Yang Chai, Ph.D. - US grants

A major issue in developmental biology is to determine how time and position-restricted instructions are signaled and received during morphogenesis of different phenotypes, of which tooth, Meckel's cartilage and tongue formation are classical examples. It is now evident that a hierarchy of growth factors and their downstream transcription factors regulate the timing, sequence and position of cells and tissues in forming different phenotypes during embryogenesis (Edelman, 1985; Lumsden and Krumlauf, 1996). We have developed an organ culture model in which mandibular explants formed tooth bud, Meckel's cartilage, tongue and osteoid-like tissue (Slavkin et al., 1989; Chai et al., 1994, 1997). Using this model, our studies have shown that transforming growth factor-beta (TGF-beta) ligands are expressed in a time and tissue specific manner and play important regulatory roles during first branchial arch morphogenesis (Chai et al., 1994). Recently, abundant molecular evidence has demonstrated that both TGF-beta type I and type II (IR and IIR) receptors as well as their downstream mediators (Smad2, Smad3 and Smad4) are required components of the TGF-beta signaling pathway (Massague, 1996). This research proposal is based on the hypotheses that (1) TGF-beta IR, IIR, Smad2, Smad3 and Smad4 are required for the signaling of TGF-beta ligands which regulate morphogenesis of the first branchial arch during craniofacial development. (2) the regulation of morphogenesis by TGF-beta is mediated by an intracellular signaling pathway that down regulates transcription factor Lef1. Three Specific Aims are designed to test the hypotheses: (1) to determine and compare the temporal and spatial expression of TGF- beta IR, IIR, Smad2, Smad3 and Smad4 during the formation of tooth, Meckel's cartilage and tongue both in vivo and in vitro using in-situ hybridization, RT-PCR, and immunohistochemistry. (II) to determine the function of TGF-beta IR, IIR, Smad2, Smad3 and Smad4 signaling in regulating first branchial arch morphogenesis during craniofacial development using gain-of-function (transgenic overexpression) and loss- of-function (antisense) experiments. (III) to determine the impact of TGF-beta signaling on the expression of Lef1 and assess the morphological changes caused by altered Lef1 expression. These studies will identify and determine the critical molecules in TGF-beta signaling pathway and how TGF-beta signaling regulates the expression of Lef1 during the specification and morphogenesis of tooth, Meckel's cartilage and tongue. Ultimately, this study will contribute to our understanding of how the TGF-beta signaling cascade regulates normal craniofacial development and how disruption in TGF-beta signaling pathway can lead to craniofacial malformations.

Embryonic development is a time- and space-ordered series of gene interactions, each in turn designating polarity, germ-layer identity, and individual cell type differentiation. Critical to our study is to determine how these time- and space-restricted instructions are signaled and received during morphogenesis of different phenotypes derived from the first branchial arch. Our previous studies on the regulation of first branchial arch morphogenesis have identical critical components of a well-accepted hierarchy of growth factors and their downstream transcription factors, which regulates the expression of genes responsible for determining cell phenotypes during embryogenesis. Here, we propose to test the hypothesis that TGF-beta signals converge on specific Smads which may alter the expression of transcription factors Msx1, Msx2 and Lef1 resulting in particular phenotypes during the morphogenesis of first branchial arch. TGF-beta ligands are expressed in a time- and tissue-specific manner and are important in regulation of tooth and Meckel's cartilage formation. Both time- and tissue-specific manner and are important in regulation of tooth and Meckel's cartilage formation. Both TGF-beta type I and type II (IR & IIR) receptors as well as their downstream intracellular mediators S Smad2, 3, 4 and 7 are required constituents of the TGF-beta signaling pathway. Activated Smads may regulate transcription factor expression, affecting target-gene transcriptional status. Our preliminary data show that TGF-beta cognate receptors, TGF-beta signaling pathway specific Smads and transcriptional factors Msx1, Msx2 & Lef1 are critical components in regulating first branchial arch morphogenesis. Our specific aims are: (1) to determine and compare the temporal and spatial expression of TGF-beta, IR, IIR, Smad2, Smad3, Smad4, Smad7 and transcription factors (Msx1,2 and Lef1) during the formation of tooth and Meckel's cartilage both in vivo and in vitro. (2) to test the function of TGF-beta IR, IIR, Smad2, Smad3 and Smad4 signaling in regulating first branchial arch morphogenesis using gain-of-function and loss of function experiments. (3) to determine the impact of TGF-beta signaling on the expression of Msx1, Msx2 and Lef1 using implantation of beads being different TGF-beta isoforms, assess the morphological changes, and evaluate the expression of Msx1, Msx2 and Lef1, using both wild type and readily available Msx1, Msx1 transgenic animals. The molecular mechanism of TGF-beta down-regulation of Msx1 and 2 expression will also be explored. (4) to define the function of TGF- beta signaling inhibitory molecule Smad7 and its regulatory effect on the expression of Msx1, Msx2 and Lef1 as well as other growth factors (EGF, PDGF-AA and FGF). Ultimately , this study will advance our understanding of how the TGF-beta signaling cascade guides normal craniofacial development and his disruption in the TGF-beta signaling pathway can lead to craniofacial malformations.

