PUBLICATION

Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino

Authors
O'Connor, M.J., Beebe, L.L., Deodato, D., Ball, R.E., Page, A.T., VanLeuven, A.J., Harris, K.T., Park, S., Hariharan, V., Lauderdale, J.D., Dore, T.M.
ID
ZDB-PUB-180913-8
Date
2018
Source
ACS Chemical Neuroscience   10(1): 266-278 (Journal)
Registered Authors
Ball, Rebecca, Lauderdale, James D.
Keywords
none
MeSH Terms
  • Animals
  • Zebrafish
  • Glutamate Decarboxylase/genetics*
  • Glutamate Decarboxylase/metabolism
  • Morpholinos/antagonists & inhibitors*
  • Morpholinos/genetics*
  • Morpholinos/metabolism
  • Craniofacial Abnormalities/enzymology*
  • Craniofacial Abnormalities/genetics*
  • Craniofacial Abnormalities/pathology
  • Microinjections
(all 11)
PubMed
30200754 Full text @ ACS Chem. Neurosci.
Abstract
γ-Amino butyric acid (GABA) mediated signaling is critical in the central and enteric nervous systems, pancreas, lungs and other tissues. It is associated with many neurological disorders and craniofacial development. Glutamic acid decarboxylase (GAD) synthesizes GABA from glutamate, and knockdown of the gad1 gene results in craniofacial defects that are lethal in zebrafish. To bypass this and enable observation of the neurological defects resulting from knocking down gad1 expression, a photoactivatable morpholino oligonucleotide (MO) against gad1 was prepared by cyclization with a photocleavable linker rendering the MO inactive. The cyclized MO was stable in the dark and toward degradative enzymes and was completely linearized upon brief exposure to 405-nm light. In the course of investigating the function of the ccMOs in zebrafish, we discovered that zebrafish possess paralogous gad1 genes, gad1a and gad1b. A gad1b MO injected at the 1-4 cell stage caused severe morphological defects in head development, which could be bypassed, enabling the fish to develop normally, if the fish were injected with a photoactivatable, cyclized gad1b MO and grown in the dark. At 1 dpf, light activation of the gad1b MO followed by observation at 3 and 7 dpf led to increased and abnormal electrophysiological brain activity compared to wild type animals. The photocleavable linker can be used to cyclize and inactivate any MO, and represents a general strategy to parse the function of developmentally important genes in a spatiotemporal manner.
Genes / Markers
Figures
Figure Gallery (3 images)
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Expression
Phenotype
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
ba2TgTransgenic Insertion
    ga2303
      Small Deletion
      1 - 2 of 2
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      Human Disease / Model
      Sequence Targeting Reagents
      Target Reagent Reagent Type
      gad1bCRISPR2-gad1bCRISPR
      gad1bMO1-gad1bMRPHLNO
      gad1bMO2-gad1bMRPHLNO
      gad2MO1-gad2MRPHLNO
      gad2MO2-gad2MRPHLNO
      1 - 5 of 5
      Show
      Fish
      Antibodies
      No data available
      Orthology
      No data available
      Engineered Foreign Genes
      Marker Marker Type Name
      EGFPEFGEGFP
      1 - 1 of 1
      Show
      Mapping
      No data available