Conditional Dync1h1 embryonic deletion and tamoxifen induced knockout in the adult mouse

Abstract

Purpose : Dynein 1 is a 1.4 MDa protein complex composed of a pair of force-generating heavy chains (DYNC1H1) and a set of non-catalytic accessory components termed intermediate, light intermediate and light chains. Cytoplasmic dynein moves cargo toward the minus end of microtubules at the basal body. Cargo includes mitochondria, membrane vesicles, lysosomes, and phagosomes. Mutations in the human DYNC1H1 gene cause spinal muscular atrophy with lower extremity predominance, Charcot-Marie-Tooth disease, and intellectual disability. Dync1h1 knockout mice do not survive past E9. Our purpose is to delete DYNC1H1 specifically in photoreceptors. We predict that loss of Dync1h1 in retinal progenitor cells will result in failure of protein trafficking and absence of outer segments. Additionally, we predict that loss of Dync1h1 in the adult retina will prevent the renewal of outer segments.

Methods : Dync1h1^tm1a(KOMP) ES cells containing a gene trap in intron 23 and a loxP site in intron 25 were used to generate mutant mice. A mouse carrying the Dync1h1 gene trap allele was bred with flp-recombinase to create the Dync1h1^flox allele. Conditional knockout in retinal progenitor cells was achieved by breeding Dync1h1^flox/flox mice with Six3Cre transgenic mice. Tamoxifen injection induced the knockout of Dync1h1 in flox/flox mice carrying CMV-CreER^T2 or Prom1-CreER^T2.

Results : DYNC1H1 (4644 amino acids) localizes to photoreceptor inner segments and the ONL. The truncated DYNC1H1 protein in the conditional knockout is non-functional as the motor domain, ATPase sites, and microtubule binding stalk are all lost. Dync1h1^flox/flox; Six3Cre mice were assessed at P8, P10, P12, P16, and P28. Loss of Dync1h1 in retinal progenitor cells results in a shrunken ONL and failure of OS formation. Scotopic and photopic ERG of retinal cKO was diminished. Loss of DYNC1H1 in the adult photoreceptor following tamoxifen injection in Dync1h1^flox/flox; ET-Cre mice is expected to result in protein mistrafficking, shortening/absence of OS, and retinal degeneration.

Conclusions : Cytoplasmic dynein 1 is necessary for postnatal development of photoreceptors, intracellular protein transport, and the formation of OS. Loss of Dync1h1 results in mistrafficking of OS proteins.

This is a 2020 ARVO Annual Meeting abstract.