Double abdomen induction by UV in Bradysia tritici (syn. Sciara ocellaris,Sciaridae): sensitive stages and conditions for photoreversal

Summary The pattern anomaly double abdomen was induced in embryos of Bradysia tritici (syn. Sciara ocellaris) by irradiation of the anterior egg pole with far UV (254 or 285 nm) using low UV fluences. The maximum yield of 18% of double abdomens was obtained when 2.5 h embryos were irradiated (late intravitelline cleavage stage); earlier irradiation failed to yield double abdomens, as did irradiations after the early syncytial blastoderm stage. Exposing irradiated embryos to photoreverting light (366 nm) reduced the yield of malformations. Most double abdomens were symmetrical and the number of segments ranged from 3 to 8 in each set, with the mean value at 6.4 segments.     

A morphogenetic determinant in the anterior pole of an insect egg (Smittia spec., Chironomidae, Diptera): localization by combined centrifugation and ultraviolet irradiation

In chironomid midges, the development of the head and thorax in the embryo requires the function of cytoplasmic determinants localized near the anterior pole of the egg. Experimental inactivation of these determinants causes a dramatic switch in the developmental program of the embryo. Instead of the normal segment pattern, the aberrant pattern “double abdomen” is formed. Head, thorax, and anterior abdominal segments are then replaced by an additional set of posterior abdominal segments jointed with reversed polarity to the original abdomen. To determine the cellular fraction which contains the effective targets for uv induction of double abdomens, Smittia eggs were centrifuged prior to uv irradiation. Accumulation of proteid spheres or lipid droplets in the irradiated anterior pole region caused a considerable decrease in the double abdomen yield. Removal of these components from the target area enhanced double abdomen formation. The maximum yield of double abdomens was obtained after uv irradiation of a cytoplasmic layer in which organelles larger than ribosomes could not be detected. The results of these and other experiments, suggest that ribosomes, ribosomal subunits, or other ribonucleoprotein particles act as effective targets for the uv induction of double abdomens in Smittia eggs.     

Proteins foretelling head or abdomen development in the embryo of Smittia spec. (Chironomidae,Diptera)

The development of the segment pattern in Smittia embryos can be manipulated experimentally. Centrifugation during intravitelline cleavage leads to a mirror image duplication of most of the head in the absence of abdominal segments (“double cephalons”). Conversely, mirror image duplications of abdominal segments in the absence of head and thorax (“double abdomens”) can be generated by UV-irradiation of the anterior pole before blastoderm formation. By subsequent exposure to blue light, UV-irradiated embryos can be reprogrammed for normal development (photoreversal). We have characterized an “anterior indicator” protein (designated AI₁; M_r ? 35,000; IEP ? 4.9). Its synthesis was restricted to anterior fragments of embryos during a late blastoderm stage (Bl_VI). This protein was synthesized, however, in both anterior and posterior fragments of prospective double cephalons. Conversely, this protein was synthesized neither in anterior nor in posterior fragments of UV-induced double abdomens. Upon photoreversal, the protein was synthesized again in anterior fragments. Thus, synthesis of this protein in a given fragment always indicated development of head and thorax there. Likewise, we have characterized a “posterior indicator protein” (designated PI₁, M_r ? 50,000, IEP ? 5.5). Its synthesis during early blastoderm stages (Bl_I and Bl_II) was restricted to posterior fragments but not to pole cells in normal embryos. In UV-induced double abdomens, PI_I was synthesized in both anterior and posterior fragments at stage Bl_II. Photoreversal again led to restriction of PI_I synthesis to posterior fragments. Thus, the synthesis of PI_I in a given fragment at stage Bl_II always foreshadowed the formation of an abdomen several hours before this can be discerned morphologically. The synthesis of two other proteins (designated a₁ and p₁) was also restricted, during certain blastoderm stages, to anterior or posterior fragments, respectively. However, UV-irradiation or centrifugation had little or no effect on the synthesis of these proteins. Conversely, programming embryos for double abdomen development by UV-irradiation caused a set of reproducible, and mostly photoreversible, changes in the pattern of proteins synthesized in anterior embryonic fragments. However, the synthesis of most of the affected proteins was not region-specific in normal embryos.     

