hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germband insect Oncopeltus fasciatus

Insects such as Drosophila melanogaster undergo a derived form of segmentation termed long germband segmentation. In long germband insects, all of the body regions are specified by the blastoderm stage. Thus, the entire body plan is proportionally represented on the blastoderm. This is in contrast to short and intermediate germband insects where only the most anterior body regions are specified by the blastoderm stage. Posterior segments are specified later in embryogenesis during a period of germband elongation. Although we know much about Drosophila segmentation, we still know very little about how the blastoderm of short and intermediate germband insects is allocated into only the anterior segments, and how the remaining posterior segments are produced. In order to gain insight into this type of embryogenesis, we have investigated the expression and function of the homolog of the Drosophila gap gene hunchback in an intermediate germ insect, the milkweed bug, Oncopeltus fasciatus. We find that Oncopeltus hunchback (Of'hb) is expressed in two phases, first in a gap-like domain in the blastoderm and later in the posterior growth zone during germband elongation. In order to determine the genetic function of Of'hb, we have developed a method of parental RNAi in the milkweed bug. Using this technique, we find that Oncopeltus hunchback has two roles in anterior-posterior axis specification. First, Of'hb is required to suppress abdominal identity in the gnathal and thoracic regions. Subsequently, it is then required for proper germband growth and segmentation. In milkweed bug embryos depleted for hunchback, these two effects result in animals in which a relatively normal head is followed by several segments with abdominal identity. This phenotype is reminiscent to that found in Drosophila hunchback mutants, but in Oncopeltus is generated through the combination of the two separate defects.     

Functional analyses in the milkweed bug Oncopeltus fasciatus (Hemiptera) support a role for Wnt signaling in body segmentation but not appendage development

Specification of the proximal-distal (PD) axis of insect appendages is best understood in Drosophila melanogaster, where conserved signaling molecules encoded by the genes decapentaplegic (dpp) and wingless (wg) play key roles. However, the development of appendages from imaginal discs as in Drosophila is a derived state, while more basal insects produce appendages from embryonic limb buds. Therefore, the universality of the Drosophila limb PD axis specification mechanism has been debated since dpp expression in more basal insect species differs dramatically from Drosophila. Here, we test the function of Wnt signaling in the development of the milkweed bug Oncopeltus fasciatus, a species with the basal state of appendage development from limb buds. RNA interference of wg and pangolin (pan) produce defects in the germband and eyes, but not in the appendages. Distal-less and dachshund, two genes regulated by Wg signaling in Drosophila and expressed in specific PD domains along the limbs of both species, are expressed normally in the limbs of pan-depleted Oncopeltus embryos. Despite these apparently paradoxical results, Armadillo protein, the transducer of Wnt signaling, does not accumulate properly in the nuclei of cells in the legs of pan-depleted embryos. In contrast, engrailed RNAi in Oncopeltus produces cuticular and appendage defects similar to Drosophila. Therefore, our data suggest that Wg signaling is functionally conserved in the development of the germband, while it is not essential in the specification of the limb PD axis in Oncopeltus and perhaps basal insects.     

多胚发育寄生蜂腰带长体茧蜂在寄主亚洲玉米螟幼虫体内的发育

多胚发育的幼虫内寄生蜂腰带长体茧蜂Macrocentrus cingulum的卵、胚胎和幼虫在寄主亚洲玉米螟Ostrinia furnacalis幼虫血腔内发育,通常1枚卵可以分裂增殖为数百只胚胎。本文通过定时解剖寄生的寄主幼虫,初步了解了腰带长体茧蜂多胚的形成过程及其在寄主体内的发育情况。结果表明：以4龄末期亚洲玉米螟幼虫为寄主时,寄生蜂卵产入寄主体内10 min左右开始卵裂,1天左右,初级胚胎从卵壳中被释放出来。之后胚胎在胚外膜内持续分裂产生大量二级胚胎形成桑葚胚。寄生后3天左右,二级胚胎从胚外膜中被释放出来,进入胚胎发育阶段。寄生后6天左右,胚胎进入胚带形成阶段。寄生后8天左右,胚带伸长,头尾形成。寄生后9天左右,身体分节完成,部分幼虫孵化,蜕去胚外膜。寄生后13天左右,蜂幼虫从寄主体内啮出。胚胎在发育初期体积变化不大,但从胚带形成开始,体积则迅速增大。腰带长体茧蜂与另一多胚发育寄生蜂佛州点缘跳小蜂Copidosoma floridanum在胚胎发育进程上明显不同,体现了它们对各自寄主的适应。     

Expression patterns of twist and snail in Tribolium (Coleoptera) suggest a homologous formation of mesoderm in long and short germ band insects

The mesodermal region in Drosophila is determined by a maternally derived morphogenetic gradient system which specifies the different cell fates along the dorsoventral axis, including the prospective mesodermal cells at the ventral side of the embryo. There are at least two zygotic target genes, twist and snail, which are required for mesoderm formation in Drosophila. To analyze whether a similar mode of mesoderm specification might also apply to short germ band insect embryos, we have cloned twist and snail- related gene fragments from the flour beetle Tri-bolium and have analyzed their expression pattern. Both genes are expressed in a ventral stripe at early blastoderm stage, which is restricted to the region of the developing germ rudiment. The cells expressing the two genes are those that invaginate during gastrulation, indicating that the early stages of mesoderm specification are indeed very similar between the two species. Interestingly, both genes are also expressed during germband extension in a subregion of the growth zone of the embryo which forms the mesodermal cells. This suggests that the expression of the two genes is required for mesoderm formation both at early blastoderm stage and during germband elongation until the end of the segmental growth process. © 1994 Wiley-Liss, Inc.     

Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum

wingless (wg)/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved [1]. Wnt signaling is also implicated in posterior patterning and germband elongation [2-4], but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions [1-3]. Tribolium contains additional Wnt family genes that are also expressed in the growth zone [5]. After depleting Tc-WntD/8 we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods [6] and vertebrates [7-9].     

Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.