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

Background Brain 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. Method 1. 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. Results 1. 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. Conclusions All 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 material The online version of this article (doi:10.1186/s13064-016-0065-y) contains supplementary material, which is available to authorized users.


Background
MicroRNAs are micro-modulators of gene expression, eliciting small changes in the expression of a wide array of targets [1]. In the last ten years, their role in almost every facet of nervous system development and function has been considered including neuronal and glial differentiation, synaptogenesis, and neuro-degeneration [2][3][4][5][6][7][8][9][10]. Yet only recently, some studies have focused on their role in modulating the dosage or duration of the most fundamental developmental moleculesmorphogens [11,12]. Given the exquisite dosage sensitivity of morphogens, an argument has been proposed that these pathways are prime substrates for microRNA micro-management [12].
Wnt and TGFβ/BMP morphogens expressed by the roof plate, or adjacent neuroepithelium, act through signaling cascades implicated in various facets of dorsal neural tube development [13]. Throughout the Central Nervous System (CNS), various studies have revealed a role for Wnt signaling in the expansion of the brain via increases in cell proliferation and survival [14][15][16][17][18].
Other studies have revealed roles in specification of key neuronal progenitor types, as well as in the timing of neurogenesis [16,[19][20][21][22]. Additionally, several studies have suggested that the dosage of the Wnt pathway is critical for normal specification, neurogenesis and differentiation [15,19,23,24]. TGFβ/BMP signaling has also been implicated in proliferation, specification, neurogenesis and gliogenesis [25][26][27][28][29][30][31]. Akin to the Wnt pathway, several studies have suggested that the TGFβ/BMP pathway is exquisitely dosage sensitive [32,33]. Despite an emerging literature on the cross talk between these two key pathways [31,34], the potential nodes of intersection and their net molecular outputs remain to be fully elucidated. It is likely that the molecular synchronization of these pathways is required for dorsal neural tube development.
The cortical hem, positioned adjacent to the hippocampus, between the choroid plexus on the medial side and the cortical neuroepithelium on the lateral side, is a Wnts and TGFβ/BMPs-rich embryonic structure [29,35,36]. The hem has been demonstrated to specify the hippocampus primordium [37][38][39][40][41][42], to serve as a source of Cajal-Retzius cells [43], to be required for choroid plexus formation [16,29,36] and to play a role in regulating the size and patterning of the neocortex [44].
Previously, we identified a microRNA, miR-135a, whose expression was correlated to the long non-coding transcript Rhabdomyosarcoma 2 associated transcript (Rmst). We deduced that miR-135a was derived from mir-135a-2 locus embedded in Rmst and we demonstrated that Rmst and miR-135a are co-expressed in the ventral midbrain, isthmus, as well as dorsal regions of the neural tube [11]. At least in the midbrain, modest and early overexpression of this microRNA yields phenotypes consistent with a reduction of Wnt signaling [11]. Given the potential importance of this microRNA, we have explored its expression, activity and induction in the dorsal forebrain as well as generated mir-135a-2 knockout and overexpressor mice. We reveal that miR-135a is strongly expressed, and is functional, in the medial wall of the telencephalon including the cortical hem and hippocampus primordium, but more weakly expressed in the choroid plexus and in the neocortex. We show that canonical Wnt/beta-catenin signaling is critical for Rmst and miR-135a expression, and also for Lmx1a expression, a key cortical hem determinant. While mir-135a-2 loss of function did not result in appreciable changes in the cortical hem and neocortex sizes or in choroid plexus complexity, its modest over-expression resulted in smaller cortical hem and neocortical domains and also in a less convoluted choroid plexus. All together, our data lead us to conclude that this Wnt induced micro-RNA is a potential modulator of the Wnt and TGFβ/BMP signaling pathways during dorsal forebrain development.

Methods
Nomenclature miRbase uses a 3 or 4 letter prefix to designate the microRNA species, such that 'mmu' refers to the mouse. The un-capitalized 'mir' refers to the pre-microRNA (mmu-mir-135). In this manuscript, we have only investigated the murine mir-135 therefore we have omitted the prefix. Distinct genomic loci that belong to the same family (mir-135) are typically indicated with an additional letter and number such as mir-135a-1, mir-135a-2 and mir-135b. The capitalized 'miR' refers to the mature form (miR-135). mir-135a-1 and mir-135a-2 give rise to only one mature form called mmu-miR-135a-5p. For simplicity, we will refer to the mature form as miR-135a. However, our experiments on mir-135a-2 knockout mice imply that the predominant mature form of miR-135a in the dorsal forebrain is produced from the mir-135a-2 locus.

