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. 2011 May;4(3):368-80.
doi: 10.1242/dmm.005744. Epub 2010 Dec 23.

Boc modifies the holoprosencephaly spectrum of Cdo mutant mice

Wei Zhang  1 , Mingi Hong, Gyu-un Bae, Jong-Sun Kang, Robert S Krauss
Affiliations

Boc modifies the holoprosencephaly spectrum of Cdo mutant mice

Wei Zhang et al. Dis Model Mech. 2011 May.

Abstract

Holoprosencephaly (HPE) is caused by a failure to form the midline of the forebrain and/or midface. It is one of the most common human birth defects, but clinical expression is extremely variable. HPE is associated with mutations in the sonic hedgehog (SHH) pathway. Mice lacking the Shh pathway regulator Cdo (also called Cdon) display HPE with strain-dependent penetrance and expressivity, implicating silent modifier genes as one cause of the variability. However, the identities of potential HPE modifiers of this type are unknown. We report here that whereas mice lacking the Cdo paralog Boc do not have HPE, Cdo;Boc double mutants on a largely Cdo-resistant genetic background have lobar HPE with strong craniofacial anomalies and defects in Shh target gene expression in the developing forebrain. Boc is therefore a silent HPE modifier gene in mice. Furthermore, Cdo and Boc have specific, selective roles in Shh signaling in mammals, because Cdo;Boc double-mutant mice do not display the most severe HPE phenotype seen in Shh-null mice, nor do they have major defects in digit patterning or development of vertebrae, which are also Shh-dependent processes. This is in contrast to reported observations in Drosophila, where genetic removal of the Cdo and Boc orthologs Ihog and Boi results in a complete loss of response to the hedgehog ligand. Therefore, there is evolutionary divergence between mammals and insects in the requirement of the hedgehog pathway for Cdo/Ihog family members, with mammalian development involving additional factors and/or distinct mechanisms at this level of pathway regulation.