DESCRIPTION (provided by applicant): Craniofacial malformations represent one of the major groups of congenital birth defects in human population. Babies born with defects often suffer multiple handicaps that significantly compromise the quality of their lives. Cranial neural crest (CNC) cells are important progenitors for the morphogenesis of craniofacial structures. Growth and transcriptional factors are critical regulators for the fate of CNC cells. Alteration of signaling by these factors results in craniofacial malformations. Our previous studies on the regulation of early craniofacial development have identified that transforming growth factor-beta (TGF-beta and transcription factors Msx1 and LEF1 are responsible for determining cell phenotypes during embryogenesis. Using the two-component genetic system for indelibly marking the progenies of neural crest cells, we provide the first in vivo evidence that mutations of TGF-beta2 or LEF1, or Msx1 affect the fate of CNC cells, indicating that each one of these molecules has a critical role in regulating CNC cells. There are significant overlaps among the expression patterns of these regulatory molecules during tooth morphogenesis. Furthermore, TGF-beta signaling can negatively regulate the expression of Msx1, which can be further repressed by the possible synergistic interaction between TGF-beta signaling Smad and LEF1. Sequence analysis of Msx1 promoter reveals multiple Smad and LEF/TCF binding sites, providing the molecular basis for the synergistic interaction between TGF-beta signaling Smad and LEF1 in regulating Msx1 expression. Msx1 is critical for proliferation and differentiation of CNC cells. Alteration of Msx1 expression may affect the proper CNC differentiation. Thus, we focus our study to explore the hierarchy of TGF-beta2, LEF1 and Msx1 in regulating CNC cells and test the hypothesis that each of the signaling molecules (TGF-beta2, LEF1 and Msx1) has specific regulatory roles for CNC cells during craniofacial development and the cooperation of TGF-beta signaling Smads and LEF1 alters the expression of Msx1, thereby, determining the fate of CNC cells during tooth morphogenesis. Ultimately, this study will contribute to our understanding on how the TGF-beta signaling cascade regulates the fate of CNC during normal craniofacial development and how pathway disruption can lead to craniofacial malformations.

DESCRIPTION (provided by applicant): This proposal is submitted to seek support to help organize and conduct a new Gordon Research Conference on Craniofacial Morphogenesis and Tissue Regeneration. This International Conference will be held January 18-23, 2004 and also in 2006, 2008 and in every two years thereafter at Ventura (near Los Angeles), California. This is a new Gordon Conference that has been proposed and awarded to recognize the increasing importance and level of research activity in the field of Craniofacial Morphogenesis and Tissue Regeneration. Previous conferences on the specific topic of craniofacial development have either been organized on an ad-hoc basis or as part of larger more general development/mineralization conferences. Previous specific meetings such as those held in Iowa 1993, NIH, Bethesda (1998) and London (1998) have been very well attended and successful and helped to bring together individuals in this field. This new GRC is a major step forward because it will provide a regular forum for the latest progress in this research area to be discussed. Tissue regeneration is an emerging field that is particular relevant to craniofacial developmental biology where bioengineering approaches to hard and soft tissue, repair and replacement are emerging. This Gordon Conference on Craniofacial Morphogenesis and Tissue Regeneration will address the current state of knowledge regarding the molecular mechanism in regulating the initiation, growth, and regeneration of craniofacial structures. Session topics will include: 1) Neural crest, ectoderm and endoderm interactions; 2) Craniofacial patterning, midline and frontonasal prominences development; 3) Placodes and their functional significance during craniofacial development; 4) Craniofacial organogenesis; 5) Suture biology; 6) Signaling interactions and gene regulation; 7) Human syndromes involving craniofacial defects; 8) Evolution biology; 9) Tissue engineering. Significantly, the themes of craniofacial evolution, morphogenesis, birth defects, and tissue engineering appear to converge at this time in history and therefore require a Gordon to assess the state-of-the-field. Undoubtedly, this newly established Gordon conference would provide a forum to spark new scientific collaborations, foster the growth of young scientists, will advance our understanding of the molecular regulatory mechanism of craniofacial development and tissue regeneration, and, ultimately, will contribute to the improvement of craniofacial health care.

DESCRIPTION (provided by applicant): The long term goal of this research project is to understand the molecular regulatory mechanism of TGF-[unreadable] signaling during palatogenesis. Cleft palate represents one of the major groups of congenital birth defects in the human population. Importantly, we and others have shown that mutations in TGF-[unreadable] signaling can cause cleft palate in both mice and humans. Specifically, TGF-[unreadable] is required for inducing apoptosis in the medial edge epithelium (MEE) and cell proliferation in the cranial neural crest (CNC)-derived palatal mesenchyme, both of which are crucial for normal palatal fusion. In this competing renewal application, we will carry out experiments to test the hypotheses that TGF-[unreadable] signaling mediator Smad4, its downstream target Msx1 and the interaction between Smad4 and Msx1 are crucial for the cell fate determination of CNC-derived palatal mesenchyme. Furthermore, TGF-[unreadable] regulates the expression of downstream target genes, such as Irf6 and Ctgf, to control the fate of MEE and CNC cells during palatogenesis. We have proposed three specific aims. In Specific Aim 1, we will investigate the functional significance of Smad4 in regulating the fate of CNC- derived palatal mesenchyme during palatogenesis. Furthermore, we will test the hypotheses that there is a CNC cell-autonomous requirement for Msx1 signaling during palatogenesis and Smad4/Msx1 interaction is crucial for regulating the fate of CNC-derived palatal mesenchyme. In Specific Aim 2, we will investigate the functional significance of TGF-[unreadable] mediated Irf6 signaling in controlling the fate of MEE cells during palatal fusion. We will test the hypotheses that TGF-[unreadable] is responsible for inducing Irf6 expression in MEE cells, Irf6 is required for MEE cells to undergo apoptosis, and TGF-[unreadable]-mediated Irf6 expression is critical for apoptosis in MEE cells and for normal palatal fusion. In Specific Aim 3, we will explore the biological significance of TGF-[unreadable] signaling mediated Ctgf expression in regulating CNC cell proliferation during palatogenesis. We will test the hypotheses that TGF-[unreadable]-induced Ctgf expression is required for CNC cell proliferation in the developing palatal shelf and TGF-[unreadable]-mediated CTGF signaling is critical for palatogenesis. Ultimately, this study will provide a better understanding of how the TGF-[unreadable] signaling cascade regulates palatogenesis and will lead to the development of methods for better diagnosis, treatment and prevention of cleft palate. PUBLIC HEALTH RELEVANCE: Cleft palate represents one of the most common congenital birth defects in the human population. Despite recent advancements in medical intervention, babies born with cleft palate often suffer multiple handicaps that significantly compromise the quality of their lives. Significantly, mutations in the Transforming Growth Factor-[unreadable] (TGF-[unreadable]) gene cause cleft palate in humans and mice. This research program is designed to further our understanding of how aberrant TGF-[unreadable] signaling may adversely affect cell fate determination during palatogenesis and causes cleft palate. Ultimately, this investigation will provide important information for the future prevention and treatment of craniofacial birth defects.

