Determining miRNA Expression Patterns in Xenopus.

Whole-mount in situ hybridization (WISH) is a technique that enables temporal and spatial visualization of RNA molecules in an embryo or whole tissue by using a complementary labelled probe. MicroRNAs are short noncoding RNAs of 20-25 nt in length mainly involved in posttranscriptional regulation of gene expression. In this chapter, we describe how to visualize miRNAs in Xenopus laevis or tropicalis by WISH using two different approaches: LNA-WISH to visualize mature miRNAs and pri-miRNA-WISH to visualize the immature form of miRNAs, the pri-miRNAs.

An Efficient CRISPR-Cas9 Method to Knock Out MiRNA Expression in Xenopus Tropicalis.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly utilized to study gene function. MicroRNAs (miRNAs) are short noncoding RNAs, 20-25 nucleotides long, which affect gene expression through posttranscriptional repression. As miRNAs are so small and due to the limitations of known PAM sequences, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore developed a novel approach using two guide RNAs to effectively "drop out" a miRNA. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of q-RT-PCR and Sanger sequencing. To show specificity of the phenotype, we provide a protocol to use miRNA mimics to rescue the KO phenotype.

The potential role of the mobile and non-coding genomes in adaptive response.

The tightly regulated feedback loops linking small RNAs (sRNAs) and transposable elements (TEs) offer the opportunity for an adaptive response to changing environments at the molecular level. Environmentally induced changes in TE and sRNA profiles may affect expression of coding genes and trigger an organismic and transgenerational response. Understanding this link may provide a mechanistic explanation for how species can adapt to changing climates and may offer novel molecular targets for biomedical and agricultural applications.

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

Characterising open chromatin in chick embryos identifies cis-regulatory elements important for paraxial mesoderm formation and axis extension.

Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior-posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.