Activin-like kinase-4 (ALK4), also termed activin receptor IB (ActRIB), is a type I transforming growth factor-beta (TGFβ) receptor that was initially identified and characterized as an activin receptor (ten Dijke et al., 1993; Willis et al., 1996). In addition to activin, other TGFβs are capable of activating the ALK4 pathway, including nodal/Xnr-1, Vg1, and derriere (Reissmann et al., 2001; Yeo and Whitman, 2001; Cheng et al., 2003; Chen et al., 2004). Based largely on studies in mouse and Xenopus laevis, two animal models in which embryonic gain-of-function and loss-of-function experiments are routinely performed, diverse activities have been proposed for ALK4 and its ligands during vertebrate development, including regulation of mesoderm induction, primitive streak formation, gastrulation, primary axis formation, and left–right axis determination (Armes and Smith, 1997; Chang et al., 1997; Gu et al., 1998; Reissmann et al., 2001; Chen et al., 2004).
Despite the accumulation of evidence indicating that ALK4 is a key regulator of many embryonic processes, very little is known about the spatiotemporal aspects of ALK4 signaling during vertebrate embryogenesis. Mice that are ALK4 null fail to form a primitive streak and are embryonic lethal before gastrulation, indicating that ALK4 signaling is required very early in development (Gu et al., 1998). Functional assays in Xenopus that use expression of a kinase-deficient, dominant-negative ALK4 receptor result in defective dorsoanterior development, mesoderm formation, and left–right pattern, demonstrating that ALK4 signaling is also necessary for other developmental events (Chang et al., 1997; Chen et al., 2004). However, as with results obtained in mouse, the studies in Xenopus uncover a requirement for ALK4 signaling but do not reveal when or where in the embryo ALK4 functions to modulate various developmental processes. To begin to address this issue, we have performed a developmental analysis of ALK4 expression in Xenopus, using embryos ranging from early blastula stages to early tail bud stages.
Temporal Analysis of ALK4 Expression
As a prelude to performing in situ hybridization studies, ALK4 mRNA expression was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) to establish when ALK4 transcripts are present during Xenopus development. We find that ALK4 is expressed in all stages examined, ranging from early blastula stages to early tail bud stages (Fig. 1). As determined by comparison to ornithine decarboxylase-1 (ODC), an enzyme that is uniformly expressed throughout Xenopus development (Cao et al., 2001), maternal (stages 7–8) and zygotic (stages 9+) expression of ALK4 appears relatively invariant across these stages.
To determine whether ALK4 expression is restricted along the animal–vegetal axis, animal or vegetal pole tissue was harvested from embryos collected at stages 8–9 for RT-PCR analysis. As shown in Figure 1B, ALK4 is detected in both tissue types, as is ODC. By comparison, a maternally expressed mRNA, Vg1, is detected only in vegetal tissue, consistent with previous findings (Weeks and Melton, 1987).
Localization of ALK4 mRNA in Blastula Stage Embryos
Whole-mount in situ hybridization of stage 7 embryos reveals that ALK4 is expressed in cells of the animal pole (Fig. 2A), with faint nuclear staining detected in some vegetal cells positioned at or near the marginal zone (Fig. 2B). At stage 8, ALK4 expression is maintained in the animal pole and marginal zone (Fig. 2C,D), and by stage 9, staining shifts posteriorly as animal cells move toward the vegetal pole in a pregastrulation process called epiboly (Fig. 2E). Examination of sectioned embryos (Fig. 2F) shows that staining is present in cells of the animal pole and the marginal zone; however, no staining is discernible in the vegetal region, despite the detection of ALK4 transcripts in this tissue by RT-PCR. Hybridization of multiple stages of embryos with a sense-strand probe was performed as a negative control to determine the extent of nonspecific background signal, which was minimal to none (Fig. 2G,H). These findings, along with results obtained by RT-PCR, indicate that, during early blastula stages, maternal ALK4 expression is present in cells of the three presumptive germ layers.
