Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution

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. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called “dorsal.” On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. 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. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. 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.</p

Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution

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. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called “dorsal.” On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. 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. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. 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

Signal transduction in frog and ascidian eggs at fertilization

At fertilization, the egg must undergo a rise in intracellular Ca 2+ so that embryogenesis may begin. How the sperm signals the egg to release Ca2+ is not well understood. This dissertation examines how the sperm stimulates Ca2+ release in frog and ascidian eggs at fertilization. ^ One secondary messenger used by somatic cells to elicit intracellular Ca2+ release is inositol trisphosphate (IP3). Results from experiments examining Xenopus oocyte maturation indicate that frog oocytes become more sensitive to IP3 as they mature into fertilizable eggs. Because this evidence suggested that IP3 may be important for Xenopus fertilization, frog eggs were injected with an antibody against the IP3 receptor and then inseminated. The inhibitory effect of this antibody on Ca2+ release after insemination indicates that the Ca2+ rise at fertilization in frog eggs requires IP3. ^ Because IP3 is generated by phospholipase C (PLC) enzymes, the role played by PLCs during Xenopus fertilization was investigated. The PLC family contains three subtypes: β, γ, and δ. Xenopus eggs injected with an antibody against Gq family G-proteins (to inhibit PLCβ activation) do not release Ca2+ in response to serotonin but do release Ca2+ at fertilization. Injecting Xenopus eggs with recombinant Src homology 2 (SH2) domains of PLCγ (to inhibit PLCγ) blocks platelet derived growth factor-induced Ca2+ release but not the Ca2+ rise at fertilization. These results indicate that the Ca2+ rise at fertilization in Xenopus eggs requires neither G q-mediated activation of PLCβ nor SH2-mediated activation of PLCγ. ^ In contrast to the Xenopus results, injecting eggs of an ascidian (an invertebrate chordate) with PLCγ SH2 domains (to inhibit PLCγ) or with Fyn SH2 domains (to inhibit Src family kinases) inhibits the Ca2+ rise at fertilization. That these SH2 domains also inhibit the Ca2+ rise in response to injecting ascidian eggs with an extract of sperm indicates that both sperm extract and fertilization stimulate Ca2+ release using a pathway requiring PLCγ and a Src family kinase. These results support the hypothesis that, during sperm-egg fusion in ascidians, the sperm signals the egg to release Ca 2+ by introducing a protein that activates the Src/PLCγ pathway in the egg.

Genes Expressed with Dorsal or Ventral Domains

<div><p>These genes were chosen as candidate targets of Bmp activation or repression. All embryos are oriented as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040291#pbio-0040291-g002" target="_blank">Figure 2</a> with anterior to the top and left of each panel and dorsal in the top right of each panel, unless otherwise specified. The telotroch or ciliated band is marked by white arrowheads. Expression of <i>dlx</i> at (A) day 2 of development, just after gastrulation and (B) at day 3 of development.</p> <p>(C) <i>Tbx 2/3</i> expression just after gastrulation, and (D) at day 3 of development.</p> <p>(E) Expression of <i>hex</i> at day 3 of development (F) Expression of <i>nk2.3/2.5</i> at day 4.</p> <p>(G) Expression of olig on day 2, just after gastrulation, and (H) at day 3.</p> <p>(I) <i>poxN</i> expression at day 4 of development.</p> <p>(J) <i>Pitx</i> expression at day 2 of development, dorsal midline toward the viewer, a glancing optical section through the dorsal-most ectoderm.</p> <p>(K) <i>Pitx</i> expression at day 4 of development. Note the two domains of expression.</p> <p>(L) <i>Netrin</i> expression, a transverse section of a post-gastrula embryo at the level of the ciliated band. Note the broad ventral expression of <i>netrin</i>, and (M) the more narrow domain at day 3 of development.</p> <p>(N) Expression of <i>lim3</i> at day 3 of development.</p> <p>(O) Expression of <i>mnx</i> at day 2 of development, and (P) at day 4. Note the ventral endodermal expression.</p> <p>(Q) Expression of <i>mox</i> (also called <i>gax</i>) 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.</p> <p>(S) Expression of <i>sim</i> at day 2 of development, and (T) at day 5.</p></div

Dorsalization of the S. kowalevskii Embryo by Application of Exogenous Bmp4 Protein

