Developmental Biology 286 (2005) 427 – 439 www.elsevier.com/locate/ydbio
hecate, a zebrafish maternal effect gene, affects dorsal organizer induction and intracellular calcium transient frequency Jamie Lyman Gingerich a, Trudi A. Westfall b, Diane C. Slusarski b, Francisco Pelegri a,* a Laboratory of Genetics, 425-G Henry Mall, University of Wisconsin - Madison, Madison, WI 53706, USA Department of Biological Sciences, 246 Biology Building, The University of Iowa, Iowa City, IA 52242, USA
b
Received for publication 17 March 2005, revised 6 July 2005, accepted 25 July 2005 Available online 9 September 2005
Abstract A zebrafish maternal effect mutation, in the gene hecate, results in embryos that have defects in the formation of dorsoanterior structures and altered calcium release. hecate mutant embryos lack nuclear accumulation of h-catenin and have reduced expression of genes specific to the dorsal organizer. We found that hecate mutant embryos exhibit a nearly 10-fold increase in the frequency of intracellular Ca2+ transients normally present in the enveloping layer during the blastula stages. Inhibition of Ca2+ release leads to ectopic expression of dorsal genes in mutant embryos suggesting that Ca2+ transients are important in mediating dorsal gene expression. Inhibition of Ca2+ release also results in the expression of dorsal-specific genes in the enveloping layer in a h-catenin-independent manner, which suggests an additional function for the Ca2+ transients in this cellular layer. The mutant phenotype can be reversed by the expression of factors that activate Wnt/h-catenin signaling, suggesting that the Wnt/h-catenin pathway, at least as activated by an exogenous Wnt ligand, is intact in hec mutant embryos. Our results are consistent with a role for the hecate gene in the regulation of Ca2+ release during the cleavage stages, which in turn influences dorsal gene expression in both marginal cells along the dorsoventral axis and in the enveloping layer. D 2005 Elsevier Inc. All rights reserved. Keywords: Wnt signaling; h-catenin; Calcium; hecate; Axis induction; Maternal effect; Zebrafish
Introduction Determination and patterning of the axes are fundamental steps in early vertebrate development that lay the framework for later developmental decisions. The molecules and processes involved in axis specification appear to be conserved among vertebrates. For example, the Xenopus dorsal lip of the blastopore, the zebrafish shield, and the mouse node all have so-called Spemann organizer activity, which induces neuroectoderm and dorsal mesoderm (Harland and Gerhart, 1997). However, the specific genes and interactions between pathways involved remain to be fully elucidated. Dorsal – ventral patterning in zebrafish depends initially on the translocation of a dorsal signal present in the cortex of the vegetal pole of the early embryo (Mizuno et al., 1999; * Corresponding author. E-mail address:
[email protected] (F. Pelegri). 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.07.031
Ober and Schulte-Merker, 1999). The alignment of a microtubule array in the yolk is required both for the transport of particles from the vegetal pole and for the formation of a dorsal axis suggesting that the initial dorsal signal must move in a microtubule-dependent manner from the vegetal pole to the presumptive dorsal region (Jesuthasan and Stahle, 1997). The translocated dorsal signal is thought to result in the localized activation of the so-called canonical Wnt/h-catenin signaling pathway (reviewed in Pelegri, 2003). Wnt proteins comprise a large class of secreted signaling molecules involved in such diverse processes as cell differentiation, cell proliferation, and cell fate determination (Huelsken and Behrens, 2002; Veeman et al., 2003). Wnt ligands that stimulate the Wnt/h-catenin signaling pathway lead to the stabilization and nuclear localization of hcatenin, which then interacts with the tcf/LEF class of transcription factors to regulate transcription of target genes. Manipulation of this pathway suggests that it is important
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for the activation of dorsal-specific genes in the zebrafish organizer (reviewed in Pelegri, 2003). In addition, both ichabod (Kelly et al., 2000) and tokkaebi (Nojima et al., 2004), zebrafish maternal-effect mutations, prevent the nuclear localization of h-catenin and lead to a loss of dorsal-specific gene expression and corresponding expansion of ventral tissues. Thus, the Wnt/h-catenin pathway plays an important role in the transduction of the dorsal signal in zebrafish embryos. Another factor involved in the regulation of dorsal patterning is calcium. Modulation of the frequency of intracellular transients has been shown to affect the expression domains of dorsal and ventral genes. In the zebrafish blastula, cells of the enveloping layer (EVL) show fluctuating levels of cytosolic Ca2+ (Slusarski et al., 1997b). Activation of heterotrimeric G-proteins and subsequent release of GhE subunits is an early step in the signaling and is required for the EVL Ca2+ transients in the zebrafish (Ahumada et al., 2002; Slusarski et al., 1997a). The GhE subunits activate phospholipase C, which in turn hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to release inositol 1,4,5-triphosphate (IP3) and diacylglycerol. IP3 binds to IP3-receptors in the endoplasmic reticulum leading to the release of Ca2+ into the cytosol (Berridge et al., 2003). In zebrafish, increasing Ca2+ levels through a chimeric serotonin receptor inhibits axis induction (Slusarski et al., 1997b) and injection of a constitutively active Ca2+-dependent calmodulin kinase (CamKII), or levels of Wnt5 that activate Ca2+ release, enlarges ventral-specific gene domains (Westfall et al., 2003a). Conversely, several pieces of data demonstrate that reduction of Ca2+ release results in dorsalization. In Xenopus, monoclonal antibodies to the IP3-receptor as well as antibodies to G-protein subunits induce a transformation