4 - Transcriptional regulatory mechanisms of SHH expression controlled by PBXs in the Frontonasal Ectodermal Zone during facial morphogenesis

Poster Board Number: 4
There are separate poster presentation times for odd and even posters.
Odd poster #s – first hour
Even poster #s – second hour

Co-authors:

Diane Hu - Orthopaedic Surgery - University of California, San Francisco; Marta Losa - Department of Orofacial Sciences and Department of Anatomy - University of California, San Francisco; Maurizio Risolino - Department of Orofacial Sciences and Department of Anatomy - University of California, San Francisco; Licia Selleri - Department of Orofacial Sciences and Department of Anatomy - University of California, San Francisco; Ralph Marcucio - Orthopaedic Surgery - University of California, San Francisco

Abstract Body :Expression of Sonic hedgehog (SHH) in the ectoderm of the frontonasal process controls facial morphogenesis and defines a signaling center, the frontonasal ectodermal zone (FEZ). Despite the important roles of SHH during face formation, little is known about its transcriptional regulatory mechanisms in the FEZ. Pre-B-cell leukemia homeobox transcription factor 1 (PBX1) is co-expressed with SHH in the ventral surface of the chick FEZ, while expression of PBX3 demarcates the dorsal boundary of the SHH domain at stage 22 (HH22). Therefore, we hypothesized that PBX1 enhances and PBX3 represses expression of SHH in the FEZ during development. To test the hypothesis, we performed gain- and loss-of-function experiments. We infected HH10 chick embryos with retroviruses encoding PBX1 or PBX3, or microRNAs targeting PBX1 or PBX3. After 72 hours (HH22), in situ hybridization showed that overexpression of PBX1 expanded the SHH expression domain while knock-down of PBX1 decreased SHH expression. In contrast, overexpression of PBX3 reduced SHH expression while knock-down of PBX3 induced premature SHH expression. To identify potential regulatory elements interacting with PBXs, we assessed open chromatin regions by ATAC-seq and binding sites of PBX1 and PBX3 by ChIP-seq from HH22 chick FEZ. We found a region of open chromatin in intron 1 of SHH that was bound by both PBX1 and PBX3. Within this region we identified a 400bp-long sequence that contained PBX consensus binding sites. We cloned this region into a reporter vector that has minimal expression in the absence of an enhancer element, and electroporated it into the chick FEZ at HH20. Expression of the reporter in the SHH domain at HH24 confirmed that the cloned element had enhancer activity. Subsequently, in vitro reporter assays using luciferase and b-galactosidase demonstrated that PBX1 activates and PBX3 represses the enhancer activity of the 400bp element. In addition, single cell multiome sequencing data were generated by a droplet-based method (10X Genomics) from HH22 chick FEZ to specify cell subpopulations expressing higher SHH. Cells expressing higher levels of SHH also have greater ATAC-seq peaks around the SHH locus. We examined eleven regions of open chromatin within ~50kb of the SHH locus. Among the eleven elements, only one element, which overlaps the 400bp PBX enriched region, showed enhancer activity. In conclusion, the present study demonstrated the complementary roles of PBX1 and PBX3 in regulating SHH expression in the FEZ and has laid foundations for further investigation of potential SHH enhancers in the genome. Elaboration of the transcriptional regulatory mechanisms of SHH expression during facial development will provide novel insights into the etiology and the clinical aspects of craniofacial birth defects.