From Phenotype to Genotype: Enter Genomics and Transformation of Primary Health Care around the World.

The progress in phenotype descriptions, measurements, and analyses has been remarkable in the last 50 years. Biomarkers (proteins, carbohydrates, lipids, hormones, various RNAs and cDNAs, microarrays) have been discovered and correlated with diseases and disorders, as well as physiological responses to disease, injury, stress, within blood, urine, and saliva. Three-dimensional digital imaging advanced how we "see" and utilize phenotypes toward diagnosis, treatment, and prognosis. In each example, scientific discovery led to inform clinical health care. In tandem, genetics evolved from Mendelian inheritance (single gene mutations) to include Complex Human Diseases (multiple gene-gene and gene-environment interactions). In addition, epigenetics blossomed with new insights about gene modifiers (e.g., histone and non-histone chromosomal protein methylation, acetylation, sulfation, phosphorylation). We are now at the beginning of a new era using human and microbial whole-genome sequencing to make significant healthcare decisions as to risk, stratification of patients, diagnosis, treatments, and outcomes. Are we as clinicians, scientists, and educators prepared to expand our scope of practice, knowledge base, integration into primary health care (medicine, pharmacy, nursing, and allied health science professions), and clinical approaches to craniofacial-oral-dental health care? The time is now.

The future of research in craniofacial biology and what this will mean for oral health professional education and clinical practice.

Today, and looking to the future, scientific discoveries from cellular, developmental and molecular biology inform our understanding of cell, tissue and organ morphogenesis as exemplified in skin, bone, cartilage, dentine, enamel, muscle, nerve and many organs such as salivary glands and teeth. Present day biomedical science yields principles for the biomimetic design and fabrication of cells, tissues and organs. Bioengineering has become a strategy that can 'mimic' biological processes, and inform clinical procedures for tissue and organ replacements. The future of regenerative craniofacial biology holds enormous promise for the diagnosis and treatment of congenital birth defects, traumatic injuries, degenerative chronic diseases as well as for Mendelian single gene and complex multigene diseases and disorders. The past 50 years have heralded the completion of the human genome and the introduction of 'personalized medicine and dentistry', the utilization of stem cell therapy for an array of diseases and disorders, the 'proof of principle' to reverse select inherited diseases such as anhidrotic ectodermal dysplasia (ED), and the fruits from interdisciplinary research drawn from the diverse biomedical sciences. Looking to the future, we can readily anticipate as major goals to emphasize the clinician's role in identifying clinical phenotypes that can lead to differential diagnosis, and rejuvenate missing or damaged tissues by establishing processes for the utilization of gene, cell and/or protein therapies. The future is replete with remarkable opportunities to enhance clinical outcomes for congenital as well as acquired craniofacial malformations. Clinicians play a pivotal role because critical thinking and sound clinical acumen substantially improve diagnostic precision and thereby clinical health outcomes.

Reflections on my journey in biomedical research: the art, science, and politics of advocacy.

Scientific Discovery often reflects the art, science, and advocacy for biomedical research. Here the author reflects on selected highlights of discovery that contributed to several aspects of our understanding of craniofacial biology and craniofacial diseases and disorders.

Salivary diagnostics and its impact in dentistry, research, education, and the professional community.

Oral fluid-based (salivary) tests have the potential to create practical, point-of-care clinical instruments that are convenient, practical, and comfortable to use in dentistry and medicine. Currently, there are no simple, accurate, and inexpensive sampling, screening, or detection methods to support definitive diagnostic platforms across dental and medical disciplines. Though the benefits from advancing screening and detection technologies seem eminent, analytical, chemical, molecular, genetic, and protein markers are still under development. Clinical applications in patient care must be validated independently to ensure that they are clinically accurate, reliable, precise, and uniformly consistent for screening and detecting specific diseases or conditions. As technology designed to improve patient care through risk assessment, prevention, and disease management is transferred into clinical practice, dentistry may need to reassess its role in general health care.

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling.

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein-DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter-luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.

Msx2 is a repressor of chondrogenic differentiation in migratory cranial neural crest cells.

During early mouse embryogenesis, cranial neural crest cells (CNCC) emigrate from the posterior midbrain and rhombomeres 1 and 2 of the anterior hindbrain into the first branchial arch-derived maxillary and mandibular processes and there provide cell lineages for several phenotypes, including cartilage, bone, and tooth. Here, we report that Sox9 and Msx2 were coexpressed in a subpopulation of CNCC during their migration. Because Sox9 is a transactivator of chondrogenesis, and Msx genes can act as transcriptional repressors, we hypothesized that Sox9 expression indicates the determination of CNCC-derived chondrogenic cell lineage and that Msx2 represses chondrogenic differentiation until CNCC migration is completed within the mandibular processes. To test whether Msx2 represses chondrogenesis, we designed experiments to inhibit Msx2 function in migratory CNCC in primary cultures through the expression of loss-of-function Msx2 mutants. We showed that infection of migratory CNCC with adenovirus Msx2 mutants accelerated the rate and extent of chondrogenesis, as indicated by the expression level of type II collagen and aggrecan, and the amount of alcian blue staining. Adenovirus infections did not apparently interfere with CNCC proliferation or migration. These findings suggest that an important early event in craniofacial morphogenesis is a transient expression of both Sox9 and Msx2 during emigration into the forming mandibular processes followed by restricted expression of Sox9 within CNCC- derived chondroprogenitor cells. We conclude that Msx2 serves as a repressor of chondrogenic differentiation during CNCC migration.

DACH: genomic characterization, evaluation as a candidate for postaxial polydactyly type A2, and developmental expression pattern of the mouse homologue.

The gene DACH is a human homologue of Drosophila melanogaster dachshund (dac), which encodes a nuclear factor essential for determining cell fates in the eye, leg, and nervous system of the fly. To investigate possible connections between DACH and inherited developmental disorders, we have characterized the human DACH genomic structure and investigated the tissue and cellular distribution of the mouse DACH1 protein during development. DACH spans 400 kb and is encoded by 12 exons. The predominant DACH transcript is 5.2 kb and encodes a 706-amino-acid protein with an observed molecular weight of 97 kDa.DACH mRNA was detected in multiple adult human tissues including kidney and heart. The mouse DACH1 protein was immunolocalized to specific cell types within the developing kidneys, eyes, cochleae, and limb buds. Data suggest genetic linkage of the limb bud patterning defect postaxial polydactyly type A (designated PAP-A2, MIM 602085) to a 28-cM interval on chromosome 13 that includes DACH. However, mutation analysis of DACH in this PAP-A2 pedigree revealed no sequence differences in the coding region, splice sites, or proximal promoter region. The data presented will allow for the analysis of DACH as a candidate for other developmental disorders affecting the limbs, kidneys, eyes, ears, and other sites of DACH expression.

The Surgeon General's Report and special-needs patients: a framework for action for children and their caregivers.

The Surgeon General's Report, Oral Health in America, is the first comprehensive assessment of oral, dental, and craniofacial health in the history of our nation. The intent of this first-ever Report is to alert Americans to the full meaning of oral health and its importance to general health and well-being across the lifespan. Moreover, the Report has been released at a time in human history of enormous changes as well as opportunities. The convergence of public health policies, "quality of life" expectations, global informatics, a new century of biotechnology, the completion of the Human Genome Project, changes in the management of health care, and the acknowledgment of enormous health disparities herald a call to action. These profound dynamics particularly affect children and their caregivers and the multitude of social, economic, and health issues associated with special patients and developmental disabilities. This paper will highlight the issues, provide recommendations, and suggest a call to action.

The human genome, implications for oral health and diseases, and dental education.

