ABSTRACT
Standard toxicological assays using the zebrafish model system evaluate lethality and teratogenicity upon exposure during the first 2 days after fertilization. We tested the biological effects of several widely used drugs on zebrafish by acute treatment for 24 h starting at late embryonic stages, between 48 and 72 h post-fertilization. For 4 out of 6 compounds, we observed a higher sensitivity of late stage zebrafish embryos for general toxicity (lethality) compared to younger embryos. Morphological defects such as edema, body curvature, delayed growth, decreased heart rate and locomotion were observed for each of the compounds tested, often at sublethal concentrations. Gene expression studies on a set of four selected genes revealed a specific regulatory pattern for the different compounds tested. Our results allow us to compare various toxicological endpoints and may contribute to the design of a rational high throughput approach using the zebrafish model.
Subject(s)
Embryo, Nonmammalian/drug effects , Psychotropic Drugs/toxicity , Water Pollutants, Chemical/toxicity , Zebrafish/embryology , Animals , Behavior, Animal/drug effects , Caffeine/toxicity , Carbamazepine/toxicity , Gene Expression Regulation, Developmental/drug effects , Heart Rate/drug effects , Lithium Chloride/toxicity , Motor Activity/drug effects , Pentobarbital/toxicity , Theophylline/toxicity , Toxicity Tests , Valproic Acid/toxicity , Zebrafish/physiologyABSTRACT
The pituitary is a complex gland comprising different cell types each secreting specific hormones. The extensive network of signaling molecules and transcription factors required for determination and terminal differentiation of specific cell types is still not fully understood. The SRY-like HMG-box (SOX) transcription factor Sox4 plays important roles in many developmental processes and has two homologs in zebrafish, Sox4a and Sox4b. We show that the sox4b gene is expressed in the pituitary anlagen starting at 24 h after fertilization (hpf) and later in the entire head region including the pituitary. At 48 hpf, sox4b mRNA colocalizes with that for TSH (tshß), glycoprotein subunit α (gsuα), and the Zn finger transcription factor Gata2a. Loss of Sox4b function, using morpholino knockdown or expression of a dominant-negative Sox4 mutant, leads to a drastic decrease in tshß and gsuα expression and reduced levels of gh, whereas other anterior pituitary gland markers including prl, slß, pomc, and lim3 are not affected. Sox4b is also required for expression of gata2a in the pituitary. Knockdown of gata2a leads to decreased tshß and gsuα expression at 48 hpf, similar to sox4b morphants. Injection of gata2a mRNA into sox4b morphants rescued tshß and gsuα expression in thyrotrope cells. Finally, sox4b or gata2a knockdown causes a significant decrease of gonadotropin expression (lhß and fshß) at 4 d after fertilization. In summary, our results indicate that Sox4b is expressed in zebrafish during pituitary development and plays a crucial role in the differentiation of thyrotrope and gonadotrope cells through induction of gata2a expression in the developing pituitary.
