Selection for increased tibia length in mice alters skull shape through parallel changes in developmental mechanisms

Abstract

Bones in the vertebrate cranial base and limb skeleton grow by endochondral ossification, under the control of growth plates. Mechanisms of endochondral ossification are conserved across growth plates, which increases covariation in size and shape among bones, and in turn may lead to correlated changes in skeletal traits not under direct selection. We used micro-CT and geometric morphometrics to characterize shape changes in the cranium of the Longshanks mouse, which was selectively bred for longer tibiae. We show that Longshanks skulls became longer, flatter, and narrower in a stepwise process. Moreover, we show that these morphological changes likely resulted from developmental changes in the growth plates of the Longshanks cranial base, mirroring changes observed in its tibia. Thus, indirect and non-adaptive morphological changes can occur due to developmental overlap among distant skeletal elements, with important implications for interpreting the evolutionary history of vertebrate skeletal form.

Keywords: Longshanks mouse; correlated evolution; cranial shape; developmental biology; endochondral ossification; evolutionary biology; geometric morphometrics; mouse.

Figure 1.. Scatter plots of the first two principal components (PC) of Procrustes shape variables in adult Longshanks and Controls throughout the selection process.

(A) Plot of sex-adjusted Procrustes shape variables (left), and vectors of shape change at each cranium landmark (magnified two times for visualization) showing shape transformations along PC1 from negative to positive scores (right). Large symbols indicate mean PC1 and PC2 scores for each respective cohort. (B) Plot of Procrustes shape variables additionally corrected for size.

Figure 1—figure supplement 1.. Boxplots of adult Longshanks and Control metrics.

(A–C) Boxplots of adult body mass, tibia length, and cranial centroid size between groups. See also Supplementary file 1.

Figure 1—figure supplement 2.. Scatter plots with regression lines by group showing the relationships between body mass and cranium size (centroid size) in adult founder mice (F01) and after 20 generations of selection (F20).

Figure 1—figure supplement 3.. Scatter plot of fitted PC1 scores (shape scores predicted by regression of shape on size) vs log (centroid size) showing within group patterns of cranium allometry.

At any given cranium size, LS1 and LS2 are predicted to have positive shape scores (longer and narrower) shapes.

Figure 1—figure supplement 4.. Adult cranium landmarks used in this study in lateral, dorsal, and ventral landmark views.

For landmark anatomical definitions, see Supplementary file 4.

Figure 2.. Scatter plots with regression lines by cohort showing the relationships between PC1 score (cranium shape) and tibia length in adult mice throughout selection.

Cranium shape data is adjusted for the effects of sex and size related allometry.

Figure 3.. Intergenerational shape changes within both Longshanks lines and Controls throughout the selection process.

Intergenerational shape changes within both Longshanks lines and Controls throughout the selection process. Top: Density plots following intergenerational shifts in mean PC1 scores within LS1 (left), LS2 (center), and CTL (right) lines for size and sex adjusted shape data. Bottom: Heatmaps showing shape transformations between mean shapes in the first 9 generations of selection (F01 to F09) and the next 11 generations (F09 to F20) after correcting for size and sex effects. Blue indicates areas of relative reduction, red indicates areas of relative expansion, and green indicates neutral areas. Longshanks independently undergo vault height reduction between generations F01 and F09, followed by snout elongation between generations F09 and F20 (blue arrows).

Figure 4.. Scatter plots of the first principal components (PC) in neonate Longshanks and Control cranium Procrustes shape variables at generation 32 (F32).

Left: Plot of litter size adjusted Procrustes shape variables (left), large symbols indicate mean PC1 and PC2 scores for each respective cohort. Shapes of individual points indicate Longshanks lines (circle = CTL, square = LS1, and triangle = LS2). Right: Neonate cranium with vectors of shape change at each cranium landmark (magnified four times for visualization) showing shape transformations along PC1 (top) and along PC2 (bottom) from negative to positive scores.

Figure 4—figure supplement 1.. Boxplots showing differences in neonate Longshanks and Control metrics.

(A) Boxplot of neonate litter sizes. (B and C) Boxplots of litter size adjusted tibia length and cranium centroid size.

Figure 4—figure supplement 2.. Neonate (P07) cranium landmarks used in this study in lateral, dorsal, dorsal cranial base, caudal, and ventral landmark views.

For landmark anatomical definitions, see Supplementary file 5.

Figure 5.. Comparison of adult and neonate cranial phenotypes through shape change heatmaps.

Heatmaps show the deformations required to transform between the mean shape of a given cohort to the mean shape of another. Blue indicates areas of relative reduction, red indicates areas of relative expansion, and green indicates neutral areas. Scale bar = 10 mm.

Figure 6.. Cranial base contribution to the Longshanks phenotype.

(A) Scatter plot of the first two PCs of litter size-adjusted Longshanks and Control cranial base Procrustes shape variables in generation 32 (F32) neonates. Large symbols indicate mean PC1 and PC2 scores for each respective cohort. (B) Neonate cranial bases with vectors of shape change at midline cranial base landmarks (magnified six times for visualization) showing shape transformations to go from the mean Control cranial base to the mean LS1 (blue) and mean LS2 (green) cranial base shapes. Views in (B) are dorsal (left) and lateral (right). Basi-occipital bone (BO), spheno-occipital synchondrosis (SOS), basi-sphenoid bone (BS), intersphenoidal synchondrosis (ISS), presphenoid bone (PS) and ethmoid (ET). (C) Sagittal midline histological sections stained in H + E (top) and safranin-o (bottom) showing differences in synchondrosis morphology of two extreme specimens in CTL and LS1 of approximately equal centroid size (indicated by dashed circles in A).

Figure 6—figure supplement 1.. Neonate (P07) cranial base landmarks on the sagittal midline used in this study.

Numbered landmarks applied to CT scan reconstruction slices at the midline (left) and landmarks numbered on a 3D cranial base mesh (right). For landmark anatomical definitions, see Supplementary file 6.