Developmental Biology 361 (2012) 364–376

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Developmental Biology

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Distinct phases of Wnt/β- signaling direct cardiomyocyte formation in zebrafish

Tracy E. Dohn a,b,c,d, Joshua S. Waxman c,d,⁎ a Molecular and Developmental Biology Graduate Program, University of Cincinnati, USA b Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA c Molecular Cardiovascular Biology Division, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, 45229, USA d The Heart Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA article info abstract

Article history: Normal heart formation requires reiterative phases of canonical Wnt/β-catenin (Wnt) signaling. Understanding Received for publication 7 February 2011 the mechanisms by which Wnt signaling directs cardiomyocyte (CM) formation in vivo is critical to being able to Revised 25 October 2011 precisely direct differentiated CMs from stem cells in vitro. Here, we investigate the roles of Wnt signaling in Accepted 27 October 2011 zebrafish CM formation using heat-shock inducible transgenes that increase and decrease Wnt signaling. We Available online 4 November 2011 find that there are three phases during which CM formation is sensitive to modulation of Wnt signaling through the first 24 h of development. In addition to the previously recognized roles for Wnt signaling during mesoderm Keywords: fi fi Cardiomyocyte speci cation and in the pre-cardiac mesoderm, we nd a previously unrecognized role during CM differentiation Differentiation where Wnt signaling is necessary and sufficient to promote the differentiation of additional atrial cells. We also Wnt signaling extend the previous studies of the roles of Wnt signaling during mesoderm specification and in pre-cardiac Zebrafish mesoderm. Importantly, in pre-cardiac mesoderm we define a new mechanism where Wnt signaling is sufficient to prevent CM differentiation, in contrast to a proposed role in inhibiting cardiac progenitor (CP) specification. The inability of the CPs to differentiate appears to lead to cell death through a p53/Caspase-3 independent mechanism. Together with a report for an even later role for Wnt signaling in restricting proliferation of differentiated ventricular CMs, our results indicate that during the first 3 days of development in zebrafish there are four distinct phases during which CMs are sensitive to Wnt signaling. © 2011 Elsevier Inc. All rights reserved.

Introduction differentiation of CMs in vitro, we need to better understand the under- lying mechanisms by which Wnt signaling directs CM formation during Proper Wnt/β-catenin (Wnt) signaling is required to direct many these different phases of development. developmental processes and for the maintenance of tissues in adults The earliest phase of development during which Wnt signaling af- through controlling specification, differentiation, and proliferation fects CM formation is during mesoderm induction, when Wnt signal- (Chien et al., 2009; Moon et al., 2004). Of the organs that Wnt signaling ing promotes cardiac progenitor (CP) specification as a consequence regulates during development, much attention has recently been paid of inducing ventro-lateral mesoderm (Naito et al., 2006; Ueno et al., to the effects of Wnt signaling during cardiomyocyte (CM) develop- 2007). Because Wnt signaling also promotes mesoderm and dorsal ment (Cohen et al., 2008; Kwon et al., 2008; Tzahor, 2007). In vivo and vessel formation in Drosophila, it has been proposed that this function in vitro, the effects of Wnt signaling have been shown to oscillate of Wnt signaling is a conserved feature of metazoan development between promoting and restricting CM formation at different develop- (Naito et al., 2006; Tzahor, 2007). After mesoderm induction, the mental steps (Kwon et al., 2009; Naito et al., 2006; Qyang et al., 2007; role of Wnt signaling then changes in the pre-cardiac mesoderm, i.e. Tzahor, 2007; Ueno et al., 2007). The ability of the effects of Wnt signal- the mesoderm after the initial phase of induction from which the ing on CM formation to be translated from developmental models to CPs will eventually arise but prior to the onset of CP marker ex- embryonic stem (ES) cell culture offers the potential for rapid genera- pression, such as nkx2.5, tbx5 and gatas 4–6. Studies in vertebrate em- tion of novel stem cell based tissue engineering and regenerative bryos and ES cells have suggested that in the pre-cardiac mesoderm therapies to combat congenital heart defects and cardiomyopathies Wnt signaling is necessary to inhibit CP specification (Foley and (Domian et al., 2009). However, to ultimately direct the precise Mercola, 2005; Foley et al., 2007; Lavery et al., 2008b; Marvin et al., 2001; Naito et al., 2006; Paige et al., 2010; Schneider and Mercola, 2001; Tzahor and Lassar, 2001; Ueno et al., 2007; Wang et al., ⁎ Corresponding author. Fax: +1 513 636 5958. 2007). One proposed mechanism for how Wnt signaling affects CP E-mail address: [email protected] (J.S. Waxman). specification in the pre-cardiac mesoderm comes from ES cells,

