How to assess Wnt activation in vivo

8 replies [Last post]

Sat, 10/09/2010 - 17:17

Naito@Cardiology

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Joined: 10/09/2010

How to assess Wnt activation in vivo

Hi. I'm Naito@Cardiology from osaka university, Japan (but not in Kikuchi-san's lab).

I am always wondering how to assess Wnt activation in vivo. Well, I know that TOPgal or BATgal mice are very useful tool,

but what is the most common method for assessing Wnt activation without using those transgenic mice.

Actually, i use qPCR for Wnt target genes (eg. Axin2..) and western blotting for active beta catenin (8E7, Millipore)

but in many cases the results are not consistennt.

For example, Axin2 is decreased while ABC is increased, or Axin2 is increased while dkk1 or Tcf7 is decreased etc...

Moreover, when i tried microarray analysis after treating many kinds of cells with Wnt3a, i become more confused..."What is the universal output for Wnt signaling???" .

I really hope for everyones idea.

Thank you,

Atsuhiko T Naito

Sat, 10/09/2010 - 22:56

Roel Nusse

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Joined: 07/12/2010

Wnt activation in vivo

The most universal outputs for wnt signaling are: axin2 expression, best measured in the axin2-LacZ mice, and beta-catenin nuclear staining.

http://www.stanford.edu/group/nusselab/cgi-bin/wnt/reporters

Sun, 10/10/2010 - 18:23

Naito@Cardiology

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Joined: 10/09/2010

Thank you

>Roel Nusse

Thank you for your response. Actually, your website is a Bible for me and i've read that page millions of times. Axin2-LacZ is very interesting for me and I will try them in the near future. I think you mean that i have to cross them to the mouse of our interest, right? Would you teach me about the sensitivity of that mouse? I am a cardiologist and have tried staining of the heart using TOP-gals, but never succeeded. I think (but not convinced) that Wnt signaling is activated in the heart under some circumstances, because the expression of Wnt targets are increasing: Axin2 (1.8-fold), c-myc (1.9-fold), (are Wisp-1&2 Wnt targets? Their expression is increased >10-fold). B-catenin staining might not be suitable for the heart, because b-catenin is extremely localized to the membrane (because of 'very tight' adherens junction?), and i have also failed in staining (or would you recommend me a good antibody?). Are the sensitivity of Axin2-LacZ greater than TOP-gals, at least in the heart? I do want to stain the heart because i want to know the cell types in which the Wnt signaling is activated. Sorry, too many questions. But needs your (and everyones) suggestions. Tom

Sun, 10/17/2010 - 12:41

eamadei

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Joined: 10/17/2010

Re: Question about role of SUFU in Wnt pathway

Dear Dr. Nusse,

My name is Elizabeth Amadei, and I am a graduate student in biomedical engineering at Georgia Tech. I am working on a project to develop a mathematical model of the Wnt pathway. I am specifically interested in the role of Suppressor of Fused (SUFU) on Wnt signaling. Meng et al. (2001) propose that Sufu binds to B-catenin and exports it from the nucleus. I noticed that the models posted on this webpage do not include SUFU. Is there a reason for this? Thank you for your help,

Elizabeth Amadei

Meng X, Poon R, Zhang X, Cheah A, Ding Q, Hui CC, Alman B (2001) Suppressor of fused negatively regulates beta-catenin signaling. J Biol Chem. 2001 Oct 26;276(43):40113-9.

Fri, 10/22/2010 - 16:00

admin

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Joined: 07/05/2010

Re: Question about role of SUFU in Wnt pathway

Thanks. By necessity, we cannot include all of the possible components of Wnt signaling in our diagrams, as there might be ~100 claims. Those components that have been established by multiple groups and papers are given preference.

Sun, 11/07/2010 - 10:30

Tomaberrant

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Joined: 11/07/2010

Wnt activity in patient material

Hi, We are trying to assess Wnt activity in patient material (tumor biopsies). We`ve got material to perform qPCR and IHC etc. Preliminary experiments have show that axin2 expression alone might not be the best parameter. Do you guys have any recommendation? Maybe look at multiple parameter?

Thanks for the help.

Best regrads,

Tom

Wed, 05/18/2011 - 10:43

EnricoP

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Joined: 05/18/2011

excellent

Thanks, Dr. Nusse. That's the exact information I was looking for.

Tue, 01/24/2012 - 18:24

Nahum (not verified)

Wnt activation

I work in the department of immunology at M.D. Anderson Cancer Center. I have generated a mouse model that reproduces the features observed in colitis-associated colorectal cancer. This mouse develops severe inflammation in the colon that leads to adenocarcinoma in few weeks. I did beta catenin staining in mutant colon samples and observed an extraordinary accumulation of this protein in the nucleus as compared with the WT (mainly in the cell membrane and cytoplasm); however, I did western blotting for TCF4/LEF and found little change in both proteins. My question is: how can I assess the disruption of the degradation complex? My hypothesis is that the knocked out protein is somehow stabilizing the complex. Is it possibe to analyze the dissociation of APC/Axin/LRP/DSH/Frz, separately? Will the dissociation serve as a read out to compare the mutant against the WT cells? I exposed deficient cells to LiCl, as a non-specific GSK-b inhibitor, to activate Wnt. The deficient and WT cells showed accumulation of beta catenin in the nucleus.

Thanks for your attention. I am not in the Wnt signaling field and I will really appreciate any help.

Best regards,

Nahum

-----------------------------------

Nahum Puebla, Ph.D.

Department of Immunology

M.D. Anderson Cancer Center

7455 Fannin. Box 902.

Houston, TX. 77054

Fri, 01/27/2012 - 09:22

Manuela

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Joined: 01/27/2012

help

Sorry to ask but I am alos lost with the activation in vivo, may you please help me.

Regards,
Manuela