Mouse lines
Animals were maintained in compliance with National Institutes of Health guidelines. The Northwestern University IACUC approved the protocols for this study. E0.5 designates the morning of the day when a vaginal plug was detected. For beta-catenin gain and loss of function experiments, Ctnnb1 lox(ex3) [45] or beta-catenin floxed mice (Ctnnb1 flox/flox ) [46], were crossed with Emx1::IRES-Cre [47] and embryos were used for in situ hybridization (ish) or RT-qPCR experiments. The miR-135a "sensor" construct was previously described [11]. To evaluate miR-135b expression, we used mir-135b flox/flox mice (Jackson lab) [48], which harbor a LacZ cassette and crossed them to wild type females. E12.5 embryos of the mir-135b flox/+ genotype were stained for Xgal as previously described [49]. To generate the mir-135a-2 knockout mice, we utilized ZFN technology (Sigma). One advantage of this approach is that no selection cassette or residual FRT, or loxP sites, will remain in the intron, and the resultant deletion will be clean. Sixteen different ZFNs were custom designed to bind and cleave the mir-135a-2 locus, within 100 bp upstream or downstream of the stem-loop precursor. The ZFN (GCCATCAGGATAGC nAACTATAGCCTGTGGAC) that demonstrated the highest activity, in an in vitro Mouse Neuro2a cell screen, was chosen for large-scale production and microinjection in mice. The ZFN mRNA was diluted to 2.5 ng/μl in injection buffer and microinjected into early stage FVB embryos. 152 mice were screened with ZFN-F: GGTC CTCGTAGCGAAGAATG and ZFN-R: AATCGGTGGT CAGGAAGATG PCR primers. Five heterozygous mice were identified with one wild type allele and one allele containing a deletion near the mir-135a-2 locus. After sequence analysis, we found that each deletion was unique and ranged from 2 bp to 294 bp. Line #4 had the largest deletion, which removed the entire mir-135a-2 precursor, and was used for the experiments here described. RT-qPCR with TaqMan primers was used to confirm drastic reduction of the mature form miR-135a.

Quantification of cortical tissue and cortical hem area
At E12.5, the length of the neocortex was measured from the pallium-subpallium boundary (PSB), chosen as a landmark, to the cortical hem. Quantification was performed at three levels of the brain, 80 μm apart, along the rostrocaudal axis. At the same stage, we additionally quantified the cortical hem area (Lmx1a+). For all of the measurements, we used ImageJ software (series 1.4, NIH, public domain). Data are expressed as a mean ± the standard error (SEM) (n = 3).

Statistical analysis
To determine statistical significance of our quantification experiments, we first determined if data followed the normal distribution by the Anderson-Darling Test for Normality. All of our data sets had a p value > 0.05, indicating normality. To assess the statistical significance of changes in the cortical hem area and neocortical domain length, the two experimental groups (control and mutant mice) were compared with two samples equal variance, two tailed, Student's t-test. To calculate the relative fold changes in miR-135a and miR-135b expression by RT-qPCR experiments, we applied the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method [51] and normalized our data to microRNA sno202. Unpaired Student's t test was applied to determine statistical significance.

Bioinformatics analysis
To determine miR-135a most predicted targeted pathways, we used the Diana-miRPath, a microRNA pathway analysis web server that combines predicted and validated microRNA targets in CDS or 3'-UTR regions with sophisticated merging and meta-analysis algorithms [52].

Results
Rmst and miR-135a expression and activity in the embryonic forebrain Previously, we identified miR-135a through a screen for microRNAs that were robustly expressed in the Wnt-rich ventral midbrain region of the embryonic Central Nervous System (CNS) [11]. We provided evidence that miR-135a expression was correlated with Rmst, and deduced that miR-135a was derived from mir-135a-2 locus located in the final detected intron of Rmst [11,53]. Since the embryonic dorsal forebrain is known to be a Wnt-rich region and dependent on Wnt signaling [36,54,55], we determined if Rmst and miR-135a were also co-expressed in this region of the CNS. We therefore first characterized Rmst expression at embryonic and adult stages ( Fig. 1 and Additional file 1: Figure S1). In embryonic forebrain sections Rmst is robustly detected in the medial wall of the telencephalon, encompassing a Wnt and TGFβ/BMP-rich signaling center, the cortical hem [35,56]. At E12.5, Rmst was strongly expressed in the dorsal telencephalon in a medial High to dorso-lateral Low gradient (Fig. 1a). Expression was robust in the cortical hem and the adjacent hippocampus primordium. A very weak hybridization signal was detected in the choroid plexus and along the neocortical domain. Rmst was also strongly expressed in the septum, diencephalon, and eminentia thalami. At later stages, Rmst expression was localized to various forebrain nuclei, and the fimbria (Additional file 1: Figure S1). We next performed Locked Nucleic Acid (LNA) ish experiments to detect mature miR-135a, focusing on the embryonic forebrain. We found that this microRNA species, like Rmst, was expressed in the medial wall of the telencephalon in a medial High to dorso-lateral Low gradient (Fig. 1b). Expression was robust in the cortical hem and the adjacent hippocampus primordium. A very weak hybridization signal was detected in the choroid plexus and along the neocortical domain. Some signal was also detected in the LGE, however, since Rmst is not expressed in this region, this signal could represent cross hybridization with a closely related microRNA. Alternatively, in this region miR-135a and Rmst expression could be uncoupled.
