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Immunohistochemical and 3D analysis of the human fetal palate Dool, Carly Jade 2016

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IMMUNOHISTOCHEMICAL AND 3D ANALYSIS OF HUMAN FETAL PALATE by  Carly Jade Dool B.Sc., McGill University, 2007 M.Sc., McGill University, 2009 DMD, McGill University, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Craniofacial Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   August 2016  © Carly Jade Dool, 2016   ii Abstract Objectives: Hard palate development occurs between 7-12 weeks post conception with the fusion of the epithelial lined maxillary prominences creating a midline epithelial seam. The failure of fusion or seam removal in hard palate leads to cleft palate or cyst formation. The mechanism of soft palate formation is less well defined. Evidence exists supporting both fusion and the alternative mechanism of merging. The aim of this study is to densely sample the late embryonic-early fetal period between 54-84days post-conception to determine the mechanism and timing of palate closure.   Methods: 28 human fetal heads aged 54-74days were serially sectioned and subjected to immunohistochemistry. Several archival specimens had the coverslips removed and were used for IHC. Seven fetal heads aged 67-84days underwent MRI and microCT with phosphotungstic acid contrast agent. Qualitative analysis of 3-dimensional shape changes during palatal development was completed using a 3D slicer program.   Results: We confirm the presence of an epithelial seam extending throughout the soft palates in 57-day specimens suggesting fusion. Cytokeratin antibody staining confirmed the epithelial character of the cells in the midline seam and that there was no difference in the intensity of staining in the endodermal versus ectodermally-derived epithelium. There was surprisingly no E-Cadherin antibody staining in the midline seam although positive signal was found in the dental lamina and dorsal surface of the tongue. MF-20 antibody staining identified the facial musculature including palatine muscles controlling movement of the soft palate. The midline seam in the soft palate is rapidly degraded prior to 64days, however MRI and PTA-microCT imaging revealed the hard palate midline seam is almost completely intact until at least 84-days.   Conclusions: The epithelial character of the midline seam in the hard and soft palate has similar cytokeratin antibody staining. There is a difference in E-Cadherin staining which, may be tied to epithelial-mesenchymal transformation that takes place in the midline epithelial seam. The 3D-imaging results were superior with PTA staining and revealed the complex anatomy of the oropharynx. PTA staining can be used in the future to comprehensively document the fusion process as well as muscular development in the soft palate.     iii Preface Human conceptuses were obtained from the University of Washington between 1988 and 1991 by Dr. V. M. Diewert.  The work was carried out under UBC Human Ethics approval # H08-02576 (Dr. Joy Richman, PI). This protocol was approved Nov. 18, 2011 and is renewed annually.  Work was supported by Faculty of Dentistry research funds to JMR. A portion of this thesis was published in the following article:  Danescu, A., Mattson, M., Dool, C., Diewert, V.M., Richman, J.M., 2015. Analysis of human soft palate morphogenesis supports regional regulation of palatal fusion. Journal of Anatomy. Volume 227, pg 474-486.  Author contributions to this publication: Danescu, A: Primary author, contributed to writing, formatting figures/ photos of slides, and editing. He also contributed to the supervision of various immunohistochemistry experiments.   Mattson, M.: Sectioning of 13 specimens and subsequent immunohistochemistry staining with Picrosirius red, Alcian blue, Hematoxylin, Eosin, MF20, as well as some light microscopy photos. 3D reconstruction of 11 sectioned and stained specimens between 54-64 days post conception. She was also involved in minor editing of the manuscript.   Dool, C.: Sectioning of 15 specimens and subsequent immunohistochemistry staining with Picrosirius red, Alcian blue, Hematoxylin, Eosin, MF-20, Anti-cytokeratin, as well as some light microscopy photos. I performed the removal of coverslips and IHC for cytokeratin as well as IHC on archival sections that were not stained previously. I was also involved in minor editing of the manuscript.   Diewert, V.M.: Provided the human specimens for the study and involved in editing.   Richman. J.M.: Primary investigator, involved heavily in the writing, formatting, editing of the manuscript, and the supervision of all students involved.        iv Table of Contents  Abstract .......................................................................................................................................... ii	Preface ........................................................................................................................................... iii	Table of Contents ......................................................................................................................... iv	List of Tables ............................................................................................................................... vii	List of Figures ............................................................................................................................. viii	List of Abbreviations ................................................................................................................... ix	Acknowledgements ...................................................................................................................... xi	Chapter 1: Introduction ................................................................................................................1	1.1	 Embryonic craniofacial development – origins of craniofacial tissues .......................... 1	1.2	 Human palatogenesis ...................................................................................................... 4	1.2.1	 Primary palate development ................................................................................... 4	1.2.2	 Secondary palate development ............................................................................... 4	1.2.3	 Soft palate development .......................................................................................... 7	1.2.4	 Soft palate myogenesis ......................................................................................... 11	1.3	 Orofacial clefts .............................................................................................................. 13	1.3.1	 Cleft lip and/or palate (CL/P) ............................................................................... 13	1.3.2	 Microforms of cleft lip and palate ........................................................................ 14	1.3.3	 Genetic factors may contribute to cleft development ........................................... 14	1.4	 3-dimensional evaluation of human embryo/fetal development ................................... 15	1.4.1	 3D imaging of the palate in vivo using ultrasound ............................................... 15	1.4.2	 Evaluation of fetal specimens by MRI in vitro ..................................................... 17	  v 1.4.3	 Evaluation of fetal specimens by microCT in vitro .............................................. 18	1.4.4	 Evaluation of specimens by reconstruction of serial histologic sections .............. 19	1.5	 Aims .............................................................................................................................. 20	1.6	 Hypotheses .................................................................................................................... 20	Chapter 2: Methods .....................................................................................................................22	2.1	 Preparation of specimens and processing into paraffin ................................................ 22	2.2	 Histochemical and immunohistochemical staining procedures .................................... 23	2.2.1	 Picosirius red and alcian blue staining .................................................................. 23	2.2.2	 Hematoxylin and eosin staining ............................................................................ 23	2.2.3	 Immunohistochemistry protocol for anti-cytokeratin, MF-20 and E-Cadherin .... 24	2.3	 Magnetic resonance imaging ........................................................................................ 25	2.4	 Micro computed tomography with phosphotungstic acid staining ............................... 25	Chapter 3: Results ........................................................................................................................27	3.1	 Structure of the midline fusion zone in the hard and soft palate boundary .................. 27	3.2	 Immunohistochemistry was successful in specimens fixed in PFA independent of storage conditions ..................................................................................................................... 28	3.3	 Markers of proliferation and apoptosis did not work on human specimens ................. 37	3.4	 59-74 day specimens display complete fusion of the hard palate and differential levels of soft palate development through histological evaluation ..................................................... 37	3.5	 MRI 3D segmentation of craniofacial structures in specimens aged 67-74 days display complete fusion of both hard and soft palate ............................................................................ 41	3.6	 Quantitative analysis of microCT imaging of the soft palate morphology in specimens aged 67-74 days ........................................................................................................................ 45	  vi Chapter 4: Discussion ..................................................................................................................49	4.1	 The human hard palatal seam persists until at least 84-days post conception .............. 49	4.2	 Variability in timing of soft palate seam degradation ................................................... 50	4.3	 The epithelial midline seam differs in phenotype in the hard and soft palate .............. 50	4.4	 Archival sections can be successfully used for immunohistochemistry ....................... 51	4.5	 Antigenicity is present after long-term storage for abundant proteins ......................... 52	4.6	 Non-destructive methods are a valuable adjunct to histology for analyzing human development .............................................................................................................................. 53	4.7	 Future directions ........................................................................................................... 54	Bibliography .................................................................................................................................57	   vii List of Tables Table 3.1 List of histology specimens used in this study…………………………….………..32     viii List of Figures  Immunohistochemical staining of a 57- day fetus with anti-cytokeratin…....33  Frontal sections of a 57-day fetal head through the hard palate stained with anti-cytokeratin, E-Cadherin and MF-20……………………………………………………..34  Immunohistochemistry of a 57-day fetal head with a positive cytokeratin, E-Cadherin and MF20 signal……………………………………………………………………..35  Immunohistochemisty carried out on archival sections of a 57-day specimen previously stained with H&E…………………………………………………………………..36  A 59-day specimen stained with H&E………………………………………...39  Mid-sagittal hard and soft palate morphogenesis in a 57-day specimen……40  Virtual slices of an MRI scan in the frontal plane with segmentation….…...43  Volume renderings of segmented craniofacial structures from MRI scans……………………………………………………………………………………………..44  PTA stained 64-day specimen displays clear hard palatal seam through microCT imaging…………………………………………………………………………….