The Halpern laboratory uses genetic approaches in the zebrafish to study how signaling pathways regulate the differentiation and patterning of the central nervous system. Previous work in collaboration with Chris Wright's group at the Vanderbilt Medical School led to the identification of the zebrafish cyclops (cyc) gene as encoding a nodal-related signaling factor. In addition to understanding how Cyclops activity in the early embryo promotes formation of the ventral midline of the neural tube, the lab has explored the function of this signaling pathway in regulating left-right differences in the brain.

It is well known that the nodal pathway mediates the asymmetric positioning and morphology of visceral organs in vertebrate embryos, such as the left-sided placement and characteristic looping of the developing heart tube. However, this is the first time this pathway has been implicated in asymmetry of the vertebrate forebrain. Since so little is known about the mechanisms underlying the characteristic anatomical and functional differences between the left and right sides of the brain, the zebrafish work is providing new insights into this intriguing neurobiological question. Comparative studies on orthologous genes of other vertebrates, such as frogs and mice, will further reveal whether asymmetric activity of the nodal pathway is a conserved feature of brain patterning.

In the zebrafish, genes encoding cyc, antivin (a presumed antagonist of Cyc), and pitx2 (a transcription factor that is an effector of Nodal-related signaling) are all transiently expressed on the left side of the dorsal diencephalon (Figure X-A). Postdoctoral fellow Jennifer Liang determined that this transcriptional asymmetry localizes to the presumptive epiphysis or pineal organ and is regulated by some of the same processes that maintain asymmetry of the visceral organs. In the brains of adult fish that lacked left-sided gene expression as embryos, the pineal stalk is displaced from its normal left-medial position (Figure X-B). This suggests that nodal function is required for correct positioning of the developing epiphysis as it grows out from the roof of the diencephalon.

Postdoctoral fellow Joshua Gamse has been examining additional regions of asymmetry in the zebrafish forebrain and has begun to construct transgenic zebrafish lines, which will be essential tools for exploring how these asymmetries arise. With the green fluorescent protein (GFP) under the control of specific promoter/enhancers, discrete regions of the brain can be labeled and the fates of cells followed. Moreover, such GFP transgenic strains of zebrafish will be extremely useful for performing genetic screens to identify other genes that regulate left-right asymmetry in the forebrain.

Other projects in the laboratory include an ongoing effort in genomics to isolate and collect mapped deletions that uncover zebrafish chromosomal regions. With the imminent sequencing of the entire zebrafish genome, these will be valuable tools for assessing gene function.

A. Frontal view of the zebrafish embryo with the eyes (e) indicated. The floating head gene is expressed on both sides of the embryonic forebrain, in the presumptive eiphysis or pineal organ (red); however, the pitx2 gene, while expressed on both sides of the brain ventrally, is only expressed on the left side dorsally (blue; arrowhead).
B. In the adult, floating head expression persists in the stalk of the pineal organ (arrowhead), which in normal fish typically emerges from the dorsal diencephalon at a left to medial position. Whole-mount RNA in situ hybridization was performed on the dissected adult brain.