Reviewer's report
Title:Wnt-reporter expression pattern in the mouse intestine during homeostasis
Version: 1Date:14 September 2008
Reviewer: Ramesh Shivdasani
Reviewer's report:
A requirement for Wnt signaling in intestinal crypt cell proliferation is well
established and constitutive activation of the Wnt pathway leads to colorectal
cancer. In spite of this knowledge, the precise cells targeted by Wnt ligands in
intestinal crypts are not known, in part because reporter mouse strains give
confusing results. In this manuscript, Davies et al take on the challenge of
defining the Wnt target cell by careful examination of reporter gene (LacZ)
activity in two independent Wnt reporter strains, TOP-GAL and BatGal. They find
rare LacZ+ cells in 0.8% (colon) to 15% (duodenum) of intestinal crypts, usually
as isolated cells. About 7% of these cells show evidence of ongoing or recent
proliferation, as judged by Ki67 and BrdU stains, and LacZ+ cells do not express
the putative stem-cell marker DCAMKL-1. Some LacZ+ cells co-express the
Paneth cell markers cryptdin1 and UEA1 but never express 5-hydroxytryptamine,
a marker of enteroendocrine cells. Exposure of reporter mice to 12 Gy
gamma-irradiation induced a modest (but statistically significant) and transient
(1-3 days) increase in the number of LacZ+ cells and the proportion of
LacZ+Ki67+ double-stained cells.
This study attempts to tackle an important outstanding question, the data are
high in quality, and the paper is written well. The question comes down to the
originality of the results and their interpretation. The findings must also be
considered in relation to published observations on Wnt activity in intestinal
crypts.
Inhibition of Wnt signaling results in rapid loss of cell proliferation in virtually all
crypts, which implies that every crypt has a continuous dependence on Wnt
signals. Therefore, the authors’ detection of LacZ expression in only 1-15% of
intestinal crypts immediately calls the validity of the experimental system into
question. Both TOP-GAL and BatGal are transgenic mouse lines with unknown
sites of integration, and expression of the transgene is undoubtedly subject to
neighboring influences. The collective data from several reports suggests that
these mouse lines can track Wnt activity in embryos or newborn mice but
probably less so in adults (the fact that the epigenome is extensively
reprogrammed in embryos may explain this difference). The Wnt activity detected
in this study might therefore represent only a fraction of the total, perhaps in cells
with the highest signal or in a stochastic distribution, making it very hard to reach
a general conclusion. The best way to get around this limitation is to detect Wnt
activity by a different method, such as staining for nuclear beta-catenin, as the
authors did. This method is, however, notoriously difficult and prone to artifact,
and although the authors overcame the difficulties, their results exactly match
published data: that beta-catenin is found in the nucleus in just a few crypt cells,
mostly of the Paneth cell lineage. Indeed, post-mitotic Paneth cells, which reside
at the lowest tier in small intestine crypts, are known to receive Wnt signals, as
attested further by the results of the present study.
Major compulsory revisions:
The authors comment meticulously on the proportion of crypts with LacZ+ cells
and the fraction of LacZ+ cells that co-express markers of cell proliferation, but
the report is vague about the fraction of LacZ+ cells that express Paneth cell
markers. That number is critical to interpret this study correctly and to distinguish
between Wnt signaling in terminally differentiated versus progenitor cells. The
authors also noted LacZ staining of mature cells in “a few” intestinal villi and
attribute this finding to the long half-life of the beta-galactosidase protein. This
explanation does not factor in the considerable dilution that would occur over
multiple cell divisions, and again, the numbers of LacZ+ villi are not provided.
Knowing these values is essential to assess the validity of the experimental
model and the significance of the findings.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Wnt-reporter expression pattern in the mouse intestine during homeostasis
Version: 1Date:14 September 2008
Reviewer: Ramesh Shivdasani
Reviewer's report:
A requirement for Wnt signaling in intestinal crypt cell proliferation is well
established and constitutive activation of the Wnt pathway leads to colorectal
cancer. In spite of this knowledge, the precise cells targeted by Wnt ligands in
intestinal crypts are not known, in part because reporter mouse strains give
confusing results. In this manuscript, Davies et al take on the challenge of
defining the Wnt target cell by careful examination of reporter gene (LacZ)
activity in two independent Wnt reporter strains, TOP-GAL and BatGal. They find
rare LacZ+ cells in 0.8% (colon) to 15% (duodenum) of intestinal crypts, usually
as isolated cells. About 7% of these cells show evidence of ongoing or recent
proliferation, as judged by Ki67 and BrdU stains, and LacZ+ cells do not express
the putative stem-cell marker DCAMKL-1. Some LacZ+ cells co-express the
Paneth cell markers cryptdin1 and UEA1 but never express 5-hydroxytryptamine,
a marker of enteroendocrine cells. Exposure of reporter mice to 12 Gy
gamma-irradiation induced a modest (but statistically significant) and transient
(1-3 days) increase in the number of LacZ+ cells and the proportion of
LacZ+Ki67+ double-stained cells.
This study attempts to tackle an important outstanding question, the data are
high in quality, and the paper is written well. The question comes down to the
originality of the results and their interpretation. The findings must also be
considered in relation to published observations on Wnt activity in intestinal
crypts.
