Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts
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Abstract
Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here, we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in the direction predicted by a hair regeneration model: Wnt10a and Grem2a increased tooth replacement rate, whereas Bmp6 and Dkk2 strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA sequencing of stickleback dental tissue showed that Bmp6 overexpression resulted in an upregulation of Wnt inhibitors. Together, these data support a model in which different epithelial organs, such as teeth and hair, share genetic circuitry driving organ regeneration.
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However, Wnt10a OE did not significantly change the total number of teeth (P=0.81), demonstrating that changes to the regeneration rate don’t necessarily alter changes to total tooth number.
Might there be physical constraints on the tissue itself that prevent an increased number of tooth fields from forming? Are there examples of total tooth number increasing without a concurrent increase in the pharyngeal tooth plate surface?
Additionally, as this experiment only marks the number of teeth at two timepoints – the start and the end of the experiment – is it possible that an entire tooth replacement cycle has occurred within the 18-day span, or is tooth replacement sufficiently slow that you would not expect teeth to be "missed" by this analysis?
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G. An overlay of the pulse-chase treatment on zebrafish teeth using the same treatment interval (see panel B, zebrafish example).
The image label in the figure seems like it should be "G" as per the figure legend, rather than "H".
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Bmp6 was expressed similarly to Wnt10a in bud-stage teeth, exhibiting focal expression in both the epithelium and mesenchyme (Fig. 1G)
If Wnt and BMP are thought to have an oppositional effect on tooth replacement/regeneration, why are these two genes expressed in such similar patterns in early regenerating teeth?
Perhaps the early expression of BMP has the effect of repressing tooth formation in nearby tissue? What is the expression of BMP receptors within Wnt-expressing cells? It seems potentially notable that the Wnt-expressing cells appear to be a subset of the BMP-expressing cells, although a lack of bicolor in situ prevents any solid conclusions about coexpression.
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D. In zebrafish, significant increases in the number of new teeth (P=0.00039), the new:retained ratio (P=0.0042), and total teeth (P=0.011) were found but retained tooth number (P=0.56) did not significantly change.
It would be nice, if available, to show images of the zebrafish teeth with corresponding staining to in panels B and C.
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In the control condition, new teeth comprised mostly mid- or late bell stage tooth germs (Fig. 4B, arrows), whereas the few unankylosed new teeth we observed under Dkk2 OE were always at or near the eruption stage (Fig. 4C, arrow)
Is there a more quantitative summary of the categories of teeth (early, mid, late, erupted, etc.) observed for each condition you could point to as evidence of this?
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While we did not observe any change in the number of tooth families in any OE individual (n=15/15), we did find an increase in the total tooth number under wnt10a OE (P=0.011), due to a higher number of tooth families undergoing early replacement.
This result seems to depend on the inclusion of nascent teeth in the total number of teeth; the number of tooth families marked by erupted teeth is unchanged.
Is there a difference in the number of nascent teeth vs. erupted teeth in the stickleback case between the heat shock and no-heat shock conditions? Might that also affect the significance of the total tooth number result?
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Fig. 5, dotted oval
Probably refers to Fig. 6C?
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Surprisingly, Bmp6 OE also resulted in a decrease in retained teeth (P=2.8e-5), suggesting that Bmp6 negatively affects new tooth formation while also promoting the shedding of existing teeth.
Might be interesting to note that BMP OE seems not to be able to eliminate nascent teeth that have already begun the process of formation, as evidenced by the stalled stain-free teeth that are inferred to have arisen just after the pulse-chase treatment.
Maybe could be related to the fact that Bmp6 and Wnt seem to be expressed simultaneously in early teeth - perhaps those cells aren't receptive to Bmp6 signalling and can't be eliminated once they get going, but new tooth germs can be inhibited before they start being formed.
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These data support a model where different epithelial organs like teeth and hair share genetic inputs driving the timing of whole organ regenerative cycles.
I appreciated the thorough phenotyping analyses of these experiments and the inclusion of multiple species in this paper! I imagine it was also a great deal of work to perform these transgenic experiments in the stickleback, especially for multiple genes and across large numbers of individuals for several different conditions. I had some questions about the implications of the results, as well as about some of the more detailed phenotypes that were discussed but I wasn't able to find figures for in the manuscript.
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Discussion
More of a stylistic choice, but some kind of summary figure would be great to help encapsulate the findings and the comparison of results between zebrafish and stickleback. Perhaps a table of some kind indicating + vs - effects for each phenotype, for each species?
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All statistical tests were performed in R, using two-sided Wilcoxon rank-sum tests in all cases
It would be nice to indicate this in the figure legends, even if it's the same in each figure, for the sake of the reader.
