1-Azakenpaullone Increases Expression of the Wnt Target dkk1b
Constitutive activation of Wnt/β-catenin signaling in mutant zebrafish expressing a non-functional form of APC has been shown to induce a dramatic increase in the level of proliferation in both leading-zone progenitors and throughout migrating primordia (Aman and Piotrowski, 2008; Aman et al., 2011). However, the effects of Wnt/β-catenin activation on proliferation in neuromasts following their deposition by the primordium have yet to be determined. In order to temporally control the activation of Wnt signaling, we use the glycogen synthase kinase 3β (GSK3β) inhibitor 1-azakenpaullone (Az), which prevents the phosphorylation and degradation of β-catenin (Kunick et al., 2004), and which we have shown is able to exogenously activate Wnt signaling in the retina of zebrafish embryos and larvae (Meyers et al., 2012). To confirm that Az was leading to activation of the canonical Wnt pathway in lateral line neuromasts, we treated zebrafish embryos with 2.5 μM Az at 24 hpf, fixed them at 36 hpf or 48 hpf, and measured the expression of dkk1b and lef1, which are downstream Wnt targets (Niida et al., 2004; Niehrs, 2006; Gamba et al., 2010; Stephens et al., 2010; McGraw et al., 2011; Valdivia et al., 2011), via in situ hybridization. In DMSO-treated controls at 48 hpf, dkk1b expression was restricted to a narrow middle domain of the lateral line primordium, which had migrated to nearly the tail of the fish (Fig. 1A, arrow). Consistent with prior reports, in embryos treated with Az, primordium migration is slowed and stalls out midway along the trunk (Fig. 1B, arrow). However, in these fish dkk1b expression was strong throughout the primordium, in the rosettes about to be deposited, and was also found in the neuromasts that have already been deposited (arrowheads in Fig. 1B,C). lef1 expression at 36 hpf is restricted to the leading edge of the primordium in DMSO-treated controls (Fig. 1D), but in fish treated with Az, lef1 is found throughout the primordium and in the protoneuromast about to be deposited (Fig. 1E), This suggests that in response to Az treatment, the cells of the developing lateral line upregulate dkk1b and lef1, as expected for hyperactivation of Wnt signaling. Further, this suggests that Az can affect cells both prior to and following deposition from the primordium.
Figure 1. Expression of the Wnt targets dkk1 and lef1 is upregulated in the lateral line primordium in response to treatment with 2.5 μM Az beginning at 24 hpf. A–C: In situ hybridization with a probe against dkk1 shows normal expression in control, DMSO-treated embryos at 48 hpf (A) restricted to a narrow domain of the lateral line primordium (arrow), which has migrated almost to the tail. In contrast, in fish at 48 hpf treated with Az (B), the primordium has only migrated to approximately halfway along the yolk-extension, and dkk1 is expressed throughout the primordium (arrow) and remains elevated in deposited neuromasts (arrowheads). The boxed area is shown at higher magnification in C. D,E: In situ hybridization in DMSO-treated control fish at 36 hpf (D) shows normal expression of lef1 at the leading edge of the primordium (outlined with the dotted line). In Az-treated fish at 36 hpf, lef1 is expressed throughout the primordium and in the rosette about to be deposited (arrowhead). Scale bars: A,B = 250 μm; C–E = 25 μm.
Download figure to PowerPoint
1-Azakenpaullone Stimulates Proliferative Growth of Neuromasts Following Their Deposition
We next examined the effect of treatment with Az on lateral line development, using the Tg(cldnb:gfp) line (Haas and Gilmour, 2006) to visualize the primordium and deposited neuromasts. Similar to what has been reported in Wnt-activating apcmcr mutants (Aman and Piotrowski, 2008; Aman et al., 2011), treatment with Az limited how far the primordium migrated but had little effect on the number of neuromasts deposited. The primordium migrates almost to the tip of the tail within 48 hpf in DMSO-treated controls, depositing 7.8 ± 0.4 neuromasts along the length (N = 7; Fig. 2A,E,F). In contrast, in fish treated with Az at 24 hpf, the primordium migrated less than half the normal distance, stalling out partway through the yolk extension (Fig. 2B,E), but depositing 7.0 ± 0.4 neuromasts within that distance (N = 7; Fig. 2B,F). Delaying the onset of Az treatment increased the distance the primordium migrated, but had little effect on the number of deposited neuromasts (Fig. 2C,D). The differences in distance traveled were statistically significant in all pairwise comparisons (P < 0.05; ANOVA with Tukey post-hoc analysis). This confirms that treatment with Az phenocopies the effects of activating Wnt mutations, leading to the expected changes in migration of the lateral line primordium. It also shows that pharmacological manipulation can be used to control the specific onset of the induction of Wnt signaling in the lateral line.
