Vandenberg LN et al. (2012), Normalized shape and location of perturbed cran...

XB-ART-44957

Dev Dyn 2012 May 01;2415:863-78. doi: 10.1002/dvdy.23770.

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Normalized shape and location of perturbed craniofacial structures in the Xenopus tadpole reveal an innate ability to achieve correct morphology.

Vandenberg LN , Adams DS , Levin M .

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Embryonic development can often adjust its morphogenetic processes to counteract external perturbation. The existence of self-monitoring responses during pattern formation is of considerable importance to the biomedicine of birth defects, but has not been quantitatively addressed. To understand the computational capabilities of biological tissues in a molecularly tractable model system, we induced craniofacial defects in Xenopus embryos, then tracked tadpoles with craniofacial deformities and used geometric morphometric techniques to characterize changes in the shape and position of the craniofacial structures. Canonical variate analysis revealed that the shapes and relative positions of perturbed jaws and branchial arches were corrected during the first few months of tadpole development. Analysis of the relative movements of the anterior-most structures indicates that misplaced structures move along the anterior-posterior and left-right axes in ways that are significantly different from their normal movements. Our data suggest a model in which craniofacial structures use a measuring mechanism to assess and adjust their location relative to other local organs. Understanding the correction mechanisms at work in this system could lead to the better understanding of the adaptive decision-making capabilities of living tissues and suggest new approaches to correct birth defects in humans.

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Species referenced: Xenopus
Genes referenced: ddx59 otc uqcc6