from ventral to dorsal cell fates (Kume et al., 1997). In zebrafish, treatment with pharmacological inhibitors including the phosphoinositide cycle inhibitor L-690,330, the PLC-inhibitor U-73122, or inhibitors of Ca2+ release from the endoplasmic reticulum results in ectopic dorsal gene expression domains (Westfall et al., 2003b). These results suggest that Ca2+ modulation can influence dorsoventral patterning. However, the precise mechanism by which Ca2+ released from the endoplasmic reticulum modulates dorsal axis induction remains unclear. Yet, it has been shown that loss of Ca2+ release leads to accumulation of nuclear h-catenin (Westfall et al., 2003b) and Ca2+ release frequency can be modulated by the noncanonical Wnt-5 (Slusarski et al., 1997b; Westfall et al., 2003a). We present the characterization of a zebrafish maternal effect mutation, in the gene hecate (hec), that affects the frequency of intracellular Ca2+ transients and the induction of the dorsal axis. We found that hec mutant embryos exhibit a marked increase in the frequency of Ca2+ transients. We show that mutant embryos lack nuclear accumulation of h-catenin and that the mutant phenotype can be reversed by the expression of factors that activate
Wnt/h-catenin signaling, indicating that the mutation is affecting Wnt/h-catenin activity. Inhibition of Ca2+ release leads to ectopic expression of dorsal genes in hec mutant embryos including, unexpectedly, in the EVL, indicating a novel role for the Ca2+ transients observed in this layer. Our results indicate a role for the hecate gene in the modulation of Ca2+ release in the blastula stage embryo and the regulation of dorsal gene expression.
Materials and methods Fish maintenance Stocks of AB (wild-type) and hec lines were raised and maintained under standard conditions at 28.5-C (Westerfield, 1993). Mutant embryos were obtained by crossing homozygous mutant females to AB males. Embryos were staged according to age and morphological criteria as in Kimmel et al. (1995). The embryonic medium (E3 saline) used is described in Pelegri and Schulte-Merker (1999). In situ hybridizations and RNA injections In situ hybridizations were carried out as described previously (Pelegri and Maischein, 1998; Knaut et al., 2000). The probes used included: bozozok (Koos and Ho, 1998; Yamanaka et al., 1998), goosecoid (Schulte-Merker et al., 1994), chordin (Schulte-Merker et al., 1994), even skipped (Joly et al., 1993), gata2 (Detrich et al., 1995), bmp2 (Kishimoto et al., 1997), no tail (Schulte-Merker et al., 1994), and axial (Odenthal and Nusslein-Volhard, 1998). Images were acquired using a dissecting microscope (Leica, FLIII) and a color camera (Diagnostic Instruments Spot Insight). RNA for injection was prepared using mMessage mMachine RNA Transcription Kits (Ambion) as previously described (Pelegri and Maischein, 1998). Constructs used included: h-catenin (Kelly et al., 1995b; Pelegri and Maischein, 1998), dominant negative GSK-3 (Pierce and Kimelman, 1995), GSK-3 binding protein (Sumoy et al., 1999), zWnt8 (Kelly et al., 1995b), zFzA (Nasevicius et al., 1998), tcfBD (Pelegri and Maischein, 1998), DNtcf (Pelegri and Maischein, 1998), h-galactosidase (Rupp et al., 1994), and the A-protomer of pertussis toxin (Slusarski et al., 1997a). RNA was diluted with phenol red and injected into the yolk of embryos prior to the 2-cell stage or, in the case of the A-protomer, after the 8-cell stage. A morpholino oligonucleotide against the published zebrafish h-catenin sequence (accession number: BC047815) was ordered from GeneTools, LLC (sequence: 5V-ATCAAGTCAGACTGGGTAGCCATGA-3V). The standard control morpholino oligonucleotide (sequence: 5V-CCTCTTACCTCAGTTACAATTTATA-3V) was used as a control. The morpholinos were dissolved in water to a concentration of 1 mM and injected into pre-1 cell embryos.
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b-catenin immunolocalization Embryos were fixed and immunostained according to Westfall et al. (Westfall et al., 2003b) with the following exceptions. Embryos were incubated overnight in a 1:500 dilution of mouse monoclonal antibody clone 15B8 (Sigma), followed by incubation with a 1:400 dilution of alexa 488-labeled rabbit anti-mouse secondary antibody (Molecular Probes). Nuclei were stained with 0.5 Ag/ml DAPI in PBS (Molecular Probes). Images were acquired using an upright fluorescence microscope (Zeiss, Axioplan II, mot i) and a black and white digital camera (Zeiss, Axiocam), and processed using image deconvolution (3Drestoration) software (OpenLab). Images from two separate experiments were scored blindly. Pharmacological reagents A solution of L-690,330 (Tocris) at a concentration of 10 mM in PBS was injected into an individual blastomere of 2cell to 8-cell stage embryos as described in Westfall et al. (Westfall et al., 2003b). Xestospongin C (Calbiochem) at a concentration of 20 AM in PBS was similarly injected into an individual blastomere after the 8-cell stage. Both L690,330 and Xestospongin C injection solutions contained dextran-conjugated Texas red lineage marker (Molecular Probes). Embryos were exposed to 10 AM thapsigargin (Molecular Probes) or 50 AM CPA (Calbiochem) solutions in embryonic medium for 60 min beginning at the 32-cell stage (Westfall et al., 2003b). Calcium image analysis Calcium image analysis was performed by injecting the ratiometric calcium-sensitive Fura-2 dye (dextran conjugated, Molecular Probes) at the one-cell stage followed by imaging and analysis as described (Slusarski et al., 1997a; Westfall et al., 2003b). Imaging was initiated between the 32- and 128-cell stage. At least three embryos were imaged for each experiment. Differences between wild-type and hec mutant embryos were analyzed statistically using the Student’s t test (two-sample assuming unequal variances) and found to be significant with a P value of 10( 4.7). Similar statistical analysis showed no significant differences between control and h-catenin MO- or tcfBD-injected wildtype embryos.