Inhibition of Wnt signaling results in rapid loss of cell proliferation in virtually all
crypts, which implies that every crypt has a continuous dependence on Wnt
signals. Therefore, the authors’ detection of LacZ expression in only 1-15% of
intestinal crypts immediately calls the validity of the experimental system into
question. Both TOP-GAL and BatGal are transgenic mouse lines with unknown
sites of integration, and expression of the transgene is undoubtedly subject to
neighboring influences. The collective data from several reports suggests that
these mouse lines can track Wnt activity in embryos or newborn mice but
probably less so in adults (the fact that the epigenome is extensively
reprogrammed in embryos may explain this difference). The Wnt activity detected
in this study might therefore represent only a fraction of the total, perhaps in cells
with the highest signal or in a stochastic distribution, making it very hard to reach
a general conclusion. The best way to get around this limitation is to detect Wnt
activity by a different method, such as staining for nuclear beta-catenin, as the
authors did. This method is, however, notoriously difficult and prone to artifact,
and although the authors overcame the difficulties, their results exactly match
published data: that beta-catenin is found in the nucleus in just a few crypt cells,
mostly of the Paneth cell lineage. Indeed, post-mitotic Paneth cells, which reside
at the lowest tier in small intestine crypts, are known to receive Wnt signals, as
attested further by the results of the present study.
Major compulsory revisions:
The authors comment meticulously on the proportion of crypts with LacZ+ cells
and the fraction of LacZ+ cells that co-express markers of cell proliferation, but
the report is vague about the fraction of LacZ+ cells that express Paneth cell
markers. That number is critical to interpret this study correctly and to distinguish
between Wnt signaling in terminally differentiated versus progenitor cells. The
authors also noted LacZ staining of mature cells in “a few” intestinal villi and
attribute this finding to the long half-life of the beta-galactosidase protein. This
explanation does not factor in the considerable dilution that would occur over
multiple cell divisions, and again, the numbers of LacZ+ villi are not provided.
Knowing these values is essential to assess the validity of the experimental
model and the significance of the findings.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Wnt-reporter expression pattern in the mouse intestine during homeostasis
Version: 1Date:14 September 2008
Reviewer: Ramesh Shivdasani
Reviewer's report:
A requirement for Wnt signaling in intestinal crypt cell proliferation is well
established and constitutive activation of the Wnt pathway leads to colorectal
cancer. In spite of this knowledge, the precise cells targeted by Wnt ligands in
intestinal crypts are not known, in part because reporter mouse strains give
confusing results. In this manuscript, Davies et al take on the challenge of
defining the Wnt target cell by careful examination of reporter gene (LacZ)
activity in two independent Wnt reporter strains, TOP-GAL and BatGal. They find
rare LacZ+ cells in 0.8% (colon) to 15% (duodenum) of intestinal crypts, usually
as isolated cells. About 7% of these cells show evidence of ongoing or recent
proliferation, as judged by Ki67 and BrdU stains, and LacZ+ cells do not express
the putative stem-cell marker DCAMKL-1. Some LacZ+ cells co-express the
Paneth cell markers cryptdin1 and UEA1 but never express 5-hydroxytryptamine,
a marker of enteroendocrine cells. Exposure of reporter mice to 12 Gy
gamma-irradiation induced a modest (but statistically significant) and transient
(1-3 days) increase in the number of LacZ+ cells and the proportion of
LacZ+Ki67+ double-stained cells.
This study attempts to tackle an important outstanding question, the data are
high in quality, and the paper is written well. The question comes down to the
originality of the results and their interpretation. The findings must also be
considered in relation to published observations on Wnt activity in intestinal
crypts.
Inhibition of Wnt signaling results in rapid loss of cell proliferation in virtually all
crypts, which implies that every crypt has a continuous dependence on Wnt
signals. Therefore, the authors’ detection of LacZ expression in only 1-15% of
intestinal crypts immediately calls the validity of the experimental system into
question. Both TOP-GAL and BatGal are transgenic mouse lines with unknown
sites of integration, and expression of the transgene is undoubtedly subject to
neighboring influences. The collective data from several reports suggests that
these mouse lines can track Wnt activity in embryos or newborn mice but
probably less so in adults (the fact that the epigenome is extensively
reprogrammed in embryos may explain this difference). The Wnt activity detected
in this study might therefore represent only a fraction of the total, perhaps in cells
with the highest signal or in a stochastic distribution, making it very hard to reach
a general conclusion. The best way to get around this limitation is to detect Wnt
activity by a different method, such as staining for nuclear beta-catenin, as the
authors did. This method is, however, notoriously difficult and prone to artifact,
and although the authors overcame the difficulties, their results exactly match
published data: that beta-catenin is found in the nucleus in just a few crypt cells,
mostly of the Paneth cell lineage. Indeed, post-mitotic Paneth cells, which reside
at the lowest tier in small intestine crypts, are known to receive Wnt signals, as
attested further by the results of the present study.
Major compulsory revisions:
The authors comment meticulously on the proportion of crypts with LacZ+ cells
and the fraction of LacZ+ cells that co-express markers of cell proliferation, but
the report is vague about the fraction of LacZ+ cells that express Paneth cell
markers. That number is critical to interpret this study correctly and to distinguish
between Wnt signaling in terminally differentiated versus progenitor cells. The
authors also noted LacZ staining of mature cells in “a few” intestinal villi and
attribute this finding to the long half-life of the beta-galactosidase protein. This
explanation does not factor in the considerable dilution that would occur over
multiple cell divisions, and again, the numbers of LacZ+ villi are not provided.
Knowing these values is essential to assess the validity of the experimental
model and the significance of the findings.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
assistant