0012-1606/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2011.10.032 T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376 365 where it has been suggested that vascular or hematopoietic fates are mesoderm. In contrast to the prevailing model that Wnt signaling specified at the expense of cardiac fates (Naito et al., 2006; Wang et inhibits CP specification through potential fate transformations, we al., 2007). However, other comparable studies using ES cells have instead find that Wnt signaling is only sufficient to prevent atrial not found this inverse relationship, clouding the mechanism behind and ventricular CM differentiation, without significant effects on CP Wnt signaling in this phase (Abu-Issa et al., 2004; Kwon et al., specification. Furthermore, the presumptive CPs that cannot differen- 2009; Paige et al., 2010; Ueno et al., 2007; Willems et al., 2011). tiate appear to die through a p53/Caspase-3 independent mechanism. Therefore, while the consensus is that Wnt signaling restricts CP spec- Finally, we also define a new role for Wnt signaling during CM differ- ification in the pre-cardiac mesoderm, the disparities between some entiation in zebrafish, where Wnt signaling is necessary and sufficient of these studies indicate we still do not have a complete picture of to promote atrial CMs. Although this is reminiscent of the role of the mechanism by which Wnt signaling affects the pre-cardiac Wnt2 in mice, our data indicate that the mechanism used to modulate mesoderm. atrial cell number in zebrafish is different than in mouse. Therefore, Most recent work has focused on the role of Wnt signaling in the these studies substantially extend our understanding of the genetic second heart field (SHF) of vertebrates. During this phase when CP and cellular mechanisms by which Wnt signaling controls CM forma- marker are expressed in the SHF, it has been proposed that Wnt tion in vivo and illuminates a previously unrecognized mechanism signaling promotes the renewal and proliferation of CPs at the expense controlling atrial CM differentiation in zebrafish. of differentiation (Klaus et al., 2007; Kwon et al., 2009; Qyang et al., Altogether with the later role of Wnt signaling in ventricular CMs 2007). Transgenic Wnt signaling reporters indicated that Wnt signaling (Rottbauer et al., 2002), these data suggest a model where there are occurs in the SHF, and pan-mesodermal depletion of Wnt signaling in- four distinct phases that CMs are sensitive to modulation of Wnt dicates that Wnt signaling is necessary and sufficient for specification signaling through the first 3 days of zebrafish development. Specifi- of CPs in the SHF (Klaus et al., 2007; Lin et al., 2007). Modulation of cally, the first two phases move from promoting to then restricting Wnt signaling using drivers specific to the SHF has indicated that Wnt CM development in the pre-gastrula embryo and pre-cardiac meso- signaling can expand the isl1 expressing CPs in the SHF through induc- derm, respectively. Then, Wnt signaling has specific effects on atrial ing proliferation (Cohen et al., 2007; Kwon et al., 2009; Lin et al., 2007; and ventricular cell development through promoting additional atrial Qyang et al., 2007). In addition, a recent study suggests that Wnt2, progenitor cell specification and subsequent differentiation, followed which is expressed in the pharyngeal mesoderm of mouse embryos, is by repressing proliferation in differentiated ventricular cells. necessary for the specification and proliferation of atrial progenitor cells in the posterior SHF (Tian et al., 2010). Therefore, in the SHF of Materials and methods mice and ES cells, it appears that Wnt signaling tightly regulates a bal- ance between specification, proliferation/renewal and differentiation Zebrafish husbandry and transgenic lines used of CPs. Studies of the zebrafish liebeskummer/ruvbl2 mutant have indicat- Zebrafish were maintained and embryos were obtained and raised ed an even later role for Wnt signaling in differentiated ventricular under standard conditions (Westerfield, 1993). The following transgenic CMs, where Wnt signaling is required to autonomously restrict ven- lines were used: Tg(hsp70l:wnt8a-GFP)w34 (Weidinger et al., 2005), Tg tricular proliferation (Rottbauer et al., 2002). The timing of this role, (hsp70l:dkk1-GFP)w32 (Stoick-Cooper et al., 2007), Tg(hsp70l:Δtcf3- where the phenotype is seen by 3 days post-fertilization, is compara- GFP)w26 (Lewis et al., 2004), Tg(−5.1myl7:nDsRed)f2 (Mably et al., tively much later than the other developmental roles of Wnt signaling 2003), Tg(−5.1myl7:EGFP)twu26 (Huang et al., 2003), Tg(kdrl:nlsEGFP)zf109 in CM formation in mice. Therefore, studies suggest that there are at (Blum et al., 2008)andTg(kdrl:mCherry)ci5 (Proulx et al., 2010). least 3 roles of Wnt signaling through the first 3 days of development in zebrafish: the first two roles may be conserved with mouse embry- os and both human and murine ES cells (Marvin et al., 2001; Naito et Heat-shock experiments al., 2006; Paige et al., 2010; Tzahor and Lassar, 2001; Ueno et al., 2007; Willems et al., 2011), while it is unclear if the later role in the For all experiments, unless otherwise indicated, the Tg(hsp70l:wnt8a- differentiated ventricular cells is conserved with other vertebrates. GFP)w34 and Tg(hsp70l:dkk1-GFP)w3 lines were used to increase and More recently, it was demonstrated that cells are added to both decrease Wnt signaling at the designated stages in embryos, respectively. poles of the zebrafish heart at time points between these stages (de For heat-shock experiments, hemizygous transgenic Tg(hsp70l:wnt8a- Pater et al., 2009; Zhou et al., 2011), which is reminiscent of later ad- GFP)w34, Tg(hsp70l:dkk1-GFP)w3 or Tg(hsp70l:Δtcf3-GFP)w26 adults were dition of CMs by the SHF in mammals and chicks (Dyer and Kirby, crossed with wild-type, homozygous Tg(−5.1myl7:nDsRed)f2, Tg 2009). However, it was not determined if Wnt signaling affects the (−5.1myl7:EGFP)twu26, Tg(kdrl:nlsEGFP)zf109 or Tg(kdrl:mCherry)ci5 adults. differentiation of the SHF as in mouse (Kwon et al., 2007, 2009; Resulting embryos were then heat-shocked at the designated stage in a Qyang et al., 2007). Therefore, while there are parallels between Biorad PCR machine by raising the temp to 37°C for 30 min, similar to heart development in zebrafish and other vertebrates, we still do what has been reported previously (Tucker et al., 2008). After ~1 h, the not have a clear understanding of the number of roles for Wnt signal- GFP from the transgenes was easily visible and the GFP positive (GFP+) ing or mechanisms that Wnt signaling uses in zebrafish to regulate sibling transgenic carriers were manually sorted from their heat- CM development. shocked GFP negative sibling embryos (Supplemental Fig. 1), which In this study, we examine the roles of Wnt signaling in CM devel- served as controls, using a Zeiss M2BioV12 Stereomicroscope. Heat- opment of zebrafish during three phases of development: during me- shocked transgenic embryos at the sphere stage produced dorsalized soderm induction, in the pre-cardiac mesoderm, and during CM and ventralized phenotypes equivalent to what has been reported previ- differentiation. With respect to the role of Wnt signaling in meso- ously (data not shown; Stoick-Cooper et al., 2007; Weidinger et al., 2005). derm induction, we extend previous studies through showing that Similarly, heat-shocks administered at the 16 somite (s) stage affected the zebrafish Wnt8a paralogs promote atrial and ventricular CM spec- axin2 expression, a Wnt signaling responsive gene, at the 20 s stage in ification similarly, although with slight dorso-ventral (DV) biases. the predicted manner for the Tg(hsp70l:wnt8a-GFP)w34 and Tg(hsp70l: With respect to the role of Wnt signaling in pre-cardiac mesoderm, dkk1-GFP)w3 embryos (Supplemental Fig. 2). Together, these data suggest we find that the pre-cardiac mesoderm is sensitive to Wnt signaling that we are effectively modulating Wnt signaling with the Tg(hsp70l: from the initiation of gastrulation through early somitogenesis, wnt8a-GFP)w34 and Tg(hsp70l:dkk1-GFP)w3 lines at all stages examined, which is longer than previously demonstrated. Importantly, we also consistent with what has been reported previously (Stoick-Cooper et al., define a new mechanism for the role of Wnt signaling in pre-cardiac 2007; Weidinger et al., 2005). 366 T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376

