Plastic Surgery Research Council

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Single Cell Transcriptome Analysis of Epithelial vs Chondrogenic Cell Types in Palate Morphogenesis
Claudio Macias-Trevino, MD-PhD Candidate1,2, Shannon H. Carroll, PhD1,2, Nora Alhazmi, DDS1,2, Edward B. Li, PhD1,2, Shawn A. Hallett, BA1,2, Dawn Truong, BA1,2, Eric C. Liao, MD/PhD1,2.
1Massachusetts General Hospital, Boston, MA, USA, 2Shriners Hospital for Children, Boston, MA, USA.

PURPOSE: IRF6 is the most frequently mutated gene in cleft lip and/or palate (CL/P) cohorts across all major populations. We hypothesized that interactions between epithelial cells and chondrocytes are essential for palate formation and craniofacial morphogenesis. We utilized advanced methods in transgenesis coupled with enhancer profiling to define the transcriptional landscape in epithelial and neural crest derived populations. Modern techniques such as single-cell RNA-sequencing enable us to probe transcriptional landscapes with high dimensionality, and are critical in defining developmental processes that can be targeted therapeutically in patients at risk for CL/P.
METHODS: One important IRF6 gene variant associated with isolated cleft-lip has been mapped to a multiple-species conserved sequence roughly 9.7kb (MCS9.7) upstream of the IRF6 gene. We cloned this MCS9.7 sequence upstream of an eGFP reporter gene and recombined the construct into the zebrafish genome by co-injecting the construct and the Tol2 transposase system into wild-type zebrafish embryos. We generated a dual-reporter irf6:eGFP;sox10:mCherry line. This transgenic reporter line will be characterized by live-imaging using two-photon confocal microscopy. In addition, we generated an epithelial-specific wntless (wls) line and a chondrocyte specific col2a line that enable us to tag and purify specific cranial neural crest cellular populations during embryogenesis. Specific lineage cell types will be isolated using FACS to generate purified cellular populations that will be analyzed using single cell RNA-sequencing to reveal dynamic lineage specific gene expressions during craniofacial morphogenesis.
RESULTS: We have generated the irf6 reporter construct using the MCS9.7 sequence and were successful in achieving germline transmission of the transgene after crossing the founder fish into a sox10:mCherry background. Imaging of developing embryos of the dual reporter line revealed expression of irf6 restricted to the ectoderm, and sox10 expression in chondrocytes of the zebrafish palate. We also delineated epithelial expression of wls in a similar wls:eGFP;sox10:mCherry line. We are using FACS to isolated oral epithelial cells and cranial neural crest cells for single-cell RNA sequencing of purified subpopulations.
CONCLUSION: This work establishes an irf6 reporter line in zebrafish that will be critical in studying developmental processes. A major strength of the zebrafish model system is the ability to image live embryos and visualize multiple stages of early development in vivo. Fate-mapping of irf6-expressing cells at different time points in zebrafish embryos using the dual reporter line will be critical in studying the interactions of epithelial and chondrocyte cells during palate morphogenesis. As new developments in gene and cellular therapies are pursued, understanding the basic transcriptional circuits is essential in designing genomic targets for gene therapy, or in the engineering of bioactive implantable materials for optimal surgical outcomes.

Figure Legend: Interactions between epithelial cells and chondrocytes are critical for palate formation in zebrafish. Images for irf6:GFP;sox10:mCh and wls:GFP;sox10:mCh lines showing epithelial expression of irf6 and wls, and chondrocyte expression of sox10. These cell types will be sorted using FACS and subjected to single-cell RNA-sequencing.


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