Alternative splicing is a diversification mechanism that allows for increases in proteome complexity via specific regulation of gene expression. Splicing may result in different protein domain combinations, alterations in tissue specificity, or dramatic changes in functional activity, all of which have profound implications for development and disease. Here, we demonstrate that a transcription factor that is important for proper cardiac development exists as two distinct isoforms that are expressed in vivo
through alternative splicing at the carboxy terminal domain. Pritsker et al. 
previously demonstrated that splicing is often weakly conserved in orthologous genes in mouse and human. Thus, the conservation of alternatively spliced ZIC3 isoforms in both human and mouse cell lines suggests functional importance.
The ZIC3-B isoform retains most of the mapped domains of ZIC3, including four zinc finger DNA binding domains, two NLS domains, and the NES domain. Thus previously identified patient mutations should be re-evaluated in terms of their functional effect not only on the ZIC3-A isoform, but also on ZIC3-B. Our past findings suggested that the NES domain of ZIC3 is cryptic, and that structural changes, possibly at the carboxy terminus, are required to expose the motif and facilitate export 
. It will therefore be important to determine the effect of the novel C-terminus identified in ZIC3-B on nuclear export. In addition, the NLS and NES motifs overlap with the zinc fingers of ZIC3, which have previously been shown to bind GLI 
. GLI transcription factors contain a functionally active bipartite NLS motif as well as an NES, and undergo complex nucleocytoplasmic trafficking in conjunction with SUFU (Suppressor of fused) to mediate hedgehog signaling 
. Physical interactions between ZIC3 and GLI may result in the coordination of several NLS sites for recruitment to the nucleus and/or alteration in exposure of NES motifs. Studies investigating possible binding partners, including GLI family members, may provide additional clues for further understanding the nucleocytoplasmic shuttling mechanism of ZIC3-A and –B during left-right patterning and cardiac development.
Studies on GLI superfamily members have also shown that post-translational modifications are a primary determinant in the protein's ability to affect transcription 
. ZIC3 can bind to consensus GLI binding sites to activate transcription, and ZIC3-A appears to act as a transcriptional co-activator in conjunction with GLI3 
. The inability of Zic3-B to also act as a transcriptional co-activator with GLI3 may be due to the previously described differences in the fifth zinc finger domain and/or third NLS domain (). More studies are necessary to compare differences in the C-termini of the two isoforms, the subcellular localization of both isoforms and GLI3 proteins when co-transfected, and the effect of potential post-translational modifications of both isoforms 
The data indicate that the developmental and tissue specific expression profiles of murine Zic3-A
overlap. Previous studies have shown that alternatively spliced isoforms that are co-expressed may act as negative regulators 
. It will be important to examine the co-regulation of the –A and –B isoforms during development. Our initial mutation analyses did not identify pathogenic mutations in exon 4. Because of the small number of families available for study, analysis of additional X-linked pedigrees will be required to establish the prevalence of the alternatively spliced isoform in familial cases. Given that ZIC3-A mutations have been identified at relatively low rates (approximately 1%) in sporadic heterotaxy cases and isolated congenital heart defects, analysis of exon 4 of the ZIC3-B isoform in larger cohorts is important to more clearly establish its contribution to disease.