Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein.

Dorsoventral polarity of the Drosophila embryo is established by a signal transduction pathway in which the maternal transmembrane protein Toll appears to function as the receptor for a ventrally localized extracellular ligand. Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, we detected a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides with antibodies to Toll. Our biochemical analyses suggest that the smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. To assay the biological activity of such a shortened form of Toll, we synthesized RNA encoding a mutant polypeptide lacking the leucine-rich repeats that comprise most of Toll's extracellular domain and injected this RNA into embryos. The truncated Toll protein elicited the most ventral cell fate independently of the wild-type Toll protein and its ligand. These results support the view that Toll is a receptor whose extracellular domain regulates the intrinsic signaling activity of its cytoplasmic domain.     

Identification of chromosomal regions involved in decapentaplegic function in Drosophila.

Signaling molecules of the transforming growth factor beta (TGF-beta) family contribute to numerous developmental processes in a variety of organisms. However, our understanding of the mechanisms which regulate the activity of and mediate the response to TGF-beta family members remains incomplete. The product of the Drosophila decapentaplegic (dpp) locus is a well-characterized member of this family. We have taken a genetic approach to identify factors required for TGF-beta function in Drosophila by testing for genetic interactions between mutant alleles of dpp and a collection of chromosomal deficiencies. Our survey identified two deficiencies that act as maternal enhancers of recessive embryonic lethal alleles of dpp. The enhanced individuals die with weakly ventralized phenotypes. These phenotypes are consistent with a mechanism whereby the deficiencies deplete two maternally provided factors required for dpp''s role in embryonic dorsal-ventral pattern formation. One of these deficiencies also appears to delete a factor required for dpp function in wing vein formation. These deficiencies remove material from the 54F-55A and 66B-66C polytene chromosomal regions, respectively. As neither of these regions has been previously implicated in dpp function, we propose that each of the deficiencies removes a novel factor or factors required for dpp function.     

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.     

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.     

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.     

Maternal-effect loci involved in Drosophila oogenesis and embryogenesis: P element-induced mutations on the third chromosome

A collection of 1609 recessive P-lethal mutations on the third chromosome was tested in germline clones for effects on egg differentiation and embryonic development. In 164 lines, normal differentiation of the egg chamber is prevented and in 841 lines, embryos develop abnormally. This latter group of maternal-effect mutations was subdivided into 23 classes based on the cuticular phenotypes. Our collection comprises new alleles of previously characterized genes (e.g. kayak, punt, string, tramtrack). For some of the genes identified in this screen, a maternal contribution to embryonic development has not been described previously (e.g. extramacrochaete, Trithorax-like, single minded, couch potato, canoe). The genes classified in our study with a dual function during oogenesis and embryogenesis not only substantially extends the existing collection of maternal-effect genes but will also aid further understanding of how patterning of the Drosophila embryo is controlled by the maternal genome.     

The maternal effect of lethal(1)discs-large-1: a recessive oncogene of Drosophila melanogaster

The maternal effect phenotypes of recessive mutations at the Drosophila zygotic lethal gene l(1)discs-large-1 (l(1)dlg-1) are described. L(1)dlg-1 is located in 10B7-8 on the salivary gland chromosome map. A complex complementation pattern is observed among the nine characterized alleles. Larvae missing zygotic l(1)dlg-1+ gene activity die due to aberrant growth of imaginal cells at the larval-pupal transition. Embryos lacking both maternal and zygotic activity of l(1)dlg-1+, i.e., embryos derived from homozygous l(1)dlg-1 germ line clones for null alleles, show neurogenesis and morphogenesis defects that result in very abnormal embryos. Although differentiated, most tissues are morphologically misshapen. This maternal effect is rescuable to some extent. One allele, l(1)dlg-1HF321, is a temperature-sensitive mutation for the zygotic lethality. Embryos derived from homozygous l(1)dlg-1HF321 females at 18 degrees C exhibit defects associated with dorsal closure and head involution. More extreme phenotypes are observed when females are shifted to higher temperatures and include defective dorsal closure, collapse of the somatic musculature, and an oversized central nervous system. The possible involvement of the recessive oncogene l(1)dlg-1 in cell adhesion is discussed.     

Plasma membrane localization of the Toll protein in the syncytial Drosophila embryo: importance of transmembrane signaling for dorsal-ventral pattern formation

Formation of the Drosophila embryo's dorsal-ventral pattern requires the maternal product of the Toll gene. DNA sequence and genetic analyses together suggested that the Toll gene product is a transmembrane protein which communicates information from an extracytoplasmic compartment to the cytoplasm. Using antibodies as probes, we show that the Toll protein is a 135 x 10(3) Mr glycoprotein which is tightly associated with embryonic membranes. During the syncytial stage when dorsal-ventral polarity is established, the maternal Toll protein is associated with the plasma membrane around the entire embryo. During later embryonic stages, the Toll protein is expressed zygotically on many cell surfaces, possibly to promote cell adhesion. The plasma membrane localization of the Toll protein in the syncytial embryo suggests that transmembrane signaling from the extracellular perivitelline space to the cytoplasm is required for establishment of the embryonic dorsal-ventral pattern.     

Graded requirement for the zygotic terminal gene, tailless, in the brain and tail region of the Drosophila embryo

We have used hypomorphic and null tailless (tll) alleles to carry out a detailed analysis of the effects of the lack of tll gene activity on anterior and posterior regions of the embryo. The arrangement of tll alleles into a continuous series clarifies the relationship between the anterior and posterior functions of the tll gene and indicates that there is a graded sensitivity of anterior and posterior structures to a decrease in tll gene activity. With the deletion of both anterior and posterior pattern domains in tll null embryos, there is a poleward expansion of the remaining pattern. Using anti-horseradish peroxidase staining, we show that the formation of the embryonic brain requires tll. A phenotypic and genetic study of other pattern mutants places the tll gene within the hierarchy of maternal and zygotic genes required for the formation of the normal body pattern. Analysis of mutants doubly deficient in tll and maternal terminal genes is consistent with the idea that these genes act together in a common pathway to establish the domains at opposite ends of the embryo. We propose that tll establishes anterior and posterior subdomains (acron and tail regions, respectively) within the larger pattern regions affected by the maternal terminal genes.     

Genetic evidence for involvement of maternally derived Wnt canonical signaling in dorsal determination in zebrafish

In zebrafish, the program for dorsal specification begins soon after fertilization. Dorsal determinants are localized initially to the vegetal pole, then transported to the blastoderm, where they are thought to activate the canonical Wnt pathway, which induces the expression of dorsal-specific genes. We identified a novel maternal-effect recessive mutation, tokkaebi (tkk), that affects formation of the dorsal axis. Severely ventralized phenotypes, including a lack of dorso-anterior structures, were seen in 5-100% of the embryos obtained from tkk homozygous transmitting females. tkk embryos displayed defects in the nuclear accumulation of beta-catenin on the dorsal side, and reduced or absent expression of dorsal-specific genes. Mesoderm and endoderm formation outside the dorsal axis was not significantly affected. Injection of RNAs for activated beta-catenin, dominant-negative forms of Axin1 and GSK3beta, and wild-type Dvl3, into the tkk embryos suppressed the ventralized phenotypes and/or dorsalized the embryos, and restored or induced an ectopic and expanded expression of bozozok/dharma and goosecoid. However, dorsalization by wnt RNAs was affected in the tkk embryos. Inhibition of cytoplasmic calcium release elicited an ectopic and expanded expression of chordin in the wild-type, but did not restore chordin expression efficiently in the tkk embryos. These data indicate that the tkk gene product functions upstream of or parallel to the beta-catenin-degradation machinery to control the stability of beta-catenin. The tkk locus was mapped to chromosome 16. These data provide genetic evidence that the maternally derived canonical Wnt pathway upstream of beta-catenin is involved in dorsal axis formation in zebrafish.     

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.     

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.     

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.     

l(1)pole hole is required maternally for pattern formation in the terminal regions of the embryo

Maternal expression of the l(1)pole hole (l(1)ph) gene product is required for the development of the Drosophila embryo. When maternal l(1)ph+ activity is absent, alterations in the embryonic fate map occur as visualized by the expression of segmentation genes fushitarazu and engrailed. If both maternal and zygotic activity is absent, embryos degenerate around 7 h of development. If only maternal activity is missing, embryos complete embryogenesis and show deletions of both anterior and posterior structures. Anteriorly, structures originating from labral and acron head regions are missing. Posteriorly, abdominal segments A8, 9 and 10, the telson and the proctodeum are missing. Similar pattern deletions are observed in embryos derived from the terminal class of female sterile mutations. Thus, the maternal l(1)ph+ gene product is required for the establishment of cell identities at the anterior and posterior poles of the Drosophila embryo.     

