Jin Y , Weinstein DC .

R03 HD077015 NICHD NIH HHS , R15 GM124577 NIGMS NIH HHS

             cement gland development

	Fig. 1. Knockdown ofpitx1andpitx2cinhibits endogenous cement gland formation andXag1expression. (A) 0% of uninjected embryos (n = 56) lack an observable cement gland. (B) 40% of tadpole stage embryos (n = 46) lack an observable cement gland following injection of Pitx1-MO and Pitx2c-MO. (C) 0% embryos (n = 36) lack an observable cement gland following injection of Pitx1–5MM and Pitx2c-5MM. (D) Cement gland in Pitx1-MO and Pitx2c-MO-injected embryo is rescued by injection of pitx1 RNA: 6% of embryos (n = 35) lack an observable cement gland. (E) 73% of uninjected neurula stage embryos (n = 22) exhibit strong Xag1 staining. (F) 36% of embryos (n = 28) display strong Xag1 staining following injection of Pitx1-MO and Pitx2c-MO. (G) 80% of embryos (n = 20) exhibit strong Xag1 staining following injection of Pitx1–5MM and Pitx2c-5MM. Embryos in (A-D) are lateral views, anterior to left; embryos in (E-G) are ventral views, anterior to top. Morpholino oligos were injected into the animal pole of early cleavage stages embryos. Black arrows indicate actual or expected sites of the cement gland; black arrowheads indicate Xag1 staining.
	Fig. 2. Pitx1 transcriptionally activates cement gland differentiation genes both directly and indirectly. (A) Injection of VP16-pitx1 RNA, or wild-type pitx1 RNA, leads to induction of cement gland markers. Indicated doses of pitx1, VP16-pitx1, and EnR-pitx1 RNA were injected at early cleavage stages. RT-PCR analysis was performed on animal caps collected at mid-neurula stages after dissection at late blastula stages. (B) nkx3.1, TGFβ1, sim2, crx, epha4 and traf4, but not Xag1, Xcg, agr2 or xa-1, are strongly induced by Pitx1-GR in both the presence and absence of cycloheximide. 400 pg pitx1-GR RNA was injected into early cleavage stages embryos. RT-PCR analysis was performed on animal caps dissected at late blastula stages and cultured with or without dexamethasone and/or cycloheximide until mid-neurula stages. Dexamethasone (10 µM) was added 10 min after cycloheximide (10 µg/ml) addition at stage 12, as listed. Dexamethasone and/or cycloheximide treatments were for approximately 5 h. EF1α is used as a loading control (Krieg et al., 1989). The -RT lane contains all reagents except reverse transcriptase, and is used as a negative control.
	Fig. 3. follistatinexpression in the cement gland is dependent on Pitx1. (A) Injection of pitx1 RNA results in the induction of follistatin. 400 pg pitx1 RNA was injected at early cleavage stages. RT-PCR analysis was performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (B) Cycloheximide treatment results in strong expression of follistatin. (C) follistatin expression is observed in anterior notochord at early neurula stages. (D) follistatin expression is observed in the hindbrain (arrow) and pronephros (arrowheads) at late neurula stages. (E) 66% of uninjected late neurula stage embryos (n = 29) have detectable follistatin staining in the presumptive cement gland region. (F) 36% of embryos (n = 33) have detectable follistatin staining in the cement gland primordium following injection of Pitx1-MO and Pitx2c-MO; (G) 77% of embryos (n = 31) have expanded follistatin staining following injection of pitx1 RNA. Morpholino oligos or pitx1 RNA were injected into the animal pole of early cleavage stages embryos. Embryos in (C, D) are dorsal views, anterior to left; embryos in (E-G) are ventral views, anterior to left. Black arrows and arrowheads indicate actual or expected follistatin staining.
	Fig. 4. Pitx1 may function as a transcriptional activator to inhibit BMP signaling. RT-PCR analysis (A, C and D) and Western blot analysis (B) were performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (A) Injection of pitx1 RNA inhibits BMP targets in a dose dependent manner. 10–400 pg pitx1 RNA was injected at early cleavage stages, as listed. Ornithine decarboxylase (ODC) is used as a loading control (Bassez et al., 1990). (B) Injection of pitx1 RNA results in decreased C-terminal phosphorylation of Smad1/5; total levels of Smad1 are not significantly affected. Embryos were injected with 20 pg, 100 pg and 400 pg pitx1 RNA at early cleavage stages, as listed. β-tubulin is used as a loading control. (C) Injection of VP16-pitx1 RNA leads to down-regulation of BMP target genes, similar to that seen with wild-type pitx1; injection of EnR-pitx1 RNA does not alter expression of BMP-responsive genes. Indicated doses of pitx1, VP16-pitx1, and EnR-pitx1 RNA were injected at early cleavage stages. (D) Pitx1-mediated inhibition of the BMP target gene sizzled is partially rescued by co-injection of Follistatin-MO. 200 pg pitx1 RNA was co-injected with 192 ng Follistatin-MO or scrambled control morpholino oligo at early cleavage stages.
	Fig. 5. Inhibition of BMP signaling is required for the expression ofpitxgenes, while BMP/Smad1 signaling is required for cement gland development. (A) Pitx1-mediated inhibition of the BMP target gene sizzled is rescued by co-expression of either Smad1 or CA-BMPRIa. Pitx1-mediated induction of two pitx2 transcript variants, pitx2b and pitx2c, as well as endogenous pitx1, are down-regulated following enhancement of BMP signaling. 50 pg pitx1 RNA was either injected alone or co-injected with 1.6 ng smad1 or CA-BMPRIa RNA at early cleavage stages. RT-PCR analysis was performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (B) 90% of neurula stage control embryos (n = 20) display strong pitx1 staining. Water was injected into the animal pole of 4-cell stage Xenopus embryos. (C) 88% of embryos (n = 16) exhibit weak and spatially restricted pitx1 staining following injection of 500 pg CA-BMPRIa RNA at early cleavage stages. Embryo views are anterior, ventral to bottom. (D) Injection of noggin RNA inhibits Pitx1-mediated induction of cement gland differentiation genes. 400 pg pitx1-GR RNA and/or 20 pg noggin RNA were injected into Xenopus embryos. RT-PCR analysis was performed on animal caps dissected at late blastula stages and cultured with or without dexamethasone until mid-neurula stages. Dexamethasone (10 µM) was added to animal caps at stage 12. (E) Pitx1 binds to Smad1. 2 ng pitx1-myc RNA was injected at early cleavage stages. Pull-down of native Smad1 from injected embryos leads to co-immunoprecipitation of exogenous Pitx1. Normal rabbit IgG antibodies was used in parallel studies as a negative control.

