The tissue was rinsed in phosphate buffered saline and immersed overnight in 30% sucrose at 4C, prior to being embedded in tissue freezing medium, frozen, and cut into 20 m transverse sections. differentiation of distinct cell types within the central nervous system and many other tissues. A study of pancreatic cell lines showed that is alternatively spliced generating a second isoform, Isl1, which is missing 23 amino acids within the C-terminal region. This study examines the expression of the canonical and alternative transcripts across other tissues, in particular, within the retina, where Isl1 is required for the differentiation of multiple neuronal cell types. The alternative splicing of is shown to occur in multiple tissues, but the relative abundance of and expression varies greatly across them. In most tissues, is the more abundant transcript, but in others the transcripts are expressed equally, or the alternative splice PIK3C1 variant is dominant. Within the retina, differential expression of the two transcripts increases as a function of development, with dynamic changes in expression peaking at E16.5 and again KI696 isomer at P10. At the cellular level, individual retinal ganglion cells vary in their expression, with a subset of small-to-medium sized cells expressing only the KI696 isomer alternative isoform. The functional significance of the difference in protein sequence between the two Isl1 isoforms was also assessed using a luciferase assay, demonstrating that the alternative isoform forms a less effective transcriptional complex for activating gene expression. These results demonstrate the differential presence of the canonical and alternative isoforms of Isl1 amongst retinal ganglion cell classes. As Isl1 participates in the differentiation of multiple cell types within the CNS, the present results support a role for alternative splicing in the establishment of cellular diversity in the developing nervous system. (Ando et al., 2003; Failli et al., 2000; Grigoriou et al., 1998; Kimura et al., 1999; Sloop et al., 2001). The alternative splicing of was identified in different pancreatic endocrine cell lines. Through the use of an alternative splice acceptor site halfway through the fifth exon, an alternative isoform, Isl1, is generated lacking 23 amino acids towards the C-terminus relative to the canonical isoform Isl1 (Figure 1A, 1B). Interestingly, both the canonical and alternative transcripts are present in a pancreatic cell line while only the canonical isoform is expressed in a pancreatic cell line. This differential pattern of splicing across these pancreatic cell lines suggests distinct roles for each isoform in the development or function of these cell types (Ando et al., 2003). The LIM and HD domains are highly conserved across all LIM-HD family members, however, the C-terminal region of these proteins is diverse and, KI696 isomer in general, not well characterized; as such, the domains and function of the C-terminal region of Isl1 corresponding to the portion absent in Isl1 had not been described when the alternative isoform was originally identified. This region of Isl1 has since been more fully characterized, independent of an examination of the alternative isoform, and contains two protein-binding domains that are separated by a short linker sequence. These two domains bind to the two LIM domains (LIM1 and LIM2) of LIM homeobox protein 3 (Lhx3), and are accordingly named Lhx3-binding domains 1 and 2 (LBD1 and LBD2) (Bhati et al., 2008). The amino acids absent in Isl1 (aa256C278) correspond to LBD1 (aa262C273) and the short linker sequence (aa274C278) that separates it from LBD2 (Figure 1B, 1C). Scanning alanine mutagenesis of this region revealed that LBD2 is dispensable for the binding of Isl1 to Lhx3, but LBD1 is critical for this interaction (Bhati et al., 2008). The absence of LBD1 in Isl1 strongly suggests there may be functional differences between the two isoforms of Isl1, such as the transcriptional complexes in which they participate and in their subsequent gene targets. Open in a separate window Figure 1 Isl1 isoform lacks a critical LIM-binding domain(A) undergoes alternative splicing through the use of an alternative 3 splice acceptor site mid-way through the 5th exon. (B) The alternative isoform, Isl1, is lacking 23 amino acids relative to the canonical isoform, Isl1, which correspond to an Lhx3-binding domain (LBD1). (C) The interaction between the LBDs of Isl1 (green) and the LIM domains of Lhx3 (blue) are.