LMO4 in Retinal Development Figure 7. Loss of Bhlhb5+ cells in P0 LMO4 cko retinas. (A) MO4 is colocalized with Bhlhb5 in amacrine cells (arrows). LMO4 mRNA in the cytoplasm was revealed by in situ hybridization with an antisense probe labeled with biotin (green). Bhlhb5 in the nuclei was revealed by an antibody to Bhlhb5 (red). Inset is enlarged in the lower panels. Scale bars = 100 and 25 mm, top and bottom panels, respectively. (B) Bhlhb5 immunostained cells were significantly fewer in the peripheral retina of LMO4 cko mice compared to littermate controls. Inset is enlarged in the lower panels. Scale bar = 100 mm. doi:10.1371/journal.pone.0013232.g007 and specifically, the development of GABAergic amacrine cells and type 2 OFF-cone bipolar cells. Our observation of coloboma in LMO4 germline deleted mice, reported here for the first time, lead us to explore the function of LMO4 in retinal development. We found that coloboma was not always linked to exencephaly, suggesting that the impaired mechanisms that underlie these phenotypes might be separable or only partially penetrant. In contrast to the increased eye size observed in zebrafish by morpholino knockdown of the LMO4b gene [27], we often observed smaller eyes in LMO4 mice. It should be noted that the sequence of the zebrafish LMO4a gene, rather than LMO4b, is more similar to LMO4 of mammals. Whether the different phenotype in zebrafish represents a species- specific difference or the partial redundancy of the two LMO4 duplicated genes of zebrafish is not known. Amacrine cells in the retina constitute an assortment of different subtypes of interneurons that perform highly complex information processing. The transcription factors that control the differentia- PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13232