…46  PTA microCT of an 84-day specimen displays a midline seam in the hard palate…………………………………………………………………………………………….47  Direct comparison of MRI to PTA-microCT imaging in the same specimen…………………………………………………………………………………………48      ix List of Abbreviations 3D: 3 dimensional  ALARA: as low as reasonably achievable BMP: bone morphogenetic protein BSA: bovine serum albumin CAR: Canadian Association of Radiologists CL/P: cleft lip and palate CPO: cleft palate only DAB: detection and visualization of antibody binding ddH2O: double distilled water EDTA: Ethylenediaminetetraacetic acid EtOH: ethanol GWAS: genome-wide association study H&E: hematoxalin and eosin IgG: Immunoglobulin G IHC: immunohistochemistry IRF6: interferon regulatory factor 6 lvp: levator veli palatine MB: mesenchymal bridge MEE/mee: medial edge epithelium mes: midline epithelial seam microCT: micro computed tomography MRI: magnetic resonance imaging   x PBS: phosphate buffered saline PCD: programmed cell death PCNA: proliferating cell nuclear antigen PFA: paraformaldehyde PTA: Phosphotungstic Acid SHH: Sonic Hedgehog SNP: single nucleotide polymorphism tvp: tensor veli palatine TESPA: 3-aminopropyltriethoxysilane TGFb: transforming growth factor beta TUNEL: terminal deoxynucleotidyl transferase nick-end labeling VPI: velopharyngeal insufficiency     xi Acknowledgements I would like to thank my parents and brother Michael for always being there for me and putting up with my nightly calls for the last 13 years of University. I could not have made it through four degrees including two master degrees without their unfailing support. I would also like to acknowledge my friends Natasha, Liat, Wendy, Wanda, Megan, Paige, and Amberene for being a truly amazing support system over the years and for giving advice regarding their respective specialties.   I would like to express my sincere appreciation and gratitude to my supervisor Dr. Joy Richman for offering me the opportunity to work with her on this exciting human study of palate development.  I would not have been able to complete this project without her support.   Many thanks to Dr. Virginia Diewert, Dr. Benjamin Pliska, Kathy Fu, Melanie Matson, Adrian Danescu, Julien Kim, and the members of the Richman lab who have helped me along the way. A very special thanks to Andrew and Barry of the UBC MRI Research centre located at the Life Sciences Institute for teaching me everything I needed to know on the 3D slicer program.     1 Chapter 1: Introduction 1.1 Embryonic craniofacial development – origins of craniofacial tissues Oral development in the human embryo begins extremely early at day 14 with the appearance of the prechordal plate (Sperber and Guttman, 2010). At this time the neural plate is still open and by stage 9 (Carnegie staging, day 19-21) neural crest cells begin to migrate away from the edges (O'Rahilly and Muller, 2007). Thus neural crest cells transform from ectoderm to mesenchyme as they break away from the neural plate (Le Douarin and Dupin, 2012). In humans, neural crest cells do not originate from the diencephalon but instead all of the facial neural crest derives from the mesencephalon (O'Rahilly and Muller, 2007). The paraxial mesoderm is derived from the middle germ layer (mesoderm) and lies on either side of the neural tube or primitive brain. Neural crest cells migrate through the paraxial mesoderm to reach the face and pharyngeal arches. The pharyngeal arches form caudal to the stomodeum beginning at Carnegie stage 12 (week 4, 26-30 days). The pharyngeal arches are paired-segmented bulges containing neural crest-derived mesenchyme surrounding a core of mesoderm (Grevellec and Tucker, 2010).  The first pharyngeal arch contributes to the lower part of the face (mandible and parts of the maxilla) (Lee et al., 2004). At the same time as neural crest cells are being specified, the foregut is forming. The cranial end of the foregut will form the oropharyngeal membrane which separates the primitive mouth from the gut (Sperber and Guttman, 2010). The bilayered oropharyngeal membrane represents the junction between the ectoderm of the oral mucosa and the endoderm of the pharyngeal mucosa (Sperber and Guttman, 2010). By day 28, the buccopharyngeal membrane disintegrates establishing a continuous structure between the oral ectoderm and the pharyngeal ectoderm (Dickinson, 2016).   2 The boundary between ectoderm and endoderm in the oral cavity remains unclear. Some case reports indicate that remnants of the buccopharyngeal membrane in adults are found connecting the soft palate and posterior third of the tongue (Kara and Kara, 2007; Ooi et al., 2005; Verma and Geller, 2009). The majority of the epithelium in the posterior palate (soft palate) is likely derived from endoderm.  The face is derived from five facial prominences surrounding the primitive stomodeum: the frontonasal, medial nasal, lateral nasal, maxillary and mandibular prominences. All the prominences are composed of neural crest-derived mesenchyme, sometimes called ectomesenchyme because of the neuroectoderm origins and are covered by ectodermally-derived epithelium. The facial midline (nasal septum, philtrum, premaxilla, bridge of the nose, 4 incisors) is formed by the medial nasal prominences which are medial to the nasal pits. The lateral nasal prominences form the nasal turbinates, sides of the nose and alae. The maxillary prominences form the cheek, sides of the upper lip, maxillary bone, the maxillary teeth posterior to the incisors, the palatine bone and soft palate. The mandibular prominences form the entire lower jaw as well as the malleus and incus of the middle ear (Thompson et al., 2012). The nasal placodes are thickened ectoderm, which divide the frontonasal process into the medial and lateral nasal prominences (Suzuki et al., 2016). By day 33, the nasal placodes invaginate into the underlying mesoderm to create the nasal pits and the future nares.  Lineage tracing of neural crest cells in chicken-quail chimeras and transgenic mice have determined that cranial neural crest cells give rise to all of the intramembranous bones in the face, the facial cartilages, connective tissues of the oral cavity, pulps of teeth, dentin, periodontal ligament (Chai et al., 2000; Grimaldi et al., 2015; Gross and Hanken, 2008). Specifically, the   3 bones and connective tissues of the hard palate are neural crest derived as is the connective tissue within the soft palate. Sixty distinct skeletal muscles comprise the extra-ocular, facial, pharyngeal, laryngeal and neck musculature of the human head (De la Cuadra Blanco et al., 2012; Sperber and Guttman, 2010). Muscles of the craniofacial complex begin to appear after 24 days following the development of the first pharyngeal arch. The origins of the skeletal muscles of facial expression and mastication lie in the head mesoderm, smooth muscle cells lining the vasculature derive from the cranial neural crest. The neural crest cells also form the fascia surrounding the muscles. First arch mesoderm gives rise to muscles of mastication, mylohyoid, anterior belly of digastric, tensor veli tympani and tensor veli palatini muscles. By day 26, the second pharyngeal arch begins to form all the muscles of facial expression, posterior belly of digastric and the stylohyoid muscles.  The third and fourth arch derivatives begin to appear at day 28 and form the stylopharyngeus and the remaining pharyngeal and palatal muscles. Occipital and cervical mesenchyme gives rise to the genioglossus and mylohyoid muscles by day 36 and they are the first identified muscle masses seen on histology (Sperber and Guttman, 2010).  Rapid craniofacial development continues during the embryonic period compared to the caudal portion. The embryonic human face appears similar to the adult face by 10 weeks. This craniocaudal growth patterning continues throughout fetal development resulting in the average human head to body length at birth as approximately 50%. By adult life the body growth and development has caught up and the head encompasses only one-eighth of the overall body length.    4 1.2 Human palatogenesis  1.2.1 Primary palate development One of the most important events in facial development takes place during the 6th week gestation, the fusion of the primary palate or lip. The medial nasal, lateral nasal and maxillary prominences meet, form a seam between the ectodermal surfaces and then this seam is degraded (Diewert and Lozanoff, 1993; Diewert et al., 1993; Diewert and Wang, 1992). A mesenchymal bridge then forms between adjacent facial prominences. The small grooves that are transiently present after mesenchymal bridge formation fill in via a process of merging leading (Abramyan and Richman, 2015) to a smooth face by 47 days post conception. Merging occurs when a furrow or groove between two partially attached structures fills in with mesenchymal cell proliferation resulting in confluence of the structures. An example of merging can be seen in the medial nasal prominences which converge in the midline to form the intermaxillary segment. In contrast, fusion occurs when two epithelial lined processes come into contact forming a bilayered epithelial seam. This seam is removed in one of three ways: mesenchymal to epithelial transformation, apoptosis or cell migration. Less is known about seam degradation in the primary palate than in the secondary palate. Lack of epithelial contact between the prominences or failure to remove the seam results in varying degrees of cleft lip (Dixon et al., 2011).  1.2.2 Secondary palate development By late week 6, two medial outgrowths from the maxillary prominence develop that are initially oriented vertically on either side of the developing tongue (Diewert, 1983). Between the 7th and 8th week the these palatal shelves re-orient to a horizontal position (Bush and Jiang, 2012; Diewert, 1983; Sperber and Guttman, 2010). Simultaneously, the mandible elongates and the head is tipped upwards, raising the upper craniofacial complex and freeing the tongue from   5 between the two palatal shelves (Diewert, 1983). Shelf elevation only occurs if mandible elongation is greater than oronasal cavity length and failure may result in clefting (Diewert, 1983).  The horizontally positioned palatal shelves covered in ectodermally-derived epithelium with the leading edge known as the medial edge epithelium (MEE) eventually contact (Nawshad, 2008). Adhesion occurs between the two layers of MEE with the aid of adhesion molecules and formation of desmosomes (Cox, 2004). Both temporal and spatial upregulated desmosomal gene expression occurs prior to fusion (Cox, 2004). The resulting bilayed epithelial structure is known as the midline epithelial seam (MES). Many growth and transcription factors have been implicated to be involved in the adhesion process between the two palatal shelves including: TGB b3, IRF 6, RUNX1 and LHX8 among others (Cui et al., 2003; Cui et al., 2005; Lan et al., 2015; Taya et al., 1999; Yu et al., 2009). Once fusion has occurred, subsequent degradation of the epithelial seam occurs in order to achieve mesenchymal confluence between the two shelves.  The mechanism of midline epithelial seam disintegration following fusion of the secondary palate has been a well-studied (Lan et al., 2015). Three major theories of seam removal have been investigated in the mouse including: oral and nasal migration of epithelial cells (Carette and Ferguson, 1992), epithelial to mesenchymal transformation (EMT) (Cuervo and Covarrubias, 2004; Nawshad, 2008) and epithelial cell apoptosis (Cuervo and Covarrubias, 2004; Cuervo et al., 2002; Vaziri Sani et al., 2005). Conflicting views still exist regarding which method(s) are responsible and this is further complicated by the fact that transforming growth factor beta 3 (TGFb3) plays an important role and is involved in all three processes (Iwata et al., 2014; Nawshad et al., 2004; Taya et al., 1999).    6 Carette and Ferguson were the first to report cell migration from the middle epithelial seam to the oral and nasal epithelia (Carette and Ferguson, 1992). This was based primarily on cell labeling with lipophilic markers and subsequent fate mapping in palatal cultures. Years later Ferguson and his research collaborators recanted this theory and suggested that EMT and/or apoptosis were more likely to occur (Martinez-Alvarez et al., 2000).  EMT occurs when epithelial cells integrated into coherent groups or layers become altered in phenotype to become less coherent and more independent in nature (Savagner et al., 1997). Recent in vivo mouse studies incorporating genetic labels into epithelial cells show that EMT is unlikely to occur within the middle epithelial seam of the hard palate (Cuervo and Covarrubias, 2004; Iwata et al., 2014; Vaziri Sani et al., 2005). Fate mapping of the labeled cells was conducted in whole palate cultures and individual cells could be followed through palatal development visually. Very few positive cells ended up in the mesenchyme, while most underwent apoptosis.  Apoptosis or programmed cell death (PCD) results from a complex signaling cascade leading to cell death. PCD has been reported in the midline epithelial seam following adhesion of the two palatal shelves (Cuervo and Covarrubias, 2004; Iwata et al., 2014; Vaziri Sani et al., 2005).Staining for nuclear DNA fragments using the TUNEL (terminal deoxynucleotidyl transferase nick-end labeling assay) method showed that TUNEL positive cells were present in the midline epithelial seam after contact, but that they disappeared progressively over time (Mori et al., 1994).  Another study confirmed that contact between the two shelves was necessary for apoptosis to be activated (Cuervo et al., 2002). However, introducing exogenous retinoic acid induced PCD regardless of contact suggesting that retinoic acid is important in regulating the process. Apoptosis mediated cell death is likely important in midline epithelial seam disruption.   