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Requirement Of Specc1lb In Facial Prominence Integration And Formation Of The Lower Jaw
Lisa gfrerer, MD1, Valeriy Shubinets, MD1, Christina Nguyen, MD1, Cynthia C. Morton, PhD2, Richard L. Maas, MD PhD2, Eric C. Liao, MD PhD1.
1MGH, boston, MA, USA, 2Brigham and Women's Hospital, boston, MA, USA.
The genetic basis of the rare oblique facial cleft (ObFC) has been unknown until the identification of SPECC1L from human translocation analysis. SPECC1L is a novel scaffolding protein where its molecular function in craniofacial morphogenesis is unknown. We explore the function of SPECC1L in craniofacial development by studying its homologs in zebrafish, where specc1lb is the ortholog.
Gene expression analysis was carried out by wholemount RNA in situ hybridization (WISH). Antisense morpholino (MO) knockdown and mRNA rescue was performed for functional analysis. Lineage tracing, cell proliferation, apoptosis assays, and molecular analysis were performed.
WISH revealed specc1lb expression to be in the epithelia juxtaposed to chondrocytes, suggesting expression in the ectoderm surrounding the neural crest cells. specc1lb may alter expression of other ectodermal molecules (edn ,wnt9, fgf8, shh) and thereby inhibit normal chondrocyte formation. Knockdown of specc1lb showed distinct craniofacial phenotypes resulting in clefts between the median and the lateral elements of the primary palate. Lineage tracing analysis revealed that CNC cells contributing to the frontonasal prominence failed to integrate or “fuse” with the maxillary prominence CNC cells. This resembles the pathogenesis of ObFC development in humans, where failure of fusion between the lateral nasal process and maxillary prominence occurs. Cells normally contributing to the lower jaw structures were able to migrate to their destined position but failed to form the mandibular elements. This defect is also observed when wnt9a, frzb and fzd7a are targeted by morpholino. Further, the same mandibular phenotype occurs in edn1−/− morphants. In specc1lb knockdown morphants, the developing ethmoid plate failed to extend fully, a process known to be regulated by wnt9a . Furthermore, genes of the wnt receptor complex (wnt9a, frzb, fzd7a) were downregulated in specc1lb morphants. In addition, ednra2 and edn1 expressions were unchanged, but homeobox transcription factor bapx1 (a downstream target of edn1) was downregulated.Co--injection of bapx1 mRNA and specc1lb MO rescued lower jaw elements, confirming that bapx1 acts downstream of specc1lb in jaw specification.
Taken together, this analysis suggests that specc1lb is required for integration of upper jaw palatal elements and convergence of mandibular prominences, potentially acting as a scaffolding protein to integrate wnt and edn pathways. Future protein chemistry experiments are underway to elucidate the cellular function and identify interactors of specc1lb. We present the first animal model of ObFC and morphogenetic insight into the mechanisms of normal palatogenesis and cleft molecular pathogenesis.
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