Structural differences between the left and right sides of the brain exist throughout the vertebrate lineage. By studying the zebrafish pineal complex, which exhibits notable asymmetries, both the genes and the cell movements that result in left-right differences can be characterized. The pineal complex consists of the midline pineal organ and the left-sided parapineal organ. The parapineal is responsible for instructing the asymmetric architecture of the bilateral habenulae, the brain nuclei that flank the pineal complex. Using in vivo time-lapse confocal microscopy, we find that the cells that form the parapineal organ migrate as a cluster of cells from the pineal complex anlage to the left side of the brain. In a screen for mutations that disrupted brain laterality, we identified a nonsense mutation in the T-box2b (tbx2b) gene, which encodes a transcription factor expressed in the pineal complex anlage. The tbx2b mutant makes fewer parapineal cells, and they remain as individuals near the midline rather than migrating leftward as a group. The reduced number and incorrect placement of parapineal cells result in symmetric development of the adjacent habenular nuclei. We conclude that tbx2b functions to specify the correct number of parapineal cells and to regulate their asymmetric migration.

Hypoxic signaling is a central modulator of cellular physiology in cancer. Core members of oxygen-sensing pathway including the von Hippel-Lindau tumor suppressor protein (pVHL) and the hypoxia inducible factor (HIF) transcription factors have been intensively studied, but improved organismal models might speed advances for both pathobiologic understanding and therapeutic modulation. To study HIF signaling during tumorigenesis and development in zebrafish, we developed a unique in vivo reporter for hypoxia, expressing EGFP driven by prolyl hydroxylase 3 (phd3) promoter/regulatory elements. Modulation of HIF pathway in Tg(phd3::EGFP) embryos showed a specific role for hypoxic signaling in the transgene activation. Zebrafish vhl mutants display a systemic hypoxia response, reflected by strong and ubiquitous transgene expression. In contrast to human VHL patients, heterozygous Vhl mice and vhl zebrafish are not predisposed to cancer. However, upon exposure to dimethylbenzanthracene (DMBA), the vhl heterozygous fish showed an increase in the occurrence of hepatic and intestinal tumors, a subset of which exhibited strong transgene expression, suggesting loss of Vhl function in these tumor cells. Compared with control fish, DMBA-treated vhl heterozygous fish also showed an increase in proliferating cell nuclear antigen-positive renal tubules. Taken together, our findings establish Vhl as a genuine tumor suppressor in zebrafish and offer this model as a tool to noninvasively study VHL and HIF signaling during tumorigenesis and development. Cancer Res; 72(16); 4017-27. Ó2012 AACR.