Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish
© Hagos and Dougan; licensee BioMed Central Ltd. 2007
Received: 11 January 2007
Accepted: 28 March 2007
Published: 28 March 2007
The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects.
We show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffer's vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated.
We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.
During vertebrate development, cells become irreversibly committed to particular fates after a series of inductive events. The first step of this process is completed during gastrulation, when cells are allocated to the three germ layers. Fate maps of vertebrate embryos show considerable organization before gastrulation, since different mesodermal and endodermal structures are derived from distinct positions along the major body axes [1–3]. In zebrafish late blastula stage embryos, for example, endoderm progenitors are restricted to the four rows of cells closest to the yolk, known as the margin, while mesoderm precursors extend further towards the animal pole [4, 5]. The germ layers are also patterned along the dorsoventral axis, such that the notochord is derived from dorsal mesoderm, the heart comes from lateral mesoderm and blood comes from ventral mesoderm [6, 7]. TGF-β signals of the Nodal-related subclass are required to induce and pattern the germ layers in vertebrates . Nodal signaling is mediated by a receptor complex containing the TGF-β Type I receptor, ALK4, the Type II receptor, ActR-IIB, and the Cripto/One- Eyed-pinhead (Oep) co-receptor [9, 10]. The Nodal receptors can also be activated by other TGF-β ligands, including Activin and Vg1 [9, 11]. For this reason, the Nodalrelated proteins, Activin and Vg1 are collectively termed Activin-like signals.
The requirement for Nodal-related proteins to induce mesoderm and endoderm is conserved throughout the vertebrate lineage . There are three nodal-related genes in zebrafish, but only two, squint (sqt/ndr1) and cyclops (cyc/ndr2), have overlapping roles in mesendoderm formation . The third nodal-related gene, southpaw (spaw/ndr3), is only expressed after gastrulation and is involved in establishing the left-right body axis . In cyc single mutants, defects in mesendoderm are first detected at mid-gastrulation and the embryos lack floorplate and ventral diencephalon at later stages [14–16]. sqt single mutants have severe deficits in dorsal mesodermal derivatives at early stages, but the embryos recover and many survive to adulthood [17, 18]. This recovery depends on cyc function, since sqt; cyc double mutants lack all derivatives of the mesoderm and endoderm in the head and trunk, including the skeletal muscle, heart, pronephros, blood and gut .
Both gain-and loss-of-function studies indicate that Activin-like signals act in a concentration-dependent manner to specify cell fates [20–23]. In explants, high doses induce marginal cell types, such as prechordal plate and endoderm, whereas lower doses induce notochord and muscle . Conversely, endoderm and prechordal plate are more sensitive to reductions in Nodal levels than are notochord and muscle [17, 23]. Zebrafish Sqt behaves like a morphogen, acting directly on cells at a distance to specify fates in a concentration dependent manner [25, 26]. These results and other data have led to the suggestion that cells adopt fates depending on their position within a gradient of Nodal-related protein .
A spatial gradient model of Nodal signaling, however, does not account for two key observations. For example, in the animal region of the mesoderm territory in pregastrula stage embryos, somite precursors are intermingled with neurectoderm progenitors, which are specified in the absence of Nodal function [5, 17]. Near the margin, by contrast, somite precursors are intermingled with endoderm precursors, which require high levels of Nodal [4, 17]. This raises the question of how adjacent cells could be exposed to different Nodal doses. Secondly, Cyc can fully compensate for loss of the Sqt morphogen despite the fact that it only acts over a short range [17, 25, 28]. This indicates that the long-range action of Nodal signals is not necessary for correct induction and patterning of the mesoderm and endoderm.
