midline represses Dpp signaling and target gene expression in Drosophila ventral leg development

Ventral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. Here we show that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus TBE site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. We further demonstrate that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, we find that mid does not affect all Dpp-target genes as we demonstrate that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid.Summary statementVentral patterning is controlled in part by the T-box Transcription factor midline blocking Dpp signaling and Dpp-activated genes, though midline does not affect the Dpp-repressed gene brk.

Extensive loss of Wnt genes in Tardigrada

Background Wnt genes code for ligands that activate signaling pathways during development in Metazoa. Through the canonical Wnt (cWnt) signaling pathway, these genes regulate important processes in bilaterian development, such as establishing the anteroposterior axis and posterior growth. In Arthropoda, Wnt ligands also regulate segment polarity, and outgrowth and patterning of developing appendages. Arthropods are part of a lineage called Panarthropoda that includes Onychophora and Tardigrada. Previous studies revealed potential roles of Wnt genes in regulating posterior growth, segment polarity, and growth and patterning of legs in Onychophora. Unlike most other panarthropods, tardigrades lack posterior growth, but retain segmentation and appendages. Here, we investigated Wnt genes in tardigrades to gain insight into potential roles that these genes play during development of the highly compact and miniaturized tardigrade body plan. Results We analyzed published genomes for two representatives of Tardigrada, Hypsibius exemplaris and Ramazzottius varieornatus. We identified single orthologs of Wnt4, Wnt5, Wnt9, Wnt11, and WntA, as well as two Wnt16 paralogs in both tardigrade genomes. We only found a Wnt2 ortholog in H. exemplaris. We could not identify orthologs of Wnt1, Wnt6, Wnt7, Wnt8, or Wnt10. We identified most other components of cWnt signaling in both tardigrade genomes. However, we were unable to identify an ortholog of arrow/Lrp5/6, a gene that codes for a Frizzled co-receptor of Wnt ligands. Additionally, we found that some other animals that have lost several Wnt genes and are secondarily miniaturized, like tardigrades, are also missing an ortholog of arrow/Lrp5/6. We analyzed the embryonic expression patterns of Wnt genes in H. exemplaris during developmental stages that span the establishment of the AP axis through segmentation and leg development. We detected expression of all Wnt genes in H. exemplaris besides one of the Wnt16 paralogs. During embryo elongation, expression of several Wnt genes was restricted to the posterior pole or a region between the anterior and posterior poles. Wnt genes were expressed in distinct patterns during segmentation and development of legs in H. exemplaris, rather than in broadly overlapping patterns. Conclusions Our results indicate that Wnt signaling has been highly modified in Tardigrada. While most components of cWnt signaling are conserved in tardigrades, we conclude that tardigrades have lost Wnt1, Wnt6, Wnt7, Wnt8, and Wnt10, along with arrow/Lrp5/6. Our expression data may indicate a conserved role of Wnt genes in specifying posterior identities during establishment of the AP axis. However, the loss of several Wnt genes and the distinct expression patterns of Wnt genes during segmentation and leg development may indicate that combinatorial interactions among Wnt genes are less important during tardigrade development compared to many other animals. Based on our results, and comparisons to previous studies, we speculate that the loss of several Wnt genes in Tardigrada may be related to a reduced number of cells and simplified development that accompanied miniaturization and anatomical simplification in this lineage.

Role of the Forkhead Transcription Factors Fd4 and Fd5 During Drosophila Leg Development

Appendage development requires the coordinated function of signaling pathways and transcription factors to pattern the leg along the three main axes: the antero-posterior (AP), proximo-distal (PD), and dorso-ventral (DV). The Drosophila leg DV axis is organized by two morphogens, Decapentaplegic (Dpp), and Wingless (Wg), which direct dorsal and ventral cell fates, respectively. However, how these signals regulate the differential expression of its target genes is mostly unknown. In this work, we found that two members of the Drosophila forkhead family of transcription factors, Fd4 and Fd5 (also known as fd96Ca and fd96Cb), are identically expressed in the ventro-lateral domain of the leg imaginal disc in response to Dpp signaling. Here, we analyze the expression regulation and function of these genes during leg development. We have generated specific mutant alleles for each gene and a double fd4/fd5 mutant chromosome to study their function during development. We highlight the redundant role of the fd4/fd5 genes during the formation of the sex comb, a male specific structure that appears in the ventro-lateral domain of the prothoracic leg.

Distinct gene expression dynamics in developing and regenerating limbs

Regenerating animals have the ability to reproduce organs that were originally generated in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned.

