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

First published online August 25, 2006
doi: 10.1242/10.1242/dev.02516

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 Zamparini, A. L. Articles by Brickman, J. M. Search for Related Content PubMed PubMed Citation Articles by Zamparini, A. L. Articles by Brickman, J. M. Social Bookmarking                         What's this?

Hex acts with ß-catenin to regulate anteroposterior patterning via a Groucho-related co-repressor and Nodal

Andrea L. Zamparini^1,*, Tim Watts¹, Clare E. Gardner², Simon R. Tomlinson¹, Geoffrey I. Johnston² and Joshua M. Brickman^1,

¹ Centre Development in Stem Cell Biology, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK.
² Discovery Biology, Pfizer Global Research and Development, Ramsgate Road, Sandwich CT13 9NJ, UK.

View larger version (58K): [in a new window]   Fig. 1. Hex anteriorises the phenotypes induced by ß-catenin and regulates its downstream targets. (A) Axis duplication phenotypes induced by Hex and ß-catenin. Embryos were injected with 500 pg Hex, ß-catenin, or both RNAs into a single-ventral blastomere at the four-cell stage. In situ hybridisation was performed for the anterior neural marker BF1 at stage 35. The inset and arrowheads indicate a small ventral outgrowth produced by Hex injection. (B) Phenotypes induced by Hex and ß-catenin in ventral marginal zone explants. Embryos were injected with the indicated RNA into both blastomeres at the two-cell stage, cultured to gastrulation, VMZ explants dissected and cultured until staging control embryos reached stage 35. BF1 in situ hybridisation was performed to highlight the phenotypes obtained. Hex RNA was injected at 500 pg. (C,D) Real-time RT-PCR analysis of Siamois and Xnr3 expression in VMZ explants analysed at stage 10.5. Embryos were injected as in B with the indicated RNA. Values were normalised to the expression level of Odc and the relative change in gene expression for the genes analysed was calculated by dividing the values from injected samples by the values from the uninjected. Data are based on three independent experiments. (E) Cell automonous induction of ß-catenin targets Siamois and Xnr3. RNA was injected into a single blastomere at the four-cell stage with the indicated RNA alongside nucGFP RNA. Dorsal injections are indicated with `D' where either 250 pg Hex or 100 pg Hex-VP2 were used. Ventral injections are indicated with `V' where 500 pg Hex and 500 pg ß-catenin were used. Embryos were processed by double in situ hybridisation and stained for both nucGFP to indicate the injected cells (light blue) and Xnr3 (dark blue). The schematic diagram in the lower right-hand corners of the lower panels indicates that the injection was carried out in both blastomeres at the two-cell stage. The insets in ß-catenin and ß-catenin co-injection with Hex show Xnr3 staining in the animal hemisphere. Arrowheads indicate the site of injection. (F) Expression of ß-catenin targets Siamois and Xnr3 in embryos depleted of endogenous Hex. Embryos were injected with a total of 40 ng Hex MO or control MO at the two-cell stage, either alone or in combination with 500 pg ß-catenin. 500 pg mouse Hex (mHex) was used to rescue the phenotypes. Siamois (upper panel) and Xnr3 (lower panel) expression was analysed by in situ hybridisation at stage 10.5. Arrowheads indicate the ectopic expression induced by ß-catenin.