DESCRIPTION (provided by applicant): Calvaria malformations represent one of the major groups of congenital birth defects in the human population. Despite recent advancements in medical intervention, babies born with calvarial defects often suffer multiple handicaps that significantly compromise the quality of their lives. The cranial neural crest (CMC) is an important population of multipotent embryonic progenitor cells which ultimately contribute to a diverse array of differentiated craniofacial tissues, including the calvarial mesenchyme, and plays an integral role during calvarial morphogenesis. An understanding of the manner in which CMC cells contribute to calvaria development and the molecular mechanism which regulates the fate of CNC are critical for understanding normal craniofacial development as well as CMC-related congenital malformations. Multiple growth and transcription factors have been identified as critical regulators for calvarial morphogenesis. Specifically, TGF-beta in the CMC-derived mesenchyme can induce premature cranial suture obliteration in postnatal calvaria development. It is not understood; however, what is the functional significance of TGF-b signaling in regulating the fate of the CMC-derived mesenchyme during initial calvaria morphogenesis. To address this issue, we have generated mice with conditional Tgfbr2 fl/fl; Wnt1-Cre gene ablation in neural crest cells. These Tgfbr2 fl/fl; mice show missing frontal bones and other craniofacial malformations with 100% phenotype penetrance. Significantly, disruption of the TGF-b signaling does not adversely affect the CNC migration, indicating that the TGF-b-mediated gene expression is specifically required locally during calvarial morphogenesis. Taking advantage of our Tgfbr2 fl/fl and other mutant animal models we design studies to investigate the hierarchy of TGF-b signaling in regulating the fate of CNC cells during frontal bone development by testing the hypothesis that TGF-beta signaling regulates the expression of Msx1, which in turn controls the progression of cell cycle to regulate the fate of CMC-derived mesenchymal cells during frontal bone morphogenesis. Ultimately, this study will provide a better understanding on how the TGF- beta signaling cascade regulates the fate of CNC cells during normal craniofacial development and how signaling disruption can lead to craniofacial malformations.

DESCRIPTION (provided by applicant): The development of the Face Base Consortium calls for a comprehensive research collaboration to facilitate data collection, organization, and optimized utilization of new and existing data on mid-facial development and malformations. Our laboratory has a long history of investigating the molecular and cellular mechanism of cleft palate. We have developed a proposal that builds on our strength and will focus on genomic and imaging analysis of selected and highly clinically relevant cleft palate animal models. Specifically, we will use the Tgfb, Tgfbr, Smad4, Msx1 and Fgfr2 mutant animal models that represent complete and sub-mucous cleft palate defects in humans as our entry point. Taking advantage of these animal models, we will work closely with several scientists to address the regulatory mechanism of CNC cell fate determination. Specifically, working with Dr. Marianne Bronner-Fraser at California Institute of Technology (Caltech), we will investigate whether the neural crest gene regulatory network of traditional vertebrate models is conserved and may exert its regulatory function during palatogenesis. In collaboration with Dr. Joseph Hacia at USC, we will discover critical components of the Tgf-b signaling network that are specifically involved in regulating the fate of CNC cells during palatogenesis. Working with Dr. Scott Fraser at Caltech, we will generate comprehensive and dynamic three-dimensional images of palatogenesis and malformations using microMRI and microCT. Finally, we have developed a strategy to screen for specific points of intervention within the gene regulatory network that will allow us to develop therapeutic strategies to prevent and rescue cleft palate. Our collective effort will not only generate tremendous resources for the Face Base Consortium but will also offer opportunities for extensive collaborations for future translational research on craniofacial birth defects. PUBLIC HEALTH RELEVANCE: Cleft palate represents one of the most common congenital birth defects in the human population. Through a collaborative approach, this research program is designed to investigate the signaling mechanism of cleft palate and to provide crucial genomic and imaging resources for future cleft palate research. More importantly, this proposal will reveal crucial points of intervention, which can be targeted for future prevention and rescue of cleft palate.