Results The maternal effect mutation hecate affects axis formation The hecate (hec) mutation was isolated in an earlypressure-based screen for recessive maternal-effect mutations (Pelegri and Schulte-Merker, 1999; Pelegri et al., 2004). Typically, over 95% of the embryos from females
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that are homozygous for the mutation (for simplicity, referred to as hec mutant embryos) exhibit defects in dorsoventral patterning. At 24 h post-fertilization (hpf), a majority of hec mutant embryos lack all dorsal anterior structures and consist of radially symmetric posterior – ventral structures (Fig. 1a). The remainder of hec mutant embryos displays a range of posterior – ventralization phenotypes. Moderately affected embryos form a trunk and have some anterior structures but lack the anteriormost head structures including the eyes and dorsal forebrain, as well as a notochord (Figs. 1b and c). Weakly affected embryos exhibit a reduction in the head and the eyes and may exhibit defects in notochord formation (Fig. 1d). A small fraction of embryos, usually less than 5%, are indistinguishable from wild-type embryos and can survive to adulthood (Fig. 1e). Mutant clutches exhibit variable expressivity of the phenotype, ranging from clutches where 100% of the embryos show the most severe, radially symmetric phenotype to clutches which include embryos from each of the phenotypic classes including wild-type. In wild-type embryos, a thickening can be observed at the dorsal side of the germ ring at 5.5 hpf just after the beginning of gastrulation (Figs. 1f and h). This morphological structure, also referred to as the shield, corresponds to the future dorsal organizer of the embryo. This thickening is not visible during gastrulation in hec mutant embryos suggesting that the embryonic organizer is reduced or absent in these mutant embryos (Figs. 1g and i). Thus, the axis formation defects observed at 24 hpf originate from defects that occur before or during early gastrulation. hecate mutant embryos show defects in early dorsoventral patterning Because the embryonic organizer region is reduced in hec mutant embryos, we wanted to ascertain whether mutant embryos have defects in the induction of genes responsible for dorsal – ventral patterning. In wild-type embryos, the homeobox domain gene bozozok (nieuwkoid/dharma), which is a direct target of the dorsal-inducing activity of the Wnt/h-catenin pathway (Ryu et al., 2001), is expressed in the presumptive dorsal region prior to the formation of a visible organizer (Fig. 2a). hec mutant embryos show reduced or absent expression of bozozok (Fig. 2b). In addition, most hec mutant embryos do not express goosecoid (Stachel et al., 1993; Schulte-Merker et al., 1994) or chordin (Fisher et al., 1997; Schulte-Merker et al., 1997), markers of the dorsal organizer, during the late blastula and early gastrula stages (Fig. 2d, compare to Fig. 2c, and data not shown—see also Fig. 5c). The degree of reduction in dorsal gene expression correlates with the severity of the phenotypes expressed within the clutch, as observed at 24 hpf (data not shown). The reduction in dorsal gene expression is accompanied by a concomitant expansion of ventrally expressed genes. In wild-type embryos, even skipped expression is restricted to
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Fig. 1. hecate mutant embryos display defects in axis induction at 24 hpf. (a – e) At 24 hpf, the majority of embryos from a given clutch have severe radial ventralization (a) while the remainder exhibit varying degrees of deficiency in dorsoanterior development (b – e). Anterior: top; posterior: bottom; dorsal: right, when identifiable. In panel a, the anterior contains a mass of apparently undifferentiated cells (asterisk). Blocky somites (arrowheads in panels b and c) are characteristic of the absence of a notochord. (f – i) Wild-type (f, h) and hec mutant (g, i) embryos at the shield stage (6 hpf). Wild-type embryos display a characteristic thickening on the dorsal side (arrows in panels f and h) while hec mutant embryos lack this thickening. (f and g: side views; h and i: animal views).