Experiments suggest that the role of Nodal signaling is quite dynamic, but it has been difficult to determine what are the functions of Nodal signals at different times. The expression pattern of nodal-related genes changes rapidly during the blastula stages in frogs, fish and mice [21, 24, 29, 30]. Efforts to determine when Nodal signals specify distinct mesodermal and endodermal cell types have been hampered by the fact that conditions which alter the timing of Nodal signaling also change the levels of nodal related gene expression. For example, levels of Nodal decrease in zygotic oep mutants as maternally supplied Oep mRNA and protein decay and eventually disappear [31, 32]. Similarly, cyc expression is both reduced and delayed in sqt mutants . Thus, it has not been possible to determine whether the fate changes observed in these mutants are due to altered timing of Nodal signaling or to the reduction in Nodal activity.
Experiments in frogs and fish have suggested two mechanisms by which Nodal signals may act to specify different tissues at different times. When Xenopus animal cap cells are exposed to Activin-soaked beads for different lengths of time, the responding cells exhibit a stepwise progression of cell fate specification as a concentration gradient of Activin is established within the explant . These results suggested that cells constantly monitor ligand levels and "ratchet-up" their response when the concentration exceeds certain threshold levels. In this view, cell fates are determined by the absolute number of receptors occupied by the ligand rather than by how long cells are exposed to the ligand [34, 35]. By contrast, experiments in zebrafish with a conditional allele of cyc determined that cells need to be exposed to Nodal signals during a two-hour window in order to become floorplate . This raised the possibility that cells respond differently to Nodal signals depending on when they are exposed. In this view, cells have intrinsically defined periods during which they are able to adopt particular fates if exposed to the proper Nodal dose.
We have utilized a pharmacological approach to determine when Nodal signals specify the different mesodermal and endodermal cell types in the zebrafish. For the first time, we have been able to block the activity of Nodal receptors during discrete blastula stages by treatment with the small molecules SB-431542 or SB-505124 and without disrupting signaling at earlier stages or altering endogenous Nodal levels [37, 38]. We find that Nodal signals specify most mesodermal and endodermal cell types between the mid-blastula (3 h) and late blastula (5 h) stages. By examining embryos with increased or decreased levels of Sqt and Cyc signals, we show that the Nodal dose controls the timing of cell fate specification. This rules out the idea that cells adopt different mesoderm and endoderm fates depending on when they are exposed to Nodal signals. We also show that embryonic cells respond to a uniform, high dose by adopting progressively more marginal fates with longer exposures to Nodal signals. This time-dependent transformation of cell fates is inconsistent with some aspects of the ratchet model. We conclude that cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.
Drug treatment at MBT prevents the response to zygotic Nodal signals in embryos
We next asked if SB-431542 could prevent the response to a mutated and constitutively-activated receptor that is active even in the absence of ligand, such as TARAM-D . TARAM-D acts in a cell-autonomous manner to induce expression of Nodal target genes, resulting in dorsalized embryos and expanded gsc expression (Fig. 3E, G; N = 30) [28, 45]. In most cases, SB-431542 completely suppresses the response to TARAM-D, consistent with its proposed mode of action (data not shown). In the course of our experiment, however, occasional embryos received higher doses of the activated receptor and displayed a milder phenotype than their siblings. These embryos have cyclopia and reduced or absent mesodermal tissues, including trunk somites and notochord (Fig. 3F). gsc expression is dramatically reduced in these embryos (Fig. 3G, H; N = 20). Thus, high levels of activated receptor can rescue the defects caused by the drug. This demonstrates the specificity of the drug, since the activated Nodal receptor would not rescue defects caused by blocking receptors for other signaling pathways. SB-431542 also blocks the response to ubiquitously expressed Sqt (Fig. 3I–L). Thus, the drug is able to effectively penetrate and act within the entire embryo. In these experiments, we injected embryos with sqt or TARAM-D mRNA at the 1–4 cell stage (1 h) and treated with the drug at 2.75 h. Therefore, SB-431542 can block the response to receptors already present during the cleavage stages. Because the drug is effective at blocking Nodal signaling when applied as late as 2.75 h, this suggests that maternally supplied Activin-like ligands normally act after MBT, if at all, to effect specification of cell fates.
