QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 16 April 2008
doi: 10.1242/dev.019323

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Herr, P. Articles by Oelgeschläger, M. Search for Related Content PubMed PubMed Citation Articles by Herr, P. Articles by Oelgeschläger, M. Social Bookmarking                         What's this?

Regulation of TGF-β signalling by N-acetylgalactosaminyltransferase-like 1

Patrick Herr^*,, Ganna Korniychuk, Yukiyo Yamamoto, Kristina Grubisic and Michael Oelgeschläger

Max-Planck Institute of Immunobiology, Stübeweg 51, D-79108 Freiburg, Germany.

View larger version (60K): [in this window] [in a new window]   Fig. 1. Sequence and expression of xGalntl-1. (A) Comparison of Xenopus Galntl-1, human GALNTL1 (hGalntl-1), zebrafish Galnt14 (zGalnt-14) and Drosophila Galnt2 (dGalnt-2) protein sequences. The transmembrane domain (TM, blue), the catalytic domain (red), the Ricin domain (green) and amino acid identities/similarities for the different protein domains are indicated. (B) Detection of mucin-type glycosylation at the cell surface and in the Golgi compartment in xGalntl-1-transfected Cos-7 cells using Helix pomatia agglutinin (HPA) conjugated to Alexa Fluor 488. (C) RT-PCR analysis of xGalntl-1, xGalnt-1, xGalnt-6 and xGalnt-7 expression, using RNA from whole embryos (left) or dorsal and ventral marginal zone explants, isolated at gastrula stages and analysed at stage 20 (right). The blood marker -globin, neural marker β-tubulin, dorsal mesodermal marker chordin and the nieuwkoop centre-specific gene siamois were used as references for the indicated developmental stages. Dorsal (ncam, myoD) and ventral (sizzled, msx1) marker genes were used as controls for the marginal zone explants. (D) Whole-mount in situ hybridisation analysis of xGalntl-1 expression at stage 13. Numbered lines indicate the positions of vibratome sections that reveal xGalntl-1 expression in the anterior mesoderm and in the deep layer of the lateral neural plate. (E) In situ analysis of xGalntl-1 expression at stage 27. The paraffin sections below show specific expression of xGalntl-1 in the anterior brain (1), neural crest (2), mediolateral spinal cord (2-4) and notochord (3,4).

View larger version (49K): [in this window] [in a new window]   Fig. 2. Inhibition of mesoderm formation and Activin signalling by xGalntl-1. (A) Stage 40 uninjected control Xenopus embryo and (B) sibling embryos microinjected radially with 400 pg xGalntl-1 mRNA at 2- to 4-cell stages. (C) In situ hybridisation for the pan-mesodermal marker xbra in gastrula stage embryo microinjected either radially with xGalntl-1 mRNA alone or into single blastomeres together with lacZ mRNA at the 4-cell stage. (D) RT-PCR analysis of dorsal (DMZ) and ventral marginal zone (VMZ) explants at stage 20. The expression of -globin (blood), gata1 and xvent1 (ventral mesoderm), msx1 (neural crest, epidermis) was reduced in VMZ from embryos microinjected with 200 pg xGalntl-1 mRNA, whereas the expression levels of xag (cement gland), otx2, rx2a (forebrain) and ncam (neural) were increased. The expression of -actin (muscle) was reduced in DMZ and slightly increased in VMZ. (E) RT-PCR analysis for chordin (dorsal mesoderm), sox17β (endoderm) and xbra (mesoderm) expression using stage 20 animal cap explants from embryos microinjected with mRNA encoding eFGF (100 pg) or Activin (2 pg), in the presence or absence of 200 pg xGalntl-1 mRNA. (F) Western blot analysis of Smad and phospho-Smad proteins from HEK 293T cells that were transiently transfected with Smad2-FLAG and xGalntl-1 expression vectors and cultured for 2 hours in the absence or presence of 2ng/ml recombinant Activin protein. (G) In vitro translation of xGalntl-1 mRNA containing (+5'-UTR) or lacking (-5'-UTR) morpholino target sequences. Two different morpholinos (Galntl-1-MO1 and Galntl-1-MO2, 2.5 µm) as well as a mixture of both (1.25 µm each) specifically interfered with translation of RNA containing the morpholino target sequences. (H) RT-PCR analysis of animal cap explants at stage 20 from embryos microinjected with 0.5 pg Activin mRNA, xGalntl-1 morpholinos (1 ng each) and 20 pg xGalntl-1 mRNA. The morpholino stimulated the induction of myoD (muscle) and xbra expression by Activin. In all RT-PCR experiments, odc served as a loading control.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Inhibition of BMP signalling by Galntl-1. (A-D) Stage 36 embryos that were injected animally at the 8-cell stage with a total of 2 ng of mRNA encoding xGalntl-1, human GALNTL1 (hGalntl-1) or secreted Galntl-1 (sGalntl-1), with the N-terminal transmembrane domain replaced by the Chordin leader peptide. (E) RT-PCR analysis of animal cap explants from embryos injected with 2 ng xGalntl-1 or 40 pg chordin mRNA at stage 20 for neural (pax6, ncam, otx2), anterior (xag), muscle (myf5) and epidermal (cytokeratin) marker genes. (F) Western blot of the supernatant (SN) and cell pellet of dissociated ectodermal cells expressing the xGalntl-1 or sGalntl-1 with a C-terminal HA-TAG. (G) RT-PCR analysis of animal cap explants from embryos microinjected with mRNA encoding human, mouse (m) or secreted forms of Galntl-1. (H) RT-PCR analysis of animal cap explants from embryos microinjected with mRNA encoding xGalntl-1 (1 ng) alone or in combination with mRNA encoding BMP7/OP-1 (400 pg), dnFGFR-4 (400 pg), dnAlk-4 (400 pg) or N-TCF-3 (400 pg). (I) Western blot for Smad1 or phospho-Smad1 using animal cap lysates from uninjected embryos or embryos microinjected with 200 pg Smad1-FLAG mRNA alone or together with 200 pg xGalntl-1 mRNA. (J) Western blot analysis for Smad1 and phospho-Smad1 from transiently transfected Hek293T cells treated for 2 hours with 40 ng/ml recombinant BMP4 protein. (K) Western blot for phospho-Smad1 or Smad1 using lysates from HEK 293T cells transiently transfected with the indicated expression plasmids. In the RT-PCR experiments -actin or myf-5 served as controls for mesoderm contamination; odc as a loading control.

