January 1, 2018;
microRNAs associated with early neural crest development in Xenopus laevis.
BACKGROUND: The neural crest (NC) is a class of transitory stem cell-like cells unique to vertebrate embryos. NC cells arise within the dorsal neural tube where they undergo an epithelial to mesenchymal transition in order to migrate and differentiate throughout the developing embryo
. The derivative cell types give rise to multiple tissues, including the craniofacial skeleton
, peripheral nervous system and skin
pigment cells. Several well-studied gene regulatory networks underpin NC development, which when disrupted can lead to various neurocristopathies such as craniofrontonasal dysplasia, DiGeorge syndrome and some forms of cancer. Small RNAs, such as microRNAs (miRNAs) are non-coding RNA molecules important in post-transcriptional gene silencing and critical for cellular regulation of gene expression.
RESULTS: To uncover novel small RNAs in NC development we used high definition adapters and next generation sequencing of libraries derived from ectodermal explants of Xenopus laevis embryos induced to form neural and NC tissue
. Ectodermal and blastula
animal pole (blastula
) stage tissues were also sequenced. We show that miR-427 is highly abundant in all four tissue
types though in an isoform specific manner and we define a set of 11 miRNAs that are enriched in the NC. In addition, we show miR-301a and miR-338 are highly expressed in both the NC and blastula
suggesting a role for these miRNAs in maintaining the stem cell-like phenotype of NC cells.
CONCLUSION: We have characterised the miRNAs expressed in Xenopus embryonic explants treated to form ectoderm
, neural or NC tissue
. This has identified novel tissue
specific miRNAs and highlighted differential expression of miR-427 isoforms.
neural crest cell migration
DIGEORGE SYNDROME; DGS
[+] show captions
Fig. 1. Size class distribution of small RNAs shows a bimodal distribution with peaks at 23 nt (miRNAs) and 29 nt (piRNAs and other sRNAs). In neural tissue 16.1% of reads are miRNA. In NC 15% of reads are miRNA. In ectoderm 18% of reads are miRNA. In blastula 5.6% of reads are miRNA
Fig. 2. Analysis of miR-427 compared to other miRNAs across all tissue types shows miR-427 is the primary miRNA expressed. a miR-427 constitutes 60–75% of all miRNA reads in all four tissue types with miR-428 the second most prevalent miRNA in NC, neural and ectoderm only b Five isoforms of miR-427 were expressed all of which showed a similar hairpin distribution over the four tissue types with isoform C the most abundant c Alignments of the five isoforms of Xenopus miR-427 and Zebrafish miR-430 hairpins. The mature 5′ and 3′ sequences are indicated with red boxes. The 3′ strand has strong conservation whilst the 5′ strand does not d Abundance plots show the isoforms of miR-427 are largely derived from changes in the 5′ end of the miRNA except in the blastula where a miR-427 isoform is generated due to changes in the 3′ end of the miRNA
Fig. 3. miRNA expression profiling shows a number of miRNAs are unique to each tissue type. Pie chart analysis shows the distribution of the top 10 most abundant miRNAs in each tissue type a Top 10 blastula miRNA b Top 10 ectoderm miRNAs c Top 10 neural miRNAs d Top 10 NC miRNAs e Heatmap of a selection of miRNAs
Fig. 4. A number of miRNAs are upregulated in NC when compared to neural tissue. a Annotation shows 11 miRNAs (in green) are upregulated in NC when compared to neural tissue. Conversely 2 miRNAs are upregulated in neural tissue when compared to NC (in blue) b Scatter plot using DeSEQ2 shows a total number of miRNAs differentially expressed between NC and neural tissue (upregulated in NC in red, upregulated in neural in green) c Expression of miR-219 is highly expressed in NC with a low expression in neural tissue d Expression of a novel miRNA, miR-nov-12a-a, is highly expressed in NC with a low expression in neural and ectoderm tissue e Expression of 130c is highly expressed in NC with a lower expression in neural, ectoderm and blastula tissue. c-e were performed in triplicate for each tissue type
Fig. 5. miR-301a and miR-338 are expressed in NC and blastula tissue suggesting a role for these miRNAs in maintaining multipotency of NC cells. a Mean read counts of miR-301a and miR-338 in NC and blastula b Enrichment of miR-338 across the four tissue types shows a high expression in NC and blastula c Enrichment of miR-301a across the four tissue types shows a high expression in NC and blastula
Fig. 6. Schematic of the structure of the four mammalian HOX gene clusters, exons numbered in yellow circles, with the location of miR-10a, miR-10b, miR-196a-1, miR-196a-2 and miR-196b located in the introns (grey lines) (Data taken from )
Additional file 1: Figure S1. Validation of tissue induction (A) PCR on RNA extracted from stage 15 animal cap tissue induced to become either neural or NC showed that tissue was induced efficiently. The NC marker Snail2 was only expressed in the NC animal cap tissue whilst the neural marker Sox2 was enriched in the neural tissue and epidermal keratin was enriched in the ectoderm (Ecto) sample. Histone H4 was used as a positive control and Bracyury for a control of mesoderm contamination Whole embryos (WE) were used a positive controls for all genes. (B) WISH for the NC marker Sox10 on stage 15 induced animal caps. This is further confirmation of induction of NC tissue as expression is only evident in NC animal caps.