Journal of Neurochemistry, 2005, 94, 1739–1745 doi:10.1111/j.1471-4159.2005.03336.x Runx1 expression defines a subpopulation of displaced amacrine cells in the developing mouse retina Lee Stewart,* Mary Anne Potok, Sally A. Camper and Stefano Stifani* *Center for Neuronal Survival, Montreal Neurological Institute, Montreal, Quebec, Canada Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA Abstract primarily in the ganglion cell layer by late embryonic/early AML1/Runx1 (Runx1) is a mammalian transcription factor that post-natal stages and were identified as a subpopulation of plays critical roles in regulating the differentiation of a number displaced amacrine cells by the continued expression of of different cell types. In the present study, we have utilized Islet1, as well as Pax6, and the coexpression of the amacrine mice expressing b-galactosidase (b-gal) under the control of cell subtype-specific markers choline acetyltransferase, cal- the Runx1 promoter to characterize the spatiotemporal retinin and the 65-kDa isoform of glutamic acid decarboxylase. expression pattern of Runx1 during retinogenesis. Expression These findings identify Runx1 as a novel marker for a of b-gal was first detected at embryonic day 13.5 in post- restricted amacrine cell subtype and suggest a role for this mitotic cells located in the inner retina and overlapped with gene in regulating the post-mitotic development of these cells. expression of the early amacrine and ganglion cell marker Keywords: amacrine cells, choline acetyltransferase, gan- protein Islet1. During subsequent developmental stages, the glion cell layer, 65-kDa isoform of glutamic acid decarboxy- number of b-gal-positive cells increased in a central-to-per- lase, retina, Runx1. ipheral gradient until late embryogenesis but then decreased J. Neurochem. (2005) 94, 1739–1745. in the early post-natal retina. b-gal-positive cells were located The differentiation of distinct neuronal subtypes at the Otto et al. 2003). In particular, Runx1 is required for correct time and place is a critical event during nervous definitive hematopoiesis in mice and mutations of its human system development. In that regard, the developing mam- homolog, AML1, are found in a high percentage of acute malian retina provides a relatively simple and well-defined myeloid leukemias and other hematological disorders experimental system to study the events that underlie the (Amman et al. 2001; Roumier et al. 2003). Recent studies generation of different cell types from common progenitors. have also identified a requirement for Runx1 function during Six main classes of neurons and one type of glial cell are the post-mitotic development of selected populations of generated in the retina and the precise spatial and temporal sensory and motor neurons in the murine hindbrain and pattern of retinogenesis is under the influence of both cranial ganglia, implicating Runx1 in the regulation of neuron extrinsic and intrinsic cues (Marquardt and Gruss 2002). subtype differentiation (Theriault et al. 2004). To determine Although a number of transcription factors have been if Runx1 is involved in the development of additional identified as important regulators of mammalian retinal development (Marquardt 2003), the mechanisms that regu- late the acquisition of specific retinal neuron traits still Received April 7, 2005; revision received May 20, 2005; accepted May 20, 2005. remain poorly defined. In particular, little is known about the Address correspondence and reprint requests to Dr Stefano Stifani, molecular characteristics of specific subtypes of each set of Center for Neuronal Survival, #F102, Montreal Neurological Institute, retinal neurons and how the differentiation of those individ- 3801 rue University, Montreal, Quebec, H3A 2B4, Canada. ual subtypes is regulated. E-mail: [email protected] The phylogenetically conserved runt/Runx gene family Abbreviations used: AMCA, Amino Methyl Coumarin Acetate; b-gal, b-galactosidase; ChAT, choline acetyltransferase; E, embryonic encodes a number of DNA-binding transcription factors that day; GAD65, 65-kDa isoform of glutamic acid decarboxylase; GAD67, play important roles during development in both inverte- 67-kDa isoform of glutamic acid decarboxylase; GCL, ganglion cell brates and vertebrates (Wheeler et al. 2000; Coffman 2003; layer; IPL, inner plexiform layer; P, post-natal day. Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 1739–1745 1739 1740 L. Stewart et al. neuronal cells, we have characterized its expression during and FITC-conjugated goat anti-mouse/rabbit (1 : 200; Jackson retinal development. Here, we demonstrate for the first time ImmunoResearch, West Grove, PA, USA). Triple-labeling experi- that Runx1 is transiently expressed in a subpopulation ments were performed using the primary antibodies indicated in the of displaced amacrine cells characterized by the expression text and the following secondary antibodies: Cy3-conjugated of choline acetyltransferase (ChAT), the 65-kDa isoform of donkey anti-goat; FITC-conjugated donkey anti-rabbit and AMCA-conjugated donkey anti-mouse (1 : 40; Jackson Immuno- glutamic acid decarboxylase (GAD65) and the calcium- Research). As controls, primary antibodies were omitted from the binding protein calretinin. These findings identify Runx1 as a incubation steps, in which case no staining was observed. new marker of a specific subset of amacrine cells. Moreover, they suggest that Runx1 may contribute during a defined In situ hybridization temporal window to the mechanisms regulating the acquisi- Sense and antisense Runx1 riboprobes were generated by first using tion of a particular terminal phenotype by post-mitotic a mouse Runx1 cDNA (Image Clone ID 4037315) to amplify by amacrine cells. PCR a 0.5-kb product corresponding to the start of the 3¢ untranslated region. The following oligonucleotides were used for PCR: Runx1-3¢-forward, 5¢-TACTGAGCTGAGCGCCATCGC- Experimental procedures CAT-3¢ and Runx1-3¢-reverse, 5¢-GACCCAAAGCTGTAGCTGTC- TCT-3¢ and the PCR product was cloned into pBluescript-SK. Tissue preparation Riboprobes were generated in the presence of digoxigenin 11-UTP lacZ/+ Runx1 mice were generated and maintained as previously by transcription with T7 or T3 RNA polymerases, respectively. lacZ/+ described (North et al. 1999). The recombined locus of Runx1 Wild-type E16.5 mouse embryos on a mixed C57/Bl6 · SJL mice encodes a fusion protein of the N-terminal 242 amino acids background were fixed in 4% paraformaldehyde (2 h), embedded of Runx1 (containing a nuclear localization sequence) and b-galac- in paraffin using a Citadel tissue processor (Thermo Electron, tosidase (b-gal). The expression of this nuclear fusion protein was Pittsburgh, PA, USA) and sectioned at 6 lm on a microtome. Tissue shown to faithfully reproduce the expression pattern of Runx1 sections were then deparaffinized and washed in phosphate-buffered transcripts (North et al. 1999; Theriault et al. 2004). For embryonic saline prior to treatment with 0.3% Triton X-100 followed by staging, the day of the appearance of the vaginal plug was considered incubation in the presence of proteinase K (7.5 lg/mL), 100 mM as embryonic day 0.5 (E0.5). Embryos or pups were collected at glycine and then 0.25% acetic anhydride in 0.1 M triethanolamine. stages E13.5 to post-natal day 5 (P5). Tail clippings were genotyped Sections were pre-hybridized at room temperature (1–2 h) in by both PCR analysis of genomic DNA (North et al. 1999) and hybridization buffer containing 50% formamide, 5· saline sodium incubation with the chromogenic substrate 5-bromo-4-chloro-3- citrate buffer, 2% blocking powder (Roche Molecular Biochemicals, indolyl-b-D-galactosidase (2 lg/mL) in phosphate-buffered saline Indianapolis, IN, USA), 0.1% Triton X-100, 0.5% CHAPS, 5 mM containing 1 M MgCl2,5mM potassium ferrocyanide and 5 mM EDTA, 50 lg/mL heparin and 1 mg/mL yeast tRNA and hybridized potassium ferricyanide. For embryonic stages, eyes were fixed in situ at 50°C overnight in the same buffer with 500 pg/lL riboprobe. in 2% paraformaldehyde containing 10 mM sodium periodate and After hybridization, sections were washed extensively and then 70 mML-lysine (PLP solution) for 2–4 h. Post-natal eyes were incubated in a blocking solution containing 10% heat-inactivated removed and immersion-fixed in 2% PLP solution for 4 h. After tissue sheep serum, 2% bovine serum albumin, 50 mM Tris-HCl (pH 7.5), freezing (Theriault et al. 2004), sections were cut on a cryostat, 100 mM NaCl and 0.1% Triton X-100 for 1 h. Sections were mounted on SuperfrostÔ slides, air-dried and stored at )20°C. All incubated with alkaline phosphatase-conjugated sheep anti-digo- experimental procedures were carried out in accordance with the xigenin Fab fragments (1 : 400) in blocking solution (3 h). guidelines set forth by the Canadian Council for Animal Care. Antibody labeling was visualized with 4.5 lg/mL 4-nitroblue tetrazolium and 3.5 lg/mL 5-bromo-4-chloro-3-indoyl-phosphate. Histochemical detection of b-galactosidase activity Representative sections from each developmental stage examined Image acquisition (E13.5 to P5) were incubated in the presence of 5-bromo-4-chloro- All images were captured with a DVC black and white camera 3-indolyl-b-D-galactosidase overnight at 37°C as described previ- mounted on an Axioskop fluorescence microscope (Zeiss, Toronto, ously (North et al. 1999) followed by counterstaining with Neutral ON, Canada). Grayscale images were digitally assigned to the Red (Sigma, St Louis, MO, USA). appropriate red (Cy3), green (FITC) or blue (AMCA) channels using Northern Eclipse software (Empix, Mississauga, ON, Canada). Immunofluorescence Tissue sections were first incubated in Image-iTÔ FX Signal Enhancer (Molecular Probes, Eugene, OR, USA) for 30 min and Results then for 1 h in a blocking solution manufactured specifically for detecting mouse primary antibodies on mouse tissue (M.O.M. Kit; Spatial and temporal Runx1 expression in the developing Vector Laboratories, Burlingame, CA, USA). The primary antibod- retina ies used to label different retinal cell types are listed in Table 1.