Nodal signals specify mesodermal tissues at different times within a three-hour period
We next treated embryos with SB-431542 at different times between 2.75 and 6 h post-fertilization. Embryos treated with SB-431542 at 3.7 h contain a small number of trunk somites, but we detected no other mesodermal tissues in the trunk (Fig. 4A1; Fig. 5). ntl was expressed in a truncated axial domain and only a small number of disorganized trunk somites are apparent, as indicated by MyoD expression (Fig. 4A4, A5). flh was expressed in two bilateral domains within the ectoderm, but not at the midline (Fig. 4A6), consistent with the lack of notochord tissue in these embryos (Fig. 4A1, Fig. 5). The expression of the pan-mesodermal marker, ntl, but not notochord marker, flh, at the midline suggests that these cells are specified to be dorsal mesoderm, but are unable to complete the differentiation program [46, 47]. The lack of pax2.1 expression in the intermediate mesoderm indicates that the pronephros was not specified at this time point (Fig. 4A7). Therefore, only trunk somites were specified following the shortest exposure time to Nodal signals.
Embryos treated with SB-431542 at later time points contain a more diverse array of mesodermal tissues (Fig. 5). Small amounts of notochord are detected in embryos treated at 4 h (Fig. 4B1, arrow; Fig. 5). Red blood cells are also apparent in live embryos examined at 48 h (Fig. 5). flh expressing cells populate the midline, but do not induce expression of MyoD in adaxial cells within the segmented mesoderm (Fig. 4B4-6). MyoD is still expressed in adaxial cells in the presomitic mesoderm (Fig. 4B5). pax2.1 expression is also apparent in embryos treated at 4 h (Fig. 4B7). The hatching gland and Kupffer's vesicle are first visible in embryos treated with the drug at 4.3 h (Fig. 4C3, arrowhead; Fig. 5). Although we observed beating hearts in embryos treated at 4.7 h, a functioning circulatory system was only established in embryos treated at 5 h (Fig. 5). Since blood is specified before the heart, we attribute the delay in circulation to the time required to specify the cells comprising the vasculature, although we have not directly examined these cell types. Tissues were specified in the same temporal order in a time course using SB-505124 (data not shown).
The total amount of mesoderm increases as embryos are treated at successively later stages. Embryos treated at 3.7 h have between 5–7 trunk somites (Fig. 4A1, 5). By contrast, embryos treated at the onset of gastrulation contain the normal complement of somites (Fig. 4E1, 5). Thus, new somite tissue is induced throughout the blastula period. Similarly, a truncated notochord forms in embryos treated at 4 h (Fig. 4B1, 6, arrow), but notochord tissue extends more anteriorly when later stage embryos are treated (Fig. 4C1-E1; C6-E6). We were unable to detect a difference in the length of notochord in embryos treated at 5 h and 6 h (Fig. 4D6, E6). Expression of flh in the neurectoderm diminishes concomitantly with its expansion along the midline, indicating that signals from the mesoderm inhibit the differentiation of some neural tissues (Fig. 4A6-C6). pax2.1 expression is weak when Nodal signaling is blocked at 4 h (Fig. 4B7), but intensifies when Nodal signaling is blocked at later stages (Fig. 4C7-E7). This demonstrates that after 4 h, Nodal signals act to specify the somites, notochord and pronephros, simultaneously. This argues against, but does not completely exclude, a model in which Nodal signals specify different mesoderm and endodermal cell types during distinct time-windows.
