based on their neurotransmitters and their synaptic partners. The bHLH genes Math3 and NeuroD are expressed in retinal progenitors at embryonic stages when amacrine cells are generated [2,5,6]. Even though Math3/NeuroD double mutant embryos exhibit selective loss of amacrine cells, misexpression of either Math3 or NeuroD only promotes the generation of rod photoreceptor cells [5]. However, misexpression of Math3 or NeuroD together with the paired box homeodomain protein Pax6 promotes amacrine cell genesis [5], suggesting that the combina- tion of bHLH and the homeobox genes is important for amacrine cell fate determination. For example, Bhlhb5 is required in the specification of GABAergic amacrine cells [7]. The transcriptional mechanisms that control Bhlhb5 expression are not well understood, but likely involve NeuroD/Math3. On the other hand, ablation of Pax6 in retinal neural progenitors leads to the production of only amacrine cells [8], indicating that amacrine cell differentiation is complex and is likely to require additional factors. Rod and cone bipolar cells transmit signals from rod and cone photoreceptor to retinal ganglion cells, respectively. There are one rod bipolar and nine cone bipolar cell types. Bipolar cell fate specification is also dependent upon the combined action of the homeodomain factor Chx10 and the bHLH factors Mash1 and Math3 [4,9]. Recent studies further showed that Bhlhb4 is required for production of rod bipolar cells [10] whereas Bhlhb5 is required for type 2 Off-cone bipolar cells [7]. The LIM domain only 4 protein is a transcription cofactor that interacts with many transcription factors and modulates their activity [11–21]. LMO4 augments the transcriptional inhibitory activity of the bHLH factor HEN1 in cultured hippocampal neurons and controls neurite outgrowth [19]. LMO4 is essential for development of the central nervous system [22,23]. Mice deficient in LMO4 die prior to birth and 50% of embryos display exencephaly [24–26]. Ablation of LMO4 in cortical neurons impairs thalamocortical projections to layer 5 [13]. In the spinal cord, LMO4 specifies the selection of interneuron excitatory and inhibitory cell fate [20]. Ablation of one of the two paralogs of LMO4 in zebrafish increases the size of the eye [27]. In the chick, LMO4 augments Wnt signaling in the progenitors of the ciliary marginal zone of the retina [28]. LMO4 is expressed in the amacrine cell layer of the postnatal day 0 mouse retina [29], but the functional importance of LMO4 in the development of the mammalian retina has not been studied. In characterizing the phenotype of LMO4 deficient mice we noticed a structural defect in the eye. This defect, coloboma, results from incomplete closure of the optic fissure during development [30]. To understand the functional significance of LMO4 in mouse retinal development, we conditionally ablated a floxed LMO4 allele in the retina using the Pax6 alpha enhancer driven Cre recombinase [8]. Whereas these mice do not develop coloboma and have normal-sized eyes, using neuron subtype- specific markers, we observed a reduction in GABAergic amacrine and OFF-cone bipolar cells. Electroretinograms revealed a functional deficit in these mice. Materials and Methods Animals All experiments in animals were approved by the University of Ottawa animal care ethics committee, adhering to the guidelines of the Canadian Council on Animal Care under the protocol permit OGH/RI-36. The Pax6 a-Cre transgenic mice with a downstream GFP reporter tag were obtained from P. Gruss [8] and were maintained on a C57BL/6 background. a-Cre/wt mice were crossed to homozygote LMO4flox/flox mice [24] to generate LMO4 in Retinal Development F1 heterozygotes that were backcrossed to produce a-Cre/ LMO4flox homozygote mutant or littermate control (LMO4- flox/flox) mice. LMO4 mice were described previously [26]. Genotyping for the a-Cre transgene, LMO4flox or LMO4 alleles was performed by PCR. Immunohistochemistry Embryos and dissected eyes plus optic nerves from animals younger than P7 were fixed in 4% paraformaldehyde (PFA) overnight in 0.1 M phosphate buffered saline (PBS) pH 7.4. Older animals were transcardially perfused with 4% PFA prior to overnight incubation. Following PBS washes, samples were cryoprotected overnight in a 30% sucrose/PBS solution. Tissues were embedded in a 1:1 mix of 30% sucrose and OCT (Tissue- Tek, Japan) and frozen by immersion in 230uC 2-methyl butane. Embedded tissue was sectioned on a cryostat at 14 mm. Sections were transferred onto Superfrost Plus coated slides (Fisher Scientific, USA) and stored at 280uC. IHC was performed in sections at the level of the optic nerve plane and immunostaining was compared at the peripheral retinal (approximately up 500 mm from the tip) according to the protocol for fluorescence detection described previously (43,44). The following primary antibodies were used: goat polyclonal anti-Bhlhb5 (1/100), anti-LMO4 (c15) (1/50), anti-Brn3b (c13) (1/100, Santa Cruz Biotechnology), anti- choline acetyltransferase (ChAT) (1/2000, Millipore); sheep polyclonal anti-Chx10 (1/2000, Exalpha Biologicals, Inc); rabbit polyclonals anti-calretinin (1/2000, Swant), anti-tyrosine-hydrox- ylase (TH) (1/2000, ImmunoStar), anti-Prox1 (1/3000, Chemicon International), anti-phosphorylated histone 3 (Ser-10) (1/400, Upstate) and anti-activated caspase 3 (Asp175) (1/100, Cell Signaling; kind gift from David Park); mouse monoclonals anti- Pax6 (1/50; Developmental Studies Hybridoma Bank), anti-PKC (1/100, BD Pharmingen), anti-calbindin (1/500, Sigma), anti- rhodopsin (1/3; Developmental Studies Hybridoma Bank), anti- peanut agglutinin (PNA) (1/500, Santa Cruz Biotechnology), anti- HPC (syntaxin)(1/2000, Sigma). Antibodies were visualized with appropriate secondary antibodies conjugated with Cy3 and Cy2 and visualized on a Zeiss Z1 fluorescence microscope. For LMO4, Pax6, Brn3b and PKC immunostaining, antigen was unmasked with microwave treatment in 10 mM citrate buffer (in 1L water add 42g citric acid, 21 g Sodium hydroxide, pH = 6) using a 1000 watt microwave set at power 6 for 10 min and sections were cooled on ice for 10 min before processing for immunostaining. To compare gene expression and differentiation, immuno- stained positive cells in the same region of the ‘‘peripheral’’ retina at the levels of the optic nerve plane were counted using Zeiss AxioVision automatic software. For every section, all immunopo- sitive cells of a square of fixed area within 500 mm from the tip of retina were counted. For PKC+ rod bipolar and Bhlhb5+ amacrine and cone bipolar cells, cells were counted manually. At least 3 comparable retinal sections from three eyes from mutant and littermate wild type mice were used for statistical analysis by Student t test and p,0.05 were considered to be significant. In situ hybridization Tissues were processed for in situ hybridization with digox- igenin-labeled antisense or sense riboprobes, as previously described [31]. For dual in situ hybridization and immunohisto- chemistry, biotin-labeled LMO4 antisense riboprobes were used, followed by biotin/avidin ABC kit (Vector laboratory) and cy2- conjugated streptavidin (Jackson laboratory) together with anti- Bhlhb5 goat antibody and cy3-conjugated anti-goat secondary antibody (Jackson laboratory). For more reliable comparisons of PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13232