7 Regardless of the method, failure of seam removal to achieve palatal confluency with underlying mesenchymal cells results in congenital midline epithelial cysts in the hard palate known as Epstein’s pearls (Kitamura, 1966).  Both Bohn’s nodules or Epstein pearls are a common occurrence in newborns and disappear over the first few months of life. They are frequently grouped together due to a similar clinical appearance but, they can be differentiated based on location and origin.  Epstein pearls are formed during palatogenesis from entrapment of the epithelial lined palatal shelves along the palatal midline (Monteleone and McLellan, 1964).  On clinical evaluation they appear as small cystic, keratin-filled nodules on the oral palatal tissue. In contrast, Bohn's nodules are located mainly at the junction of the hard and soft palate along alveolar ridges away from the midline (Singh et al., 2012). They most likely form from salivary gland tissue (Singh et al., 2012) Completion of hard palate development occurs with the fusion of the palatal shelves with the nasal septum and the primary hard palate through a similar mechanism to midline palatal shelf fusion (Dixon et al., 2011). Failure of nasal septum fusion to the palatal shelves on both sides results in bilateral cleft palate, while failure of union on one side results in unilateral cleft palate (Dixon et al., 2011).   1.2.3 Soft palate development The soft palate extends from the secondary hard palate as a posterior muscular portion of the secondary palate. Its function is essential for proper swallowing and speech development. Dysfunction of the soft palate can lead to velopharyngeal insufficiency (VPI). Prior to speech development, VPI may present as feeding difficulties, nasal regurgitation and chronic otitis media with associated conductive hearing loss due to Eustachian tube dysfunction (Ha et al., 2013). When speech development occurs, VPI is associated with hypernasal speech, nasal   8 turbulence and air emission through the nose during speech (Ha et al., 2013). Many symptomatic patients require surgical intervention (Ha et al., 2013). Palatogenesis of the soft palate is believed to occur after the hard palate with initiation reported between 9-12 weeks. Until the early 1990’s myogenesis of the soft palate was hailed as the marker for completion of soft palate formation around 16 weeks (Cohen et al., 1993). Our study suggests that soft palate completion is in fact much earlier at 10 weeks (Danescu et al., 2015). While the mechanism of hard palate development is well defined in evidence based literature, the formation of the soft palate is less understood. Several studies of human soft palate development have taken place since the 1960’s, but controversy remains on whether the soft palate closes by fusion or merging. The timing of the different stages of soft palate closure is also not very clear (Burdi and Faist, 1967; Rood, 1973; Smiley, 1975; Smiley and Koch, 1975; Suga et al., 2010).  Burdi and Faist (Burdi and Faist, 1967) examined serial sections of 31 human specimens aged 7-12 weeks post-conception based on staging from crown-rump length. However, only 2 of these specimens were between 41-60 days of development where soft palate formation is most likely to occur. They confirmed reports that the hard palate formed primarily by the fusion of the two palatal shelves with the formation and subsequent removal of a midline epithelial seam. After hard palate completion, the soft palate was hypothesized to form primarily by proliferation of two subepithelial mesenchymal growth centers leading to displacement of the epithelium and eventual merging. They believed that merging is the primary mechanism of soft palate formation in humans. This theory was supported with the absence of a midline epithelial seam, epithelial remnants, and the fact that only one case of a midline cyst in the soft palate had been reported at the time of their study (Andrews, 1962). This concept gained popularity through citing of   9 prominent embryologists who further spread the merging theory of soft palate formation (O'Rahilly and Muller, 2000). However, with such a limited sample size during the window of soft palatogenesis, Burdi and Faist could have missed the presence of an epithelial seam or cysts within the soft palate. Additionally, evidence suggests that staging of embryos through crown-rump length alone is unsatisfactory and leads to the underestimation of stage due to fetal curling especially after 13 weeks. Crown-rump length along is not as accurate as total height without lower limbs, so there may be some error in the staging of their specimens (O'Rahilly and Muller, 2000). I focused earlier stages (up to 12 weeks) when the embryo/fetus is straighter.  Some researchers have proposed that mammalian soft palate development occurs through a hybrid mechanism of both merging and fusion. Poswillo reported epithelial seam formation followed by degradation in the anterior two thirds of the soft palate (Poswillo, 1974). The posterior third including the developing uvula displayed no seam during palatogenesis and formed primarily through merging. To date, no human studies support the hybrid model of soft palate formation.  In contrast, many studies suggest that soft palate formation occurs primarily by a mechanism of fusion similar to the hard palate (Kitamura, 1966; Smiley, 1972; Smiley, 1975; Smiley and Koch, 1975; Suga et al., 2010; Wood and Kraus, 1962). The first study to propose fusion examined 5 human specimens aged 7-11 weeks but they provided no proof of a midline epithelial seam (Wood and Kraus, 1962). They reported the presence of epithelial masses or pearls solely in the hard palate. Transient hard palatal cysts known as Bohn nodules or Epstein pearls, which arise from entrapped epithelial tissue during normal palatal development are well-documented to occur in up to 80% of newborns (Monteagudo et al., 2012). Additionally, they hinted at increased layers of epithelial cells within the posterior soft palate especially in the   10 midline region (Wood and Kraus, 1962). A second study suggested a mechanism of fusion with rapid degradation of the midline epithelial seam (Kitamura, 1966). They indicated the clear presence of midline epithelial islands in the soft palate of specimens aged 53-55 days but not past 60 days. The presence of epithelial islands in the soft palate suggests a mechanism of fusion with entrapped epithelial tissue similar to the hard palate. Smiley further supported the primary mechanism of fusion in the soft palate through many published reports (Smiley, 1972; Smiley, 1975; Smiley and Koch, 1975; Suga et al., 2010). He proposed different rates of seam degradation between hard and soft palate, with the soft palate seam forming later but degrading much faster than the hard palate seam (Simley, 1975). More recently, a graduate student in our lab analyzed serial sections of 13 specimens between ages 54-74 days (Fig. 1.1)(Mattson, 2013). The presence of an epithelial seam in the soft palate of 7 specimens was reported in the thesis. The seam was difficult to distinguish from the surrounding mesenchyme using routine histological staining. The hard and soft palate both likely develop from a similar mechanism of fusion and act as a single continuous structure after birth. A recent concept has emerged that there are molecular differences in the anterior and posterior palate (Hilliard et al., 2005). This work originated in the mouse model where regionally restricted gene expression patterns were observed (Bush and Jiang, 2012). In addition, knockout of anterior-restricted genes such as Shox2, causes a cleft between the primary and secondary palate. This mouse data leaves open the possibility that signaling between epithelium and mesenchyme is different in the hard and soft palate and thus the mechanism of epithelial seam formation may be distinct.    11 1.2.4 Soft palate myogenesis The soft palate musculature plays a vital role in opening and closing the boundary between the oral and nasal cavities during function. Improper function of these muscles may lead to defects in swallowing, speech and hearing. The human soft palate consists of five pairs of muscles: tensor veli palatini, levator veli palatini, palatoglossus, palatopharyngeus and musculus uvula (Perry, 2011). While the anatomy of the human soft palate post birth is well defined, little published data exists on the timing of muscle development and morphologic changes in utero. Most of the existing knowledge on soft palate myogenesis and morphogenesis stems from genetic research on mouse models (Grimaldi et al., 2015; Hilliard et al., 2005; Iwata et al., 2014). The orientation and attachments of the mouse soft palate musculature is similar to the human soft palate (Cho et al., 2013). The main difference in the mouse soft palate is the absence of both musculus uvula and flexation of the soft palate and pharyngeal tissues as humans are upright (Grimaldi et al., 2015).  Tensor veli palatini is the first muscle to appear pre-fusion of the soft palate at 6 weeks in humans (De la Cuadra Blanco et al., 2012). Tensor veli palatini is the only palatal muscle derived from the first pharyngeal arch along with tensor tympani (De la Cuadra Blanco et al., 2012; Grimaldi et al., 2015). Both muscles are innervated by the mandibular branch of the trigeminal nerve (Norton, 2012). Thus, many first arch syndromes including Treacher Collins (OMIM: 154500) and auricular oculovertebral spectrum (OMIM: 164210) may present with defects in the tensor muscles resulting in VPI and/or submucous clefting (Breckpot et al., 2016; Sperber and Guttman, 2010).The two heads of the tensor veli palatini originate from the pterygoid plate of the sphenoid bone and the cartilage of the eustachian tube (De la Cuadra Blanco et al., 2012). Both heads of the tensor veli palatini envelope the hamulus of the sphenoid   12 and are continuous with the pharyngeal aponeurosis by week 9 (De la Cuadra Blanco et al., 2012). The palatine aponeurosis forms the oral side of the soft palate as a thin fibrous sheath. The aponeurosis allows a site of insertion for the tensor veli palatini and levator veli palatini, while palatopharyngeal, palatoglossus and musculus uvula all originate from the aponeurosis (Norton, 2012).  The remaining four pairs of palatal muscles are derivatives of the fourth pharyngeal arch and occipital somites 1 and 2 (Sperber and Guttman, 2010) . They receive innervation from the superior laryngeal branch of the vagus nerve (Norton, 2012). Levator veli palatini is the largest and most studied muscle of the soft palate and primarily functions to elevate the soft palate during swallowing and speech. Defects in thickness and tone of the levator veli palatinin may result in velopharyngeal incompetency. This muscle originates from the petrous temporal bone and eustachian tube with insertion into the palatine aponeurosis. The palatoglossus muscle originates from the palatine aponeurosis and inserts into the lateral surface of the tongue, where its fibers mix with the intrinsic muscles of the tongue (Norton, 2012). According to prior reports the levator veli palatini and palatoglossus muscles appear similar in morphology and fully developed at the time of soft palate fusion initiation (Grimaldi et al., 2015). The palatopharyngeal muscle also originates from the palatine aponeurosis near the posterior edge of the hard palate and inserts into the posterior surface of the thyroid cartilage and plays an important role in the elevation of the pharynx and aids in nasopharynx closure during swallowing (Norton, 2012). Levator veli palatini, palatoglossus and palatopharyngeal myogenesis lags behind in cleft patients involving the soft palate (Cohen et al., 1994) . Again, many fourth arch syndromes likely have underlying submucous cleft or bifid uvula due to the embryonic origin of the palatal muscles.    13 For the purpose of this study, the palatine aponeurosis will serve as a marker for the mid soft palate region (Danescu et al., 2015; Kraus et al., 1966). When the tensor veli palatini and the aponeurosis appear continuous soft palate myogenesis is complete. We previously reported that tensor veli palatini formation and aponeurosis completion occurs between 59- and 67-days post-conception (Danescu et al., 2015). By day 74, soft palate myogenesis reached its post-natal appearance. 