mesendodermal precursors of the ventral margin, and is absent from the dorsal and dorsolateral mesoderm (Joly et al., 1993; Fig. 2e). In hec mutant embryos, even skipped expression is expanded around the entire margin of the embryo (Fig. 2f). In addition, expression of bmp2 (Kishimoto et al., 1997) and gata2 (Detrich et al., 1995), normally confined to the ventrolateral regions in more animal regions of the blastula, corresponding to the prospective ectoderm, is expanded in hec mutant embryos (Figs. 2h and j, compare to Figs. 2g and i). Other than the observed changes in dorsoventral patterning, we do not detect a reduction in the levels of expression of genes that are markers for the various germ layers, such as bmp2 and gata2 (ectoderm), no tail (mesendodermal precursors, Schulte-Merker et al., 1994; Fig. 2l, compare to Fig. 2k), and foxa2 (endoderm, Odenthal and Nusslein-Volhard, 1998; Fig. 2n, compare to Fig. 2m). A landmark in the formation of the zebrafish dorsal organizer is the nuclear localization of h-catenin at the prospective dorsal side of the blastula embryo. Because hec mutant embryos show defects in the formation of a dorsal organizer, we investigated whether mutant embryos also display defects in the nuclear accumulation of h-catenin. In blind scorings of fixed embryos labeled using an anti-hcatenin antibody, nuclear localization of h-catenin protein was not detected in most hec mutant embryos at the sphere
stage (4 hpf, Figs. 3d – h, 17% with detectable nuclear localization, n = 18), while nuclear h-catenin is detectable in the presumptive organizer of most wild-type embryos (Figs. 3a– c, 76% with detectable nuclear localization, n = 17). Thus, the morphological data combined with the gene expression patterns show that hec mutant embryos exhibit a global reduction in dorsal cell fates and a concomitant expansion of ventral cell fates. hecate mutant embryos exhibit an increased frequency of intracellular calcium release The zebrafish embryo exhibits various periods of increased cytosolic Ca2+ during cleavage, gastrulation, and early segmentation, including Ca2+ transients occurring in the EVL (reviewed in Creton et al., 1998; Webb and Miller, 2003) (Figs. 4a and g). Although no correlation has been shown between the location of the EVL transients during the blastula stages and the site of the future dorsal axis, Ca2+ transients have been shown to antagonize the Wnt/h-catenin pathway and proposed to promote ventral cell fates (Kuhl et al., 2001; Westfall et al., 2003b). We, therefore, asked whether the frequency of Ca2+ transients during the blastula stages is altered in hec mutant embryos by visualizing intracellular free Ca2+ using the ratiometric indicator fura-2-
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Fig. 2. Gene expression analysis of hecate mutant embryos. Whole mount in situ hybridization of embryos shows a reduction in the expression of dorsal genes: nieuwkoid (nwk), at sphere [4 hpf; (a, b)]; goosecoid (gsc), at 50% epiboly [5.3 hpf; (c, d)]; as well as an expansion in the expression of ventral genes: even skipped 1 (eve), at 50% epiboly (e, f); bmp2, at 80% epiboly [7.3 hpf; (g, h)]; gata2, at 80% epiboly (i, j). Expression of the mesendodermal marker gene no tail at 50% epiboly (k, l) and the endodermal marker foxa2 (axial) at 80% epiboly (m, n) show no reduction in germ layer specific staining, although as expected the dorsal domains of expression of these genes are reduced. All panels are animal views with dorsal side to the right (when identifiable), except panels m, which is a dorsal view.
dextran. Intracellular Ca2+ EVL transients during the blastula stages occur at a 5- to 10-fold higher frequency in hec mutant embryos than in wild-type embryos (Figs. 4b, e, and g). Not only do hec mutant embryos exhibit an increased frequency of transients, but the transients continue beyond the 1000-cell stage, when transients in wild-type embryos have started to subside (Fig. 4g). Considering the reduction in nuclear h-catenin, we examined whether the increase in Ca2+ transient frequency is a secondary consequence of the inhibition of h-catenin at the embryonic organizer. To address this possibility, we tested whether the inhibition of h-catenin activity results in increased Ca2+ transients in wild-type embryos. Unilateral injection with mRNA coding for a fragment of tcf3 that binds h-catenin and interferes with its activity (tcfBD) (Pelegri and Maischein, 1998) does not lead to ectopic Ca2+ transients in the injected side relative to the uninjected side (Figs. 4i and j). Similar results are obtained after injection of a morpholino oligonucleotide (MO) against h-catenin (Fig. 4k), a reagent expected to inhibit translation of maternally derived h-catenin mRNA (Nasevicius and Ekker, 2000) and which leads to the specific inhibition of dorsal gene expression (Table 1). The inhibition of dorsal gene
expression by h-catenin MO can be reversed by the injection of h-catenin mRNA lacking the MO target sequence (Table 1), suggesting that the effect of the MO is specific. Thus, inhibition of Wnt/h-catenin activity at the level of h-catenin function does not lead to an increase in the frequency of intracellular Ca2+ transients. To examine potential pathway components involved in the induction of Ca2+ transients, we measured the frequency of Ca2+ transients following treatment of hec mutant embryos with pharmacological agents. First, we investigated whether the increased Ca2+ transients present in hec mutants were dependent on endoplasmic reticulum Ca2+ stores. In vivo image analysis of Ca2+ release in hec mutant embryos after unilateral injection of Xestospongin C, an inhibitor of the IP3-receptor, showed a decrease in the frequency of the Ca2+ transients specifically in the injected side (Fig. 4c, injected on the left side). Similarly, unilateral injection of L690,330, an inhibitor of the phosphatidylinositol pathway, also reduces the frequency of Ca2+ transients in hec mutant embryos, further indicating that the increased Ca2+ release depends on IP3 signaling (Fig. 4d, injected on the right side). Unilateral injection into hec mutant embryos of the Aprotomer of pertussis toxin, which inhibits the function of a
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Fig. 3. h-catenin protein does not localize to the nuclei of hec mutant embryos. Nuclear localization of h-catenin protein was observed in wild-type embryos on the presumptive dorsal side (a, c) but not the presumptive ventral side (b). No nuclear localization was apparent in hec mutant embryos (d – h), h-catenin localization at the membrane can be detected in both wild-type and hec mutant embryos. Animal views of sphere stage embryos (4 hpf). Panels b, c and e – h are higher magnifications of panels a and d, respectively, as indicated by the red boxes in panels a and d.
