RNA in situ hybridization (RISH) allows for validation and characterization of the long non-coding (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 (TF) in axial skeleton development.Objective.
Validation of Klhl14as in the developing murine IVD.Summary of Background Data.
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 non-coding antisense transcript as down regulated in embryos devoid of key transcription factors during axial skeleton development. Here, primarily the antisense transcript is analyzed in this loss-of-function context.Methods.
4930426D05Rik and 6330403N15Rik were identified as Klhl14as and sense, respectively, two transcripts down regulated in the vertebral column of midgestation Pax1 and Pax9 mutant mouse embryos. RISH on wildtype and mutant embryos for the transcription factor encoding genes Pax1/Pax9, Sox5/Sox6/Sox9 and Bapx1 was used to further analyze Klhl14as in the developing IVD.Results.
Klhl14as and Klhl14 were the top down regulated transcripts in Pax1-/-; Pax9-/- E12.5 embryos. Our data demonstrates 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 transcription factors Pax1/Pax9, Sox6, Sox9 and Bapx1.Conclusion.
We are the first to suggest a role for the lncRNA Klhl14as in the developing IVD. Our data links Klhl14as to a previously established gene regulatory network during axial skeleton development and contributes further evidence that lnc NATs are involved in crucial gene regulatory networks in eukaryotic cells.Conclusion.
Level of Evidence: N/A.