Targeted ablation of Crb2 in photoreceptor cells induces Retinitis Pigmentosa

Abstract

Purpose: In humans, the Crumbs homologue-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and consequent removal from Müller glia and photoreceptor cells, results in severe and progressive retinal degeneration with concomitant loss of retinal function that mimics retinitis pigmentosa due to mutations in the CRB1 gene. We studied the effects of cell-type specific loss of CRB2 from the developing and from the adult mouse retina using targeted conditional deletion of Crb2 in photoreceptors or Müller cells.

Methods: The Crb2 conditional knockout mice were crossed with Crx-Cre or Pdgfrα-Cre transgenic mice, in order to ablate CRB2 during retinal development, from photoreceptor or Müller cells, respectively. Retinas from animals with different ages were analyzed. In vivo analyses of retinal function were performed using optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO) and electroretinography (ERG). To ablate CRB2, in the adult mouse retina, we generated adeno-associated viral (AAV) vectors encoding Cre recombinase and short hairpin RNA against Crb2.

Results: In vivo analyses by OCT and SLO on retinas lacking CRB2 in photoreceptors showed progressive thinning of the photoreceptor layer and cellular mislocalization. ERG under scotopic conditions showed severe attenuation of the a-wave, suggesting degeneration of photoreceptors resulting in severe retinitis pigmentosa. Retinas lacking CRB2 in developing photoreceptors showed early onset of abnormal lamination. Retinas lacking CRB2 in developing Müller cells showed late onset retinal disorganization, resulting in mild retinitis pigmentosa. Short-term removal of CRB2 from the adult retina did not result in major abnormalities in retinal structure.

Conclusions: Our data show that CRB2 has a redundant function in mouse Müller glia cells, however in photoreceptor cells CRB2 is crucial for proper retinal development. Removal of CRB2 from photoreceptors leads to severe and progressive degeneration with a concomitant loss of retinal function. Furthermore, under normal physiological conditions, CRB2 is not essential for maintenance of adhesion between adult photoreceptors or Müller glia cells.

Keywords: 740 transgenics/knock-outs • 648 photoreceptors • 540 glia