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

Lindsey L. Beebe, Davide Deodato, Rebecca E. Ball, A. Tyler Page, Ariel J. Vanleuven, Kyle T. Harris, Sungdae Park, Vani Hariharan, James D. Lauderdale, Timothy Dore, Matthew O'Connor

Research output: Contribution to journalArticle

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 day post fertilization (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.

Original languageEnglish (US)
JournalACS Chemical Neuroscience
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

Morpholinos
Glutamate Decarboxylase
Defects
Zebrafish
Genes
Fertilization
Fish
gamma-Aminobutyric Acid
Fishes
Observation
Gene Knockdown Techniques
Enteric Nervous System
Light
Wild Animals
Butyric Acid
Cyclization
Neurology
Nervous System Diseases
Glutamic Acid
Pancreas

Keywords

  • (8-bromo-7-hydroxyquinolin-2-yl)methyl
  • (8-cyano-7-hydroxyquinolin-2-yl)methyl
  • GABAergic signaling
  • Glutamic acid decarboxylase
  • photoactivated morpholino
  • γ-amino butyric acid

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Cognitive Neuroscience
  • Cell Biology

Cite this

Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino. / Beebe, Lindsey L.; Deodato, Davide; Ball, Rebecca E.; Page, A. Tyler; Vanleuven, Ariel J.; Harris, Kyle T.; Park, Sungdae; Hariharan, Vani; Lauderdale, James D.; Dore, Timothy; O'Connor, Matthew.

In: ACS Chemical Neuroscience, 01.01.2018.

Research output: Contribution to journalArticle

Beebe, Lindsey L. ; Deodato, Davide ; Ball, Rebecca E. ; Page, A. Tyler ; Vanleuven, Ariel J. ; Harris, Kyle T. ; Park, Sungdae ; Hariharan, Vani ; Lauderdale, James D. ; Dore, Timothy ; O'Connor, Matthew. / Bypassing Glutamic Acid Decarboxylase 1 (Gad1) Induced Craniofacial Defects with a Photoactivatable Translation Blocker Morpholino. In: ACS Chemical Neuroscience. 2018.
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