Abstract
Purpose :
The transcripts encoding the circadian clock genes are present in the embryonic retina. Their function in retinal neurogenesis is, however, relatively unknown. We recently identified a molecular function for the circadian clock genes in cone photoreceptors (Sawant et al., 2017, Cell Reports). Based on these findings, we are investigating the role of the molecular clock in regulating both development and function of other retinal neurons.
Methods :
We deleted Bmal1 from retinal progenitor cells (RPCs) using the Chx10-cre mouse line. Immunofluorescence technique was used to label specific retinal cell types in flat mounts and cryosections. Cell cycle kinetics of RPCs were studied using uridine (EdU/BrdU) DNA labelling technique where EdU or BrdU was injected (i.p. 100 µg/g) to label proliferating cells. The integrity of the visual system conduction pathway was assessed using visual evoked potentials (VEPs). Western blot and qRT-PCR techniques were used to validate the Bmal1 deletion and other immunofluorescence data.
Results :
Expression of Bmal1 was observed as early as embryonic day 13 in mouse retina. Embryonic deletion of Bmal1 from RPCs decreased the number of early born neurons. The densities of Brn3b+ ganglion cells and Calretinin+ amacrine cells were decreased by 19 and 28%, respectively, in conditional mutants (CKO) of Bmal1 compared to their littermate controls (P=0.029 and <0.001, respectively). The density of displaced ChAT+ amacrine cells was decreased by 15% (P=0.021). Our preliminary data suggest that loss of Bmal1 from RPCs results in constitutively high levels of proliferation and delayed cell cycle exit. We observed a significant increase in number of EdU+ cells in non-proliferative region in Bmal1 CKO retinas compared to littermate controls at E15 (EdU injection on E14) (P=0.005). Alterations in cell cycle kinetics also resulted in lamination defects in terminally differentiated Müller glia (MG) cells. A significant increase in mislocalized MG in outer nuclear and ganglion cell layers was observed (P<0.01). Light-adapted VEPs were significantly delayed (P<0.01) in Bmal1 CKO.
Conclusions :
Bmal1 regulates retinal neurogenesis by coordinating the timing of cell cycle entry and exit of the proliferating RPCs, thus leading to proper lamination and a functional retina.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.