Pax8 regulates development of dopaminergic neurons in diencephalon

^○Takanori Ikenaga¹, Nichole Gebhart², Jason Urban¹, Koichi Kawakami³, Fumihito Ono¹

1) Laboratory of Molecular Physiology, NIH/NIAAA, Bethesda, MD, 2) Whitney Laboratory, University of Florida, St Augustine, FL, 3)National Institute of Genetics, Mishima, Shizuoka, Japan

  During vertebrate development, transcription factors exert a strict control on gene expression, both spatially and temporally. Pax genes comprise an important family of transcription factors involved in diverse aspects of development, such as cell specification, differentiation, growth, survival, migration and morphogenesis. Using a gene trap technique, we established a stable line of zebrafish in which the red fluorescent protein (RFP) trapped the pax8 gene. In this line, RFP insertion occurred in the first intron of the pax8 gene. Afusion transcript of pax8 and RFP is made under the control of the pax8 promoter, which gives rise to a functional RFP. At 1day post fertilization (dpf), a heterozygous embryo expresses RFP in the midbrain-hindbrain boundary (MBH) and the otic vesicle. RFP expression spreads to hindbrain, spinal cord, and retina after 2 dpf.
  Real-time PCR showed that the RFP insertion results in a significant knockdown of pax8 expression in homozygous embryos. Unlike morpholino studies in which knockdown of pax8 is transient, the knockdown in homozygous embryos continues throughout development. Despite the significant pax8 knockdown, homozygous fish can survive to adulthood. Gross morphology of brain and distribution of RFP expressing cells were intact in homozygous embryos. Otic vesicles were smaller compared to wild type at 1 dpf, a finding also observed in previous morpholino studies. This abnormal size of the otic vesicles persists up to 10 days, reflecting the lasting effect of pax8 knockdown.
  MBH is a key center in specifying the cell fate of a majority of dopaminergic neurons. We performed tyrosine hydroxylase immunohistochemistry to examine the effect of pax8 knockdown on the development of dopaminergic neurons. In pax2 or fgf8 mutants that show pronounced defects in MHB, certain populations of dopamenergic or noradrenergic neurons disappear. In spite of a normal MHB morphology in pax8 homozygous embryos, a subset of dopaminergic neurons in the ventral diencephalon were absent at 4 dpf. In contrast noradrenergic neurons were intact. A corresponding defect of dopaminergic neurons was also observed in adult brains. These missing dopaminergic neurons do not express pax8 in heterozygotes, suggesting the non cell-autonomous nature of the pax8 effect. This highly specific effect of pax8 on dopaminergic neurons indicates a new role of pax8 in the development of catecholaminergic neurons in vertebrate brains.