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Figures

Fig. 1.
Fig. 1.
Boc mutant mice are viable. (A) Strategy for targeted mutagenesis of the Boc locus via homologous recombination. The wild-type allele (Boc), targeting vector and resulting targeted alleles (BocAP-1 and BocAP-2) are shown with relevant restriction sites and exons (black boxes). BocAP-1 and BocAP-2 were generated by homologous recombination at one of two regions of homology present in the 5′ end of the targeting vector (designated crossover points C1 and C2, respectively); the 3′ crossover point (C3) is the same for both alleles. Note that BocAP-1 deletes exon 1 and contains an insertion of a human alkaline phosphatase (AP) reporter gene [under the control of an internal ribosome entry site (IRES); not shown] linked to a PGK-Neo selectable marker gene. BocAP-2 contains only the insertion. (B) Genotype analysis of ES cell clones by Southern blot. Lane numbers represent ES clone numbers, and bands corresponding to wild-type (wt), BocAP-1 and BocAP-2 alleles are indicated. (C) PCR genotyping analysis of tail DNA from offspring of intercrosses of Boc+/− animals, showing all three possible genotypes. (D) Western blot analysis of Boc expression in wild-type and mutant animals. E13.5 embryos from intercrosses of Boc+/AP-1 (left panel) and Boc+/AP-2 (right panel) mice were genotyped, and extracts from embryonic heads were analyzed by immunoblotting with antibodies against Boc. Genotypes are indicated above the lanes. (E) Whole E11.5 embryos of all three genotypes were stained for AP activity, which mirrors normal Boc expression. Notably, Boc is expressed in dorsal telencephalon (arrowhead), facial primodia (black arrow) and anterior limb buds (white arrow). Scale bar: 1 mm.
Fig. 2.
Fig. 2.
HPE phenotypes in Cdo–/−;Boc–/− mice on a 129 background. (A) Frontal views of E11.5 embryos of the indicated genotypes. Note that the distance between the left and right nasal pits (denoted by brackets) is dramatically reduced but somewhat variable in Cdo–/−;Boc–/− embryos (panels d, e) compared with the control embryo (panel a). Scale bar: 0.5 mm. (B) Frontal views of forebrains and faces of E13.5 (upper panel) and E15.5 (lower panel) embryos of the indicated genotypes. Note that the Cdo–/−;Boc–/−embryos have fused nostrils (panels d, h; black arrows) and upper lip (panels d, h; red arrows) but display separation of the left and right forebrain hemispheres. A higher magnification view of the E13.5 Cdo–/−;Boc–/−embryo highlights the defects. Scale bars: 1 mm; 0.25 mm for enlargement. (C) H&E staining of frontal forebrain sections of E13.5 embryos. Cdo–/−;Boc–/−embryos display lobar HPE as indicated by continuity across the ventral midline (panels d, e; red arrows) and reduced medial ganglionic eminences. M, medial ganglionic eminence; L, lateral ganglionic eminence. Scale bar: 0.5 mm. (D) In situ hybridization analysis of Ptch1 (middle panel) and Nkx2.1 (lower panel) expression, revealing diminished expression in Cdo–/−;Boc–/− embryos. Scale bars: 0.5 mm.
Fig. 3.
Fig. 3.
Cranial bone defects in Cdo–/−;Boc–/−embryos. Ventral views of cranial bone preparations of E18.5 embryos stained with Alizarin Red and Alcian Blue for bone and cartilage, respectively. Mandibles have been removed for visualization of the palate. Some Cdo–/−;Boc+/+ embryos have cranial bone defects including fused premaxillary bones (c; white arrow), missing premaxillary shelves, dysmorphogenesis of the maxillary bones and underdeveloped maxillary shelves (c; white asterisk). Cdo–/−;Boc+/− and Cdo–/−;Boc–/−embryos also show fused premaxillary bones (e–h; white arrows), as well as more severe cranial bone defects than Cdo–/−;Boc+/+ embryos, in a Boc dosage-dependent manner. These latter defects include open palatal processes of the palatine bones (f; red asterisk), highly dysmorphic maxillary bones (e,g,h; red arrows), and underdeveloped basisphenoid bones (f,h; red arrowheads). The larger image in the lower right corner (a’) is an enlargement of the control Cdo+/−;Boc+/−preparation (a) for the purpose of labeling structures. PM, premaxillary bone; M, maxillary bone; MS, maxillary shelves; PP, palatal process of the palatine; PS, premaxillary shelves; BS, basisphenoid bone; BO, basioccipital bone. Scale bar: 1 mm.
Fig. 4.
Fig. 4.
Expression of Shh and Fgf8 correlate with the strain-dependent severity of forebrain phenotypes in Cdo and Boc mutants. (A–C) Whole-mount RNA in situ hybridization analyses. (A) Fgf8 expression in E8.75 Cdo+/+ and Cdo–/− embryos on a B6 background. Note that Fgf8 expression is induced properly in the Cdo–/− embryo in both the anterior neural ridge (arrow) and isthmus (arrowhead). Scale bar: 0.5 mm. (B) Lateral view of Shh expression in E9.5 embryos of the indicated genotype on a 129 background. Note that Shh is expressed in the ventral telencephalon of the Cdo–/−;Boc–/−embryo (d, arrow) but signal strength is reduced compared with the control embryo (a). Scale bar: 0.2 mm. (C) Lateral view of Fgf8 expression in E9.5 embryos of the indicated genotype on a 129 background. Note that Fgf8 is expressed in the commissural plate of the Cdo–/−;Boc–/− embryo (d; arrow). Scale bar: 0.2 mm.
Fig. 5.
Fig. 5.
Reduction of Shh signaling in the ventral forebrain and facial primodia of 129.Cdo–/−;Boc–/−embryos at E10.5. (A–D) Whole-mount RNA in situ hybridization analyses. (A) Lateral and ventral views of Shh expression in the ventral forebrain and facial epithelium of E10.5 embryos of the indicated genotype on a 129 background. Note the dramatic reduction of Shh expression in the ventral forebrain of the Cdo–/−;Boc–/− embryo (denoted by the bracket, telencephalon indicated with an arrow). Scale bars: 0.5 mm (lateral); 0.2 mm (ventral). (B) Lateral and ventral views of Ptch1 expression in the ventral forebrain and facial epithelium of E10.5 embryos of the indicated genotype on a 129 background. Scale bars: 0.5 mm (lateral); 0.2 mm (ventral). (C) Lateral and ventral views of Gli1 expression in the ventral forebrain of E10.5 embryos on a 129 background. Scale bars: 0.5 mm (lateral); 0.2 mm (ventral). (D) Ventral view of Fgf8 expression in commissural plates of E10.5 embryos on a 129 background. The head of the Cdo–/−;Boc–/−embryo is smaller than the control Cdo+/−;Boc+/−embryo but was alive, and the two embryos were of similar somite number and size (supplementary material Fig. S1). Scale bar: 0.5 mm. (E) Frontal view of Nkx2.1 expression in E10.5 embryos on a 129 background. Scale bar: 0.5 mm.
Fig. 6.
Fig. 6.
qPCR analysis of Shh, Fgf8 and Nkx2.1 expression in control and Cdo–/−;Boc–/− embryonic heads at E10.5. (A) Shh expression. (B) Fgf8 expression. (C) Nkx2.1 expression. Signals were normalized to Gapdh expression, which was the same in all embryos, and the mean values for control embryos were set to 1.0. Control embryos were of the Cdo+/+;Boc+/−, Cdo+/−;Boc+/+ or Cdo+/−;Boc+/−genotypes. *P<0.05 in Student’s t-test, n≥3 embryos.
Fig. 7.
Fig. 7.
Cdo–/−;Boc–/− mice display defects in palatogenesis. (A) Ventral views of E17.5 palates of embryos of the indicated genotype on a 129 background. Lower jaws have been removed for visualization of palates. PP, primary palate; SP, secondary palate. Black arrows indicate an open palate in Cdo–/−;Boc+/− and Cdo–/−;Boc–/− embryos (f,h). The red arrow indicates abnormal rugae in the secondary palate of a Cdo–/−;Boc–/− embryo (g). The red arrowheads indicate misshapen primary palates in Cdo–/−;Boc+/− and Cdo–/−;Boc–/− embryos (e–h). Scale bar: 1 mm. (B) H&E staining of palate sections from E13.5, E14.5 and E15.5 embryos of a control embryo and three different Cdo–/−;Boc–/− embryos; note that the double-mutant embryos display various palatogenesis defects. See text for detailed description. ps, palatal shelves; T, tongue; asterisk, molar; arrow in e indicates the medial epithelial seam (MES). Scale bars: 0.5 mm. (C) RNA in situ hybridization of Ptch1 expression in E13.5 palate sections. Scale bar: 0.5 mm.
Fig. 8.
Fig. 8.
Limb, digit and cervical vertebrae development in Cdo–/−;Boc–/−mice. (A) Whole E15.5 Cdo+/−;Boc+/− (control, a) and Cdo–/−;Boc–/− embryos (f) and their four limbs (b–e and g–j). RF, right forelimb; LF, left forelimb; RH, right hindlimb; and LH, left hindlimb. Scale bars: 1 mm. (B) Frontal view of cervical vertebrae of E18.5 mice of the indicated genotype. Note the lack of ossification of intervertebral disks in the Cdo–/−;Boc–/− embryo (arrow). Scale bar: 1 mm.
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