LMO4 in Retinal Development Figure 8. Electroretinography reveals a deficit in the b-wave in LMO4 cko mice. ERGs were recorded from 8 week old littermate control (diamonds and solid line) and LMO4 cko (squares and dashed line) mice. (A) b-wave average amplitude was significantly reduced at all light intensities. (B) a-wave amplitude tended to be lower in LMO4 cko mice, but was not significantly different. (C) a-wave latency was not different between control and LMO4 cko mice. All measures are means 6 SEM for n = 13 control (WT) and n = 12 LMO4 cko mice. doi:10.1371/journal.pone.0013232.g008 tion of amacrine cells are gradually being revealed from genetic models. Here, we showed that LMO4 is co-expressed with Pax6, a pan amacrine cell marker, and by selective ablation of LMO4 in retinal progenitor cells we demonstrated that LMO4 is required for Bhlhb5+ amacrine cell development. A 30% reduction in the number of Bhlhb5+ amacrine cells was observed in the peripheral retina of LMO4 cko mice. This suggests the existence of two subsets of Bhlhb5+ neurons, one that is LMO4-dependent and another that is LMO4-independent. In addition, the deficit appears to affect the postnatal wave of Bhlhb5+ neurons suggesting a temporal requirement for lmo4 in retinal neuron development. Despite the loss of Bhlhb5+ (GABAergic) amacrine cells, immunostaining for glutamic acid decarboxylase (GAD), an enzyme that catalyzes the decarboxyl- ation of glutamate to GABA neurotransmitter, did not reveal any difference between LMO4 cko and littermate control mice. This might reflect a compensatory effect of existing Bhlhb5 neurons. In either case, the 30% reduction of Bhlhb5+ amacrine cells in LMO4 cko mice was sufficient to impact the retinal visual signals as shown by ERG. The reduced b-wave amplitude is consistent with loss of amacrine cells (and cone bipolar cells). Although, given that Bhlhb5+ amacrine cells are typically GABAergic inhibitory interneurons, the reduction, rather than the elevation of b-wave amplitude, was unexpected. However, it is possible that the function of these Bhlhb5+ GABAergic interneurons is to inhibit other inhibitory interneurons. This is the first report that links LMO4 with GABAergic amacrine cell development and function. A previous study reported a role for LMO4 in regulating the balance of inhibitory and excitatory interneurons that develop in the spinal cord; ablation of LMO4 lead to a loss of V2b GABAergic inhibitory interneurons and favored V2a glutamatergic excitatory interneu- rons [20]. However, unlike in the spinal cord, we did not observe an increase in the excitatory cholinergic amacrine cells despite the loss of GABAergic inhibitory amacrine cells. Thus, LMO4 appears to have different functions in different regions of the nervous system. Similar to its requirement for inhibitory amacrine interneuron development in the eye, Bhlhb5 is also required for the survival of a subtype of inhibitory interneurons in the spinal cord that regulates pruritis; loss of inhibitory synaptic input in the Bhlhb5 mice results in an excessive abnormal itch [35]. In contrast, in the cerebral cortex Bhlhb5 specifically regulates the differentiation of excitatory glutamatergic projection neurons of neocortical layers II–V and is required for structural organization in the somatosensory and caudal motor cortices [36]. LMO4 is also expressed in the layer II/III and V cortical neurons [37,38] and is critical for patterning thalamocortical connections during devel- opment [13]. In light of our finding that LMO4 is required for Bhlhb5+ retinal neuron development, the similar spatial and temporal expression patterns of LMO4 and Bhlhb5 make it plausible that LMO4 regulates Bhlhb5 expression in the developing cortex and participates in the development of glutamatergic projection neurons. Our study found no deficit in photoreceptor cell number and function as reflected by a normal a-wave amplitude and latency. Consistent with our finding, the Bhlhb5-null mutation does not affect photoreceptor cell development [39]. Another important finding of our study is that LMO4 contributes to the differentiation of Bhlhb5+ type 2 OFF-cone bipolar cells, but not the rod bipolar cells whose development, on the other hand, depends on bHLHb4 [10]. Despite a 30% reduction of Bhlhb5+ cone bipolar cells observed in LMO4 cko mice, the response of the ON-OFF ERG was not different between LMO4 cko and littermate controls. Although this result would argue against a role for LMO4 in cone bipolar cell function, alternatively, there is loss in both ON- and Off- bipolar cells, as reflected in 30% reduction in the number of Chx10+ bipolar cells. The basic helix-loop-helix transcription factors Math3+ and NeuroD+ are important for amacrine cell development, whereas the Math3+ and Mash1+ are for the type 2 OFF-cone bipolar cells [39]. Previous studies showed that LMO4 forms a complex with the basic helix-loop-helix transcription factor SCL and GATA2 and to regulate interneuron differentiation in the spinal cord [20]. Our studies show that the Bhlhb5 promoter is dependent upon LMO4 for expression in neuronal cells in culture. However, since LMO4 is not known to bind DNA directly, the effects of LMO4 on Bhlhb5 promoter activity are likely to be indirect. Future studies will be required to determine whether LMO4 interacts with these bHLH transcription factors to activate Bhlhb5 expression in the retina. Supporting Information Figure S1 Expression of the green fluorescent protein (GFP) reporter from the Pax6 a-enhancer in aCre-IRES-EGFP mice. At embryonic day 14.5 (E14.5), GFP is highly expressed in the peripheral retina, predominantly in the ventral compartment. By postnatal day 0 (P0), GFP expression is widespread in the PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13232