Eivers E , Fuentealba LC , Sander V , Clemens JC , Hartnett L , De Robertis EM .

HD21502-23 NICHD NIH HHS , Howard Hughes Medical Institute , R01 HD021502-23 NICHD NIH HHS , R01 HD021502 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Figure 7. Smad5/8 Is Required for Segment Border Formation in Xenopus Embryos. (A) Illustration of C3 Xenopus blastomere injection at the 32 cell stage. The fate of C3 cells in the somites is indicated in a stage 28 tadpole. (B and C) In Xenopus, microinjection of Smad8-MO at the 16 or 32 cell stage (in C2 or C3 blastomeres) erased segmental somite borders on the injected side. Somites are composed mostly of segmental muscles, which were stained for myosin light chain (a-MLC). (D-F0) Smad8-MO effects on segment borders were cell autonomous (co-injection at 32-cell stage with rhodamine dextran amine lineage tracer), and were rescued by human Smad1 mRNA (n = 55, 44 with somite fusions, n = 19, with 17 completely rescued, n = 24, all normal, respectively, 3 independent experiments).
	The Mad12 mutant, which lacks the C-terminal phosphorylation sites, retains posteriorizing activity in Xenopus, in particular when the GSK3 sites are also mutated. (A) Whole mount in situ hybridization of tail bud stage Xenopus embryos (n = 16). Embryos are stained for Rx2a (eye), Krox20 (Hindbrain) and Sizzled (Ventral/Belly). (B) Microinjection of Mad12 mRNA reduced the anterior head region of the embryo, indicated by decreased Rx2a expression (n = 15). (C) Elimination of the anterior head structures in MGM12 microinjected mRNAs (mutation of the GSK3 phosphorylation sites mimics Mad receiving a maximal Wg signal) resulted in a severely posteriorized embryo with almost complete loss-of Rx2a expression (n = 13). (D) Similar posteriorized phenotypes are generated when Wnt10b DNA is microinjected into Xenopus embryos. In addition to this, there is an increase in the expression of the BMP responsive gene sizzled, presumably because it affects the stability of endogenous Smad1/5/8 (n = 7).
	Knockdown of xSmad8 Dorsalizes Xenopus Embryos. (A) Whole-mount in situ hybridization for the pan-neural marker Sox2 in an uninjected control embryo, stage 22, dorsal view. (B) Injection of xSmad8 morpholino (0.5 mM, 4 nl injected four times radially) leads to expansion of the neural plate. (C) Control embryo stained for Otx2 (forebrain and midbrain marker) and Krox20 (hindbrain, rhombomeres 3 and 5), lateral view. (D) Smad8-depleted embryos are dorsalized (anti-BMP phenotype) and show expansion of head structures. It should be mentioned here that the original depletion of Xenopus laevis Smad8 by Miyanaga et al [30] yielded a very different result, namely apoptosis via activation of caspases. However, it should be noted that their methods for depletion were different. They used DNA oligonucleotides to deplete Smad8 transcripts in oocytes that were then subjected to maternal transfer and fertilization. We used morpholino oligos (of a different sequence) injected at the 4-cell stage, and therefore the depletion of maternal transcripts must have been less extensive. This explains why we did not observe apoptosis, but instead dorsalization (anti-BMP phenotype) of the embryo. The morpholino described here provides a useful reagent for knockdown of the maternal Xenopus laevis BMP-Smad. Smad8 probably corresponds to the homolog of zebrafish Smad5 [10] and is therefore referred to below as Smad5/8.
	GSK3-resistant Activated Forms of Smad1 Disrupt Segmentation in Xenopus Embryos. (A and B) Immunostainings for myosin light chain showing loss of segmental borders in somites in the injected side (B) of a Xenopus embryo (stage 26), compared to the uninjected side. C2 or C3 blastomeres were injected with 100 pg of hSmad1 resistant to GSK3 phosphorylation [5] and 50 pg of nuclear LacZ mRNA (n = 32/42). (C) In situ hybridizations for the somite marker MyoD shows a disruption of the segmental pattern in Xenopus embryos (stage 30) injected with activated forms of Smad1. Uninjected control embryos express MyoD in the somitic segments in a typical chevron shape (n = 27). Overexpression of Smad1 wild-type mRNA (SWT) does not change this pattern (n = 21). An activated mutant of Smad1 that has phospho-mimetic amino acid substitutions on the C-terminus (the two most c-terminal serines mutated into glutamic acids, designated EVEmutant [5]) displays mild disruptions of the somite pattern (n = 9/12), while the same phospho-mimetic form of Smad1 with an additional mutation in the GSK3-phosphorylation site in the linker (named EVE-GM exhibits strongly impaired segmentation (n = 14/18). Nuclear LacZ marks the injected cells.