subset of G-proteins and leads to a decrease in the frequency of Ca2+ transients in wild-type zebrafish embryos (Slusarski et al., 1997a), also decreases the hec-induced Ca2+ transient frequency in the injected side (Fig. 4f, injected on the left side). These data suggest that the increased frequency of Ca2+ transients observed in hec mutant embryos are due to G-protein-dependent IP3R-mediated Ca2+ release from the endoplasmic reticulum. The ability of these pharmacological agents to inhibit Ca2+ transients is limited. For example, wild-type embryos injected with L-690,330 resume a normal frequency of Ca2+ transients approximately 20 to 40 min after injection (Westfall et al., 2003b). Ca2+ imaging of injected hec embryos likewise demonstrate a recovery from exposure to the pharmacological reagent in that embryos resume the prototypical hec-specific increased Ca2+ transients (Fig. 4h, this specific example, injected with the A-protomer of pertussis toxin, recovered 45 min after initiating image analysis). A reduction in intracellular Ca2+ levels alters gene expression patterns both at the margin and in the EVL In light of reports of the ability of the Wnt/Ca2+ pathway to antagonize the Wnt/h-catenin pathway (Slusarski et al., 1997b; Westfall et al., 2003a), we investigated whether decreasing the frequency of Ca2+ transients would lead to the activation of dorsal gene expression in wild-type and hec embryos. Thapsigargin and cyclopiazonic acid are cell-
permeable drugs that inhibit the uptake of Ca2+ into the endoplasmic reticulum, thus depleting stores essential for localized Ca2+ release (Thastrup et al., 1990). Treatment of wild-type zebrafish embryos with thapsigargin has been previously shown to lead to the reduction of calcium transients and to expanded expression of the dorsal marker chordin in marginal cells at 50% epiboly (5.3 hpf; Westfall et al., 2003b). We confirmed these results in wild-type embryos at the 50% epiboly stage (Figs. 5a and b, Table 2). In thapsigargin-treated wild-type embryos, we also note the presence of endogenous dorsal domain of chordin expression at sphere stage (4 hpf) as previously reported (Fisher et al., 1997; Schulte-Merker et al., 1997), which is responsive to h-catenin activity (Pelegri and Maischein, 1999). However, there is no apparent expansion of this early dorsal domain in these embryos (data not shown). Treatment with cyclopiazonic acid gave embryonic phenotypes at 24 hpf similar to those obtained with thapsigargin in both wild-type and hec mutant embryos (data not shown) suggesting that CPA has a similar effect on dorsal gene expression. Thus, treatments that interfere with Ca2+ transient release can result in the expansion of dorsal gene expression in both wild-type and hec mutant embryos. In both the thapsigargin-treated hec mutant embryos and wild-type embryos, we also observed ectopic chordin and goosecoid expression in the animal cap in cells whose external position and large, flattened morphology correspond to those of the EVL (Fig. 5f, compare to Fig. 5e, Table 2). Optical sections of embryos fluorescently labeled
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Fig. 4. Ca2+ transients occur more frequently in hec mutant embryos than in wild-type embryos. (a – f) Surface plot representations showing the number of Ca2+ transients in individual embryos during the cellular blastoderm stage. Wild-type (a) has less activity when compared to untreated hec embryos (b and e: hec mutant embryos from different females). Sibling embryos from clutches with similar activity to untreated embryo (b) were unilaterally injected with XeC on the left side (c) and L-690,330 on the right side (d). Sibling embryo from a clutch with similar activity to untreated embryo (e) was injected with A-protomer of pertussis toxin on the left side (f). White arrows in panels c, d, and f indicate side of injection and the inset in panel d demonstrates the location of Texas red lineage marker coinjected with the reagent. Embryos were oriented in a lateral view relative to the long axis. The image represents a composite of all the transient activity in a single embryo. The color and peak size represent the total number of Ca2+ transients that occurred in that location in the embryo. The color bar shows the frequency range with low numbers as cooler colors (purple = 0 – 1) and high numbers as hotter colors (orange – red = 25). Composites in panels a – f were accumulated from 200 data frames, 50 min. (g – h) Graphical output of the number of Ca2+ transients as a function of time (after image analysis was initiated) for wild-type (black) and hec (red) (g). Graphical plot of hec mutant embryo injected with A-protomer of pertussis toxin demonstrating recovery of Ca2+ transients after 45 min (h). Imaging was initiated at the 32- to 128-cell stage (1.75 – 2.25 hpf = time 0 in plots). (i – k) Surface plot representation showing the number of Ca2+ transients in individual embryos during the cellular blastoderm stage. Wild-type control (i). Wildtype embryo with unilateral injection of tcfBD (j). Wild-type embryo with unilateral injection of h-catenin MO (k). Composites in panels i – k were accumulated from 260 data frames, 65 min. Note that control embryo (i) was imaged for a longer time than the wild-type embryo in panel a; thus, it has more transients in total (and hence the peaks are taller) but a similar frequency of transients.