Localization of ALK4 mRNA in Gastrula Stage Embryos
At stage 10, ALK4 is detected in cells of the animal pole and the marginal zone (Fig. 3A). Although cells within the blastopore do not express ALK4, pronounced staining is detected in the dorsal lip (Fig. 3A). Sections of hybridized embryos show that ALK4 is present in involuting mesoderm and in cells of the blastocoele roof (Fig. 3B). In addition, endoderm and mesoderm cells that are beginning to migrate over the blastocoele roof show staining for ALK4 expression (Fig. 3B,F, arrowheads). These localization patterns persist during stage 11 (Fig. 3C), with no obvious asymmetry detected in the left–right or dorsoanterior axes (Fig. 3D). At stage 12–12.5, ALK4 is detected in the entire ectoderm of whole-mount embryos (Fig. 3E). Sections of hybridized embryos show that ALK4 is expressed in cells of the entire blastocoele roof, the entire outer ectoderm, the involuting dorsal and ventral mesoderm, and to a lesser extent, cells of the ventral endodermal yolk mass (Fig. 3F). By stage 13–13.5, ALK4 signal begins to resolve to broad domains encompassing the dorsoanterior and midline regions of the embryo as viewed in whole-mount (Fig. 3G). Transverse and sagittal sections (Fig. 3H,I) show that ALK4 is expressed in the primitive notochord, paraxial mesoderm, prechordal plate, and ectoderm. No expression is detected in endoderm cells at this stage (Fig. 3H,I), and no staining is observed in embryos hybridized with sense-strand probe (Fig. 3J).
Localization of ALK4 mRNA in Neurula and Tail Bud Stage Embryos
At stage 15 (Fig. 4A,B), ALK4 continues to be detected in the dorsoanterior and midline (arrowheads) regions of the embryo where expression is present in the neural plate, the neural and non-neural ectoderm, and the neural folds. Other tissues in this region, including the somitic mesoderm and the notochord, also express ALK4. At stage 18 (Fig. 4C,D), ALK4 midline expression (Fig. 4C, arrowheads) is localized in the neural tube, neural folds, and the notochord. Staining also is detected in the somitic mesoderm and the dorsoanterior region of the developing head. Midline expression is maintained in stage 20 embryos (Fig. 4E, arrowheads), and stronger staining is detected in the outer ectoderm (Fig. 4F). Control embryos hybridized with a sense-orientation probe did not show discernible staining (Fig. 4G,H). At stage 22, dorsoanterior ALK4 expression is detected in all three regions of the developing brain as well as in the developing eye; at the posterior end of the embryo, ALK4 is detected in the unsegmented paraxial (somitic) mesoderm and the tail blastema (not shown). Transverse sections of hybridized embryos show that midline structures that express ALK4 include the spinal cord, neural crest, notochord, and somites (Fig. 4I). At stage 24, the midline distribution of ALK4 transcripts is maintained, and staining is additionally detected in the cement gland, branchial arches, eye vesicle, otic placode, lateral plate mesoderm, and in the bilateral heart fields (Fig. 4J–L).
In the early blastula stage embryo, ALK4 expression is detected in both the animal and vegetal pole regions of the embryo. These results are not unexpected, as longstanding models of germ layer formation in Xenopus have shown that TGFβ-related signaling (most likely Xnr-1 and/or Vg1) emanating from vegetal cells induces adjacent cells at the marginal zone to become mesoderm (Joseph and Douglas, 1998; Kofron et al., 1999; Osada and Wright, 1999; Agius et al., 2000). Cells that are located at the anterior-most region of the animal pole are normally fated to become ectoderm, although they too are competent to respond to the inductive signal if brought into proximity with vegetal cells. Our finding that ALK4 is expressed by the presumptive ectoderm and mesoderm indicates that ALK4 is expressed at an appropriate site to function as a receptor for the TGFβ-related signal(s) emitted from the vegetal cells. Moreover, the ALK4 expression in blastula stage embryos is consistent with functional studies in which widespread expression of a dominant-negative ALK4 receptor in Xenopus causes axial defects resulting from deficient mesoderm formation (Chang et al., 1997).