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	Figure 1. Four examples of tadpoles with perturbed craniofacial structures. In all four examples, the malformed jaw and branchial arch structures appear to “normalize” over time. In contrast, eyes change somewhat in shape but are never completely normalized. In all panels, blue arrowheads indicate malformed eyes, red arrowheads indicate malformed branchial arches, and red arrows indicate malformed jaws. All images are dorsal views except two panels showing ventral views marked with a V. Inset: schematics of the dorsal (top) and ventral (bottom) structures of the head and face. Olf, olfactory bulbs (nostrils); Br, brain; Otc, otocyst; Otl, otolith; MC, Meckel's cartilage (mandible/jaw); H, heart; BA, branchial arches.Download figure to PowerPoint
	Figure 2. Abnormalities in placode-derived tissues persist throughout tadpole stages. Placode-derived structures including eyes, nose, and otolith/otocyst were imaged in tadpoles over a range of ages. A: In a tadpole with conjoined eye–nose tissue at d9, a stalk of tissue clearly connects these two malformed structures. As the animal ages, the nostril moves away from the eye to its normal position, but a small strand of tissue remains connecting these structures. The eye is severely malformed at all ages examined. Pink arrow indicates eye, blue arrowhead indicates nostril, and yellow arrowhead points to the ectopic tissue connecting these two structures. B: A tadpole with a malformed left side is shown. The left otocyst is missing the otolith stones at all ages. Additionally, there is a pocket of abnormal ectopic tissue that is never resorbed. Green arrowheads indicate a normal otolith/otocyst, red arrowheads indicate an otocyst that is missing its otoliths, and the blue arrow indicates ectopic abnormal tissue.Download figure to PowerPoint
	Figure 3. Dorsal and ventral landmarks used for morphometric analysis. A total of 20 ventral (V) and 12 dorsal (D) landmarks were selected on the images collected of unaffected and perturbed tadpoles at several ages. Only perturbed tadpoles with an affected left or right side were used for morphometric analysis; tadpoles with visible defects on both sides were not included. This figure shows the dorsal and ventral landmarks on unaffected and perturbed tadpoles at 23 days postfertilization (d23). Green, unaffected; red, perturbed on left side; blue, perturbed on right side.Download figure to PowerPoint
	Figure 4. Outlines from principal components analysis (PCA) -derived landmark locations indicate that the average face appears to be normalized. A: PCA of dorsal landmarks for each treatment group and age reveal differences in the centroid placement for many landmarks at early stages. However, by later stages, the outlines derived from animals with craniofacial perturbations largely resemble the outlines derived from unaffected tadpoles. Green, unaffected; red, perturbed on the left side; blue, perturbed on the right side. B: PCA of ventral landmarks reveal centroid placements that do not appear to change considerably over time in unaffected tadpoles (green), with the exception of a lengthening of the anterior part of the face at later stages. In contrast, tadpoles with perturbed left (red) and right (blue) sides have branchial arch landmarks that are inappropriately close together and misplaced jaw landmarks at early stages. By late stages, the two halves of these faces are visibly more symmetrical and closely resemble the outlines of unaffected tadpoles.Download figure to PowerPoint
	Figure 5. Canonical variate analysis indicates that tadpoles with perturbed craniofacial features become indistinguishable from unaffected tadpoles. A: Analysis of ventral landmarks reveals significant differences between the three groups (unaffected [n = 10], perturbed left side [n = 17], perturbed right side [n = 19]) at early ages (d9, d23, d30). However, in later stages, these groups are no longer statistically distinguishable (P > 0.01). B: Canonical variate analysis reveals more complicated statistical relationships for dorsal landmarks. At early stages (d9, d23, d30) the three treatment groups are clearly different from each other. However, at d51–90 and d91–131, there are no longer any statistical differences distinguishing the perturbed groups from the unaffected controls (P > 0.01). Finally, at d131–167, both groups with perturbed faces were statistically distinguishable from unaffected controls (P < 0.01). For all panels, P values indicated in red are the differences between animals with perturbed left sides and unaffected controls. P values indicated in blue are the differences between animals with perturbed right sides and unaffected controls.Download figure to PowerPoint
	Figure 6. Anterior structures “calculate” their distances from each other during the normalization process. A: Landmarks for the left and right jaws, and the left and right nostrils were located in images collected from all three groups at d9, d23, d30, d51–d90, d91–d130, and d131–d167. This image shows the location of jaws (X) and nostrils (O) in unaffected and perturbed tadpoles at the earliest and latest ages examined. B: To calculate the relative differences in location of each landmark along the anterior–posterior and left–right axes, the coordinates describing the position of each landmark at day 167 were subtracted from the coordinates describing the location of the same landmark at d9. Shown here are two examples of the relative differences in location of the left jaw and left nostril in unaffected tadpoles. Clearly, between d9 and d167, the left nostril moves further in the anterior and right directions compared with the jaw on the same side. C: Quantification of movements in the anterior–posterior and left–right axes for left and right jaws and nostrils. In unaffected tadpoles (green), the majority of movements between d9 and d131–167 were in the anterior direction with little movement along the left–right axis. Animals with perturbations on the left side (red) had extensive anterior movements of the left jaw that were more than twice the distance moved in unaffected tadpoles, and decreased movements in the anterior direction for structures on the right side (jaw and nostril). Animals with perturbations on the right side (blue) had heightened movements of the right jaw that were largely in the anterior direction. Sample sizes for all analyses: unaffected n = 10, perturbed left side n = 10, perturbed right side n = 9.Download figure to PowerPoint
	Figure 7. Correct placement of craniofacial structures defined relative to the distance from the brain and the angle from the midline. A: Distances from the front of the brain (yellow square) were measured for nostrils (green dotted lines) and jaws (pink dotted lines) at each age. The angle from the midline (yellow line) was also calculated. B: As an example, the location of the left jaw is shown here at each timepoint measured for unaffected tadpoles (green) and tadpoles with perturbed left sides (red). The movements of the jaw in controls are relative minimal. In contrast, the position of the jaw is highly variable in animals with perturbed left sides. However, the final position of the jaw is very similar between these two groups. The movements of the jaw in controls are relative minimal. In contrast, the position of the jaw is highly variable in animals with perturbed left sides. However, the final position of the jaw is very similar between these two groups. C, D: When the distance from the brain and angle from the midline are quantified, it is clear that animals with perturbations in their craniofacial structures (red, perturbed left sides, n = 10; blue, perturbed right sides, n = 9) eventually achieve normal values for these parameters similar to those observed in unaffected controls (green, n = 10).Download figure to PowerPoint
	Figure 8. Craniofacial structures following metamorphosis of unaffected and perturbed tadpoles. A: Froglets were raised from unaffected tadpoles or tadpoles with craniofacial perturbations. Tadpoles with eye malformations metamorphosed into frogs with severe eye deformities. In contrast, tadpoles with defects in their jaws and/or branchial arches developed into froglets with normal exterior morphologies. The one exception was a froglet with a slight protrusion of the skin under the left eye. B: High resolution CT scans reveal remarkably normal craniofacial bone structures in the faces of froglets raised from tadpoles with craniofacial malformations. One froglet had slightly narrowed jaw bones (orange arrows) and a small section of the parasphenoid bone was absent (white arrow). Three orientations are shown from each sample. C: High resolution CT images of soft tissues reveal eye deformities (yellow arrow) only in those froglets raised from tadpoles with eye malformations. These images show similar coronal planes through the heads of froglets. D: Canonical variate analysis indicates that animals with either left eye perturbations (red) or right eye perturbations (blue) are significantly different from unaffected controls (green). However, froglets that were raised from tadpoles with malformed jaws and/or branchial arches (black) were not distinguishable from unaffected controls. P values indicated in red are the differences between animals with perturbed left eyes and unaffected controls, P values indicated in blue are the differences between animals with perturbed right eyes and unaffected controls, and P values in black are the differences between animals with jaw/branchial arch defects during tadpole stages and unaffected controls.Download figure to PowerPoint
	Figure 9. An algorithmic model for the mechanism dictating normalized position of craniofacial structures. A: During the first few hours of life, perturbations in bioelectricity can produce abnormal face states that manifest within a few days of development. Over a period of several weeks/months, a “ping” signal is sent between an “organizing center” and each craniofacial structure. If the organ is positioned properly, the ‘organizing center’ communicates a “stop” signal, migration of the organ halts, and the resulting face is normal in both tadpole and froglet stages. If the organ is not positioned appropriately, no “stop” signal is given, so the organ moves/migrates and the cycle of “ping” and “move/migrate” continues until the organ is properly localized. B: This system of ping/move/stop occurs independently in each organ/structure. Thus, if for example the ‘organizing center’ is located in the forebrain, a ping (blue arrow) is communicated between the brain and each structure (for example, each nostril). If the location of a structure is correct (i.e. on the unaffected side of an individual), the “stop” signal is sent (red arrow). But if this stop signal is not sent, the organ continues to move (shown here as a mislocalized nostril on the perturbed side, yellow arrow).Download figure to PowerPoint
	COVER PHOTOGRAPH: Visualization of the developing neural tube and craniofacial structures using pH and membrane voltage reporter dyes in Xenopus laevis embryos. Manipulation of pH and membrane voltage via misexpression of subunit c of the H(+) -V-ATPase produces craniofacial abnormalities that are capable of normalizing themselves as the animal develops and ages. From Vandenberg et al., Developmental Dynamics 241:863-878, 2012.

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