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Formation of the head during vertebrate embryogenesis has been a hot topic in developmental biology since the discovery of the head organizer by Spemann and Mangold (Spemann, 1924). Embryological and genetic evidence indicates that vertebrate head induction requires the concerted inhibition of Nodal, Wnt, and bone morphogenetic protein (BMP) signaling (Piccolo et al., 1999). During anterior–posterior (AP) patterning, the Spemann organizer produces a group of factors that inhibit the posteriorizing effects of Wnt and BMP signaling (Glinka et al., 1997). This so-called “Two Inhibitor Model” proposes that inhibition of both pathways is responsible for the regional specification of vertebrate head induction.
RNA binding proteins (RBPs) are proteins containing one or more RNA binding domain, the most common being the RNA recognition motif (RRM) (Lunde et al., 2007). RBPs are gene regulators required throughout early vertebrate development. They exert their effects through interactions with gene transcripts, thus modulating their activity. There are a multitude of mechanisms through which RBPs can regulate gene expression (Colegrove-Otero et al., 2005). These effects may be exerted at all levels of posttranscriptional regulation: nonsense-mediated decay (e.g., UPF3; Ruiz-Echevarria et al., 1998), splicing (e.g., U2AF; Ruskin et al., 1988), and alternative splicing (e.g., hnRNPA1; Allemand et al., 2005), mRNA stability (e.g., HuD; Lazarova et al., 1999), RNA editing (e.g., ACF; Dance et al., 2002), RNA localization (e.g., HuR; Gallouzi et al., 2001), pre-rRNA complex formation (e.g., Nucleolin; Chen et al., 2012), and translation (e.g., PABP; Tarun and Sachs, 1996).
RBPs are important regulators during development of various organs, and tissues including germ cells, heart, and ear (Jiang et al., 1997; Beck et al., 1998; Gerber et al., 2002; Rowe et al., 2006). Several RBPs have been identified for their roles in neural development. For example, Vg1-RBP, expressed in embryonic and neoplastic cells, is required for the migration of cells forming the roof plate of the neural tube, and plays essential roles in neural crest migration (Yaniv et al., 2003). Quaking homolog, also known as KH domain RNA binding (QKI), regulates distinct mRNA targets to promote oligodendrocyte differentiation and myelin formation, which is associated with schizophrenia (Bockbrader and Feng, 2008). Depletion of cold-inducible RNA binding protein (CIRP), a Xenopus transcription factor 3 (XTcf-3)-specific target gene, by antisense morpholino oligonucleotide injection leads to an enlargement of the anterior neural plate (van Venrooy et al., 2008). There is emerging evidence to suggest the importance of RBPs in head development. For example, the putative RBP cellular nucleic acid binding protein (CNBP) controls neural crest cell expansion during rostral head development by affecting levels of cellular proliferation and apoptosis as well as fate determination (Weiner et al., 2011).
RBM proteins possess one or more RRMs, highly conserved RNA interaction motifs consisting of a four-stranded antiparallel β-sheet packed against two α-helices (Nagai et al., 1990). By regulating posttranscriptional processes, RBMs are capable of functioning through diverse mechanistic pathways. For example, RBM4, possessing two RRMs and a CCHC-type zinc finger, functions in several cellular processes including alternative splicing of pre-mRNA, translation, and RNA silencing (Lin and Tarn, 2005; Kar et al., 2006; Markus et al., 2006; Markus and Morris, 2006, 2009; Lin et al., 2007). RBM5, which contains 2 RRMs, is a modulator of apoptosis (Mourtada-Maarabouni and Williams, 2002). Some RRM domains are capable of protein–protein interaction, such as in the RBM protein heterogeneous ribonucleoprotein A1 (hnRNPA1), whose first RRM domain interacts with the cap region of topoisomerase I through a hydrophobic pocket on its β-surface, and thus may be involved in DNA relaxation (Trzcinska-Daneluti et al., 2007). Several RBMs appear to be important for vertebrate development. RBM19 is reported to play a role in digestive organ development in zebrafish (Mayer and Fishman, 2003) and preimplantation development in mice (Lorenzen et al., 2005; Borozdin et al., 2006; Zhang et al., 2008). RBM24a and b are involved in vasculogenesis, early angiogenesis, and vascular maintenance in the developing zebrafish (Maragh et al., 2011).
RNA Binding Motif Protein 47 (RBM47) (aka Ribonucleoprotein-47, NCBI Accession #AF262323) is an uncharacterized, putative RBP. In the current study, we have characterized human and zebrafish RBM47 (Rbm47), and explored its role in zebrafish embryonic development, demonstrating that it plays a pivotal role in head formation and early embryonic patterning through a pathway involving Wnt8a signaling.
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In the present study, we have characterized human and zebrafish RBM47 (Rbm47), and investigated its role as a putative RNA binding protein involved in zebrafish embryogenesis. RBPs mediate their effects by altering posttranscriptional events of specific gene transcripts. RBPs interact with target transcripts through RNA binding domains, such as RNA Recognition Motifs. Zebrafish Rbm47 possesses three RRMs with high homology to those found in the human orthologue. We found that human rbm47 strongly interacts with poly-A, -C, and -U RNAs, while binding with poly-G RNA occurs with low affinity, demonstrating its ability to bind to RNA. rbm47 is not capable of interaction with ssDNA. Additionally, we created an rbm47-GFP fusion protein to determine its subcellular localization, which was found to be within the HeLa cell nuclei. Based on this information, we propose that rbm47 is a novel RNA Binding Protein.