View larger version (46K): [in a new window]   Fig. 2. Downstream response to the induction of early embryonic signalling by Hex. (A) Real-time RT-PCR of Cerberus expression in VMZ explants analysed at stage 10.5. Embryos were injected with the indicated RNA and Cerberus expression quantified as in Fig. 1C. Data are based on three independent experiments. (B) In situ hybridisation for Cerberus expression and double in situ for Cerberus (purple) and nucGFP (light blue) expression at stage 10.5. Embryos were injected with 500 pg Hex, ß-catenin or both at the two-cell stage into both blastomeres (indicated with a schematic diagram in the lower right-hand corner) or into a single-ventral blastomere at the four-cell stage (indicated by `V') alongside a nucGFP RNA. Arrowheads indicate the site of injection. (C,D) Real-time RT-PCR analysis of Goosecoid and Chordin. Embryos were injected as in A and RNA from either animal caps (C) or VMZ (D) explants extracted and analysed at stage 10.5. Values are normalised to the expression level of Odc and the relative change in gene expression for the genes analysed was calculated by dividing the values from injected samples by the values from the uninjected. Data is based on three independent experiments. (E) In situ hybridisation for Goosecoid and Chordin expression at stage 10.5. Embryos were injected with 500 pg Hex, ß-catenin, or both, at the four-cell stage into a single-ventral blastomere alongside nucGFP. Double staining was performed. Arrowheads indicate the injected cells. High-magnification views of embryos co-injected with Hex and ß-catenin are shown for both Goosecoid (indicated as Gsc) and Chordin (indicated as Chd) expression. (F) Depletion of Hex by Hex MO. Embryos were injected as in Fig. 1F and in situ hybridisation for Cerberus, Goosecoid and Chordin performed at stage 10.5. Arrowheads indicate the ectopic domain induced by ß-catenin injection.

View larger version (18K): [in a new window]   Fig. 3. Hex amplifies the transcriptional activity of ß-catenin in HEK 293T cells. (A) Reporter plasmids used in the transfections: TOPflash, a reporter gene containing reiterated Tcf-binding sites upstream of the tk promoter and FOPflash, a mutated version of TOPflash used as a negative control. (B,C) Wnt reporter gene activity in response to transfection of Hex and Wnt pathway components. The indicated DNA was co-transfected with either TOPflash or FOPflash. Hex, Hex-VP2 and ß-catenin were used at 100 ng. Stabilised ß-catenin (stab. ß-catenin), LefdNß-CTA and Xtcf3-VP2 at 90 ng. Values are represented as fold induction of the indicated reporters. Experiments were carried out in triplicate.

View larger version (48K): [in a new window]   Fig. 4. Identification of Hex targets in ES cells. (A) Schematic representation of the ES cell lines used in the Hex target screen. (a) cDNAs inserted into the Rosa26 locus. (b) Strategy for the generation of clonally related cell lines. Top line illustrates the two Rosa26 targeting constructs used to generate parental control cell lines that do not express the transgene because of the triple PolyA stop cassette. Transfection of these lines with CRE-recombinase induces recombination between the two LoxP sites, removing the stop cassette and allowing expression of the indicated cDNA. This strategy ensures that all the transgenic lines are derived from a clonally related control. SA refers to Rosa26 splice acceptor. (B) Outline of the Hex target screen. RRosa is recombined Rosa. (C) Profile of gene expression across a series of treatment comparisons. RHETVPcl1+/-4-OHT refers to the average ratio of gene expression levels in Rosa HexERT2-VP2 in the presence and absence of 4-OHT for clone 1. RRHETVPcl1+/-4-OHT refers to the average ratio of gene expression levels in the recombined Rosa HexERT2-VP2 in the presence or absence 4-OHT for clone 1. RHETVPcl2+/-4-OHT refers to the average ratio of gene expression levels in Rosa HexERT2-VP2 in the presence or absence of 4-OHT for clone 2. RRHETVPcl2+/-4-OHT refers to the average ratio of gene expression levels in the recombined Rosa HexERT2-VP2 in the presence or absence of 4-OHT for clone 2. RH+/-CRE refers to the average ratio of gene expression levels in Rosa Hex in the presence or absence of CRE. The five candidate genes obtained are shown in black against the background of all gene expression profiles. The gene expression averages in this plot are based on samples considered to represent undifferentiated cultures as judged by expression of key markers such as Oct4. (D) List of candidate Hex target genes. (E) Tle4 and Nodal expression is induced by the addition of 4-OHT to the cultured HexERT2-VP2-expressing cells. Cells were cultured and treated with 600 nM 4-OHT in serum-free media and expression of the genes analysed by real-time RT-PCR. Removal of serum from ES cell cultures appears to remove a suppressor of HexERT2-VP2 activity and consequently we observe more robust target gene induction. Values are normalised to the expression level of ß-actin and presented as relative copy number.

