If this developmental axis were inverted during very early development, without moving the mouth-forming agency, all aspects of definitive dorsoventral organization would necessarily invert. We cloned the S. kowalevskii ortholog of admp from an expressed sequence tag collection.

(E) pitx, (F) hex, (G) olig, (H) pox neuro. Note the three parts of the body: the prosome (proboscis) at the anterior end, the narrow mesosome (collar) in the middle, and the elongated metasome to the posterior (only half the length is shown). (G) Expression of olig on day 2, just after gastrulation, and (H) at day 3. E-mail: gerhart@socrates.berkeley.edu, 29 Dec 2015: Thus, uniform exposure of the embryo to excess Bmp protein activates the widespread expression of those genes normally expressed dorsally, and represses those normally expressed ventrally. For injection of the siRNAs, several eggs were added to a small dish containing freshly diluted sperm for 15 s and then transferred to a silicon rubber injection chamber. (A) Transforming growth factor class genes admp, bmp2/4, and bmp5/8, (B) tsg, (C) cv2, (D) tbx2/3. In the dorsoventral dimension, the basic Bmp-Chordin axis would have been just as fundamental in the common lineage of bilaterians, but considerable modification of the subdivisions of this axis has certainly occurred in deuterostomes. Also, it is expressed in a uniform speckled pattern in the prosome ectoderm, as reported earlier [45].

The final gene expressed in the dorsal endoderm is pax1/9. RNA interference; Shh, A central question would be whether the segregation of epidermis and neurogenic ectoderm, as a means of centralizing the nervous system, dates back to the bilaterian ancestor or arose independently in several lines of descendents including chordates and arthropods [19]. This insensitivity of neurogenesis to Bmp in hemichordates, despite the presence of a Bmp-Chordin axis in the embryo, implies that the anti-neurogenic phase of Bmp signaling is absent in hemichordates. To further these inquiries, we plan to examine the means by which the Bmp and Chordin stripes are placed opposite one another in the ectoderm of the hemichordate embryo, and the means of placement of the mouth. As concluded previously for chordates and arthropods, and now supported by the hemichordate findings, this Bmp-Chordin axis is truly a developmental axis underlying the definitive anatomical dorsoventral axis, and it may date back to the earliest bilateral animals [1,27]. The highest expression occurs just anterior to the telotroch ciliated band. In hemichordates, neurogenic and epidermal cells are finely intermixed throughout the ectoderm [21]. In vertebrates, dlx paralogs have multiple developmental roles, but all share a role in patterning neural crest cells on the dorsal midline where the neural folds meet, a site of high Bmp levels [46,47]. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. The centralized nervous system of arthropods and vertebrates, so important in the previous theories of anatomical evolution of the bilateral organisms, may itself have arisen independently in both groups but built on the pre-existing Bmp-Chordin gradient.

Orthologs encoding seven transcription factors exhibit dorsal domains of expression in the different germ layers, providing evidence of a dorsoventral pattern of transcriptional regulation in hemichordates, previously unreported. Black arrowheads indicate thick condensations of mesenchyme around the anterior gut. The parameters of the analysis are as follows: 10,000,000 generations, burn in 7,500, sampling frequency 100, and four chains, under a mixed model of evolution. Citation: Lowe CJ, Terasaki M, Wu M, Freeman RM Jr, Runft L, Kwan K, et al. https://doi.org/10.1371/journal.pbio.0040291.g004. (I) Expression of bambi at the late gastrula stage. (2015) Expression begins during gastrulation as a circumferential ectodermal stripe in the prospective anterior metasome (Figure 4E). Low levels of expression are also detected throughout the ectoderm. All embryos have a similar orientation: anterior in the top left and dorsal in the top right of each panel, unless otherwise specified.