Nodal signals pattern the animal-vegetal axis in a time-dependent manner
Nodal levels control when cell fates are specified
According to the "ratchet-model", cells generate a response appropriate to the highest dose to which they are exposed independently of the duration of exposure . If true, then cells should always adopt the most marginal fate when they are exposed to a uniformly high Nodal dose, regardless of how long the exposure lasts. In contrast to this prediction, however, we found that cells in Sqt-injected embryos are transiently specified to the more animal flh expressing fate (Fig. 9A4). As the duration of exposure increases, flh expression gradually diminishes (Fig. 9A4-A7), and gsc and sox17 expression increase concomitantly (Fig. 9B4-C7). This demonstrates that cells adopt progressively more marginal identities in response to increasing exposure times to Nodal signals. These results rule out the possibility that presumptive mesoderm and endodermal cells respond to Nodal signals by a ratcheting-type mechanism.
Chemical inhibitors of ALK 4/5/7 are new tools for dissecting the roles of Nodal signals
In this study, we addressed the question of when members of the Nodal-related subclass of the TGF-β superfamily act to pattern the mesoderm and endoderm. We took a pharmacological approach to inactivate Nodal signaling at different times, and examined the resulting cell fates by an extensive analysis of gene expression and morphology. Three lines of evidence show that we were able to inhibit zygotically expressed Nodal signals. Firstly, we generated a phenocopy of sqt; cyc double mutants by treating embryos with 800 μM SB-431542 at the mid-blastula stage, when zygotic expression of sqt and cyc initiates (Fig. 1). Secondly, we could phenocopy cyc single mutants by treating embryos at the onset of gastrulation, when cyc expression predominates (Fig. 4). These two experiments demonstrate that our treatment reduces receptor activity to at least the levels in the respective mutants. We confirmed our results with a second drug, SB-505124, which is more potent and soluble than SB-431542 (Fig. 1; data not shown), which rules out possible artefacts due to the high dose of SB-431542. Finally, drug treatment in the late blastula stages inhibited expression of a Nodal target gene within 30 minutes (Fig. 2).
Our results differ markedly from those of earlier studies, in which 50 μM SB-431542 was unable to reproduce the sqt; cyc phenotype when added to embryos older than the 8-cell stage [40, 41]. Two technical aspects of our treatment protocol may account for our different results. First, we used a much higher dose of SB-431542 (800 μM) than the other groups. Secondly, we perforated the embryos to ensure the drug fully penetrated the embryos. Perforation was not necessary with SB-505124, which was also effective at a much lower dose (50 μM). We conclude that the milder effects of the drug reported by others are due to the poor ability of SB-431542 to penetrate the embryo as the number of cells increases during the cleavage stages. Even though multiple ligands can activate the ALK4/5 and 7 receptors, our phenotypes all resemble those resulting from reductions of nodal-related gene function [17, 23]. This indicates that the other Activin-like ligands are either not expressed during the stages we examined or act downstream of Nodal signals.
Time-dependent patterning of the animal-vegetal axis by Nodal signals
Previous attempts to determine when Nodal signals specify different mesoderm and endoderm cell types have focused on the analysis of oep mutant embryos. In Zoep mutants, late Nodal signaling is blocked due to the absence of an essential co-receptor, and prechordal plate and endoderm do not form [32, 51]. It is not clear, however, whether these defects are due to the absence of late Nodal-signaling activity, or to the reduction of signaling levels caused by the decay of maternally supplied Oep protein. In an alternate approach to determine the role of Nodal signals at different times, oep function was restored to MZoep mutants at different stages, rescuing the ability of mutant cells to respond to Nodal signals [31, 52]. In these experiments, restoring Nodal signaling at early stages completely rescued MZoep mutants. By contrast, prechordal plate and endoderm was missing when Nodal signaling was restored at later stages. Although these results are apparently consistent with our findings, we found that sqt and cyc expression are expressed at very low levels when oep function is supplied at late stages (4 h; Hagos and Dougan, submitted). Since the defects in late-rescued MZoep mutants result from aberrant nodal-related gene expression, these experiments do not address the question of when Nodal signals are required to specify cell fates.