Parameters influencing reversal of segment sequence in posterior egg fragments ofCallosobruchus (Coleoptera)

Summary The double abdomen type of embryonic segment pattern can develop in posterior fragments ofCallosobruchus eggs. In this type of pattern, a series of posterior segments is joined in reversed polarity to an equal set from the original pattern persisting in normal polarity. Reversed and non-reversed sets are fused in a plane of mirror symmetry, which shows in the larval cuticle as a symmetry line. This line may be located anywhere in the posterior thorax or the anterior abdomen. The reversed abdomen may be incomplete caudally due to secondary causes. Polarity reversal and concomitant double abdomen formation occurred only when temporary constriction was terminated before cellularization of the blastoderm, and only when the anterior fragment was degenerating. Maximum reversal frequency was 94% of analyzable posterior partial larvae when the constriction was applied slightly anterior to the middle of the egg when the egg contained 4–32 nuclei. Reversal was often restricted to longitudinal strips of the larval cuticle. The longitudinal borderlines between the reversed and the non-reversed strips ran predominantly along the larval midlines. Such borderlines probably existed in the blastoderm anywhere around its circumference, but borderlines in the future mesoderm and serosa would be internalized during gastrulation and dorsal closure, respectively, and the embryonic midlines would then become secondary borderlines visible in the larval cuticle. If a morphogen is involved in segment pattern formation, its transport in the egg must be polarized longitudinally in order to account for reversals restricted to longitudinal cuticular strips.     

Double abdomen induction with low UV-doses inSmittia spec. (Chironomidae,diptera): Sensitive period and complete photoreversibility

Summary In embryos of the chironomid midgeSmittia spec., UV-irradiation of anterior pole regions results in double abdomen formation. In this abnormal body pattern, head and thorax are replaced by a mirror image of the abdomen. Irradiation at a particular stage between nuclear migration and blastoderm formation, and with the anterior pole facing the UV-beam, yields 100% double abdomens after a minimum UV-dose (140 J·m^–2 at 285 nm wavelength). By subsequent exposure to light of longer wavelength, embryos can be reprogrammed so that they develop normally again. The irradiation procedure described is suitable for programming large numbers of embryos for double abdomen formation, with a minimum of side effects and virtually complete photoreversibility.     

Dicephalic — ADrosophila mutant affecting polarity in follicle organization and embryonic patterning

Summary The mutationdicephalic (dic) affects follicle development and thereby alters the antero-posterior polarity of embryonic patterning. It maps at a single locus (3–46.0±1.0) and can be characterized as a semi-dominant maternal effect mutation with low penetrance. Indic follicles, the 15 nurse cells form two clusters located at opposite poles of the oocyte; the numerical distribution of the nurse cells among the clusters varies from 7:8 to 1:14. Thedic egg shell carries a micropyle (anterior marker) at either pole, but the misshapen respiratory appendages are restricted to one of the two poles in most eggs. The malformed eggs rarely yield larvae and these are always abnormal anteriorly and/or posteriorly. The segment pattern expressed in their cuticle may represent two anterior parts of opposite polarities (double head type), two posterior parts of opposite polarities (double abdomen type, rare) or show uniform polarity. Lability of organization at the cystocyte stage appears as the primary developmental defect of the mutant.     

Changes in the apparent localization of anterior determinants during early embryogenesis (Smittia spec., Chironomidae,Diptera)

Summary InSmittia embryos, anterior UV irradiation causes the formation of double abdomens in which head and thorax are replaced by a mirror-image duplication of abdominal segments. The transformation is ascribed to the inactivation of cytoplasmic ribonucleoprotein particles acting as anterior determinants. During the first 5 h after egg deposition (h.a.d.), high yields of double abdomens are produced by microbeam irradiation of a small target area (diameter 20 m) behind the anterior pole. This localization coincides with a yolk-free cytoplasmic cone observed behind the anterior pole of newly depositedSmittia eggs (Zissler and Sander 1973). Irradiation of this area becomes less efficient between 5 and 7 h.a.d., while double abdomen yields increase after irradiation of adjacent target areas. A dramatic increase in the double abdomen yield is also observed after irradiation of the entire anterior surface. Under these conditions, the UV fluence required for a given yield decreases by a factor of 3.9 between 5.5. and 7 h.a.d. This can be explained quantitatively by the assumption that the effective targets move an average distance of 8 from the strongly absorbing endoplasm towards the egg surface. During the period of apparent target deshielding, the distance of nuclei from the egg surface actually decreases from 11 to 3 m. However, the timing and direction of the nuclear migration and of the apparent deshielding differ, indicating that the shift of effective targets may be triggered by, but does not exactly follow, the nuclear movement. The data suggest that a major fraction of anterior determinants shifts from a small volume behind the anterior pole to a spread-out distribution over the surface of the anterior half.     

