Neurod1 Control of Gene Expression in the Mammalian Pineal and Retina: Results from In Vivo cre/loxp-Mediated Gene Inactivation

M. OCHOCINSKA; E.M. MUÑOZ; S. GOEBBELS; N. POZDEYEV; P.M. IUVONE; FURUKAWA; K.A. NAVE; D.C. KLEIN.

Chicago, USA.

Congreso; Neuroscience 2009; 2009

Abstract: NeuroD1 encodes a basic helix-loop-helix (bHLH) transcription factor involved in cell cycle regulation, retinal cell genesis, and in development of neural and endocrine structures.  NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to that in the retina, consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. While both tissues express a common set of transcription and phototransduction genes, the pineal gland is composed 95% of pinealocytes, whereas the retina is far more heterogeneous. The greater degree of cellular homogeneity makes the pineal gland a useful model to use to understand developmental mechanisms shared by both tissues. Previous attempts to study the role of NeuroD1 in the retina and pineal gland have been limited by the fact that NeuroD1-/- animals die shortly after birth. To overcome this limitation, we used a Cre/loxP approach, in which Cre recombinase was expressed in pinealocytes and retinal photoreceptors, under control of the promoter for Crx, which is selectively expressed at high levels in these cells. Crx-cre mice were crossed with NeuroD1flox mice to obtain the conditional NeuroD1 knockout (cKO, NeuroD1flox/Crx-cre+/-) and control (NeuroD1flox/Crx-cre-/-) mice. NeuroD1 expression was undetectable in the pineal gland and retinas of the cKO mice, but was expressed at normal levels in other tissues tested. The cKO animals were visually blind by 4 months of age, based on rod and cone-related electroretinogram responses; however the nonvisual melanopsin system appeared to be functional based on evidence of entrained rhythmic gene expression in the pineal gland. Gene expression in the cKO and control adult pineal gland and retina was assessed by Affymetrix microarray analysis using the GeneChip Mouse Genome Array 430_2.0 and GeneSpring GX10 software; qRT-PCR was used to confirm results. The microarray results identified several potential Neurod1-regulated genes. In the cKO retina, the expression of 20 genes was altered > 2-fold, including genes linked to phototransduction and the immune response. In the cKO pineal gland, the expression of 10 genes was reduced > 2-fold. The rhythmic expression of several genes was also eliminated. These findings provide clear evidence of the role NeuroD1 plays in controlling gene expression in the mammalian pinealocyte and retina. Moreover, strategies designed to produce retinal photoreceptors following photoreceptor degeneration must consider the importance of NeuroD1.