We are living in an extraordinary time in human history punctuated by the convergence of major scientific and technological progress in the physical, chemical, and biological ways of knowing. Equally extraordinary are the sparkling intellectual developments at the interface between fields of study. One major example of an emerging influence on the future of oral health education is at the interface between the human genome, information technology, and biotechnology with miniaturizations (nanotechnology), suggesting new oral health professional competencies for a new century. A great deal has recently been learned from human and non-human genomics. Genome databases are being "mined" to prompt hypothesis-driven "postgenomic" or functional genomic science in microbial models such as Candida albicans related to oral candidiasis and in human genomics related to biological processes found in craniofacial, oral, and dental diseases and disorders. This growing body of knowledge is already providing the gene content of many oral microbial and human genomes and the knowledge of genetic variants or polymorphisms related to disease, disease progression, and disease response to therapeutics (pharmacogenomics). The knowledge base from human and non-human genomics, functional genomics, biotechnology, and associated information technologies is serving to revolutionize oral health promotion, risk assessment using biomarkers and disease prevention, diagnostics, treatments, and the full range of therapeutics for craniofacial, oral, and dental diseases and disorders. Education, training, and research opportunities are already transforming the curriculum and pedagogy for undergraduate science majors, predoctoral health professional programs, residency and specialty programs, and graduate programs within the health professions. In the words of Bob Dylan, "the times they are a-changing."

Expanding the boundaries: enhancing dentistry's contribution to overall health and well-being of children.

The Surgeon General's Report, Oral Health in America, is the first comprehensive assessment of oral, dental, and craniofacial health in the history of our nation. The intent of this report is to alert all Americans to the full meaning of oral health and its importance to general health and well-being across the life span. Moreover, the report has been released at a time of enormous changes in human history as well as opportunities. The convergence of public health policies, "quality of life" expectations, global informatics, a new century of biotechnology, the advent of nanotechnology, the completion of the human genome project, changes in the management of health care, and the acknowledgment of enormous health disparities heralds a call to action. These changes affect children and their caregivers and the elderly. They also affect the social, economic, and health issues associated with special patients, including those with developmental disabilities. This paper highlights dentistry's future and how oral health is broadening the impact on patient and community health and dental practice, with a focus on children's oral health. The paper provides recommendations and suggests a call to action.

Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2.

Cranial neural crest cells emigrate from the posterior midbrain and anterior hindbrain to populate the first branchial arch and eventually differentiate into multiple cell lineages in the maxilla and mandible during craniofacial morphogenesis. In the developing mouse mandibular process, the expression profiles of BMP4, Msx2, Sox9, and type II collagen demonstrate temporally and spatially restrictive localization patterns suggestive of their functions in the patterning and differentiation of cartilage. Under serumless culture conditions, beads soaked in BMP4 and implanted into embryonic day 10 (E10) mouse mandibular explants induced ectopic cartilage formation in the proximal position of the explant. However, BMP4-soaked beads implanted at the rostral position did not have an inductive effect. Ectopic chondrogenesis was associated with the up-regulation of Sox9 and Msx2 expression in the immediate vicinity of the BMP4 beads 24 hours after implantation. Control beads had no effect on cartilage induction or Msx2 and Sox9 expression. Sox9 was induced at all sites of BMP4 bead implantation. In contrast, Msx2 expression was induced more intensely at the rostral position when compared with the proximal position, and suggested that Msx2 expression was inhibitory to chondrogenesis. To test the hypothesis that over-expression of Msx2 inhibits chondrogenesis, we ectopically expressed Msx2 in the mandibular process organ culture system using adenovirus gene delivery strategy. Microinjection of the Msx2-adenovirus to the proximal position inhibited BMP4-induced chondrogenesis. Over-expression of Msx2 also resulted in the abrogation of endogenous cartilage and the down-regulation of type II collagen expression. Taken together, these results suggest that BMP4 induces chondrogenesis, the pattern of which is positively regulated by Sox9 and negatively by Msx2. Chondrogenesis only occurs at sites where Sox9 expression is high relative to that of Msx2. The combinatorial action of these transcription factors appear to establish a threshold for Sox9 function and thereby restricts the position of chondrogenesis.