Identification of microRNAs and microRNA targets in Xenopus gastrulae: The role of miR-26 in the regulation of Smad1.

MicroRNAs (miRNAs) are known to play diverse roles in the regulation of vertebrate development. To investigate miRNA-target mRNA relationships in embryonic development, we have carried out small-RNA sequencing to identify miRNAs expressed in the early gastrula of Xenopus laevis. We identify a total of 180 miRNAs, and we have identified the locations of the miRNA precursor sequences in the X. laevis genome. Of these miRNAs, 141 represent miRs previously identified in Xenopus tropicalis. Alignment to human miRNAs led to the identification of 39 miRNAs that have not previously been described for Xenopus. We have also used a biochemical approach to isolate mRNAs that are associated with the RNA-Induced Silencing Complex (RISC) in early gastrulae and thus candidate targets of miRNA-dependent regulation. Interrogation of this RISC-associated mRNA pool by RT-PCR indicates that a number of genes essential for early patterning and specification may be under regulation by miRNAs. Smad1 transcripts are associated with the RISC; target prediction algorithms identify a single miRNA-binding site for miR-26, which is common to the 3'UTRs of Smad1a and Smad1b. Disruption of the interaction between miR-26 and the Smad1 3'UTR via a Target Protector Morpholino Oligonucleotide (TPMO) leads to a 2-fold increase in Smad1 protein accumulation, moderate increases in the expression of BMP4/Smad1 target genes, and a reduction in organizer gene expression, as well as a partially ventralized phenotype in approximately 25% of embryos. Overexpression of miR-26 resulted in moderately decreased expression of Smad1-dependent genes and an expansion of the region expressing the Organizer gene not1. Our findings indicate that interactions between miR-26 and the Smad1 3'UTR modulate Smad1 function in the establishment of axial patterning; they also establish a foundation for the functional analysis of miRNAs and their regulatory interactions during gastrulation.

TAK1 promotes BMP4/Smad1 signaling via inhibition of erk MAPK: a new link in the FGF/BMP regulatory network.

FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-ß Activated Kinase 1 (TAK1) in the maintenance of Smad1 activity in Xenopus embryos, via the inhibition of erk MAPK. Up- or downregulation of TAK1 levels produces an inverse alteration in the amount of activated erk MAPK. The inhibition of erk MAPK by TAK1 is mediated by p38 and a corresponding decrease in phosphorylation of MEK. TAK1 morphant embryos show a decrease in the nuclear accumulation of Smad1. Conversely, reduction of erk MAPK activity via overexpression of MAP Kinase Phosphatase1 (MKP1) leads to an increase in nuclear Smad1. Both TAK1 morphant ectoderm and ectoderm treated with FGF show a decrease in the expression of several Smad1-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm coexpressing noggin and TAK1, suggesting that TAK1 is sufficient to inhibit neural specification. Introduction of TAK1 morpholino oligonucleotide expands the expression of organizer genes, disrupts formation of the boundary between organizer and non-organizer mesoderm, and increases the spatial range of MAPK activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4 effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.