The majority of transcripts in the mammalian cell do not code for proteins. Two major classes of non-coding RNAs (ncRNAs) are long non-coding RNA (lncRNA) and microRNA, with members of both having been shown to strengthen gene regulatory networks (GRNs) to promote robust embryogenesis. The branchial arches (BAs) serve as a developmental paradigm. These transient structures arise during mid-embryonic vertebrate development, and comprise a series of reiterated outgrowths on either side of the head and pharynx. They develop into various adult derivatives including craniofacial bone, cartilage and muscle, in addition to vasculature belonging to the great cardiac vessels. To identify functional roles for lncRNAs and microRNAs during BA development, we generated BA microRNA-seq libraries, and utilised previously generated total RNA-seq and ChIP-seq from equivalent BA samples. LncRNAs are challenging to group functionally. We adopted a contextual classification approach, annotating mouse BA lncRNAs by their overlap or proximity to protein-coding genes. We then considered their co-expression with the respective overlapping mRNAs. We also interrogated lncRNA association with H3K27me3, a histone mark that has previously been implicated with lncRNA-mediated regulation. We identified 74 pairs of overlapping antisense lnc and protein-coding genes, which demonstrated enrichment for positively correlated expression, promoter H3K27me3, and G-quadruplex (G4) forming structures. We find that many of these protein-coding genes encode transcriptional regulators. We therefore hypothesise that upstream antisense lncRNA transcription removes local H3K27me3, functioning as a cis-regulatory mechanism to facilitate timely expression of its overlapping developmental gene during embryogenesis. MicroRNAs are a comparatively well-characterised class of ncRNA. To identify microRNAs that regulate BA development, we generated a mouse microRNA-seq dataset. We combined these data with previously generated BA total RNA-seq to predict BA expressed microRNA target genes. We identified candidate posterior BA and outflow tract (PBA/OFT) regulatory microRNAs, and validated interactions between one of these, miR-92b-3p, and two key developmental transcription factors, Gata6 and Tbx20. Our findings place miR-92b-3p into a cardiovascular GRN, whereby it functions in a microRNA-mediated coherent feedforward loop. The work we have presented in this thesis extends our knowledge of BA development into the ncRNA field. We have generated datasets that can be utilised by others in the microRNA field, whilst providing key examples of ncRNA cis-regulatory and trans-regulatory mechanisms during mammalian embryogenesis.
- Embryonic development
- Branchial Arches