RNA in situ hybridization (RISH) allows for validation and characterization of the long noncoding (lnc) natural antisense RNA (NAT) Klhl14as in the embryonic murine intervertebral disc (IVD) in the context of loss-of-function mutants for key transcription factors (TFs) in axial skeleton development.
Validation of Klhl14as in the developing murine IVD.
The IVD is a focus of regenerative medicine; however, processes and signaling cascades resulting in the different cell types in a mature IVD still require clarification in most animals including humans. Technological advances increasingly point to implications of lnc NATs in transcription/translation regulation. Transcriptome data generation and analysis identified a protein encoding transcript and related noncoding antisense transcript as downregulated in embryos devoid of key TFs during axial skeleton development. Here, primarily, the antisense transcript is analyzed in this loss-of-function context.
4930426D05Rik and 6330403N15Rik were identified as Klhl14as and sense, respectively, two transcripts downregulated in the vertebral column of midgestation Pax1 and Pax9 mutant mouse embryos. RISH on wildtype and mutant embryos for the TF encoding genes Pax1/Pax9, Sox5/Sox6/Sox9, and Bapx1 was used to further analyze Klhl14as in the developing IVD.
Klhl14as and Klhl14 were the top downregulated transcripts in Pax1-/-; Pax9-/- E12.5 embryos. Our data demonstrate expression of Klhl14as and sense transcripts in the annulus fibrosus (AF) and notochord of the developing IVD. Klhl14as expression in the inner annulus fibrosus (iAF) seems dependent on the TFs Pax1/Pax9, Sox6, Sox9, and Bapx1.
We are the first to suggest a role for the lncRNA Klhl14as in the developing IVD. Our data link Klhl14as to a previously established gene regulatory network during axial skeleton development and contribute further evidence that lnc NATs are involved in crucial gene regulatory networks in eukaryotic cells.
Level of Evidence: N/A.
For the first time, a role in intervertebral disc development is demonstrated for the novel long noncoding RNA Klhl14as. Implications for Klhl14as with an extensive vertebral-IVD gene regulatory network are established through loss-of-function models. Klhl14as is differentially regulated in cells giving rise to the future annulus fibrosus and nucleus pulposus.
∗Department of Biology, Clarkson University, Potsdam, NY
†Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA.
Address correspondence and reprint requests to Thomas Lufkin, PhD, Bayard and Virginia Clarkson Endowed Chair in Biology, Department of Biology, Clarkson University, 8 Clarkson Avenue, Potsdam, NY 13699; E-mail: email@example.com
Received 23 May, 2018
Accepted 17 July, 2018
This work was supported by the Bayard and Virginia Clarkson Endowment to Professor Thomas Lufkin.
The manuscript submitted does not contain information about medical device(s)/drug(s).
No relevant financial activities outside the submitted work.