THE EFFECTS OF IN UTERO THYROXINE EXPOSURE ON MANDIBULAR SHAPE IN MICE by Matthew J. Kesterke B.A., Anthropology, University of Wyoming, 2003 M.A., Anthropology, University of Wyoming 2008 Submitted to the Graduate Faculty of the Kenneth P. Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Anthropology University of Pittsburgh 2016 UNIVERSITY OF PITTSBURGH DIETRICH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Matthew J. Kesterke It was defended on October 21st, 2016 and approved by James M. Cray, PhD, Assistant Professor Mark P. Mooney, PhD, Professor Michael I. Siegel, PhD, Professor Co-Chair: Seth M. Weinberg, PhD, Associate Professor Dissertation Advisor: Margaret M. Judd, PhD, Associate Professor ii Copyright © by Matthew J. Kesterke 2016 iii THE EFFECTS OF IN UTERO THYROXINE EXPOSURE ON MANDIBULAR SHAPE IN MICE Matthew J. Kesterke, PhD University of Pittsburgh, 2016 An estimated 3% of U.S. pregnancies are affected by maternal thyroid dysfunction, with between one and three of every 1,000 pregnancies being complicated by overactive maternal thyroid levels. Overactive maternal thyroid hormones have been linked to neurological impairment and craniofacial development dysmorphogenesis, affecting both endochondral and intramembranous bone. Using both Euclidean Distance Matrix Analysis (EDMA) and geometric morphometric approach, this study evaluates the role of in utero thyroxine overexposure on the growth of offspring mandibles in a sample of 241 mice. Principle component analysis (PCA) and canonical variate analysis (CVA) utilized 16 unilateral mandibular landmarks obtained from 3D microCT to assess shape changes between unexposed controls (n=63) and exposed mice (n=178). By evaluating shape changes in the mandible between different age groups (15, 20, and 25 days postnatal) and different dosage levels (low, medium, and high), this study found that maternal thyroxine alters offspring mandibular shape in both age- and dosage-dependent manners, particularly within the high dosage individuals in the oldest age group. The EDMA results demonstrate marked shape changes throughout the mandible, with the gonial angle and alveolus undergoing significant (p <0.10) changes. Geometric morphometric analysis revealed that group differences in overall shape were significant (p <0.001 for both PCA and CVA) and showed major changes in regions of the mandible associated with muscle attachment (coronoid process, gonial angle) and regions of growth largely governed by articulation with the cranial base (condyle) and occlusion (alveolus). These results compliment recent studies demonstrating that maternal thyroxine levels can alter the cranial base and cranial vault of offspring, contributing to a better understanding of both normal and abnormal mandibular development and facilitating a fuller understanding of evolutionary and medical implications of craniofacial growth and development. iv TABLE OF CONTENTS TABLE OF CONTENTS .............................................................................................................V LIST OF TABLES ................................................................................................................... VIII LIST OF FIGURES ......................................................................................................................X PREFACE ................................................................................................................................. XIII 1.0 INTRODUCTION ........................................................................................................ 1 1.1 RESEARCH RATIONALE ................................................................................ 2 1.2 LITERATURE REVIEW ................................................................................... 2 1.2.1 Normal Bone Growth ................................................................................... 2 1.2.2 Role of the Thyroid ..................................................................................... 14 1.2.3 Thyroid and Craniofacial Studies ............................................................. 19 1.2.4 History of Mandibular Research ............................................................... 21 1.2.5 Normal Prenatal Growth and Development of the Mandible ................ 33 1.2.6 Postnatal Mandibular Growth and Development. ................................... 40 1.3 RESEARCH GOALS AND DESIGN .............................................................. 44 1.3.1 Hypotheses ................................................................................................... 44 1.4 RESEARCH SIGNIFICANCE......................................................................... 45 2.0 MATERIALS AND METHODS .............................................................................. 48 2.1 ANIMAL MODEL............................................................................................. 48 2.2 MICRO-CT SCANNING .................................................................................. 50 2.2.1 Landmarking ............................................................................................... 51 2.2.2 Observer Error............................................................................................ 54 v 2.3 EUCLIDEAN DISTANCE MATRIX ANALYSIS ......................................... 56 2.3.1 Application of Euclidean Distance Matrix Analysis ................................ 59 2.4 GEOMETRIC MORPHOMETRIC ANALYSIS ........................................... 60 2.4.1 Application of Geometric Morphometric Analysis .................................. 64 2.5 DATA MANAGEMENT PROTOCOL ........................................................... 73 3.0 RESULTS.................................................................................................................... 74 3.1 EUCLIDEAN DISTANCE MATRIX RESULTS ........................................... 74 3.1.1 Age-Based EDMA Results .......................................................................... 74 3.1.2 EDMA Results Summary ........................................................................... 94 3.2 GEOMETRIC MORPHOMETRIC RESULTS ............................................. 94 3.2.1 ANOVA ........................................................................................................ 95 3.2.2 Principle Component Analysis Results ..................................................... 