventral axis.     

Heat-Induced Reversal of Dorsal-Ventral Polarity in Xenopus Eggs

Heat-treatment of fertilized Xenopus laevis eggs at 30°C induced; 1. conspicuous concentration of the pigment toward the sperm entry point (SEP), 2. eccentric first cleavage furrow formation, and 3. reversal of the dorsal-ventral polarity of the embryos. The optimal treatment was for 2.5 min applied at 20 min postfertilization (p.f.). The rotation movement of the Nile-blue stained spots in the vegetal hemisphere of the heated eggs accurately located the future dorsal midline as in untreated embryos (ref. 22). Exposure of eggs to D₂O also reversed the dorsal-ventral polarity of the embryo suggesting that stabilization of microtubules is involved in the dorsal-ventral axis reversal.     

The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal

Twelve maternal effect loci are required for the production of Drosophila embryos with a correct dorsoventral axis. Analysis of mosaic females indicates that the expression of the genes nudel, pipe, and windbeutel is required in the somatic tissue, presumably in the follicle cells that surround the oocyte. Thus, information coming from outside the egg cell influences dorsoventral pattern formation during embryogenesis. In transplantation experiments, the perivitelline fluid from the compartment surrounding the embryo can restore dorsoventral pattern to embryos from females mutant for nudel, pipe, or windbeutel. The positioning of the transplanted pervitelline fluid also determines the polarity of the restored dorsoventral axis. We propose that the polarizing activity, normally present at the ventral side of the egg, is a ligand for the Toll receptor. Presumably, local activation of the Toll protein by the ligand initiates the formation of the nuclear concentration gradient of the dorsal protein, thereby determining dorsoventral pattern.     

Modification of Dorsal-Ventral Polarity in Xenopus laevis Embryos Following Withdrawal of Egg Contents before First Cleavage

When fertilized Xenopus laevis eggs were pricked just beneath the marginal zone with a thick glass needle prior to the first cleavage, a small amount of cytoplasm escaped into the exudate. Those eggs were placed in a poly L-lysine-coated plastic dish filled with 10% Ficoll solution. The location of the sperm entrance site (SES) of each egg was marked by scratching the surface of the plastic dish. The pricked embryos were anchored to the dish through poly L-lysine, and developed, therefore, without changing their original position. Consequently, development of the dorsalventral polarity was conveniently monitored with respect to the location of the SES. Embryos which developed from eggs pricked on the side opposite the SES showed modification of the dorsal-ventral polarity: Semi-quantitative studies showed that an exudation approximately 1.5–12.5% of the whole egg contents from the presumptive dorsal side caused a reversal of the dorsal-ventral polarity. That is, the dorsal lip of the blastopore formed on the same side of the SES, whereas the dorsal lip formed on the side opposite the SES in the normal control and sham-operated embryos. Half of the embryos which had larger cytoplasmic exudates more than 12.5% of the whole egg contents failed to form the dorsal lip by the time all controls and the embryos with smaller exudates showed normal dorsal lip formation. When eggs were pricked on the SES side, the normal topographic relationship between the SES and future dorsal lip side was reinforced.     

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.     

Genetic Characterization of Tube and Pelle, Genes Required for Signaling between Toll and Dorsal in the Specification of the Dorsal-Ventral Pattern of the Drosophila Embryo

tube and pelle are two of the maternally transcribed genes required for dorsal-ventral patterning of the Drosophila embryo. Females homozygous for strong alleles of tube or pelle produce embryos that lack all ventral and lateral embryonic pattern elements. By analyzing the phenotypes caused by 24 pelle and 9 tube alleles, we have defined characteristic features of the two genes, including the extremely variable phenotypes of a number of tube alleles and the antimorphic character of a number of pelle alleles. Double mutant females carrying dominant ventralizing alleles of Toll and dorsalizing alleles of tube or pelle produce dorsalized embryos, suggesting that tube and pelle act downstream of the membrane protein Toll in the signaling pathway that defines the embryonic dorsal-ventral pattern. Both tube and pelle are also important zygotically for survival: at least 30% of the zygotes lacking either tube or pelle die before adult stages, while 90-95% of tube(-) pelle(-) double mutant zygotes die. We discuss the phenotypes of tube-pelle double mutants in the context of whether the two proteins interact directly.