Robert B. Beckstead

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis.

BackgroundThe steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription.ResultsHere we identify the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. We show that genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. We provide evidence that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. We also present an initial characterization of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes.ConclusionThis study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development.

HIF- and Non-HIF-Regulated Hypoxic Responses Require the Estrogen-Related Receptor in Drosophila melanogaster

Low-oxygen tolerance is supported by an adaptive response that includes a coordinate shift in metabolism and the activation of a transcriptional program that is driven by the hypoxia-inducible factor (HIF) pathway. The precise contribution of HIF-1a in the adaptive response, however, has not been determined. Here, we investigate how HIF influences hypoxic adaptation throughout Drosophila melanogaster development. We find that hypoxic-induced transcriptional changes are comprised of HIF-dependent and HIF-independent pathways that are distinct and separable. We show that normoxic set-points of carbohydrate metabolites are significantly altered in sima mutants and that these animals are unable to mobilize glycogen in hypoxia. Furthermore, we find that the estrogen-related receptor (dERR), which is a global regulator of aerobic glycolysis in larvae, is required for a competent hypoxic response. dERR binds to dHIFa and participates in the HIF-dependent transcriptional program in hypoxia. In addition, dERR acts in the absence of dHIFa in hypoxia and a significant portion of HIF-independent transcriptional responses can be attributed to dERR actions, including upregulation of glycolytic transcripts. These results indicate that competent hypoxic responses arise from complex interactions between HIF-dependent and -independent mechanisms, and that dERR plays a central role in both of these programs.

Avian-Induced Pluripotent Stem Cells Derived Using Human Reprogramming Factors

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.

Indicted : Worms caught using steroids

Three recent papers provide new insights into endocrinology in the worm Caenorhabditis elegans. These studies identify natural steroid ligands for the DAF-12 nuclear receptor, define a new enzyme in the hormone biosynthetic pathway, and clarify the role of endocrine signaling in adult longevity.

Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism

Significance The ubiquitin–proteasome system (UPS) is required for Drosophila class IV dendritic arborization neuron dendrite pruning. We found that mutants in the ubiquitylation machinery, the ubiquitin-dependent chaperone Valosin-Containing Protein (VCP), and the 19S regulatory particle of the proteasome—but not the 20S core particle—showed defects in pruning gene expression and mislocalization or overexpression of specific mRNA-binding proteins. In the case of VCP inhibition, we were able to detect a specific change in the splicing pattern of a pruning gene that likely contributes to pruning defects. A link between VCP and mRNA-binding proteins had been observed in the context of human neurodegenerative disease. This study implicates a specific function of VCP and ubiquitin in mRNA metabolism. The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process—also known as dendrite pruning—depends on the ubiquitin–proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. Here, we show that mutation or inhibition of Valosin-Containing Protein (VCP), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, we find that VCP inhibition causes an altered splicing pattern of the large pruning gene molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR–DNA-binding protein of 43 kilo-Dalton (TDP-43). Our data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, our results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism.

Molecular Characterization of Histomonas meleagridis and Other Parabasalids in the United States Using the 5.8S, ITS-1, and ITS-2 rRNA Regions

abstract:  Extracted DNA from 28 Histomonas meleagridis-infected avian tissue samples from multiple hosts and geographic locations was analyzed for variation in the 5.8S rRNA and the flanking internal transcribed spacer regions (ITS 1 and ITS 2). Samples were amplified by polymerase chain reaction, sequenced, and compared with known sequences from GenBank accessions of H. meleagridis and other related protozoa. The analyses revealed significant genetic variation within H. meleagridis sequences and suggested the possibility of multiple genotypes within the samples or a possible misdiagnosis. Related protozoa found in some samples were mostly identified as Tetratrichomonas spp. However, 1 sample had a 93% identity to Simplicimonas similis, a newly described organism, suggesting the possibility of a new pathogen in poultry. A phylogenetic tree analyzing the 5.8S and flanking ITS regions was inconclusive and we were unable to resolve all H. meleagridis into a single grouping. In contrast, a tree constructed only on the 5.8S rRNA grouped all but 1 H. meleagridis sample into 1 clade, including GenBank accessions submitted from Europe. This suggests that the 5.8S region alone is more reliable in identifying H. meleagridis than are the combined 5.8S and flanking ITS regions. There was no correlation between genotypes and host species or geographic location, suggesting that H. meleagridis moves freely between multiple avian species in the sampled regions.

