Nanos is the localized posterior determinant in Drosophila

Segmental pattern in the Drosophila embryo is established by two maternal factors localized to the anterior and posterior poles of the egg cell. Here we provide molecular evidence that the localized posterior factor is the RNA of the nanos (nos) gene. nos RNA is localized to the posterior pole of early embryos, and nos protein acts at a distance to direct abdomen formation. Synthetic nos RNA has biological activity identical to that of the posterior pole plasm. Injection of nos RNA rescues the segmentation defect of embryos derived from females mutant for all nine known posterior group genes. Injection of nos RNA into the anterior is able to direct formation of ectopic posterior structures. Our results demonstrate that a localized source of nos RNA is sufficient to specify abdominal segmentation and imply that other posterior group genes are required for localization, stabilization, or distribution of the nos gene product.     

A pupal lethal mutation with a paternally influenced maternal effect on embryonic development in Drosophila melanogaster

The maternal effect and zygotic phenotype of l(1)pole hole (l(1)ph) is described. l(1)ph is a zygotic lethal mutation which affects cell division of adult precursor cells in Drosophila larvae. The locus is located in 2F6 on the salivary gland chromosome map and four alleles have been characterized. Germ-line clonal analysis of amorphic alleles indicates that l(1)ph has a maternal effect lethal phenotype. Two lethal phenotypes are observed among embryos derived from female germ-line clones homozygous for amorphic alleles dependent upon the zygotic activity of l(1)ph⁺ introduced via the sperm. Class 1: If no wild-type dose of the gene is introduced, embryos form abnormal blastoderms in which nuclear migration and cell formation is disrupted leading to an ill-defined cuticular pattern. Class 2: If a wild-type copy of the gene is introduced, blastoderm cells do not form beneath the pole cells (the pole hole phenotype); subsequently such embryos are missing cuticular structures posterior to the seventh abdominal segment (the torso phenotype). When the zygotic activity l(1)ph⁺ is modulated using position effect variegation a new phenotype is observed among class 2 embryos in which torso embryos are twisted along their longitudinal axis.     

Analysis of cytoplasmic activity dependent on the Drosophila terminal pattern gene torso

Cytoplasm from wildtype Drosophila embryos was transplanted into torso (tor) mutant embryos to determine the distribution of terminal rescuing activity at the cleavage stage. Although posterior and lateral wildtype cytoplasm contained rescuing activity that restored posterior terminal (telson) structures Klingler et al. (1988, Nature (London) 335, 275-277) this rescuing activity was not found in anterior cytoplasm. Similarly, transplantation of anterior and lateral wildtype cytoplasm into the anterior of tor embryos rescued anterior terminal (acron) structures, whereas posterior cytoplasm did not. This failure of reciprocal rescue is due to the presence of the products of the anterior and posterior classes of genes, because anterior cytoplasm from bicoid mutant embryos restored the telson in the posterior as well as the acron in the anterior of tor embryos, and because posterior cytoplasm from nanos embryos rescued the acron in the anterior as well as the telson in the posterior of tor embryos. Therefore terminal rescuing activity is evenly distributed throughout the cleavage stage embryo as anticipated from molecular studies.     

Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos.

The mago nashi (mago) locus is a newly identified strict maternal effect, grandchildless-like, gene in Drosophila melanogaster. In homozygous mutant mago females reared at 17 degrees C, mago+ function is reduced, the inviable embryos lack abdominal segments and 84-98% of the embryos die. In contrast, at 25 degrees C, some mago alleles produce a novel gene product capable of inducing the formation of symmetrical double abdomen embryos. Reciprocal temperature-shift experiments indicate that the temperature-sensitive period is during oogenetic stages 7-14. Furthermore, embryos collected from mago1 homozygous females contain no apparent functional posterior determinants in the posterior pole. In viable F1 progeny from mago mutant females, regardless of genotype and temperature, polar granules are reduced or absent and germ cells fail to form (the grandchildless-like phenotype). Thus, we propose that the mago+ product is a component of the posterior determinative system, required during oogenesis, both for germ cell determination and delineation of the longitudinal axis of the embryo.     

Different requirements for l(1) giant in two embryonic domains of Drosophila melanogaster

l(1) giant is a zygotic lethal mutation which affects the embryonic development of both the labial/thoracic segments and a subset of posterior abdominal segments. Using antibodies specific for proteins encoded by several Drosophila genes to identify the compartmental origin of the defects, we show that the requirement of giant activity is different in these two embryonic domains. Anteriorly, the posterior compartment of the labial segment is missing at the blastoderm stage. Posteriorly, cells are specifically deleted by cell death within the anterior compartments of abdominal segments 5-7 during germ band elongation. In mature embryos, posterior compartment structures of the peripheral nervous system of A5-7 are fused. In addition to a different pattern of defect in the two parts of the embryo, the kind of action appears different. Anteriorly, giant resembles a gap mutation in that a particular region is missing from the blastoderm fate map, whereas in the abdominal domain, giant affects the development of anterior compartment-specific structures.     