for thapsigargin-induced chordin expression and the DNA dye propidium iodide confirm that the chordin-expressing cells are contained both in the outermost cellular layer and the underlying deeper cells (Figs. 5k– r). This expression was evident at the sphere stage, before the expansion of
expression in the margin was visible, as well as during early gastrulation (30 –50% epiboly, Table 2). Because the Ca2+ transients observed during the blastula stages in both the wild-type and the hec mutant embryos occur in the cells of the EVL, our observations suggest a role for Ca2+
Table 1 Dose-dependent reduction of dorsal gene expression (cho) at 30% (4.7 hpf) epiboly by a morpholino against h-catenin and reversal of the effect by b-catenin mRNA lacking the morpholino target site Treatment
No expression (%)
Reduced expression (%)
Wild-type expression (%)
Ectopic expression (%)
n
Uninjected 0.47 pmol hcatMO 1.6 pmol hcatMO 2.9 pg hgal RNA + 7.3 pmol Std. Ctl. MO 3.5 pg hcat RNA + 7.3 pmol Std. Ctl. MO 2.9 pg hgal RNA + 7.3 pmol hcat MO 3.5 pg hcat RNA + 7.3 pmol hcat MO
0 0 65 0 11 50 24
6 23 32 9 5 37 28
94 77 3 91 37 13 31
0 0 0 0 47 0 17
52 13 37 22 19 24 29
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Fig. 5. Decreasing the frequency of Ca2+ transients leads to ectopic dorsal gene expression in hecate mutant embryos. (a – d) Both wild-type and hec mutant embryos show an expansion of cho expression in the margin at 50% epiboly (a and c: DMSO-treated control; b and d: Thapsigargin-treated, animal views). (e – j) Ectopic cho expression in the EVL is not dependent on h-catenin function. Thapsigargin (f, h, j)- and DMSO (e, g, i)-treated wild-type embryos. hcatenin MO effectively suppresses endogenous wild-type cho expression but not the ectopic expression in the EVL in wild-type embryos (g and h). Standard control MO does not affect the level of cho expression (data not shown). Injection of DNtcf suppresses both ectopic thapsigargin-induced cho expression and its endogenous expression in the dorsal region (i, j) (animal views, sphere stage, arrows denote expression in EVL). (k – r) Thapsigargin-induced ectopic cho expression occurs in cells of the EVL. Double labeling of cho mRNA using fluorescent situ hybridization (green in panels n and r) and the DNA stain DAPI (pseudocolored red for clarity in panels n and r). Under these labeling conditions, cho expression is observed diffusely in the cytoplasm and in two bright spots likely representing new transcription at the endogenous loci. (k – n) Control (DMSO)-treated embryo (k) and higher magnification views of the endogenous dorsal cho expression domain (red box in panel k). cho is expressed only in the dorsal region, where it is detected in deep cells and a small fraction of EVL cells. Arrows indicate EVL cells, distinguished by their external position and larger, flattened nuclei. Asterisks next to the arrows indicate EVL cells with some detectable expression. (o – r) Thapsigargin-treated embryos show ectopic cho expression in cells of the EVL layer. White arrows and asterisks as in (k – n). Panels are animal views of embryos at 30% epiboly (4.7 hpf).
transients in suppressing gene expression in this embryonic cell layer. Dependence of dorsal gene expression in the EVL on b-catenin and tcf function Expression analysis suggests that the gene chordin may be a direct transcriptional target of the h-cat/Tcf complex in
the dorsal marginal cells (Fisher et al., 1997; SchulteMerker et al., 1997). We therefore wanted to test the effects of interfering with these transcriptional regulators on the thapsigargin-induced chordin ectopic expression. mRNA coding for tcfBD effectively reduces the endogenous dorsal gene expression in wild-type embryos (Pelegri and Maischein, 1998), as well as any remaining dorsal gene expression in hec mutants (data not shown). As stated
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Table 2 Effect of thapsigargin, a Ca2+-depleting drug, on dorsal marginal and EVL chordin expression at sphere stage (4 hpf), and its dependence on b-catenin and tcf function Treatment (line)
Endogenous (dorsal) domain No expression (%)
DMSO (wt) Thap (wt) DMSO (hec) Thap (hec) DMSO + hcatMO (wt) DMSO + TcfBD (wt) DMSO + DNtcf (wt) Thap + hcatMO (wt) Thap + TcfBD (wt) Thap + DNtcf (wt)
Reduced expression (%)
Normal expression (%)
40
37
30 100 86
67
100 24 23 4 3
69
10 17
Ectopic expression of the Wnt/b-catenin pathway components can override the hec mutant phenotype To determine if the defect caused by the mutation in the hec gene affects a specific step in the Wnt/h-catenin pathway, we investigated whether injecting mRNA coding for factors expected to activate the Wnt/h-catenin pathway could reverse the hec mutant phenotype. The injected embryos were subsequently analyzed both by examination of the live phenotype and by testing for the induction of the dorsalspecific gene, goosecoid, by in situ hybridization. Injection of h-catenin mRNA (Kelly et al., 1995b; Pelegri and Maischein, 1998) into one-cell stage embryos results in the induction of
76 96
4 83 100 16
15
above, injection of a MO against h-catenin, leads to the reduction of dorsal gene expression in the margin (Fig. 5g, Table 2), which is consistent with the proposed role for this factor in axis induction (reviewed in Pelegri, 2003). However, the thapsigargin-induced chordin expression in the EVL was not suppressed by either tcfBD or the hcatenin MO (Fig. 5h, Table 2). On the other hand, injection of mRNA coding for DNtcf, which lacks the h-cateninbinding activating domain and acts as a constitutive repressor of transcription at TCF-binding sites (Molenaar et al., 1996), suppressed both the endogenous dorsal gene expression in the dorsal region and the ectopic thapsigargininduced chordin expression in the EVL (Figs. 5i and j). These data indicate that the thapsigargin-induced chordin expression in the EVL is regulated independently of hcatenin function but may still be dependent on tcf-dependent expression. The thapsigargin-induced marginal expression only becomes apparent at 50% epiboly, a time when both TcfBD and DNtcf dominant negative constructs induce ectopic dorsal gene expression on their own (Pelegri and Maischein, 1998) and when our evidence indicates that the h-cat MO ceases to have an effect (data not shown). Thus, we have been unable to test whether h-catenin or tcf function is essential for the thapsigargin-induced expansion of dorsal gene expression in the marginal zone at 50% epiboly.