To demonstrate that miR-135a is functional in these domains, we generated double transgenic embryos harboring a "sensor" and a control transgene. A "sensor" construct contains a constitutively expressed reporter gene (eGFP), under control of a CAG promoter, and multiple binding sites for miR-135a in the 3' UTR region ( Fig. 1c, cartoon, yellow bars). In cells expressing miR-135a, its perfect complementarity to sequences in the 3' UTR should result in suppression of the eGFP reporter. A control transgene construct contains a tdTomato reporter, but lacks the miR-135a binding sites and, therefore, should be LGE, lateral ganglionic eminences; ncx, neocortex; d, diencephalon. Scale bar 100 μm constitutively active. In E12.5 double transgenic embryos, we found that the eGFP reporter (Fig. 1d), but not tdTomato (Fig. 1e), was selectively reduced in the medial wall of the telencephalon where miR-135a is strongly expressed, as well as in the choroid plexus and in the dorsal neocortical domain, where Rmst and miR-135a are more weakly expressed. eGFP was also not detected in the diencephalon (Fig. 1d). Thus, miR-135a is expressed, and displays activity, in the embryonic dorsal forebrain.

beta-catenin signaling induces Rmst and miR-135a expression
Previously, we reported that in the embryonic midbrain, the transcription factor Lmx1b induces Wnt1/Wnt signaling as well as Rmst and mir-135a-2 locus [11]. Since Lmx1b and Lmx1a are partially redundant, we postulated that Lmx1a might also be a regulator of Rmst and mir-135a-2. However, since, in the dorsal forebrain, Rmst and miR-135a expressions exceed the Lmx1a domain, one possibility is that in addition to Lmx genes, Wnt signaling abets the induction of the Rmst and mir-135a-2 locus. To address this question, we performed gain and loss of function experiments of beta-catenin, a key effector of Wnt signaling pathway [57]. Emx1::Cre mouse line was used to drive recombination throughout the dorsal, but not ventral, forebrain [47]. For gain of function experiments, by conditionally deleting betacatenin exon 3 (Ctnnb1 lox(ex3) ), which encompasses GSK3β phosphorylation sites, we effectively elevated Wnt signaling [23,45]. In mutants (Emx1::Cre;Ctnnb1 lox(ex3) ) ( Fig. 2b), but not in controls (Fig. 2a), Rmst was detected throughout the dorsal forebrain. Conversely, for loss of function experiments, we conditionally removed exons 2-6 of beta-catenin to prevent formation of a functional beta-catenin protein thus impairing Wnt signaling [46]. In mutants (Emx1::Cre;Ctnnb1 cKO) (Fig. 2c), but not in controls (Fig. 2a), we observed drastic loss of Rmst expression throughout the dorsal forebrain and in the remaining cortical hem tissue (Fig. 2c, asterisk). miR-135a expression levels, quantified by RT-qPCR on E12.5 dissected dorsal telencephalon, were strongly induced in Emx1::Cre;Ctnn-b1 lox(ex3) mutants, and reduced in Emx1::Cre;Ctnnb1 cKOs ( Fig. 2d). Overall, these data demonstrate that betacatenin signaling is necessary and sufficient for Rmst and miR-135a expression in the dorsal forebrain.