Cytoplasmic factors determining anteroposterior polarity in Drosophila embryos

Summary Cytoplasm removal/transplant techniques applied to Drosophila cleavage-stage embryos induced changes in anteroposterior polarity. Removal of anterior cytoplasm or anterior transplantation of posterior cytoplasm caused the anterior formation of posterior (telson) structures, and the replacement of anterior cytoplasm with posterior cytoplasm induced double-abdomen embryos, as reported by Frohnhöfer et al. [J Embryol Exp Morphol 97 (suppl):169–179 (1986)]. Changing the conditions of anterior cytoplasm removal we showed that greater volumes, earlier stages, and removal from the periphery were efficient. In addition we found that double-cephalon embryos are induced by replacing posterior cytoplasm with anterior cytoplasm, while removal of posterior cytoplasm or the posterior transplantation of anterior cytoplasm was without effect. However, introduction of anterior cytoplasm into the posterior of nanos embryos, which are mutants not developing abdominal segments, caused the formation of double-cephalon embryos. Similarly, double-abdomen embryos are produced by introducing posterior cytoplasm into the anterior of bicoid embryos, which are mutants not forming cephalic and thoracic structures. These results are compatible with the initial involvement of separate anterior, posterior and terminal cytoplasmic factors deduced from mutant analysis (Nüsslein-Volhard and Roth 1989).     

Genetic analysis of pattern-formation in the embryo ofDrosophila melanogaster

Summary The mutationbicaudal (Bull, 1966) causes embryos to develop a longitudinal mirror image duplication of the posteriormost abdominal segments, while head and thorax are missing. These embryos occur with varying frequencies among eggs laid by mutant females, irrespective of the paternal genotype. Recombination and deletion mapping indicate thatbicaudal (bic) is a recessive, hypomorphic, maternal-effect mutation mapping at a single locus on the second chromosome ofDrosophila melanogaster close tovg (67.0±0.1). The frequency of bicaudal embryos depends on the age of the mother, her genetic constitution and the temperature at which she is raised. Best producers are very young females hemizygous forbic (bic/Df(2)vg ^B ) at 28° C. Under these conditions 80% to 90% of the eggs which differentiate can show the bicaudal embryo phenotype. Upon ageing of the mother the frequency of bicaudal embryos declines rapidly, and most of the eggs develop the normal body pattern. Temperature shift experiments suggest a temperature-sensitive period at the onset of vitellogenesis.The mutation causes several types of abnormalities in the segment pattern of theDrosophila embryo, which are interpreted as various degrees of expression of the mutant character. The most frequent abnormal phenotype is the symmetrical bicaudal embryo with one to five abdominal segments duplicated. Less frequent are asymmetrical types, in which the smaller number of segments is always in the anterior reversed part. Other phenotypes are embryos with missing or rudimentary heads, and embryos with irregular gaps in the segment pattern. In bicaudal embryos, the pole cells, formed at the posterior pole of the egg prior to blastoderm formation, are not duplicated at the anterior. The significance of thebicaudal phenotypes for embryonic pattern-formation inDrosophila is discussed.     

A scanning electron microscope study of the developing embryo of Manduca sexta (L.) (Lepidoptera : Sphingidae)

Scanning electron microscopy of the developing Manduca sexta (Lepidoptera : Sphingidae) embryo reveals that the body wall of the insect undergoes considerable morphogenesis beginning at 20 hr post-oviposition. The elongated 19 hr embryo contracts in length, which gives rise to the formation of rudimentary segments. By 33 hr, many of the appendage anlagen are visible, the presumptive spiracles appear as bifurcate pits and the proctodeum begins invagination. During this same period, prior to katatrepsis, the body walls become established, and the segments and appendages develop. Between 50 and 60 hr post-oviposition, involution of the oral cavity and reorientation of the associated gnathal appendages occurs. During this same period, katatrepsis and provisional dorsal closure take place. Developmental polarity is evident as a distinctive wave of specialization proceeding posterior to anterior in the thorax/abdomen, and anterior to posterior in the head. Configuration of the oral cavity is strikingly prognathous until just prior to eclosion. Two embryonic molts are apparent, as determined by the remnants of ecdysed “embryonic cuticles”.     

Switch in pattern formation after puncturing the anterior pole of Smittia eggs (Chironomidae, Diptera).

The aberrant pattern, “double abdomen,” previously induced in the egg of Smittia by uv irradiation of anterior pole regions was also produced by puncturing of the egg at the anterior pole. Double abdomens and embryos with anterior defects developed in eggs in which puncturing had locally prevented the regular arrangement of cleavage nuclei in the periplasm. The resulting gap in the blastoderm at the anterior pole was subsequently closed under exclusion of a small amount of egg material. Double abdomens did not develop in eggs where exclusion of anterior egg material was not observed. Thus a basic switch in the developmental program of the egg appears to depend upon the functional elimination of some crucial components in the anterior egg region.     

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.     

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.     

