Chronic up-regulation of sonic hedgehog has little effect on postnatal craniofacial morphology of euploid and trisomic mice

Abstract

Background: In Ts65Dn, a mouse model of Down syndrome (DS), brain and craniofacial abnormalities that parallel those in people with DS are linked to an attenuated cellular response to sonic hedgehog (SHH) signaling. If a similarly reduced response to SHH occurs in all trisomic cells, then chronic up-regulation of the pathway might have a positive effect on development in trisomic mice, resulting in amelioration of the craniofacial anomalies.

Results: We crossed Ts65Dn with Ptch1(tm1Mps/+) mice and quantified the craniofacial morphology of Ts65Dn;Ptch(+/-) offspring to assess whether a chronic up-regulation of the SHH pathway rescued DS-related anomalies. Ts65Dn;Ptch1(+/-) mice experience a chronic increase in SHH in SHH-receptive cells due to haploinsufficiency of the pathway suppressor, Ptch1. Chronic up-regulation had minimal effect on craniofacial shape and did not correct facial abnormalities in Ts65Dn;Ptch(+/-) mice. We further compared effects of this chronic up-regulation of SHH with acute pathway stimulation in mice treated on the day of birth with a SHH pathway agonist, SAG. We found that SHH affects facial morphology differently based on chronic vs. acute postnatal pathway up-regulation.

Conclusions: Our findings have implications for understanding the function of SHH in craniofacial development and for the potential use of SHH-based agonists to treat DS-related abnormalities.

Keywords: Down syndrome; Ts65Dn; development; geometric morphometrics; patched.

© 2015 Wiley Periodicals, Inc.

Figures

Fig 1

Forty landmarks used in the study. Definitions provided in Table 2. A) Oblique supero-lateral view; B) Anterior view; C) Inferior view.

Fig 2

Principal component (PC) analysis of forty 3D cranial landmarks. PC1 primarily captures the differences between the euploid and trisomic morphology, with some distinction between the Ptch+/− (dashed convex hulls) and wildtype (solid convex hulls) individuals in the respective euploid and triosmic groups. The shape changes along the PCs are represented by surface reconstructions computed from the grand mean of the total dataset and warped according to the variation defined along the respective PC scores. PC1 shows shape changes consistent with ploidy in the neurocranium, which is markedly more rounded and raised in the Ts;Ptch+/− and Ts;WT compared to the Eu;Ptch+/− and Eu;WT. PC2 mainly accounts for the within-group variation in all four groups, more so than in PC1, and also shape changes between the Ts;WT and Ts;Ptch+/− . The morphological changes on PC2 relate to the orientation of the snout and width of neucrocranium (not shown).

Fig 3

Principal component analysis of twenty-three facial landmarks. PC1 separates the euploid and trisomic groups, showing little difference between the Ptch+/− (dashed convex hulls) and their wildtype (solid convex hulls) individuals along this axis. The wireframe diagrams illustrate the shape changes (in black) from the negative to the positive end along each PC relative to the grand mean shape (in gray) computed from all the specimens in the sample. PC1 captures mediolateral expansion and contraction of the face (narrow and elongated in euploids and laterally expanded in both the trisomic groups), marked by the position of the orbital margin. PC2 accounts for changes between the Ptch+/− and WT mice, particularly between the Ts;Ptch+/− and Ts;WT. Shape changes along PC2 captures differences in the relative length of the snout, being shorter and retracted in the Ptch+/− mice compared to their WT counterparts.

Fig 4

Multivariate regression analysis of all forty Procrustes shape coordinates on centroid size. The plot illustrates differences in allometric variation between the euploid and trisomic mice. Both Ts;Ptch+/− (dashed convex hull) and Ts;WT (solid convex hull) mice are considerably smaller than the euploids, and show more variation in aspects of shape than size. The euploids are distinct from the trisomic mice in shape and are larger in size, with the Eu;Ptch+/− (dashed convex hull) being slightly larger in size than the Eu;WT (solid convex hull).

Fig 5

Principal component analysis on the multivariate regression residuals. The euploid and trisomic mice are less distinct from one another after the effects of size have been removed from the analysis. The Eu;Ptch+/− and Ts;Ptch+/− (dashed convex hulls) both occupy the positive end of PC1, showing slight overlap and subtle similarities along this axis.

Fig 6

Principal component analysis of all Ptch+/− mice (the Eu;Ptch+/− and Ts;Ptch+/− groups are indicated by the dashed convex hulls) and their wildtype littermates (solid convex hulls), and the SAG and Vehicle (Veh) treated euploid and trisomic mice (solid convex hulls). The plot illustrates the difference between an acute and chronic up-regulation of SHH on craniofacial morphology. The shape changes (in black) along the respective PCs are represented by wireframe diagrams of the extreme shapes at the end of each axis relative to the grand mean shape (in gray) computed from all the specimens in the dataset. PC1 separates a subset of affected SAG-treated mice from all the other groups in the sample. PC2 distinguishes all the trisomic mice, irrespective of gentoype, from all the euploid mice in the sample.