Lrp2 +/+ and Lrp2 +/− littermates served as controls ( n=66). Dorsal view of the head revealed a shortened and widened skull with impaired suture formation and an open fontanelle (arrowhead). (A) Lrp2 −/− C57BL/6N E18.5 embryos ( n=30) with craniofacial defects: underdeveloped or missing eyes (asterisk), cleft lip (arrow) and hypertelorism. Rescue of craniofacial malformations, HPE and heart defects in Lrp2 −/− FVB/N mice. Corpus callosum and the anterior commissure were present in the LRP2-deficient FVB/N mice ( Fig. 1B). In contrast, HPE was fully rescued in all Lrp2 −/− FVB/N mutants, which presented with correctly separated cortical hemispheres, olfactory bulbs and clearly defined lateral ventricles. HPE in Lrp2 −/− C57BL/6N embryos, resulting from impaired ventral midline specification, was fully penetrant and characterized by a fusion of the cortical hemispheres ( Fig. 1B, arrowheads) with a single lateral ventricle ( Fig. 1B, arrow) and absent olfactory bulbs ( Fig. 1B, asterisks). In contrast, Lrp2 −/− congenic mice with a FVB/N background showed 100% penetrant rescue of all the above described craniofacial defects ( Fig. 1A), except for a mild suture phenotype ( Fig. 1A, arrowhead). Further characteristic features in these mutants were a shortened skull and an open anterior suture ( Fig. 1A, arrowhead). In particular, they showed cleft lip ( Fig. 1A, arrow) and incompletely developed or even bilaterally or unilaterally absent eyes ( Fig. 1A, asterisk). Lrp2 −/− mice on a C57BL/6N background at embryonic stage (E) 18.5 displayed severe HPE-associated craniofacial dysgenesis ( Fig. 1A). Such intrafamilial variability of HPE phenotypes could be due to both environmental and genetic factors ( Heussler et al., 2002 Hong and Krauss, 2018 Ming and Muenke, 2002 Muenke and Beachy, 2000 Muenke and Cohen, 2000 Roessler et al., 1996). However, even within pedigrees carrying the same SHH mutation, HPE phenotypes vary among relatives and can range from alobar HPE, to facial abnormalities typical of HPE, to asymptomatic appearance of the carrier. Single mutations causing HPE have been identified ( Hayhurst and McConnell, 2003 Roessler and Muenke, 2010 Roessler et al., 1996) and, among those, mutations in human sonic hedgehog ( SHH) and its downstream effector genes account for at least 5% of autosomal dominant nonsyndromic HPE cases ( Wallis and Muenke, 1999, 2000 Wallis et al., 1999). During embryonic forebrain development, HPE can be induced by exposure to environmental toxins ( Krauss and Hong, 2016 Weiss et al., 2018) or can be due to genetic predisposition with mono- or polygenic contributions ( Krauss and Hong, 2016 Roessler and Muenke, 2010). HPE is characterized by impaired separation of the cerebral hemispheres and is typically accompanied by craniofacial malformations ( Geng and Oliver, 2009 Hong and Krauss, 2018 Krauss, 2007 Ming and Muenke, 2002 Muenke and Beachy, 2000). Holoprosencephaly (HPE) is the most common structural defect of human forebrain development. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity.
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To identify such modifiers, we established novel congenic mouse models. Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes.