Negative regulation of Smad2 by PIASy is required for properXenopusmesoderm formation

Abstract

Mesoderm induction and patterning are primarily regulated by the concentration of locally expressed morphogens such as members of the TGFβsuperfamily. Smad2 functions as a transcription factor to regulate expression of mesodermal genes downstream of such morphogens. We have identified Xenopus PIASy (XPIASy), a member of the PIAS family, by yeast two-hybrid screening using Xenopus Smad2 (XSmad2) as a bait. During mesoderm induction, XPIASy is expressed in the animal half of embryos with a ventral high-dorsal low gradient at the marginal zone. XPIASyexpression is positively and negatively regulated by activities of the XSmad2 and Wnt pathways, respectively. Interestingly, inhibition of XPIASy by morpholinos induces elongation of animal caps with induction of mesoderm genes even in the absence of their morphogen-mediated activation. In addition, their introduction into the ventral marginal zone results in a secondary axis formation. Gain-of-function analysis revealed that XPIASy inhibits mesoderm induction by specific and direct downregulation of XSmad2 transcriptional activity. These observations indicate that XPIASy functions as an essential negative regulator of the XSmad2 pathway to ensure proper mesoderm induction at the appropriate time and in the appropriate region, and suggest that both the initial step of morphogen-mediated activation of the XSmad2 pathway and regulation of the final downstream transcription step have crucial roles in mesoderm induction and patterning.

XSUMO‐1 is required for normal mesoderm induction and axis elongation during early Xenopus development

“…Similar effects were seen with injection of activin/ nodal pathway inhibitors such as dnSmad2 and XPIASy ( Fig. 2K; Hoodless et al, 1999;Daniels et al, 2004). These results suggested XSUMO1 is involved in the activin/nodal signal transduction pathway.…”

“…3A and B), similar to the phenotype caused by injection with dominantnegative Smad2 (dn-Smad2) or XPIASy, both negative regulators of activin/nodal signaling ( Fig. 3C; Hoodless et al, 1999;Daniels et al, 2004). These phenotypes were rescued by coinjection with myc-XSUMO-1 mRNA in a dose-dependent manner ( Fig.…”

“…We first proposed Smad proteins as candidate targets, because previous studies showed that overexpression of SUMO E3 ligases upregulated Smad4-or TGF-␤-mediated transcriptional activity (Lee et al, 2003;Lin et al, 2003;Liang et al, 2004), while another report showed that SUMO-1 conjugation of Smad4 recruits the binding of transcriptional corepressor, Daxx, and this binding downregulated the transcriptional activity of Smad4 (Chang et al, 2005). Moreover, in Xenopus, XPIASy interacts with XSmad2, enhances its SUMOylation, and suppresses XSmad2 activity for proper mesoderm induction (Daniels et al, 2004). These findings together suggested that SUMOylation of Smads is important for mesoderm formation in Xenopus development.…”

“…Some Smad proteins that transduce signals in the TGF-␤ signaling pathway are conjugated by SUMO. Smad6 and Smad7 also can interact with the SUMOylation E3 enzyme PIASy, although it is unclear whether these factors are conjugated by SUMO (Muller et al, 2000;Ross et al, 2002;Lee et al, 2003;Lin et al, 2003;Imoto et al, 2003;Daniels et al, 2004). TGF-␤ signaling is crucial in cell proliferation and differentiation.…”

FoxD3 regulation of Nodal in the Spemann organizer is essential forXenopusdorsal mesoderm development

“…Misregulation of Nodal activity can result in gastrulation defects, expansion of mesodermal lineages into the ectodermal domain, loss of head structures, and situs inversus. Furthermore, as the Nodal positive feedback loop can amplify Nodal expression and signaling, mechanisms that negatively regulate Nodal expression and activity are essential for normal development.Multiple Nodal antagonists have been identified that act at each step of the Nodal signal transduction cascade; Cerberus, Coco and Lefty/Antivin block Nodal signaling at the extracellular level (Thisse and Thisse, 1999;Piccolo et al, 1999;Cheng et al, 2000;Bell et al, 2003;Branford and Yost, 2004), whereas Dapper2, Smad7, Ectodermin and PIASy act intracellularly by stimulating receptor turnover or inhibiting Smad function (Nakao et al, 1997;Casellas and Brivanlou, 1998;Daniels et al, 2004;Zhang et al, 2004b;Dupont et al, 2005 To assess the dependence of Xnr1 and VegT activity on FoxD3, the animal pole was injected at the one-cell stage embryo with VegT (500 pg) or Xnr1 (100 pg) alone, or in combination with FoxD3MO or mismatch MO (50 ng). Animal explants were analyzed by RT-PCR at the gastrula stage (stage 11) for the expression of Brachyury (Xbra), MyoD, Goosecoid (Gsc), Xnr1 and Xnr2.…”