View larger version (75K): [in this window] [in a new window]   Fig. 4. Xenopus Galntl-1 loss-of-function phenotype. (A) Stage 40 uninjected control embryo and embryo injected radially with the xGalntl-1-specific morpholinos at the 2- to 4-cell stage. Numbered lines indicate the positions of the paraffin sections shown on the right that reveal a reduction of neural tissue in the morpholino-injected embryos. (B) Uninjected control embryo and embryos microinjected with xGalntl-1 morpholinos stained with the notochord-specific antibody MZ15 at stage 40 (left) and analysed by in situ hybridisation for slug expression at stage 30. The morpholino-injected embryos display defects in the anterior notochord and reduced anterior expression of slug. (C) Whole-mount in situ hybridisation for slug of stage 15 embryos after radial microinjection of the xGalntl-1 morpholinos at the 2- to 4-cell stage, and stage 13 embryo stained for msx1 after single injections of the xGalntl-1 morpholinos into a dorsal-animal blastomere at the 8-cell stage together with lacZ mRNA. (D) RT-PCR analysis of stage 20 dorsal marginal zone explants from uninjected embryos or embryos microinjected with xGalntl-1 morpholinos alone or together with xGalntl-1 mRNA (20 pg). The expression of otx2 and six3 (forebrain) as well as of snail (neural crest) was inhibited by the xGalntl-1 morpholinos, whereas the expression of dlx5 (epidermis), xvent2 (ventral mesoderm) and xbra (mesoderm) was unaffected.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Inhibition of ActR-IIB by Galntl-1. (A) RT-PCR analysis of animal cap explants analysed at stage 20 from embryos microinjected with 400 pg xGalntl-1 mRNA alone or together with 200 pg mRNA encoding human ALK3, mouse Alk6, human BMPR-II or Xenopus ActR-IIB. (B) RT-PCR analysis of stage 20 animal caps microinjected with 400 pg ActR-IIB mRNA either alone or together with 1 ng xGalntl-1 mRNA. The mesodermal marker genes xbra and wnt8 indicated ActR-IIB activity, whereas otx2 and xag indicated xGalntl-1 activity, and xvent2 served as a marker for ventral mesoderm and odc as a loading control. (C) RT-PCR analysis of stage 20 animal caps microinjected with 40 pg ActR-IIB mRNA alone, together with xGalntl-1 morpholinos, and xGalntl-1 morpholinos and 100 pg xGalntl-1 mRNA. (D) Western blot analysis of Smad and phospho-Smad levels from HEK 293T cells transiently transfected with Smad1-FLAG, BMPR-II and xGalntl-1 (left) or Smad2-FLAG, ActR-IIB and xGalntl-1 (right). (E) Western blot analysis of phospho-Smad2 and Smad2 levels from HEK 293T cells transiently transfected with Smad2-FLAG, ActR-IIB and xGalntl-1 and incubated overnight in the presence or absence of 2 mM benzyl-GalNAc. (F) Whole-mount in situ hybridisation for msx1 using stage 13 embryos microinjected with xGalntl-1 morpholinos, 100 pg xActR-IIB mRNA and 100 pg xGalntl-1 mRNA into dorsal-animal blastomeres at the 4- to 8-cell stage.

View larger version (47K): [in this window] [in a new window]   Fig. 6. Galntl-1 inhibits the binding of ActR-IIB to type I receptors. (A) Immunohistochemical staining of transiently transfected Cos-7 cells for ActR-IIB containing a C-terminal HA-TAG and xGalntl-1 containing a C-terminal FLAG-TAG. (B) Western blot analyses of stage 11 Xenopus animal cap cells expressing HA-tagged ActR-IIB, Alk-3, Alk-4 and Alk-6 and xGalntl-1. (C) Western blot analyses of transiently transfected HEK 293T cells expressing HA-tagged xActR-IIB and xGalntl-1 and incubated overnight in the presence or absence of 2 mM benzyl-GalNAc. (D) Co-immunoprecipitation of HA-tagged Alk-3, Alk-4 and Alk-6 with MYC-tagged ActR-IIB in the presence or absence of FLAG-tagged xGalntl-1 from lysates of transiently transfected HEK 293T cells. (E) Co-immunprecipitation of Alk-3 with Myc-tagged ActR-IIB or FLAG-tagged BMPR-II in the presence or absence of untagged xGalntl-1. The binding of BMPR-II to Alk-3 does not seem to be inhibited by xGAlntl-1. (F) Co-immunprecipitation of Alk-4-HA with ActR-IIB-Myc in the presence of xGalntl-1, sGalntl-1 or xGalnt-6. Only xGalntl-1 interfered with the formation of the heteromeric complex. In all experiments, 10% of the lysates used for co-immunprecipitation was subjected to direct immunprecipitation and the quantity of protein analysed by western blotting for the different epitopes.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter