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Research to Prevent Blindness
Title: Neurod1 control of gene expression in the mammalian pineal and retina: Results from in
vivo cre/loxp-mediated gene inactivation
Authors: *M. J. OCHOCINSKA 1 , E. M. MUÑOZ 2 , N. POZDEYEV 3 , S. GOEBBELS 4 , P. M.
IUVONE 3 , T. FURUKAWA 5 , K.-A. NAVE 4 , D. C. KLEIN 1 ;
1 NIH, Bethesda, MD; 2 Inst. of Histology and Embryology, Sch. of Medicine, Natl. Univ. of
Cuyo, Natl. Council of Research, Sci. and Technol. (CONICET), Mendoza, Argentina; 3 Dept. of
Ophthalmology and Pharmacol., Emory Univ. Sch. of Med., Atlanta, GA; 4 Dept. of
Neurogenetics, Max-Planck-Institute of Exptl. Med., Göttingen, Germany; 5 Osaka Biosci. Inst.,
Osaka, Japan
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. NeuroD1 flox/+ /Crx-cre +/- mice were crossed
with NeuroD1 flox/flox mice to obtain the conditional NeuroD1 knockout (cKO,
NeuroD1 flox/flox /Crx-cre +/- ) and control (NeuroD1 flox/flox /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 altered > 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.
Disclosures: M.J. Ochocinska, None; E.M. Muñoz, None; N. Pozdeyev, None; S. Goebbels,
None; P.M. Iuvone, None; T. Furukawa, None; K. Nave, None; D.C. Klein, None.