1.3 Orofacial clefts 1.3.1 Cleft lip and/or palate (CL/P) Cleft lip with or without cleft palate (CL/P) has the highest incidence with 1/700 individuals being affected, while cleft palate only (CPO) affects approximately 1/2500 live births (Bell et al., 2013; Dixon et al., 2011).The rate of CL/P varies greatly among various ethnic and geographic populations (Dixon et al., 2011). A recent American population study showed that the ratio of unilateral to bilateral affected in CL/P was 2:1, while CL was 10:1 respectively with the majority being left-sided (Genisca et al., 2009). CPO were up to 61% more likely to have other common defects affecting the heart, limb, digits, CNS, musculoskeletal and are more likely to be syndromic (Bell et al., 2013; Genisca et al., 2009). Indeed 45.9% of CPO cases are syndromic (Watkins et al., 2014). Up to 25% of live births with CPO are associated with Pierre-Robin sequence (Genisca et al., 2009). Less severe forms of CPO such as velopharyngeal insufficiency, submucous clefts or bifid uvula are frequently seen as part of the 22q11.2 deletion syndrome (DiGeorge syndrome or velocardiofacial syndrome)(Watkins et al., 2014). However the vast majority of CL/P occurs without any other associated abnormalities and is therefore termed non-syndromic (Watkins et al., 2014).   14 1.3.2 Microforms of cleft lip and palate There are three microforms of cleft palate: submucous clefting, bifid uvula and velopharyngeal insufficiency due to muscular abnormalities in the soft palate. Submucous clefting is estimated to occur in 0.02-0.08% of individuals (Weatherley-White et al., 1972). In the past, submucous cleft palate was defined as a triad of occurrences: a bifid uvula, a visible furrow in the midline of the soft palate and a posterior notch on the border of the hard and soft palate (Kaplan, 1975). More recently, a fourth characteristic of submucous clefting has been added,  the absence of the posterior nasal spine (Ha et al., 2013). Typically submucous clefting has minor functional impacts unless the soft palate is involved. Similarly cleft uvula has minimal impact on function. VPI on the other hand causes speech abnormalities that can significantly impact the quality of life. The cause may be improper sling formation across the midline that normally occurs with the union of the right and left levator veli palatini muscles (Kaplan, 1975). Levator veli palatini dysfunction results in the uncoordinated elevation of the soft palate during swallowing and speech. This may be the result of improper or incomplete fusion or merging during palatogenesis. 1.3.3 Genetic factors may contribute to cleft development Genetics plays an important role in orofacial clefting, including in non-syndromic CL/P. Multiple genes are likely involved and mutations may lead to a loss of protein function or other minor defects. With the advent of the genomics era, many approaches have been used the study of genetic influences of CL/P including: association studies in family and populations, twin studies, analysis of chromosome anomalies with FISH analysis and candidate gene studies through direct sequencing of DNA from affected individuals (Marazita, 2012). Several recent   15 unbiased genome-wide association studies (GWAS) comparing families with a history of CL/P (Beaty et al., 2011; Birnbaum et al., 2009; Grant et al., 2009; Mangold et al., 2011)  or CP (Beaty et al., 2011) to controls have allowed some insight into specific single nucleotide polymorphisms (SNP). The SNPs may be either in coding or non-coding regions of the genome and the functional effects of the variants may be difficult to determine. Nevertheless, statistical power comes from studying large groups of affected individual and their immediate relatives. Some of the SNPs have been replicated across populations of different ethnic backgrounds (Beaty et al., 2011; Mangold et al., 2011). Examples of locations of SNPs associated with clefting that have been replicated include in chromosome 8q24 (Beaty et al., 2011; Gowans et al., 2016; Grant et al., 2009; Leslie et al., 2016), 17q22 and 10q25.3 (Mangold et al., 2010). One such SNP heavily associated with CL/P in Central Europeans was identified as a variant of the interferon regulatory factor 6 (IRF6) gene (Birnbaum et al., 2009). Some SNPs may be further influenced by maternal factors such as: exposure to alcohol, smoking and multivitamins during the embryonic period of development leading to an increased risk of cleft (Beaty et al., 2011). Thus, there appears to be a complex interaction and heterogeneous involvement of both genetic and environmental factors leading to cleft development. Gene studies may lead to potential prenatal testing options, while known environmental risks aid in developing appropriate guidelines during pregnancy.  1.4 3-dimensional evaluation of human embryo/fetal development 1.4.1 3D imaging of the palate in vivo using ultrasound Much of our existing knowledge on human soft palate shape changes over time stem from cadaver or ultrasound studies (Captier et al., 2008; Wong et al., 2008; Wong et al., 2009; Zajicek et al., 2013). Cleft lip with or without cleft palate (CL/P) can be detected as early as 11 weeks in utero by ultrasound, but isolated clefts of the hard and soft palate are more difficult to   16 identify in utero. If they are identified during pregnancy, they are most likely to be detected in the second trimester. A recent prospective study analyzed the secondary palate of routine ultrasounds of 49 human fetuses aged 12-16 weeks by 3-D ultrasound (Zajicek et al., 2013). Images were acquired with the fetus head facing the transducer at an angle of 30-40 degrees to the ultrasound transducer. They determined that only 39% of the secondary palates were visualized, whereas only 34% acquired images of the soft palate. This is the first report of ultrasound analysis of the soft palate in the first trimester. There exists conflicting views on the ideal time for soft palate ultrasound evaluation with some investigators suggesting earlier evaluation at 12-24 weeks post-conception when the bone formation of the hard palate has commenced but there is less shadowing from adjacent tissues (Campbell, 2007)while others suggest evaluation during 20 weeks of gestation and beyond due to an increased ease of palate identification (Faure et al., 2008). Many ultrasound studies of the palate of the developing fetus utilize the uvula as a marker for viewing the soft palate during the second and third trimester (Wong et al., 2008). This is especially useful in 3-dimensional ultrasounds using an oblique view of the secondary palate, however, a recent study shows that the uvula is not clearly detectable in all views of the soft palate until week 19 (Wong et al., 2009). Thus, the completion of soft palate closure is difficult to determine. This retrospective study evaluated 31 stored 3D ultrasound volumes of human fetal faces between 15-35 weeks of gestation. They showed that by week 19-21, the soft palate is straight and flat on sagittal view with the uvula displaying a slight elevation at this stage. By week 23, the uvula had elongated and drooped downward towards the oropharynx, which was easily identified by ultrasound. From week 23-35 the uvula becomes longer in length with the developing soft palate. Another evaluation of 87 human fetuses at 21-25 weeks with the same   17 methodology allowed the detection of the uvula and velum between 80-90% of scans (Faure et al., 2008). The main limitation of soft palate visualization in this study with 2- and 3-dimensional ultrasound remains to be image quality. Image quality of the soft palate is greatly reduced by acoustic shadowing from the adjacent alveolar processes, tooth buds, maxillary and palatal bones (Wayne et al., 2002). Additionally, the soft palate does not lie in the same horizontal plane as the hard palate, rather the muscular soft palate hangs downwards off of the posterior hard palate leading to poor localization and acquisition.  Ultrasound still holds promise of being a safe way to diagnose clefts of the palate and possibly in the future submucous cleft identification can be made in utero. 1.4.2 Evaluation of fetal specimens by MRI in vitro Magnetic resonance imaging (MRI) is currently the gold standard for soft tissue evaluation in both in vivo and ex vivo specimens. MRI uses magnetic fields and radio waves to create a 3D image of soft tissue structures (Kuijpers et al., 2014). The advantage of this type of imaging is that there is low risk to the patient as there is no radiation dose. Disadvantages of MRI include the high cost, as well as low resolution of cartilage. There are limited studies using MRI to examine human fetal craniofacial development in vivo. One study evaluated craniofacial deformities in 28 human fetuses retrospectively between 23-33 weeks in vivo in pregnant women (Arangio et al., 2013). Without contrast agent they were able to adequately visualize facial and palatal clefts in these fetuses. Another study showed the value of MRI fetal analysis of anomalies in the fetal head and neck, and provides a valuable tool for evaluation of fetal airway obstruction (Mirsky et al., 2012). Another study evaluate the size of the maxillary sinus in vivo in fetuses aged 16-38 weeks by non-contrast MRI retrospectively (Ozcan et al., 2014). MRI may be indicated during pregnancy when suspected fetal head and neck anomalies exist such as goiter,   18 encephalopathy, and large masses which may alter the delivery plan. MRI is safe during pregnancy and it is relatively contraindicated to avoid contrast agents (Runge, 2000). For example, gadolinium a commonly used magnetic intravenous contrast agent that passes the placenta and has unknown effects on the developing fetus so it remains a class C pregnancy drug and is not used (Runge, 2000). There is hope for potential use of Gadolinium during pregnancy since it should decrease scan time. The fetus may move during imaging so a shorter scan time is a major advantage. A recent study showed Gadolinium contrast improves MRI diagnosis of placental abnormalities in the third trimester with no fetal abnormalities reported (Millischer et al., 2016). None of these studies evaluate early fetal palatal development but likely a longer scan would be necessary to resolve the details of the palate. 1.4.3 Evaluation of fetal specimens by microCT in vitro 3D X-ray microtomography (micro computed tomography) for prenatal fetuses cannot be carried out due to high radiation exposure. However micro CT can be used for cadaveric specimens. The advantages of micro-CT is that it is method a non-destructive and slices are in perfect registration. It is possible to reorient the specimen and reslice virtually in any plane. Relatively high resolution can be achieved and quantification on bone size and density carried out. However, since x-rays are the basis of CT imaging, only mineralized tissues can be seen. The natural low contrast of soft tissue and partially mineralized structures such as cartilage means the x-rays pass through these structures. Fortunately, more recently advances in micro-CT imaging have focused on using contrast agents that increase radiodensity of non-mineralized or partially mineralized tissues. A recent report shows that using inorganic iodine or phosphotungstic acid provides sufficient radio-opacity to visualize soft tissues of mouse and chicken embryos in micro-CT (Metscher, 2009). Inorganic iodine staining is rapid and stable for   19 several months. Phosphotungstic acid penetrates the embryo within 24 hours and remains stable for several months. Both inorganic iodine and phosphotungstic acid compounds did not interact with preserving agents such as Bouin, formaldehyde or ethanol commonly used to store embryonic tissue.   MicroCT analysis can attain resolutions as high at 8 µm or less for small specimens such as mice, which is comparable to microtome sections thickness (Johnson et al., 2006). To achieve such high resolution, scan times may be as high as 12 hours for mouse embryos between 9.5 and 12.5 days (Johnson et al., 2006). It is unclear whether microCT on larger human specimens using contrast agent will provide sufficient resolution to see small structures such as the midline epithelial seam. There are limited studies on human cadaveric fetal specimens (Neumann et al., 1997; Sastre et al., 2011; Tejszerska et al., 2011)and of these only a few use contrast agent(Siebert et al., 2013)  1.4.4 Evaluation of specimens by reconstruction of serial histologic sections Microscopic imaging of serial sectioned slides can be used to produce stacked images for visualizing structures in 3D (Radlanski et al., 1998; Radlanski et al., 2003; Radlanski et al., 2016). This involves serially sectioning a specimen by microtome. Then, serial photographs of serial sections in the area of interest are taken by microscope and imported to a 3D reconstruction program. Our lab has used WinSurf 3D Reconstruction program (SURFdriver Software, Kailua, HI, USA developed by Scott Lozanoff)(Buchtová et al., 2008; Danescu et al., 2015; Handrigan et al., 2010). The software is powerful for visualization in 3 planes but does not have quantitative capabilities. Histological reconstructions have the advantage of providing cellular detail. For example, a study on human mandibular development was able to map the locations of osteoclasts and   20 osteoblasts onto the 3D structure of the alveolar bone. However reconstruction is tedious and this limits the sample size that can be studied.. Additionally, histological sectioning commonly results in voids/spaces, wrinkles, or tears present between anatomical structures as distortion may occur as the specimen is processed, sectioned and preparation of slides. Thus quantification from 3D histological reconstructions cannot be done. In this thesis, the aim is to test out two methods of 3D imaging in order to determine if they have sufficient resolution to address our research questions.  