Gill histopathologies following exposure to nanosilver or silver nitrate.

Fish gill is the site for many crucial physiological functions. It is among the first sites of xenobiotic exposure, and gill histopathological alterations may be detected soon after toxicant exposure. Silver (Ag) is one of the most toxic metals to aquatic organisms mainly due to its ability to disrupt ionic regulation. The goal of this study was to determine the effect of ionic and nanoscale Ag on fathead minnow gills by examining gill histology and Na(+)/K(+)-ATPase immunoreactivity. Fathead minnows were exposed to two measured concentrations of silver nitrate (AgNO3: 1.3 or 3.7 µg/L as Ag(+)), citrate silver nanoparticles (citrate-AgNP: 15 or 39 µg/L), and polyvinylpyrrolidone-AgNP (PVP-AgNP) (AgNP: 11 or 50 µg/L). Circulatory disturbances were the most prevalent gill alterations detected and were significantly increased in all Ag treatment groups compared to control. AgNO3 (1.3 µg/L) was the only treatment that significantly elevated the number of total mucous goblet cells present. In all other Ag treatments, the percent of degenerated goblet cells was significantly increased compared to control. When the sum of all histopathological abnormalities (weighted index) was calculated, all Ag groups displayed a significantly higher index, with citrate-AgNP having the highest toxicity (index of 10 ± 0.32 versus 2.4 ± 0.6 in controls). Gill Na(+)/K(+)-ATPase immunoreactivity was decreased by Ag. These results indicated that both AgNO3 and AgNP created similar disruptions in gill structure and ionic regulation, possibly due to the ionic Ag portion of each treatment.

Characterization of missense alleles of the glial cells missing gene of Drosophila.

Glial cells missing (Gcm) is the primary regulator of glial cell fate in Drosophila. Gcm belongs to a small family of transcriptional regulators involved in fundamental developmental processes found in diverse animal phyla including vertebrates. Gcm proteins contain the highly conserved DNA-binding GCM domain, which recognizes an octamer DNA sequence. To date, studies in Drosophila have primarily relied on gcm alleles caused by P-element induced DNA deletions at the gcm locus, as well as a null allele caused by a single base pair substitution in the GCM domain that completely abolishes DNA binding. Here I characterize two hypomorphic missense alleles of gcm with intermediate glial cells missing phenotypes. In embryos homozygous for either of these gcm alleles the number of glial cells in the central nervous cystem (CNS) is reduced approximately in half. Both alleles have single amino acid changes in the GCM domain. These results suggest that Gcm protein activities in these mutant alleles have been attenuated such that they are operating at threshold levels, and trigger glial cell differentiation in neural precursors in the CNS in a stochastic fashion. These hypomorphic alleles provide additional genetic resources for understanding Gcm functions and structure in Drosophila and other species.

Characterization of cis-regulatory elements controlling repo transcription in Drosophila melanogaster.

The glial cells missing (gcm) gene has been identified as a "master regulator" of glial cell fate in the fruit fly Drosophila. However, gcm is also expressed in and required for the development of larval macrophages and tendon cells. Thus, the Gcm protein activates the transcription of different sets of genes in different developmental contexts. How the Gcm protein regulates these different outcomes is not known. Our goal is to identify proteins that collaborate with Gcm to promote the transcriptional activation of Gcm target genes specifically in glial cells, or prevent their activation in the other tissues in which Gcm is expressed. To address this, we have focused on the transcriptional regulation of a well-characterized glial-specific Gcm target gene, the transcription factor reversed polarity (repo). We aim to understand how the transcription of the glial-specific Gcm target gene repo is regulated by Gcm and other factors. Previously we defined a 4.3 kb cis-regulatory DNA region that recapitulates the endogenous Repo expression pattern dependent on multiple Gcm binding sites. We proposed that there may be multiple cis-regulatory sub-regions that drive cell-specific expression independent of Gcm binding sites. Here, using lacZ reporter activity in transgenic lines, we have characterized three cis-regulatory elements: 1) a distal element that promotes expression in dorsolateral epidermis; 2) a repressor element that suppresses expression in the epidermis; and, 3) a proximal element that promotes expression in a subset of cell body glia. Most significantly, we have defined a minimal cis-regulatory element that recapitulates the endogenous repo expression pattern dependent on a single Gcm binding site.