Zebrafish embryo injections Acridine orange

Zebrafish embryos were injected at the one cell stage with morpho- Embryos were placed in 10 μg/ml acridine orange (AO; Sigma) for linos (MOs) or mRNA. wnt8a.1 and wnt8a.2 MOs and wnt8a.1 mRNA 30 min in embryo water (without methylene blue) (Westerfield, were reported previously (Lekven et al., 2001). For MOs, a mixture of 1993). Embryos were then washed 3× for 10 min with embryo both MOs containing 2 ng each was injected. For wnt8a.1 mRNA, water while gently shaking on a nutator. Embryos were visualized 50 pg was injected. Capped mRNA was made using a Message Machine and photographed using a Zeiss M2BioV12 Stereo microscope and Kit (Ambion). p53 MO was reported previously (Robu et al., 2007)and Zeiss Axio Imager Z1 with ApoTome. injected at 5 ng into one cell stage Tg(hsp70l:wnt8a-GFP)w34 embryos. Whole mount immunohistochemistry In situ hybridization Detection of activated Caspase-3 at 20 s was performed as previously In situ hybridization (ISH) was performed as reported previously described (Sidi et al., 2008). (Oxtoby and Jowett, 1993). All probes were reported previously: myl7 (formerly called cmlc2; ZDB-GENE-991019-3), amhc (ZDB-GENE- Quantitative real time PCR 031112-1), vmhc (ZDB-GENE-991123-5), nkx2.5 (ZDB-GENE-980526- 321), gata4 (ZDB-GENE-980526-476), gata6 (ZDB-GENE-000622-1), Total RNA was isolated from staged embryos homogenized in aldh1a2 (ZDB-GENE-011010-3), etv2 (ZDB-GENE-050622-14), kdrl TRIzol (Ambion) and collected using RNeasy mini columns (Qiagen). (ZDB-GENE-000705-1), fli1a (ZDB-GENE-980526-426), tbx5a (ZDB- TURBO DNA-free kit (Applied Biosystems) was used to remove geno- GENE-991124-7), tbx6 (ZDB-GENE-980526-171), and flh (ZDB-GENE- mic contamination. 1 μg RNA was used for cDNA synthesis using the 990415-75). ThermoScript Reverse Transcriptase kit (Invitrogen). Quantitative real time PCR (qPCR) for myl7, amhc, vmhc, nkx2.5, tbx5, gata4, and ß- was performed using standard PCR conditions in a Bio-Rad Area measurements CFX PCR machine with Power SYBR Green PCR Master Mix (Applied Biosystems). Expression levels were standardized to ß-actin expres- Areas of myl7, vmhc and amhc expressing cells were measured as sion. All experiments were performed in triplicate. Primer sequences previously reported (Waxman et al., 2008). Briefly, ImageJ was used and PCR protocol are available upon request. to measure the areas of the total amount of cells expressing myl7 and vmhc at the 20 s stage and amhc and tbx5a at the 22 s stage. Be- cause slight variation can occur between ISH of different experiments, Results only samples from individual experiments were compared to each other. Measurements were made using arbitrary units. Student's Zebrafish Wnt8a paralogs are necessary and sufficient to promote atrial t-test was used to determine if the means from the individual exper- and ventricular CM specification in the pregastrula embryo iments were statistically different (pb0.05). Previous studies have not determined if Wnt signaling, an effector of dorso-ventral patterning (DV) (Baker et al., 2010; Lekven et al., Cell counting and imaging of zebrafish hearts 2001; Ueno et al., 2007), affects ventricular and atrial cells similarly in the pregastrula embryo. BMP signaling, also an effector of DV Cell counting was done as previously described (Waxman et al., patterning (Nguyen et al., 1998), differentially affects the formation 2008). Briefly, hemizygous transgenic Tg(hsp70l:wnt8a-GFP)w34 or of these CMs consistent with their relative positions within the pre- Tg(hsp70l:dkk1-GFP)w3 adults were crossed to homozygous Tg gastrula embryo (Keegan et al., 2004; Marques and Yelon, 2009). (−5.1myl7:nDsRed)f2 adults. After selection of heat-shocked control Therefore, we hypothesized that Wnt signaling may differentially sibling embryos (HCSEs) and GFP+ embryos, embryos were har- affect ventricular and atrial CM formation during this early patterning vested at 48 hours post-fertilization (hpf), fixed and processed using event. To determine the role of Wnt signaling in ventricular and atrial an α-DsRed (Clontech) and S46 antibody (Stainier and Fishman, CM formation prior to gastrulation, we used the heat-shock inducible 1992). Embryos were then gently flattened underneath a cover slip. Tg(hsp70l:wnt8a-GFP)w34 and Tg(hsp70l:dkk1-GFP)w3 lines to modu- The hearts were photographed and the number of cardiac cells in late Wnt signaling at the sphere stage. We then examined the earliest each chamber was counted. Because slight variation in cell number ventricular and atrial specific differentiation markers, ventricular can occur between different clutches of embryos and experiments, heavy chain (vmhc) and atrial myosin heavy chain (amhc), at only counts from individual experiments were compared to each the 20 s and 22 s stages, respectively (Berdougo et al., 2003; Yelon other. Student's t-test was then used to determine if the means et al., 1999). We also examined myl7 at the 20 s stage, which at this from the individual experiments were statistically different stage primarily marks ventricular cells though later it is expressed (pb0.05). For imaging of hearts, without the nuclear DsRed trans- in all differentiated CMs (Berdougo et al., 2003; Yelon et al., 1999). gene, the MF20 antibody (Stainier and Fishman, 1992) was used to Similar to what has been previously reported (Ueno et al., 2007), visualize all cardiac cells. we found that increasing and decreasing Wnt signaling prior to gastrulation, using Tg(hsp70l:wnt8a-GFP)w34 and Tg(hsp70l:dkk1- Phospho Histone H3 (pHH3) staining GFP)w3 embryos respectively, resulted in increased and decreased myl7 (Figs. 1B, 2B and Supplemental Table 1). With respect to vmhc An anti-pHH3 antibody (Ser10; Millipore) was used to identify and amhc, we found that modulation of Wnt signaling had similar proliferating cells. To be able to distinguish between atrial and endo- effects on both these markers (Figs. 1H, N, S; 2H, N, S and Supplemental cardial cells, we crossed the transgenic Wnt and Dkk lines to the Tg Table 1), though quantification of the amount of cells revealed potential (kdrl:nlsEGFP)zf109 line, which allows for the visualization of the DV biases (Figs. 1S, 2S and Supplemental Table 1). The more ventrally nuclei in endothelial cells. After fixation and processing as above, located atrial cells had a greater fold increase when Wnt signaling was except at 24 and 26 hpf, we used the α-AMHC (S46) antibody. To increased relative to the more dorsally located ventricular cells enhance the visualization of GFP after fixation, we used an α-GFP (Fig. 1S; Keegan et al., 2004). Conversely, the ventricular cells were antibody (Millipore). slightly more sensitive to loss of Wnt signaling (Fig. 2S). T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376 367

Fig. 1. Effects of increased Wnt signaling on CM differentiation markers. (A–F) myl7,(G–L) vmhc and (M–R) amhc expression in representative HCSEs and embryos when Wnt signaling is increased using the Tg(hsp70l:wnt8a-GFP)w34 line at the designated stages. Increased Wnt signaling promoted (B,H,N) and then inhibited (C–E,I–K,O–Q) the expression of CM differentiation markers when Wnt signaling was increased prior to gastrulation at the sphere (SP) stage and from the initiation of gastrulation [shield (SH)] through early somitogenesis. (F,L,R) Increasing Wnt signaling at 16 s does not overtly affect CM differentiation markers. (S) Graph indicating the fold difference in areas of cardiac differentiation marker expression. Asterisks indicate a statistical difference in the areas from HCSEs and GFP+ embryos in Supplemental Table 1 according to Student's t-test. Images in all figures are dorsal views with anterior up, unless otherwise indicated.

To corroborate the ISH analysis, we also used qPCR to examine the would be predicted. Therefore, we tested if the Wnt8as are required expression of the CM differentiation markers in embryos where Wnt for CM formation through co-injection of wnt8a.1 and wnt8a.2 MOs signaling had been modulated prior to gastrulation. Similar to the (Lekven et al., 2001). We found that depletion of these wnt8as causes trends that we observed with the ISH analysis (Figs. 1S and 2S), we a reduction in cells expressing myl7, vmhc and amhc (Figs. 4B,E,H). found that increasing Wnt signaling promoted the expression of the While the trends on the cardiac differentiation markers were similar cardiac differentiation markers (Fig. 3A), while decreasing Wnt sig- to what we observed using the Tg(hsp70l:dkk1-GFP)w32 line (Figs. 2B, naling reduced the expression of the cardiac differentiation markers H,N), the effects on the cardiac differentiation markers were less strong. (Fig. 3D). Like with the ISH, amhc expression was more sensitive to The minor differences in effects on CM differentiation markers could be increased Wnt signaling, while vmhc was more sensitive to decreased due to differences in efficacy of Wnt signaling inhibition from the Wnt signaling. We note that in contrast to the ISH analysis we did not hsp70l:dkk1-GFP transgene vs.thewnt8a MOs. Alternatively, another observe a significant effect on amhc expression using qRT-PCR. How- possibility is that wnt3a also plays a minor role promoting cardiac ever, this could be due to that amhc expression was more variable in progenitor specification, consistent with its limited role in DV patterning control sibling and transgenic embryos. Altogether, our data suggest of the mesoderm compared to wnt8a.1 and wnt8a.2 (Thorpe and Moon, that Wnt signaling is necessary and sufficient for the formation of 2004). Gain-of-function experiments with injected wnt8a.1 mRNA led to both atrial and ventricular CMs, which exhibit slight DV biases in sen- the opposite effect, an increase in the amount of cells expressing myl7, sitivity to Wnt modulation. vmhc and amhc (Figs. 4C,F,I). Therefore, these results further support We next wanted to determine which endogenous Wnt(s) control that increased Wnt signaling prior to gastrulation is sufficient to promote CM formation through patterning the mesoderm. In zebrafish, two the formation of differentiated CMs, while the wnt8a paralogs are neces- Wnt8a paralogs (wnt8a.1 and wnt8a.2) are required for ventro-lateral sary for the formation of differentiated CMs in zebrafish embryos. mesoderm induction (Baker et al., 2010; Lekven et al., 2001; Ramel Wnt8a paralogs in zebrafish are necessary for ventro-lateral and Lekven, 2004), yet it has not specifically been shown that these mesoderm specification (Baker et al., 2010; Lekven et al., 2001; Wnt8as are necessary for CM specification and differentiation, as Martin and Kimelman, 2008; Ueno et al., 2007). The subtle differences 368 T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376

