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.     

even-skipped is not a pair-rule gene but has segmental and gap-like functions in Oncopeltus fasciatus, an intermediate germband insect

The pair-rule gene even-skipped is required for the initiation of metameric pattern in Drosophila. But Drosophila segmentation is evolutionarily derived and is not representative of most insects. Therefore, in order to shed light on the evolution of insect segmentation, homologs of the pair-rule gene even-skipped have been studied in several insect taxa. However, most of these studies have reported the expression eve but not its function. We report the isolation, expression and function of the homolog of Drosophila even-skipped from the intermediate germband insect Oncopeltus fasciatus. We find that in Oncopeltus, even-skipped striped expression initiates in a segmental and not pair-rule pattern. Weak RNAi suppression of Oncopeltus even-skipped shows no apparent pair-rule like phenotype, while stronger RNAi suppression shows deletion of nearly the entire body. These results suggest that in Oncopeltus, even-skipped is not acting as a pair-rule gene. In almost all insects, prior to its striped expression, even-skipped is expressed in a conserved broad gap-like domain but its function has been largely ignored. We find that this early broad domain is required for activation of the gap genes hunchback and Krüppel. Given the large RNAi deletion phenotype and its regulation of hunchback and Krüppel, even-skipped seems to act as an über-gap gene in Oncopeltus, indicating that it may have both upstream and downstream roles in segmentation.     

The LATD gene of Medicago truncatula is required for both nodule and root development

The evolutionary origins of legume root nodules are largely unknown. We have identified a gene, LATD, of the model legume Medicago truncatula, that is required for both nodule and root development, suggesting that these two developmental processes may share a common evolutionary origin. The latd mutant plants initiate nodule formation but do not complete it, resulting in immature, non-nitrogen-fixing nodules. Similarly, lateral roots initiate, but remain short stumps. The primary root, which initially appears to be wild type, gradually ceases growth and forms an abnormal tip that resembles that of the mutant lateral roots. Infection by the rhizobial partner, Sinorhizobium meliloti, can occur, although infection is rarely completed. Once inside latd mutant nodules, S. meliloti fails to express rhizobial genes associated with the developmental transition from free-living bacterium to endosymbiont, such as bacA and nex38. The infecting rhizobia also fail to express nifH and fix nitrogen. Thus, both plant and bacterial development are blocked in latd mutant roots. Based on the latd mutant phenotype, we propose that the wild-type function of the LATD gene is to maintain root meristems. The strong requirement of both nodules and lateral roots for wild-type LATD gene function supports lateral roots as a possible evolutionary origin for legume nodules.     

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration [1]. In bilaterians, there are at least 12 subfamilies of Wnt genes [2]. Wnt3 and Wnt8 are required for somitogenesis in vertebrates [3-7] and are thought to be involved in posterior specification in deuterostomes in general [8]. Although TCF and beta-catenin have been implicated in the posterior patterning of some short-germ insects [9, 10], the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. Here we investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum[11]. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. Our results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, we propose that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes.     

Functional analyses of tiptop and antennapedia in the embryonic development of Oncopeltus fasciatus suggests an evolutionary pathway from ground state to insect legs

In insects, selector genes are thought to modify the development of a default, or 'ground state', appendage into a tagma-specific appendage such as a mouthpart, antenna or leg. In the best described example, Drosophila melanogaster, the primary determination of leg identity is thought to result from regulatory interactions between the Hox genes and the antennal-specifying gene homothorax. Based on RNA-interference, a functional analysis of the selector gene tiptop and the Hox gene Antennapedia in Oncopeltus fasciatus embryogenesis is presented. It is shown that, in O. fasciatus, tiptop is required for the segmentation of distal leg segments and is required to specify the identity of the leg. The distal portions of legs with reduced tiptop develop like antennae. Thus, tiptop can act as a regulatory switch that chooses between antennal and leg identity. By contrast, Antennapedia does not act as a switch between leg and antennal identity. This observation suggests a significant difference in the mechanism of leg specification between O. fasciatus and D. melanogaster. These observations also suggest a significant plasticity in the mechanism of leg specification during insect evolution that is greater than would have been expected based on strictly morphological or molecular comparisons. Finally, it is proposed that a tiptop-like activity is a likely component of an ancestral leg specification mechanism. Incorporating a tiptop-like activity into a model of the leg-specification mechanism explains several mutant phenotypes, previously described in D. melanogaster, and suggests a mechanism for the evolution of legs from a ground state.     

smedinx-11 is a planarian stem cell gap junction gene required for regeneration and homeostasis

The largely unknown mechanisms that regulate adult stem cells probably involve signals from neighboring differentiated cells. Gap junction channels providing direct cell-cell communication via small molecules are a crucial component of morphogenesis and normal physiology. However, no specific gap junction protein has yet been functionally linked to adult/somatic stem cell behavior in vivo or to organ regeneration. We report the identification and characterization of smedinx-11--an innexin gap junction channel gene expressed in the adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. smedinx-11 RNAi treatment inhibits regeneration and abrogates neoblast maintenance. Moreover, smedinx-11 expression is enriched in an irradiation-sensitive subpopulation (;X2') and is required for proper expression of other stem cell-specific markers. Analyses of the smedinx-11 downregulation phenotype revealed a striking anterior-posterior neoblast gradient. Our data demonstrate a novel role for gap junction proteins and suggest gap junction-mediated signaling as a new and tractable control point for adult, somatic stem cell regulation.     

The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc finger motifs.

We have discovered a reporter gene insertion that is expressed in the trunk region of Drosophila embryos. Genetic and molecular details of a new regulatory gene neighboring the reporter gene insertion, which we call teashirt (tsh), are described. In situ hybridization of a tsh probe to embryos shows that this gene is expressed in a way similar to the reporter gene. Mutations of tsh show that the gene is required for normal development of the ventral trunk region of embryos, which correlates with the spatial expression of the gene in the anteroposterior axis but not in the dorsoventral axis. Sequencing of a tsh cDNA shows that the putative protein possesses three distantly spaced CX2CX12HX5H zinc finger motifs.     

Stella is a maternal effect gene required for normal early development in mice

stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella null offspring, stella-deficient females displayed severely reduced fertility due to a lack of maternally inherited Stella-protein in their oocytes. Indeed, we demonstrate that embryos without Stella are compromised in preimplantation development and rarely reach the blastocyst stage. stella is thus one of few known mammalian maternal effect genes, as the phenotypic effect on embryonic development is mainly a consequence of the maternal stella mutant genotype. Furthermore, we show that STELLA that is expressed in human oocytes is also expressed in human pluripotent cells and in germ cell tumors. Interestingly, human chromosome 12p, which harbours STELLA, is consistently overrepresented in these tumors. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumors.     

The wingless gene is required for embryonic brain development in Drosophila

 We have studied the role of the wingless gene in embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional wingless protein during embryonic stages 9–10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in dramatically oversized CNS. We conclude that wingless is required for the development of the anterior protocerebral brain region in Drosophila. We propose that an important role of wingless in this part of the developing brain is the determination of neural cell fate. Received: 7 October 1997 / Accepted: 30 December 1997     

Anti-apoptotic gene Bcl2 is required for stapes development and hearing

In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2^−/− mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2^−/− mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2^−/−Bim^−/− mice and partially rescued in Bcl2^−/−Bim^+/− mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2^−/− defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development.     

Distribution and activity of a Dippu DH31-like peptide in the large milkweed bug Oncopeltus fasciatus

The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.     

The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm

The 95F myosin, a class VI unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three- dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions lead to aberrant nuclear divisions and to the nuclear defects observed. We propose that 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form.     

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.