Background
Equine sarcoids (ES) are the most common cutaneous tumors in equids. Systemic treatment options are sparse. Subcutaneous (SC) injections of Viscum album extract (VAE) demonstrate efficacy ...as a systemic treatment directed against ES.
Objectives/Aim
To critically assess the therapeutic efficacy of orally administered VAE.
Animals
Forty‐five ES‐affected, privately owned, 3–12 year‐old horses.
Methods
A 3‐armed randomized placebo‐controlled, double‐blinded study was conducted in a double‐dummy design. Horses were subjected to oral administration and SC injections of either VAE or placebo (VAE oral/placebo SC, VAE SC/placebo oral, placebo oral/placebo SC) over a 7‐month treatment period. Primary endpoint was the change of baseline of a composite index of ES number and ES area after 14 months. Second endpoint was the clinical response.
Results
No statistically significant difference in the composite endpoint between the 3 study arms was found. The primary endpoint showed 4 (27%) horses in the VAE oral group with complete ES regression, 3 (21%) in the VAE SC injection group, and 2 (13%) in the placebo group. The clinical response revealed complete or partial regression in 6 horses of the oral VAE group (40%), 4 of the SC injection group (29%), and 4 of the placebo group (25%). Direct comparison of oral VAE and placebo showed an odds ratio, stratified for prognosis of 2.16 (95%‐CI: 0.45–10.42) and a P‐value of 0.336.
Conclusion and Clinical Importance
Oral administration of VAE is well tolerated. No statistically significant difference in the effectiveness of systemic VAE versus placebo against ES was found.
In both vertebrates and invertebrates, the Wnt-signaling pathway is essential for numerous processes in embryogenesis and during adult life. Wnt activity is fine-tuned at various levels by the ...interplay of a number of Wnt-agonists (Wnt ligands, Frizzled-receptors, Lrp5/6 coreceptors) and Wnt-antagonists (among them Axin, Secreted frizzled and Lrp4) to define anterior-posterior polarity of the early embryo and specify cell fate in organogenesis. So far, the functional analysis of Wnt-pathway components in insects has concentrated on the roles of Wnt-agonists and on the Wnt-antagonist Axin. We depict here additional features of the Wnt-antagonist Axin in the flour beetle
. We show that
is dynamically expressed throughout embryogenesis and confirm its essential role in head development. In addition, we describe an as yet undetected, more extreme
RNAi-phenotype, the ectopic formation of posterior abdominal segments in reverse polarity and a second hindgut at the anterior. For the first time, we describe here that an
ortholog is involved in axis formation in an insect. The
Lrp4 ortholog is ubiquitously expressed throughout embryogenesis. Its downregulation via maternal RNAi results in the reduction of head structures but not in axis polarity reversal. Furthermore, segmentation is impaired and larvae develop with a severe gap-phenotype. We conclude that, as in vertebrates,
functions as a Wnt-inhibitor in
during various stages of embryogenesis. We discuss the role of both components as negative modulators of Wnt signaling in respect to axis formation and segmentation in
.
Unlike
but similar to other arthropod and vertebrate embryos, the flour beetle
develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia ...are only poorly understood.
Here we show that the newly discovered genes
and
are involved in regulating the directional budding of appendages. RNAi-knockdown of
results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The
genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that
, a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a
-like RNAi-phenotype.
The similarity of the inverted appendage phenotype in both the
- and the
RNAi gene knockdown led us to conclude that the
orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.