Fig. 2. Wnt signaling is necessary for CM formation in the pregastrula embryo. (A–F) myl7,(G–L) vmhc and (M–R) amhc expression in representative HCSEs and embryos when Wnt signaling is decreased using the Tg(hsp70l:dkk1-GFP)w32 line at the designated stages. (B,H,N) Wnt signaling is necessary prior to gastrulation to promote ventricular and atrial marker expressions. (C, I) Decreasing Wnt signaling at the initiation of gastrulation (shield stage) causes aberrant morphology of CMs expressing myl7 and vmhc. (O) Decreasing Wnt signaling at the initiation of gastrulation reduces the amount of cells expressing amhc.(D–F,J–L,P–R) The expression of CM differentiation markers is not affected when Wnt signaling is decreased from TB through 16 s. (S) Graph indicating the relative difference in area of cells expressing these markers. Asterisks indicate a statistical difference in the areas from HCSEs and GFP+ embryos in Supplemental Table 1 according to Student's t-test.

in atrial and ventricular sensitivity could be consistent with effects on region was expanded (Supplemental Figs. 4A,B). Moreover, floating ventro-lateral mesoderm specification and the more ventral location head (flh), a marker of dorsal mesoderm (Talbot et al., 1995), and of atrial progenitors compared to the ventricular progenitors in the tbx6, a marker of ventro-lateral mesoderm (Hug et al., 1997), were pregastrula embryo (Keegan et al., 2004), like what has been found expanded and reduced in the dorsal embryo respectively (Supplemental with BMP signaling (Marques and Yelon, 2009). To confirm that our Figs. 4E,F,I,J), consistent with what has been reported previously for loss abrogation of Wnt signaling is affecting mesoderm specification, we of Wnt signaling in the early embryo (Baker et al., 2010; Lekven et al., first examined aldh1a2, an early Wnt responsive gene that is initially 2001). Therefore, based on the effects upon DV markers, we conclude expressed throughout the ventro-lateral mesoderm (Weidinger et al., that zebrafish wnt8a paralogs affect atrial and ventricular specification 2005), in wnt8a morphants and wnt8a.1 mRNA injected embryos. as a consequence of their role in dorso-ventral patterning of the Consistent with effects on ventro-lateral mesoderm specification, mesoderm. we found that decreasing and increasing Wnt8a signaling reduced and expanded aldh1a2 expression, respectively, at the margin in Wnt signaling is sufficient to inhibit CM differentiation during gastrulation shield stage embryos (Supplemental Fig. 3). and early somitogenesis We next examined markers of mesodermal DV patterning at ~80% epiboly in Tg(hsp70l:dkk1-GFP)w3 embryos heat-shocked at the We next wanted to better understand the role of Wnt signaling in sphere stage. Shortly after its initial expression in the ventro-lateral the pre-cardiac mesoderm. Previously, the length of this second mesoderm, aldh1a2 is no longer directly sensitive to Wnt signaling phase, when Wnt signaling was proposed to inhibit CM progenitor and its expression becomes excluded from the ventral and dorsal re- specification, was not determined (Ueno et al., 2007). Therefore, to gions of the embryo, making it an excellent marker for DV patterning precisely define the time period when the pre-cardiac mesoderm is events by 80% epiboly. In embryos with decreased Wnt signaling at sensitive to modulation of Wnt signaling, we modulated Wnt signaling the sphere stage, we found that shortly before the end of gastrulation using the aforementioned Wnt8 and Dkk transgenic lines at the shield aldh1a2 was now expressed on the ventral most side while the dorsal and tail bud (TB; end of gastrulation) stages, as well as stages that T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376 369

Fig. 3. qPCR analysis of myl7, vmhc and amhc expression after modulation of Wnt signaling. (A) Increasing Wnt signaling at the sphere stage causes an increase in CM differentiation marker . (B) Increasing Wnt signaling at the TB stage causes a decrease in CM differentiation marker gene expression. (C) Increasing Wnt signaling at the 16 s stage does not have a significant effect on CM differentiation marker gene expression. (D) Decreasing Wnt signaling causes a decrease in myl7 and vmhc expression. (E,F) Decreasing Wnt signaling at the TB and 16 s stages does not affect CM differentiation marker gene expression. Analysis of gene expression was performed on embryos at the 20–22 s stages. All trends of expression examined with qPCR, except for amhc after decreasing Wnt signaling at sphere, are similar to those observed in Figs. 1 and 2 with quantification by ISH area. The inability to detect a decrease in amhc expression could be due to the higher variability of amhc observed in both control and transgenic embryos. Although vmhc is also expressed in the somites, trends with respect to expression closely parallel those observed in the heart. However, we cannot rule out that the trends observed reflect additional effects of Wnt signaling on vmhc in the somites. Error bars for all qPCR analysis indicate the standard deviation of the triplicates used for analysis. Asterisks for all qPCR experiments indicate a statistically significant difference (pb0.05) in expression using Student's t-test. were not examined previously: the 8 s stage (initiation of robust CP O–Q, S). The CM differentiation markers were no longer sensitive to gene expression) and 16 s stage (initiation of cardiac differentiation increased Wnt signaling at the 16 s stage (Figs. 1F, L, R, S). The loss of gene expression). We then examined the areas of myl7, vmhc,and CM differentiation marker expression after increasing Wnt signaling amhc expression, as described above. We found that increasing Wnt at the TB stage and lack of effect on CM differentiation marker expres- signaling inhibited CM formation through 8 s and embryos were most sion after increasing Wnt signaling at the 16 s stage were confirmed sensitive to increased Wnt signaling at the TB stage (Figs. 1C–E, I–K, using qPCR (Figs. 3B and C). Therefore, in zebrafish embryos, the pre- cardiac mesoderm is sensitive to increased Wnt signaling over a rela- tively long period of time, extending from the initiation of gastrulation through early somitogenesis. Because of the lack of inverse relationship observed with Wnt signaling in the pre-cardiac mesoderm, we speculated that there could be effects on the specification of CPs, which are then compensated for during differentiation leading to a normal number of CMs. To explore this possibility, we examined the CP markers nkx2.5 and gata4, which also mark more anterior mesoderm progenitors, at the 8 s stage after modulating Wnt signaling at the TB stage. In contrast to what has been reported previously (Ueno et al., 2007), we did not find a discernible effect on either of the CP markers in embryos with decreased Wnt signaling (Supplemental Figs. 6A,B,E,F), suggesting that decreased Wnt signaling is not affecting CP specification. When we examined embryos with increased Wnt signaling, we were also sur- prised that the majority (89%; n=45) had normal nkx2.5 expression (Supplemental Figs. 6C,D) and no discernible effect on gata4 expression (Supplemental Figs. 6G,H). The lack of effects on CP markers via ISH after modulation of Wnt signaling at the TB stage was confirmed using qPCR (Supplemental Figs. 6I,J). Because we did not observe a strong effect of increased Wnt signaling at the TB stage upon nkx2.5 expression at 8 s, we next examined nkx2.5 expression at the 14 s stage via ISH. In this case, 100% (n=27) of embryos had nkx2.5 expression that was indistinguishable from HCSEs (Figs. 5A,B), further supporting that increased Wnt signaling in the pre-cardiac mesoderm does not Fig. 4. Zebrafish Wnt8a paralogs are required to promote proper CM formation. (A,D,G) affect CP specification. Altogether, these results suggest that increased Control uninjected embryos. (B,E,H) Embryos depleted of wnt8a.1 and wnt8a.2 display a fi reduced mount of cells expressing myl7, vmhc and amhc. (C,F,I) Embryos injected with Wnt signaling does not affect CP speci cation nor is Wnt signaling wnt8a.1 mRNA display increased numbers of cells expressing myl7, vmhc and amhc. required to limit the number of CPs in the pre-cardiac mesoderm. 370 T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376