Figure 2. Pharmacological inhibition of GSK3β with Az limits migration of the lateral line primordium. Whole-mount confocal examination of Tg(cldnb:gfp) zebrafish at 48hpf shows the extent of lateral line primordium migration and the location of deposited neuromasts (arrowheads). A: Embryos treated with DMSO as a vehicle control from 24–48 hpf deposit 7–9 neuromasts extending from the ear to the end of the tail. B: Embryos treated with 2.5 μM Az from 24–48 hpf show greatly reduced migration of the primordium, though 7–9 neuromasts were still deposited. C,D: Delaying the onset of Az treatment to 26 hpf (C) or 30 hpf (D) allowed the primordium to migrate further down the tail, but had little effect on the number of deposited neuromasts. In addition to the distance migrated, the size of the neuromasts in Az-treated fish appears larger than controls (insets show magnified view of the central-most neuromast for each fish). E: Quantification of the distance along the lateral midline beginning at the otic vesicle that the primordium had migrated by 48 hpf (n = 6 embryos per condition) in fish treated at varying times with Az or DMSO as a control. All pairwise comparisons between conditions are statistically significant (P < 0.05; ANOVA with post-hoc Tukey). F: Quantification of the number of deposited neuromasts at 48 hpf in fish treated with DMSO or Az at 24 hpf. Though Az significantly reduced the migration of the primordium, there was no significant effect on the number of deposited neuromasts (P > 0.05; t-test). Scale bar = 500 μm.
Download figure to PowerPoint
In addition to the distance the primordium migrated, we noted that neuromasts in all of the Az-treated embryos appeared much larger than those in DMSO-treated controls (Fig. 2A–D, insets). To examine this more specifically, we treated cldnb:gfp zebrafish with either 0.025% DMSO or 2.5 μM Az beginning at 24 hpf and fixed the fish at 72 hpf. When we measured the area of the second neuromast deposited in 7 fish, Az-treated neuromasts were significantly larger at 72 hpf than the DMSO-treated controls (Fig. 3A–C; 1,936.9 ± 81.8 μm2 vs. 830.7 ± 37.9 μm2; P < 0.001; t-test). To test whether this increase was due to changes in cell shape/size or cell number, we quantified the number of cells in the widest cross-section of each neuromast. DMSO-treated controls had 42.3 ± 1.8 cells in a single cross-section through the neuromast while Az-treated fish had 107.3 ± 7.0 cells (Fig. 3A',B',C; P < 0.0001; t-test). Notably, the ratio of cells to cross-sectional area shows that each cell in a DMSO-treated control takes up approximately 19.7 ± 0.6 μm2 while each cell in an Az-treated neuromast takes up 18.3 ± 0.7 μm2, which was not a significant difference in cell size (P > 0.1).
Figure 3. Az leads to increased size of deposited neuromasts and increased growth of the neuromasts following deposition. Tg(cldnb:gfp) zebrafish were treated with either 2.5 μM Az or DMSO as a vehicle control from 24–72 hpf. A,B: Projections of z-stacks showing the difference in size of the second-most anterior neuromast from DMSO-treated (A) or Az-treated (B) zebrafish. A',B': Individual z-sections from different neuromasts from DMSO- (A') and Az-treated (B') zebrafish showing the cross-sectional area of the neuromast (labeled with cldnb1:gfp) and the nuclei (DAPI; blue) in that cross-section. C: Quantification of the cross-sectional area of the neuromasts and number of nuclei in a cross-section through the neuromast reveals that Az treatment leads to a more than doubling in the area and number of cells in the neuromast (*P < 0.0001 for both; t-test). D–H: Time-lapse microscopy of Tg(cldnb:GFP) zebrafish treated from 26–72 hpf with DMSO (D,E) or 2.5 μM Az (F,G). Individual frames from one representative time-lapse are shown at 36 hpf (D,F; shortly after the deposition of the imaged neuromast) and at 72 hpf (E, G; approximately 36 hr after deposition of the imaged neuromast). H: Quantification of neuromast size at 36 and 72 hpf. The size of the deposited neuromast at 36 hpf is significantly larger in Az-treated fish compared to DMSO-treated controls. While control neuromasts decrease slightly in area over the next 36 hr as the neuromast condenses, Az-treated neuromasts increase in size by more than 33% (*P < 0.001), suggesting that activation of Wnt signaling triggers an increase in the number of cells in the deposited neuromast leading to growth of the neuromast following deposition. Scale bars: B' = 10 μm; G = 20 μm.
