RIPPLY3 is a retinoic acid-inducible repressor required for setting the borders of the pre-placodal ectoderm

Abstract

SUMMARYRetinoic acid signaling is a major component of the neural posteriorizing process in vertebrate development. Here, we identify a new role for the retinoic acid receptor (RAR) in the anterior of the embryo, where RAR regulates Fgf8 expression and formation of the pre-placodal ectoderm (PPE). RAR2 signaling induces key pre-placodal genes and establishes the posterolateral borders of the PPE. RAR signaling upregulates two important genes, Tbx1 and Ripply3, during early PPE development. In the absence of RIPPLY3, TBX1 is required for the expression of Fgf8 and hence, PPE formation. In the presence of RIPPLY3, TBX1 acts as a transcriptional repressor, and functions to restrict the positional expression of Fgf8, a key regulator of PPE gene expression. These results establish a novel role for RAR as a regulator of spatial patterning of the PPE through Tbx1 and RIPPLY3. Moreover, we demonstrate that Ripply3, acting downstream of RAR signaling, is a key player in establishing boundaries in the PPE. represses the ability of TBX1 to activate reporter gene constructs in vivo and this inhibition depends on the association of RIPPLY3 with its co-repressor GROUCHO and with TBX1. In agreement with our predictions, RIPPLY3 knockdown perturbs the borders of PPE marker expression. These results demonstrate a novel role for RAR in the precise positioning of the PPE boundaries and establish RIPPLY3 as a key factor that demarcates the boundaries of the PPE. MATERIALS AND METHODS Ripply3 alignment and construction of a phylogenetic treeRipply sequences were obtained from Genbank and Uniprot databases (Benson et al., 2008;Uniprot Consortium, 2009), aligned with MAFFT (L-INS-i algorithm) (Katoh et al., 2009;Katoh et al., 2005) and a phylogenetic tree constructed with PROml, version 3.69 (Protein Maximum Likelihood) (Felsenstein, 2005). Default settings were used, global rearrangements (-G) were performed, and the outgroup (-O) was set to amphioxus. The resultant tree was drawn with FigTree (Rambaut, 2007). Conserved domains of the Ripply gene family were visualized with WebLogo (Crooks et al., 2004;Schneider and Stephens, 1990). EmbryosXenopus eggs were fertilized in vitro as described previously (Blumberg et al., 1997;Koide et al., 2001) and embryos staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Embryos were maintained in 0.1ϫ MBS until appropriate stages or treated with 1 M agonist (TTNPB) and 1 M antagonist (AGN193109) as described (Arima et al., 2005). MicroinjectionEmbryos were injected bilaterally or unilaterally at the two-cell stage with combinations of gene specific morpholinos (MO), mRNAs and 100 pg/embryo -galactosidase mRNA lineage tracer. MOs used for this study are found in supplementary material Table S1. Control embryos were injected with 20 ng standard control MO: CCT CTT ACC TCA GTT ACA ATT TAT A (GeneTools). The following plasmids were constructed by PCR amplification of the protein-coding regions of the indicated genes and cloning into the expression vector pCDG1: xRARa2.2 (Sharpe, ...

RARβ2 is required for vertebrate somitogenesis

“…We found that overexpression of Ripply2 double mutants (WRPW→AAAA; FPVQ→AAAA), which presumably cannot interact with Tbx or Groucho, are phenotypically normal in somite morphology, marked by Myod (Fig. S13D) We have previously shown that Ripply3 inhibits Tbx1 transcriptional activity in vivo (Janesick et al, 2012). Similarly, zebrafish Ripply1 converts Tbx6 from a transcriptional activator to a repressor in vitro (Kawamura et al, 2008) and this protein interaction is essential for establishing the posterior somite boundary (Morimoto et al, 2007;Takahashi et al, 2010).…”

“…RARα or RARγ knockdown in the frog, Xenopus laevis, produces specific defects in neuronal differentiation, pre-placodal ectoderm formation and axial elongation (Janesick et al, 2014(Janesick et al, , 2013(Janesick et al, , 2012Koide et al, 2001). We have shown that RARγ is required for axial elongation and for maintenance of the caudal progenitor pool (Janesick et al, 2014).…”

“…6A). We and others showed that developing organisms are exquisitely sensitive to misexpression of Ripply genes (Janesick et al, 2012;Li et al, 2013;Kawamura et al, 2005). Ripply1 overexpression eliminates notochord and myoseptum (Kawamura et al, 2005) and (Sucov et al, 1990), upstream DR5 and upstream half-site (HS).…”

“…Ripply proteins commonly associate with T-BOX proteins, converting them to transcriptional repressors in the presence of Groucho (Hitachi et al, 2009;Janesick et al, 2012;Kawamura et al, 2005Kawamura et al, , 2008Kondow et al, 2006Kondow et al, , 2007Windner et al, 2015). Tbx6 interacts with Ripply1 in zebrafish (Kawamura et al, 2008) and inhibits adaxial Myod expression, suppressing notochord formation (Goering et al, 2003).…”

“…S13A). Xenopus Ripply1 has apparently been lost during evolution (Janesick et al, 2012). Ledgerline and Bowline are both expressed in a Ripply2-like pattern.…”