By conditionally inactivating the Nodal receptors, we were able to determine the specification state of the presumptive mesoderm and endoderm at different embryonic stages. We found a time-dependent progression of cell fate specification along the animal-vegetal axis, consistent with earlier studies demonstrating that Nodal signals pattern the animal-vegetal axis, but not the dorsoventral axis . Blocking Nodal signals at late stages inhibits formation of tissues derived from the margin, such as prechordal plate and endoderm, but not from more animal regions, such as notochord or somites (Figs. 4, 5, 6). Previous studies have determined that endoderm and prechordal plate require higher doses of Nodal signals than somites [17, 31]. This suggests a linkage between Nodal dosage and the length of exposure.
Nodal levels control when cells are specified to become mesoderm and endoderm
Our results place Nodal signals at the top of a developmental program that determines the fates of responding cells and controls when these fates are specified. We considered the possibility that Nodal signals pattern the mesoderm and endoderm by acting in fixed time windows to specify different cell types. When Nodal levels are low, as in sqt mutants, specification of endoderm does not begin until early gastrulation (7 h; Fig. 8C7). By contrast, when Nodal levels are high, specification of endoderm begins 1.7 h earlier (Fig. 9C4). We conclude that cell identities are specified at different times depending on the Nodal dosage (Figs. 8, 9). These results exclude the possibility that cells have fixed time windows during which they can adopt particular mesoderm and endodermal fates in response to Nodal signals. To the contrary, the level of Nodal signalling determines when cells are specified to adopt particular mesoderm and endodermal identities.
Previous cell transplant experiments defined a broad window of competence during which cells can respond to mesoderm and endoderm inducing signals, which we now know to be the Nodal-related proteins [53, 54]. Experiments in Xenopus animal caps demonstrated that this window of competence is controlled by an intrinsic timing mechanism and ends by mid-gastrulation . Our results show that within this broad window, cells have a considerable degree of flexibility as to when they can become mesoderm and endoderm that depends on the levels of Nodal signals. At the molecular level, the loss of the ability to respond to Nodal signals could reflect the Nodal-dependent induction of a feedback inhibitor of the pathway. Consistent with this idea, expression of the secreted Nodal antagonist Lefty is under the control of Nodal signaling [23, 56]. Thus, one role of Lefty could be to place a temporal limit on when cells can respond to Nodal signals. In support of this, Nodal signals persist well into gastrulation when lefty function is depleted, and act during this time to convert ectoderm into mesoderm and endoderm (X. Fan and S. Dougan, unpublished data) [26, 57].
The length of exposure to Nodal signals determines cell fate choices
Generating a response to a cumulative Nodal dose
We found that cells respond to the cumulative dose of Nodal signals to which they are exposed. In embryos exposed to a uniform, high Nodal dose, cells exhibit a time dependent transformation towards more marginal fates as the length of exposure increases (Fig. 9). This means that cells must have a mechanism to record the duration of their exposure to Nodal signaling and to generate a response to the cumulative dose. Although this regulation may occur at many different levels, the ultimate readout is at the level of gene transcription. Of the marker genes we analyzed, gsc is a likely direct target of the Nodal pathway . gsc expression initiates at 4 h in the absence of both sqt and cyc function, but quickly decreases . This indicates that Nodal signals are required for maintenance, but not for the induction of gsc expression. In this study, we showed that gsc expression is lost when Nodal signaling is inactivated at 4.3 h, but continues when Nodal signaling is blocked at 5 h (Fig. 6). Thus, Nodal input is required for about an hour in order to maintain gsc expression. After this transient maintenance phase, gsc expression continues independently of Nodal, by an unknown mechanism. In sqt mutants, it takes a longer period of time for the gsc promoter to transit to the Nodal independent phase, whereas the gsc promoter reaches this state more rapidly when Sqt is overexpressed. Other genes have been shown to undergo similar phases of gene regulation, most notably the Drosophila engrailed gene , but this is the first case to our knowledge in which the levels of a secreted factor control the length of the maintenance phase of a target gene.