Members of the Sp gene family are involved in a variety of developmental processes in both vertebrates and invertebrates. We identified the ortholog of the Drosophila Sp-1 gene in the red flour ...beetle Tribolium castaneum , termed T-Sp8 because of its close phylogenetic relationship to the vertebrate Sp8 genes. During early embryogenesis, T-Sp8 is seen in segmental stripes. During later stages, TSp8 is dynamically expressed in the limb buds of the Tribolium embryo. At the beginning of bud formation, TSp8 is uniformly expressed in all body appendages. As the limbs elongate, a ring pattern develops sequentially and the expression profile at the end of embryogenesis correlates with the final length of the appendage. In limbs that do not grow out like the labrum and the labium, T-Sp8 expression remains uniform, whereas a two-ring pattern develops in the longer antennae and the maxillae. In the legs that elongate even further, four rings of T-Sp8 expression can be seen at the end of leg development. The role of T-Sp8 for appendage development was tested using RNAi. Upon injection of double stranded T-Sp8 RNA, larvae develop with dwarfed appendages. Affected T-Sp8 RNAi legs were tested for the presence of medial and distal positional values using the expression marker genes dachshund and Distal-less , respectively. The results show that a dwarfed TSp8 RNAi leg consists of proximal, medial and distal parts and argues against T-Sp8 being a leg gap gene. Based on the differential expression pattern of T-Sp8 in the appendages of the head and the thorax and the RNAi phenotype, we hypothesise that T-Sp8 is involved in the regulation of limb-length in relation to body size - a process called allometric growth.
Short germ embryos elongate their primary body axis by consecutively adding segments from a posteriorly located growth zone. Wnt signalling is required for axis elongation in short germ arthropods, ...including Tribolium castaneum, but the precise functions of the different Wnt receptors involved in this process are unclear. We analysed the individual and combinatorial functions of the three Wnt receptors, Frizzled-1 (Tc-Fz1), Frizzled-2 (Tc-Fz2) and Frizzled-4 (Tc-Fz4), and their co-receptor Arrow (Tc-Arr) in the beetle Tribolium. Knockdown of gene function and expression analyses revealed that Frizzled-dependent Wnt signalling occurs anteriorly in the growth zone in the presegmental region (PSR). We show that simultaneous functional knockdown of the Wnt receptors Tc-fz1 and Tc-fz2 via RNAi resulted in collapse of the growth zone and impairment of embryonic axis elongation. Although posterior cells of the growth zone were not completely abolished, Wnt signalling within the PSR controls axial elongation at the level of pair-rule patterning, Wnt5 signalling and FGF signalling. These results identify the PSR in Tribolium as an integral tissue required for the axial elongation process, reminiscent of the presomitic mesoderm in vertebrates. Knockdown of Tc-fz1 alone interfered with the formation of the proximo-distal and the dorso-ventral axes during leg development, whereas no effect was observed with single Tc-fz2 or Tc-fz4 RNAi knockdowns. We identify Tc-Arr as an obligatory Wnt co-receptor for axis elongation, leg distalisation and segmentation. We discuss how Wnt signalling is regulated at the receptor and co-receptor levels in a dose-dependent fashion.
The appendages of an insect are subdivided into distinct segments or podomeres. Many genes responsible for the regionalization of the growing limb into subdomains have been isolated from Drosophila. ...So far, only one gene is known in the leg that is solely required for specifying the distal-most pattern element-the pretarsal claw. In Drosophila, the gene aristaless is expressed in the centre of the antennal and leg imaginal disc that represents the most distal position of appendages, and in a proximal region. When Drosophila aristaless function is impaired, antennae and legs develop without their distal-most structures-the arista and the claw. We describe here the analysis of aristaless in the beetle Tribolium-an insect that shows a different, more ancestral mode of appendage formation than Drosophila. In Tribolium, appendages grow out continuously during embryogenesis, and no imaginal discs are formed. Tribolium aristaless (Tc-al) expression starts midway during appendage elongation, and is seen in a distal and a proximal position of head and trunk appendages. At the end of embryogenesis, Tc-al is seen in four expression domains in the leg, in the dorsal epidermis, and ventrally in every segment in lateral groups of cells, presumably the histoblasts. Like in the Drosophila adult, Tc-al is required in the larva for the formation of the most distal structures of the leg and the antenna as revealed by RNAi experiments. We conclude that aristaless is evolutionarily robust, meaning that it has retained its expressional and functional characteristics, although a heterochronic change of the process of appendage elongation took place towards the evolution of the highly derived diptera.
Fibroblast growth factor (FGF) signalling plays a key role in early embryonic development and cell migration in vertebrates and in invertebrates. To gain novel insights into FGF signalling in an ...arthropod, we characterized the fgf1b ortholog in the beetle Tribolium that is not represented in the Drosophila genome.