Ectopic expression (%)
n
44 43 43 53 27 19 21 18 23 13
dorsal and anterior structures, as can be observed in live embryos at 24 hpf (Fig. 6b, compare to Fig. 6a). In situ hybridization analysis of embryos fixed at the 30% epiboly stage (4.7 hpf) revealed that the expression of dorsal genes such as goosecoid is increased in embryos injected with hcatenin mRNA (Fig. 6c). Injection of a h-galactosidase construct as a control did not result in any differences in either goosecoid expression or the 24 hpf phenotype compared to uninjected embryos (data not shown). Injection of mRNA coding for other factors expected to activate the Wnt/hcatenin pathway, including a dominant negative GSK-3 construct (Pierce and Kimelman, 1995), the GSK inhibitory factor GBP/Frat (Sumoy et al., 1999), the ligand zWnt8 (Kelly et al., 1995a), and its receptor FzA (Nasevicius et al., 1998), leads to the reversal of the mutant phenotype as measured by an increase in expression of goosecoid in injected hec mutant embryos (Figs. 6d– f). Thus, the hec mutant phenotype can be reversed by the activation of the Wnt/h-catenin signaling pathway at various levels within the pathway, including at the level of the Wnt ligand.
Discussion We have found that the maternal gene hec has a role in the establishment of the dorsoventral axis in the developing zebrafish embryo. Three other maternal-effect mutations affecting the induction of the dorsal axis have been reported: ichabod (Kelly et al., 2000), tokkaebi (Nojima et al., 2004), and brombones (Wagner et al., 2004). We have mapped the hec locus to linkage group 8 (JLG and FP, unpublished data), a distinct linkage group from the previously identified mutations, indicating that hec represents a new gene involved in the induction of the embryonic organizer. Females homozygous for a mutation in this gene produce a high proportion of embryos displaying defects in dorsal – anterior structures and an increased frequency of Ca2+ transients. The frequency of Ca2+ transients may affect proper Wnt/h-catenin signal transduction and consequently, the establishment of dorsal structures. Our analysis of hec
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Fig. 6. Dorsal gene expression increases in hec mutant embryos upon injection of upstream components of the Wnt/h-catenin pathway. (a – b) Injection of hcatenin mRNA leads to the appearance of dorsal structures in hec mutant embryos (24 hpf; anterior to left). (c – f) The frequency of embryos expressing goosecoid increases after injection with h-catenin RNA (c), dominant negative GSK-3 RNA (XG114) (d), GSK-3 binding protein RNA (e), and zWnt8 + FzA RNAs (f). ‘‘Wild-type gsc’’ refers to the presence of staining in a pattern similar to that seen in similarly staged uninjected wild-type embryos. ‘‘Ectopic gsc’’ refers to the presence of staining in regions in the margin outside the normal wild-type pattern. Presence of staining in a portion of the uninjected hec mutant embryos is due to the differences in expressivity of the phenotype. Injection of h-galactosidase mRNA does not lead to an increase in dorsal gene expression (data not shown).
mutant embryos provides the first genetic correlation between increased Ca2+ signaling and defects in the induction of the embryonic organizer. The increased frequency of Ca2+ transients during the blastula stages in the hec mutant embryos can be partially suppressed using pharmacological reagents. The ability of the A-protomer of pertussis toxin, L-690,330, and Xestospongin C to partially suppress the increased Ca2+ transients suggests that the wild-type hec gene product regulates Ca2+ transients that emanate from the endoplasmic reticulum upstream or at the level of G-protein function. These reagents and thapsigargin, have been shown to induce ectopic h-catenin protein nuclear localization and dorsal gene expression in wild-type embryos (Westfall et al., 2003b). Thapsigargin and CPA led to the expanded expression domains of dorsal genes in hec mutant embryos, further suggesting that the frequency of Ca2+ transients affects dorsal axis induction in both wild-type and hec
mutant embryos. Contrary to the result using CPA and thapsigargin, the other inhibitors of Ca2+ used in our experiments did not lead to the expression of dorsal genes in hec mutant embryos. This difference may be due to the manner in which these reagents act and the duration of their efficacy within the cell. Thapsigargin and CPA are cellpermeable reagents that can be applied to the embryos by immersion during the stages of development thought to be critical for dorsal induction. Exposure to the other inhibiting reagents, which are cell impermeable, can only be accomplished by injection into the embryos at early stages and, as we show (Fig. 4h), their effects in mediating Ca2+ transients are only temporary. It is possible that the injected reagents may not perdure in the embryos long enough to fully span the window of development important for dorsal axis determination. Alternatively, the effects of thapsigargin and CPA on Ca2+ concentrations could be qualitatively different from those induced by the other reagents used in