A spatio-temporal gradient model for patterning by Nodal signals
Any model for how Nodal signals act to pattern the mesoderm and endoderm must account for four observations. First, the model must explain how adjacent cells become exposed to different levels of Nodal signals. Fate mapping studies show that precursors of cell types that require different levels of Nodal signaling, such as somites and endoderm, are juxtaposed in the pre-gastrula stage embryo [5, 17]. Second, the model must account for our observation that the blastomeres are highly dynamic during the period they respond to Nodal signals. We found that Nodal signals act primarily before 5 h (40% epiboly) (Figs. 4, 5, 6), a period in which cells divide rapidly and frequently change positions with respect to each other . This presents a particular challenge to classic morphogen gradient models, which generally assume a static field of responding cells. Third, the model must explain how a short-range signal, like Cyc, can specify the same cell types as a long-range signal, like Sqt. Finally, the model must account for our observation that cells respond to the cumulative dose of Nodal signals.
We propose that the total Nodal dose is a function of both the length of time a cell is exposed to Nodal signals and the distance of a cell from the Nodal source (Fig. 10B). Key predictions of this model remain to be tested, but it accounts for all these observations. In this view, cells that remain near the Nodal source for an extended period would receive a high dose and adopt a marginal cell fate, such as prechordal plate or definitive endoderm. Conversely, cells that move away from the source after a short time would receive a lower dose and become somites. Specification of mesoderm and endoderm is delayed in sqt mutants because it takes longer than in wild type for cells to accumulate the necessary Nodal dosage. Because the gradient of positional information is influenced by the length of time responding cells are exposed to the signaling source and their distance from the source, we call this the spatio-temporal gradient model. In other species, Nodal signals also pattern tissues comprised of dynamic cell populations, such as the node and primitive streak in mice and Hensen's node in the chicken [61, 62]. Thus, cell movements could provide a general mechanism for generating a gradient of exposure to Nodal signals during mesoderm patterning in all vertebrates.
Our model predicts that a stable source of Nodal signals exists in the embryo that is independent of the dynamic cell movements of the responding cell population. We propose that the extraembryonic yolk syncytial layer (YSL) acts as this source. Sqt is normally expressed in this tissue and can induce fate changes in overlying blastomeres when overexpressed in the YSL [19, 24]. We suggest that Nodal signals in the YSL act to induce and/or maintain nodal-related gene expression in the overlying blastomeres via the autoregulatory pathway. If a cell that is initially close to the YSL moves away, it will lose expression of sqt and cyc. Conversely, sqt and cyc expression will be induced in a cell as it moves closer to the YSL. Thus, the autoregulatory pathway provides a mechanism by which a stable zone of Nodal signaling can be imposed upon the dynamic, intermixing population of cells at the embryo margin.
Our data indicate that Nodal signals act in a time-dependent manner to pattern the mesoderm and endoderm. Three lines of evidence support the idea that cells respond to the cumulative dose of Nodal signals. First, marginal cell types, which are specified by the highest Nodal dose, require the longest exposure to Nodal signals. Second, cell fate specification is delayed when Nodal levels are reduced, and accelerated when Nodal levels are increased. Finally, in response to a uniform, high Nodal dose, cell fates transform toward progressively more marginal identities as the length of exposure increases. These results rule out the possibility that Nodal signals act during discrete time windows to specify different mesodermal and endodermal cell types. They are also inconsistent with the "ratcheting-up" model, in which the absolute number of occupied receptors determines cell fates, not the duration of exposure. We conclude that cells respond to the cumulative Nodal dose, which we suggest is a product of the distance of the responding cell from the signaling center and the length of exposure.
Zebrafish strains and staging
We used the WIK strain to obtain wild type embryos. Embryos homozygous for the sqt cz35 null allele were obtained from crossing mutant adults. oep tz57 mutant adults were obtained by mRNA injection, as previously described . In all experiments, the embryonic stages were determined by morphology and are reported as hours postfertilization (h) at 28.5°C, according to Kimmel et al. (1995).