beta-catenin signaling is necessary for Lmx1a expression
We also determined if Lmx1a, a key cortical hem determinant [58], is a target of Wnt/beta-catenin signaling in the forebrain, as in other brain regions [11,22,23]. While in Emx1::Cre;Ctnnb1 lox(ex3) embryos (Fig. 2f), we did not observe a drastic change in the cortical hem size (Lmx1a+, Fig. 2f and Wnt3a+, Additional file 2: Figure S2) or in Lmx1a expression (Fig. 2f) with respect to controls (Fig. 2e), in Emx1::Cre;Ctnnb1 cKOs, we observed a drastic reduction of Lmx1a signal in the remaining cortical hem tissue (Fig. 2g, asterisk). Lmx1a was detected in the choroid plexus of both mutants ( Fig. 2f and g). These results suggest that Wnt/beta-catenin signaling is necessary, but not sufficient, for Lmx1a expression in the dorsal forebrain. Given that Lmx1a in part functions to repress Lhx2 [58], a negative regulator of the hem [39], Wnt/beta-catenin induction of Lmx1a is likely to be an important event in the cortical hem establishment and/or maintenance. miR-135a is predicted to target the Wnt and TGFβ/BMP pathways, but loss of function does not affect dorsal forebrain development To begin to elucidate miR-135a functions, we performed bioinformatics analysis to determine the most common pathways targeted by miR-135a. To do that, we took advantage of the Diana-miRPath software [52], which Fig. 2 Rmst, miR-135a and Lmx1a are altered in beta-catenin mutants. a-c Rmst expression in E12.5 wild type control (a), Emx1::Cre;Ctnnb1 lox(ex3) (b) and Emx1::Cre;Ctnnb1 cKO mutant (c) brains. d Quantification of miR-135a expression levels by RT-qPCR on E12.5 dissected dorsal telencephalon of control (wild type), Emx1::Cre;Ctnnb1 lox(ex3) and Emx1::Cre;Ctnnb1 cKO mutants (n = 3). miR-135a fold induction was normalized to microRNA sno202. e-g Lmx1a expression in E12.5 controls (e) and beta-catenin mutant (f and g) brains. Asterisks in c and g highlight remaining cortical hem tissue in Emx1::Cre;Ctnnb1cKO mutants. ***, p value <0.001; **, p < 0.01; *, p < 0.05 cp, choroid plexus; ncx, neocortex; LGE, lateral ganglionic eminences; d, diencephalon. Scale bar 100 μm utilizes predicted, and validated, microRNA targets to perform a hierarchical clustering of microRNA and pathways based on their interactions. We found that Wnt and TGFβ/BMP signaling pathways are among the top pathways targeted by miR-135a with an extremely high statistical significance (p value of approximately 2.9E-07 and 3.6E-10, respectively) (Additional file 3: Figure S3A). It is worth noticing that the genes targeted by miR-135a in the TGFβ/BMP (Additional file 3: Figure S3B) and Wnt pathways (Additional file 4: Figure S4) include ligands, receptors and downstream transcriptional regulators suggesting that this microRNA likely acts through multiple levels of the Wnt and TGFβ/BMP cascades to modulate the outcome of their signaling.
To determine the role of miR-135a in embryonic dorsal forebrain development, we generated a mouse line in which mir-135a-2 was deleted by pronuclear injection of a specific Zn finger nuclease ( Fig. 3a and b), designed to cleave <100 bp from the mature sequence. Of~60 pups examined, 1 harbored a~294 bp deletion ( Fig. 3c and d) and was used to generate a line for the experiments here described. RT-qPCR on dissected E12.5 dorsal forebrain tissue confirmed a drastic reduction in miR-135a expression levels (Fig. 3e). We thus deduced that, in the dorsal forebrain akin to the ventral midbrain, miR-135a is predominantly produced from mir-135a-2 locus.
Because a growing literature has described interactions between microRNAs and long non-coding RNAs (lncRNAs) as a novel mechanism to regulate gene expression as well as microRNA function [59][60][61], we investigated the possibility of an interaction between Rmst and miR-135a. A search on miRcode web site, which represents a comprehensive map of putative microRNA target sites across the GENCODE long non-coding transcriptome [62], indicated lack of miR-135a responsive elements in Rmst. To confirm the bioinformatics prediction, we performed ish for Rmst on E12.5 coronal sections of mir-135a-2 knockout mice and we did not observe any change in its expression ( Fig. 4a and b). These data seem to therefore exclude the possibility of Rmst being a potential target of miR-135a. Next, we performed in situ hybridization with Lmx1a ( Fig. 4c and d), a marker for the cortical hem and the choroid plexus to investigate any possible change in proper development of these Wnt and TGFβ/BMP responsive domains. We did not, however, observe any apparent change in the cortical hem size (Fig. 4d) as was also confirmed by quantification analysis at three levels of the forebrain, equally spaced along the rostro-caudal axis (referred to as Rostral, Mid and Caudal) (Fig. 4e). No difference was detected in the extent of the cortical domain (Fig. 4d), which was again measured at three levels of the forebrain, equally spaced along the rostro-caudal axis (Rostral, Mid and Caudal) from the inflection that marks the pallium-subpallium boundary (PSB), chosen as a landmark, to the cortical hem (Fig. 4f). No change in the choroid plexus complexity was observed (Fig. 4d).