The specification of metameric order in the insectCallosobruchus maculatus Fabr. (Coleoptera)

Summary Mechanically dividing an insect egg into anterior and posterior fragments results in a segment gap (Sander 1976), a loss of non-terminal segments in the constricted region. By altering the stage and duration of constriction, we produced different types of egg fragments in the pea beetleCallosobruchus. The patterns formed by these fragments suggest the existence of interactions between anterior and posterior egg regions that influence segment patterning and placement. Segments in excess of the numbers expected on the basis of permanent constrictions were produced in fragments when: (1) the constriction was released before cellularization occurred and (2) in addition the complementary fragment degenerated. Apparently the degenerating fragment induced the formation of excess segments in the developing fragment. Differences in the time and extent of excess segment formation in anterior versus posterior fragments suggest an asymmetric distribution of prerequisites for segment formation. This conclusion is consistent with our finding that a partial reversal of segment sequence (double abdomen formation) can be induced only in posterior fragments by a degenerating fragment, but not in anterior fragments (see companion paper).The formation of excess segments shows that the segment gap observed after permanent separation cannot be due to non-specific damage, caused by the process of constriction as such, to the egg or to localized putative segment precursors.     

Maternal Co-ordinate Gene Regulation and Axis Polarity in the Scuttle Fly Megaselia abdita

Axis specification and segment determination in dipteran insects are an excellent model system for comparative analyses of gene network evolution. Antero-posterior polarity of the embryo is established through systems of maternal morphogen gradients. In Drosophila melanogaster, the anterior system acts through opposing gradients of Bicoid (Bcd) and Caudal (Cad), while the posterior system involves Nanos (Nos) and Hunchback (Hb) protein. These systems act redundantly. Both Bcd and Hb need to be eliminated to cause a complete loss of polarity resulting in mirror-duplicated abdomens, so-called bicaudal phenotypes. In contrast, knock-down of bcd alone is sufficient to induce double abdomens in non-drosophilid cyclorrhaphan dipterans such as the hoverfly Episyrphus balteatus or the scuttle fly Megaselia abdita. We investigate conserved and divergent aspects of axis specification in the cyclorrhaphan lineage through a detailed study of the establishment and regulatory effect of maternal gradients in M. abdita. Our results show that the function of the anterior maternal system is highly conserved in this species, despite the loss of maternal cad expression. In contrast, hb does not activate gap genes in this species. The absence of this activatory role provides a precise genetic explanation for the loss of polarity upon bcd knock-down in M. abdita, and suggests a general scenario in which the posterior maternal system is increasingly replaced by the anterior one during the evolution of the cyclorrhaphan dipteran lineage.     

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.     

Biology of Pseudopotamilla reniformis (Müller 1771) in the White Sea,with description of asexual reproduction

The tube-dwelling polychaete Pseudopotamilla reniformis (Sabellidae) forms dense and complex aggregations of flexible tubes on hard substrates in the subtidal zone of the White Sea. No sexual reproduction was observed in this study and recruitment appeared to be due to asexual reproduction by architomy in winter, from October to March. The posterior part of the abdomen undergoes spontaneous fission into from 2 to 4 fragments and depending on their position, the fragments regenerate their anterior ends or both anterior and posterior ends. Regeneration in P. reniformis takes place via a combination of epimorphosis (replacement of missing parts by cell proliferation and the growth of new tissue) and morphallaxis (the remodelling of pre-existing structures without cell proliferation). The morphogenetic events during regenerative restoration include de novo formation of branchial crown, formation of thoracic segments and restoration of the posterior end. Asexual reproduction appears to play a crucial role for formation of P. reniformis aggregations and is very important for the population in the White Sea, at the margin of the species’ range.     

The genital disc of Drosophila melanogaster

 The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The results show that the three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs, and the absence of engrailed and invected cause duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development. Received: 4 February 1997 / Accepted: 22 May 1997     

Abd-B suppresses lepidopteran proleg development in posterior abdomen

Pterygotes lack abdominal appendages except for pleuropods and prolegs. The larvae of some holometabolous insects develop prolegs, which are used for locomotion. We analyzed the role of the homeotic genes abd-A and Abd-B in lepidopteran proleg development using mutant analysis and embryonic RNAi in the silkworm Bombyx mori. The E^Mu mutant developed extra prolegs in its posterior abdomen and showed the misexpression of both genes, suggesting their involvement in proleg formation. The depletion of Abd-B by embryonic RNAi caused the development of extra prolegs on all segments posterior to A6, indicating the suppressive function of Abd-B. The abd-A RNAi animals failed to develop prolegs. These results indicate that abd-A and Abd-B are involved in proleg development in B. mori.     

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.