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Published February 3, 2021 | Supplemental Material + Published
Journal Article Open

Transcriptomic Identification of Draxin-Responsive Targets During Cranial Neural Crest EMT

Hutchins, Erica J. ORCID icon
Piacentino, Michael L. ORCID icon
Bronner, Marianne E. ORCID icon

Abstract

Canonical Wnt signaling plays an essential role in proper craniofacial morphogenesis, at least partially due to regulation of various aspects of cranial neural crest development. In an effort to gain insight into the etiology of craniofacial abnormalities resulting from Wnt signaling and/or cranial neural crest dysfunction, we sought to identify Wnt-responsive targets during chick cranial neural crest development. To this end, we leveraged overexpression of a canonical Wnt antagonist, Draxin, in conjunction with RNA-sequencing of cranial neural crest cells that have just activated their epithelial–mesenchymal transition (EMT) program. Through differential expression analysis, gene list functional annotation, hybridization chain reaction (HCR), and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we validated a novel downstream target of canonical Wnt signaling in cranial neural crest – RHOB – and identified possible signaling pathway crosstalk underlying cranial neural crest migration. The results reveal novel putative targets of canonical Wnt signaling during cranial neural crest EMT and highlight important intersections across signaling pathways involved in craniofacial development.

Additional Information

© 2021 Hutchins, Piacentino and Bronner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Received: 30 October 2020; Accepted: 07 January 2021; Published: 03 February 2021. We thank A. Collazo and G. Spigolon for imaging assistance at the Caltech Biological Imaging Facility; P. Cannon and R. Diamond of the Caltech Flow Cytometry Cell Sorting Facility for cell sorting assistance; I. Antoshechkin of the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory for sequencing of our RNA-seq libraries; and M. Martik and S. Gandhi for assistance with data processing. This work was supported by the National Institutes of Health (R01DE027538 and R01DE027568 to MB, K99DE028592 to EH, and K99DE029240 to MP). Author Contributions: EH, MP, and MB conceived the project and conducted the experimental design and data interpretation. EH and MP performed the cell dissociations, library preparations, and RNA-seq analyses. EH performed the functional annotation, hybridization chain reaction experiments, imaging, quantitation, and statistical analyses. EH and MB wrote the manuscript with editing by MP. All authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Statement: Ethical review and approval was not required for the animal study because our study uses chicken embryos at E1-2. These are not considered vertebrate embryos until E10, and thus we do not require ethics committee approvals or protocols, as they are not considered vertebrates at the stages we work.

Attached Files

Published - fphys-12-624037.pdf

Supplemental Material - Image_1_Transcriptomic_Identification_of_Draxin-Responsive_Targets_During_Cranial_Neural_Crest_EMT.TIF

Files

Image_1_Transcriptomic_Identification_of_Draxin-Responsive_Targets_During_Cranial_Neural_Crest_EMT.TIF

Additional details

Identifiers Funding Dates Caltech Custom Metadata
Created:
August 20, 2023
Modified:
December 22, 2023