To characterize the spatiotemporal expression of rbm47, whole mount in situ hybridization for rbm47 mRNA was performed on developing zebrafish embryos. rbm47 is expressed ubiquitously throughout early embryonic development. To study its function during zebrafish development, we used a morpholino-based knockdown approach to target either the rbm47 translation start codon or exon-1/intron-1 splicing. We demonstrated that a high percentage of rbm47 knockdown embryos had incomplete head formation or total loss of head development. This striking phenotype was rescued upon co-injection of rbm47 mRNA, supporting our conclusion that defective head development is a consequence of rbm47 knockdown, and that rbm47 expression is required for normal head development.
Vertebrate head induction requires the concerted inhibition of both Wnt and BMP signaling pathways (Glinka et al., 1997; Piccolo et al., 1999). Indeed, the headless phenotype as a consequence of single gene knockdown is an important observation that has only be seen as a result of altering the regulation of a small set of master regulatory genes involved in early vertebrate development, including foxA3 and gsc (Yao and Kessler, 2001; Seiliez et al., 2006), tcf3 (Kim et al., 2000), and dkk1 (Glinka et al., 1998). Wnt8 is the key transcriptional motivator to act on the anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the central nervous system (Erter et al., 2001). The graded Wnt8 activity mediates overall neuroectodermal posteriorization and thus determines the location of the midbrain–hindbrain boundary organizer (Rhinn et al., 2005). Wnt8 expression is inhibited within the organizer, but is found in the lateral margin of the zebrafish gastrula (Kim et al., 2000). Thus, excess Wnt8 activity due to overexpression or loss of inhibition leads to loss of anterior structure. Our observation that morpholino knockdown of rbm47 causes headless and reduced head phenotypes suggests that it may act through a pathway involving Wnt8.
In investigating Rbm47's mechanistic pathway, we used microarray analysis to screen the expression levels of 15,619 zebrafish genes from rbm47 MO-knockdown embryos at 75% epiboly. We found 92 genes with increased expression in both splice- and translation-blocked knockdown groups, compared with MO-control embryos, with 20 genes having a minimum four-fold increase. epcam was identified as the most up-regulated gene by this screen. epcam regulates cell adhesion, integrity, plasticity and morphogenesis as a partner of E-cadherin during zebrafish epiboly and skin development (Slanchev et al., 2009). Two Tcf-binding elements were identified in the epcam promoter and epcam was found to be a Wnt-β-catenin target gene in hepatocellular carcinoma cells (Yamashita et al., 2007). These findings support the idea that rbm47's effect on head development occurs through the canonical Wnt8 signaling pathway (Lu et al., 2011).
In accordance with this, rescue experiments demonstrated that a wnt8a-blocking morpholino can partially rescue the rbm47 knockdown phenotype (Fig. 2D). In addition to epcam, several other genes involved in Wnt signaling were up-regulated, including gsk3a, otx2, and chordin. This further supports our conclusion that the effect of rbm47 knockdown on head development occurs through an overactive Wnt pathway.
Meanwhile, of the genes with decreased expression, 26 exhibited a minimum four-fold expression reduction by microarray analysis, with a2ml being the most severely affected. The qRT-PCR results confirmed reduced gene expression of a2ml in rbm47 knockdown embryos. A2M, the human homologue of zebrafish a2ml, is a plasma protease inhibitor, cytokine carrier, and ligand for cell-signaling receptors (Roberts, 1985). A2M in the human and rat brain is an acute-phase protein synthesized primarily by astrocytes, and is associated with Alzheimer's disease due to its ability to mediate the clearance and degradation of amyloid β (Cavus et al., 1996; Kovacs, 2000). The activated forms of A2M can bind to neurotrophic factors and directly inhibit neurotrophic factor-receptor signal transduction to repress neurite outgrowth of central neurons (Koo and Liebl, 1992; Liebl and Koo, 1993; Koo et al., 1994; Hu and Koo, 1998). Most importantly, human A2M is reported to regulate β-catenin signaling though the Wnt inhibitory co-receptor low-density lipoprotein receptor-related protein-1 (LRP1) (Lindner et al., 2010). An A2M conformational intermediate is capable of regulating peripheral nerve injury response by a mechanism that requires LRP1 (Arandjelovic et al., 2007). These previous studies have demonstrated that A2M plays an essential role in neurogenesis. In this study, zebrafish a2ml's down-regulation by rbm47 knockdown provides an important insight into the mechanism of rbm47 on development, suggesting that it is also involved in neural development. However, to identify the RNA binding partners of rbm47, detailed mechanistic evaluation is required in future investigations.
As Rbm47 is ubiquitously expressed during zebrafish embryonic development, the finding that its knockdown results in a tissue-specific phenotype requires explanation. We hypothesize that the rbm47 target gene(s) and/or its binding partner(s) are tissue-specific regulators. Our preliminary study indeed demonstrates that a2ml is expressed in the anterior head region during embryonic patterning by RNA in situ hybridization (data not shown).
In summary, the present study demonstrates that Rbm47 is an RNA binding protein that plays an important role in head development during zebrafish embryogenesis.