Background: Background: Early embryonic development and growth is governed by intricate cell signaling mechanisms. Hierarchy of a typical metazoan cell signaling cascade includes, the extra-cellular signaling molecules and cell surface receptors interaction, transmission of signals from cellular surface receptors to inside the nucleus, the conclusive outcome of molecular signals through changes in expression pattern of target gene bodies. The GLI gene family of transcriptional factor (GLI1, GLI2 and GLI3) is important mediator of one such evolutionary conserved pathway, the sonic hedgehog (shh) cascade. Previously the GLI gene family members have been intensely investigated through, molecular, genetic and biochemical means. Therefore, a great deal of understanding exists, regarding their cellular localization, interacting partners, response of GLIs to Shh signal, and a subset of their target genes. Furthermore, developmental and disease relevant roles of GLI gene family members have been explored extensively. However, despite of all these important advancements in genetic, molecular, development and disease relevant aspects of GLI family members, little is known as for as their cis-regulatory expression control is concerned. Human GLI3 is known to be an indispensable primary signaling transducer of Sonic hedgehog (Shh). GLI3 is known to contribute in the development of multiple organs, including limbs and the central nervous system (CNS). Previous attempts, employing computational approaches in conjunction with in vitro and in vivo assays have identified a subset of cis-regulators for this developmentally important gene. Present study is an effort to decipher further cis-regulatory elements of human GLI3. Furthermore, this investigation exploits the availability of in vivo functionally confirmed enhancers to elucidate the genomic features of human limb specific enhancers. II Results: The present study identifies novel anciently conserved non coding sequences (CNEs) within intronic regions of human GLI3 gene, and are named as CNE13 (intron4) and CNE14 (intron3).The regulatory potential of CNE13 and CNE14 was investigated in transgenic zebrafish embryos by employing independent strategies: co-injection methodology and by direct cloning in Tol2 vector. Both of these elements, up-regulates reporter expression at already reported domains of endogenous Gli3 transcription. The CNE13 region induces reporter expression notably in the hindbrain region, muscle cells and pectoral fin whereas the CNE14 triggered reporter expression was confined only to the pectoral fin. CNE13 and CNE14 intronic elements depicted different activities the cell culture based luciferase reporter assays, supporting the belief that GLI3 expression regulation is complex and cellular context-dependent. The Tol2 based transgenic zebrafish assay of earlierreportedGLI3 limb-specific enhancers (CNE6 and CNE11; tested in the chicken limb-buds and mice) suggest that limb activity of a subset of GLI3-associatedconserved non-coding elements might be a tetrapod specific evolutionary innovation. Furthermore, the present study reports regulatory communications for large numbers of genes involve in limb growth and patterning. This data suggests that long-distance regulatory contacts are rather widespread during limb patterning. This observation emphasizes the importance of chromosomal aberrations in limb deformities. Furthermore, transcription factors (TF) examination predicts that developing limb bud differentiation into future different territories might involve distinct TF networks. Conclusions: Results presented in the current study when taken in combination with previously published data, clearly propose that over the course of evolution, the vertebrateGLI3 expression pattern has obtained a complex catalog of cis-regulatory elements for the development of CNS and limbs. Comparative evaluation of resulted experimental data from mice III and fish suggest that functions of such regulatory controls have diverged exceptionally among these two highly diverged lines of animals. In fact these enhancers will assist in pinpointing the molecular aspects which administer the space and time availability of effectors molecules of Shh cascade during early development. For instance, this catalog of GLI3 specific cis-regulators could help elucidate the gene expression underpinning of precise balance among SHH and GLI3gene products in complementary developmental domains (e.g., limb and neural tissue) and during organogenesis. This complex network of GLI3 enhancers could help understand the genetics of human birth abnormalities that cannot be ascribed to mutations in coding intervals of GLI3. In these cases, the enhancers that can potentially affect the expression pattern of GLI3can be scrutinized during early embryonic development. Furthermore, human limb specific gene regulatory networks established in the current study would be useful to elucidate the role of enhancers in developmental gene expression, disease and evolution.