DESCRIPTION (provided by applicant): The tongue is an important muscular organ and carries out crucial physiological functions. Despite the important functions of the tongue in our daily life, we know very little about the regulatory mechanism of mammalian tongue muscle development. The long term goal of this project is to understand the molecular and cellular mechanisms that control tissue-tissue interactions during tongue myogenesis. Specifically, our preliminary studies show that cranial neural crest (CNC) cells contribute to the interstitial connective tissue, which ultimately compartmentalizes both intrinsic and extrinsic tongue muscles and serves as their attachments. Occipital somite-derived cells migrate into tongue primordium and give rise to muscle cells in the tongue. The intimate relationship between CNC- and mesoderm-derived cells suggests that tissue-tissue interaction may play an important role in regulating tongue development. Transforming growth factor-[unreadable] (TGF- [unreadable]) and its signaling mediator Smad are expressed in both CNC- and mesoderm-derived cells in the tongue. Significantly, disruption of TGF-[unreadable] signaling in either CNC or mesoderm-derived cells does not adversely affect cell migration into the tongue primordium, indicating that TGF-[unreadable] signaling is specifically required locally during tongue morphogenesis. We discovered that mutation of Tgfbr[unreadable] in CNC cells results in a defect in tongue muscle patterning and microglossia, whereas loss of Tgfbr[unreadable] in mesoderm-derived cells results in a myogenic differentiation defect with 100% phenotype penetrance. Taking advantage of our neural crest- or mesoderm- specific Tgfbr2 mutant animal models, we designed studies to test the hypothesis that TGF-[unreadable] signaling controls the fate of CNC as well as mesoderm-derived cells and regulates tissue-tissue interaction during tongue development. Ultimately, this study will provide a better understanding of how the TGF-[unreadable] signaling cascade regulates the fate of the CNC- and mesoderm derived cells during normal craniofacial development and how signaling pathway disruption can lead to craniofacial malformations. This study will have a broad impact on our understanding of the regulatory mechanism of skeletal muscle development and regeneration. PUBLIC HEALTH RELEVANCE: The tongue is a muscular organ and plays a critical role in speech, deglutition, and taste. Tongue development defects severely affect the physiological function of the tongue. Despite these important functions of the tongue, we know very little about the control of tongue development. This research program is designed to obtain important information in order to have a clear understanding how different gene mutations adversely affect tissue-tissue interactions during tongue development and lead to tongue malformation. Ultimately, this investigation will provide important information on future prevention and treatment of skeletal muscle defects.

DESCRIPTION (provided by applicant): The University of Southern California (USC) P30 project is directed at recruiting junior faculty members whose research interests are complementary to our goal of enhancing our understanding of the polydisciplinary events shaping craniofacial morphogenesis, their malformations and the use of stem cell biology for their regeneration. These new faculty members will be jointly recruited into the USC School of Dentistry (USCSD) and Keck School of Medicine (KSOM), permitting their access to university wide infrastructural research resources. These resources include technical support core laboratories and full access to graduate students and postdoctoral fellows. The most important resource we will supply them with is a highly talented, vertically stratified faculty cohort whose interests parallel their own research interests and who are dedicated to mentoring these new colleagues towards a path of independent biomedical research. The leadership of the two USC schools commits to space resources and faculty salaries that will permit our new colleagues to devote greater than 75% of their professional time over the next four years to their scholarship efforts. Year-end goal setting and reporting mechanisms are in place that will provide metrics of performance for these two new colleagues. These metrics will be reviewed and a plan created to enhance their strengths while attenuating any weaknesses as they achieve a path towards tenure and promotion at the University of Southern California. Ultimately, the success of this P30 project is measured by the career success of these two newly recruited faculty members, their integration with the existing distinguished faculty experts and the enrichment and expansion of the craniofacial biology program at USC. PUBLIC HEALTH RELEVANCE: This collaborative project between the University of Southern California (USC) School of Dentistry (SD) and Keck School of Medicine (KSOM) will provide crucial support for the development of junior faculty members and academic programs at the USC and will certainly have a long lasting impact towards the improvement of oral health care for all Americans.

DESCRIPTION (provided by applicant): This study is designed to elucidate the molecular regulatory mechanism of dental root development. Despite its important physiological and biological functions, dental root development and its regulation are not well understood. The root development involves continuous interactions between the dental epithelium (Hertwig's Epithelial Root Sheath, HERS) and the cranial neural crest derived mesenchyme, hence the root is an excellent model to investigate tissue-tissue interactions in regulating organogenesis. Recent studies show that Bmp/Tgf-¿, Shh, Fgf, Wnt, and Nfic may be involved in regulating root development. We have discovered that Smad4-mediated Bmp/Tgf-¿ signaling plays a crucial role in regulating root development. However, it is unclear whether the inductive signal(s) of root formation resides in the dental epithelium or mesenchyme and how the Bmp/Tgf-¿ signaling network controls root development. In this study, we will carry out experiments to test the hypotheses that Smad4 mediated Bmp/Tgf-¿ signaling and their downstream target genes, such as Fgf10, Wnt, Nfic, and Bmps/Tgf-¿s, are crucial for mediating tissue-tissue interactions and controlling the fate of HERS and dental mesenchymal cells during root development. We have proposed three Specific Aims. In Specific Aim 1, we will test the hypothesis that epithelial Smad4 is required for HERS and dental mesenchymal cell fate determination during root development. We will investigate Smad4- mediated Fgf10 signaling and its functional significance in the induction of root development. In Specific Aim 2, we will investigate the functional significance of the Smad4-mediated Bmp/Tgf-¿ and Wnt signaling cascades in regulating the fate of the dental mesenchyme and root development. We will elucidate the molecular mechanism(s) by which signals from the dental mesenchyme control root development. We will investigate mesenchymal Smad4/Msx2 interaction and its role in regulating root development. In Specific Aim 3, we will investigate the biological significance of Nfic-mediated Bmp and/or Tgf-¿ signaling during root development. We will elucidate the regulatory mechanism(s) that control mesenchymal-epithelial interaction during root development. Ultimately, this study will provide a better understanding of the molecular regulatory mechanism of root development. The knowledge gained from this study will serve as the foundation for stem cell mediated tooth regeneration. PUBLIC HEALTH RELEVANCE: Dental roots are crucial for oral health because they provide anchorage for our dentition, transmit occlusal force to the jaw bone and send bio-feedback to our central nervous system. Despite recent advancements in research on the regulation of early tooth development, we still have limited knowledge of the regulatory mechanism of root development. Our research program is designed to further our understanding of how aberrant growth factor signaling networks may adversely affect cell fate determination, tissue-tissue interactions and cause root development defects.