	Figure 1. Phosphorylation-Resistant Mad Proteins are Hyperactive.(A) Model summarizing the integration of Dpp, EGFR and Wg signaling at the level of Mad phosphorylations in Drosophila. (B) Diagrams of Mad Wild Type (MWT), Mad MAPK Mutant (MMM) and Mad GSK3 Mutant (MGM) proteins. (C–F) Microinjection of MMM and MGM mRNAs into Xenopus embryos had stronger ventralizing activity than MWT, causing upregulation of sizzled (n = 17, 32, 26, and 30, two independent experiments). Brain markers otx2 and krox20 were repressed. (G–J) Driving MMM and MGM with patched-Gal4 in the anterior wing compartment caused formation of ectopic crossvein-like tissue. This tissue links longitudinal veins two and three in both proximal and distal regions, pulling the two veins closer together. (K–N) Driving phosphorylation-resistant Mads with apterous-Gal4 induced ectopic vein tissue and blistering, indicating increased Dpp signaling. (O) Polyubiquitinylation of Mad requires GSK3 and MAPK phosphorylation sites. Lane 1, 293T cells cotransfected with MWT-Flag, Drosophila Smurf and HA-ubiquitin all cloned in pCS2. The strong smear represents polyubiquitinylated Mad tagged with HA-ubiquitin. Lanes 2 and 3, polyubiquitinylation was greatly decreased in the MMM and MGM mutant proteins. The lower panel shows equal levels of immunoprecipitated Mad (α-Flag).
	Figure 2. Mad GSK3 Mutants Mimic Wg Overexpression.(A–C) Additional bristles are induced in the wing margin by MGM driven by scalloped-Gal4. Arrows indicate chemosensory bristles and arrowheads stout mechanosensory bristles. Insets show that the number of Senseless-expressing bristle precursors in wing imaginal discs is increased by MGM, but not MWT.(D–G) Expression domains of sd-Gal4 driver, Wg protein, Armadillo stabilized by Wg (expression in the proveins is noted), and Senseless in wing discs. (H and I) Senseless protein expression was inhibited in Mad RNAi clones marked by GFP. (J–O) Quantitative RT-PCR of wing discs showing that MGM increased both a Wg target gene (senseless) and Dpp target genes (spalt and optomotor blind), while not affecting wg or hh levels. dpp was inhibited by MGM RNA. Samples were normalized for rp49, except for the inset in L in which Gal4 mRNA was used. (P and Q) Clonal overexpression of MGM does not change Wg levels. (R and S) MGM clone in the anterior margin causes ectopic expression of Senseless within the clone (inset). (T) Overexpression of MGM in clones marked by yellow induced duplications of the wing margin (n = 24). Yellow arrowheads indicate ectopic margins and a black arrowhead the wild type one. (U) Knockdown of Mad in clones partially eliminates the wing margin In this large clone the remaining margin bristles are yellow (y).
	Figure 3. Phospho-Specific Antibodies Reveal Wg-regulated Segmental Expression Patterns of pMadMAPK and pMadGSK3.(A and B) Western blot analysis of pMadMAPK and pMadGSK3 antibodies demonstrating that they were phospho-specific, and that GSK3 phosphorylation had an absolute requirement for MAPK priming. Drosophila S2 cells were transiently transfected with the plasmids indicated. (C) Cultured 293T cells stably transfected with Mad-Flag treated with L-cell control conditioned medium (CMed), Wnt3a medium, control DMEM (Con), or 30 mM LiCl in DMEM for 2 hours. Wnt3a and LiCl inhibited the MadGSK3 phosphorylation band and increased β-Catenin levels (indicating that the Wnt treatment was effective). (D and E) pMadMAPK and pMadGSK3 antibodies stain the entire blastoderm and a Dpp-dependent dorsal stripe (inset). (F) pMadMAPK tracks ventral EGFR-activated MAPK (inset shows diphospho-Erk staining). (G and H) Segmental staining of pMadMAPK (Stage 9) and pMadGSK3 (Stage 17). (I) In Wg null mutants segmental expression is lost. Mutant embryos were identified by lack of staining with Wg antibody. Inset shows same embryo stained with DAPI to indicate that, despite its abnormal shape, it reached late stages of development. (J–L) Wg stabilizes pMadMAPK, overlapping with Wg stripes. (M–O) Nuclear pMadGSK3 accumulates in between Wg stripes, indicating that Wg inhibits Mad phosphorylation at GSK3 sites in vivo. (P–R) Wg overexpression driven with prd-Gal4 stabilizes pMadMAPK over a broader domain compared to just MWT alone (compare brackets in P and R). This experiment shows that Wg expression stabilizes pMADMAPK.
	Figure 4. Mad10 and Mad12 Alleles Are not Nulls; Mad RNAi Is an Effective and Specific Loss-of-Function Reagent.A) Schematic representation of Mad, showing RNAi and mutant sites. (B–E) Mad10 and Mad12 mutants are phosphorylated in the linker region in the absence of C-terminal phosphorylation. (F) UAS-Mad RNAi depleted stage 15 embryos of endogenous pMadCter when driven by daughterless-Gal4. (G) Repression of brinker-LacZ reporter by Dpp (demarcated by hatched lines) was inhibited by Mad RNAi in wing imaginal discs. (H–K) Mad RNAi driven by MS1096-Gal4 causes complete vein loss at room temperature, which was rescued by UAS-hSmad1. (L and M) Anterior margin mechanosensory bristles are lost when two copies of Mad RNAi were driven with A9-Gal4; this phenocopies Wg loss-of-function. (N–Q) Epistasis by QRT-PCR showing that Wg overexpression in the wing pouch, driven by sd-Gal4, increased transcript levels of the reporter genes optomotor blind, senseless, distalless and vestigial, and that this induction required Mad Samples were normalized for Gal4 mRNA levels Inset shows that levels of Wg transcripts were not affected by Mad RNAi.
	Figure 5. Mad Is Epistatic to Wg Signaling During Neurogenic Induction.(A) Mad was phosphorylated at its MAPK sites in developing CNS neuroblasts. (B) pMadCter was excluded from the neurogenic ectoderm marked by SoxNeuro (stage 8). (C and D) Mad RNAi driven in the egg by pUASp knocked down pMadMAPK staining (stage 7). (E and F) Maternal Mad RNAi knocked down pMADMAPK centrosomal staining (stage 7, see Figure S5 for asymmetric centrosomal staining). (G–J) Mad RNAi increased neurogenic ectodermal nuclei marked by SoxNeuro at stage 8, Wg overexpression reduced it, and the double Wg;RNAi embryos displayed the Mad depletion phenotype. All images were taken at identical exposure conditions. This experiment shows that Mad is epistatic to Wg.
	Figure 6. Mad Is Required for Segmentation in Drosophila.(A–B) Early depletion of Mad caused wider (ventralized) denticle belts and internalized posterior spiracles in embryonic cuticles (n = 259 cuticles, 20% ventralized and 34% ventralized with denticle belt fusions). (C and C') Denticle belt fusions showing large (row 5-like) denticles. (D and D') Wg loss-of-function caused a ventral lawn of denticles. Note that these are large denticles with a small refringent spot (row 5 denticles) resembling those seen in Mad RNAi depletion. (E) DppH46 mutant embryo showing fusion of two denticle belts. (F and F') Overexpression of UAST-MGM driven by mat-Gal4-VP16 caused patches of naked cuticle at the expense of denticle rows. (G–I) Embryos stained for Engrailed at stage 9, showing that Mad depletion disrupts abdominal segmental bands, while MGM overexpression does not.