Genetic Analysis of the Additional Sex Combs Locus of Drosophila Melanogaster

Additional sex combs (Asx) is a member of the Polycomb group of genes, which are thought to be required for maintenance of chromatin structure. To better understand the function of Asx, we have isolated nine new alleles, each of which acts like a gain of function mutation. Asx is required for normal determination of segment identity. AsxP1 shows an unusual phenotype in that anterior and posterior homeotic transformations are seen in the same individuals, suggesting that AsxP1 might upset chromatin structure in a way that makes both activation and repression of homeotic genes more difficult. Analysis of embryonic and adult phenotypes of Asx alleles suggests that Asx is required zygotically for determination of segment number and polarity. The expression pattern of even-skipped is altered in Asx mutant embryos, suggesting that Asx is required for normal expression of this gene. We have transposon-tagged the Asx gene, and can thus begin molecular analysis of its function.     

Maternal and zygotic activity of the zebrafish ogon locus antagonizes BMP signaling.

The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone morphogenetic protein (BMP) activity, which, in part, arises through the interaction of dorsally expressed antagonists Chordin and Noggin with the ventralizing BMPs. The zebrafish mercedes(tm305), ogon(m60), and short tail(b180) mutations produce ventralized phenotypes, including expanded bmp2b/4 expression domains. We find that the three mutations are allelic and that the locus they define, renamed ogon (ogo), maps to linkage group 25. The ogo(m60) and ogo(b180) mutations are deficiencies and thus represent null alleles, whereas the ENU-induced allele ogo(tm305) retains partial function. Aspects of the ogo(m60) and ogo(tm305) mutant phenotypes are fully suppressed by overexpression of BMP antagonists. Moreover, swirl(tc300), a null mutation in bmp2b, is epistatic to ogo(m60) mutation, providing further evidence that ogo normally functions in a BMP-dependent manner. Embryonic patterning is highly sensitive to maternal and zygotic ogo gene dosage, especially when the level of zygotic chordin activity is also reduced. However, elimination of the zygotic activity of both genes does not result in a completely ventralized embryo. Thus, while ogo and chordin are required to limit activity of BMPs, additional mechanisms must exist to block these ventralizing signals. We have ruled out zebrafish noggin homologues as candidates for the ogo gene, including a newly identified gene, nog1, which is specifically expressed in the gastrula organizer. The results suggest that ogo encodes an as yet unidentified dorsalizing factor that mediates dorsoventral patterning by directly or indirectly antagonizing BMP activity.     

Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo.

Seven zygotically active genes are required for normal patterning of the dorsal 40% of the Drosophila embryo. Among these genes, decapentaplegic (dpp) has the strongest mutant phenotype: in the absence of dpp, all cells in the dorsal and dorsolateral regions of the embryo adopt fates characteristic of more ventrally derived cells (Irish and Gelbart (1987) Genes Dev. 1, 868-879). Here we describe the phenotypes caused by alleles of another of this set of genes, tolloid, and show that tolloid is required for dorsal, but not dorsolateral, pattern. Extragenic suppressors of tolloid mutations were isolated that proved to be mutations that elevate dpp activity. We studied the relationship between tolloid and dpp by analyzing the phenotypes of tolloid embryos with elevated numbers of the dpp gene and found that doubling the dpp+ gene dosage completely suppressed weak tolloid mutants and partially suppressed the phenotypes of tolloid null mutants. We conclude that the function of tolloid is to increase dpp activity. We also examined the effect of doubling dpp+ gene dosage on the phenotypes caused by other mutations affecting dorsal development. Like tolloid, the phenotypes of mutant embryos lacking shrew gene function were suppressed by elevated dpp, indicating that shrew also acts upstream of dpp to increase dpp activity. In contrast, increasing the number of copies of the dpp gene enhanced the short gastrulation (sog) mutant phenotype, causing ventrolateral cells to adopt dorsal fates. This indicates that sog gene product normally blocks dpp activity ventrally. We propose that the tolloid, shrew and sog genes are required to generate a gradient of dpp activity, which directly specifies the pattern of the dorsal 40% of the embryo.     

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.