Download figure to PowerPoint
This increase in neuromast size and cell number stimulated by Az could be explained either by an increased number of cells in the protoneuromast as it is being deposited, or by stimulation of proliferative growth following deposition. To help distinguish between these two hypotheses, we conducted time-lapse microscopy of neuromasts from DMSO- or Az-treated zebrafish measuring the size of neuromasts shortly after deposition at 36 hpf and 36 hr after deposition at 72 hpf. Az-treated neuromasts were significantly larger than DMSO-treated controls shortly after deposition (Fig. 3D,F,H; 989.3 ± 128.2 μm2 vs. 707.5 ± 48.4 μm2; P < 0.001), suggesting that the activation of the Wnt/β-catenin pathway was triggering an increase in the number of cells being deposited within one protoneuromast. Furthermore, while DMSO-treated controls decreased in size slightly over the next 36 hr as the neuromast condensed into its round shape (to 654.8 ± 7.2 μm2), Az-treated neuromasts had increased in size by more than a third (Fig. 3D–H; 1,366.2 ± 105.4 μm2; P < 0.001). Thus, Az treatment leads to both an increase in the size of the protoneuromast being deposited and in growth of the neuromast post-deposition.
In order to test more directly whether Az stimulation was leading to growth of the neuromast following its deposition, we treated fish with DMSO or Az beginning at 24 hpf, then gave them a 10-min pulse of BrdU at either 48 or 96 hpf, fixing them 2 hr after BrdU-treatment. Az led to a more than doubling in the number of BrdU+ cells per neuromast when the BrdU pulse was given at 48 hpf (Fig. 4C; 31.75 ± 0.6 BrdU+ cells per neuromast for Az compared with 11.1 ± 0.5 BrdU+ cells per neuromasts for DMSO-treated controls, N = 20; P < 0.001). A BrdU pulse at 96 hpf, when the cells of the control neuromasts have become largely quiescent (Harris et al., 2003; Laguerre et al., 2005), labels only 5.6 ± 0.4 BrdU+ cells per neuromast, but Az-treated fish still maintained more than twice as many proliferating cells (12.5 ± 0.6 BrdU+ cells per neuromast; P < 0.001 compared to DMSO at 96 hpf; Fig. 4A–C). Thus, Az is capable of maintaining lateral line progenitors in a proliferative state long after deposition of the neuromast from the primordium.
Figure 4. Az stimulates a persistent increase in proliferation of lateral line progenitors dependent on the canonical Wnt signaling pathway. A–C: Zebrafish were treated with DMSO or 2.5 μM Az beginning at 24 hpf, then given a 10-min pulse label of BrdU either at 48 or 96 hpf with fixation following 2 hr after BrdU-labeling. Images show BrdU labeling (red) or BrdU overlaid on DAPI (blue) labeling all nuclei. Dashed lines represent the extent of the neuromast. A: DMSO-treated zebrafish labeled with BrdU at 96 hpf show few dividing supporting cells. B: Az-treated zebrafish labeled with BrdU at 96 hpf show supporting cells throughout the neuromast labeled with BrdU. C: Quantification of the number of BrdU-positive cells in two anterior neuromasts from each of 7 fish treated with BrdU at either 48 or 96 hpf and fixed 2 hr later. Az stimulates more than twice as many dividing cells as DMSO-treated controls at both time-points (*P < 0.001). D–F: Inhibition of the canonical Wnt/β-catenin signaling pathways by heat-shock induction of dominant-negative TCF3 at 48 hpf suppresses the Az-induced increase in the number of proliferating progenitors at 72 hpf. Wild-type siblings that do not express the dominant negative transgene show the significant increase in the number of BrdU+ cells at 72 hpf induced by Az (D,F), but this increase is completely suppressed in fish expressing the transgene (E,F; *P < 0.001). G–I: Inhibition of supporting cell proliferation by the dkk1 transgene. Following heat shock, wildtype fish show normal levels of proliferation (G,I), but transgenic siblings expressing the dkk1 transgene have largely suppressed the proliferation of their supporting cells (H,I; *P < 0.001; t-test). Scale bars: B, E, H = 10 μm.