DESCRIPTION (provided by applicant): In this application, we propose to integrate our research in functional genomics and craniofacial morphology/dysmorphology within the FaceBase Consortium. Specifically, we will focus on the development of the mandible and maxilla. Congenital malformations involving these facial bones significantly impact quality of life because our face is our identity. For example, mandibular dysmorphogenesis ranging from agenesis of the jaw to micrognathia is a common malformation and appears in multiple syndromes. Micrognathia not only presents as a facial deformity but can also cause cleft palate and airway obstruction, such as in Pierre-Robin sequence. The maxilla contributes to mid-facial formation. Maxillary hypoplasia is often associated with cleft palate and has been described in more than sixty different syndromes. Despite their importance, the mechanisms that regulate facial bone development are relatively uncharacterized. This is a significant gap in our knowledge and an important opportunity to generate invaluable resources for the research community. The proposed work is a logical progression from our current spoke project within the FaceBase Consortium on palatal development. Over the past five years, we have deposited nearly 200 hard and soft tissue scans and 125 microarray gene expression datasets in the FaceBase hub. These datasets have demonstrated their utility, as shown by other researchers' presentations at major international conferences and publications. Equally importantly, our team has played a significant role in the FaceBase Consortium, the hub website design, data organization and presentation. Building on our experience and in alignment with RFA-DE-14-004, we propose to investigate facial bone development and malformations. In Specific Aim 1, we will perform global and specific gene expression profiling analysis of mandible development, and will integrate these datasets with cell lineage and quantitative 3D dynamic imaging analyses. In collaboration with the ontology group within the FaceBase consortium, we will define anatomical landmarks and morphometric parameters of the developing mandible. In Specific Aim 2, we will expand our gene expression profile analyses in the developing maxilla. We will correlate this information with 3D imaging of the maxilla and define anatomical landmarks and parameters in collaboration with the ontology group within the FaceBase consortium. Our data will facilitate the investigation of the molecular regulatory mechanism of facial bone formation. In Specific Aim 3, using the data generated here, we will investigate the role of the TGF? and Msx1 signaling network in regulating mandible development and test how manipulation of TGF? downstream target genes can prevent and rescue mandible defects in mutant animal models. This study will showcase how our datasets at the hub can facilitate the generation of hypothesis-driven research and collaborations. Because of the prevalence of facial bone defects in orofacial clefting patients and the lack of quantitative studies in this are, our proposed study will fill a void and provide a significant resource for the research community.

? DESCRIPTION (provided by applicant): Craniofacial and dental defects have devastating effects on patients not only because vital sense organs and the brain are housed within the cranium, but also because our face represents an important aspect of our identity. Furthermore, such defects can lead to compromised general health. In recent years, stem cells combined with sophisticated scaffolds have shown promise as bio-inspired solutions to biological problems in dental, oral, and craniofacial regenerative medicine. Our vision is to assemble a comprehensive team of clinicians, research scientists, biostatisticians, regulatory science and pre-clinical/clincal trial experts to enable the development and clinical implementation of innovative approaches for dental, oral, and craniofacial tissue regeneration. Towards this end, we will develop a multi-institutional Center for Dental, Oral, and Craniofacial Tissue and Organ Regeneration (C-DOCTOR) that is driven by clinical needs, as defined by practicing clinicians. In Specific Aim 1, we will assemble a team of experts with integrated clinical, scientific/technical, and regulatory science expertise required to form the C-DOCTOR and enable us to successfully develop, manufacture, and apply mesenchymal stem cell-mediated tissue regeneration therapies. The team will evolve based on newly identified clinical and research opportunities. We will host two working retreats and monthly teleconferences to develop and refine the overall vision, roadmap, organizational structure, and operational procedures for the C-DOCTOR. The capacity of our center will match public needs in order to provide clinical benefits for patients. In Specific Aim , we will enable clinicians to define clinical needs for craniofacial tissue and organ regeneration, and to engage with research scientists to assess feasibility. According to these needs, we will develop the necessary technical approaches, statistical methods, manufacturing and quality control procedures, vertebrate animal protocols, and human subject protections. In support of this aim, we will develop a website to foster communications with the public, potential interdisciplinary translational project teams, and other resource centers funded through this RFA throughout the nation. Ultimately, our collective expertise at the C-DOCTOR will provide comprehensive services to meet patient needs for tissue regeneration by establishing well-defined procedures for successful experimental design, large animal models, product validation, manufacturing scale-up, regulatory approval, clinical testing, and implementation.