Fig. 5. Increased Wnt signaling at the TB stage causes a loss of nkx2.5 expression after the 14 somite stage. (A,C) HCSEs. (B,D) GFP+ sibling embryos. Expression of nkx2.5 is normal in embryos at the 14 s stage after increasing Wnt signaling at the TB stage (B; 100%, n=27), compared to HCSEs (A; n=38). (C) nkx2.5 expression in HCSEs (n=21). (D) Expression of nkx2.5 is lost by 20 s (100% had severely reduced nkx2.5 expression, n=27) in GFP+ embryos after heat-shock at the TB stage. There are diffuse nkx2.5 expressing cells at the midline (large arrowhead). Posterior pharyngeal arch staining appears increased in embryos with increased Wnt signaling at the TB stage (small arrowheads). (E) qPCR analysis of nkx2.5 expression indicates that total nkx2.5 expression is reduced in 20–22 s stage GFP+ embryos after increasing Wnt signaling at the TB stage.

Increased Wnt signaling in pre-cardiac mesoderm prevents CM causing these cells to die. To assay apoptotic cell death in embryos differentiation leading to cell death with increased Wnt signaling at the TB stage, we treated embryos with the vital dye acridine orange at the 20 s stage (Furutani-Seiki We have found that increased Wnt signaling in the pre-cardiac et al., 1996). In contrast to HCSEs, where there were only sporadic mesoderm eliminates expression of cardiac differentiation markers, dying cells throughout the embryo and never large clusters of apopto- yet we find that CP specification initially is unchanged. One potential tic cells, embryos with increased Wnt signaling at the TB stage had model that could explain this is similar to what has been found for large bilateral populations of dying cells (Figs. 6A,B; 7A–F). The clus- Wnt signaling and cardiac cell differentiation of the SHF, where it has ters of apoptotic cells were exactly where CPs would be expected to been proposed that Wnt signaling promotes cell renewal/proliferation reside in the lateral plate mesoderm (LPM) if they had not migrated of CPs at the expense of differentiation (Qyang et al., 2007). Because to the midline and begun to differentiate (Figs. 7A–F and Supplemental we did not observe an increase in nkx2.5 expression at 14 s (Figs. 5A, Figs. 8A–F; Bussmann et al., 2007). From one representative experiment, B), we examined nkx2.5 expression later at the 20 s stage in embryos 100% (n=24) of embryos with increased Wnt signaling at the TB stage after Wnt signaling had been increased at the TB stage. In contrast to a had clusters of apoptotic cells in the LPM at 20 s, while 0% (n=14) of model where CPs are maintaining a proliferative state, we found that their HCSEs had clusters of apoptotic cells. Dying cells were also observed there were a small number of cells flanking the midline that expressed in the anterior neural tissue where Wnt repression is also required (Kim nkx2.5 at a low level in these embryos (Figs. 5C,D). Interestingly, we also et al., 2000). However, in other regions of the transgenic embryos, there found that there appeared to be a modest increase in pharyngeal cells was only minimal sporadic cell death equivalent to what was observed in expressing nkx2.5 (Figs. 5C,D), yet overall nkx2.5 expression was HCSEs. Furthermore, the analysis of hematopoietic and vascular markers decreased (Fig. 5E). The pharyngeal expression is thought to initiate (Supplemental Figs. 7A,B,E,F,I,J) and the lack of AO positive endothelial later and not be derived from the cardiac population of nkx2.5 expres- cells (Figs. 7A–F) suggest that these other LPM fates are not significantly sing cells (Schoenebeck et al., 2007). Thus, our data do not support a affected and would be unlikely to contain a significant amount of dying model where increased Wnt signaling in the pre-cardiac mesoderm cells. causes CPs to renew/proliferate at the expense of differentiation. One mechanism by which these cells could be dying is through It has been proposed that in pre-cardiac mesoderm of mouse p53-mediated apoptosis (Pyati et al., 2007). To determine if the CP embryoid bodies (EBs) Wnt signaling can drive the formation of cells are dying through a p53-dependent mechanism and if inhibition hematopoietic/vascular lineages at the expense of cardiac lineages of p53 can rescue CM differentiation, we injected Tg(hsp70l:wnt8a- (Naito et al., 2006; Wang et al., 2007). To test this model, we exam- GFP)w34 embryos with a p53 MO and then assayed CM differentiation ined etv2, which marks vascular lineages and anterior myeloid line- markers in transgenic and HCSEs after heat-shock at the TB stage. ages (Sumanas and Lin, 2006; Sumanas et al., 2008) and the However, depletion of p53 was not able to restore the expression of vascular markers kdrl (Liao et al., 1997)andfli1a (Thompson et al., CM differentiation marker (data not shown). One potential model 1998) at the 20 s stage in embryos when Wnt signaling was modulated that the inability to rescue CM differentiation suggested is that at the TB stage. However, we did not observe a discernible effect on these cells are dying through a p53-independent mechanism. To these markers when increasing or decreasing Wnt signaling (Supple- test this hypothesis, we examined activated Caspase-3 after increas- mental Figs. 7A–L). Therefore, these results suggest that increased ing Wnt signaling at the TB stage because activated Caspase-3 in Wnt signaling is not sufficient to promote an overtly detectable fate many contexts is a downstream mediator of p53-dependent apoptosis transformation between cardiac and hematopoietic or vascular lineages (Pyati et al., 2007; Sidi et al., 2008). In transgenic embryos with in zebrafish embryos. increased Wnt signaling at the TB stage, we did not observe activated Because it appeared that increased Wnt signaling in pre-cardiac Caspase-3 in the LPM where we observed dying cells after AO treatment mesoderm did not induce CPs to increase nor to differentiate as (Fig. 6C,D). However, we did observe activated Caspase-3 in the anterior another cell type, we asked if the increased Wnt signaling could be neural tissue (Figs. 6C,D), which provided a positive internal control and T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376 371