(A) Expression of bmp2/4 at late gastrula stage and (B) at day 3 of development. (J) Expression of crossveinless at the late gastrula stage and (K) at day 3 of development, with the dorsal midline oriented toward the viewer. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other. Such segregation was presumably a key evolutionary step in the centralization of the nervous system, which is a major dorsoventral innovation. For example, since arthropods and chordates have central nervous systems, and since new molecular data reveal commonalities of their development and organization, the central nervous system has been considered a dorsoventral differentiation already present in the bilateral ancestor [1]. pituitary homeobox Since we find no such correspondence in S. kowalevskii; either the domains and roles were lost in the lineage leading to hemichordates (and perhaps echinoderms), or they were independently co-opted in arthropods and chordates. This expanded expression may imply an auto-activating circuitry of the Bmp synexpression group, as is known for Drosophila and vertebrates [90]. (L) Control expression of hex at day 4 of development, and (M) following treatment with Bmp4 at 100ng/ml. In Bmp protein-exposed embryos, the expression of elv is not down-regulated; it is similar to controls along the anteroposterior axis and is expressed at uniform high levels around the embryo's circumference (Figure 5G and 5H). In the case of pitx, which has two ectodermal domains normally, the dorsal spot at the base of the proboscis expands in the presence of excess Bmp to encircle the base of the proboscis, along with underlying mesenchyme expression, as if repressed by Bmp. 1,000 bootstrap replicates were carried out, and nodes with less than 50% support were collapsed. However, chordates appear to achieve much more patterning of neuronal cell types than do hemichordates. The classical inversion hypothesis, despite its attractions, does not require the nervous system to have been already centralized before inversion occurred, even though such centralization is usually assumed. Thus like chordin expression, admp does not identify organizer-like mesoderm in hemichordates. Like bambi, its dorsal expression declines after day 2 of development to undetectable levels. At early stages, its breadth of expression extends almost to the dorsal side in the mid-level ectoderm (Figure 2M2P), though less extensively in the anterior and posterior ectoderm. The same developmental stage is shown in panel E and F. (E) Expression of admp expands strongly throughout the ectoderm of the detached prosome.

White arrowhead indicates the submerged domain in the collar (mesosome) similar to that of bmp2/4 in panel D. (G) Expression of bmp5/8 in the far posterior of a late juvenile at day 13 of development. Whatever the means by which the mouth and the central nervous system are produced in chordates, these studies reinforce the prominence of the Bmp-Chordin axis as a patterning mechanism in perhaps all bilaterian animals. Similar expression is found for the S. kowalevskii T-box gene, tbx2/3. These three phyla along with the recently reclassified Xenoturbella constitute the deuterostome supertaxon [20]. Yes pox neuro; RNAi, Three genes were chosen for study in S. kowalevskii because of their well-known roles in dorsoventral patterning of the chordate neural tube: shh, nk2.2, and msx. Yes Two cDNA libraries were used in this study, one from mixed blastula and gastrula stages and another from mixed gastrula and neurula stages. In Drosophila and vertebrates, Bmp represses neurogenesis, but in hemichordates there is no apparent segregation of ectoderm into neurogenic and epidermal territories. Presumably, intrinsic to the evolution of a restriction of neural and epidermal fates in the different lineages was the use in all lineages of the pre-existing Bmp-Chordin axis. The ventral side, assigned because of the mouth location, is down in this schematic figure. Like bmp2/4 and bmp5/8, its domain is submerged in the anterior mesosome, where the dorsal axon tract is internalized (unpublished data). As a second example, pitx expression is altered in the embryos from eggs injected with anti-bmp siRNAs; the posterior ventral domain of pitx expands uniformly throughout the ectoderm (Figure 6D) whereas the anterior dorsal spot of expression is absent from the detached prosome. White arrowhead shows the position of the telotroch. If so, the circuitry and its role in segregating neural and epidermal territories must have arisen independently in the chordate and arthropod lines, and perhaps in other lines as well [19].

After staining, samples were fixed overnight in Bouins fixative and then rinsed multiple times in 80% EtOH/ 0.1M TrisHCl [pH 8] until the picric acid color was gone. Bmp5/8 is the S. kowalevskii ortholog of an ancestral gene that duplicated and diverged in the chordate lineage to bmps 5,6, 7, and 8 and in the Drosophila lineage to screw and glass bottom boat [30]. The evidence for a conserved role of the encoded protein in Drosophila and chordates remains weak. et al.

