Establishment of dorsal-ventral polarity in the Drosophila embryo: genetic studies on the role of the Toll gene product

Within the group of maternal effect genes necessary for the establishment of the dorsal-ventral pattern of the Drosophila embryo, the Toll gene mutates to give a singular variety of embryonic phenotypes. Lack of function alleles produce dorsalized embryos as a recessive maternal effect. Dominant gain of function alleles result in ventralized embryos. Other recessive alleles cause partial dorsalization or lateralization of the embryonic pattern. Gene dosage studies indicate that the dominant ventralized phenotype results from an altered activity of the Toll product. Complementation studies show specific trans interactions between copies of the Toll product. Double mutant phenotypes suggest that the products of several other dorsal-group genes regulate the activity of Toll.     

Activation of the easter zymogen is regulated by five other genes to define dorsal-ventral polarity in the Drosophila embryo.

The product of the Drosophila easter gene, a member of the trypsin family of serine proteases, must be more active ventrally than dorsally to promote normal embryonic polarity. The majority of the easter protein in the embryo is present in the unprocessed zymogen form and appears to be evenly distributed in the extracellular space, indicating that the asymmetric activity of wild-type easter must arise post-translationally. A dominant mutant form of easter that does not require cleavage of the zymogen for activity (ea delta N) is active both dorsally and ventrally. The ea delta N mutant bypasses the requirement for five other maternal effect genes, indicating that these five genes exert their effects on dorsal-ventral patterning solely by controlling the activation of the easter zymogen. We propose that dorsal-ventral asymmetry is initiated by a ventrally-localized molecule in the vitelline membrane that nucleates an easter zymogen activation complex, leading to the production of ventrally active easter enzyme.     

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.     

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.     

The Maternal Nudel Protein of Drosophila Has Two Distinct Roles Important for Embryogenesis

The nudel gene is maternally required to define dorsoventral polarity of the Drosophila embryo. It encodes an unusual mosaic protein with a protease domain that may trigger the protease cascade required for ventral development. We describe phenotypic and molecular analyses of nudel mutations that provide further insight into nudel protein function. Surprisingly, nudel mutations primarily cause either dorsalized embryos in which dorsal cell fates are expanded over ventral and lateral cell fates or fragile eggs that fail to develop beyond early embryonic stages. The nudel protein is therefore required not only for embryonic dorsoventral polarity but also for structural integrity of the egg. Complementation and antagonistic interactions between nudel alleles suggest that the nudel protein is functionally modular and that protein-protein interactions are important for nudel protein function. Three nudel mutations that produce dorsalized embryos map to the protease domain of nudel, suggesting that this domain is specifically required for defining embryonic dorsoventral polarity. Finally, certain combinations of nudel alleles simultaneously produce completely dorsalized and normal embryos yet very few embryos of intermediate mutant phenotypes. The unusual biphasic distribution of phenotypes may indicate that nudel activity above a threshold is required to generate embryonic dorsoventral polarity.     

Regulation of Easter activity is required for shaping the Dorsal gradient in the Drosophila embryo

Dorsoventral polarity of the Drosophila embryo requires maternal sp?tzle-Toll signaling to establish a nuclear gradient of Dorsal protein. The shape of this gradient is altered in embryos produced by females carrying dominant alleles of easter (ea(D)). The easter gene encodes a serine protease that generates processed Sp?tzle, which is proposed to act as the Toll ligand. By examining the expression domains of the zygotic genes zen, sog, rho and twist, which are targets of nuclear Dorsal, we show that the slope of the Dorsal gradient is progressively flattened in stronger ea(D) alleles. In the wild-type embryo, activated Easter is found in a high M(r) complex called Ea-X, which is hypothesized to contain a protease inhibitor. In ea(D) embryo extracts, we detect an Easter form corresponding to the free catalytic domain, which is never observed in wild type. These mutant ea(D) proteins retain protease activity, as determined by the production of processed Sp?tzle both in the embryo and in cultured Drosophila cells. These experiments suggest that the ea(D) mutations interfere with inactivation of catalytic Easter, and imply that this negative regulation is essential for generating the wild-type shape of the Dorsal gradient.     

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.     

Genetic Interactions between the Drosophila Abelson (Abl) Tyrosine Kinase and Failed Axon Connections (Fax), a Novel Protein in Axon Bundles

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, disabled, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase.     

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.     