The Broad Transcription Factor Links Hormonal Signaling, Gene Expression, and Cellular Morphogenesis Events During Drosophila Imaginal Disc Development

Imaginal disc morphogenesis during metamorphosis in Drosophila melanogaster provides an excellent model to uncover molecular mechanisms by which hormonal signals effect physical changes during development. The broad (br) Z2 isoform encodes a transcription factor required for disc morphogenesis in response to 20-hydroxyecdysone, yet how it accomplishes this remains largely unknown. Here, we use functional studies of amorphic br5 mutants and a transcriptional target approach to identify processes driven by br and its regulatory targets in leg imaginal discs. br5 mutants fail to properly remodel their basal extracellular matrix (ECM) between 4 and 7 hr after puparium formation. Additionally, br5 mutant discs do not undergo the cell shape changes necessary for leg elongation and fail to elongate normally when exposed to the protease trypsin. RNA-sequencing of wild-type and br5 mutant leg discs identified 717 genes differentially regulated by br, including a large number of genes involved in glycolysis, and genes that encode proteins that interact with the ECM. RNA interference-based functional studies reveal that several of these genes are required for adult leg formation, particularly those involved in remodeling the ECM. Additionally, br Z2 expression is abruptly shut down at the onset of metamorphosis, and expressing it beyond this time results in failure of leg development during the late prepupal and pupal stages. Taken together, our results suggest that br Z2 is required to drive ECM remodeling, change cell shape, and maintain metabolic activity through the midprepupal stage, but must be switched off to allow expression of pupation genes.

Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology

Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, we further analysed the role and regulation of Ultrabithorax (Ubx) during mesothoracic (T2) leg development in Drosophila melanogaster. Ubx represses leg trichomes in the proximal posterior region of the T2 femur (the so-called naked valley) and we found that it likely does so through activating the expression of microRNA-92a. We also identified a T2 leg enhancer of Ubx that recapitulates the temporal and regional activity of this Hox gene in these appendages. Analysis of motifs in this enhancer predicted that it is bound by Distal-less (Dll) and we found that knockdown of Dll results in the loss of trichomes on the T2 femur. This suggests that while Ubx activates microRNA-92a to repress trichomes in the naked valley region of the proximal femur, Dll may repress Ubx more distally to enable formation of trichomes. Taken together our results provide insights into how Ubx is integrated into a postembryonic gene regulatory network to determine fine-scale leg morphology.

An Investigation of the Prevalence of Giardia agilis in Anuran Amphibians Reveals Special Parasitic Adaptations of this Giardia Species

Background: Giardia agilis is a Giardia species with a very narrow and elongated body distinct from others. It was first reported in 1882, and was detected in several species of anuran amphibians. Although there were some studies about its morphology, no investigations about its prevalence have ever been reported to date.Methods: We detected G. agilis in frogs and tadpoles from some areas of China based on its distinct morphology. Statistical analysis was performed using mid-P exact probability tests and differences were considered significant when p-values ≤0.05 were obtained.Results: We investigated the prevalence of G. agilis in 25 anuran amphibian species and found that 195 of the 463 (42.1%) samples were detected to be positive. Our molecular phylogenetic analysis indicated all the detected G. agilis were from the same species. The 195 positive frog samples were from 9 frog species, which are distributed scatteredly in four families that are not closely related rather than just restricted to a narrow lineage. The statistical prevalence among adults of different frog species showed no significant difference, and so did among tadpoles, but the prevalence in the tadpoles is significantly higher than in their adults. More interestingly, although the prevalence in Kaloula verrucosa from the same area showed no significant differences between its tadpoles without legs and the ones with two legs, but is significantly higher in these two developmental stages than in the four-legged stage, which is still much higher than in the adults. Moreover, all the positive samples were found to be from the areas with relatively high altitude (more than 870 meters).Conclusions: G. agilis is probably able to infect all anuran amphibians without species-bias. The turning point of prevalence in the period of front leg development might be related with the development of immune system of the hosts. That G. agilis tends to infect easily the frogs living in high altitude areas means that it has adapted to the dramatic temperature change in the poikilothermal animal hosts. Therefore, G. agilis has evolved some special successful parasitism strategies for parastizing the poikilothermal hosts with metamorphosis development -- anuran amphibians.

MicroRNA clusters integrate evolutionary constraints on expression and target affinities: the miR-6/5/4/286/3/309 cluster in Drosophila leg development

A striking feature of microRNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a microRNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this microRNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of microRNA cluster members were also constructed. Expression of individual microRNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient microRNAs together (miR-5/4/286/3/309) or more recently evolved clustered microRNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed down-regulation of leg patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of microRNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct microRNAs. Considering together the microRNA targets and the evolutionary ages of each microRNA in the cluster demonstrates the importance of microRNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing microRNAs.