Transcriptional regulation of the Drosophila glial gene repo.

reversed polarity (repo) is a putative target gene of glial cells missing (gcm), the primary regulator of glial cell fate in Drosophila. Transient expression of Gcm is followed by maintained expression of repo. Multiple Gcm binding sites are found in repo upstream DNA. However, while repo is expressed in Gcm positive glia, it is not expressed in Gcm positive hemocytes. These observations suggest factors in addition to Gcm are required for repo expression. Here we have undertaken an analysis of the cis-regulatory DNA elements of repo using lacZ reporter activity in transgenic embryos. We have found that a 4.2 kb DNA region upstream of the repo start site drives the wild-type repo expression pattern. We show that expression is dependent on multiple Gcm binding sites. By ectopically expressing Repo, we show that Repo can regulate its own enhancer. Finally, by systematically analyzing fragments of repo upstream DNA, we show that expression is dependent on multiple elements that are responsible for activity in subsets of glia, as well as repressing inappropriate expression in the epidermis. Our results suggest that Gcm acts synergistically with other factors to control repo transcription in glial cells.

Transcriptional control of glial cell development in Drosophila.

Neurons and glia are generated from multipotent neural progenitors. In Drosophila, the transcriptional regulation of glial vs. neuronal fates is controlled by the expression of the transcription factor encoded by the glial cells missing gene (gcm) in multiple neural lineages. The cis-regulatory control of gcm transcription serves as a nodal point to translate a complex array of spatially and temporally regulated transcription factors in distinct neural lineages into glial-specific expression. Gcm acts synergistically with several downstream transcription factors to initiate and maintain glial-specific gene expression. The identification of a large set of glial-specific genes through the application of computational and whole genome tools provides the opportunity to analyze the transcriptional regulation of glial cell development at the genomic level in a relatively simple genetic model system.

Transcriptional control of glial and blood cell development in Drosophila: cis-regulatory elements of glial cells missing.

In Drosophila, glial cell differentiation requires the expression of glial cells missing (gcm) in multiple neural cell lineages, where gcm acts as a binary switch for glial vs. neuronal fate. Thus, the primary event controlling gliogenesis in neural progenitors is the transcription of gcm. In addition, gcm is also required for the differentiation of macrophages, and is expressed in the hemocyte lineage. This dual role of gcm in glial cell and blood cell development underscores the need for the precise temporal and spatial regulation of gcm transcription. To understand how gcm transcription is regulated, we have undertaken an analysis of the cis-regulatory DNA elements of gcm using lacZ reporter activity in transgenic embryos, testing the activity of approximately 35 kilobases of DNA from the gcm locus. We have identified several distinct DNA regions that promote most of the elements of gcm expression. These include elements for general neural expression, gcm-independent and gcm-dependent glial-specific expression, as well as early and late hemocyte expression. We show that expression of an abdominal glial-specific element is dependent on the homeotic gene abdominal-A. Our results indicate that gcm transcription is controlled by a combination of general and lineage-specific elements, positive autoregulation, and neuronal repression.

gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila.

glial cells missing (gcm) is the primary regulator of glial cell fate in Drosophila. In addition, gcm has a role in the differentiation of the plasmatocyte/macrophage lineage of hemocytes. Since mutation of gcm causes only a decrease in plasmatocyte numbers without changing their ability to convert into macrophages, gcm cannot be the sole determinant of plasmatocyte/macrophage differentiation. We have characterized a gcm homolog, gcm2. gcm2 is expressed at low levels in glial cells and hemocyte precursors. We show that gcm2 has redundant functions with gcm and has a minor role promoting glial cell differentiation. More significant, like gcm, mutation of gcm2 leads to reduced plasmatocyte numbers. A deletion removing both genes has allowed us to clarify the role of these redundant genes in plasmatocyte development. Animals deficient for both gcm and gcm2 fail to express the macrophage receptor Croquemort. Plasmatocytes are reduced in number, but still express the early marker Peroxidasin. These Peroxidasin-expressing hemocytes fail to migrate to their normal locations and do not complete their conversion into macrophages. Our results suggest that both gcm and gcm2 are required together for the proliferation of plasmatocyte precursors, the expression of Croquemort protein, and the ability of plasmatocytes to convert into macrophages.