A Genetic Screen Identifies New Regulators of Steroid-Triggered Programmed Cell Death in Drosophila

The steroid hormone ecdysone triggers the rapid and massive destruction of larval tissues through transcriptional cascades that culminate in rpr and hid expression and caspase activation. Here we describe the use of genetic screens to further our understanding of this steroid-triggered programmed cell death response. Pupal lethal mutants were screened for specific defects in larval salivary gland destruction. A pilot screen using existing P-element collections resulted in the identification of mutations in known cell death regulators, E74 and hid, as well as multiple alleles in CBP (nejire) and dTrf2. A large-scale EMS mutagenesis screen on the third chromosome resulted in the recovery of 48 mutants. These include seven multiallelic complementation groups, at least five of which do not map to regions or genes previously associated with cell death. Five mutants display defects in the transcriptional induction of rpr and hid, and all display a penetrant block in caspase activation. Three were mapped to specific genes: CG5146, which encodes a protein of unknown function, Med24, which encodes a component of the RNA polymerase II mediator complex, and CG7998, which encodes a putative mitochondrial malate dehydrogenase. These genetic screens provide new directions for understanding the regulation of programmed cell death during development.

Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species

Birds display remarkable diversity in the distribution and morphology of scales and feathers on their feet, yet the genetic and developmental mechanisms governing this diversity remain unknown. Domestic pigeons have striking variation in foot feathering within a single species, providing a tractable model to investigate the molecular basis of skin appendage differences. We found that feathered feet in pigeons result from a partial transformation from hindlimb to forelimb identity mediated by cis-regulatory changes in the genes encoding the hindlimb-specific transcription factor Pitx1 and forelimb-specific transcription factor Tbx5. We also found that ectopic expression of Tbx5 is associated with foot feathers in chickens, suggesting similar molecular pathways underlie phenotypic convergence between these two species. These results show how changes in expression of regional patterning genes can generate localized changes in organ fate and morphology, and provide viable molecular mechanisms for diversity in hindlimb scale and feather distribution. DOI: http://dx.doi.org/10.7554/eLife.12115.001

Sox14 is required for transcriptional and developmental responses to 20-hydroxyecdysone at the onset of drosophila metamorphosis

The steroid hormone 20‐hydroxyecdysone (20E), by means of a heterodimer consisting of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (Usp), triggers the major developmental transitions in the Drosophila life cycle through the regulation of genetic hierarchies. We have previously demonstrated that the Sox14 transcription factor is a primary response gene to 20E/EcR/Usp complex. In this study, we show that mutations in sox14 result in prepupal and pupal lethality with animals displaying a multitude of defects in 20E developmentally regulated pathways. In addition, through Northern blot and microarray analyses of sox14 mutant animals, we demonstrate that Sox14 is required for the proper expression of 20E‐ and non–20E‐regulated genes at the onset of metamorphosis. We also show that the Sox14‐regulated gene set correlates well with Sox14 expression in a variety of larval and adult tissues. Thus, Sox14 is a critical transcription factor required for 20E signaling at the onset of metamorphosis. Developmental Dynamics 239:2685–2694, 2010.

Diagnosis of Parelaphostrongylus spp. infection as a cause of meningomyelitis in calves

Migration of Parelaphostrongylus spp. has been documented to cause central nervous system damage in a number of aberrant host species but appears to be uncommon in cattle. The current report describes the clinical and laboratory findings, antemortem and definitive diagnosis, and response to treatment of Parelaphostrongylus spp. infection in five 3–7- month-old Limousin calves from 2 farms. All calves had signs of acute (n = 2) and chronic (n = 3) progressive spinal cord dysfunction. Cerebrospinal fluid analysis revealed a marked eosinophilic (acute cases) or lymphocytic (chronic cases) pleocytosis and elevated protein in all calves. A necropsy and histopathologic evaluation was performed on 2 euthanized calves, and histopathology revealed lymphoplasmacytic and eosinophilic meningomyelitis with multiple intradural and intramedullary expansile hyperplastic lymphoid nodules containing germinal centers and nematode fragments. DNA sequencing was performed on nested polymerase chain reaction products amplified with parasite-specific primers obtained from formalin-fixed and frozen spinal cord; PCR products from these 2 calves were 100% identical to Parelaphostrongylus species on DNA sequencing, confirming the diagnosis. Surviving calves rapidly improved following treatment with anthelmintics and corticosteroids. This case series identified Parelaphostrongylus spp. (likely P. tenuis) as a cause of spinal cord disease in calves and highlights the need for vigilance against aberrant parasite migration in calves grazing wet, snail-infested pastures. Cerebrospinal fluid eosinophilia is useful for supporting an antemortem diagnosis of Parelaphostrongylus in calves with acute neurologic disease; however, a lymphocytosis is observed in chronic or treated cases.