Localization of nanos RNA controls embryonic polarity.

Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.     

Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus

The segmental plan of the Drosophila embryo is already established at the blastoderm stage through the action of maternal effect genes which determine the polarity of the embryo and zygotically active genes involved in segmentation. We have analyzed the first example of a group of maternally acting genes which are necessary for establishing the developmental potential of the posterior 25% of the blastoderm. Females, homozygous for the X-linked maternal-effect mutation female sterile(1)Nasrat211 [fs(1)N211], produce embryos, characterized as torso-like, which lack all posterior endodermal derivatives as well as structures characteristic of abdominal segments 8 to 10. In addition, anterior endodermal derivatives are deficient and the absence of pharyngeal musculature causes a collapse of the cephalopharyngeal apparatus. The columnar blastoderm cell layer is defective at the posterior tip below the pole cells in these embryos. This defect, however, is presumably secondary to some abnormal feature of pole cell formation since in double mutants of fs(1)Nasrat211; tudor3 the blastoderm is normal but the embryos still show the torso-like phenotype. In situ hybridization with RNA probes derived from the fushi tarazu gene establishes that the cellular determination of the posterior blastoderm of embryos produced by fs(1)N211 is changed. This represents the first direct demonstration that a maternal-effect mutation alters the spatial distribution of a zygotic gene product involved in the segmental patterning of the embryo.     

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.     

Biochemical analysis of torso and D-raf during Drosophila embryogenesis: implications for terminal signal transduction.

Determination of anterior and posterior terminal structures of Drosophila embryos requires activation of two genes encoding putative protein kinases, torso and D-raf. In this study, we demonstrate that Torso has intrinsic tyrosine kinase activity and show that it is transiently tyrosine phosphorylated (activated) at syncytial blastoderm stages. Torso proteins causing a gain-of-function phenotype are constitutively tyrosine phosphorylated, while Torso proteins causing a loss-of-function phenotype lack tyrosine kinase activity. The D-raf gene product, which is required for Torso function, is identified as a 90-kDa protein with intrinsic serine/threonine kinase activity. D-Raf is expressed throughout embryogenesis; however, the phosphorylation state of the protein changes during development. In wild-type embryos, D-Raf is hyperphosphorylated at 1 to 2 h after egg laying, and thereafter only the most highly phosphorylated form is detected. Embryos lacking Torso activity, however, show significant reductions in D-Raf protein expression rather than major alterations in the protein's phosphorylation state. This report provides the first biochemical analysis of the terminal signal transduction pathway in Drosophila embryos.     

A gradient of bicoid protein in Drosophila embryos

The maternal gene bicoid (bcd) organizes anterior development in Drosophila. Its mRNA is localized at the anterior tip of the oocyte and early embryo. Antibodies raised against bcd fusion proteins recognize a 55-57 kd doublet band in Western blots of extracts of 0-4 hr old embryos. This protein is absent or reduced in embryonic extracts of nine of the 11 bcd alleles. The protein is concentrated in the nuclei of cleavage stage embryos. It cannot be detected in oocytes, indicating temporal control of bcd mRNA translation. The bcd protein is distributed in an exponential concentration gradient with a maximum at the anterior tip, reaching background levels in the posterior third of the embryo. The gradient is probably generated by diffusion from the local mRNA source and dispersed degradation.     

Defects of the body plan of mutant embryos lacking Lim1, Otx2 or Hnf3beta activity

The orientation of the anterior-posterior (A-P) axis was examined in gastrula-stage Hnf3beta, Otx2 and Lim1 null mutant embryos that display defective axis development. In situ hybridization analysis of the expression pattern of genes associated with the posterior germ layer tissues and the primitive streak (T, Wnt3 and Fgf8) and anterior endoderm (Cer1 and Sox17) revealed that the A-P axis of mutant embryos remains aligned with the proximo-distal plane of the gastrula. Further analysis revealed that cells which express Chrd activity are either absent in Hnf3beta mutant embryos or localised in heterotopic sites in Lim1 and Otx2 null mutants. Lim1-expressing cells are present in the Hnf3beta mutant embryo albeit in heterotopic sites. In all three mutants, Gsc-expressing cells are missing from the anterior mesendoderm. These findings suggest that although some cells with organizer activity may be present in the mutant embryo, they are not properly localised and fail to contribute to the axial mesoderm of the head. By contrast, in T/T mutant embryos that display normal head fold development, the expression domains of organizer, primitive streak and anterior endoderm genes are regionalised correctly in the gastrula.     

Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo

Summary Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.     

The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo

A group of maternal genes, the posterior group, is required for the development of the abdominal region in the Drosophila embryo. We have used genetic as well as cytoplasmic transfer experiments to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity is synthesized during oogenesis and the gene nanos most likely encodes this activity. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. Staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. We suggest that the products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants.     