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this study, resulting in the differential effects on dorsal gene expression observed. Ca2+ transients at the blastula stages do not show any difference in their frequencies relative to the dorsal –ventral axis. Thus, it remains unclear how they contribute to the normal establishment of the dorsal – ventral axis. It is possible that a basal level of negative regulation of dorsal gene expression in the marginal cells by intracellular Ca2+ may contribute to the sharpening of the expression boundaries of genes activated by regionally localized Wnt/ h-catenin activity. While the Ca2+ transients originate in the EVL layer, they appear to influence dorsoventral patterning in cells of the deep layer. The Ca2+ release signal originating in the EVL may be transmitted via gap junctions present in zebrafish blastula cells to more internal cells, either through transmission of Ca2+ itself or secondary messengers such as IP3 (reviewed in Webb and Miller, 2003). Our analysis also suggests that Ca2+ transients are important for the regulation of gene expression within the cells of the EVL, although in this case this regulation appears to occur independently of h-catenin function. Previous studies have shown that Wnt/h-catenin signaling activity is essential for the induction of the dorsal axis (reviewed in Pelegri, 2003). hec mutant embryos do not show an accumulation of h-catenin protein in any region of the margin, suggesting a defect in Wnt/h-catenin signaling activity. Previous reports have suggested that the intracellular Ca2+ produced by Wnt/Ca2+ pathway activity can negatively regulate Wnt/h-catenin activity and the stabilization of cytoplasmic h-catenin protein (Slusarski et al., 1997b; Westfall et al., 2003b). Recent studies have implicated Wnt5 as the potential endogenous ligand that triggers Wnt/Ca2+ activity in early development, as a fraction of zebrafish embryos lacking maternal and zygotic Wnt-5a protein exhibit an expansion of dorsal cell fates at the expense of ventral fates (Westfall et al., 2003a). Previous reports have also linked FGF signaling to Ca2+ release in the Xenopus blastula (Dı´az et al., 2002, 2005) and the regulation of Bmp expression and dorsoventral patterning in zebrafish embryos (Fu¨rthauer et al., 1997, 2001, 2004; Cao et al., 2004). It is possible that the hec mutation is affecting a step within the Wnt/Ca2+ or FGF signaling pathways, which affects the release of intracellular Ca2+. Alternatively, the hec gene product could be required for a more general process involved in Ca2+ regulation in the early embryo that affects dorsal –ventral patterning. Previous reports have indicated that Ca2+ can act at multiple levels within the Wnt/h-catenin pathway. For example, in Xenopus, the Wnt/Ca2+ pathway activates PKC, which phosphorylates Dishevelled, thus potentially providing a level of regulation of the Wnt/h-catenin pathway (Kuhl et al., 2001). Similarly, research in cell culture and the mouse limb suggests that Wnt-5a, the ligand that activates the Wnt/Ca2+ pathway, may act by promoting GSK-3 independent degradation of h-catenin
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(Topol et al., 2003). Our data are consistent with multiple levels of regulation of Wnt/h-catenin signaling by Ca2+. The reduction in nuclear h-catenin staining observed in marginal cells of hec mutant embryos suggests that in these cells the effect of Ca2+ on the Wnt/h-catenin pathway occurs upstream of h-catenin stabilization. On the other hand, the h-catenin-independence of thapsigargin-induced dorsal gene expression in the EVL suggests that in this cell layer, Ca2+ regulation may occur downstream of h-catenin function, reminiscent of the direct, hcatenin-independent, effect of TAK1-NLK-MAP kinase activity on Tcf/Lef transcription factors (Ishitani et al., 2003). hec mutant embryos exhibited an increase in dorsal gene expression upon injection of all tested Wnt/h-catenin pathway components, including coinjection of zWnt8 and FzA. It is possible that the increased Ca2+ levels may induce a block in the Wnt/h-catenin pathway and that overexpression of Wnt/h-catenin activating factors may override this block. An alternative scenario to explain why exogenous activation of the Wnt/h-catenin pathway by ligand expression is independent of hec function is that the effect of the increased frequency of Ca2+ transients in hec mutant embryos may be on a pathway involved in the expression or function of the endogenous activator of the Wnt/h-catenin signaling pathway, and the exogenous Wnt ligand bypasses this endogenous pathway. A recent report has implicated Wnt11 as the endogenous ligand involved in Wnt/hcatenin activation in early Xenopus embryos (Tao et al., 2005), although the identity of such a putative signal in the zebrafish remains unknown. It will be of interest to test a potential role for hec function on such early activating signals. In conclusion, we report the functional characterization of a recessive maternal-effect mutation in the gene hecate. The wild-type hec gene product is required for the proper modulation of G-protein-dependent Ca2+ transients emanating from the endoplasmic reticulum and the establishment of dorsal anterior structures. Mutant embryos display an increase in Ca2+ transients during the blastula stages that appear to affect dorsal gene expression, and the Wnt/hcatenin pathway is not activated in these embryos. We also report that the Ca2+ transients in the EVL during the blastula stages may be important for the regulation of gene expression in the EVL itself.
Acknowledgments We are grateful to Stephen Ekker, David Kimelman, Randall Moon, and Michael Rebagliati for constructs. We also thank the members of the Pelegri lab for their comments and helpful discussions. Work in the Pelegri lab was funded by a March of Dimes Basil O’Connor grant and NIH, and in the Slusarski lab, by the American Cancer Society and NIH.
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