1.5 Aims The specific aims of this study are: 1. To extend our previous sample age from 74-days to 84-days post conception. The goal was to go up to a stage when the soft palate is complete and only remnants of the midline seam persist in the hard palate. 2. To characterize the epithelial seam in the hard and soft palate using immunohistochemical techniques. 3. To visualize epithelial-mesenchymal transformation or apoptosis in the midline epithelial seam using molecular techniques. 4. To test whether storage of human specimens for over 25 years has any effect on antigenicity.  5. To develop high-resolution3D imaging methods for the fetal head.  1.6 Hypotheses 1. The hard palate has an intact seam up to at least 84 days, which is long after the fusion of the soft palate   21 2. There is no difference in the epithelium between the ectodermal and endodermal epithelia of the palate.  3. Epithelial-mesenchymal transformation is the primary mechanism of human medial edge epithelium removal in the palate.  4. Antigenicity of human specimens is preserved after long periods of storage in 4% PFA 5. MRI and phosphotungstic acid micro-computed tomography offer novel 3D imaging for assessing the developing human palate.          22 Chapter 2: Methods 2.1 Preparation of specimens and processing into paraffin The 35 human specimens obtained for this study originated from elective pregnancy terminations carried out at the University of Washington between 1988-1991. All specimens were of European decent and deemed to have a normal pattern of development based on external examination by pathologist at the time of termination, combined with the mother’s health history. At the time, screening for chromosomal abnormalities was not completed on fetuses that appeared normal at termination. Staging of fetuses was completed at the time of termination by pathologist using combined methods of last reported menstrual cycle, crown-rump length and foot length and the average between them. The specimens ranged from 53-84 days post-conception and all were past stage 23 on the Carnegie embryo staging system, so I will refer to ages as days post-conception instead of numbered stages of development. The specimens consisted of a partial head, lacking brain and mandible in some cases. They were stored in the lab of Dr. V. Diewert as either paraffin sections, paraffin blocks or as specimens in 4% paraformaldehyde.   Wet specimens in 4% paraformaldehyde were washed twice with PBS for 1 hour. They were photographed for overall morphology using a Leica stereoscope in frontal, dorsal, sagittal and sub-mental views before decalcification. Then specimens were transferred to 7% EDTA to decalcify for at least 8 weeks. Decalcification was considered successful once the skull could be pierced easily with a pin. Twenty-eight decalcified specimens were washed in PBS and dehydrated in a series of 25%, 50%, 75%, 100% EtOH in water and sent for paraffin block processing at the UBC Department of Histopathology. Paraffin embedded specimens were sectioned by microtome in the transverse/coronal or horizontal plane at a thickness of 7µm.   23 Sections were stored in boxes as ribbons or mounted on TESPA-coated glass slides and stored at room temperature.  2.2 Histochemical and immunohistochemical staining procedures Archival unstained slides were immersed in xylene for up to one week for coverslip removal. Next, sections were rehydrated in a series of EtOH washes (100% EtOH for 2x10 mins, 90% EtOH in ddH2O for 5 mins, 70% EtOH in ddH2O for 5 mins, 50% EtOH in ddH2O for 5 mins, ddH2O for 5 mins). Newly fabricated slides were de-waxed through 45 mins of heat treatment in the 60-degree oven, cooled at room temperature, and followed by two washes of xylene for 20mins each. Next, sections were rehydrated in a series of EtOH washes (100% EtOH for 2x10 mins, 90% EtOH in ddH2O for 5 mins, 70% EtOH in ddH2O for 5 mins, 50% EtOH in ddH2O for 5 mins, ddH2O for 5 mins). 2.2.1 Picosirius red and alcian blue staining After coverslip removal on archival slides or de-wax/rehydration of newly created slides was followed by sequential immersion of slides in 1% Alcian blue stain in acetic acid for 30mins, 1% acetic acid for 5 mins, ddH2O for 5mins was completed. Slides were counter stained with Picrosirius red for 1hr in the dark, following by 1% acetic acid for 5 mins, and ddH2O for 5 mins. Once staining completed, slides were dehydrated using the reverse series of EtOH as that for rehydration. Slides were then coverslipped for microscope analysis.  2.2.2 Hematoxylin and eosin staining Coverslip removal on archival slides or de-wax/rehydration of newly created slides was completed and slides were sequentially immersed in Shandon hematoxylin (Thermo Scientific) diluted in 50% ddH2O for 2 mins, tap water for 5 mins, saturated lithium carbonate solution for 1   24 min, dipped in 1% Eosin Y 30 times and rinsed in tap water for 5 mins. Once staining completed, slides were dehydrated through the EtOH series and coverslipped for microscope analysis.  2.2.3 Immunohistochemistry protocol for anti-cytokeratin, MF-20 and E-Cadherin The polyclonal rabbit anti-cytokeratin wide spectrum antibody  (Dako, Z0622) cross-reacts with mucosal keratins (K4, K13, K19) and keratins present in the oral mucosa (K5, K6, K16) (Presland and Dale, 2000). We also used the monoclonal antibody to MF-20, myosin heavy chain from the Developmental Studies Hybridoma bank. Finally, we used a monoclonal antibody to E-Cadherin from BD transduction (catalogue number: 610181). The E-Cadherin was a mouse monoclonal antibody that works on human cells (Graves et al., 2016). The antibody was raised to amino acids 735-883 of  human E-cadherin.  The pan-cytokeratin primary antibody was diluted 1:1000, the monoclonal MF-20 antibody was used neat and the E-Cadherin antibody was diluted1:100. Newly fabricated slides were dewaxed and rehydrated in EtOH series, followed by antigen retrieval with steaming for 20 mins at 99oC in 10% DIVA decloaker in ddH2O (Biocare Medical) for 20 mins. Slides were cooled at room temperature for 30 mins, then rinsed in PBS twice for 5 mins. Slides were marked with a PAP pen outlining sections and blocking was performed with 1%BSA, 0.05% Tween 20, in PBS and stored for 1 hr at room temperature in a humidified box. Primary antibodies were diluted in blocking buffer, added to the slides, and left for overnight at 4oC. Slides were immersed twice for 5 mins in PBS. Biotinylated anti-rabbit, Jackson Immuno Research (711-065-152) or anti-mouse secondary antibody from the ABC kit was added to slides and incubated for 1 hr at room temperature. Slides were rinsed 2x5 mins in PBS. The Vector ABC kit (1 drop of A, 1 drop of B, 5ml PBS) added to slides and incubated for 1 hr at room temperature. Slides were rinsed 2x5 mins in PBS. Diaminobenzidine-horseradish peroxide (DAB) detection was completed in a dark incubation box at room temperature. Once   25 detection was completed, slides were counterstained in Hematoxylin for 30 secs. Slides were rinsed in running tap water for 5 mins, xylene for 2x20 mins and then mounted.  2.3 Magnetic resonance imaging Specimens stored in 70% EtOH were rehydrated in 50% EtOH in ddH2O for 1hr, PBS twice for 30 mins, then transferred to 3mM Gadolinium in PBS for storage at room temperature for several weeks. Gadolinium both increased the contrast and decreased the scanning time needed. Prior to scanning, specimens were placed in a plastic tube with Styrofoam chips to aid in stabilizing the specimen. Specimens were positioned chin down with the Frankfort plane (porion to orbitale) parallel to the base of the tube. Scanning was completed using the 7 Tesla magnet magnetic resonance imaging (MRI) machine at the UBC MRI Research centre located at the Life Sciences Institute. Scanning resolution was best at a slice thickness of 53-75nM. Sectioning software (3D Slicer 4.4.0) provided by the centre was used to trace and calculate volumetric data. The structures of interest included: the dorsal tongue, hard palate, soft palate, midline palatal seam, palatine aponeurosis, nasal cartilage and nasal septum. These structures were traced manually on sequential slices for each specimen. The tracings were then stacked together by the 3D Slicer program to form a 3D model of each structure. Screen captures were created from the various views of the 3D model and imported into Adobe Photoshop.  2.4 Micro computed tomography with phosphotungstic acid staining The same seven specimens aged 67-84 days previously used for MRI scanning with Gadolinium contrast were washed in a series of alcohol washes (see below) and re-used for microCT. Our specimens were subsequently stained with phosphotungstic acid as described  protocol for micro computed tomography (microCT) scanning with a modified protocol developed by Tesarova (Tesařová et al., 2016). Specimens were re-fixed after gadolinium in 4%   26 formaldehyde in PBS for 72hrs, washed in 1x PBS for 1 hr, and dehydrated in 30% EtOH in ddH2O for 24hrs, 50% EtOH for 24hrs, 70% EtOH for 2x24hrs, EtOH:MeOH:H2O (4:4:3)  for 1hr, 80% MeOH for 1hr and 90% MeOH for 1hr. Next, the specimens were soaked in 0.7% phosphotungstic acid (PTA) in 90% MeOH solution for 6 days, with fresh stain daily. Specimens were then rehydrated in a methanol series: 90% MeOH for 10mins, 80% MeOH for 10mins, 70% MeOH for 10mins, 50% MeOH for 10mins, 30% MeOH for 10mins, and sterile water. Voxel size was 17.2-24.6 microns3.    27 Chapter 3: Results 3.1 Structure of the midline fusion zone in the hard and soft palate boundary In order for proper analysis of palate fusion in 2D, frontal sections, specific anatomic markers were used to accurately determine the transition of hard palate to soft palate  and these were based on the Atlas of Developmental Anatomy of the Face (Kraus et al., 1966). Several large anatomic structures aided in defining the depth of section including the orbits and nasal septum. When sections were past the orbit (past the lens and posterior wall of the eye) and the nasal septum was no longer in contact with the palate we determined that we were close to the boundary of the hard and soft palate. Two other features marked the transition to the soft palate; the absence of the horizontal palatine bones in the palatine shelves, as well as the absence of the primary molar tooth buds. I used the aponeurosis, a tendinous band, as a marker of the mid-soft palate. The uvula marked the posterior soft palate; however, it was challenging to orient the plane of section if the block was tilted during cutting. In extreme samples, one side of the head was cut through the soft palate while the other side was cut through the hard palate.  Our previous study found that all the 54-day specimens and some 57-day specimens displayed incomplete hard palate and open soft palates (Danescu et al., 2015). Therefore, I focused mainly on 57-day specimens and older when there was full contact in the soft palate. In the previous study using histochemical stains there was obvious demarcation of the hard palate midline seam from the surrounding mesenchyme however in the soft palate, the distinction was much fuzzier. This difference in staining may be due to a lower level of keratinization in the soft palate epithelium. Therefore, for my study I decided to use an immunohistochemistry approach to better distinguish the keratinized from non-keratinized epithelium. Additionally, I wanted to determine when the palatine muscles began to invade the soft palate. In order to confirm the   28 presence of muscle versus tendon I used antibody to muscle that was used in our previous study (Danescu et al., 2015). 3.2 Immunohistochemistry was successful in specimens fixed in PFA independent of storage conditions  I decided to test whether immunohistochemistry would be successful on tissues that were stored under varying conditions. The 4 variables that I tested were 1) type of fixation PFA versus Carnoys 2) Length of time of storage prior to paraffin embedding 3) length of time since sections were cut and 4) whether previous H and E staining would still preserve antigenicity. The distribution of samples in each category is listed in table 3.1. In order to determine whether the hard and/or soft palate midline seam was epithelial derived I used a polyclonal pan-cytokeratin antibody that cross-reacts with mucosal keratins and keratins present in the oral mucosa (Presland and Dale, 2000). I also used a monoclonal antibody the rod-like tail region of myosin heavy chain protein (MF20) (Bader et al., 1982) to identify muscle in the soft palate and to distinguish the muscle from the aponeurosis. Finally, I used an antibody to E-Cadherin which marks epithelial cells. E-cadherin is a calcium dependent adhesion molecule with a role in growth and development, as well as metastasis. Several studies identified a link between E-cad germ-line SNPs that result in a phenotype of gastric cancer and non-syndromic cleft lip and palate (Ittiwut et al., 2016; Vogelaar et al., 2013). E-cadherin is expressed in the midline epithelial seam of the hard palate (Vogelaar et al., 2013). One study showed that TGFβ3 signaling induces EMT in the midline epithelial cells by forming activating transcription complexes that directly inhibit E-cad gene expression (Nawshad et al., 2007). Therefore, E-Cadherin may be downregulated at the time that EMT is taking place.    