Modeling the complex etiology of holoprosencephaly in mice

Abstract

Holoprosencephaly (HPE) is a common developmental defect caused by failure to define the midline of the forebrain and/or midface. HPE is associated with heterozygous mutations in Nodal and Sonic hedgehog (SHH) pathway components, but clinical presentation is highly variable, and many mutation carriers are unaffected. It is therefore thought that such mutations interact with more common modifiers, genetic and/or environmental, to produce severe patterning defects. Modifiers are difficult to identify, as their effects are context-dependent and occur within the complex genetic and environmental landscapes that characterize human populations. This has made a full understanding of HPE etiology challenging. We discuss here the use of mice, a genetically tractable model sensitive to teratogens, as a system to address this challenge. Mice carrying mutations in human HPE genes often display wide variations in phenotypic penetrance and expressivity when placed on different genetic backgrounds, demonstrating the existence of silent HPE modifier genes. Studies with mouse lines carrying SHH pathway mutations on appropriate genetic backgrounds have led to identification of both genetic and environmental modifiers that synergize with the mutations to produce a spectrum of HPE phenotypes. These models favor a scenario in which multiple modifying influences-both genetic and environmental, sensitizing and protective-interact with bona fide HPE mutations to grade phenotypic outcomes. Despite the complex interplay of HPE risk factors, mouse models have helped establish some clear concepts in HPE etiology. A combination of mouse and human cohort studies should improve our understanding of this fascinating and medically important issue.

Keywords: gene-environment interaction; hedgehog signaling; holoprosencephaly; modifier genes; mouse; mutation; nodal signaling; teratogen.

FIGURE 1

Schematic of Nodal and Shh Signaling. (a) Simplified schematic of Nodal signaling. Nodal binds a receptor complex that includes ActRII, Alk4, and Cripto, leading to Smad2/3 phosphorylation. P-Smad2/3 binds Smad4 and, with FoxH1, activates pathway target genes. (b) Simplified schematic of Shh signaling. In the absence of Shh (far right cell), Ptch1 inhibits Smo activity, leading to proteolysis of Gli2 and Gli3 (Gli2/3) to transcriptional repressor forms (GliR), which inhibit pathway target gene expression. Shh is produced as a precursor product (Shh(pre); far left cell). Upon autoproteolysis, cholesterol adduction, and palmitoylation by Skn, mature Shh ligand is produced. Shh is released from producing cells in a Disp1-dependent manner. Upon binding of Shh to a complex of Ptch1 and at least one coreceptor (Cdon, Boc, or Gas1) (center cell), Smo inhibition by Ptch1 is relieved. Smo signals to block Gli2/3 proteolysis, allowing formation of transcriptional activator forms (GliA), which induce pathway target gene expression. Factors indicated in color have been identified as targets of mutation in HPE.

FIGURE 2

129S6 Cdon^−/− mice display modifier-dependent HPE phenotypes. (a1–a3) Cdon^−/− mice on a 129S6 genetic background are largely normal, displaying two nostrils and normally partitioned upper lip (a1; arrow); two optic vesicles (a2; arrowheads); and normally partitioned forebrain (a3). We note that a minority of these mice show mild midfacial defects (not shown, see text). (b1) Genetic removal of Boc enhances this phenotype in a dosage sensitive manner, resulting in pronounced midfacial midline hypoplasia, fused upper lip, and single nostril (red arrow). (c1–c3) In utero exposure of 129S6 Cdon^−/− mice to alcohol leads to a full spectrum of HPE phenotypes, including in: (c1) single nostril (a mid-severity phenotype; arrow); (c2) unpartitioned forebrain plus cyclopia (the most severe phenotypes; arrow and arrowhead, respectively); and (c3) partitioned forebrain with ventral continuity (a mild phenotype; arrow). (d1) Genetic removal of one copy of the negative Shh pathway regulator Ptch1 rescues HPE phenotypes in alcohol-treated 129S6 Cdon^−/− mice. (e1, e2) C57BL/6 Cdon^−/− have more severe HPE than 129S6 Cdon^−/− mice, and show single nostril (e1; arrow) and unpartitioned forebrain (e2; arrow). (f1, f2) Genetic removal of Boc enhances this phenotype in a dosage sensitive manner, resulting in severely hypoplastic midface and hypotelorism (f1) and cleft lip (f2; arrow). All whole mount embryos are at E14.5 or E15.5. Sections are at E10.5 (a2, c2, e2) and E14.5 (a3, c3).