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Principal Investigator: Licia Selleri
Organization: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
Fiscal Year: 2024
Award: $567,205
Funding agency: National Institute of Dental and Craniofacial Research
PROJECT SUMMARY
Human cleft lip with or without cleft palate (CL/P), the most common craniofacial birth defect, is caused by im-
paired fusion of the facial prominences. During normal morphogenesis, the frontonasal and maxillary promi-
nences fuse at a three-way seam, the lambdoidal junction (l), to form the upper lip/primary palate. If the epithe-
lium that covers the prominences persists at the l, orofacial clefting ensues, as we previously reported in mouse
embryos deficient for PBX transcription factors (TFs). We described that: 1) prominence fusion requires coordi-
nation of two distinct cellular behaviors, apoptosis and epithelial-to-mesenchymal transition (EMT), respectively,
in two discrete subpopulations within the l epithelium; 2) these two subpopulations are characterized by different
molecular signatures; and 3) mice deficient for PBX TFs with CL/P lack both of these epithelial subpopulations.
Our preliminary evidence in the mouse indicates that: I) the λ epithelium is heterogeneous and comprises 6 main
cell subpopulations prior to prominence fusion, as demonstrated by single cell RNA sequencing; II) one of the
subpopulations, that we termed “l fusion effectors”, is enriched for genes that have been associated with human
or mouse orofacial clefting, for genes encoding pro-apoptotic factors, and for inhibitors of cell cycle progression;
III) “l fusion effector” cells are located at the tip of the prominences; and IV) the proportion of “l fusion effectors”
is perturbed in compound Pbx1/2 mutant embryos with CL/P compared to controls. Based on these results, we
posit that the “l fusion effector” cluster comprises cells that are prime executors of prominence fusion; is halted
in the cell cycle, a prerequisite for subsequent cellular changes, like apoptosis and EMT, to achieve fusion; and
is quantitatively and/or qualitatively perturbed in mouse models of CL/P. We will test this hypothesis via the
following Specific Aims: 1) Determine the spatiotemporal dynamics of the epithelial “l fusion effector”
subpopulation throughout midface prominence fusion. We will establish whether the “l fusion effector” clus-
ter is transient or if it persists after fusion, as well as track the progenitors and descendants of “l fusion effector”
cells in vivo across space and developmental time. 2) Establish the molecular mechanisms underlying cell
cycle arrest in “l fusion effector” cells during upper lip/primary palate fusion. We will uncover in vivo cell
cycle dynamics of “l fusion effector” cells and assess whether PBX-dependent regulation of cell cycle genes
mediates cell cycle control in this cluster. 3) Determine whether the perturbations of the “λ fusion effector”
cluster in Pbx1/2 mutants are recapitulated in p63- or Bmpr1a-deficient mice with CL/P. We will establish
whether the cellular and transcriptional changes of the l epithelium resulting in CL/P in Pbx1/2 mutants are
recapitulated in all three mouse models, or if they are distinct. This research will lead to discover new genes for
prenatal diagnostics of CL/P and open strategies for CL/P repair through reactivation of developmental programs
that are defective in orofacial clefting. Broadly, this work will foster mechanistic studies testing whether other
fusion processes are mediated by cell cycle arrest.
Terms: <Apoptosis><Apoptosis Pathway><Apoptotic><Assay><BUdR><Basal Transcription Factor><Basal transcription factor genes><Bio-Informatics><Bioassay><Bioinformatics><Biological Assay><Birth Defects><Body Tissues><BrdU><Bromodeoxyuridine><Bromouracil Deoxyriboside><Broxuridine><CUT&RUN><Cell Body><Cell Cycle><Cell Cycle Arrest><Cell Cycle Control><Cell Cycle Genes><Cell Cycle Progression><Cell Cycle Regulation><Cell Division Cycle><Cell Division Cycle Genes><Cell Growth in Number><Cell Multiplication><Cell Proliferation><Cells><Cellular Proliferation><Cephalic><Cleavage Targets and Release Using Nuclease><Cleavage Under Targets and Release Using Nuclease><Cleft Palate><Cleft lip with or without cleft palate><Congenital Abnormality><Congenital Anatomical Abnormality><Congenital Defects><Congenital Deformity><Congenital Malformation><Cranial><Cryosectioning><Cryoultramicrotomy><Development><Diagnostic><Effector Cell><Embryo><Embryonic><Epithelial Cells><Epithelium><Exhibits><Face><Fostering><Frontonasal Prominence><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic><Genetic Transcription><Gestation><Grant><Histone H3><Human><Image><Immunofluorescence><Immunofluorescence Immunologic><Impairment><In Situ Hybridization><Individual><Lip><Lip structure><Live Birth><Maxillary Prominence><Mediating><Mesenchymas><Mesenchyme><Mice><Mice Mammals><Microdissection><Modern Man><Molecular><Molecular Fingerprinting><Molecular Profiling><Morphogenesis><Murine><Mus><Neural Crest><Pathogenesis><Pathway interactions><Population><Pregnancy><Premaxillary palate><Primary Palate><Process><Programmed Cell Death><Publishing><Quantitative RTPCR><Quantitative Reverse Transcriptase PCR><RNA Expression><Reporting><Research><Societies><Staining method><Stains><Testing><Time><Tissues><Transcript><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><Work><Wound Repair><cdc Genes><cell behavior><cellular behavior><cl/p><cleft of the lip and/or palate><compare to control><comparison control><craniofacial><craniofacies><developmental><epithelial to mesenchymal transition><experiment><experimental research><experimental study><experiments><faces><facial><global gene expression><global transcription profile><imaging><in situ Hybridization Genetics><in situ Hybridization Staining Method><in vivo><inhibitor><molecular profile><molecular signature><morphogenetic process><mouse model><murine model><mutant><orofacial cleft><orofacial clefting><palate repair><palatoplasty><pathway><prenatal><progenitor><programs><qRTPCR><repair strategy><scRNA-seq><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><spatiotemporal><tissue repair><transcription factor><transcriptome><unborn><wound healing><wound recovery><wound resolution>