Pax3 is required for enteric ganglia formation and functions with Sox10 to modulate expression of c-ret
Deborah Lang, … , Min Min Lu, Jonathan A. Epstein
Deborah Lang, … , Min Min Lu, Jonathan A. Epstein
Published October 15, 2000
Citation Information: J Clin Invest. 2000;106(8):963-971. https://doi.org/10.1172/JCI10828.
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Article

Pax3 is required for enteric ganglia formation and functions with Sox10 to modulate expression of c-ret

Abstract

Hirschsprung disease and Waardenburg syndrome are human genetic diseases characterized by distinct neural crest defects. Patients with Hirschsprung disease suffer from gastrointestinal motility disorders, whereas Waardenburg syndrome consists of defective melanocyte function, deafness, and craniofacial abnormalities. Mutations responsible for Hirschsprung disease and Waardenburg syndrome have been identified, and some patients have been described with characteristics of both disorders. Here, we demonstrate that PAX3, which is often mutated in Waardenburg syndrome, is required for normal enteric ganglia formation. Pax3 can bind to and activate expression of the c-RET gene, which is often mutated in Hirschsprung disease. Pax3 functions with Sox10 to activate transcription of c-RET, and SOX10 mutations result in Waardenburg-Hirschsprung syndrome. Thus, Pax3, Sox10, and c-Ret are components of a neural crest development pathway, and interruption of this pathway at various stages results in neural crest–related human genetic syndromes.

Authors

Deborah Lang, Fabian Chen, Rita Milewski, Jun Li, Min Min Lu, Jonathan A. Epstein

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A Pax3- and Sox10- responsive enhancer in the c-RET gene. (a) Cotransfec...
A Pax3- and Sox10- responsive enhancer in the c-RET gene. (a) Cotransfection of 293T cells with reporter constructs composed of portions of the c-RET upstream genomic sequence (see Methods) cloned upstream of a luciferase gene with pCMV-Pax3, pCMV-Sox10, or both reveals a 750-bp enhancer element (open box) and a more proximal repressor element (compare full-length Ret promoter to construct D15). All transfections were normalized for transfection efficiency (see Methods) and are expressed as fold activation compared with transfection without Pax3 or Sox10 (mean ± SD; n =12 for each condition). (b) Further analysis of the 750-bp enhancer was performed by modifying construct D15. Deletion of 45 bp at the 3′ end significantly reduced activity (compare constructs 615 and 570). Mutation in a putative Pax3 binding site (indicated by “X” in construct 615mut) had a similar effect, although activation by Sox10 alone was not affected by this mutation. Residual Pax3 responsiveness present in construct 570 is eliminated by deletion of 20 bp at the 5′ end of the enhancer (compare constructs 570 and 550). The 3′ and 5′ response elements are indicated by dotted lines and filled bars labeled A and B, respectively. Cotransfection results are expressed as in a, n=12 for each condition. (c) The response of construct 615 (depicted in b) is specific for Pax3 and is activated to a much lesser degree by Pax2, Pax6, and Pax9. Although low-level activation is apparent after cotransfection with Pax2 or Pax6, no synergistic activation with cotransfected Sox10 is evident. Cotransfection results are expressed as in a; n=8 for each condition. Pax3 and Sox10 bind to the c-RET enhancer. (d) EMSA using a 45-bp probe derived from the 3′ response element in the 750-bp c-RET enhancer (site A in part b) reveals specific binding by Pax3 (lane 3). Pax3 does not bind to a similar probe in which the putative Pax3 binding site has been replaced by an AscI restriction site (lane 4; and see Methods). Unlabeled competitor wild-type probe is able to compete for binding (lane 5), whereas mutated probe competes far less efficiently (lane 6), indicating binding specificity. (e) Sox10 also binds to site A, and mutation in the putative Sox10 binding site significantly reduces binding. In vitro translated Sox10 forms two complexes in EMSA experiments with probe A (lanes 2 and 3), whereas the reticulocyte lysate without Sox10 does not bind (lane 1). Mutation in the putative Sox10 binding site (see Methods) reduces Sox10 binding (lane 4). Lanes 1 and 2 are from a different gel than lanes 3 and 4. (f) The sequence of probe A is shown with Sox10 and Pax3 binding sites boxed.