29  I first carried out immunohistochemistry on sections that were recently cut, from specimens stored in PFA for 25 years, decalcified and processed into wax by a previous graduate student (Table 3.1; 57d-9; Fig. 3.1). The student decalcified the specimen using 7% EDTA for several months prior to processing into wax. This specimen had a fully fused soft palate with no remaining midline seam. In tests with pre-immune serum followed by secondary antibody and detection with diamino-benzidine (DAB), there was no background staining in the tissue (Fig. 3.1A, A’). The primary antibody detected cytokeratin staining in the hard (right side of section in Fig. 3.1B) and soft palate as well as in the lining of the naso and oropharynx (Fig. 3.1B,C). The dental lamina is present on the right side of the maxilla (Fig. 3.1B) and has strong staining. This epithelium is ectodermal in origin. The posterior-soft palate, which was well into the endodermally derived part of the oral epithelium (Fig. 3.1C,C’), had similar intensity staining to the sections that were through the hard palate (Fig. 3.1B,B’). The thickened epithelium covering the oral side of the soft palate is positive for cytokeratin (Fig. 3.1C’). The thickening may be due to bending of the soft palate and therefore the section cuts at a tangent through the epithelium.  Another specimen that was stored wet in 4% PFA for 25 years, (57d-6) was studied. This time sections were through the hard palate where a robust midline epithelial seam was present. In addition, the dental lamina was present on both sides of the maxilla, which would allow us to test if there were differences in staining between the dental epithelium and other oral epithelium. In this sample we tested 3 antibodies, anti-cytokeratin, E-Cadherin and MF20. The cytokeratin antibody successfully detected keratin in the external head ectoderm, oral and nasal ectoderm as well as the dental lamina and midline seam (Fig. 3.2A,A’). The E-Cadherin antibody did cross react with epithelial antigens in the dental lamina and oral epithelium but not the midline epithelial seam (Fig. 3.2B,B’). The MF20 antibody was able to detect ocular muscles however   30 soft palatine musculature was not included in this section (Fig. 3.2C,C’). In summary, positive cytokeratin staining but absence of E-Cadherin staining of the midline seam does not resolve the question of whether EMT is taking place. Thus, at the stage we have examined, the seam is still epithelial in character.  The next specimen examined was one that had been processed into paraffin in the 1980’s and sectioned but not stained (57d-4). Here the antibodies to cytokeratin, E-Cadherin and MF20 all worked with the best being the cytokeratin antibody. There was strong positive signal for cytokeratin in the fungiform papillae of the tongue, the lining of the Eustachian tube, the naso and oropharynx (Fig. 3.3A,A’). The small epithelial islands present in the soft palate were also stained with the cytokeratin antibody (Fig. 3.3A’ and inset). The E-Cadherin antibody was less robust, very light brown signal was present in the fungiform papillae (Fig. 3.3B). The midline epithelial seam remnants were not included in the section used for E-Cadherin or perhaps the antibody did not detect signal (Fig. 3.3B’). This could be resolved in future studies staining sequential sections with both antibodies (E-Cadherin is raised in mouse and Pan-cytokeratin is raised in rabbit). The MF20 antibody stained the ocular muscles but background was high. No counterstaining was carried out due to the light stain. The levator veli palatini or tensor palatini muscles did not appear to have specific staining (Fig. 3.3C,C’).  Having had fairly good success with 3 antibodies on specimens that were stored for many years in PFA, we next tested whether previously stained and coverslipped archival sections could be used for immunostaining. We used the most robust antibody to cytokeratin on a section through the soft palate, where midline seam was visible (57d-2; Fig. 3.4 A, A’). We were pleased   31 to see that specific staining of the epithelium was visible (Fig. 3.4B,B’). This opens up the possibility of retrieving additional information from archival tissues.      32 Table 3.1 List of histology specimens used in the study IHC Specimen Hard palate Seam Soft palate seam Embedded in wax in the 80’s Embedded in wax after 2010 Previously coverslipped  57d-1 + open  +  57d-2 + Seam, apo +  + 57d-3 + Seam ant, apo +   57d-4 + Remnants, apo +   57d-5 + Open   +  57d-6 + R ant/mid, O post +   57d-7 + R ant/mid, O post  + + 57d-8 + Seam ant, O mid/post  +  57d-9 + Seam ant, R mid, apo  +  57d-10 + Seam ant, apo  +  57d-11 + Seam ant, O mid/post  +  57d-12 + Seam ant, R mid, apo  +  57d-13 + Sagittal  +  57d-14 + Seam, apo  +   Key: ant=anterior, apo=aponeurosis, mid=middle, o=open,  post=posterior, R=remnants          33   Immunohistochemical staining of a 57- day fetus with anti-cytokeratin  Fig. 3.1 This specimen was stored wet in PFA for 25 years. (A,A’) Preimmune control through the mid-posterior soft palate displaying lack of non-specific stain. The epithelium on the oral side  has multiple layers. The soft palate has a void in the midline which may be an artifact of sectioning or a real dehiscence. The aponeurosis is developing in the centre of the soft palate. (B,B’) A frontal section of the same specimen stained with anticytokeratin. The beginning of the anterior soft palate is distinguished by the presence of the dental lamina and the disconnected nasal septum and the absence of the palatal bone. The antibody stains the midline epithelial seam in the soft palate with epithelial remnants (arrow in B’). Oral epithelium lining the oral side of the soft palate also displays strong staining. In addition, the endoderm, dental lamina, dorsum of the tongue and the pharynx lining are positive. (C,C’) An adjacent section through the soft palate stained with anticytokeratin. Positive antibody stain is present in the oropharyngeal and nasopharyngeal epithelium. A multilayered positive seam is visible on the oral side. Key: ap=aponeurosis, d=dentary bone, dl=dental lamina, en=endoderm, ep=epithelium, mc=meckel’s cartilage, np=nasopharynx, op=oropharynx t=tongue. Scale bar= 2mm for panels A-C and 500µm for panels A’-C’.     34   Frontal sections of a 57-day fetal head through the hard palate stained with anti-cytokeratin, E-Cadherin and MF-20  Fig. 3.2 This specimen was stored wet in PFA for 25 years. All sections are frontal through the hard palate. Fusion has taken place and a midline seam is present. (A,A’) The midline seam/epithelial islands (blue arrows), nasopharyngeal lining, oral epithelium and epithelium on the dorsum of the tongue display positive stain. Additionally, the ectodermally-derived dental lamina has strong cytokeratin stain. (B,B’) A sequential frontal section of the same specimen stained for E-Cad. E-Cad positive cells are present in the dental lamina but not in the midline seam. (C,C’) A sequential frontal section of the same specimen stained for MF-20. All extraocular muscles, genioglossus and buccinator are present and clearly stained with antibody. Key: d=dentary bone, dl=dental lamina, en=endoderm, gg=genioglossus, ir=inferior rectus muscle, lps=levator palpebrae superioris, lr=lateral rectus, mc=meckel’s cartilage, mr= medial rectus, ns=nasal septum, pb=palatal bone, t=tongue. Scale bar=2mm for panel A-C and 500µm for A’, 500µm for B’, 500µm for C’.         35    Immunohistochemistry of a 57-day fetal head with a postive cytokeratin, E-Cadherin and MF20 signal   Fig. 3.3 This specimen was processed 25 years ago into wax and sectioned at that time. Unstained sections were used. All sections are in the frontal plane through the soft palate.  The specimen was embedded crooked in the paraffin resulting in eustachian tube being present on the right side. Though the palatal bone is present on the right side of the section, the majority of the section is in the soft palate. The nasal septum is disconnected with the palate and the uvula is present. (A,A’) Positive cytokeratin stain is present throughout the fragmented soft palatal seam. Numerous positive epithelial islands are present within the seam (insert with blue arrows). Positive cytokeratin stain is also seen in the nasopharyngeal lining, oral epithelium, endodermal lining of the eustachian tube and the fungiform papillae of the tongue. (B,B’) A sequential frontal section of the same specimen stained for E-Cadherin. The midline seam displayed negative E-Cad stain while the tongue epithelium has some light staining. (C,C’) A sequential frontal section stained with MF-20. Positive stain is present in the extraocular and buccinators muscles. Key: ap=aponeurosis, bu=buccinator, et=eustachian tube, fp=fungiform papillae, ir=inferior rectus muscle, lps=levator palpebrae superioris, lr=lateral rectus, mr=medial rectus muscle, np=nasopharyngeal lining, ns=nasal septum, pb=palatal bone, sr=superior rectus muscle, tf=temporal fascia, u=uvula. Scale bar= 2mm for panels A-C and 500µm for A’, 100µm for insert, 500µm for B’, 1mm for C’   36     Immunohistochemisty carried out on archival sections of a 57-day specimen previously stained with H&E  Fig. 3.4 These sections were previously stained and coverslipped 25 years ago. Coverslips were soaked off in xylene leaving residual hematoxylin staining. This head is incomplete, lacking the mandible and tongue. These frontal sections are at the level of the anterior soft palate. (A,A’) In the original stained sections, a midline seam is present within the soft palate and fragmenting towards the oral side (A’). The stain in the seam is stronger than the adjacent mesenchyme. The palatine aponeurosis is also developing on the right and left shelves (asterisks). Thick layers of epithelium can be seen on the oral side of the pre-fusion soft palatal shelves. (B.B’) A section from the next slide in the series. The coverslip was removed and IHC staining performed. The midline epithelial seam stains positive for anticytokeratin. The nasopharyngeal lining, oral epithelium and the endoderm of the buccal mucosa also display moderate cytokeratin stain. Key: ap=aponeurosis, en=endoderm, np= nasopharyngeal lining, oe=oral epithelium, pb=palatal bone, s=seam. Scale bar= 2mm for panels A-B, 500µm for panels A’-B’.      37 3.3 Markers of proliferation and apoptosis did not work on human specimens The interesting lack of E-Cadherin staining in the midline seam suggested that possibly epithelial-mesenchymal transformation was underway. However to rule out the other major mechanism, apoptosis, I wanted to examine cell death in the midline seam. TUNEL analysis is a commonly used to identify DNA fragments due to apoptosis.  A terminal deoxynucleotide transferase (TdT) enzyme is used to locate nicks in DNA and catalyze the addition of dUTPs that are labeled with a secondary label. I completed TUNEL analysis on three specimens with seam present throughout the soft palate. Unfortunately, TUNEL analysis did not work on any of our specimens, likely due to the state of the tissue and the length of specimen storage (data not shown). Thus, the exact fate of the epithelial cells in the midline epithelial seam of our specimens cannot be defined conclusively without molecular data. Similarly we tested the antibody to proliferating nuclear cell antigen (PCNA, N = 2) and phosphohistone H3 (N = 2). Once again no nuclear staining was observed (data not shown). More rapid fixation would most likely preserve integrity of nuclear antigens. Alternatively, pretreatments of sections may need to be optimized for detection of antigens that are internalized or intracellular such as E-Cadherin.  