Abreu, Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. 2002, Pubmed, Xenbase

Abreu, Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. 2002, Pubmed , Xenbase
Ambrosio, Crossveinless-2 Is a BMP feedback inhibitor that binds Chordin/BMP to regulate Xenopus embryonic patterning. 2008, Pubmed , Xenbase
Bassez, Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. 1990, Pubmed , Xenbase
Bradley, Positive and negative signals modulate formation of the Xenopus cement gland. 1996, Pubmed , Xenbase
Campione, The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. 1999, Pubmed , Xenbase
Chang, xPitx1 plays a role in specifying cement gland and head during early Xenopus development. 2001, Pubmed , Xenbase
Chung, Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut. 2010, Pubmed , Xenbase
Coumailleau, Characterization and developmental expression of xSim, a Xenopus bHLH/PAS gene related to the Drosophila neurogenic master gene single-minded. 2000, Pubmed , Xenbase
Deglincerti, Coco is a dual activity modulator of TGFβ signaling. 2015, Pubmed , Xenbase
Fainsod, The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4. 1997, Pubmed , Xenbase
Faucourt, The pitx2 homeobox protein is required early for endoderm formation and nodal signaling. 2001, Pubmed , Xenbase
Gammill, otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation. 2001, Pubmed , Xenbase
Gammill, Coincidence of otx2 and BMP4 signaling correlates with Xenopus cement gland formation. 2000, Pubmed , Xenbase
Gammill, Identification of otx2 target genes and restrictions in ectodermal competence during Xenopus cement gland formation. 1997, Pubmed , Xenbase
Hama, The molecular basis of Src kinase specificity during vertebrate mesoderm formation. 2002, Pubmed , Xenbase
Hawley, Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction. 1995, Pubmed , Xenbase
Hemmati-Brivanlou, Inhibition of activin receptor signaling promotes neuralization in Xenopus. 1994, Pubmed , Xenbase
Hemmati-Brivanlou, Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. 1994, Pubmed , Xenbase
Hollemann, Xpitx-1: a homeobox gene expressed during pituitary and cement gland formation of Xenopus embryos. 1999, Pubmed , Xenbase
Iemura, Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo. 1998, Pubmed , Xenbase
Jamrich, Differential gene expression in the anterior neural plate during gastrulation of Xenopus laevis. 1989, Pubmed , Xenbase
Kalkan, Tumor necrosis factor-receptor-associated factor-4 is a positive regulator of transforming growth factor-beta signaling that affects neural crest formation. 2009, Pubmed , Xenbase
Kessler, Siamois is required for formation of Spemann's organizer. 1997, Pubmed , Xenbase
Kim, TGF-beta-induced transcriptional activation of MMP-2 is mediated by activating transcription factor (ATF)2 in human breast epithelial cells. 2007, Pubmed
Knecht, Mechanisms of dorsal-ventral patterning in noggin-induced neural tissue. 1997, Pubmed , Xenbase
Kondaiah, Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis. 1990, Pubmed , Xenbase
Krieg, The mRNA encoding elongation factor 1-alpha (EF-1 alpha) is a major transcript at the midblastula transition in Xenopus. 1989, Pubmed , Xenbase
Lanctôt, The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm. 1997, Pubmed
Lee, Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. 2006, Pubmed , Xenbase
Lim, TGF-beta1 induces cardiac hypertrophic responses via PKC-dependent ATF-2 activation. 2005, Pubmed