We found that FGF1b dependent signalling organizes the anterior to posterior axis of the early embryo. The loss of Tc-fgf1b function in Tribolium by RNA interference resulted in the reduction of the anteriormost extraembryonic fate, in an anterior shift of embryonic fate and in the loss or malformation of anterior embryonic structures. Without intact extraembryonic membranes the serosa and the amnion, Tc-fgf1bRNAi embryos did not undergo morphogenetic movements and remained posteriorly localized throughout embryogenesis. Only weakly affected embryos developed into a cuticle that show dorsally curved bodies with head defects and a dorsal opening. Except for the posterior dorsal amnion, the overall topology of the dorsal–ventral axis seemed unaffected. Moreover, FGF signalling was not required for the onset of mesoderm formation but for fine-tuning this tissue during later development. We also show that in affected embryos the dorsal epidermis was expanded and expressed Tc-dpp at a higher level.
We conclude that in the Tribolium blastoderm embryo, FGF1-signalling organizes patterning along the AP-axis and also balances the expression level of Dpp in the dorsal epidermis, a tissue critically involved in dorsal closure.
•First study showing the requirement of FGF signalling for blastoderm patterning in an insect.•In Tribolium, fgf1b gene is crucial for anterior patterning in the blastoderm.•Intact serosa and amnion are required for morphogenetic movements of the embryo.•Tc-fgf1b balances Dpp-level and regulates the process of dorsal closure.
•We identify an early amnion precursor within the anterior blastoderm embryo.•We describe hindsight (hnt) as a reliable serosa marker in Tribolium.•We show that Tc-dpp expression starts as an ...asymmetric domain.•We describe an anterior ventral domain of pMAD in the blastoderm embryo.
Most insect embryos develop with two distinct extraembryonic membranes, the serosa and the amnion. In the insect beetle Tribolium the early origin of the serosa within the anterior blastoderm is well established but the origin of the amnion is still debated. It is not known whether this tissue develops from a blastodermal precursor or originates de novo later from embryonic tissue during embryogenesis.
We undertook an in-depth analysis of the spatio-temporal expression pattern profile of important extraembryonic membrane marker genes with emphasis on early blastoderm development in Tribolium.
The amnion marker iroquois (Tc-iro) was found co-expressed with the serosa marker zerknüllt1 (Tc-zen1) during early blastoderm formation in an anterior cap domain. This domain later resolved into two adjacent domains that likely represent the precursors of the serosa and the amnion. In addition, we found the hindsight ortholog in Tribolium (Tc-hnt) to be a serosa-specific marker. Surprisingly, decapentaplegic (Tc-dpp) expression was not seen as a symmetric cap domain but detected asymmetrically first along the DV- and later also along the AP-axis. Moreover, we found a previously undescribed domain of phosphorylated MAD (pMAD) protein in anterior ventral serosal cells.
This is the first study showing that the anterior-lateral part of the amnion originates from the anterior blastoderm while the precursor of the dorsal amnion develops later de novo from a dorsal-posterior region within the differentiated blastoderm.
Insect embryogenesis is best understood in the fruit fly Drosophila. However, Drosophila embryogenesis shows evolutionary-derived features: anterior patterning is controlled by a highly derived Hox ...gene bicoid, the body segments form almost simultaneously and appendages develop from imaginal discs. In contrast, embryogenesis of the red flour beetle Tribolium castaneum displays typical features in anterior patterning, axis and limb formation shared with most insects, other arthropods as well as with vertebrates. Anterior patterning depends on the conserved homeobox gene orthodenticle, the main body axis elongates sequentially and limbs grow continuously starting from an appendage bud. Thus, by analysing developmental processes in the beetle at the molecular and cellular level, inferences can be made for similar processes in other arthropods. With the completion of sequencing the Tribolium genome, the door is now open for post-genomic studies such as RNA expression profiling, proteomics and functional genomics to identify beetle-specific gene circuits.
Scanning electron microscopy (SEM) is a valuable method for examining surface structures. Taking wax impressions of plant structures, such as leaves, is a nondestructive procedure that makes it ...possible to view changes in surface structures over time, such as during development. This protocol describes a method for making dental wax impressions of plant tissues.
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