This is a proposal to investigate the role of stem cell regulation in cranial suture development and maintenance, and its pathophysiology in craniosynostosis. More broadly, this proposal focuses on how stem cells are controlled in space and time to promote the development and maintenance of vertebrate organs. Our recent results show that heterozygous loss of either of two related transcription factors, TWIST1 and TCF12, account for coronal suture defects in the majority of Saethre-Chotzen patients. Using sophisticated in vivo imaging and genetics in mice and zebrafish, we will test that Tcf12 modifies the function of Twist1 to maintain skeletal progenitors during both the specification and maintenance of sutures. A common role for Twist1 and Tcf12 in the developing and postnatal coronal suture would have the potential to explain both the initial synostosis and the high recurrence rate of postoperative synostosis in patients. A particular strength of our research plan is the complementary expertise of three accomplished investigators in craniofacial genetics. Rob Maxson has long-standing expertise in mouse models of synostosis, having contributed to the identification of TWIST1 and TCF12 as the two most affected genes in Saethre-Chotzen syndrome. Yang Chai recently identified a population of Gli1+ stem cells in the suture that are required for long-term suture patency and calvarial bone growth. Gage Crump has pioneered in vivo imaging techniques in zebrafish to examine the cellular basis of craniofacial defects. First, this team will test that Twist1 and Tcf12 function in the same tissues to repress the Ihh-driven differentiation of sutural progenitors into osteoblasts, as predicted if Tcf12 serves as a suture-specific dimerization partner for Twist1. Second, we will examine continuous requirements for Twist1 and Tcf12 in suture maintenance by conditionally deleting these genes in postnatal Gli1+ sutural stem cells. Third, we will use new knock-in tagged alleles of Twist1 and Tcf12 to identify the direct genomic targets of Twist1-Tcf12 dimers in postnatal sutural stem cells, as well as how Tcf12 modifies the ability of Twist1 to engage regulatory regions necessary for suture maintenance. Fourth, we will use powerful imaging techniques to reveal the in vivo spatial patterns of Twist1-Tcf12 dimers within sutures. Fifth, we will take advantage of the first zebrafish model of Saethre-Chotzen syndrome to directly visualize over time how changes in the pattern and timing of osteoblast differentiation result in later coronal suture defects. The results of these aims will test our model that Tcf12 functions as a suture-specific partner for Twist1, in part by guiding Twist1 to particular genomic regions necessary to inhibit premature osteoblast differentiation in suture mesenchyme. These new insights into the long-term requirements of synostosis genes in suture maintenance will have the potential to lead to new ways of preventing post-operative synostosis, thus reducing the number of risky operations currently performed on young children with Saethre-Chotzen syndrome.

? DESCRIPTION (provided by applicant): Stem cells are remarkable. They form tissues during development, maintain tissue homeostasis and perform injury repair in adults. The mouse incisor provides an excellent model for stem cell study because it grows continuously throughout life. Stem cells residing in the proximal region of the incisor in adult mice can replenish all incisor cells within one month. We have recently shown that the mesenchymal stem cells (MSCs) in the adult mouse incisor are a Gli1+ cell population surrounding the neurovascular bundle (NVB) near the cervical loop region and that they govern tissue homeostasis and repair. The NVB secretes Shh and provides a niche for MSCs in the incisor. However, the functional significance of Shh secreted from the sensory nerve within the NVB still needs to be investigated. During normal homeostasis, MSCs exit from their niche and become transit- amplifying (TA) cells, undergoing a series of divisions before terminal differentiation. Te transition process from MSC to TA cell is a common feature in diverse organs but little is known about the presumably complex signaling network that governs this transition. Based on our data and taking advantage of well-established animal models, we hypothesize that (i) secretion of Shh by the sensory nerve supports MSCs in the adult mouse incisor and that (ii) Wnt signaling and Wnt/Bmp interactions control the MSC to TA cell transition and the fate of MSCs to maintain mesenchymal cell homeostasis in the adult mouse incisor. To test our hypotheses, we will perform studies under the following specific aims. In Specific Aim 1, we will investigate whether incisor Gli1+ MSCs contribute to the TA cell population to maintain mesenchymal tissue homeostasis. We will test the differentiation property of Gli1+ MSCs and ascertain whether secretion of Shh by the sensory nerve supports MSCs in the adult mouse incisor. In Specific Aim 2, we will determine the role of Wnt signaling in the regulation of the MSC to TA cell transition and maintenance of incisor mesenchymal cell homeostasis. We will further explore the molecular and cellular mechanisms of the altered fate of TA cells in ?-catenin mutant mice. In Specific Aim 3, we will investigate whether Bmp signaling interacts with Wnt signaling in the dental mesenchyme to control the fate of MSCs. Ultimately, this study will provide important knowledge of the signaling network that regulates the transition from MSCs to TA cells in maintaining tissue homeostasis. The understanding gained from this study will serve as the foundation for future studies in MSC biology and stem cell-mediated tissue regeneration.

The overall goal of this U24 is to operationalize the Center for Dental, Oral and Craniofacial Tissue and Organ Regeneration (C-DOCTOR). Through an integrated series of collaborative planning activities funded during Stage I, eight renowned centers of translational research excellence - UCSF, USC, UCLA, UCB, UCD, UCSD, CoH, and Stanford - have partnered to form a public-private consortium focused on accelerating promising tissue engineering/regenerative medicine therapies for dental, oral, and craniofacial (DOC) tissue to human clinical trials. C-DOCTOR will recruit Interdisciplinary Translational Project (ITP) teams with promising DOC regeneration therapies and then provide scientific, technical, regulatory, financial, and managerial resources necessary to facilitate large animal model studies and promote a cost-effective transition to Stage III. C- DOCTOR will efficiently leverage an extensive array of existing resources that resonate with the Center mission. Three proposed Aims are to: 1) Recruit and select ITP teams that align with our clinical indication priorities; 2) Coordinate and customize our broad resource infrastructure along ITP team needs.; and 3) Cultivate, train, triage, and collaborate with ITP teams to assemble a balanced ITP portfolio ready for Stage III. To accomplish these aims, we will build on our preliminary needs assessment through a strong partnership with our diverse network of C-DOCTOR clinical advisors. We will then adapt best practices from a number of existing innovation programs across our multi-institutional network to select promising therapies with high potential for clinical adoption. C-DOCTOR Resource Directors will match selected ITP teams with domain and resource experts to refine their business case and address their technical needs through interactive collaboration, where continued funding and successful progression through Stage II will be dependent on meeting bi-annual milestones and specific go/no-go decision gates. In this manner, C-DOCTOR will efficiently and deliberately triage from a large number of promising ITPs into active partnership with only those that have maximum likelihood for successful transition through Stage III, which includes an FDA filing and commercial partnering that together support future Phase I clinical testing.