SB-431542 (4- [4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl-1H-imidazol-2- yl]benzamide), was obtained from Tocris (Ellisville, MO) and stored as a 100mM stock in DMSO at -20°C. SB-505124 (2-(5-benzo[1, 3]dioxol-5-yl-2-tert-butyl-3Himidazol- 4-yl)-6-methylpyridine hydrochloride) was a kind gift from GlaxoSmithKline (King of Prussia, PA) and is stored at 10 mM in DMSO at 4°C. For the drug time course studies shown in Figs. 1 and 4, approximately 1000 embryos equivalently staged embryos from 3–4 single pair matings were pooled, split into 10 dishes at a density of 100 embryos/dish, and raised in an incubator at 28.5°C. For drug treatment, embryos from one dish were removed at the desired stage, perforated near the margin with a pulled capillary tube, and split into glass dishes containing the drug in 5 ml embryo medium, at a density of 25 embryos/dish. Embryos were fixed at 10h and split into three groups for analysis of ntl, flh or shhb expression, or fixed at 14h and split into two groups for analysis of MyoD or pax2.1. Time courses depicted in other figures followed the same protocol, but embryos were fixed at the stages indicated for analysis of marker gene expression. In each figure, representative images are shown, and all embryos were treated on the same day. Embryos damaged by the perforation were discarded. Embryos treated with SB-505124 did not require perforation. In all experiments, some embryos in each experiment were allowed to develop until 24 h and examined morphologically to verify the efficacy of the treatment. All experiments were performed at least two times. The effective dose on 2.75 h embryos SB-431542 was determined in a titration of 5 μM-1mM SB-431542 or 3 μM–75 μM SB-505124. SB-431542 treatment was always associated with the formation of a dark precipitate in the solution. At 800 μM, all embryos resembled sqt; cyc mutants, whereas lower doses generated milder phenotypes similar to Zoep mutants . This milder phenotype is also observed by treating cleavage stage embryos with 50 μM SB-431542 (data not shown) [40, 41]. The previously described toxic effects of SB-431542 in cell culture are apparent at doses above 800 μM on blastula stage embryos and above 100 μM on cleavage stage embryos (data not shown) . For SB-505124, the lowest dose that produced the sqt; cyc phenotype ranged from 30–50 μM, depending on the age of the drug.
Microinjections and whole-mount in situhybridization
The sOep, sqt and TARAM-D cDNAs were described previously [19, 32, 45]. Sense transcripts were synthesized using the Message Machine kit (Ambion, Inc., Austin TX). We injected 10pg sqt, TARAM-D or β-galactosidase mRNA into chorionated embryos at the 1–4 cell stage. 100pg sOep mRNA was co-injected into the YSL of MZoep mutants with the Oregon Green 488 lineage tracer dye (Invitrogen, Inc., Carlsbad, CA) to verify the targeting of the injection, as described . In situ hybridizations were performed as in Dougan, et al., 2003. We used the following probes: sqt, cyc, gsc, ntl, flh, MyoD, pax2.1, shhb, sox17, mezzo, cyp26, cmlc2, amhc and vmhc [16, 19, 23, 46–49, 63–69].
We thank Benjamin Feldman, Jim Lauderdale, Walter Schmidt, Howard Sirotkin, Will Talbot and an anonymous reviewer for valuable comments on the manuscript; Steve Stice for suggesting the use of SB-431542; Nicholas J. Laping for advice on the use of SB-505124; Xiang Fan, Katie Holcombe and Chari Jefferson for fish care; Steve Dalton and members of the Dougan and Lauderdale laboratories for helpful discussions. S. Dougan is a Georgia Cancer Coalition Distinguished Investigator. This work was supported by the Georgia Cancer Coalition and a grant from the University of Georgia Research Foundation.
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