In S. kowalevskii, cv-2 is expressed on the dorsal midline (Figure 2J) of the embryo. Dorsal is at the top of the panel. However, many modulators are known to affect the local level of active Bmp, such as the complexes it forms with Chordin, Tsg, and perhaps Cv-2, and its release from those complexes by the Tolloid protease [95]. At day 4, admp is still expressed along the ventral midline of the embryo; however, its level is much lower in the mesosome and anterior prosome (Figure 2S). (Q) Expression of mox (also called gax) at day 3 of development, and (R) a close up of the ventral domain at day 3, ventral midline toward the viewer, displaying the metasome and part of the mesosome. In anti-bmp siRNA embryos, admp expression expands uniformly, especially in the proboscis where it becomes very intense (Figure 6E). Yes In treated embryos, these spots expand to a ring of expression around the body (Figure 5N). crossveinless; S. kowalevskii indeed expresses many orthologs in a topology that would support the hypothesis of conserved roles in patterning along a Bmp-Chordin axis. We might expect very different effects of Bmp in specifying the nervous system of hemichordates. Its early ring-like domain resembles that of engrailed [45] except that the ring of the latter is interrupted on the dorsal midline. e291. Still, whatever its shape, we propose that the Bmp profile constitutes the fundamental developmental axis for the selection and placement of components of the eventual anatomical dorsoventral axis. The ortholog is expressed on the ventral midline of embryonic ectoderm and also in some regions of ventral midline endoderm (Figure 2Q2T). Top amino acid blast hits were aligned with additional sequences in ClustalX (http://www.embl.de/~chenna/clustal/darwin/index.html). These shared features, including their similar placements along the Bmp-Chordin axis, can be ascribed to the deuterostome ancestor, and some perhaps even to the bilateral ancestor. Whether these domains in the ectoderm are really associated with the specification of motorneurons is unknown; currently no information is available on neuronal cell type specification in this animal. In later developmental stages (day 3), the expression in the endoderm splits into two domains, an anterior one in the ventral pharynx and a posterior one in the ventral endoderm (prospective gut). Correction: Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution. We see the initial expression of the olig ortholog of S. kowalevskii in a dorsal spot at the base of the prosome, in a cluster of cells (Figure 3G). The prosome has detached from the mesosome at this stage, as the mouth indentation encircles the embryo. Over-expression of Bmp2/4 was achieved by addition of recombinant Zebrafish Bmp4 protein (R&D Systems, Minneapolis, Minnesota, United States) at four concentrations (10, 100, 250, and 500 ng/ml) from the blastula stage until day 4 of development. Unlike the genes of other Bmps, admp appears to be co-expressed with chordin, not opposite it, and to be repressed by Bmps. Alternatively, hemichordates (and perhaps echinoderms) may have lost the central nervous system of a deuterostome ancestor, rendering it diffuse and presumably simplified.

(A) Side view of a control embryo cultured without Bmp4. Comparative studies of netrin show that the encoded protein plays a conserved role in axonal guidance [67,68]. In juveniles with 23 gill slits (14 d post fertilization), expression continues in a tight midline reaching to the anus, but not extending into the post-anal tail (unpublished data). Posterior to the anus is the post anal tail. We thus find significant correlative evidence of Bmp signaling from this midline in hemichordates. It is on the Chordin side, as in Drosophila, but in chordates it is on the Bmp side. In mid-stage embryos (day 4), its expression is uneven along the ventral midline, forming patches with large gaps at the telotroch and in the mesosome (unpublished data). As a final case, we chose admp as a candidate for Bmp repression since this gene is normally expressed only on the ventral midline of the ectoderm and endoderm. (B) Embryos treated with 250ng/ml of Bmp 4, fixed at the same time as the control in panel A. Bayesian analysis was carried out using Mr Bayes (v3.0B4) (http://mrbayes.csit.fsu.edu). Nerve cells and epidermal cells are finely intermixed [21]. Adult S. kowalevskii were collected intertidally in September near Woods Hole, Massachusetts. Preceding the dorsoventral axis of anatomical and physiological specializations in the course of development is a molecular axis. As in Drosophila the proteins elicit either commissural axon attraction or repulsion depending on the receptor expressed by the responding growth cone. If true, the dorsoventral axis would have been a locus of much more evolution in chordates than was the anteroposterior axis since, as we showed previously [45], the gene expression domains of this axis are extensively similar in both groups, hence in the deuterostome ancestor. Of particular interest is the development of the nervous system in treated embryos, which is normally diffuse throughout the ectoderm even though the embryo possesses a Bmp-Chordin axis. https://doi.org/10.1371/journal.pbio.0040291.sg002.