In addition to mesoderm formation, endoderm formation is dependent upon TGFβ-related signaling emitted by vegetal cells. A role for ALK4 in mediating the latter is supported by functional studies showing that ALK4 can induce endodermal marker expression (endodermin, GATA-4) in animal caps (Armes and Smith, 1997; Reissmann et al., 2001) and that expression of constitutively active ALK4 in zebrafish blastomeres converts them to an endodermal fate (David and Rosa, 2001). Consistent with these studies, we find by RT-PCR that ALK4 is expressed in vegetal pole tissue at levels comparable to those detected in animal pole tissues. However, because the sensitivity of whole-mount in situ hybridization in detecting endodermally expressed transcripts in Xenopus whole-mounts is much lower than RT-PCR (Smith and Harland, 1992; Ku and Melton, 1993), we cannot report its precise localization.
As gastrulation begins, ALK4 expression continues to be detected in the ectodermal and mesodermal germ layers, and a little later, in the ventral endoderm. These findings are similar to a report of ALK4 expression by all three germ layers in the early mouse embryo (Gu et al., 1998), and it is likely that ALK4 signaling is necessary for migration of cells as they involute. Mouse embryos that are ALK4 null fail to form a primitive streak, and chimeric embryos show that ALK4 null cells cannot contribute to the mesodermal layer during gastrulation (Gu et al., 1998). Consistent with these findings, we show that ALK4 is expressed in sites corresponding to the involuting dorsal and ventral mesoderm as well as in nascent mesendoderm cells that are migrating over the roof of the blastocoele. It is notable that ALK4 expression also was detected in the dorsal lip, a structure that is functionally analogous to the node in avians and mammals and the shield in fish. We find that the dorsal lip in Xenopus shows prominent ALK4 expression, suggesting that ALK4 signaling could be involved in dorsoventral patterning of the body plan. Because animal cap assays have thus far only been performed by using pan-mesodermal markers (Armes and Smith, 1997; Reissmann et al., 2001), it is not known whether this is the case; however, consistent with this possibility, ectopic expression of dominant-negative ALK4 results in embryos that exhibit highly disorganized axial structure with respect to dorsoanterior development (Chang et al., 1997).
Mesodermal ALK4 expression during neurula and tail bud stages is observed in midlines structures, including the notochord, neural tube, paraxial (somitic) mesoderm, and the neural crest. Ectodermal ALK4 expression is detected in both neural and non-neural regions and the significance of this expression is not known, as it has been suggested that ALK4 is not involved in neural induction (Chang et al., 1997). Nevertheless, the prominent ALK4 expression in the neural tube and its derivatives—brain and spinal cord—as well as its conserved expression in these tissue types in mouse (Verschueren et al., 1995) suggests that this issue is still open to investigation. Whether ALK4 additionally plays a role in induction of non-neural ectodermal tissue types also remains to be determined. With regard to ALK4 expression by paraxial (somitic) mesoderm, and later by developing somites, these results indicate a possible role for ALK4 signaling in somitogenesis. Although this role has not yet been directly investigated, a recent report indicates that activin, a known ALK4 ligand, is capable of inducing ectopic somite formation in the chick embryo (Patwardhan and Ghaskadbi, 2001) and analysis of ALK4 expression in later stage mouse embryos indicates that ALK4 is expressed in tissues that are derived from somites, e.g., ribs, limb muscles, and the body wall (Verschueren et al., 1995). Moreover, studies in Xenopus indicate that TGF-β5 is expressed in somites and other areas that overlap with the sites of ALK4 expression reported here (Kondaiah et al., 2000). Additional sites of ALK4 expression in neurula stage embryos are the neural folds, the neural crest, and the branchial arches, through which neural crest cells migrate. Although this is the first report of ALK4 expression in these embryonic regions, there is abundant evidence that TGFβ signaling regulates neural crest cell emigration (Chai et al., 2003), and by analogy to ALK4 expression in other migrating cell types (involuting mesendoderm), our results suggest that ALK4 could play a role in this process.
Other notable sites of ALK4 expression in tail bud stage embryos include the developing eye, otic placode, and the bilateral heart fields. ALK4 expression in the retina of the embryonic rat eye has been reported previously and described in more detail (Yamada et al., 1999). With regard to expression in the otic placode, no requirement has been identified for ALK4 in ear morphogenesis, although studies do indicate not only that TGFβs are expressed in the ear but also that TGFβ signaling is necessary in promoting its normal development (Ito et al., 2001; Chang et al., 2002; Somi et al., 2003). The finding that ALK4 additionally is expressed in the bilateral heart fields is consistent with the expression of Vg1 in the linear heart tube of the chick (Somi et al., 2003) and raises the possibility of yet another role for ALK4 signaling—cardiogenesis—in vertebrate embryogenesis.