Affolter, Multiple requirements for the receptor serine/threonine kinase thick veins reveal novel functions of TGF beta homologs during Drosophila embryogenesis. 1994, Pubmed

Affolter, Multiple requirements for the receptor serine/threonine kinase thick veins reveal novel functions of TGF beta homologs during Drosophila embryogenesis. 1994, Pubmed
Baena-Lopez, Control of growth and positional information by the graded vestigial expression pattern in the wing of Drosophila melanogaster. 2006, Pubmed
Bejsovec, Segment polarity gene interactions modulate epidermal patterning in Drosophila embryos. 1993, Pubmed
Bilic, Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. 2007, Pubmed
Brand, Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. 1993, Pubmed
Brunner, pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila. 1997, Pubmed
Couso, The wingless signalling pathway and the patterning of the wing margin in Drosophila. 1994, Pubmed
Coussens, Genetic and cellular mechanisms of oncogenesis. 2008, Pubmed
Crémazy, Sox neuro, a new Drosophila Sox gene expressed in the developing central nervous system. 2000, Pubmed
Damen, Evolutionary conservation and divergence of the segmentation process in arthropods. 2007, Pubmed
De Robertis, Evo-devo: variations on ancestral themes. 2008, Pubmed
De Robertis, The molecular ancestry of segmentation mechanisms. 2008, Pubmed
Eivers, Integrating positional information at the level of Smad1/5/8. 2008, Pubmed , Xenbase
Estella, Molecular integration of wingless, decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development. 2008, Pubmed
Ferguson, Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo. 1992, Pubmed
Fuentealba, Asymmetric mitosis: Unequal segregation of proteins destined for degradation. 2008, Pubmed
Fuentealba, Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. 2007, Pubmed , Xenbase
Gabay, MAP kinase in situ activation atlas during Drosophila embryogenesis. 1997, Pubmed
Garrity, Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase. 1999, Pubmed
Holley, The Xenopus dorsalizing factor noggin ventralizes Drosophila embryos by preventing DPP from activating its receptor. 1996, Pubmed , Xenbase
Hu, Smad1, beta-catenin and Tcf4 associate in a molecular complex with the Myc promoter in dysplastic renal tissue and cooperate to control Myc transcription. 2005, Pubmed
Kintner, Monoclonal antibodies identify blastemal cells derived from dedifferentiating limb regeneration. , Pubmed , Xenbase
Kramps, Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. 2002, Pubmed
Kretzschmar, Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. 1997, Pubmed
Labbé, Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-beta and wnt pathways. 2000, Pubmed , Xenbase
LaLonde, A role for Phospholipase D in Drosophila embryonic cellularization. 2006, Pubmed
Lecuit, Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. 1996, Pubmed
Lee, Making a better RNAi vector for Drosophila: use of intron spacers. 2003, Pubmed
Little, Extracellular modulation of BMP activity in patterning the dorsoventral axis. 2006, Pubmed , Xenbase
Logan, The Wnt signaling pathway in development and disease. 2004, Pubmed
Marquez, Transgenic analysis of the Smad family of TGF-beta signal transducers in Drosophila melanogaster suggests new roles and new interactions between family members. 2001, Pubmed
Miyanaga, A maternal Smad protein regulates early embryonic apoptosis in Xenopus laevis. 2002, Pubmed , Xenbase
Mizutani, EvoD/Vo: the origins of BMP signalling in the neuroectoderm. 2008, Pubmed
Müller, Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient. 2003, Pubmed
Niehrs, Regionally specific induction by the Spemann-Mangold organizer. 2004, Pubmed
Nishita, Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer. 2000, Pubmed , Xenbase
Nüsslein-Volhard, Mutations affecting segment number and polarity in Drosophila. 1980, Pubmed
O'Keefe, Spitz and Wingless, emanating from distinct borders, cooperate to establish cell fate across the Engrailed domain in the Drosophila epidermis. 1997, Pubmed
Pera, Integration of IGF, FGF, and anti-BMP signals via Smad1 phosphorylation in neural induction. 2003, Pubmed , Xenbase
Persson, The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation. 1998, Pubmed , Xenbase
Pourquié, The segmentation clock: converting embryonic time into spatial pattern. 2003, Pubmed
Pueyo, Ancestral Notch-mediated segmentation revealed in the cockroach Periplaneta americana. 2008, Pubmed
Rørth, Gal4 in the Drosophila female germline. 1998, Pubmed
Sander, The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning. 2007, Pubmed , Xenbase
Sapkota, Balancing BMP signaling through integrated inputs into the Smad1 linker. 2007, Pubmed , Xenbase
Schwyter, The decapentaplegic core promoter region plays an integral role in the spatial control of transcription. 1995, Pubmed
Sekelsky, Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. 1995, Pubmed
Shi, Mechanisms of TGF-beta signaling from cell membrane to the nucleus. 2003, Pubmed
Struhl, Hedgehog acts by distinct gradient and signal relay mechanisms to organise cell type and cell polarity in the Drosophila abdomen. 1997, Pubmed
Struhl, Organizing activity of wingless protein in Drosophila. 1993, Pubmed
Szüts, Antagonism between EGFR and Wingless signalling in the larval cuticle of Drosophila. 1997, Pubmed
Takaesu, A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles of Drosophila. 2008, Pubmed
Takaesu, DNA-binding domain mutations in SMAD genes yield dominant-negative proteins or a neomorphic protein that can activate WG target genes in Drosophila. 2005, Pubmed
Zecca, Sequential organizing activities of engrailed, hedgehog and decapentaplegic in the Drosophila wing. 1995, Pubmed
Zeng, Inhibition of Drosophila Wg signaling involves competition between Mad and Armadillo/beta-catenin for dTcf binding. 2008, Pubmed
Zeng, Drosophila Nemo antagonizes BMP signaling by phosphorylation of Mad and inhibition of its nuclear accumulation. 2007, Pubmed