These features were just brought forward in the chordate lineage (hemichordate, too), not evolved anew within the lineage. Copyright: 2006 Lowe et al. , A venerable explanation of the inverse relationship is the inversion hypothesis (dating back to E. Geoffroy Saint-Hilaire [26]) in which a chordate ancestor inverted its entire body dorsoventrally and then moved the mouth to the opposite side (or the reverse order of events). Samples were fixed in MEMFA and stored in ethanol [91]. This pattern of expression of chordin, centered on the ventral midline, strongly suggests that its encoded protein and Bmp on the dorsal side are involved in an antagonistic interaction, as found in other bilateral animals. No, Is the Subject Area "Embryos" applicable to this article? There are two prominent exceptions to the list of targets of the Bmp-Chordin axis shared by hemichordates and chordates, namely, the nervous system and the mouth. Particularly noteworthy are the nk2.2 and msx domains since these are thought to have similar expression in Drosophila neurectoderm and the chordate neural tube [15]. (S) Admp expression at day 5 of development, and (T) surface view of an uncleared embryo, ventral midline toward the viewer. Expression of hex in S. kowalevskii begins very early in the gastrula and shows a tight restriction to the prospective dorsal endoderm in a region of expression directly beneath the bmp2/4 stripe (unpublished data). Yet, despite its diffuse nervous system, hemichordates have a well-developed dorsoventral polarity in all three germ layers, as if phase 2 patterning occurs. (O) Like pax1/9, the nk23/25 domain expands from a short dorsal stripe to a ring in the endoderm, after Bmp4 treatment. Dlx, as described earlier (Figure 3A and 3B), is expressed in the ectoderm of the entire prosome [45] and on the dorsal midline of the meso- and metasome. In the anterior metasome near the first gill slit, ectodermal expression extends ventrally and encircles the embryo as a band. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, Affiliation In S. kowalevskii as well as Drosophila, chordin is only expressed in the ectoderm, whereas in Xenopus, it is expressed in both the ectoderm and mesoderm [42]. Said in other words, they predicted that the hemichordate mouth falls on the Chordin side of the animal, whereas in chordates it is on the Bmp side. Pox neuro is a pax gene of Drosophila and other arthropods, closely related to the pax 2/5/8 group of genes and the cnidarian paxA group [56], but there is no clear ortholog in vertebrates.