View larger version (67K): [in a new window]   Fig. 5. Validation of Hex targets in Xenopus embryos. (A) Expression of candidate Hex target genes in response to depleting endogenous Hex. In situ hybridisation for Xtle4, Xnr1, Xnr2, Xnr5 and Xnr6 in Hex MO depleted and rescued embryos. Embryos were injected as in Fig. 1F with the indicated MO/RNA combination and analysed by in situ hybridisation at stage 10.5. Where CHX is indicated, embryos were transferred to media containing CHX before MBT and collected 90 minutes later for fixation. As CHX slightly delays development, some of these embryos do not present evident dorsal lips. (B) Expression of candidate Hex target genes in response to Hex-VP2. In situ hybridisation of embryos injected with Hex-VP2 and assayed for the expression of the same set of candidate genes as in A. Embryos were injected at the four-cell stage on the dorsal side alongside nucGFP RNA. Embryos were bisected for better observation of the internal expression. Dorsal is towards the right.

View larger version (58K): [in a new window]   Fig. 6. Xtle4 overexpression antagonises Hex-amplification of Wnt signalling. (A)In situ hybridisation for the expression of Xnr3 and Cerberus at stage 10.5. Embryos were injected at the two-cell stage in both blastomeres with 500 pg Hex, ß-catenin and/or 3000 pg Xtle4. (B) Activity of Hex and ß-catenin from the TOPflash Wnt reporter gene in the presence of Xtle4. The indicated DNA was co-transfected with either TOPflash or FOPflash in HEK 293T cells. Hex, Hex-VP2 and ß-catenin were used at 100 ng, Xtle4 at 90 ng. Values are represented as fold induction of the indicated reporters. Experiments were carried out in triplicate.

View larger version (67K): [in a new window]   Fig. 7. Hex suppresses the activity of Nodal-related TGFß proteins. (A) Axis duplication phenotypes induced by Hex and Xnr1. Embryos were injected into a single ventral-blastomere at the four-cell stage with 500 pg Hex, 250 pg Xnr1 or both and in situ hybridisation carried out at stage 35 using BF1. (B) Phenotypes induced by Hex and Xnr1 in ventral marginal zone explants. Embryos were injected with the indicated RNA at the two-cell stage, explants dissected at stage 10.5 and in situ hybridisation for BF1 carried out when staging control embryos reached stage 35. Hex was injected at 500 pg. Arrowheads indicate patches of BF1 expression. (C) In situ hybridisation for the expression of Cerberus at stage 10.5. Embryos were injected at the two-cell stage in both blastomeres with 500 pg Hex and/or 250 pg Xnr1 mRNA. (D) In situ hybridisation of Hex-depleted embryos for the mesendodermal markers Cerberus, Goosecoid and Chordin. Embryos were injected as in Fig. 1F. (E) Hex suppresses the induction of mesoderm in animal cap explants. Embryos were injected as in B with 500 pg Hex. Animal caps were dissected at blastula stage and cultured in 8 U/ml Activin protein until control embryos reached stage 18. (F) Molecular marker analysis in Hex-injected and Activin-treated animal cap explants. Animal caps were isolated from embryos injected as in B, cultured to stage 10.5 in 12 U/ml Activin, and RNA extracted for real-time RT-PCR analysis for Goosecoid, Chordin, Xbra and Mixer. Values are normalised to the expression level of Odc. Experiments were carried out in triplicate.

View larger version (28K): [in a new window]   Fig. 8. Schematic representation of Hex interactions with the Wnt and Nodal-related pathways. At blastula stage, Hex amplifies Wnt signalling through the repression of the Wnt antagonist Tle4. The Nieuwkoop centre genes Siamois and Xnr3 are markers of this process and this signalling centre is responsible for the induction of both anterior endoderm and axial mesoderm. Following mesendoderm induction, Hex is expressed in the anterior endoderm where it antagonises the propagation of the Nodal signal to prevent mesoderm formation in the head field. Broken lines indicate the induction of defined domains in the mesendoderm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter