Molecules and Therapies for Craniofacial and Dental Disorders
- Rena D’Souza, MS, DDS, PhD, Head, Section on Craniofacial Genetic Disorders, Director of the National Institute of Dental and Craniofacial Research
- Fahad K. Kidwai, BDS, MSc, PhD, Clinical Staff
- Resmi Raju, PhD, Postdoctoral Fellow
- Parna Chatteraj, Lab Manager
- Jeremie Oliver Piña, MS, MBA, Predoctoral Fellow
Embryonic development of the craniofacial complex requires tightly controlled molecular crosstalk between proliferating and differentiating cell networks to form the most intricate structures in the human body. During embryogenesis, perturbations in the environmental and/or genetic milieu can negatively affect craniofacial development. While considerable progress has been made in studying isolated genetic mutations leading to syndromic and non-syndromic craniofacial disorders, the broad molecular-genetic mechanisms driving morphogenetic events have yet to be sufficiently explored and understood. Such gaps in our understanding have restricted clinical treatment options for patients affected by common developmental anomalies, such as cleft palate or tooth agenesis. Thus, there is a strong biologic rationale for a thorough investigation of basic molecular mechanisms driving craniofacial structure morphogenesis, which may pave the way toward translatable therapeutic developments for patients.
The overarching goal of our research program is to conduct basic and translational studies of genetic and molecular mechanisms involved in craniofacial development, with the primary aim of unveiling novel regulatory molecules and putative patient-centric therapeutic solutions for craniofacial and dental disorders. Among the molecular pathways known to control craniofacial development is Wnt/β-catenin signaling. Wnts are also well known as upstream effectors of osteogenesis and odontogenesis. Our lab demonstrated, for the first time, the successful in utero correction of cleft palate defects in a Pax9–/– mouse genetic model by small-molecule neutralizing therapy targeting Wnt–antagonizing proteins. We are now actively investigating additional drug-delivery approaches and molecular targets for the modulation of Wnt signaling in vivo to permit targeted correction of both cleft palate and tooth agenesis. Our research group employs basic principles of developmental biology, next-generation sequencing, regenerative medicine, tissue engineering, and drug delivery models to identify and validate novel approaches to restore molecular equilibrium in genetic models of the highly relevant human diseases cleft palate and tooth agenesis. Actively fostering collaborations with both intramural and extramural investigators, from basic scientists, engineers, to clinicians, the lab aims to pioneer innovative approaches toward the treatment of patients affected by craniofacial disorders of development. Through these rigorous and robust preclinical, proof-of-principle studies, we hope to open up pathways toward novel clinical trials for the treatment of previously unpreventable disorders affecting the craniofacial complex.
Figure 1.
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Characterization of palate osteogenesis begins with fusion.
Toward a novel in utero therapy for the prenatal cure of cleft palate in a mouse genetic model
Cleft palate (CP), together with cleft lip, is among the most common birth defects in humans, occurring in up to 1 in 500 live births. Such birth defects can inflict heavy physical, mental, psychosocial, and financial burdens on patients and their caregivers throughout life, often requiring many stages of complex surgical correction with varying success rates. Hence, there remains a substantial need for innovative approaches to alleviate the burden of postnatal care for these patients. While considerable progress has been made in studying isolated genetic mutations leading to syndromic and non-syndromic cleft disorders, the broad molecular-genetic mechanisms driving osteogenic differentiation from palatal shelf out-growth, elevation, and fusion events have yet to be sufficiently explored and understood. Such gaps in our understanding have restricted clinical treatment options for patients affected by cleft disorders. Given that prenatal molecular diagnostics have made CP identifiable earlier in gestation, the advancement of safe and efficacious interventions in utero to correct CP has become feasible.
Figure 2.
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Wnt signaling drives palate osteogenesis and fusion.
