A distant global control region is essential for normal expression of anterior HOXA genes during mouse and human craniofacial development.

Craniofacial abnormalities account for approximately one third of birth defects. The regulatory programs that build the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Clusters of coactivated enhancers and their target genes, known as superenhancers, are important in determining cell identity but have been largely unexplored in development. In this study we identified superenhancer regions unique to human embryonic craniofacial tissue. To demonstrate the importance of such regions in craniofacial development and disease, we focused on an ~600 kb noncoding region located between NPVF and NFE2L3. We identified long range interactions with this region in both human and mouse embryonic craniofacial tissue with the anterior portion of the HOXA gene cluster. Mice lacking this superenhancer exhibit perinatal lethality, and present with highly penetrant skull defects and orofacial clefts phenocopying Hoxa2-/- mice. Moreover, we identified two cases of de novo copy number changes of the superenhancer in humans both with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and copy number changes are pathogenic in human patients.

Spatial Multiomics Reveal the Role of Wnt Modulator, Dkk2, in Palatogenesis.

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which constitutes the most common among the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study utilized the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis.

The terminal differentiation of osteoblasts and subsequent formation of bone marks an important phase in palate development that leads to the separation of the oral and nasal cavities. While the morphogenetic events preceding palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms driving the formation of this bony union of the fusing palate. Through bulk, single-nucleus, and spatially resolved RNA-sequencing analyses of the developing secondary palate, we identify a shift in transcriptional programming between embryonic days 14.5 and 15.5 pinpointing the onset of osteogenesis. We define spatially restricted expression patterns of key osteogenic marker genes that are differentially expressed between these developmental timepoints. Finally, we identify genes in the palate highly expressed by palate nasal epithelial cells, also enriched within palatal osteogenic mesenchymal cells. This investigation provides a relevant framework to advance palate-specific diagnostic and therapeutic biomarker discovery.

Integrative analysis of transcriptome dynamics during human craniofacial development identifies candidate disease genes.

Craniofacial disorders arise in early pregnancy and are one of the most common congenital defects. To fully understand how craniofacial disorders arise, it is essential to characterize gene expression during the patterning of the craniofacial region. To address this, we performed bulk and single-cell RNA-seq on human craniofacial tissue from 4-8 weeks post conception. Comparisons to dozens of other human tissues revealed 239 genes most strongly expressed during craniofacial development. Craniofacial-biased developmental enhancers were enriched +/- 400 kb surrounding these craniofacial-biased genes. Gene co-expression analysis revealed that regulatory hubs are enriched for known disease causing genes and are resistant to mutation in the normal healthy population. Combining transcriptomic and epigenomic data we identified 539 genes likely to contribute to craniofacial disorders. While most have not been previously implicated in craniofacial disorders, we demonstrate this set of genes has increased levels of de novo mutations in orofacial clefting patients warranting further study.

Integration of multimodal data in the developing tooth reveals candidate regulatory loci driving human odontogenic phenotypes.

Human odontogenic aberrations such as abnormal tooth number and delayed tooth eruption can occur as a symptom of rare syndromes or, more commonly, as nonsyndromic phenotypes. These phenotypes can require extensive and expensive dental treatment, posing a significant burden. While many dental phenotypes are heritable, most nonsyndromic cases have not been linked to causal genes. We demonstrate the novel finding that common sequence variants associated with human odontogenic phenotypes are enriched in developmental craniofacial enhancers conserved between human and mouse. However, the bulk nature of these samples obscures if this finding is due to the tooth itself or the surrounding tissues. We therefore sought to identify enhancers specifically active in the tooth anlagen and quantify their contribution to the observed genetic enrichments. We systematically identified 22,001 conserved enhancers active in E13.5 mouse incisors using ChIP-seq and machine learning pipelines and demonstrated biologically relevant enrichments in putative target genes, transcription factor binding motifs, and in vivo activity. Multi-tissue comparisons of human and mouse enhancers revealed that these putative tooth enhancers had the strongest enrichment of odontogenic phenotype-associated variants, suggesting a role for dysregulation of tooth developmental enhancers in human dental phenotypes. The large number of these regions genome-wide necessitated prioritization of enhancer loci for future investigations. As enhancers modulate gene expression, we prioritized regions based on enhancers' putative target genes. We predicted these target genes and prioritized loci by integrating chromatin state, bulk gene expression and coexpression, GWAS variants, and cell type resolved gene expression to generate a prioritized list of putative odontogenic phenotype-driving loci active in the developing tooth. These genomic regions are of particular interest for downstream experiments determining the role of specific dental enhancer:gene pairs in odontogenesis.