Untersuchungen zu funktionell konservierten sowie divergenten Mechanismen des Segmentierungsprozesses der Arthropoden am Beispiel der Spinne Cupiennius salei

Das Ziel der vorliegenden Arbeit war es, die molekularen Segmentierungsmechanismen der Spinne Cupiennius salei zu untersuchen, um so funktionell konservierte und divergente Aspekte der Arthropoden Segmentierung zu bestimmen und Aussagen über die Evolution des Segmentierungsprozesses zu ermöglichen. Hierbei wurden die Expressionsmuster verschiedener Cupiennius Paar-Regel und Gap Gen Homologe untersucht und funktionelle Analysen dieser Gene durchgeführt. Zudem wurde auch die Rolle der Notch-Signalkaskade während der Spinnen Segmentierung untersucht, um mögliche Gemeinsamkeiten mit dem Segmentierungsprozess der Vertebraten zu bestimmen. Die vorliegenden Ergebnisse zu den Paar-Regel und Gap Gen Homologen der Spinne lassen vermuten, dass die aus Drosophila bekannte Segmentierungsgenkaskade nicht vollständig konserviert ist. So konnte für die Cupiennius Krüppel Gene keine klassische Gap Funktion nachgewiesen werden. Im Fall des Paar-Regel Gen Homologs pairberry-3 lassen die Daten darauf schließen, dass die Segmente des Opisthosomas einzeln von der Wachstumszone abgespalten werden. Somit scheint zumindest für das Opisthosoma kein doppelsegmentaler Mechanismus vorzuliegen. Eine wichtige Rolle beim Segmentierungsprozess der Spinne übernimmt die Notch-Signalkaskade. Diese ist nicht nur an der Etablierung der Segmente sowie deren Grenzen beteiligt, sondern reguliert auch die dynamische Expression des Cupiennius Paar-Regel Gen Homologs hairy. Obwohl nicht ausgeschlossen werden kann, dass die Beteiligung der Notch-Signalkaskade an der Segmentierung der Spinne und der Vertebraten auf ein unabhängiges Rekrutierungsereignis zurückzuführen ist, liefern diese Ergebnisse doch einen starken Hinweis auf einen gemeinsamen Ursprung des Segmentierungsprozesses

Duplicated Hox genes in the spider Cupiennius salei

BACKGROUND: Hox genes are expressed in specific domains along the anterior posterior body axis and define the regional identity. In most animals these genes are organized in a single cluster in the genome and the order of the genes in the cluster is correlated with the anterior to posterior expression of the genes in the embryo. The conserved order of the various Hox gene orthologs in the cluster among most bilaterians implies that such a Hox cluster was present in their last common ancestor. Vertebrates are the only metazoans so far that have been shown to contain duplicated Hox clusters, while all other bilaterians seem to possess only a single cluster. RESULTS: We here show that at least three Hox genes of the spider Cupiennius salei are present as two copies in this spider. In addition to the previously described duplicated Ultrabithorax gene, we here present sequence and expression data of a second Deformed gene, and of two Sex comb reduced genes. In addition, we describe the sequence and expression of the Cupiennius proboscipedia gene. The spider Cupiennius salei is the first chelicerate for which orthologs of all ten classes of arthropod Hox genes have been described. The posterior expression boundary of all anterior Hox genes is at the tagma border of the prosoma and opisthosoma, while the posterior boundary of the posterior Hox genes is at the posterior end of the embryo. CONCLUSION: The presence of at least three duplicated Hox genes points to a major duplication event in the lineage to this spider, perhaps even of the complete Hox cluster as has taken place in the lineage to the vertebrates. The combined data of all Cupiennius Hox genes reveal the existence of two distinct posterior expression boundaries that correspond to morphological tagmata boundaries

Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium

Tribolium resembles C. elegans in showing a robust systemic RNAi response, but does not have C. elegans-type RNAi mechanisms; insect systemic RNAi probably uses a different mechanism

Opposing effects of Notch-signaling in maintaining the proliferative state of follicle cells in the telotrophic ovary of the beetle Tribolium