Download figure to PowerPoint
As GSK3β is a component of many signaling pathways, we next wanted to confirm that the Az-mediated induction of proliferation was due to its effects on the canonical Wnt/β-catenin signal transduction cascade. We used fish expressing dominant negative TCF, which lacks a β-catenin binding site, under control of the heat shock promoter Tg(hsp70:dnTCF-GFP); Lewis et al., 2004) to test whether we could suppress the Az-stimulated augmentation of proliferation by blocking the output of the canonical Wnt pathway. We heat shocked transgenic and wild-type sibling embryos at 48 hpf for 1 hr, then treated them with DMSO or Az until 72 hpf followed by pulse labeling with BrdU and fixation. While wild-type siblings showed a similar Az-mediated increase in the number of proliferating cells in each neuromast compared to DMSO-treated controls (Fig. 4D,F), this effect was completely blocked in transgenic fish expressing dominant negative TCF (Fig. 4E,F).
As a method for blocking upstream activation of the Wnt cascade, we also used Tg(hsp70:dkk1b-GFP) zebrafish (Stoick-Cooper et al., 2007), which express the secreted inhibitor of the Wnt ligand, dkk1b, following heat shock. We allowed both wildtype and dkk1b transgenic fish to develop to 48 hpf, then gave them a 2-hr heat shock to induce expression of the transgene. Fish were reared until 60 hpf, given a 10-min pulse of BrdU, then fixed 3 hr after BrdU labeling. While wildtype siblings had 4.6 ± 0.8 BrdU-positive nuclei per neuromast (range 2–10 BrdU+ nuclei), transgenic fish expressing the dkk1b transgene had only 0.8 ± 0.4 BrdU-positive nuclei per neuromast (Fig. 4G–I; range 0–3 BrdU+ nuclei; P < 0.001). Thus, expression of dkk1b to block activation of the Wnt cascade significantly suppresses proliferation in the lateral line neuromasts just after their deposition.
Treatment With 1-Azakenpaullone Leads to Overproduction of Hair Cells
Prior work by Aman and Piotrowski (2008) suggested that constitutive activation of Wnt signaling in mutant embryos expressing a non-functional APC during lateral line development does not prevent the differentiation of precursor cells into mature hair cells. In contrast, pharmacological stimulation of Wnt signaling in the retina of fish at similar ages maintains progenitors in a proliferative fate at the expense of production of differentiated progeny (Yamaguchi et al., 2005; Meyers et al., 2012). To test whether the maintenance of lateral line progenitors in a proliferative state by Az treatment blocks differentiation, we examined the number of hair cells differentiated at 96 hpf in fish where Az was applied beginning at 2 dpf, such that all effects of Az were after deposition of the primary neuromasts. At 4 dpf, DMSO-treated fish had 10.3 ± 0.5 HCS-1-positive hair cells per neuromast in the two anterior-most neuromasts, while fish treated with Az had 16.3 ± 0.9 hair cells per neuromast in the two anterior-most neuromasts, a statistically significant increase (Fig. 5C; N = 12; P < 0.001). Thus, the initial maintenance of progenitors in a proliferative state leads to increased production of differentiating hair cells in Az-treated fish. To test whether this increased production could be maintained over longer incubations with Az, we counted HCS-1-positive hair cells in fish treated with DMSO or Az from 2–7 dpf. The two anterior-most neuromasts in DMSO-treated controls had 16.1 ± 1.6 hair cells per neuromast (a 56% increase from 4 dpf), while those treated with Az had 34.6 ± 2.3 hair cells per neuromast at 7 dpf (a 112% increase from 4 dpf; Fig. 5A–C). We were also able to observe the increase in hair cell production using the Tg(pou4f3:gap43-GFP) line of fish, which express membrane-bound GFP under control of the Brn3c promoter and thus have constitutively fluorescent hair cells (Xiao et al., 2005; Fig. 5D,E). Thus, Az-treatment not only led to an increase in the number of hair cells that were produced, but led to a sustained overproduction of hair cells, leading to a more than doubling of the normal number by 7 dpf. This demonstrates that the increase in proliferation triggered by Az does not come at the expense of hair cell differentiation, consistent with the work of Aman and Piotrowski (2008). In fact, these data suggest that Az is able to sustain an increased rate of hair cell production long after normal production has slowed.