PROJECT SUMMARY The major goal of the FaceBase Consortium is to advance research by creating comprehensive datasets of craniofacial development and dysmorphologies, and to disseminate these datasets to the wider craniofacial research community. The FaceBase 3 Data Management and Integration Hub builds on the existing and successful scientific and technical team that has lead the development, deployment, operation and community engagement of the FaceBase 2 data hub. Looking forward to the future impact of the FaceBase Consortium, we face major challenges that include: (1) how to annotate large datasets to empower the biomedical research community; (2) how to improve data integration and facilitate data search and retrieval from the hub; (3) how to use the data from FaceBase to design studies and otherwise inform our future research; and (4) how to translate our knowledge from animal model studies to improve human craniofacial health. Importantly, the projects we propose here are designed to address these issues as we develop anatomy-based comprehensive data search and display, incorporate additional datasets from the community, and promote the usage of FaceBase data. In pursuit of these goals, we propose the following three Specific Aims: Specific Aim 1: Create a FAIR data repository and resource hub for the craniofacial research community. Specific Aim 2: Promote and enable data contributions to FaceBase by NIDCR-supported researchers. Specific Aim 3: Foster a community of active FaceBase users through outreach activities and dissemination of new features and available datasets. We will create educational materials and provide training opportunities for the next generation of craniofacial researchers. Ultimately, our proposed projects will not only contribute to our comprehensive understanding of the molecular regulatory mechanisms of craniofacial development but will also demonstrate how others can use the datasets from the FaceBase Hub to develop innovative approaches to improve human health.

PROJECT SUMMARY The goal of the FaceBase III Hub is to create a FAIR data repository to serve the entire community of dental and craniofacial researchers by sharing diverse data related to craniofacial development and dysmorphia. To meet this goal, FaceBase is built on Deriva, an open-source data management system designed with FAIR data principles in mind. This platform has allowed FaceBase to evolve with changing requirements for data on new experimental methodologies and instruments, additional model organisms, cell characterization, integration of computational pipelines, and visualization interfaces. Currently, we implement Deriva on private and public clouds using a ?data center-in-the-cloud? format; i.e., treating the cloud like a traditional remote computer, to run virtual machine images and conventional data storage. However, cloud platforms such as Amazon Web Services offer a wide range of cloud-native services beyond virtual machines which if fully leveraged would drastically improve important aspects of Deriva that would directly benefit FaceBase. Hence, we propose to enhance Deriva for cloud-based operations to address three key aspects of Deriva in support of FaceBase and its other NIH communities: scalability, reliability, and sustainability. Specifically, we plan to use AWS native services to improve its scalability (Aim 1), decouple Deriva services to run in containerized execution environments to ensure its reliability (Aim 2), and develop cost management dashboards to monitor and predict costs of operating in the cloud to achieve sustainability (Aim 3). The AWS native services are fully managed and highly-scalable, and offload much of the overhead of system operations and maintenance. Improvements in Deriva scalability, reliability, and sustainability achieved by these Aims will allow the FaceBase Hub to provide the growing community of data contributors and users with better service. In addition, many other user communities such as GUDMAP, (Re)Building a Kidney, the Kidney Precision Medicine Project (NIDDK) and the Common Fund Data Environment (OD) rely on Deriva, and all of the improvements resulting from these Aims would yield a direct and immediate benefit to thousands of additional users.

Project Summary/Abstract This supplement will address the unintended consequences and collateral damage that arise when facial recognition software is used for medical purposes, such as for syndrome diagnosis as in our Facebase-funded project. In Aim 1, we will determine whether the accuracy of this technology varies based on self-reported race, sex and age. In this aim, we examine our existing database for evidence of bias based on self-reported race, sex or age. We further determine the extent to which these variables influence classification performance. To the extent sample sizes allow, we will carry this analysis to the level of specific syndromes. Finally, we will use anonymized reference datasets of non-syndromic faces to compare false positive rates based on NIH race definitions, sex and age. The outcome of this aim is to objectively establish bias and estimate the effects of under-representation across race, age and sex categories within our data. In Aim 2, we will determine how the reports of race-, sex- and age-based bias in facial recognition technology may influence views of the technology and its application amongst researchers and clinicians. This aim will establish the extent to which the storing of large databases of facial images and the application of machine learning processes to them for diagnostic purposes may raise privacy concerns. The concerns investigated will include potential hacks into protected health information; fear relating to the bias in some facial recognition software (and, potentially, in the Facebase database); and fear of discrimination in the application of the technology, such as by insurers. The outcome will be a white paper that targets a high-profile journal, summarizing the findings and defining crucial issues that should guide the development of facial imaging for disease diagnosis and clinical usage.