Mercurio, Connective-tissue growth factor modulates WNT signalling and interacts with the WNT receptor complex. 2004, Pubmed , Xenbase
Murato, Xhairy2 functions in Xenopus lens development by regulating p27(xic1) expression. 2009, Pubmed , Xenbase
Newman, Xenopus bagpipe-related gene, koza, may play a role in regulation of cell proliferation. 2002, Pubmed , Xenbase
Novoselov, Expression zones of three novel genes abut the developing anterior neural plate of Xenopus embryo. 2003, Pubmed , Xenbase
Nudi, Bone morphogenic protein (Smad)-mediated repression of proopiomelanocortin transcription by interference with Pitx/Tpit activity. 2005, Pubmed
Pascal, Cloning and developmental expression of STAT5 in Xenopus laevis. 2001, Pubmed , Xenbase
Pera, Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. 2003, Pubmed , Xenbase
Proctor, The physiology of salivary secretion. 2016, Pubmed
Reversade, Regulation of ADMP and BMP2/4/7 at opposite embryonic poles generates a self-regulating morphogenetic field. 2005, Pubmed , Xenbase
Rosa, Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos. 1989, Pubmed , Xenbase
Schweickert, Pitx1 and Pitx2c are required for ectopic cement gland formation in Xenopus laevis. 2001, Pubmed , Xenbase
Schweickert, Differential gene expression of Xenopus Pitx1, Pitx2b and Pitx2c during cement gland, stomodeum and pituitary development. 2001, Pubmed , Xenbase
Sive, Progressive determination during formation of the anteroposterior axis in Xenopus laevis. 1989, Pubmed , Xenbase
Sive, A sticky problem: the Xenopus cement gland as a paradigm for anteroposterior patterning. 1996, Pubmed , Xenbase
Smith, Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. 1992, Pubmed , Xenbase
Smith, The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells. 1997, Pubmed , Xenbase
Suri, Xema, a foxi-class gene expressed in the gastrula stage Xenopus ectoderm, is required for the suppression of mesendoderm. 2005, Pubmed , Xenbase
Suzuki, Smad5 induces ventral fates in Xenopus embryo. 1997, Pubmed , Xenbase
Suzuki, Regulation of epidermal induction by BMP2 and BMP7 signaling. 1997, Pubmed , Xenbase
Suzuki, MID1 and MID2 are required for Xenopus neural tube closure through the regulation of microtubule organization. 2010, Pubmed , Xenbase
Szeto, Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development. 1999, Pubmed
Varley, Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures. 1998, Pubmed
Vignali, Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis. 2000, Pubmed , Xenbase
Vonica, The left-right axis is regulated by the interplay of Coco, Xnr1 and derrière in Xenopus embryos. 2007, Pubmed , Xenbase
Wardle, What's your position? the Xenopus cement gland as a paradigm of regional specification. 2003, Pubmed , Xenbase
Wardle, Cement gland-specific activation of the Xag1 promoter is regulated by co-operation of putative Ets and ATF/CREB transcription factors. 2002, Pubmed , Xenbase
Weinstein, Neural induction. 1999, Pubmed , Xenbase
Wilson, Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its signal transducer Smad1. 1997, Pubmed , Xenbase
Wilson, Induction of epidermis and inhibition of neural fate by Bmp-4. 1995, Pubmed , Xenbase
Xu, A dominant negative bone morphogenetic protein 4 receptor causes neuralization in Xenopus ectoderm. 1995, Pubmed , Xenbase
Yuan, IRE1beta is required for mesoderm formation in Xenopus embryos. 2008, Pubmed , Xenbase