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Vanderbilt Vision Training Seminar

Friday, February 01, 2019,

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  • Location: Vanderbilt Eye Institute (VEI) • 2311 Pierce Ave. • Nashville , TN 37232
  • Room: Elliott Conference Room

Amy Whitener, PhD

Postdoctoral Fellow (Kuchtey Lab)

"Investigation of ADAMTS10 in Retinal Development"

 Purpose: Mutations in the gene encoding ADAMTS10 (A Disintegrin And Metalloproteinase domain with ThromboSpondin type-1 motifs 10) cause glaucoma and a rare connective tissue disorder, Weill-Marchessani Syndrome, characterized by short stature.  ADAMS10 plays a role in the formation and function of microfibrils, which are components of the extracellular matrix that regulate activation of transforming growth factor β (TGFβ) signal transduction.  TGFβ signaling promotes development of retinal ganglion cells (RGCs) and likely plays a role in glaucoma pathogenesis. This study examines the role of adamts10 in RGC development and TGFβ signaling using zebrafish as a model organism.

 

Methods: Two fluorescent zebrafish lines were used; one which specifically labels RGCs with GFP expression driven by a brn3a promoter (brn3a-GFP) and the other which expresses GFP in response to TGFβ signaling driven by a smad-binding element (12xSBE-GFP).  Embryos at the one-cell stage were injected with a translation-blocking morpholino designed to knock down adamts10 expression, or a 5-bp mismatch control morpholino, or left un-injected.  Phenotypes were determined by microscopy at 2 days post fertilization.  A rescue experiment was performed by co-injecting adamts10 morpholino with normal ADAMTS10 mRNA or ADAMTS10 mRNA carrying the Gly661Arg mutation, previously shown to cause glaucoma in dogs.

Results: The body length of adamts10 morpholino-injected embryos was much shorter than controls, while eye diameter was less affected.  Injection of adamts10 morpholino in Brn3a-GFP embryos resulted in reduction of GFP in the RGC layer of the retina and in the optic nerve.   adamts10 morpholino-injected 12XSBE-GFP displayed reduced GFP fluorescence in the retina.  Normal ADAMTS10 mRNA prevented the short body phenotype and restored RGC development, while Gly661Arg ADAMTS10 mRNA did not.

Conclusions: These data suggest that adamts10 is required for normal RGC development and TGFβ signaling in the retina.  The glaucoma-causing mutation of ADAMTS10 has functional impact on its developmental roles.  To investigate further the impact of glaucoma-causing mutations, adamts10 mutant lines will be generated using CRISPR-Cas9.