PROJECT SUMMARY / ABSTRACT Craniosynostosis is a craniofacial disorder characterized by the premature fusion of cranial sutures with defective mesenchymal stem cells (MSCs). Patients with severe craniosynostosis often have intellectual disabilities (IDs). Both genetic mutations and environmental factors have been linked to craniosynostosis coupled with MSC depletion. We propose to determine gene-environment interaction mechanisms in craniosynostosis by addressing how craniosynostosis disease genes Twist1 and Tcf12 interplay with an environmental risk factor, namely maternal usage of the antidepressant citalopram. Importantly, we aim to establish a MSC-based therapeutic strategy to mitigate both skull dysmorphology and neurocognitive dysfunctions in craniosynostosis. This is innovative and significant because we have little understanding of environmental factors and gene-environment interactions in craniosynostosis, and new treatments for this devastating disorder are urgently needed. Neurocognitive functions have been largely neglected in studies of animal models of craniosynostosis, although cognitive abnormalities such as IDs have been frequently observed in craniosynostosis patients. The only current treatment option for craniosynostosis is complex surgery, which is invasive and often requires re-operation due to the calvarial bones fusing again. Our MSC- based cranial suture regeneration approach is less invasive, avoids re-fusion, corrects skull dysmorphology, restores elevated intracranial pressure, and reduces neurocognitive dysfunctions later in life in a clinically relevant Twist1+/- mouse model of craniosynostosis. Gli1+ MSC depletion is observed both in Twist1+/- mice and in those with maternal exposure to citalopram. Citalopram is a selective serotonin reuptake inhibitor (SSRI), which is the most commonly prescribed class of antidepressant drugs. Maternal SSRI usage is also known as an environmental risk factor for craniosynostosis in humans. These results lead to the hypothesis that Twist1 and Tcf12 mutations may interplay with citalopram in exacerbating skull and neurocognitive defects in craniosynostosis, which will be tested in Aim 1. Aim 2 will determine cellular and molecular mechanisms by which gene mutations and maternal citalopram exposure act together to cause craniosynostosis. Aim 3 will use our newly developed MSC-based suture regeneration approach to determine whether and how MSC implantation mitigates skull and neurocognitive dysfunctions in craniosynostosis caused by gene mutations, citalopram, and their interactions. Collectively, our proposed studies build upon our previous discoveries, and our findings will be highly significant for improving the understanding of mechanisms underlying gene- environment interplay in craniosynostosis; it offers a unique opportunity for improving treatment of infants with craniosynostosis.

PROJECT SUMMARY The goal of the FaceBase III Hub was created by the National Institute for Dental and Craniofacial Research (NIDCR) to create a data repository to serve the entire community of dental and craniofacial researchers by sharing diverse data related to craniofacial development and dysmorphia, as well as other research communities that can leverage the diverse data that is in the FaceBase repository. One particularly unique and important element of FaceBase III is that it has over 22,000 facial images from over 11,000 human subjects, many of which are labeled with syndromes based on clinical and genomic diagnoses. Facial images are a critical resource for studying the correlation between genotype and phenotype and have received intense interest within the Artificial Intelligence (AI) and Machine Learning (ML) research field with notable advances in automated phenotyping. While FaceBase embraces the FAIR (Findable, Accessible, Interoperable, and Reusable) principles, there are unique concerns specific to AI/ML research including: presence of noise, uncertainty of labels, and bias within datasets. It is imperative that we remedy any limitations in the utility of FaceBase?s facial imaging data for AI/ML research. In this project, we propose to unlock the tremendous potential of FaceBase facial scans by identifying gaps in how data is characterized, formated, and preprocessed from the perspective of its use in AI/ML research and algorithm development. To accomplish this, we propose to initiate a pilot application that applies existing deep learning algorithms developed by investigators in this proposal to existing FaseBase data (Aim 1). The goal of the pilot is to identify how curation, organization and preparation of FaceBase data might be improved so as to streamline their use in ML/AI based investigations. Based on what we learn from the pilot, we will modify the current FaceBase self curation processes specifically around Facial Scans (Aim 2). This will require us to streamline our process associated with curation of human subject data, so that we have the necessary rich descriptive elements while maintaining required restrictions on data handling. Ultimately, the goal is to position the FaceBase Hub so that the existing facial scan resources become more broadly useful to AI/ML researchers. More significantly, we expect to see an increased availability with facial scan data and other associated data types, such as genotyping and neurofunctional data. By making the proposed improvements to our data ingest procedures, we anticipate that this proposal will allow FaceBase to scale to significantly larger data set sizes, and consequently, cementing and expanding its position as a unique resource to the broader NIH community of ML and AI researchers.

PROJECT SUMMARY The goal of the FaceBase III Hub is to create a data repository to serve the entire community of dental and craniofacial researchers by sharing diverse data related to craniofacial development and dysmorphia, as well as other research communities that can leverage the diverse data that is in the FaceBase repository. FaceBase was designed to follow FAIR data principles: that data be Findable, Accessible, Interoperable, and Reusable. To date, we have had significant success getting new research teams to submit data to FaceBase in a manner that adheres to FAIR. However, since the start of FaceBase III, the digital repository community has developed a new set of principles designed to promote trust in the data contained in a repository with the goal of promoting broader reuse of the data within the repository. The TRUST principles of Transparency, Responsibility. User Focus, Sustainability, and Technology have been identified as core to increasing users? confidence in a data-repository and hence promoting data use and reuse. Surveys and interviews of FaceBase users indicate that FaceBase is perceived within the community as being a high-quality trustworthy repository. However, a deliberate application of TRUST guidelines has the advantage of making FaceBase commitment to these principles more explicit, increasing our ability to promote the use of FaceBase data outside of the core craniofacial research community, and better serving our users across the NIH research community. In this proposal, we seek to achieve these goals by explicitly identifying, defining, and adopting operational principles in FaceBase to meet community agreed-upon standards for TRUST (Aim 1). A critical part of TRUST is to define specific metrics that enable one to evaluate the impact of the data repository. To this end, we will leverage the extensive built-in analytics that FaceBase collects to define key performance metrics of impact and produce a reporting structure to disseminate those metrics to the FaceBase user community (Aim 2). Ultimately, the goal is for the FaceBase Hub to become a certified trusted data repository. Certification will establish greater credibility for the FaceBase Hub with our user community and with users less familiar with FaceBase. Long term, the adoption of TRUST principles will ensure that datasets deposited with us and entrusted to our care will be maintained at the highest level of quality.