3.4 59-74 day specimens display complete fusion of the hard palate and differential levels of soft palate development through histological evaluation   Our recent publication analyzed eleven specimens aged 59-74 days to look at retention of the midline seam and development of the soft palate (Danescu et al., 2015). Complete fusion of the hard palate had taken place and all fetuses had epithelial remnants except for a single 59-day specimen. Ten specimens displayed continuous mesenchyme throughout all regions of the soft palate. One of the more challenging aspects of working on the older specimens was that if the   38 block was angled it was difficult to be certain about whether the section was in relation to the different regions of the palate (Fig. 3.5A-B’). The tonsillar fauces are forming on the lateral sides of the oropharynx and these also form medial projections similar to the palate (Fig. 3.5A’,B’).. While it can be informative to have two different depths through the head in a single section, the angulation can make it challenging to identify the soft palate. In particular, the bending of the soft palate makes it difficult to recognize the three regions (anterior, middle, posterior). A typical fetus as young as 57 days will have a horizontal hard palate while the soft palate forms an angle of 130° to the hard palate (Fig. 3.6A-C’). Thus we initiated 3D imaging of whole specimens to gain a better appreciation of the palate and oropharynx.      39   A 59-day specimen stained with H&E  Fig. 3.5 (A,A’) A frontal section of a 59-day specimen embedded and sectioned crooked. The sections are stained with H&E. The frontal section is not parallel to the coronal suture and only the right eye and maxillary bone is present. On the left side the palatine process of the maxillary bone is seen. A faint midline seam is present in the fused hard palate (blue arrows). (B,B’) A frontal section more posterior to A. The plane of section is difficult to assess since the specimen was crooked. The eyes are asymmetric and there is developing aponeurosis in the soft palate even though the nasal septum is continuous with the palate. Key: ap=aponeurosis, d=dentary bone, e=eye, mb=maxillary bone, pp=palatine process of the maxillary bone. Scale bar=2mm for panels A-B and 1mm for panels A’-B’.    40   Mid-sagittal hard and soft palate morphogenesis in a 57-day specimen  Fig. 3.6 (A,A’) A mid-sagittal section of the fetal head of a 57-day specimen stained with H&E. Both hard and soft palates are present and boundary can be differentiated (solid black line). The angle of the developing soft palate with the uvula projecting posterior and inferior (solid blue line). The pituitary adenohypophysis is developing dorsal to the palatal boundary. The tongue, palatal bone and vestibule are readily discernable. (B,B’) A sagittal section of the same specimen lateral to the midline stained with H&E. The seam near the boundary of the hard and soft palate represents the hard palate and anterior soft palate seam cut obliquely (black arrowheads). (C,C’)  A sagittal section of the same specimen more lateral with more of the nasal septum present. Key: gg=genioglossus, nc=nasal concha, ns=nasal septum, pa=pituitary adenohypophysis, pb=palatal bone, pmx=pre-maxilla, t=tongue, v=vestibule. Scale bar= 2mm for panels A-C, 1mm for panels A’-C’.        41 3.5 MRI 3D segmentation of craniofacial structures in specimens aged 67-74 days display complete fusion of both hard and soft palate Imaging of soft tissues in 3D requires either MRI or µCT scanning with contrast agents. We planned on using the whole specimens that were already decalcified and wanted to preserve their use for future histological sectioning. Therefore, we began with MRI since the likelihood was that staining with gadolinium would not affect tissue integrity.  Gadolinium acts to increase the contrast and reduce the scanning time. Seven whole specimens aged 67-84 days (64-days-1, 67-days-2, 74-days-1, 75-days-1, 76-days-1, 84-days-1) were examined with MRI scan. Resolution was adequate at a slice thickness of 53-75µm, which was 7-10 times greater in thickness than our histology sections. Two of the larger specimens (75- and 84-day) lacked internal definition and could not be used. The larger specimens likely did not have adequate perfusion of the contrast agent. Post-processing was carried out to denoise the image stack. We were then able to visualize different tissues in the head with sufficient detail to segment out the tongue, palatal shelves, nasal passages and even the midline epithelial seam (Fig. 3.7A-B’). Bone and epithelium were both dark whereas empty spaces in the eyes and nasopharynx were light. Soft tissues were also light. Some muscles could also be visualized such as the genioglossus and ocular muscles as a medium grey intensity (Fig. 3.7A; Fig. 3.8A,B). Tooth buds were recognizable although not very distinct (Fig. 3.8A). Hard palate fusion was complete in the five specimens between 64 and 75 days as expected (Fig. 3.8A-E and data not shown).  All five specimens displayed the presence of epithelial remnants or partial seam within the hard palate on MRI (Fig. 3.7A-B’; data not shown). Additionally, soft palate fusion was complete in all specimens. One 64-day specimen displayed seam remnants in the anterior soft palate (data not   42 shown). This result confirms the histological sections published by our group which a soft palate seam persists by another 10 days to what was published by our group (Danescu et al., 2015).   In order to visualize both the hard and soft palate maturation in 3-dimensions, 3D models were fabricated using digital segmentation. A volumetric model of the completed specimen was created. Specimens were then transformed in all three planes of space by rotating the volume plane first. Sequential slices were traced for anatomic structures adjacent and including the hard and soft palate in the frontal view. Tracings were meshed together to create a 3D model of each traced structure. MRI was adequate to trace and fabricate a 3D model of the epithelial remnants in the palate (fig 3.7C-E). Resolution was not sufficiently detailed enough to trace muscle insertions and individual nerves. No overall conclusions could be made on the palatine muscle insertion of specimens examined by MRI. The relationship of the midline epithelial remnants in the hard palate of all and soft palate of one specimen was consistent with our histological analysis. Additionally, the shape of the soft palate tapered from anterior to posterior similar to histological 3D reconstructions from our previous study (Fig. 3.8E).      43   Virtual slices of an MRI scan in the frontal plane with segmentation   Fig. 3.7  A 64 day specimen was reconstructed, transformed to a new orthogonal plane and then segmentation was carried out on the image stack. (A,A’) Slice through the hard palate included the orbits (golden), primary tooth buds (yellow), the tongue (brick red), palatal shelves (purple), nasal passages (blue) and midline seam (green). Bones, cartilages and epithelia appear dark. Spaces and mesenchyme appear light grey, muscle medium grey. The horizontal plates of the palatine bone were used to define the superior border of the palate mesenchyme. Note that he tongue is in direct contact with the palate in this wet specimen. Scale= 1mm for all panels         44                   Volume renderings of segmented craniofacial structures from MRI scans   Fig. 3.8  A) A slice through the hard palate. B) a slice through the soft palate. C) frontal view of segmented regions onto a projection of the slices. D) Subtracting the slice reveals the outlines of each slice in the stack. E) Rotated view from the sagittal aspect showing the bend in the soft palate. Note that the midline seam is not visible through the volume since the seam is not transparent. Key: e =eye, hp =hard palate, sp =soft palate, t= tongue, tb=tooth bud, ve =vestibule, v =vomer     45 3.6 Quantitative analysis of microCT imaging of the soft palate morphology in specimens aged 67-74 days Soft palate morphogenesis was challenging to study using MRI due to poor contrast. I therefore washed and stained the seven MRI specimens using a phosphotungstic acid staining protocol by Tesarova (Tesařová et al., 2016) as modified from Metscher et al. (Metscher, 2009).  The seven decalcified specimens aged 67-84 days (64-days-1, 67-days-2, 74-days-1, 75-days-1, 76-days-1, 84-days-1) were soaked in 0.7% phosphotungstic acid (PTA) contrast agent. PTA dramatically improved resolution in all seven specimens over the MRI imaging (Fig. 3.9A-F; Fig. 3.10A-D). In contrast, conventional CT scanning of unstained specimens only permits visualization of radiopaque bone (data not shown). PTA differentially stains cartilage, muscle, nerve and bone. Individual muscles and vessels which could not be distinguished through MRI were readily discernable with microCT (Fig. 3.9 A-F). This method holds the best promise for detailed visualization of 3D anatomical structures.   One of the issues with contrast staining that can arise is tissue shrinkage (Metscher, 2009). Therefore, I compared a paraffin embedded 64d specimen to a specimen imaged with MRI and PTA-µCT (Fig. 3.11A-C’). As suspected, the paraffin processing introduced large spaces into the oral cavity. These gaps in between the tongue and palate are absent in the MRI specimen and thus must be an artifact of dehydration and heating the specimen in wax. Similar space in the oral cavity is seen in other paraffin specimens (Fig. 3.1, 3.2, 3.3). The PTA-µCT specimen is intermediate between the paraffin and MRI (Fig. 3.10C,C’). Interestingly, this is exactly the same specimen, reimaged following MRI and yet more space is visible in the oral cavity than with MRI. Nevertheless, the shrinkage following PTA staining is less than for paraffin.   46    PTA stained 64-day specimen displays clear hard palatal seam through microCT imaging   Fig. 3.9 PTA staining of the same specimen as in Figure 3.7. (A-C, F) Virtual slices through the hard palate. The images are taken from the 3 orthogonal planes. The frontal view corresponds to the green line in B. The midsagittal view in B corresponds to the red line in A and C. The transaxial view in C and F corresponds to the blue line in B. The angled soft palate is visible in the sagittal view and the midline seam in the hard palate is visible in A,C and F. The bend in the soft palate means that it appears to be disconnected from the hard palate in C,F. From the frontal section there is clear delineation of the muscles, tongue, nasal septum, nasal and oral cavity. The hard palate has radiopaque palatal bones present bilaterally. The seam is only present on the oral side of the developing soft palate palate (F). D,E) More posterior section through the head within the soft palate shows the separation of the oro and nasopharynx by the soft palate. The aponeurosis is visible in the frontal slice. Key: HP=hard palate, SP=soft palate     47       PTA microCT of an 84-day specimen displays a midline seam in the hard palate  Fig 3.10 PTA staining of an 84-day specimen. (A,B) Virtual slices taken through the hard and soft palate in an axial view. The red line indicates the level of section in the sagittal view shown in (C). (B) Displays a magnified view of the hard palatal seam (blue arrowheads. (C) Parasagittal slice with the green line representing the level of the axial sections in A-B. (D) Frontal slice at the level of the hard palate. Key: HP=hard palate, ns=nasal septum, SP=soft palate, t=tongue   48    Direct comparison of MRI to PTA-microCT imaging in the same specimen   Fig. 3.11 The left column is through the hard palate and right column is through the soft palate. A,A’) A representative 64 day specimen fixed in PFA, dehydrated through ethanol series and then processed into paraffin. Note the large spaces between the tongue and palate as well as between the nasal septum and nasal turbinates. Also this is an example of a specimen that was not cut in the ideal orthogonal plane. The left side is deeper than the right side and is cutting through the soft palate. B,B’) Much the same anatomy is visible but the specimen is oriented along the true orthogonal planes. Note the direct contact between the tongue and palate and very small space in the nasal passages. C,C’) PTA staining involves some dehydration steps which has led to shrinkage of the tissue. There are more spaces between the tongue and palate than in the MRI specimen but not as much as in the paraffin embedded specimen. Key: e=eye, hp=hard palate, ns=nasal septum, pb=palatine bone, sp=soft palate, t =tongue, v=vomer    49 Chapter 4: Discussion 4.1 The human hard palatal seam persists until at least 84-days post conception Our previous study examined 28 human specimens aged 54-74 days post-conception through general IHC techniques for palatal development (Danescu et al., 2015). Only 9 of these specimens were 64-days or older and 8/9 specimens displayed what appeared to be remnants in the hard palate midline. In the present study, I extend the sample ages for observing the midline seam to at least 84 days using PTA-µCT. I conclude that the oral side of the midline epithelial seam in the hard palate persists in all specimens studied by both MRI and µCT however the nasal side is obliterated by the palatine bone and palatine process of the maxillary bone. This contradicts prior studies by Wood and Kraus reported 56-days as the latest date for hard palate seam visualization (Wood and Kraus, 1962). A recent histological study in human specimens aged 12-18 weeks showed that epithelial like pearls were developed in the palatal midline by 70 days (Kim et al., 2016). These pearls which contain keratin and are not lined by epithelial cells became larger by 98-days, when the ossification of the palatal bones was complete. In contrast, Kitamura showed a definite midline epithelium in the hard palate at 77-days but after that the epithelium was not present and instead there were keratin cysts in the midline (Kitamura, 1966). These cysts can persist until birth.   