Roles of vertebrate Gli3 gene in brain development and evolution

Abstract

Background The Gli3 protein is a zinc finger transcription factor and is the primary transducer of Shh signaling, serving as both transcription activator and repressor. During early embryonic development Gli3 participate in patterning and growth of central nervous system, face, limb and skeleton. It exhibits allelic heterogenicity, as mutations in GLI3 gene as associated a variety of dominant developmental defect syndromes subsumed under the term “GLI3 morphopathies,” including Greig cephalopolysyndactyly syndrome and Pallister Hall syndrome. It is extensively studied by biochemical, molecular and genetic means. In this study, the importance of Gli3 protein in brain development and evolution has been explored by answering the following questions. • What are the consequences of a mutation in Gli3 on vertebrate brain functioning and morphology? • What are the patterns along which the expression of Gli3 protein in brain evolved within and between vertebrate species? Results Gli3 has a wide expression pattern in vertebrate brain functioning and morphology. However, given the embryonic lethality in homozygous knockout mice, no murine model studies have presented enough evidence to explore the extensive role of Gli3 in brain functioning and morphology. Here in this study we generated a gli3 knockout zebrafish line using CRISPR/Cas9 genome editing technology and analysed the morphological anomalies of these knockout lines by histology and computed tomography. Our results showed that gli3 loss of function in zebrafish largely phenocopies the human brain malformations: the telencephalon is enlarged, the diencephalon is reduced, the cerebellum is vertically extended and enlarged, and cerebrovascular morphology and architecture is altered. As compared to coding sequence the non-coding DNA is the major target of evolutionary innovations. Previously, we and others have uncovered a set of enhancers that mediate many of the known aspects of Gli3 expression during neurogenesis. Here we presented that subset of these Human GLI3 central nervous system enhancers have undergone an accelerated rate of molecular evolution in the human lineage in comparison to other primates/mammals. These fast-evolving enhancers have acquired human-specific changes in transcription factor binding sites (TFBSs). These human-unique changes within subset of GLI3 associated CNS specific enhancers were further validated as single nucleotide polymorphisms through 1000 Genome Project Phase 3 data. Conclusion Consistent with the complex role of Gli3 in a multitude of patterning steps during embryonic development, in this study a new model is presented to explore the extensive role of Gli3 in brain functioning and morphology. Congruent with the hypothesis of King and Wilson’s that regulatory changes drove the difference between and within the species, the subset of GLI3 associated CNS-specific enhancers evolve under positive selection in all human populations. In this subset of positively selected GLI3 associated CNS-specific enhancers, the single nucleotide polymorphisms led to the changes in transcriptional factor binding sites. Evolution of TFBSs, in pre-existing enhancers may allow a gene to acquire a novel expression patterns without compromising the ancestral expressions/functions. Therefore, the results of this study open avenues for further studies aimed at understanding the possible roles of GLI3 associated enhancers in brain evolution and inter-population variability of gene expression in humans