98 3.2.3 Canonical Variate Analysis Results ........................................................ 109 3.2.4 GM RESULTS SUMMARY .................................................................... 115 4.0 DISCUSSION ........................................................................................................... 116 4.1.1 EDMA Shape Changes ............................................................................. 117 4.1.2 GM Shape Changes................................................................................... 123 4.1.3 Comparison of EDMA and GM Results ................................................. 129 4.1.4 Overall Shape Changes and Existing Literature ................................... 131 4.1.5 Strengths and Limitations ........................................................................ 134 4.1.6 Future Directions ...................................................................................... 135 APPENDIX A: AMIRA PROTOCOL .................................................................................... 138 APPENDIX B: EDMA OUTPUTS .......................................................................................... 148 vi APPENDIX C: GM OUTPUTS ............................................................................................... 158 BIBLIOGRAPHY ..................................................................................................................... 165 vii LIST OF TABLES Table 1. Major hormones/growth factors affecting skeletal growth ............................................. 13 Table 2. Stages of mandibular development in both humans and mice........................................ 35 Table 3. Sample sizes of C57BL/6 individuals. ............................................................................ 50 Table 4. Order of mandibular landmarks (left side). .................................................................... 53 Table 5. Intra-observer error calculations. .................................................................................... 55 Table 6. Mean form matrix for the 20-day low dose sample. ....................................................... 77 Table 7. Mean form matrix for 20-day high dose sample............................................................. 78 Table 8. Mean shape matrix for the 20-day low dose sample. ..................................................... 79 Table 9. Mean shape matrix for the 20-day high dose sample. .................................................... 80 Table 10. Sorted shape difference matrix for the 20-day samples ................................................ 81 Table 11. Mean form matrix for the 25-day control sample. ........................................................ 86 Table 12. Mean form matrix for the 25-day high dose sample. .................................................... 87 Table 13. Mean shape matrix for the 25-day control sample. ...................................................... 88 Table 14. Mean shape matrix for the 25-day high dose sample. .................................................. 89 Table 15. Sorted shape difference matrix for the 25-day samples ................................................ 90 Table 16. ANOVA results for treatment for both size and shape for the combined data. ............ 96 Table 17. ANOVA results for both size and shape within the 20-day and 25-day samples. ........ 97 Table 18. PCA results within the 15-day old sample.................................................................... 99 Table 19. PCA results within the 20-day old sample.................................................................. 102 Table 20. PCA results a within the 25-day old sample. .............................................................. 105 Table 21. Between group distance statistics from CVA ............................................................. 110 viii Table A1. Setting a threshold and visualizing the AmiraMesh file............................................. 144 ix LIST OF FIGURES Figure 1. Frost's 'mechanostat' feedback loop ............................................................................... 11 Figure 2. The ‘Utah paradigm’ ..................................................................................................... 14 Figure 3. Location and blood supply of the human thyroid hormone .......................................... 15 Figure 4. The role of thyroid hormones in the human body. ........................................................ 17 Figure 5. Enlow’s “V” Principle. .................................................................................................. 30 Figure 6. Meckel’s cartilage within the pharyngeal arches (lateral view). ................................... 37 Figure 7. Development of the alveolar process of the mandible (superior view). ........................ 37 Figure 8. Development of Meckel’s cartilage within the first pharyngeal arch (lateral view towards midline). ........................................................................................................................................ 39 Figure 9. Development of Meckel’s cartilage within the first pharyngeal arch (lingual view) .... 39 Figure 10. Development of Meckel’s cartilage within the first pharyngeal arch (lateral view towards midline). .......................................................................................................................... 40 Figure 11. Development of Meckel’s cartilage with the first pharyngeal arch (lingual view). .... 40 Figure 12. Postnatal growth of the human mandible. ................................................................... 43 Figure 13. Landmarks utilized for this study (left mandible). ...................................................... 54 Figure 14. Visual explanation of the steps involved in geometric morphometric analysis. ......... 64 Figure 15. MorphoJ output for identification of outliers .............................................................. 65 Figure 16. MorphoJ output of all landmarks ................................................................................ 66 Figure 17. Visual depiction of principle component analysis ....................................................... 68 Figure 18. Sample eigenvalues ..................................................................................................... 69 Figure 19. Sample visualizations of PCA from MorphoJ software. ............................................. 70 x