An early role of maternal mRNA in establishing the dorsoventral pattern in pelle mutant Drosophila embryos

Mutations of the maternal effect locus pelle (pll) cause dorsalized Drosophila embryos. In extreme mutants, the embryo develops into a long hollow tube of dorsal cuticular structures with no sign of ventral pattern elements. Injection of wild-type cytoplasm or poly(A)+RNA into mutant pll embryos partially restores the normal pattern. Rescuing activity is present in the wild-type cytoplasm until the late blastoderm stage, but is already absent from the poly(A)+RNA fraction by the time of pole cell formation. At the same time, pll embryos fail to respond to injected biologically active poly(A)+RNA. This indicates that pll+ mRNA is lost early from the pool of maternal RNA and that there is a non-RNA component of rescue. This component, most likely the pll+ protein, appears to be unequally distributed in wild-type embryos.     

hunchback, a gene required for segmentation of an anterior and posterior region of the Drosophila embryo

The locus hunchback (hb) is a member of the gap class of segmentation genes of Drosophila. A number of X-ray-induced deletions locate the hb locus at the chromosomal site 85A3-B1, to the right of the pink locus, which maps in the same interval. A total of 14 EMS and 3 X-ray-induced hb alleles have been studied. Homozygous mutant embryos show deletions of segments in two separate regions. In the six strong alleles, the labium and all three thoracic segments are deleted anteriorly while posteriorly the 8th abdominal segment and adjacent parts of the 7th abdominal segment are lacking. The eight weak alleles show smaller deletions both in the thoracic and posterior abdominal region. In the weakest allele only part of the mesothorax is deleted. Three hb alleles produce a homoeotic transformation: superimposed on a strong or weak deletion phenotype, head or thoracic segments are transformed into abdominal segments, respectively. This suggests that hb might also be involved in the regulation of genes in the Bithorax complex (BX-C). Fate mapping of the normal-appearing segments in strong mutant embryos using the UV-laser beam ablation technique (Lohs-Schardin et al., 1979) shows that these segments arise from the normal blastoderm regions. The mutant phenotype can be recognized soon after the onset of gastrulation in a failure to fully extend the germ band. In 6-hr-old mutant embryos, two clusters of dead cells are observed in the thoracic and posterior abdominal region. These observations indicate region specific requirement of hb gene function. The analysis of germ line chimeras by transplantation of homozygous mutant pole cells shows that hb is already expressed during oogenesis. Homozygous mutant embryos derived from a homozygous mutant germ line have a novel phenotype. The anterior affected region is enlarged, including all three gnathal segments and the anterior three abdominal segments. In addition three abdominal segments with reversed polarity are formed between the remaining head structures and the posterior abdomen. Heterozygous mutant embryos derived from a homozygous mutant germ line develop normally, indicating that maternal gene expression is not required for normal development.     

High bicoid levels render the terminal system dispensable for Drosophila head development

In Drosophila, the gradient of the Bicoid (Bcd) morphogen organizes the anteroposterior axis while the ends of the embryo are patterned by the maternal terminal system. At the posterior pole, expression of terminal gap genes is mediated by the local activation of the Torso receptor tyrosine kinase (Tor). At the anterior, terminal gap genes are also activated by the Tor pathway but Bcd contributes to their activation. Here we present evidence that Tor and Bcd act independently on common target genes in an additive manner. Furthermore, we show that the terminal maternal system is not required for proper head development, since high levels of Bcd activity can functionally rescue the lack of terminal system activity at the anterior pole. This observation is consistent with a recent evolution of an anterior morphogenetic center consisting of Bcd and anterior Tor function.     

Requirements for zygotic gene activity during gastrulation in Drosophila melanogaster

Mutations at the folded gastrulation (fog) and twisted gastrulation (tsg) loci interfere with early morphogenetic movements in Drosophila melanogaster. fog embryos do not form a normal posterior midgut and although their germbands do elongate, they do not extend dorsally. As a result, when normal embryos have fully extended germbands, the germbands in mutant embryos are folded into the interior on the ventral side of the embryo. tsg embryos have abnormally deep dorsal folds during early gastrulation, associated with the failure of dorsal cells to slip laterally to make way for the expanding germband. Both fog and tsg embryos continue to develop, but form disorganized first instar larvae. fog and tsg are zygotically active genes expressed at least by 10 and 20 min after the onset of gastrulation. Both mutations are viable in homozygous germ cells and the wild-type genes need not be expressed during oogenesis for survival of heterozygous progeny. Elimination of fog+ gene product from maternal germ cells does, however, affect the extent of folding observed during gastrulation in viable heterozygotes. Analysis of fog adult and larval gynandromorphs indicates that normal folded gastrulation gene function is only required at the posterior region of the embryo, most probably in the cells giving rise to the posterior midgut or proctodeum. The relative survival of fog mosaics suggests that embryos with mosaic "lethal foci" also die during embryogenesis, although the typical fog phenotype is only produced when the entire focus is mutant. In contrast to the fog focus, no particular cell must be wild type in tsg mosaics for survival. Wild-type cells on the dorsal side of the embryo, however, are most effective in rescuing the embryo. This indicates that normal tsg gene product may be required only on the dorsal side of the embryo, potentially in the region which gives rise to the amnion serosa.