The cortical hem is a signaling center known to induce and pattern the adjacent hippocampus [38,41]. We therefore performed in situ hybridization with neuronal marker NeuroD2 to assess any morphological changes in the hippocampal complex at post-natal and adult stages of mir-135a-2 knockouts. No changes were observed (Fig. 4g-j). These findings clearly demonstrate that, at least by these criteria, mir-135a-2 loss of function does not alter forebrain development.
microRNAs often display redundancy with family members [63,64]. To determine whether miR-135b, a closely related microRNA was expressed in a similar domain, we obtained mice in which LacZ had been inserted into the mir-135b locus. In E12.5 whole mount   (Fig. 5a) and forebrain sections (Fig. 5b and c), we did not observe any LacZ staining in the dorsal forebrain ( Fig. 5a and b) whereas we found LacZ expression in cells emanating from the ganglionic eminences in the ventral forebrain (Fig. 5c). Consistent with this, RT-qPCR experiments for miR-135b on wild type dorsal and ventral forebrain dissected tissues, revealed a ventral enrichment of this microRNA (Fig. 5d). These data suggest that miR-135b does not serve a redundant function in the dorsal forebrain.

Early mir-135a-2 overexpression affects dorsal forebrain development
To complement our loss of function results, we took advantage of previously generated transgenic mice to conditionally express mir-135a-2 for gain of function experiments. This time, we reasoned that if the Wnt/miR-135a circuitry identified in the midbrain [11] is functionally conserved in the forebrain, mir-135a-2 overexpression might result in Wnts related phenotypes. To test this hypothesis, mice in which mir-135a-2 precursor expression is under control of a CAG promoter [11] (henceforth mir-135a-2OE), were crossed with Emx1::Cre line [47] to overexpress mir-135a-2 throughout the Emx1 domain of the dorsal, but not ventral, forebrain as early as E9.5 [43]. RT-qPCR on dissected E12.5 dorsal forebrain tissue confirmed that miR-135a expression levels, in Emx1::Cre;mir-135a-2OE mutants, were 1.5 fold more than in controls (Additional file 5: Figure S5). In such mutants we observed a clear reduction in the size of the cortical hem (Wnt3a+, Wnt8b+) and the hippocampus primordium (Wnt8b+) (Fig. 6a-d). Additionally, we observed a reduction in size of the neocortical domain ( Fig. 6a-b, dashed  lines), a phenotype previously shown in mice with genetic ablation of the cortical hem [44,65], but not reported in BmpRIA/IB cKOs which display a smaller cortical hem [42]. The choroid plexus, normally specified by TGFβ/ BMPs [29,42] and demarcated by rTtr1, was overall less (See figure on previous page.) Fig. 4 mir-135a-2 loss of function characterization. a-b E12.5 coronal sections of control (mir-135a-2 +/-) and mir-135a-2KO brains (mir-135a-2 -/-) processed for ish for Rmst showing no change in its expression. c-d ish for Lmx1a in control (c) and mir-135a-2KO (d) brains. Dashed lines highlight the cortical hem area and the neocortical domain size. e Quantification of the cortical hem area (μm 2 ± SEM) along the rostro-caudal axis (n = 3). f Quantification of the neocortex length (μm ± SEM) from the PSB to the hem at the same levels (n = 3). g-j ish for NeuroD2 on coronal sections of post-natal P1 (g-h) and adult (i-j) control and mutant brains showing no change in hippocampus morphology (boxed in g and h). ch, cortical hem; cp, choroid plexus; hp, hippocampus; PSB, pallium-subpallium boundary; ncx, neocortex; CA1, CA3, hippocampus fields; DG, dentate gyrus. Scale bar 100 μm in panels a-d, 400 μm in panels g and h; 200 μm in panels i and j  Figure S6). Altogether, Emx1::Cre;mir-135a-2OE mutants show characteristics previously described in mice deficient for Wnt or TGFβ/ BMP activity, or in which the cortical hem has been genetically ablated [16,29,42,44,65,66], although they display a milder phenotype.
Next, we quantified the cortical hem area along the rostro-caudal axis. We estimated a reduction in its size of~34 % at rostral level,~38 % at mid level and~18 % at caudal level (Fig. 6e) in mutants compared to controls. Finally, we quantified the extent of the neocortical domain and found a significant reduction in size of the mutant neocortices in comparison to controls (Fig. 6f ). Since the cortical hem and the hippocampus primordium are reduced in Emx1::Cre;mir-135a-2OE embryos, these mutants showed a significant reduction of all hippocampal structures (CA fields and dentate gyrus) from postnatal stage P1 (Additional file 7: Figure S7, A-B and E) to adulthood (Additional file 7: Figure S7C and D) when compared to controls.
Finally, we examined the expression of several bioinformatically predicted miR-135a targets. Of these, only phospho-Smad (1/5/8) and Msx2 revealed consistent changes, showing apparent reduction in their level and expression domain extent (Additional file 8: Figure S8). Their reduction might be due to direct repression, overall net down regulation of these pathways, or both.
mir-135a-2 overexpression in mir-135a-2 +/mice does not result in forebrain abnormalities To further demonstrate the specificity of our results, we removed one copy of endogenous mir-135a-2 from embryos conditionally overexpressing mir-135a-2. We reasoned that if the phenotype observed in Emx1::Cre;mir-135a-2OE mutants is due to mir-135a-2 overexpression, then, removal of one copy of the endogenous mir-135a-2 should alleviate this phenotype. E12.5 embryos of Emx1::Cre;mir-135a-2OE;mir-135a-2 +/-(mutants) and Emx1::Cre;mir-135a-2 +/-(controls) genotypes were analyzed for the expression of Lmx1a ( Fig. 7a and b) and Wnt3a (Additional file 10: Figure S10). The cortical hem area and the extent of the cortical domain were quantified as previously described ( Fig. 7c and d). No alterations in these domains and in the choroid plexus were observed, suggesting that normal miR-135a levels are important for embryonic forebrain development.

Discussion
From the present study we deduced six prominent conclusions. First, miR-135a, and Rmst, are expressed and functional in the embryonic dorsal forebrain. Second, in the dorsal forebrain, mature miR-135a is predominantly derived from the mir-135a-2 locus. Third, miR-135a is dispensable for forebrain development. Fourth, modest mir-135a-2 overexpression, within an early but not late time window, results in a phenotype consistent with Wnt and TGFβ/BMP signaling deficitsa reduced cortical hem, hippocampus primordium and neocortex, and a less convoluted choroid plexus. Fifth, Wnt/beta-catenin pathway is necessary and sufficient to induce Rmst and miR-135a expression pointing to 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 pathway. Finally, Wnt/beta-catenin pathway is also necessary Fig. 7 mir-135a-2OE in mice heterozygous for endogenous mir-135a-2. a-b ish showing Lmx1a expression in E12.5 control (Emx1::Cre;mir-135a-2 +/-) (a) and mutant (Emx1::Cre;mir-135a-2OE;mir-135a-2 +/-) (b) brains. Dashed lines highlight the cortical hem area and the neocortical domain size. c Quantification of the cortical hem area (μm 2 ± SEM) along the rostro-caudal axis (n = 3). d Quantification of the neocortex length (μm ± SEM) from the PSB to the hem at the same levels (n = 3). ch, cortical hem; cp, choroid plexus; PSB, pallium-subpallium boundary; ncx, neocortex. Scale bar 100 μm in panels a and b for Lmx1a expression, a key cortical hem determinant. A model of these interactions is depicted in Fig. 8.
Several studies have suggested that the dosage of the Wnt and TGFβ/BMP pathways is critical and has to be tightly controlled through intricate networks of positive and negative feedback loops [15,19,23,32,33]. It is therefore challenging to understand how these pathways are modulated in time and space during embryonic development, how cells receive and integrate multiple signals and whether potential nodes of intersection exist. Recently, it has been demonstrated that microRNAs contribute to gene networks that transform the graded activity of a morphogen in robust cell fate decisions by establishing contextdependency, threshold responses and sharpening temporal and spatial expression patterns [4,12,72]. miR-135a and its host long non-coding transcript, Rmst, were shown to be expressed and functional in the embryonic Wnt-rich domain of the midbrain [11]. Here, we have shown that miR-135a and Rmst are also co-expressed in the Wnt and TGFβ/BMP-rich domains of the embryonic dorsal forebrain suggesting a correlation between this microRNA, the Wnts and TGFβ/BMPs-rich domains across the embryonic CNS. Interestingly, akin to several Wnts and TGFβ/BMPs, strong expression of Rmst in the embryonic hippocampal primordium declines over time and, at post-natal stages, become restricted to the fimbria and virtually undetectable in the adult hippocampus. Rmst expression has also been detected in human fetal cortical radial glia, suggesting a conserved role for this locus [73]. Taking together, the Rmst/miR-135a expression pattern in mice and humans, the finding that Wnt signaling is indeed able to induce their expression and the strong bioinformatics predictions, we postulate that this microRNA might play a role in finetuning the Wnt pathway and, possibly, the TGFβ/BMP pathway.