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Effects of Wnt10a overexpression in stickleback and zebrafish.
Where possible, it would be great to include the exact number of individuals phenotyped for each condition plotted (basically, state the number of data points for each plot).
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Effects of Wnt10a overexpression in stickleback and zebrafish.
Where possible, it would be great to include the exact number of individuals phenotyped for each condition plotted (basically, state the number of data points for each plot).
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These data support a model where different epithelial organs like teeth and hair share genetic inputs driving the timing of whole organ regenerative cycles.
I appreciated the thorough phenotyping analyses of these experiments and the inclusion of multiple species in this paper! I imagine it was also a great deal of work to perform these transgenic experiments in the stickleback, especially for multiple genes and across large numbers of individuals for several different conditions. I had some questions about the implications of the results, as well as about some of the more detailed phenotypes that were discussed but I wasn't able to find figures for in the manuscript.
-
Discussion
More of a stylistic choice, but some kind of summary figure would be great to help encapsulate the findings and the comparison of results between zebrafish and stickleback. Perhaps a table of some kind indicating + vs - effects for each phenotype, for each species?
-
All statistical tests were performed in R, using two-sided Wilcoxon rank-sum tests in all cases
It would be nice to indicate this in the figure legends, even if it's the same in each figure, for the sake of the reader.
-
Surprisingly, Bmp6 OE also resulted in a decrease in retained teeth (P=2.8e-5), suggesting that Bmp6 negatively affects new tooth formation while also promoting the shedding of existing teeth.
Might be interesting to note that BMP OE seems not to be able to eliminate nascent teeth that have already begun the process of formation, as evidenced by the stalled stain-free teeth that are inferred to have arisen just after the pulse-chase treatment.
Maybe could be related to the fact that Bmp6 and Wnt seem to be expressed simultaneously in early teeth - perhaps those cells aren't receptive to Bmp6 signalling and can't be eliminated once they get going, but new tooth germs can be inhibited before they start being formed.
-
Fig. 5, dotted oval
Probably refers to Fig. 6C?
-
In the control condition, new teeth comprised mostly mid- or late bell stage tooth germs (Fig. 4B, arrows), whereas the few unankylosed new teeth we observed under Dkk2 OE were always at or near the eruption stage (Fig. 4C, arrow)
Is there a more quantitative summary of the categories of teeth (early, mid, late, erupted, etc.) observed for each condition you could point to as evidence of this?
-
While we did not observe any change in the number of tooth families in any OE individual (n=15/15), we did find an increase in the total tooth number under wnt10a OE (P=0.011), due to a higher number of tooth families undergoing early replacement.
This result seems to depend on the inclusion of nascent teeth in the total number of teeth; the number of tooth families marked by erupted teeth is unchanged.
Is there a difference in the number of nascent teeth vs. erupted teeth in the stickleback case between the heat shock and no-heat shock conditions? Might that also affect the significance of the total tooth number result?
-
D. In zebrafish, significant increases in the number of new teeth (P=0.00039), the new:retained ratio (P=0.0042), and total teeth (P=0.011) were found but retained tooth number (P=0.56) did not significantly change.
It would be nice, if available, to show images of the zebrafish teeth with corresponding staining to in panels B and C.
-
However, Wnt10a OE did not significantly change the total number of teeth (P=0.81), demonstrating that changes to the regeneration rate don’t necessarily alter changes to total tooth number.
Might there be physical constraints on the tissue itself that prevent an increased number of tooth fields from forming? Are there examples of total tooth number increasing without a concurrent increase in the pharyngeal tooth plate surface?
Additionally, as this experiment only marks the number of teeth at two timepoints – the start and the end of the experiment – is it possible that an entire tooth replacement cycle has occurred within the 18-day span, or is tooth replacement sufficiently slow that you would not expect teeth to be "missed" by this analysis?
-
G. An overlay of the pulse-chase treatment on zebrafish teeth using the same treatment interval (see panel B, zebrafish example).
The image label in the figure seems like it should be "G" as per the figure legend, rather than "H".
-
Bmp6 was expressed similarly to Wnt10a in bud-stage teeth, exhibiting focal expression in both the epithelium and mesenchyme (Fig. 1G)
If Wnt and BMP are thought to have an oppositional effect on tooth replacement/regeneration, why are these two genes expressed in such similar patterns in early regenerating teeth?
Perhaps the early expression of BMP has the effect of repressing tooth formation in nearby tissue? What is the expression of BMP receptors within Wnt-expressing cells? It seems potentially notable that the Wnt-expressing cells appear to be a subset of the BMP-expressing cells, although a lack of bicolor in situ prevents any solid conclusions about coexpression.
-