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BackgroundSplit Hand/Foot Malformation (SHFM) is a congenital limb disorder presenting with limb anomalies, such as missing, hypoplastic, or fused digits, and often craniofacial defects, including a cleft lip/palate, microdontia, micrognathia, or maxillary hypoplasia. We previously identified three novel variants in the transcription factor,PRDM1, that are associated with SHFM phenotypes. One individual also presented with a high arch palate. Studies in vertebrates indicate that PRDM1 is important for development of the skull; however, prior to our study, human variants inPRDM1had not been associated with craniofacial anomalies.MethodsUsing transient mRNA overexpression assays inprdm1a-/-mutant zebrafish, we tested whether thePRDM1SHFM variants were functional and could lead to a rescue of the craniofacial defects observed inprdm1a-/-mutants. We also mined a CUT&RUN and RNA-seq dataset to examine Prdm1a binding and the effect of Prdm1a loss on craniofacial genes.Resultsprdm1a-/-mutants exhibit craniofacial defects including a hypoplastic neurocranium, a loss of posterior ceratobranchial arches, a shorter palatoquadrate, and an inverted ceratohyal. Injection of wildtypehPRDM1inprdm1a-/-mutants partially rescues these structures. However, injection of each of the three SHFM variants fails to rescue the skeletal defects. Loss ofprdm1aleads to a decreased expression of important craniofacial genes, such asdlx5a/dlx6a, hand2, sox9b, col2a1a, andhoxbgenes.ConclusionThese data suggest that the three SHFM variants are not functional and may have led to the craniofacial defects observed in the humans. Finally, they demonstrate how Prdm1a can directly bind and regulate craniofacial gene expression.

Rhabdomyosarcoma (RMS) is a pediatric muscle sarcoma characterized by expression of the myogenic lineage transcription factors (TFs) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their functions. Here, we demonstrate that the developmental TF SIX1 is highly expressed in RMS and critical for maintaining a muscle progenitor-like state. SIX1 loss induces differentiation of RMS cells into myotube-like cells and impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at loci more permissive to tumor growth over muscle differentiation. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status and predicts RMS progression in human disease. Our findings demonstrate a master regulatory role of SIX1 in repression of RMS differentiation via genome-wide alterations in MYOD1 and MYOG-mediated transcription.

Defects in craniofacial development represent a majority of observed human birth defects, occurring at a rate as high as 1:800 live births. These defects often occur due to changes in neural crest cell (NCC) patterning and development and can affect non-NCC-derived structures due to interactions between NCCs and the surrounding cell types. Proper craniofacial development requires an intricate array of gene expression networks that are tightly controlled spatiotemporally by a number of regulatory mechanisms. One of these mechanisms involves the action of microRNAs (miRNAs), a class of noncoding RNAs that repress gene expression by binding to miRNA recognition sequences typically located in the 3' UTR of target mRNAs. Recent evidence illustrates that miRNAs are crucial for vertebrate facial morphogenesis, with changes in miRNA expression leading to facial birth defects, including some in complex human syndromes such as 22q11 (DiGeorge Syndrome). In this review, we highlight the current understanding of miRNA biogenesis, the roles of miRNAs in overall craniofacial development, the impact that loss of miRNAs has on normal development and the requirement for miRNAs in the development of specific craniofacial structures, including teeth. From these studies, it is clear that miRNAs are essential for normal facial development and morphogenesis, and a potential key in establishing new paradigms for repair and regeneration of facial defects.