Identification of initially appearing glycine-immunoreactive neurons in the embryonic zebrafish brain

Moly, P.K., Ikenaga, T., Kamihagi, C., Tariqul Islam, A.F., and Hatta, K.
Developmental Neurobiology   74(6): 616-32 (Journal)
Registered Authors
Hatta, Kohei
GABA, GlyT2, MiD2cm, MiD3cm, electroporation, reticulospinal neuron
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • Axons/physiology
  • Brain/cytology*
  • Brain/embryology*
  • Choline O-Acetyltransferase/metabolism
  • Dextrans/metabolism
  • Electroporation
  • Embryo, Nonmammalian
  • Glycine/metabolism*
  • Glycine Plasma Membrane Transport Proteins/metabolism
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Microtubule-Associated Proteins/metabolism
  • Neural Pathways/physiology
  • Neurons/cytology
  • Neurons/metabolism*
  • RNA, Messenger/metabolism
  • Rhodamines/metabolism
  • Zebrafish
  • Zebrafish Proteins/metabolism
  • gamma-Aminobutyric Acid/metabolism
24318965 Full text @ Dev. Neurobiol.

Glycine is a major inhibitory neurotransmitter in the central nervous system of vertebrates. Here, we report the initial development of glycine-immunoreactive (Gly-ir) neurons and fibers in zebrafish. The earliest Gly-ir cells were found in the hindbrain and rostral spinal cord by 20 h post-fertilization (hpf). Gly-ir cells in rhombomeres 5 and 6 that also expressed glycine transporter 2 (glyt2) mRNA were highly stereotyped; they were bilaterally located and their axons ran across the midline and gradually turned caudally, joining the medial longitudinal fascicles in the spinal cord by 24 hpf. Gly-ir neurons in rhombomere 5 were uniquely identified, since there was one per hemisegment, whereas the number of Gly-ir neurons in rhombomere 6 were variable from one to three per hemisegment. Labeling of these neurons by single-cell electroporation and tracing them until the larval stage revealed that they became MiD2cm and MiD3cm, respectively. The retrograde labeling of reticulo-spinal neurons in Tg(glyt2:gfp) larva, which express GFP in Gly-ir cells, and a genetic mosaic analysis with glyt2:gfp DNA construct also supported this notion. Gly-ir cells were also distributed widely in the anterior brain by 27 hpf, whereas glyt2 was hardly expressed. Double staining with anti-glycine and anti-GABA antibodies demonstrated distinct distributions of Gly-ir and GABA-ir cells, as well as the presence of doubly immunoreactive cells in the brain and placodes. These results provide evidence of identifiable glycinergic (Gly-ir/glyt2-positive) neurons in vertebrate embryos, and they can be used in further studies of the neurons' development and function at the single-cell level.

Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes