I use zebrafish as a model system, in combination with molecular approaches, to answer questions focused on neural development. My laboratory consists of three research projects that broadly examine the role of neurotransmitter systems, specifically focused on glycine and neuronal nicotinic acetylcholine receptors.
Background for Project 1 and 2: Approximately 20% of pregnant women in the western world continue to smoke despite a number of fetal complications linked with cigarette smoking such as premature birth, low-birth-weight infants, stillbirth and infant death, in addition to, long-term anatomical, behavioral and cognitive deficits. Nicotine mediates its actions through nicotinic acetylcholine receptors, nAChRs (chrnas in zebrafish), which are widely distributed throughout the body. In various animal models, exposure to nicotine during fetal development has resulted in decreased size and weight, cognitive and behavior abnormalities, changes in motor behavior, reduced eye growth, cataracts, delayed eye opening, and abnormal sensory function. It remains unclear as to how specific nAChR subtypes function during normal developmental processes and how exposing the fetus to chronic nicotine affects fetal development at the cellular level.
Project 1: It has been established that exposure of zebrafish to low concentrations of nicotine induces abnormal motor neuron development in the spinal cord and that high concentrations of nicotine induce paralysis. My laboratory will be focused on chrna2 (Figure 1), chrna4, chrna6 as candidate genes involved in spinal cord development. We will employ PCR-based cloning, in situ hybridization to localized mRNA expression, and morpholino-mediated gene knockdown to determine which gene(s) is/are associated with motor neuron pathfinding and swim behavior.
Figure 1: Colormetric In situ hybridization analysis of chrna2 mRNA expression (purple) reveals chrna2 in hindbrain and spinal cord at both 24 (left column) and 48 hpf (right column) using Islet 1 (brown/red) as a motor neuron marker in the brain and spinal cord (A,B,E,F). Fluorescent in situ hybridization of chrna2 (red) paired with immunohistochemistry reveals that chrna2 is not localized to Zn12-positive rohon beard sensory neurons (C,G: green) or HB9-positive motor neurons (D,H: green).
Project 2: The neuronal nicotinic receptor subtype chrna6 is expressed in ganglion and amacrine cells of the developing zebrafish retina (Figure 2). It has been established in developing animals that nicotine exposure reduces eye size, but alterations at the cellular level have not yet been described. We will take a pharmacological approach to establish the concentration of nicotine necessary to reduce eye size in the zebrafish. Next, using a gene knockdown approach we will examine the role of chrna6 in basic cellular mechanisms such as cell death, proliferation, differentiation, progenitor cell formation, migration, etc.
Figure 2: In situ hybridization analysis of chrna6 RNA expression (purple) in 48 hours post fertilization (hpf) embryos reveals chrna6 in the pineal gland (pin), tectum (tec), trigeminal ganglion (tg), diencephalic catecholaminergic cluster (dcc) in the midbrain, locus coeruleus, and the retinal ganglion cells (rgc) in the eye.
Background Project 3: Approximately 285 million individuals worldwide are visually impaired, of these 39 million are blind. The leading cause of late-stage visual impairments involves permanent photoreceptor cell death and limited treatments exist to cure degeneration/blindness. Efforts in regenerative medicine are focused on either: 1) implanting exogenous cells/tissues (retinal progenitors, rod-like cells, or retinal graphs) that must integrate and replace the lost photoreceptor cells or 2) the induction of the remaining endogenous neuronal progenitors or photoreceptors to produce new cells. It is well documented in in vitro model systems using induced pluripotent stem cells (iPS), embryonic stem cells (ES) and developing retinal explants that taurine is critical for the differentiation of progenitor cells into rod or rod-like photoreceptors.
Project 3: Limited studies address the mechanisms by which taurine induces rod photoreceptor differentiation. Until recently, taurine-induced differentiation had not be demonstrated in an animal model. Using the developing zebrafish, I have determined that taurine-induced rod photoreceptor differentiation indeed occurs in vivo (Figure 3) and that photoreceptor differentiation is mediated via changes in the transcription factors, Ath5 and NeuroD. I have also determined that taurine signals through a glycine receptor subtype, not a taurine transporter, to induce rod photoreceptor generation. Using in situ hybridization, immunohistochemistry, gene knockdown and pharmacological approaches, my laboratory will be focused on determining which of the 7 glycine receptor subtypes (a1, a2, a3, a4a, a4b, ba, and bb) that taurine can signal through to induce this taurine-mediated differentiation process.
Figure 3: Transgenic zebrafish embryos labeling rod photoreceptors with GFP (green) treated with 30 mM taurine from 36 hpf to 72 hpf display a robust increase in the number of rod photoreceptors (H,K) evident in both whole mount larvae (G,H) and confocal retinal sections (J,K) relative to wild-type (WT) control larvae.