Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression
Mayumi Nagashimada, … , Teruhiko Wakayama, Hideki Enomoto
Mayumi Nagashimada, … , Teruhiko Wakayama, Hideki Enomoto
Published August 27, 2012
Citation Information: J Clin Invest. 2012;122(9):3145-3158. https://doi.org/10.1172/JCI63401.
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Research Article

Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression

Abstract

The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse Phox2b locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine β-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.

Authors

Mayumi Nagashimada, Hiroshi Ohta, Chong Li, Kazuki Nakao, Toshihiro Uesaka, Jean-François Brunet, Jeanne Amiel, Delphine Trochet, Teruhiko Wakayama, Hideki Enomoto

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Figure 1

Generation of NPARM PHOX2B knockin mice.

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Generation of NPARM PHOX2B knockin mice. (A) Protein structure of WT a...
(A) Protein structure of WT and NPARM (931 del5 and 693–700 del8) PHOX2B. Red arrows indicate the regions of the deletions. Dark gray boxes depict aberrant 42 amino acids shared by both del5 and del8 mutant proteins. HD, homeodomain; 9, 20, polyalanine stretches. (B) Schematics showing the gene-targeting strategy. Gray boxes indicate exons of the Phox2b gene. Red boxes show human genomic fragments spanning from the deletion mutations to the aberrant stop codon due to the shift in the ORF. A floxed (white triangles) Tn5-neo cassette was removed in vivo by Cre recombination. Bars depict the location of the probe for Southern blot analysis and PCR primers for screening the recombined ES clones. (C) Southern blot and PCR analysis of WT and the targeted allele of the Phox2b gene. Properly targeted ES clones (Mut) display both WT (14.5 kb) and mutant (7 kb) fragments. (D) ISH analysis of embryonic gut (E13.5). NPARM PHOX2B–specific riboprobes detected signals in enteric ganglion progenitors of del8 mutant but not in WT gut (bottom panels). Anti-WT Phox2b antibodies were used to visualize enteric ganglion progenitors (top panels). Scale bar: 50 μm.