modulate ventricular CM proliferation (Rottbauer et al., 2002). There- fore, we next wanted to determine if there were additional roles of Wnt signaling in CM formation between 8 s and 48 hpf. To do so, we modulated Wnt signaling in the transgenic embryos at the 16 s stage, 24 hpf and 30 hpf and examined the hearts at 48 hpf. Interest- ingly, even though we did not observe a noticeable effect of Wnt sig- naling on the expression of CM differentiation markers at 20 s or 22 s (Figs. 1F,L,R,S; 2F,L,R,S; 3C,F), by 48 hpf the hearts from embryos receiving increased Wnt signaling at 16 s had significantly larger atria (Fig. 8D; Supplemental Movies 1 and 2). As would be expected from our analysis of earlier markers, hearts from embryos with increased Wnt signaling at TB and 8 s were almost absent and signif- icantly smaller, respectively (Figs. 8B,C), while decreased Wnt signal- ing did not discernibly disturb the hearts (Figs. 8G,H). We also found that increased Wnt signaling at 24 hpf caused hearts with slightly enlarged atria (Fig. 8E), while hearts appeared normal after increas- ing Wnt signaling at 30 hpf (Fig. 8F). At 48 hpf, after Wnt signaling w32 Fig. 6. Increased Wnt signaling at the TB stage causes cell death independent of was decreased with either the Tg(hsp70l:dkk1-GFP) or Tg(hsp70l: w26 Caspase-3 activation. (A,C) HCSEs. (B,D) GFP+ sibling embryos. (A,B) Embryos at Δtcf3-GFP) lines at 16 s, 24 hpf, and 30 hpf the hearts appeared 20 s with AO staining to detect dying cells. There is increased cell death in the LPM at relatively normal, though occasionally the atria looked smaller after 20 s after increasing Wnt signaling at TB (B; small arrowhead and inset) compared to decreasing Wnt signaling at the 16 s stage (Figs. 8I–K; Supplemental HCSEs (A; arrow and inset). Significant cell death was also observed in the anterior neural tissue (large arrowhead in B). (C,D) Embryos at 20 s with whole mount Figs. 5C,D). Thus, we have uncovered a previously unrecognized role immunohistochemical analysis of activated Caspase-3. (D) Activated Caspase-3 is not for Wnt signaling in zebrafish where inappropriate Wnt signaling detected in the LPM where AO staining is observed at 20 s after increasing Wnt signaling during CM differentiation can affect atrial size. at the TB stage (small arrowhead). However, there is substantial activated Caspase-3 To determine if the Wnt induced increase in atrial size is due to an detected in the anterior neural tissues (large arrowhead), which is also observed with increase in cell number, as opposed to only changes in morphology, AO staining (B). Views in A and B are dorso-lateral. Views C and D are lateral. Lines in A–D indicate the position of the midbrain–hindbrain boundary. CPs/CMs are located we counted the number of cells in each chamber (Waxman et al., immediately posterior to this boundary in the LPM. 2008). Indeed, we found that increasing Wnt signaling at 16 s caused an ~20% increase in atrial cell number, while not affecting ventricular suggests that the Wnt induced neural cell death is via Caspase-3 mediat- cell number (Fig. 8L; Supplemental Table 3). Based on these results, ed apoptosis. Altogether, these results suggest that increased Wnt signal- we expected that decreased Wnt signaling might cause a decrease in ing in the pre-cardiac mesoderm does not affect CP specification, but atrial cell number, so we also complemented the gain-of-function instead prevents them from differentiating, which potentially results in experiments by counting the number of atrial and ventricular CMs their death through a p53/Caspase-3 independent apoptotic mechanism. after decreasing Wnt signaling at 16 s. Although only 1 out of 3 experi- ments indicated a statistically significant decrease in atrial cell number relative to HCSEs (Fig. 8M), all 3 experiments demonstrated a modest fi Wnt signaling is suf cient to promote the additional differentiation of decrease in atrial cell number relative to HCSEs (Supplemental Table atrial cells 3), suggesting a trend where Wnt signaling is necessary, although min- imally, during CM differentiation for appropriate atrial CM number. There is substantial developmental time and events between the Therefore, our results suggest that Wnt signaling is necessary and suffi- 8 s stage and 48 hpf, when Wnt signaling has been shown to cient to specifically promote proper atrial cell number during CM differentiation. We next wanted to explore the mechanism by which Wnt signaling affected atrial cell development at the 16 s stage resulting in increase cell number. We hypothesized that Wnt signaling could be promoting additional atrial cell development through an effect on differentiation and/or proliferation. We began by exploring if Wnt signaling was affecting differentiation through modulating gata gene expression, because recently it was shown that Wnt2 in mice promotes posterior SHF/atrial specification and proliferation through positively regulating gata6 expression (Tian et al., 2010). However, when we modulated Wnt signaling at the 16 s stage and examined gata6 or gata4 expression at 26 hpf, we did not find an effect on the expression of either of these markers (Figs. 9C,D; Supplemental Figs. 9C–F). The atrial region of the heart is already morphologically expanded by this stage, as easily seen by the endocardial expression of gata4 (Figs. 9E,F). By comparison, increasing Wnt signaling at 8 s significantly reduced gata6 expression, consistent with an expected inhibitory effect on the unspecified pre- Fig. 7. Increased Wnt signaling at the TB stage induces cell death in the anterior LPM. cardiac mesodermal cells (Figs. 9A,B). Decreasing Wnt signaling at 8 s (A–C) Representative HCSE. (D–F) Representative GFP+ embryo after increasing Wnt signaling at the TB stage. Hemizygous Tg(hsp70l:wnt8a-GFP)w34 fish were crossed did not discernibly affect gata6 expression (Supplemental Figs. 9A,B). to hemizygous Tg(kdrl:mCherry)ci5 fish to examine the position of apoptotic cells We also examined gata6 expression at 20 s after Wnt signaling was relative to endothelial cells in the LPM. Images are from a single optical plane taken modulated at 16 s and found no difference between HCSEs and embryos with a Zeiss Axio Imager and ApoTome. In the transgenic embryo with increased with modulated Wnt signaling (Supplemental Figs. 10A–D). Therefore, Wnt signaling at the TB stage, there are AO positive apoptotic cells adjacent to the Wnt signaling does not appear to modulate atrial cell formation through endothelial cells (arrows in D and F), while the HCSE does not have a significant cluster of apoptotic cells in the same region of the anterior LPM (A and C). Images are dorso- regulation of gata6 or gata4. These results suggest that despite the lateral views with anterior to the top and dorsal to the left. similarity of the later sensitivity of atrial cell formation to Wnt signaling 372 T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376

Fig. 8. Wnt signaling is necessary and sufficient to promote atrial cell development during CM differentiation. (A) Representative HCSE. (B–F) GFP+ embryos with increased Wnt signaling. (G–K) GFP+ embryos with decreased Wnt signaling. Decreasing Wnt signaling from TB through 30 hpf had inconsistent and modest effects on morphology. (B,C) Increasing Wnt signaling at TB or 8 s almost eliminates and greatly reduces the size of the hearts, respectively, at 48 hpf. Arrow in C indicates the severely smaller . (D) When Wnt signaling is increased at 16 s, the is significantly enlarged (arrow). Although the ventricles were dismorphic, we did not find an effect on ventricular cell number. (E) Increasing Wnt signaling at 24 hpf causes modestly enlarged atria. (F) Increasing Wnt signaling at 30 hpf no longer has a discernible effect on the heart. (L) Increasing Wnt signaling at 16 s causes a specificincreaseinatrialcell number. (M) Decreasing Wnt signaling at 16 s causes a modest reduction in atrial cell number. Images are frontal views. Red indicates ventricle and green indicates atrium. Asterisks indicate asignificant difference in the cell number from HCSEs and GFP+ embryos in Supplemental Table 3 according to Student's t-test.