The objective of our work is to investigate the molecular-genetic mechanisms driving palatal osteogenesis and to optimally manipulate key signaling environments in utero to promote CP correction in a mouse genetic model. If properly explored, such information can be applied to the development of novel therapeutics that can benefit individuals with isolated or syndromic CP defects, who face complex surgeries and the arduous burden of life-long care. The long-term goal of this research is to investigate the spatio-temporal molecular mechanisms driving osteogenic differentiation in normal palate development and in CP dysmorphogenesis, using Pax9–/– as a model. Differential multiomic profiles of expression in spatial biological context will unveil the molecular framework for the development of novel therapeutic strategies to optimize signaling environments during development. The proposed research will test the following hypotheses: (1) Wnt signaling effector function is critical for osteogenesis of the embryonic palate; and (2) loss of the up-stream master regulator of Wnt signaling homeostasis, Pax9, results in the disruption of palatal osteogenesis via up-regulation of sclerostin (Sost), a potent inhibitor of Wnt signaling and bone formation (Figures 1, 2). The specific aims of this proposal are as follows: (1) to define the spatio-temporal transcriptomic profile of embryonic palate osteogenesis via unbiased stage-specific signature mapping of cell populations in the normal murine secondary palate; (2) to differentially compare epigenomic, proteomic, and gene expression signatures of Wnt–related osteogenesis in Pax9–/– CP; and (3) to pilot novel in utero drug delivery approaches and molecules, based on the molecular profiles observed in situ, for the prenatal cure of CP. The proposed research will add novel foundational knowledge of multiomic morphogenetic expression gradients of key Wnt signaling regulators within the embryonic palate and will propose an innovative therapeutic model whereby palatal clefts may be corrected in utero.
Figure 3.
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Wnt signaling drives palate osteogenesis and fusion.
The proposed research will improve unbiased mechanistic understanding of palatal bone development and lead to the first preclinical study of intra-amniotic small-molecule and antibody-replacement drug delivery for targeted palatal osteogenesis. The validation of a translational in utero drug delivery system for reversal of single-gene CP disorders will lead to preventive and corrective prenatal therapeutic interventions in humans.
Profiles of Wnt pathway gene expression during tooth morphogenesis
Mouse and human genetic studies indicate key roles of the Wnt10a ligand in odontogenesis. Despite the advances, little is known about the temporo-spatial modulation of Wnt10a and whether its expression profiles explain the reciprocal epithelial-mesenchymal signaling events that drive morphogenesis and cell differentiation. We systematically compared the profiles of Wnt10a in developing murine molars and incisors with the Wnt signaling pathway inhibitors Sost and Dkk1, using multiplex in situ hybridization and single-cell RNA-sequencing (scRNA-seq). During tooth bud morphogenesis, Wnt10a transcripts were restricted to the epithelium, in contrast to the localization of Sost and Dkk1 to the dental mesenchyme. At E15.5, there was a marked shift of Wnt10a from dental epithelium to mesenchyme, while Sost and Dkk1 expression remained enriched in the mesenchyme. By E18.5 and P0 (postnatal day 0), Wnt10a expression coincided with the gradients of ameloblast and odontoblast differentiation from cusp to apical regions. Interestingly, Sost and Dkk1 co-expressed with Wnt10a in odontoblasts at these stages. At P7 and 14, following dentin and enamel mineralization, Wnt10a was confined to odontoblasts, while Wnt modulators were reduced or absent in the molars, but intense signals were continuously present in ameloblasts (Wnt10a) and odontoblasts (Wnt10a, Sost, and Dkk1) towards the proximal end of incisors near the cervical loop. scRNA-seq confirmed in situ expression signatures of target genes to clusters containing dental cells. These data provide cell type–specific insight into the role of Wnt signaling mediators during epithelial-mesenchymal interactions in odontogenesis. Our results provide a framework for future research on well timed therapies that target Wnt signaling for the reversal of tooth agenesis and for regenerating the dentin-pulp complex after injury.
Figure 4.