A genetic and developmental analysis of mutations in the Deformed locus in Drosophila melanogaster

Individuals expressing recessive mutations in the Deformed (Dfd) locus of Drosophila melanogaster were examined for embryonic and adult defects. Mutant embryos were examined in both scanning electron microscope and light microscope preparations. The adult Dfd recessive mutant phenotype was assessed in somatic clones and in survivors homozygous for hypomorphic alleles of the gene. The time of Dfd+ action was determined by studying a temperature conditional allele. Dfd+ is required in three embryonic cephalic segments to form a normal head. Mutant embryos of Dfd display defects in derivatives of the maxillary segment, of the mandibular segment, and of some more anterior segments. In the adult fly, defects are seen in the posterior aspect of the head when the gene is mutant. A transformation from head to thoracic-like tissue is seen dorsally and a deletion of structures is seen ventrally. Shift studies utilizing a temperature conditional allele have shown that the gene product is necessary during at least two periods of development, during embryonic segmentation and head involution and during the late larval and pupal stages. From these studies we conclude that Dfd is a homeotic gene necessary for proper specification of both the embryonic and the adult head.     

The Thick Veins Gene of Drosophila Is Required for Dorsoventral Polarity of the Embryo

We have discovered a new member of the class of genes controlling embryonic dorsoventral patterning. Mutants of the thick veins (tkv) gene have been described previously (as slater alleles) as embryonic lethal, lacking dorsal epidermis, but not as showing a recognizable dorsoventral phenotype. We show here that maternal alteration of function coupled with zygotic reduction of function of tkv is strongly ventralizing. In addition, in double heterozygous combinations in the mother, tkv mutations increase the ventralizing effect of dominant, weakly ventralizing alleles of the maternal effect, dorsoventral genes easter and cactus. An interaction is also seen with zygotic dorsoventral genes: tkv interacts maternally and zygotically in double heterozygotes with decapentaplegic and zygotically with screw in double homozygotes. We conclude that both maternally and zygotically supplied wild-type tkv product can play a role in dorsoventral patterning of the early embryo. On the basis of the phenotype of trans-heterozygous adult escapers, we propose that tkv might act by potentiating the activity of the zygotically acting decapentaplegic gene.     

Dominant negative mutations in the α-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product.     

The segment polarity gene costal-2 in Drosophila. I. The organization of both primary and secondary embryonic fields may be affected

A series of loss of function alleles at the costal-2 locus is described. Embryos mutant for lethal alleles that are derived from a mutant female germ line display polarity defects on the larval segments. A posterior part of the segmental denticle belt is missing and in its place is a mirror-image duplication of the anterior part including the segment boundary. Maternally rescued embryos are lethal but have normal morphology. Hypomorphic alleles escape to adults that display pattern duplications on the wings and halteres. Dominant gain of function alleles at the Costal-1 locus are also described and data are presented that argue that these are neomorphic and act in trans to impair functioning of costal-2. Some wild-type isoalleles of costal-2 are particularly sensitive to interference from Costal-1 mutations and different combinations of these alleles with Costal-1 can lead to embryos in which the primary embryonic field is disrupted (bicaudal phenotype) and adults with pattern duplications on the anterior compartment of most body segments.     

Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae.

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product. Received: 15 January 1999 / Accepted: 25 March 1999     

The Homeotic Gene Sex Combs Reduced of Drosophila Melanogaster Is Differentially Regulated in the Embryonic and Imaginal Stages of Development

The Sex combs reduced (Scr) locus is unique among the genes contained within the Antennapedia complex (ANT-C) of Drosophila melanogaster in that it directs functions that are required for both cephalic and thoracic development in the embryo and the adult. Antibodies raised against protein encoded by Scr were used to follow the distribution of this gene product in embryos and imaginal discs of third instar larvae. Analysis of Scr protein accumulation in embryos hemizygous for breakpoint lesions mapping throughout the locus has allowed us to determine that sequences required for establishment of the Scr embryonic pattern are contained within a region of DNA that overlaps with the identified upstream regulatory region of the segmentation gene fushi tarazu (ftz). Gain-of-function mutations in Scr result in the presence of ectopic sex comb teeth on the first tarsal segment of mesothoracic and metathoracic legs of adult males. Heterozygous combinations of gain-of-function alleles with a wild-type Scr gene exhibit no evidence of ectopic protein localization in the second and third thoracic segments of embryos. However, mesothoracic and metathoracic leg imaginal discs can be shown to accumulate ectopically expressed Scr protein, implying a differential regulation of the Scr gene during these two periods of development. Additionally, we have found that the spatial pattern of Scr gene expression in imaginal tissues involved in the development of the adult thorax is governed in part by synapsis of homologous chromosomes in this region of the ANT-C. However, those imaginal discs that arise anteriorly to the prothorax do not appear to be sensitive to this form of gene regulation. Finally, we have demonstrated that the extent of Scr expression is influenced by mutations at the Polycomb (Pc) locus but not by mutant alleles of the zeste (z) gene. Taken together, our data suggests that Scr gene expression is differentially regulated both temporally and spatially in a manner that is sensitive to the structure of the locus.