Finally, with regard to formation of the body plan, it should be noted that there are several sites of ALK4 expression that are interesting in the context of left–right axis determination. These locations include involuting mesendoderm cells; midline structures such as the dorsal lip, notochord, and neural tube; and the lateral plate mesoderm. Because these tissues are involved in relaying left–right pattern in the embryo and moreover, because they are thought to do so, at least in part, by means of TGFβ-related signaling (Yost, 1998; Mercola and Levin, 2001; Whitman and Mercola, 2001; Hamada et al., 2002), the detection of ALK4 at these sites suggests that this TGFβ receptor could play multiple, important roles in left–right development in addition to its other roles in vertebrate embryogenesis.
Eggs were obtained from PMSG- and hCG-injected adult albino Xenopus laevis females (fertilized with minced testes) and were de-jellied with 2.5% cysteine. Embryos were reared in 1/3× MMR and staged according to Nieuwkoop and Faber (1967).
Total RNA was extracted from embryos of stages 7–24 as indicated in the figure legends using TriReagent (Molecular Research Center, Inc.) according to the manufacturer's protocol. Two micrograms of total RNA from each stage was used for first-strand cDNA synthesis using SuperScript III first-strand synthesis kit from Invitrogen, and one tenth of the cDNA served as template for PCR. Twenty-two cycles (94°C, 30 sec; 55°C, 1 min; 72°C, 30 sec) were carried out for both ODC and Xenopus ALK4 (XALK4). Primers for ODC (forward 5′-CAA CGT GTG ATG GGC TGG AT-3′ and reverse 5′-CAT AAT AAA GGG TTG GTC TCT GA-3′) were synthesized according to Altmann et al. (2002). Primers for XALK4 (forward 5′-GCG GAG CTA CCG GCC TTC TTC-3′ and reverse 5′-TGG GAT TGC AAT AAC AGC TAC-3′) were synthesized according to Chang et al. (1997). The primer sequences for Vg1 (forward 5′-GAT GTT GGA TGG CAA AAC TGG-3′ and reverse 5′-CCA CAC TCA TCT ACT GCC ATG-3′) were synthesized according to Alarcon and Elinson (2001). For comparison of transcripts between animal and vegetal regions, animal caps and vegetal cells were dissected out separately from 40 stage 8–9 embryos by using #5 forceps. Total RNA extraction and RT-PCR were performed as described above except that the PCR was run for 25 cycles.
Whole-Mount In Situ Hybridization
A BglII insert from truncated ALK4 (tALK4; Chang et al., 1997) encoding the extracellular, transmembrane, and kinase-deficient cytoplasmic domains of Xenopus ALK4 was ligated into BamHI digested CS2+. Digoxigenin-labeled riboprobes were synthesized with the MAXIscript kit from Ambion using HindIII linearized CS2-tALK4 template and T7 RNA polymerase for antisense probe and XbaI linearized CS2-tALK4 template and SP6 RNA polymerase for sense probe. Whole-mount in situ hybridization was performed as described previously (Harland, 1991) with the following modifications: preabsorption of the anti–digoxigenin-alkaline phosphatase antibody with matched stage embryos for 24 hr at 4°C and probe hybridization at 68°C to reduce background. The BCIP/NBT color reaction was stopped just before corresponding sense-hybridized embryos began to stain.
After whole-mount in situ hybridization, the stained embryos were photographed and then sectioned as described (Germroth et al., 1995) with the following modifications: embryos were soaked in 15% polyacrylamide for 30 min and oriented according to the plane of sectioning after the addition of ammonium persulfate. Sections were cut with a Lancer Vibratome at 200 μm thickness.
We thank Carlene Brandon (MUSC) for photographic assistance, Cara Baldwin from Dr. Joseph Yost's lab (University of Utah) for assistance with in situ hybridization, and Chiffvon Stanley and Dr. Robert Thompson (MUSC) for assistance with polyacrylamide sectioning.