in zebrafish and mouse embryos, zebrafish likely use a different mech- not shown). However, the proliferation rate in HCSEs and embryos anism to regulate the addition of atrial cells. with increased Wnt signaling was not statistically different at Our results examining myl7, gata4 and gata6 expression indicate 24 hpf (Supplemental Table 5) or at 26 hpf, when we could distin- that there is a morphological difference in the atria between HCSEs guish the difference between the endocardial and atrial cells (Sup- and embryos with increased Wnt signaling at 16 s that arises between plemental Table 6; Supplemental Figs. 11A–C). Therefore, we 20 and 26 hpf, which suggests that the increase in atrial cell number presently conclude that increased Wnt signaling during CM differen- may be occurring sometime between 20 s and 26 hpf. To define the tiation is not discernibly affecting proliferation leading to more atrial earliest time point that we can see a difference in atrial size, we ex- cells. The lack of evidence to support an effect on proliferation makes amined the CP markers nkx2.5 and tbx5a at 22 s and amhc at 24 hpf. us currently favor a model where during atrial CM differentiation Although we did not find an increase in nkx2.5 expression (Figs. Wnt signaling promotes the additional specification of CPs leading 10A,B,G,H), which is understandable because at this stage it to excess atrial cells. primarily marks ventricular cells in zebrafish, we did find an increase in the amount of tbx5a expression via analysis of ISH and qPCR (Figs. 10C,D,G,H,I; Supplemental Table 4). Interestingly, the ~30% fold Discussion increase in area of tbx5a expressing cells is highly reminiscent of the ~20% fold increase in atrial cell number at 48 hpf. Furthermore, by In the present study, we examined the role of Wnt signaling in CM 24 hpf, the atria were expanded when Wnt signaling was increased formation during 3 distinct phases of CM development in zebrafish. at 16 s (Figs. 10E,F) similar to what we observed with gata6 (Figs. First, we provide evidence that zebrafish Wnt8a paralogs regulate the 9E,F). Therefore, our results suggest that increasing Wnt signaling specification of both ventricular and atrial CMs through promoting during CM differentiation causes an increase in the expression of ventro-lateral mesoderm. Second, we define a mechanism where Wnt the CP marker tbx5a by 22 s, which precedes the enlarged atrial signaling is sufficient, but is not necessary, to inhibit CM differentiation, morphology visible at 24 hpf. which subsequently may lead to their death through a p53/Caspase-3 We next wanted to understand if the increase in atrial progenitors and independent mechanism. Third, we define a mechanism where Wnt CMs is also occurring through an effect on proliferation. To test if Wnt signaling promotes a surplus of atrial cells during CM differentiation signaling is promoting increased proliferation during atrial CM differenti- through promoting the additional specification and differentiation of ation, we increased Wnt signaling in Tg(kdrl:nlsEGFP)zf109 embryos at 16 s, atrial progenitors. Taken together with a previous study that defined a to be able to distinguish the endocardium from the myocardium, and role for Wnt signaling in preventing proliferation of differentiated used antibodies for AMHC (S46) and phospho-Histone H3 (pHH3) ventricular cells (Rottbauer et al., 2002), we propose that Wnt signaling (Supplemental Figs. 11A–C). Unfortunately, prior to or at 24 hpf we has 4 distinct roles in regulating CM formation in the early zebrafish could not visualize the GFP in these embryos after fixation (data embryo (Fig. 11). T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376 373

Fig. 10. Effects of increased Wnt signaling during atrial CM differentiation are evident by the 22 somite stage. (A,C,E) HCSEs. (B,D,F) GFP+ embryos with increased Wnt signaling at the 16 s stage. (B) The amount of cells expressing nkx2.5, which at 22 s Fig. 9. Modulating Wnt signaling does not affect gata4 or gata6 expression in differentiating primarily marks ventricular cells, is not increased when Wnt signaling is increased CMs. (A,C,E) HCSEs. (B,D,F) GFP+ embryos with increased Wnt signaling at the 8 s and 16 s during cardiac differentiation. (D) The amount of cells expressing tbx5a, which is stages. (B) Increasing Wnt signaling at 8 s decreases the amount of gata6 expression expressed in both ventricular and atrial cells, is modestly increased by 22 s. (F) When (arrow). (D,F) Increasing Wnt signaling at 16 s did not discernibly affect gata6 or gata4 Wnt signaling is increased at 16 s, amhc expression indicates that the atria are wider expression at 26 hpf. However, the endocardial morphology of gata4 expression indicates compared to controls (E) by 24 hpf. (G) Areas of the amount of cells expressing nkx2.5 that the venous pole is expanded by 26 hpf (E,F; dashed outline). and tbx5a in arbitrary units. (H) Fold difference in the areas of cells expressing nkx2.5 and tbx5a. (I) qPCR analysis of nkx2.5 and tbx5a expression at 22 s. There is an increase in tbx5a greater than what is observed with the ISH analysis. However, we did observe an increase in tbx5a is expressed in the forelimb mesenchyme (data not shown). Therefore, Wnt signaling effects on the pre-cardiac mesoderm of vertebrates the increase in tbx5a expression observed with qPCR likely reflects an increase in tbx5a expression from both populations. No difference in tbx5a expression in the eye was With respect to the pre-cardiac mesoderm, previous studies have observed between HCSEs and embryos with increased Wnt signaling at the 16 s stage at suggested that Wnt signaling in the pre-cardiac mesoderm is necessary the 22 s stage (data not shown). Asterisks indicates significant difference using Student's t-test. and sufficient to inhibit CP specification and differentiation (Foley and Mercola, 2005; Foley et al., 2007; Lavery et al., 2008b; Marvin et al., 2001; Naito et al., 2006; Schneider and Mercola, 2001; Tzahor and Although studies in mouse EBs and in Xenopus embryos have suggested Lassar, 2001; Ueno et al., 2007). However, our results suggest that that inhibition of Wnt signaling alone is required to increase CPs (Foley Wnt signaling is sufficient, but not required, to prevent cardiac differen- and Mercola, 2005; Foley et al., 2007; Lavery et al., 2008b; Marvin et al., tiation and importantly that increased Wnt signaling is not sufficient to 2001; Naito et al., 2006; Schneider and Mercola, 2001), these studies do affect CP specification. Unfortunately, we presently do not understand not rule out that BMP signaling is concomitantly required to expand the the reasons behind the differences seen with the previous study using CM population. Altogether, these disparities indicate that future studies zebrafish (Ueno et al., 2007). Although we used the same transgenic are clearly necessary to be able to thoroughly understand and compare lines as those reported in Ueno et al. (2007), it is possible that the com- the individual vs. dual requirements of Wnt and BMP signaling in the bination of technical differences, including the PCR machine based pre-cardiac mesoderm of vertebrate embryos and ES cells. method of heat-shock, quantitative ISH/area and qPCR analysis, which Our data also suggest a new mechanism where increased Wnt sig- were not previously used, and examining additional stages has allowed naling in the pre-cardiac mesoderm does not affect CM specification, for the different observations and conclusions. as previously suggested (reviewed in Kwon et al., 2008; Tzahor, When comparing our results to other vertebrate models, however, 2007). Instead, increased Wnt signaling in the pre-cardiac mesoderm there is significant similarity. For instance, the loss-of function results prevents CM differentiation, which we hypothesize eventually leads reported here with zebrafish are consistent with chick explant experi- to apoptotic death of the CPs via a p53/Caspase-3 independent mech- ments, which indicated that inhibition of Wnt signaling alone could not anism. One possible explanation for this discrepancy is that in previ- expand the amount of CMs (Tzahor and Lassar, 2001). Instead, Wnt sig- ous studies the CP and differentiation markers were assayed naling depletion needed to be coupled with BMP addition to stimulate concurrently at later time points, not progressively over time points an increase in CMs. Similarly, in mouse ES cells, both inhibition of Wnt when the progenitor markers are first expressed (Afouda and and activation of BMP signaling downstream of Notch is required to pro- Hoppler, 2009; Afouda et al., 2008; Foley and Mercola, 2005; Foley mote cardiac cell development instead of hemangioblast development et al., 2007; Lavery et al., 2008a, 2008b; Martin et al., 2010; Marvin (Chen et al., 2008). In human ES cells, it was found that Wnt signaling is et al., 2001; Naito et al., 2006; Tzahor and Lassar, 2001; Ueno et al., required to limit CM differentiation in the context of BMP addition, 2007). This leaves open the possibility that exploring additional which is used to direct the mesodermal differentiation of these cells time points could explain differences between this and previous stud- (Paige et al., 2010; Willems et al., 2011).Inaddition,inanendodermal ies. In addition, although cell culture studies have suggested that conditional β-catenin KO mouse that leads to ectopic hearts, there is cardiac fates may be inhibited at the expense of vascular or hemato- also ectopic BMP expression (Lickert et al., 2002), further implicating poietic fates in the pre-cardiac mesoderm (Chen et al., 2008; Marvin the connection of these two pathways in restricting CM formation. et al., 2001; Naito et al., 2006; Wang et al., 2007), we did not find 374 T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376

