Alternative Splicing and Nonsense-Mediated Decay of a Zebrafish GABA Receptor Subunit Transcript.

The superfamily of Cys-loop ionotropic neurotransmitter receptors includes those that detect GABA, glutamate, glycine, and acetylcholine. There is ample evidence that many Cys-loop receptor subunit genes include alternatively spliced exons. In this study, we report a novel example of alternative splicing (AS): we show that the 68-bp exon 3 in the zebrafish gabrr2b gene-which codes for the ρ2b GABAAR subunit-is an alternative cassette exon. Skipping of gabrr2b exon 3 results in a downstream frame shift and a premature termination codon (PTC). We provide evidence in larval zebrafish that transcripts containing the PTC are subject to degradation through nonsense-mediated decay. We also compile reports of AS of homologous exons in other Cys-loop receptor genes in multiple species. Our data add to a large body of research demonstrating that exon 3 in Cys-loop receptor genes is a conserved site for AS, the effects of which can vary from novel splice-isoform generation to downregulation of gene expression through transcript degradation.

Modulating the Zebrafish Camouflage Response Pathway to Illustrate Neuroendocrine Control Over a Robust and Quantifiable Behavior.

Zebrafish detect the light levels of their surroundings and adjust their coloration in response. By controlling the location of melanosome pigment granules within melanocytes in their dermis, fish can lighten or darken their appearance to blend in with their environment. This camouflage response pathway, which begins in the retina and ends in the melanocyte, involves both neuronal and endocrine signaling. Ultimately, two hormones, α-melanocyte stimulating hormone and melanin concentrating hormone, converge on the melanocyte and cause dispersion or aggregation of melanosomes, respectively; the camouflage behavior can therefore be modulated both environmentally and pharmacologically. Here, we describe a two-part protocol designed for use in an undergraduate laboratory. Students induce the camouflage response by exposing zebrafish larvae to darkness or bright light, in conjunction with pharmacological treatments that alter the ability of the larvae to properly respond to these environmental cues. Students then fix the larvae, take photographs of their samples using their smartphones and dissecting microscopes, and directly measure the camouflage response by quantifying the size of melanocytes using ImageJ software. Finally, students present their data in a single professional-quality figure with an accompanying detailed figure legend. This protocol enables students to gain unique laboratory experiences in which they modulate and quantify a hormone-driven behavior, observable on a cellular level. It can therefore complement course topics in neurobiology, endocrinology, animal physiology, animal behavior, and cell biology classes.