Referee 2's Review (Paul Marker):

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication, the manuscript has been revised in light of these comments and to address other editorial requirements.
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The manuscript, "regulation of epithelial branching morphogenesis and cancer cell growth of the prostate by WNT signaling," presents a series of experiments that implicate canonical WNT signaling as important for the regulation of prostate developmental growth and branching morphogenesis as well as cancer cell growth and invasion. This is an interesting study, and it has the potential to further clarify the molecular basis for prostatic development. The data within the manuscript do support a regulatory role for WNT signaling during prostate development. However, the author's interpretation that the effects of manipulating WNT signaling are due to specific regulation of epithelial stem/progenitor cells over-interpret the data presented. Additional details are also needed to determine the reproducibility and statistical validity of the study.

General comments:
This study uses relatively few differentiation markers to identify the cell types affected by WNT signaling in the study. A marker of differentiated basal cells, p63, is examined in several of the figures. A second marker of differentiated luminal cells, CK8, is mentioned as data collected but not shown. These markers are also expressed by undifferentiated epithelial cells of the urogenital sinus. The authors interpret changes in the expression of these two markers to be an indication that WNT signaling regulates stem/progenitor cells. They also refer to p63-positive cells as progenitor cells. This is not appropriate. The markers used in this study are not adequate to determine if stem/progenitor cells are the target for regulation by WNT signaling. The molecular phenotype of prostate stem/progenitor cells has not been fully established. Recent studies suggest that sorting prostate epithelial cells for a CD45-CD31-Ter119-Sca-1+CD49f+ antigenic profile achieves a 60-fold enrichment of cells with stem cell properties. However, only 1 in 35 cells within the resulting cell population is estimated to be a stem cell. These studies suggest both that more work needs to be done to define the stem/progenitor cell population in the prostate and true stem/progenitor cells are relatively rare in the prostate epithelium. The authors of the current study would need to do substantially more to establish whether WNT signaling was affecting stem/progenitor cells as they claim rather than having more general affects on differentiated and/or lineage committed epithelial cells.

Specific Comments:
Figure 1: It is not clear from the legend, results, or methods how many organs were treated. Are the results shown in the figure reproducible? What is the variability among treated organs? Can any of the findings be quantified and statistically analyzed?

Figure 2: It is not clear from the figure how many organs were analyzed or cells counted to yield the data in the graph. The meaning of "**" on the graph is also not stated.

Figure 3: This figure evaluates proliferation rates at the end of a 7 day organ culture period. This analysis should be repeated earlier in the culture (for example, after 48 hours) to determine if the affects of changing WNT signaling on proliferation are similar throughout the culture period. The 7-day time point is problematic because the proliferation potential of actively-growing organ cultures becomes exhausted near the end of a 7-day culture period. This means that a culture that proliferates rapidly early in the culture period may yield an artificially low value near the end.

Figure 5: The graphs presented in M and N are not adequately explained in the figure legend. What does "number of beta-gal+ cells/duct" mean? Was the entire duct reconstructed from serial sections, or is this an average in a typical cross-section?