Fig. 11. Model depicting the roles of Wnt signaling in CM formation at the four distinct phases of development. (A) Wnt signaling in the pregastrula embryo promotes the formation of CMs indirectly through its role in ventro-lateral mesoderm induction. (B) Wnt signaling is sufficient (red inhibitory sign), but alone is not necessary, to restrict cardiac differentiation in the pre-cardiac mesoderm. (C) Wnt signaling is necessary and sufficient to promote the addition of atrial cells after the initiation of CM differentiation. (D) Wnt signaling is required to repress proliferation in differentiated ventricular cells (Rottbauer et al., 2002).

an inverse relationship between these fates, in agreement with Ueno signaling can promote a relatively substantial increase in atrial cell num- et al. (2007). Though we think it is unlikely, we cannot rule out that ber, there appears to be a minimal requirement for Wnt signaling in pro- there is a small portion of cells that do make this fate transformation. moting atrial cell formation during the same time period. Furthermore, Therefore, further evaluation is necessary to determine the extent of our results indicate that Wnt signaling does not discernibly regulate conservation of this previously unrecognized mechanism on the gata6 or gata4 expression. Therefore, these results suggest that one pre-cardiac mesoderm of vertebrates. possibility is that despite similarities in the ability of Wnt signaling to As AO labels apoptotic cells in zebrafish (Furutani-Seiki et al., 1996; affect atrial CM development in zebrafish and mice, these similarities Pyati et al., 2007), our results suggest a model where the inability of do not reflect a conserved role of Wnt signaling. Alternatively, it is the CPs to differentiate appears to result in apoptosis through a p53 possible that Wnt regulation of atrial CM development is conserved in and Caspase-3 independent mechanism. However, we do not yet under- vertebrates, but a requirement for Wnt signaling and downstream com- stand the mechanisms that lead to their inability to differentiate or what ponents has either been lost/reduced in zebrafish or gained/expanded in apoptotic pathway that is being triggered. We hypothesize that the in- mouse. ability of the CPs to differentiate is not a direct consequence of excess Recent studies in zebrafish have also suggested that isl1 regulates Wnt signaling at the 20 s stage, because Wnt responsive genes are no a progressive addition of atrial cells to the venous pole of the longer affected at 20 s after the heat-shock at the TB stage (data not zebrafish heart (de Pater et al., 2009), naturally drawing further shown). Rather, we propose that the inability to differentiate is a down- comparisons to the mammalian SHF. Furthermore, multiple studies stream consequence of the increased Wnt signaling upon the CPs in the in mice and stem cells have indicated that Wnt signaling controls pre-cardiac mesoderm. With respect to the apoptosis, it is surprising that the differentiation of isl1 cells (Klaus et al., 2007; Kwon et al., 2009; a p53/Caspase-3 independent pathway is being employed. However, Lin et al., 2007; Qyang et al., 2007), prompting us to explore the pos- there are many ways to trigger apoptosis as both cell culture and sibility that Wnt signaling was upstream of isl1 in zebrafish. However, zebrafish studies have uncovered p53-independent apoptotic pathways we found that the function of isl1 does not appear to be epistatic to (Fujita and Ishikawa, 2011; Pyati et al., 2007; Sidi et al., 2008). Moreover, Wnt signaling as depletion of isl1 does not affect the number of atrial arecentstudyinzebrafish elucidated a conserved Chk1/Caspase-2 cells when Wnt signaling is increased in differentiating CMs (data not dependent and p53/Caspase-3 independent apoptotic pathway (Sidi et shown). Therefore, our results currently support the notion that Wnt al., 2008). Therefore, it is conceivable that this Caspase-2 apoptotic path- must be influencing atrial CM specification and differentiation using way or another apoptotic mechanism is what is being utilized. Future different mechanisms than described in the mammalian SHF. studies will be aimed at elucidating the mechanisms by which Wnt If Wnt signaling is affecting atrial CM specification and not prolif- signaling prevents CP differentiation and the apoptotic pathway that is eration, this suggests that Wnt signaling is influencing cells to take on being triggered. an atrial cell identity that would otherwise become another fate. We speculate that these additional atrial cells could be coming from two Wnt signaling and atrial cell differentiation sources: the additional atrial cells could be derived from a population of cells capable of responding that resides within the differentiating A role for Wnt signaling during differentiation that expands the atrial atrial cell field; alternatively, Wnt signaling could be influencing a cell population in zebrafish is immediately reminiscent of the character- nearby cell type to take on an atrial cell fate. Future lineage studies ization of Wnt signaling in the SHF of mice (Dyer and Kirby, 2009). aimed at understanding the origin of these additional cells will Specifically, Wnt2 in mice is required to promote specification and pro- allow us to better understand how the size of this chamber is ulti- liferation of atrial CPs in the posterior SHF through positively regulating mately regulated during development, which will impact our ability gata6 (Tian et al., 2010). However, our results suggest that while Wnt to precisely derive atrial cells in culture. T.E. Dohn, J.S. Waxman / Developmental Biology 361 (2012) 364–376 375

Conclusions Huang, C.J., Tu, C.T., Hsiao, C.D., Hsieh, F.J., Tsai, H.J., 2003. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac 2 promoter of zebrafish. Dev. Dyn. 228, 30–40. Altogether, this is the first study to define a comprehensive picture Hug, B., Walter, V., Grunwald, D.J., 1997. tbx6, a brachyury-related gene expressed by of the distinct phases of Wnt signaling in vivo during early develop- ventral mesendodermal precursors in the zebrafish embryo. Dev. Biol. (NY 1985) 183, 61–73. ment in a vertebrate. The relatively clean distinctions of these four Keegan, B.R., Meyer, D., Yelon, D., 2004. Organization of cardiac chamber progenitors in phases are likely to provide a basis to reconcile the conservation of the zebrafish blastula. 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