Gene Regulatory Network for Zebrafish Eye Development
Eye development is orchestrated by scores of genetic pathways and gene families that control cell proliferation, specification and differentiation processes. A thorough understanding of the structure of the network and its regulation provides the foundation for the investigation of abnormal processes that lead to human eye diseases. To this end, we study eye development in zebrafish by various experimental approaches.
The research interests of our lab are currently focused in two directions. First, we hope to identify components of the gene regulatory network that regulate zebrafish eye development by gene expression profiling. Gene expression levels in microdissected eye tissues including retina, retinal pigment epithelium, and lens from wild-type and eye mutant embryos at different stages of development are being measured by microarray analysis or next generation sequencing (Figure 1 and 2). Novel statistical and bioinformatic analyses are being built for the identification of the relevant gene regulatory network and gene families.
Figure 1. Histology of eye development in wild-type (WT) and yng mutant. The WT retinas in zebrafish develop rapidly and begin differentiation by day 1 (36hpf). The differentiating retinal neurons undergo terminal differentiation by day 2 (52hpf) and become functional at around day 3 (3dpf). A functional retina is a laminated neuronal tissue with the ganglion cell layer (GCL) on the inside, inner nuclear layer (INL) in the middle, and the outer nuclear layer (ONL) on the outside. In between these layers are the synaptic layers, the inner (IPL) and outer plexiform layer (OPL). Zebrafish retinas also retain a proliferative region called the marginal zone (MZ) throughout adulthood. The yng mutant (bottom row)carries a mutation in brg1, the ATPase of a chromatin remodeling complex. The retinas in yng fail to terminally differentiate and laminate. Scale bar = 50µm.
Figure 2. Microarray analysis of zebrafish eye development. Our laboratory studies expression profiles in different zebrafish eye tissues by microarray analysis. The image shows a small section of an Affymetrix Zebrafish Genome Array that can analyze expression levels of approximately 15,000 genes in one experiment.
“Second, we hope to investigate the connectivity of these candidate genes to elucidate the sub-circuits of the gene regulatory network, as well as the relationship of the sub-circuits to the eye developmental processes. The sub-circuits are being identified by methodologies including Chromatin Immunoprecipitation (ChIP) – microarrays and/or sequencing analyses (ChIP-Chip or ChIP-Seq). The identified sub-circuits including signal transduction pathways, transcription factor and cell cycle regulator families will be characterized by various genetic, biochemical, molecular, cellular and histological techniques (Figure 3).”
Figure 3. Characterization of eye developmental pathways. The microarray analysis of zebrafish eye tissues reveals interesting signaling pathways and transcription factor families that give insights on the subcuits of the gene regulatory networks of eye development. For example, we have identified candidate genes in the yng retinas for the deregulation of cellular differentiation (shown by the decrease in the green immunostaining domain for the zn5 antigen, a GC marker), as well as the deregulation of cell cycle control (shown by the increase in the red immunostaining domain for the pH3 stain, a mitotic cell maker). Our laboratory will elucidate the underlying gene regulatory network that controls these processes by various experimental tools.