Embryos lacking Wnt3a [16], functional LEF1 [66], or with genetic ablation of the cortical hem [44,65], display loss of the hippocampus and shrinkage of the neocortex [44,65], while mice with disrupted TGFβ/BMP signaling fail to develop or properly differentiate the choroid plexus [29]. Modest mir-135a-2 overexpression appears to recapitulate these phenotypes, albeit in a milder fashion. Coupled with the findings that this microRNA is bioinformatically predicted to target both positive and negative regulators in the Wnt and TGFβ/BMP pathways, the Wnt related phenotypes observed in the midbrain [11], and proven interactions between miR-135a and targets like GSK, Tcf7l2, Ccnd1 and APC in heterologous systems or cancer cell lines [11,[74][75][76][77][78], we posit that miR-135a modulates the Wnt and TGFβ/BMP signaling cascade in the developing forebrain.
mir-135a-2 loss of function embryos did not display overt forebrain phenotypes, at least by the criteria that we assayed. One plausible explanation comes from the observation that members of a microRNA family are often predicted to target the same or overlapping sets of Fig. 8 Schematic representation of interactions between Lmx1 genes, the Wnt pathway and Rmst/mir-135a-2 [miR-135a]. a In the embryonic forebrain, canonical Wnt/beta-catenin pathway induces Rmst/mir-135a-2 [miR-135a], as well as the cortical hem determinant Lmx1a. miR-135a, in turn, negatively targets Wnt pathway mRNAs establishing an auto-regulatory loop. miR-135a is also predicted to target TGFβ/BMP pathway mRNAs as well as other targets. It is possible that the TGFβ/BMP pathway is also able to induce Rmst/mir-135a-2 [miR-135a] and establish a miR-135a/TGFβ/BMP auto-regulatory loop. It is also possible that Lmx1a contributes to Rmst/mir-135a-2 [miR-135a] expression. However, these interactions remain to be demonstrated. Ultimately, this complex network of positive and negative interactions plays a role in determining proper dorsal forebrain size (cortical hem, hippocampus primordium, choroid plexus and neocortex). b A similar scenario was previously identified in the embryonic midbrain [11] where Lmx1b drives the expression of Wnt1/Wnt pathway and of Rmst/mir-135a-2 [miR-135a], which in turn negatively modulates the levels of Lmx1b, Wnt1/Wnt pathway and other targets. Additionally, Wnt1/Wnt pathway and Lmx1a interactions have been demonstrated. All together these interactions are critical for midbrain dopamine progenitor pool patterning and expansion genes and therefore to act in a functionally redundant manner [63]. Supporting this idea, single microRNA loss of function or single microRNA silencing, through antisense oligonucleotides and sponge techniques, typically results in subtle or no phenotypes [64,[79][80][81]. Because mir-135b was expressed in the embryonic ventral forebrain, we ruled out any compensatory effect due to this microRNA. We considered the possibility that the other member of this family, mir-135a-1 might be expressed in a similar and overlapping pattern. Because mir-135a-1 and mir-135a-2 produce an identical mature form, it is not possible to analyze their differential expression by standard techniques. However, RT-qPCR experiments for mature miR-135a on dorsal forebrain tissue of mir-135a-2 null embryos revealed greatly reduced expression levels. Therefore, we concluded that the main miR-135a mature form in the dorsal forebrain could be attributed to mir-135a-2 precursor. Redundancy, on the other hand, can either be attributed to very low levels of miR-135a produced by the mir-135a-1 precursor or to some other microRNA having a similar seed sequence. This latter, more likely, possibility has been raised in the recent literature [82].
Identifying regulators of the Wnt pathway has been highlighted as an important goal [83]. miR-135a is up regulated in several tumor types characterized by high levels of Wnt/beta-catenin signaling, including colorectal tumors as well as certain subtypes of medulloblastomas [74,76]. It is possible that, in these tumors, Wnt signaling, in accordance with our data in the forebrain, induces miR-135a. If so, miR-135a would serve as a useful marker for aberrant Wnt signaling in these tumors, and possibly others, and in therapeutic protocols designed to circumscribe unrestrained Wnt signaling. In colorectal cancers, miR-135a has been proposed to act as a positive regulator of Wnt signaling by targeting APC, a key molecule of the beta-catenin destruction complex [74]. Thus, while it is emerging that this microRNA is intimately correlated with Wnt signaling in the embryonic CNS ( [11]and this work), in tumors [74][75][76] and in other cell types with high Wnt signaling [77,78], it is possible that the net effect of miR-135a depends on the physiological milieu. Elucidating the targets may reveal how a single microRNA, predicted to target both positive and negative regulators of a signaling pathway, may have distinct outcomes depending on the context. Ultimately, an array of biochemical and genetic approaches will be required to accurately define direct miR-135a targets and the molecular underpinnings of mir-135a-2 mutant phenotypes.