Molecular and developmental comparisons have been impossible given the paucity of data from hemichordates. This is a residual spot (thus showing that the staining procedure has worked), whereas the entire ventral domains of ectoderm and endoderm have disappeared. (D) Section of a juvenile of a similar stage to (C). The endoderm is divided into two sections, the pharyngeal region in the anterior, divided from the posterior gut region by a posterior constriction shown by blue arrows. Bambi, which encodes an inhibitory pseudoreceptor of Bmp that is co-expressed with bmp4 in Xenopus and mouse [33,34], is similarly co-expressed in S. kowalevskii, on the dorsal midline shortly after gastrulation (Figure 2I). These seven were not difficult to find, and we expect that many more genes exhibit such dorsoventral expression, perhaps all dependent on the Bmp-Chordin axis. All embryos are shown as optical sections, and oriented in a similar manner as in Figure 2 with anterior to the top and left of each panel and dorsal in the top right of each panel, unless otherwise specified. Anteroposterior organization is normal, as reflected by the fact that the prosome, mesosome, and metasome are set off by anterior and posterior grooves. The dorsoventral orientation is not possible to determine since they are cylindrically symmetric. Furthermore, when siRNA injected embryos were then treated with Bmp protein, they undertook Bmp-dependent gene expression, indicating that the siRNAs did not cause systemic damage unrelated to Bmp. Competing interests: The authors have declared that no competing interests exist. https://doi.org/10.1371/journal.pbio.0040291.g005. The site of Bmp signaling in the embryos of chordates and arthropods defines one pole of the Bmp-Chordin developmental axis. Unlike chordates, the hemichordate expression is in the ectoderm and endoderm, rather than in the mesoderm. The first is an nk homeobox gene, we have named nk-2.3/2.5 that is related to Drosophila tinman, amphink-tin, and a range of nk2 class vertebrate genes that all play roles in cardiac development (see gene tree in S-4P). It is expressed in neurogenic regions, such as the ventral-lateral neural tube of chordates, and its encoded transcription factor may play a conserved role in motor neuron development [73]. This is in part because of the functional independence of the dorsal-ventral and anterior-posterior axes and the fact that patterning genes often appear dedicated to a single use and not used successively in new patterns. Tsg, a modifier of Bmp signaling, binds to Chordin and Tolloid to agonize or antagonize Bmp signaling, depending on the presence of yet other modifiers [3537]. (O) Day 3 of development, a surface view of the lateral ectoderm, (P) and day 4 of development, in sagittal section. The phenotypes of the resulting embryos are identical and highly reproducible from all three kinds of materials and between injection runs. Expression seems entirely down-regulated in the mid endoderm region, possibly at the level where gill slits would normally begin to develop (Figure 5M). One appears at gastrulation in the posterior ventral ectoderm in a broad band of scattered cells, just anterior to the telotroch (unpublished data). These differences suggest that much of the regulatory architecture involved in dorsoventral patterning of chordate nervous system evolved subsequent to the divergence of hemichordates and chordates. In the other case, the sequences were: sense: 5-CUCGACCAAUCAUGCGAUAUU-3 and antisense: 3-UUGAGCUGGUUAGUACGCUAU-5 which were directed to positions 13961416. In cladograms, poxN clusters with Pax A and C of cnidarians and poxN of Drosophila; it is distinct from the pax 2/5/8 group of genes (Figure S-2H). (H) Expression of tolloid/xolloid at day 3 of development.

(I) lim3, (J) sim, (K) netrin1/2, (L) mox. , We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. During those later stages, as the stomochord projects into the prosome directly underneath this spot, pitx expression extends into the mesoderm of the heart/kidney complex, just dorsal to the stomochord. Eggs were injected immediately following fertilization with bmp siRNA and development was allowed to continue. Then we examined three genes normally expressed asymmetrically in the endoderm. However, as shown here, the exposure of the hemichordate embryo to a uniform excess of exogenous Bmp protein, which up-regulates endogenous bmp gene expression around the body, does not cause the territory of neurogenesis to contract, as shown by the persistence of widespread pan-neural gene expression (Figure 5H).

Another gene from the Bmp sub-family of transforming growth factor-beta signaling molecules, which encodes a protein with a key role in vertebrate organizer function, is anti-dorsalizing morphogenetic protein (admp). Anterior is to the left. This segregation of ectodermal territories along the Bmp-Chordin axis in the first phase accomplishes the centralization of the nervous system in chordates and Drosophila. However, the ventral axon tract is more associated with motor function, and the body wall musculature of the trunk is primarily ventral [75]. We have succeeded in cloning only one member of this related group of vertebrate genes, as was also reported in amphioxus [62], suggesting that the vertebrate nk2 class cardiac genes represent a diversification of an ancestral copy of a tinman-related gene in basal chordates.

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