Figure 5. Azakenpaullone triggers an increase in the number of hair cells differentiating in developing lateral line neuromasts. (A-B) Fish treated with 1-azakenapaullone beginning at 48 hpf show a dramatic increase in the size of the neuromast and in the number of HCS-1 positive hair cells at 7 dpf compared to DMSO-treated controls. (C) Quantification of the number of HCS-1 positive hair cells in an anterior neuromast from 7 fish treated with Az or DMSO beginning at 48 hpf. At both 4 dpf and 7 dpf, Az stimulated a significant increase in the number of differentiated hair cells, with more than twice as many hair cells produced by 7 dpf (*P < 0.01). (D-E) The increase in hair cell number can also be seen using the Brn3c:GFP transgenic line that express GFP in all of their hair cells. In all images, the dashed line represents the extent of the neuromast. Scale bar: 10 μm.
Download figure to PowerPoint
Treatment With 1-Azakenpaullone Stimulates Proliferation in Mature Neuromasts
The findings reported above demonstrate that exogenous activation of Wnt signaling stimulates progenitor proliferation in protoneuromasts and recently deposited neuromasts, but those experiments all initiated the stimulation of Wnt signaling when the progenitors/precursors were still proliferative, either as part of the proliferative primordium or shortly after deposition. However, the initially high levels of proliferation decrease during cell differentiation between 48–96 hpf, leading to quiescence of most cells in the neuromast, such that there are only 1–2 proliferative cells at any one time per neuromast in uninjured fish by 4–5 dpf (Williams and Holder, 2000; Harris et al., 2003; Laguerre et al., 2005; Ma et al., 2008). To test whether stimulation of Wnt signaling by Az would be able to trigger proliferation in these normally quiescent supporting and mantle cells, we added 2.5 μM Az, or DMSO as a vehicle control, to fish from 6–8 dpf, with BrdU present throughout to label all cells that pass through S-phase during the incubation period. To determine whether there was any distinction between the stimulation of peripheral supporting cells or internal supporting cells, we counted proliferative cells based on their position in the neuromast according to the definitions of Ma et al. (2008): the outermost row of cells and cells 2–3 layers past the neuromast border were defined as peripheral and cells inside that boundary were defined as internal. We counted the number of BrdU-positive cells for two anterior neuromasts in each of 6 fish per condition. Az led to a more than doubling of the number of proliferative cells during those two days, both for peripheral and internal supporting cells, compared to control, a statistically significant increase (Fig. 6A–C; P < 0.001). Thus, activation of Wnt-signaling by Az is sufficient to stimulate normally quiescent supporting cells in mature neuromasts to return to the cell cycle.
Wnt-Activation Increases Proliferation During Hair Cell Regeneration
Although uninjured neuromasts have a relatively low rate of ongoing proliferation, quiescent supporting cells readily return to the cell cycle to regenerate lost hair cells (Harris et al., 2003; Hernández et al., 2007; Ma et al., 2008). Since Az was sufficient to increase the number of normally quiescent supporting cells that proliferate in mature, uninjured neuromasts, we next asked whether it would also increase the number of cells that proliferate in response to hair cell lesion. We lesioned lateral line hair cells in zebrafish at 5 dpf with a 1-hr incubation in 500 μM neomycin (Harris et al., 2003), then incubated them in either DMSO or 2.5 μM Az for 2 days following the lesion with BrdU present continuously in the media. We quantified the number of BrdU+ cells in three anterior neuromasts for each of 13 fish. Az again stimulated a more than doubling in the number of BrdU-labeled supporting cells per neuromast from 16.8 ± 1.8 BrdU+ cells per neuromast in DMSO controls to 36.7 ±1.8 BrdU+ cells in fish treated with Az (Fig. 7A–C; P < 0.001). When we distinguished between internal and peripheral supporting cells, Az led to a doubling for each population (Fig. 7C), suggesting that, as seen above in unlesioned neuromasts, it is not selectively affecting only internal or peripheral supporting cells, but triggers both populations to proliferate.
As our data from the developmental studies suggest that stimulating Wnt/β-catenin signaling can maintain progenitors in a proliferative state for several days and over several rounds of division, it is possible that the increase in proliferating cells we see over the 48 hr following a lesion is not due to an increase in the number of supporting cells recruited to proliferate in response to the lesion, but merely maintenance of a limited number of cells in a proliferative state. To test whether Az actually stimulated an increase in the number of supporting cells returning to the cell cycle, we examined the number of BrdU+ cells in two anterior neuromasts 18 hr after lesion, a point when the number of cells returning to the cell cycle is peaking but without enough time for multiple rounds of mitosis. Az-treated fish had 13.0 ± 0.6 BrdU+ cells per neuromast (8.0 ± 0.3 internal supporting cells and 5.0 ± 0.5 peripheral supporting cells), a significant increase compared with only 5.5 ± 0.5 BrdU+ cells in DMSO-treated controls (Fig. 7D; 2.4 ± 0.3 internal supporting cells and 3.1 ± 0.4 peripheral supporting cells; P < 0.001 for each). Thus, Az stimulates a significant increase in the number of supporting cells that have returned to the cell cycle within 18 hr of the lesion, suggesting that Wnt/β-catenin signaling is sufficient to trigger supporting cells to re-enter the cell cycle.
In order to determine whether the canonical Wnt pathway was being activated in response to neomycin-induced hair cell loss, we examined expression of the TOP:gfp reporter transgene as a readout for active Wnt signaling (Dorsky et al., 2002). Control fish at 6 dpf that were not treated with neomycin show little expression of GFP within the neuromast (Fig. 7E). In contrast, 12 hr after 500 μM neomycin exposure to lesion the hair cells, the TOP:gfp transgene became strongly expressed in the neuromast (Fig. 7F). This suggests that the canonical Wnt pathway is being activated in neuromasts at the same time that the cells are beginning to proliferate in response to hair cell loss.
To test whether Wnt signaling was required for regenerative proliferation, we again used the Tg(hsp70:dkk1b-GFP) transgenic fish line to suppress activation of the Wnt pathway. The fish were heat-shocked at 39.5°C for 2 hr at 5 dpf, then placed in 500 μM neomycin for 1 hr to lesion the hair cells. Fish were maintained in BrdU for 18 hr, then fixed for staining. Wild-type siblings had 8.8 ± 1.5 BrdU+ cells per neuromast while transgenic fish expressing dkk1b had 3.9 ± 1.0 BrdU+ cells per neuromast, a significant decrease (Fig. 7G–I; P < 0.05; t-test).
1-Azakenpaullone Increases the Number of Regenerated Hair Cells
Finally, we asked whether the Wnt-stimulated increase in the number of supporting cells proliferating during hair cell regeneration led to a functional increase in the number of hair cells regenerated. We counted the number of GFP-positive hair cells from the Brn3c:gfp line for 12 fish per condition 72 hr after neomycin lesioning of the hair cells. While DMSO-treated controls had 8.7 ± 0.6 hair cells per neuromast, fish treated with Az had 14.1 ± 1.1 hair cells per neuromast, a statistically significant increase (Fig. 8A–C; P < 0.001). Thus, as during development, the stimulation of proliferation by Az results in an increased number of differentiating hair cells during regeneration.
Together these data demonstrate a novel role for Wnt/β-catenin signaling in controlling the proliferative capacity of lateral line progenitors during the development of the neuromasts and also of supporting cells in more mature neuromasts during their normal growth and regeneration.