Finally, our study has highlighted a role for Wnt/ beta-catenin signaling in the expression of cortical hem determinant Lmx1a, a LIM-homeodomain transcription factor. Wnt1/Wnt signaling has been shown to form an autoregulatory loop with Lmx1a to control dopaminergic neurons differentiation in the embryonic ventral midbrain [22,23,84,85]. In the dorsal embryonic forebrain, Lmx1a is expressed in the cortical hem, a Wnt-rich region. While Lmx1a is not required for cortical hem induction, it is critical for proper regulation of cell fate decisions [58]. In the absence of Lmx1a, the hippocampal selector gene Lhx2 is ectopically expressed in the cortical hem leading to excessive production of hippocampal cells and decreased production of Cajal-Retzius cells [58]. Here, through gain and loss of function experiments of beta-catenin, we show that, in the dorsal forebrain akin to the ventral midbrain [22,24], Wnt/beta-catenin signaling is necessary, but not sufficient, for Lmx1a expression. Wnt/beta-catenin induction of Lmx1a is likely to be a key event in the proper cortical hem cell fate establishment and/or maintenance.

Additional files
Additional file 1: Figure S1. Rmst expression during forebrain development. (A-F) Rmst ish on coronal sections of wild type brains from E11.5 to adult stage. (A) At E11.5, Rmst is expressed in the epithalamus, medial wall of the dorsal telencephalon, the eminentia thalami, and the septum. (B) At E12.5 (as in Fig. 1), Rmst was detected in the medial wall of the telencephalon and in the diandephalon. (C) At E14.5, Rmst is still highly expressed in the cortical hem and hippocampus primordium, thalamus and hypothalamus. (D) At E16.5, Rmst expression is high in the hippocampus and fimbria but low in scattered cells of the migratory stream. Expression in the thalamus is maintained. (E) At post-natal stages P1, Rmst is restricted to the fimbria, yet expressed outside of the hippocampal formation to become virtually undetectable in the hippocampus of adult mice (F) (hippocampal staining is not considered specific to the Rmst probe). dm, dorso-medial wall of telencephalon; emT, eminentia thalami; Et, epithalamus; tel, telencephalon; v3, third ventricle; lv, lateral ventricle; Sp, septum; d, diencephalon; hp, hippocampus; ch, cortical hem; cp, choroid plexus; ncx, neocortex; LGE, lateral ganglionic eminences; th, thalamus, hy, hypothalamus; fm, fimbria; ms, migratory stream; CA1 and CA3, hippocampal fieds; DG, dentate gyrus. Scale bar 400 μm in panels A, Additional file 3: Figure S3. miR-135a bioinformatics analysis. A) Top pathways targeted by miR-135a. TGFβ/BMP and Wnt signaling pathways rank at position number 2 and 3 with high statistical significance. P values and number of genes targeted in each pathway are indicated. B) Overview of TGFβ/BMP cascade with highlighted, and listed, 16 genes targeted by miR-135a. It is worth noticing that the number of listed genes reflects only the data available on the Diana web site and the algorithm used for the search, as a higher number of putative miR-135a targets have already been reported using multiple search engines [11]. (TIF 1865 kb) Additional file 4: Figure S4. miR-135a targets several mRNAs in the Wnt pathway. Schematic representation of miR-135a predicted targets in the Wnt signaling pathway, as generated by the Diana-miRPath software [52]. The 22 genes predicted to be miR-135a targets are highlighted and listed. As for the TGFβ/BMP pathway, the number of genes here listed reflects the data available on the Diana web site and the algorithm used for the search. (TIF 1741 kb) Additional file 5: Figure S5. Quantification of miR-135a expression in Emx1::Cre;mir-135a-2OE mice. RT-qPCR showing a 1.5 fold change in miR-135a expression level in E12.5 dissected dorsal forebrain tissue from mutant mice compared to controls (n = 3). Data are shown as a fold change and have been normalized to microRNA sno202. ***, p value <0.001. (TIF 145 kb) Additional file 6: Figure S6. Early mir-135a-2 overexpression affects choroid plexus development. (A-B) Coronal sections of E12.5 control (mir-135a2-OE) and mutant brains (Emx1::Cre;mir-135a-2OE) showing expression of choroid plexus specific marker rTtr1. The dashed red line is used to highlight the change in choroid plexus complexity. cp, choroid plexus. Scale bar 100 μm. (TIF 2429 kb) Additional file 7: Figure S7. Early mir-135a-2 overexpression affects hippocampus development. (A-D) Coronal sections of post-natal stage P1 and adult brains processed for ish for NeuroD2. Panels A, B show overall morphology and size of the hippocampus. Asterisks point towards the pallium-subpallium boundary. Insets show the extent of the hippocampus from the CA1 field to the tip of the dentate gyrus, quantification of which, at mid and caudal level of the brain, is reported in panel E. Length is expressed in μm ± SEM (n = 4). Consistent with a reduced cortical hem size at embryonic stages, the hippocampus was significantly reduced in its extent in mutant brains with respect to controls. ***, p <0.001. Abbreviations: pSub, para subiculum; Sub, subiculum; DG, dentate gyrus; Cng, cingulate cortex. Scale bar 400 μm in panels A and B; 200 μm in panels