Loss of LMO4 in the Retina Leads to Reduction of GABAergic Amacrine Cells and Functional Deficits Philippe M. Duquette1, Xun Zhou 1 , Nida Lerma Yap1, Erik J. MacLaren1, Jesse J. Lu1, Valerie A. Wallace2,3,5,6, Hsiao-Huei Chen1,3,4* 1 Centre for Stroke Recovery, Neuroscience, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada, 2 Molecular Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada, 3 Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada, 4 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada, 5 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada, 6 University of Ottawa Eye Institute, University of Ottawa, Ottawa, Ontario, Canada Abstract Background: LMO4 is a transcription cofactor expressed during retinal development and in amacrine neurons at birth. A previous study in zebrafish reported that morpholino RNA ablation of one of two related genes, LMO4b, increases the size of eyes in embryos. However, the significance of LMO4 in mammalian eye development and function remained unknown since LMO4 mice die prior to birth. Methodology/Principal Findings: We observed the presence of a smaller eye and/or coloboma in ,40% LMO4 mouse embryos. To investigate the postnatal role of LMO4 in retinal development and function, LMO4 was conditionally ablated in retinal progenitor cells using the Pax6 alpha-enhancer Cre/LMO4flox mice. We found that these mice have fewer Bhlhb5- positive GABAergic amacrine and OFF-cone bipolar cells. The deficit appears to affect the postnatal wave of Bhlhb5+ neurons, suggesting a temporal requirement for LMO4 in retinal neuron development. In contrast, cholinergic and dopaminergic amacrine, rod bipolar and photoreceptor cell numbers were not affected. The selective reduction in these interneurons was accompanied by a functional deficit revealed by electroretinography, with reduced amplitude of b-waves, indicating deficits in the inner nuclear layer of the retina. Conclusions/Significance: Inhibitory GABAergic interneurons play a critical function in controlling retinal image processing, and are important for neural networks in the central nervous system. Our finding of an essential postnatal function of LMO4 in the differentiation of Bhlhb5-expressing inhibitory interneurons in the retina may be a general mechanism whereby LMO4 controls the production of inhibitory interneurons in the nervous system. Citation: Duquette PM, Zhou X, Yap NL, MacLaren EJ, Lu JJ, et al. (2010) Loss of LMO4 in the Retina Leads to Reduction of GABAergic Amacrine Cells and Functional Deficits. PLoS ONE 5(10): e13232. doi:10.1371/journal.pone.0013232 Editor: Amit Singh, University of Dayton, United States of America Received July 1, 2010; Accepted September 14, 2010; Published October 7, 2010 Copyright: 2010 Duquette et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Early Researcher Award from the Ontario Ministry of Research and Innovation (HHC), by infrastructure support from the Ontario Research Fund (HHC) and the Canada Foundation for Innovation (HHC), and by CIHR grants (MOP-179197). HHC is a recipient of a New Investigator Award from the Heart and Stroke Foundation of Canada. VAW is supported by CIHR (RMF92100), Foundation Fighting Blindness and Stem Cell Network of Canada. PMD is a recipient of an Ontario Graduate Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hchen@uottawa.ca Introduction The vertebrate retina derives from common, multipotent progenitor cells that give rise to six classes of neurons and one type of glial cell organized into three nuclear layers. The rod and cone photoreceptors reside in the outer nuclear layer (ONL), three types of interneurons including the horizontal, bipolar and amacrine neurons, together with the M u¨ller glial cells are found in the inner nuclear layer (INL) and the displaced amacrine and ganglion cells are in the ganglion cell layer (GCL) [1,2]. The photoreceptors are the primary sensory neurons responding to light stimuli, the interneurons process a highly complex array of spatial and frequency information and convey this information to the retinal ganglion cells, the final output neurons of the retina. Mammalian retinal development starts at mid-gestation (embry- onic day 12 for the mouse) and continues into the first two weeks after birth. Retinal ganglion cells, amacrine cells, cone photore- ceptors, and horizontal cells differentiate first and the process is almost complete at birth (gestation period being 21 days in mice). In contrast, bipolar cells, rod cells and M u¨ller glial cells continue to be generated from neural precursors up to two weeks after birth (see review [2]). Many transcription factors specify retinal cell fate, and their precise hierarchy is gradually yielding to genetic analysis (for review, see [2]). Homeodomain factors are thought to regulate the layer specificity but not the neuronal fate, while basic helix loop helix (bHLH) transcription factors determine the neuronal fate within homeodomain factor-specified layers [2–4]. Thus, combi- nations of proper bHLH and homeodomain factors are required for neuronal subtype specification. Amacrine cells are the most complex component of the vertebrate retina, comprising almost 40 different functional types PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13232