Expression and functional analysis of the Wnt/beta-catenin induced mir-135a-2 locus in embryonic forebrain development

Abstract

BackgroundBrain size and patterning are dependent on dosage-sensitive morphogen signaling pathways – yet how these pathways are calibrated remains enigmatic. Recent studies point to a new role for microRNAs in tempering the spatio-temporal range of morphogen functions during development. Here, we investigated the role of miR-135a, derived from the mir-135a-2 locus, in embryonic forebrain development.Method1. We characterized the expression of miR-135a, and its host gene Rmst, by in situ hybridization (ish). 2. We conditionally ablated, or activated, beta-catenin in the dorsal forebrain to determine if this pathway was necessary and/or sufficient for Rmst/miR-135a expression. 3. We performed bioinformatics analysis to unveil the most predicted pathways targeted by miR-135a. 4. We performed gain and loss of function experiments on mir-135a-2 and analyzed by ish the expression of key markers of cortical hem, choroid plexus, neocortex and hippocampus.Results1. miR-135a, embedded in the host long non-coding transcript Rmst, is robustly expressed, and functional, in the medial wall of the embryonic dorsal forebrain, a Wnt and TGFβ/BMP-rich domain. 2. Canonical Wnt/beta-catenin signaling is critical for the expression of Rmst and miR-135a, and the cortical hem determinant Lmx1a. 3. Bioinformatics analyses reveal that the Wnt and TGFβ/BMP cascades are among the top predicted pathways targeted by miR-135a. 4. Analysis of mir-135a-2 null embryos showed that dorsal forebrain development appeared normal. In contrast, modest mir-135a-2 overexpression, in the early dorsal forebrain, resulted in a phenotype resembling that of mutants with Wnt and TGFβ/BMP deficits - a smaller cortical hem and hippocampus primordium associated with a shorter neocortex as well as a less convoluted choroid plexus. Interestingly, late overexpression of mir-135a-2 revealed no change.ConclusionsAll together, our data suggests the existence of a Wnt/miR-135a auto-regulatory loop, which could serve to limit the extent, the duration and/or intensity of the Wnt and, possibly, the TGFβ/BMP pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-016-0065-y) contains supplementary material, which is available to authorized users.

Antagonism between the transcription factors NANOG and OTX2 specifies rostral or caudal cell fate during neural patterning transition

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microRNA profiling of mouse cortical progenitors and neurons reveals miR-486-5p as a novel regulator of neurogenesis

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Long Non-Coding RNA H19 Regulates Human Lens Epithelial Cells Function

“…Previous studies identified a microRNA, miR-135a, whose expression was correlated to the lncRNA RMST. miR-135a was derived from miR-135a-2 locus embedded in RMST [49,50]. Functional enrichment analysis with StarBase software identified 17 pathways [51].…”