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Wnt10a and Wnt modulators
In vivo molecular consequences of Sosdc1 deficiency on Pax9– and/or Msx1–dependent signaling events that control maxillary vs. mandibular incisors
Tooth agenesis is a developmental anomaly defined by the lack of one or more teeth (excluding third molars) resulting from to early failures in tooth formation. Several potential genes causing tooth agenesis have been studied. A familial autosomal dominant hypodontia (tooth agenesis) was demonstrated to be caused by a point mutation in the MSX1 and PAX9 genes. However, how MSX1 and PAX9 control the patterning of tooth agenesis requires further investigation. Interestingly, Pax9+/–/Msx1+/– mutant mice exhibit selective oligodontia (missing lower incisors only) and offer an excellent opportunity to study the Pax9– and Msx1–dependent tooth agenesis patterning. Concurrently, we and others have shown that the transcription factors Msx1 and Pax9 modulate the functions of Bmp and Wnt during odontogenesis in a mouse model. Interestingly, we found that transgenic inhibition of a known bifunctional BMP and Wnt antagonist, Sosdc1, was able to rescue the lower incisor in Pax9– and Msx1–deficient mice (Pax9+/–; Msx1+/–/Sosdc1–/–). However, how exclusively Msx1 and/or Pax9 interact with Sosdc1 in the mandibular incisor’s domain vs. the maxillary incisor’s domain requires further investigation. Therefore, our ongoing experiments (bulk RNA, multiplex in situ hybridization, and multiomics analysis) will explore the molecular consequences of Sosdc1 deficiency on Pax9– and/or Msx1–dependent signaling events in the maxillary vs. mandibular incisors domain.
Figure 5.
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In vivo molecular consequences of Sosdc1 deficiency on Pax9– and/or Msx1–dependent signaling events that control maxillary vs. mandibular incisors
Genetic interaction between Msx1 and the Wnt signaling pathway during mandibular incisor development
Tooth development is a distinct embryological process, which can be used as a model to study the multi-stage epithelial-mesenchymal interaction. The MSX1 transcription factor is essential in early tooth morphogenesis through bud-to-cap transitioning. Deleting the Msx1 gene causes tooth arrest at the bud stage in mice. In mice deficient in Msx1, Bmp4 mRNA expression in the dental mesenchyme is abated at E13.5 and E14.5. Alternatively, deleting the Bmp and Wnt modulator Sosdc1 acts as an impetus to forming supernumerary teeth as a result of dysregulated Bmp and Wnt signaling activity in the rudimentary dental tissues. However, Msx1 interaction with Wnt modulators (Wise and/or Dkk2) in incisors has not been reported. Our multiplex in situ hybridization results confirm the distinct interactions between Msx1 and the expression patterning of Wnt modulators (Sosdc1 and Dkk2) during lower incisor development (E13.5 and E14.5). Interestingly, at E14.5, Sosdc1 was found to be drastically downregulated in the Msx1–/– embryos. Alternatively, Dkk2 was found to be regulated differently than Sosdc1 in the lower incisiform of Msx1–deficient embryos (Msx1–/–) at E14.5. Moreover, no alteration in the Dkk2 expression pattern at E14.5 was found in the lower incisiform of Msx1– and Sosdc1–deficient embryo (Msx1–/– Sosdc1–/–) compared with Msx1–/– embryos. These data confirm that Msx1 is upstream of the Wnt modulator (Dkk2 and Sosdc1) and regulates Sosdc1 and DKK2 independently during the bud-to-cap stage transition.
Figure 6.
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Genetic interaction between Msx1 and the Wnt signaling pathway during mandibular incisor development
Publications
- Piña JO, Raju R, Roth DM, Winchester EW, Chattaraj P, Kidwai F, Faucz FR, Iben J, Mitra A, Campbell K, Fridell G, Esnault C, Cotney JL, Dale RK, D'Souza RN. Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis. Nat Commun 2023 14:5687.
- Raju R, Piña JO, Roth DM, Chattaraj P, Kidwai F, Faucz FR, Iben J, Mitra A, Campbell K, Fridell G, Dale RK, D'Souza RN. Profiles of Wnt pathway gene expression during tooth morphogenesis. Front Physiol 2023 14:1316635.
- Reddy MS, D'Souza RN, Webster-Cyriaque J. A call for more oral health research in primary care. JAMA 2023 330:1629–1630.
- Weintraub JA, Kaeberlein M, Perissinotto C, Atchison KA, Chen X, D'Souza RN, Feine JS, Ghezzi EM, Kirkwood KL, Ryder M, Slashcheva LD, Touger-Decker R, Wu B, Kapila Y. Geroscience: aging and oral health research. Adv Dent Res 2023 31:2–15.
Collaborators
- Fabio R. Faucz, PhD, Molecular Genomics Core, NICHD, Rockville, MD
- Steven L. Goudy, MD, MBA, FACS, Emory University School of Medicine, Atlanta, GA
Contact
For more information, email rena.d'souza@nih.gov or visit https://irp.nih.gov/pi/rena-dsouza.
