Molecular evidence for a functional ecdysone signaling system in Brugia malayi.

BACKGROUND: Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle). METHODS AND FINDINGS: We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. CONCLUSIONS: Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

Strongylocentrotus drobachiensis oocytes maintain a microtubule organizing center throughout oogenesis: implications for the establishment of egg polarity in sea urchins.

Although it has been known for over a century that sea urchin eggs are polarized cells, very little is known about the mechanism responsible for establishing and maintaining polarity. Our previous studies of microtubule organization during sea urchin oogenesis described a cortical microtubule-organizing center (MTOC) present during germinal vesicle (GV) migration in large oocytes. This MTOC was localized within the future animal pole of the mature egg. In this study we have used electron microscopy and immunocytochemistry to characterize the structure of this MTOC and have established that this organelle appears prior to GV migration. We show that the cortical MTOC contains all the components of a centrosome, including a pair of centrioles. Although a centrosome proper was not found in small oocytes, the centriole pair in these cells was always found in association with a striated rootlet, a structural remnant of the flagellar apparatus present in precursor germinal cells (PGCs). The centrioles/striated rootlet complex was asymmetrically localized to the side of the oocyte closest to the gonadal wall. These data are consistent with the previously proposed hypothesis that in echinoderms the polarity of the PGCs in the germinal epithelium influences the final polarity of the mature egg.

Effects of ethanol on photoreceptors and visual function in developing zebrafish.

PURPOSE: Children born to mothers who have consumed alcohol during pregnancy have an array of retinal abnormalities and visual dysfunctions. In the past, rodent systems have been used to study the teratogenic effects of ethanol on vertebrate embryonic development. The exact developmental windows in which ethanol causes specific developmental defects have been difficult to determine because rodents and other mammals develop in utero. In this study, we characterized how ethanol affects the function and development of the visual system in an ex utero embryonic system, the zebrafish. METHODS: Zebrafish embryos were raised in fish water containing various concentrations of ethanol from 2 to 5 days after fertilization. The effects of ethanol on retinal morphology were assessed by histologic and immunohistochemical analyses and those on retinal function were analyzed by optokinetic response (OKR) and electroretinography (ERG). RESULTS: Zebrafish embryos exposed to moderate and high levels of ethanol during early embryonic development had morphological abnormalities of the eye characterized by hypoplasia of the optic nerve and inhibition of photoreceptor outer segment growth. Ethanol treatment also caused an increased visual threshold as measured by the OKR. Analysis with the ERG indicated that there was a severe reduction of both the a- and b-waves, suggesting that ethanol affects the function of the photoreceptors. Indeed, low levels of ethanol that did not cause obvious morphologic changes in either the body or retina did affect both the OKR visual threshold and the a- and b-wave amplitudes. CONCLUSIONS: Ethanol affects photoreceptor function at low concentrations that do not disturb retinal morphology. Higher levels of ethanol inhibit photoreceptor development and cause hypoplasia of the optic nerve.

Sphedgehog is expressed by pigment cell precursors during early gastrulation in Strongylocentrotus purpuratus.

We have sequenced the Sphedgehog (Sphh) gene from the sea urchin Strongylocentrotus purpuratus. Sphh transcripts are detected first at the mesenchyme blastula stage, and they accumulate throughout early embryogenesis. The Sphh protein is produced by precursor pigment cells during early and midgastrulation. NiCl2 inhibits pigment cell differentiation in sea urchins. Here, we show that, in S. purpuratus, nickel affects a process(es) between 17 and 24 hr of development, corresponding to the time period when Sphh mRNA is first detected. However, nickel treatment does not alter the early expression of Sphh.