Introduction Establishment of distinct follicle cell fates at the early stages of Drosophila oogenesis is crucial for achieving proper morphology of individual egg chambers. In Drosophila oogenesis, Notch-signaling controls proliferation and differentiation of follicular cells, which eventually results in the polarization of the anterior-posterior axis of the oocyte. Here we analyzed the functions of Tribolium Notch-signaling factors during telotrophic oogenesis, which differs fundamentally from the polytrophic ovary of Drosophila. Results We found Notch-signaling to be required for maintaining the mitotic cycle of somatic follicle cells. Upon Delta RNAi, follicle cells enter endocycle prematurely, which affects egg-chamber formation and patterning. Interestingly, our results indicate that Delta RNAi phenotypes are not solely due to the premature termination of cell proliferation. Therefore, we monitored the terminal/stalk cell precursor lineage by molecular markers. We observed that upon Delta RNAi terminal and stalk cell populations were absent, suggesting that Notch-signaling is also required for the specification of follicle cell populations, including terminal and stalk precursor cells. Conclusions We demonstrate that with respect to mitotic cycle/endocycle switch Notch-signaling in Tribolium and Drosophila has opposing effects. While in Drosophila a Delta-signal brings about the follicle cells to leave mitosis, Notch-signaling in Triboliumis necessary to retain telotrophic egg-chambers in an “immature” state. In most instances, Notch-signaling is involved in maintaining undifferentiated (or preventing specialized) cell fates. Hence, the role of Notch in Tribolium may reflectthe ancestral function of Notch-signaling in insect oogenesis. The functions of Notch-signaling in patterning the follicle cell epithelium suggest that Tribolium oogenesis may - analogous to Drosophila - involve the stepwise determination of different follicle cell populations. Moreover, our results imply that Notch-signaling may contribute at least to some aspects of oocyte polarization and AP axis also in telotrophic oogenesis

Insertional mutagenesis screening identifies the zinc finger homeodomain 2 (zfh2) gene as a novel factor required for embryonic leg development in Tribolium castaneum

The genetic control of leg development is well characterized in the fly Drosophila melanogaster. These control mechanisms, however, must differ to some degree between different insect species to account for the morphological diversity of thoracic legs in the insects. The legs of the flour beetle Tribolium castaneum differ from the Drosophila legs in their developmental mode as well as in their specific morphology especially at the larval stage. In order to identify genes involved in the morphogenesis of the Tribolium larval legs, we have analyzed EGFP enhancer trap lines of Tribolium. We have identified the zfh2 gene as a novel factor required for normal leg development in Tribolium. RNA interference with zfh2 function leads to two alternative classes of leg phenotype. The loss of a leg segment boundary and the generation of ectopic outgrowths in one class of phenotype suggest a role in leg segmentation and segment growth. The malformation of the pretarsal claw in the second class of phenotype suggests a role in distal development and the morphogenesis of the claw-shaped morphology of the pretarsus. This suggests that zfh2 is involved in the regulation of an unidentified target gene in a concentration-dependent manner. Our results demonstrate that enhancer trap screens in T. castaneum have the potential to identify novel gene functions regulating specific developmental processes

A dual role for nanos and pumilio in anterior and posterior blastodermal patterning of the short-germ beetle Tribolium castaneum

AbstractAbdominal patterning in Drosophila requires the function of Nanos (nos) and Pumilio (pum) to repress posterior translation of hunchback mRNA. Here we provide the first functional analysis of nanos and pumilio genes during blastodermal patterning of a short-germ insect. We found that nos and pum in the red flour beetle Tribolium castaneum crucially contribute to posterior segmentation by preventing hunchback translation. While this function seems to be conserved among insects, we provide evidence that Nos and Pum may also act on giant expression, another gap gene. After depletion of nos and pum by parental RNAi, Hunchback and giant remain ectopically at the posterior blastoderm and the posterior Krüppel (Kr) domain is not being activated. giant may be a direct target of Nanos and Pumilio in Tribolium and presumably prevents early Kr expression. In the absence of Kr, the majority of secondary gap gene domains fail to be activated, and abdominal segmentation is terminated prematurely. Surprisingly, we found Nos and Pum also to be involved in early head patterning, as the loss of Nos and Pum results in deletions and transformations of gnathal and pre-gnathal anlagen. Since the targets of Nos and Pum in head development remain to be identified, we propose that anterior patterning in Tribolium may involve additional maternal factors

Power bleaching enhances resin infiltration masking effect of dental fluorosis. A randomized clinical trial