My data showing the remnant of a midline seam on the oral side of the hard palate up to 84 days raises the question of how this epithelium is removed. Most babies are born with Epstein’s pearls or Bohn’s nodules (up to 80%) (Monteagudo et al., 2012). Without exception these keratinized structures are within the connective tissue rather than in the midpalatal suture. One possibility is that radial migration is taking place (T. Cox, personal communication), gradually moving the epithelial cells in the triangle of epithelium towards the oral surface,   50 ultimately leading to a flattened epithelium. Alternatively, apoptosis or EMT may be taking place. I would argue that since the keratin cysts are so prevalent it seems unlikely that EMT or apoptosis can be taking place. Instead it seems that the epithelial character has been retained since keratin is deposited. A large cell population remains to generate the keratin cysts arguing against apoptosis as the mechanism. 4.2 Variability in timing of soft palate seam degradation This study confirms and adds support to the results from our previous study that fusion is the primary mechanism of soft palate development in humans (Mattson, 2013) I have extended her work by reporting histological sections with the midline seam present in the soft palate of a 64- and a 67-day specimen (Danescu et al., 2015). The interesting point is that not all 64-day specimens had a soft palate seam, as shown by my PTA-stained specimen as well as one specimen in our previous study (Danescu et al., 2015). I also added 2, 67-day specimens that were scanned with PTA-µCT. Neither of these had a seam in the soft palate. It will be important to expand the sample further to determine if these 2 specimens are outliers.  4.3 The epithelial midline seam differs in phenotype in the hard and soft palate Although both the hard and the soft palate are shown to form primarily by a mechanism of fusion, with the formation and subsequent removal of a midline seam, several differences were observed. The hard palate seam formed when the epithelial lined palatal shelves touch is uniform and displays consistent width in appearance. We observed the hard palate seam to be highly organized and bilayered in appearance with darkly stained cells following H&E staining. These cells are discernable from the adjacent mesenchymal cells. Staining with the anti-cytokeratin antibody showed that the cells forming the seam were positive and darkly stained providing evidence of their epithelial nature. In contrast, the cells forming the soft palate seam   51 were disorganized, irregular, clumpy and pale in appearance. The soft palate seam is formed by multilayered pseudostratified cells, which range from 2-20 cells thick. This may be the result of epithelial seam outgrowth similar to the mouse or epithelial sloughing may occur (Diewert, 1982). This seam was more difficult to distinguish from the underlying mesenchyme with both H&E and Picosirius Red staining when compared to the hard palate seam. However, anti-cytokeratin staining showed that the cells in the soft palate seam were positive for stain. This could be seen on the same specimen when hard and soft palate seam or remnants coexist, with slides treated similarly. We cannot rule out other antibodies that recognize forms of keratin found primarily in endodermal epithelium could detect differences in the hard and soft palate epithelium. A second epithelial marker was also similar in the hard and soft palate, E-Cadherin. In both regions of the palate staining was absent in the midline seam.  This negative data can be interpreted as a transition to a mesenchymal phenotype. The positive staining found in other epithelia that do not undergo EMT (dental lamina, oral epithelium) suggests the antibody was working. The negative data however is not conclusive and additional markers of EMT need to be studied. 4.4 Archival sections can be successfully used for immunohistochemistry All histological specimens used in this study were preserved in 4% PFA made up in phosphate buffered saline prior to decalcification and fixation in paraffin wax for sectioning. Some specimens were plated and coverslipped many years ago. Since my sample was unique and irreplaceable, I decided to attempt IHC on some of these slides. Coverslips were carefully removed avoiding tissue damage after soaking in xylene. The slides were rehydrated and used for typical IHC. I report that many robust antibodies displayed positive stain compared to   52 controls. Archival slides with coverslip removal and previous staining from the 1980’s did not stain as well as fresh slides in both the hard and soft palatal tissues.  Thus, for long-term preservation of human tissues in PFA does not have significant deleterious effects on human tissue. Archival material can be mined for molecular information years after routine sectioning and coverslip application has been carried out. Only one study to my knowledge exists whereby 90 slides that were de-coverslipped with salivary gland tissue for malaria detection were used to identify the presence of spores by ELISA analysis (Beier et al., 1991). The slides were de-coverslipped, left to dry for thirty days before rehydration and molecular studies were completed. The authors report similar results with de-coverslipped specimens compared to freeze dried samples. The present study is the first time, to my knowledge that IHC has been attempted on archival material stored for such a long period of time. 4.5 Antigenicity is present after long-term storage for abundant proteins The human specimens that comprised our sample are rare and irreplaceable. The collection of these unique specimens was done at the time of abortion and dates back to the 1980’s. As such, they have been in storage for several decades, which may lead to their deterioration of proteins over time. The majority of specimens were stored long-term at room temperature in 4% paraformaldehyde. Even with long-term storage my specimens readily stained with cytokeratin, MF-20 and E-Cadherin antibodies for IHC.  Due to the excessive length of storage, the RNA and DNA integrity was compromised. I completed many trials of TUNEL analysis, proliferating nuclear cell antigen (PCNA) and histone 3 phosphatase (H3P) staining without any results. The previous graduate student tried radioactive in situ hybridization with several probes to no avail (Mattson, 2013). Had the TUNEL detection   53 worked, I would have been able to rule in or out apoptosis as a mechanism involved in human midline epithelial seam removal. PCNA or H3P staining would allow the characterization of cell proliferation within the seam. A recent study on the palates of human embryos from 6 -10 weeks of gestation used antibodies to Ki67 for proliferation and Caspase 3 for apoptosis (Vukojevic et al., 2012).  These authors also used TUNEL on their specimens. The primary palate seam between the medial nasal and maxillary prominences showed proliferation and apoptosis at 6 weeks. The secondary palate was not shown in the study but they do report apoptosis being present. These authors suggest that a certain level of apoptosis is taking place in the hard palate but do not investigate other mechanisms. The quality of preservation appeared to vary between specimens, an issue that is probably the variability in my study 4.6 Non-destructive methods are a valuable adjunct to histology for analyzing human development As shown in my study different imaging methods have their strengths and weaknesses. Although, I pioneered using MRI for imaging on fixed, wet specimens and tissue shrinkage was completely avoided, the resolution was significantly lower than expected. Also, the cost for imaging per hour was 4-fold higher than µCT. Alternatively, PTA-µCT was very successful.  Previous to my work, only two publications used contrast staining of human cadaveric or pathological tissues. One study used PTA to enhance the soft tissue contrast of the inner ear morphology of 6 human cadaver temporal bones prior to µCT scanning (De Greef et al., 2015). The second study utilized PTA to evaluate collagen distribution in osteochondral samples from 2 men undergoing total joint replacement of the knee (Nieminen et al., 2015).  The only human fetal study published so far is on the urinary tract (Siebert et al., 2013). The present study is the first study to my knowledge to utilize PTA-µCT in human fetal craniofacial tissue and offers an   54 excellent alternative to MRI. One caveat is that I do not know whether histology is preserved after PTA staining, or whether PTA can be removed from tissues prior to embedding and sectioning. In the future, synchrotron scanning in combination with PTA staining will provide subcellular resolution equivalent to histology.  4.7 Future directions Outstanding questions raised by my study include 1) Whether or not EMT is taking place in the midline seam during fusion? 2) How is the oral side of the midline seam removed in the fetal hard palate? 3) Can the IHC technique be optimized so that other antibodies will work on our specimens? 4) Does the midline seam persist beyond 84 days? 5) What underlies the structural differences in the soft palate versus hard palate epithelium? Are there differences in the basement membrane or tight junctions in different regions of the palate? 6) Is the mouse a good experimental model for studying epithelial differences in the hard and soft palate? Going forward, I would test more rigorously whether EMT is taking place in the human palate. There are many markers identified in mouse studies, particularly in the epithelium that could be studied (Yu et al., 2009). Tgfb3 is required for palate fusion in the mouse and is specifically expressed in medial edge epithelia at the time of adhesion (Kaartinen et al., 1997; Proetzel et al., 1995). It is likely that this molecule is downregulated once adherence of the palatal shelves has occurred. There is a much longer time interval in humans compared to mice in which to study this question.  Syndecan-1 (Sun et al., 1998; Vukojevic et al., 2012) is a good epithelial marker in both mouse and human palatal epithelium.  Interferon regulatory factor 6 (IRF-6) a transcription factor that is expressed in the oral epithelium and which is implicated in cleft formation (Beaty et al., 2010; Kondo et al., 2002; Leslie et al., 2016) should also be studied.  In addition to loss of epithelial markers (negative data), it is essential to investigate cytoskeletal   55 changes that indicate cells have become mesenchymal in nature (positive data).  Use of an expanded set of markers for apoptosis, EMT and differentiation status of the epithelial cells would also address our second question of what happens to the oral epithelial seam in the hard palate after the midline suture has formed. Before any future immunohistochemistry can be performed, we need to test other pre-treatment conditions such as use of detergents, acids or enzymes to unmask antigens.  A new hypothesis that was raised by my study was that there are differences in the basement membrane and cell-cell junctions that correlate with the multilayered epithelium of the soft palate versus the highly organized bilayered hard palate epithelium. The lack of tight junctions in soft palate epithelium could explain why the seam is so rapidly degraded. Perhaps cell contacts are looser allowing cells to migrate between different inner and outer layers. A different panel of antibodies would be needed to answer this question. Alternatively, transmission electron microscopy would be able to resolve tight junctions at the ultrastructural level.  There are studies in mouse suggesting this could be a useful model for looking at soft palate development (Grimaldi et al., 2015; Iwata et al., 2014). These authors demonstrated that there is a seam in the soft palate and that Tgfb signaling is specifically required for soft palate fusion. While some aspects of the anatomy of a mouse are different to human such as the lack of a uvula and the mouse soft palate morphology is flatter, the mouse could be used for future work on epithelial differences in the hard and soft palate. No one has investigated the epithelial nature specifically in the mouse soft palate, therefore this would be a fruitful line of research. 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