Abstract
Truncating mutations in Gli3, an intracellular effector in the SHH-SMO-GLI signaling pathway, cause renal aplasia/dysplasia in humans and mice. Yet, the pathogenic mechanisms are undefined. Here, we report the effect of decreased SHH-SMO signaling on renal morphogenesis, the expression of SHH target genes and GLI binding to Shh target genes. Shh deficiency or cyclopamine-mediated SMO inhibition disrupted renal organogenesis, decreased expression of GLI1 and GLI2 proteins, but increased expression of GLI3 repressor relative to GLI3 activator. Shh deficiency decreased expression of kidney patterning genes (Pax2 and Sall1) and cell cycle regulators (cyclin D1 and MYCN). Elimination of Gli3 in Shh(-/-) mice rescued kidney malformation and restored expression of Pax2, Sall1, cyclin D1, MYCN, Gli1 and Gli2. To define mechanisms by which SHH-SMO signaling controls gene expression, we determined the binding of GLI proteins to 5' flanking regions containing GLI consensus binding sequences in Shh target genes using chromatin immunoprecipitation. In normal embryonic kidney tissue, GLI1 and/or GLI2 were bound to each target gene. By contrast, treatment of embryonic kidney explants with cyclopamine decreased GLI1 and/or GLI2 binding, and induced binding of GLI3. However, cyclopamine failed to decrease Gli1 and Gli2 expression and branching morphogenesis in Gli3-deficient embryonic kidney tissue. Together, these results demonstrate that SHH-SMO signaling controls renal morphogenesis via transcriptional control of Gli, renal patterning and cell cycle regulator genes in a manner that is opposed by GLI3.
Highlights
Regulation of GLI3 repressor formation via cleavage of GLI3 is crucial during mammalian morphogenesis
The significance of GLI3 repressor formation was demonstrated by our finding that homozygous GLI3 deficiency in Shh deficient mice rescued the dysplastic renal phenotype and the expression of Pax2, Sall1, cyclin D1 and Mycn
Binding of GLI1 and GLI2 to the Pax2, Sall1, cyclin D1 and Mycn promoters established these genes as direct targets of SHH
Summary
Regulation of GLI3 repressor formation via cleavage of GLI3 is crucial during mammalian morphogenesis. Gli mutations that generate a putative truncated protein similar in size to GLI3 repressor are found in humans with Pallister-Hall syndrome (PHS) and malformations including polydactyly, imperforate anus, hypothalamic harmartoma and renal dysplasia/aplasia (Kang et al, 1997). Mice homozygous for a targeted mutation that generates a 699 amino acid N-terminal GLI3 protein exhibit numerous malformations, including renal aplasia/dysplasia as observed in PHS (Bose et al, 2002). GLI3 is an intracellular transcriptional effector in the sonic hedgehog (SHH) signaling pathway. In the absence of a Hh signal, Ci is processed by proteolysis into an N-terminal fragment that includes the zinc finger region and acts to repress gene transcription
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