Objectives: Patients with moderate dental fluorosis often feel esthetically compromised. Aim of this RCT was to evaluate the objectively and self-assessed masking effect of resin infiltration alone or in combination with inoffice bleaching on dental fluorosis in adults. Methods: Twenty-seven patients (9 male, 18 female, 24.81 +/- 3.7 yrs) with 410 fluorotic teeth (TF 1-4) were randomly assigned to a treatment (BLI) or control group (NBLI). Patients underwent in-office bleaching (25% H2O2) in the BLI or a placebo bleaching (ACP gel) in the NBLI group followed by resin infiltration after two weeks. Standardized digital photographs were obtained at baseline; after bleaching; before and after resin infiltration and after 1, 3, and 6 months. Color differences (AE) between sound and fluorotic areas were calculated and patient satisfaction was evaluated using a VAS (1-10). Results: Statistical analysis revealed significant differences in the mean AE values 6 months after resin infiltration between the BLI (Delta E = 1.41) and the NBLI group (Delta E = 4.33) (p = 0.024). VAS values increased after resin infiltration (p < 0.05) in both groups. After 3 months patients in the BLI group had higher VAS values than in the NBLI group (p = 0.029). Conclusions: Findings of this study suggest that resin infiltration alone can effectively mask mild to moderate dental fluorosis in young adults. In-office bleaching with 25% H2O2 before resin infiltration provides significantly better masking effects. Clinical Significance: Resin infiltration is a safe and efficient treatment option for masking fluorotic opacities. A priori in-office bleaching with 25% H2O2 enhances the masking effect

TC003132 is essential for the follicle stem cell lineage in telotrophic Tribolium oogenesis

Abstract Background Stem cells are undifferentiated cells with a potential for self-renewal, which are essential to support normal development and homeostasis. To gain insight into the molecular mechanisms underlying adult stem cell biology and organ evolution, we use the telotrophic ovary of the beetle Tribolium. To this end, we participated in a large-scale RNAi screen in the red flour beetle Tribolium, which identified functions in embryonic and postembryonic development for more than half of the Tribolium genes. Results We identified TC003132 as candidate gene for the follicle stem cell linage in telotrophic Tribolium oogenesis. TC003132 belongs to the Casein Kinase 2 substrate family (CK2S), which in humans is associated with the proliferative activity of different cancers. Upon TC003132 RNAi, central pre-follicular cells are lost, which results in termination of oogenesis. Given that also Notch-signalling is required to promote the mitotic activity of central pre-follicular cells, we performed epistasis experiments with Notch and cut. In addition, we identified a putative follicle stem cell population by monitoring the mitotic pattern of wild type and TC003132 depleted follicle cells by EdU incorporations. In TC003132 RNAi these putative FSCs cease the expression of differentiation makers and are eventually lost. Conclusions TC003132 depleted pre-follicular cells neither react to mitosis or endocycle stimulating signals, suggesting that TC003132 provides competence for differentiation cues. This may resemble the situation in C. elegans were CK2 is required to maintain the balance between proliferation and differentiation in the germ line. Since the earliest effect of TC003132 RNAi is characterized by the loss of putative FSCs, we posit that TC003132 crucially contributes to the proliferation or maintenance of follicle stem cells in the telotrophic Tribolium ovary

Using the modified Schirmer test for dry mouth assessment: A cross-sectional study

This study aimed to establish whether the modified Schirmer test could serve as a diagnostic tool for dry mouth, that is, whether it could reliably measure salivary film at selected locations within the oral cavity, and to identify levels of sensitivity/specificity and determine reference values. Therefore, a cross-sectional study (N = 120, mean age 63.5 [SD 13.9] years) was performed. The test was used at five locations (hard palate; buccal mucosa in molar region at 4 mm above occlusal plane; anterior tongue; lower lip; mouth floor), and results were recorded after 1, 2 and 3 min. A statistically significant discriminatory ability of the Schirmer test for the unstimulated salivary flow rates could be shown for the palate (at 3 min), buccal mucosa (at 1 min), mouth floor (at 1 min), and tongue (at 2 and 3 min) (areas under the curve 0.64-0.68), with individual sensitivity/specificity values depending on test location/time points. Thus, the modified Schirmer test has potential to become a simple and reproducible instrument for the detection of dry mouth based on low unstimulated salivary flow rates in dentistry and especially outreach care. Care must be taken concerning intraoral test location and measurement time

Additional file 1: of TC003132 is essential for the follicle stem cell lineage in telotrophic Tribolium oogenesis

Table S1. EdU positive cells in wildtype. Table S2. PH3 positive cells. Table S3. EdU positive cells in TC003132 RNAi. Table S4. EdU positive cells in Cut RNAi. Methods. (DOCX 26 kb