Study of Mitochondrial Diseases

The YFP transgene is fused to the mitochondrial localization signal of mouse COX8 (the N-terminal 34 amino acids). Defective mitochondrial transport within retinal neurons is speculated to underlie diseases of both retinal ganglion cells and retinal photoreceptors [12]. The differential expression of YFP in subsets or all RGCs and photoreceptors offers a tractable system by which researchers studying these processes can visualize mitochondria either in vivo or in vitro. Such approaches have been applied to motor neurons, and facilitate studies of mitochondrial morphology, trafficking, and fusion. In the eye, autosomal dominant optic atrophy 1 is caused by mutation of the OPA1 gene. OPA1 encodes a mitochondrial inner membrane protein involved in mitochondrial fusion. For studies of diseases such as optic atrophy, the mice described in this study would provide an excellent research tool.

Study of Retinal Neuron Subtype Development

The expression of various combinations of transcription factors are required for specification of different populations of retinal neurons. Transcription factors required for generation of the various basic types of retinal neurons, such as rods or amacrine cells, have been identified; however, it is largely unknown how subtypes of retinal neurons are specified. The availability of transgenic labels that are expressed in specific subtypes of retinal neurons will simplify identification of such factors by allowing the isolation of specific subtypes of retinal neurons. Purified cell populations can also be used for gene expression profiling to obtain a better molecular definition of the differentiation state of the cells, to provide additional markers for studying the cells, and to better understand the molecular code of surface proteins that confer cell identity. Furthermore, being able to visualize specific cell types in vivo is a great boon to studies of retinal patterning and mosaic formation.

Study of Clonal Proliferation and Lateral Migration

X-inactivation in the optic cup occurs by approximately embryonic day 9.5 (E9.5), during which retinal progenitor cells have not yet begun to differentiate into retinal neurons [13, 14]. Production of retinal neurons occurs in a columnar clonal fashion, with a given population of retinal precursor cells giving rise to all populations of retinal neurons in a horizontal column [11, 15]. Because the retina forms in a columnar fashion during development, with limited lateral migration, patches of YFP-positive and YFP-negative retina are observed resulting from either neural progenitors in which the X-Chromosome carrying the Mito-Z transgene is expressed or inactivated, respectively, making this line a useful marker of clonal proliferation (Figure 3B). The clonal columns generated by hemizygous female Mito-Z mice are not as obvious as those generated by other transgenic lines because YFP is not widely expressed in bipolar cells and photoreceptors, which do not migrate across the horizontal plane of the retina, while the amacrine and ganglion cells that YFP is expressed in do, making clonal borders less sharp [16]. The X-Chromosome location of the Mito-Z transgene will add another useful reagent, that can be used by itself or in combination with other X-Chromosome reporters such as a β-Gal reporter, for example by using both reporters in tandem to compare horizontal migration into and out of differentially labeled clonal columns [17]. The X-Chromosome insertion site of the transgene also simplifies maintaining Mito-Z mice, as male carries crossed to wild type females will sire only transgenic female and wild type male offspring.

In this report we characterize the retinal expression pattern of three transgenic mouse lines. These lines will be useful for a number of various studies, including analysis of mitochondrial trafficking, and will complement a similar line of mice that express of mitochondrial localized CFP in different populations of retinal neurons [18]. An increasing number of transgenic mouse lines offers the ability to label more retinal cell types, permitting an increasing amount of experimental flexibility and control [3, 7, 9, 19]. The mice described here add value by labeling new subsets and by labeling mitochondria allowing new fields of study.

Animal care and handling

Mice were used in accordance with protocols approved by the Animal Care and Use Committee at The Jackson Laboratory. The Jackson Laboratory is accredited by AAALAC. Animals were housed in PIV caging and given food and water ad libitum, and maintained on a 14 hour:10 hour light:dark cycle.

Mouse strains

The following mouse strains were used in this study: Mito-R: Tg(Eno2-YFP/Cox8a)RRwb/J [8], Mito-Y: Tg(Eno2-YFP/Cox8a)YRwb/J (Jax stock number 007857) [8], Mito-Z: Tg(Eno2-YFP/Cox8a)/ZRwb (Jax stock number (007860) [8].

Tissue Preparation and staining

Mice were transcardially perfused with PBS followed by perfusion with 4% paraformaldahyde buffered with PBS. Eyes were then fixed for an additional one hour in 4% paraformaldahyde buffered with PBS. Eyes were enucleated and sunk in 30% sucrose overnight and then frozen in cyropreservation media. Sections of retina were cut at 8 μm thickness with a cyrostat. Sections were rinsed in PBS and mounted with antifade or stained with antibodies.

Immunofluorescence was performed by blocking sections in PBS with 3% normal horse serum and 0.1% triton x-100, and then incubating sections overnight at 4°C with primary antibodies diluted in blocking buffer. Sections were then washed three times for ten minutes in PBS and incubated with secondary antibodies diluted in blocking buffer. Sections were washed three times for ten minutes in PBS, with DAPI included in the last wash, and mounted with antifade media. The following commercially available antibodies were used: mouse anti-nonphosphorylated neurofilament (1:200; Covance Research Products), rabbit anti-tyrosine hydroxylase (1:500 Invitrogen), goat anti-ChAT (1:500, Chemicon) and goat anti-BRN3b (1:200; Santa Cruz Biotechnology).

Whole retinas were prepared by fixing as described above. Retinas were then blocked in PBS, 3% normal horse serum and 0.4% triton X-100. Retinas were incubated in primary antibodies diluted in the blocking solution for four days at 4°C and then washed overnight in blocking buffer. Retinas were incubated overnight with secondary antibodies diluted in blocking buffer and washed in blocking buffer for an additional day and then mounted with antifade media. All images were collected on a Leica SP5 confocal microscope.