<div><p>Exogenous Bmp4 protein was applied to the late blastula embryo (14 h post fertilization), and development was allowed to continue. All embryos are shown at a similar developmental stage, day 3 of development.</p> <p>(A) Side view of a control embryo cultured without Bmp4. The mouth is indicated by a black arrowhead on the ventral side. The normally developing endoderm shows a dorsal, anterior projection of the gut called the stomochord, indicated with a white arrowhead. One of the mesocoels is clearly visible on the dorsal midline, indicated by a yellow arrowhead. 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. The first gills slit is indicated by a green arrowhead, just anterior to the gut division.</p> <p>(B) Embryos treated with 250ng/ml of Bmp 4, fixed at the same time as the control in panel A. The dorsoventral orientation is not possible to determine since they are cylindrically symmetric. Black arrowheads indicate thick condensations of mesenchyme around the anterior gut.</p> <p>(C) Embryos fixed at a similar development stage following a treatment with 500 ng/ml Bmp4 displaying a consistent phenotype between samples. Note the flattened anterior end and the thick connection of prosome and mesosome.</p> <p>(D) Expression of <i>bmp2/4</i> following treatment with Bmp4 protein showing activation of endogenous expression throughout the ectoderm.</p> <p>(E) Stereomicrographs of uncleared embryos showing the expression of <i>chordin</i> in control embryos, with broad ventral expression, and (F) embryos following treatment with Bmp4 protein at 100 ng/ml.</p> <p>(G) Stereomicrographs of uncleared embryos showing the expression of <i>elv</i> in control embryos, with broad expression, but stronger at the midlines, and (H) embryos following treatment with Bmp4 protein at 100 ng/ml. Note the persistence of <i>elv</i> expression; it is not repressed by Bmp4.</p> <p>(I) Ubiquitous ectodermal expression of <i>dlx</i> following treatment with 100 ng/ml of Bmp4.</p> <p>(J) Expression of <i>tbx2/3</i> expands throughout the ectoderm following treatment of the embryo with Bmp4 protein (250 ng/ml). White arrowheads indicate the position of the telotroch/ciliated band.</p> <p>(K) Expression of <i>pitx</i> expands from a spot to a ring around the base of the prosome in both the ectoderm and underlying mesenchyme, after Bmp4 protein treatment.</p> <p>(L) Control expression of <i>hex</i> at day 4 of development, and (M) following treatment with Bmp4 at 100ng/ml.</p> <p>(N) <i>Pax1/9</i> expression expands from a dorsolateral spot to a circumferential ring in the endoderm following Bmp4 treatment at 100 ng/ml.</p> <p>(O) Like <i>pax1/9,</i> the <i>nk2–3/2–5</i> domain expands from a short dorsal stripe to a ring in the endoderm, after Bmp4 treatment.</p> <p>(P) Expression of <i>admp</i> in the most anterior endoderm following treatment with 500 ng/ml of Bmp4. This is a residual spot (thus showing that the staining procedure has worked), whereas the entire ventral domains of ectoderm and endoderm have disappeared.</p></div

Summary of Inferred Evolutionary Changes of the Dorsoventral Axis in Deuterostome Evolution, with Emphasis on Hemichordates and Chordates

<p>Mesoderm is shown in red, endoderm in yellow and ectoderm in blue (neural) and grey (epidermis). An ancestral secreted Bmp axis was involved in dorsoventral patterning of the three germ layers in the bilaterian ancestor. This ancestor we propose was characterized by a diffuse organization of its nervous system, shown by blue dots. A Bmp gradient was involved in dorsoventral patterning of all three germ layers. In the basal deuterostome and hemichordates the role of the Bmp gradient is conserved in general dorsoventral patterning. During chordate (and protostome) evolution, the existing Bmp/Chordin axis was co-opted for an additional developmental role in nervous system centralization.</p

Expression in S. kowalevskii of Orthologs of Chordate Genes Important in Dorsoventral Patterning of the Neural Tube

<div><p>All embryos are shown as optical sections, and oriented in a similar manner as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040291#pbio-0040291-g002" target="_blank">Figure 2</a> with anterior to the top and left of each panel and dorsal in the top right of each panel, unless otherwise specified.</p> <p>(A) <i>Hh</i> expression in the apical tip of a day 3 embryo, and (B) at day 5 at the same location.</p> <p>(C) Endodermal expression of <i>nk2–2</i> (also called <i>nkx2.2</i>) in the late gastrula, with a sharp delineation between presumptive proboscis mesoderm and definitive endoderm, and (D) at day 4, with expression continuing in the endoderm but down-regulated in the pouches of the forming gill slits (black arrowhead).</p> <p>(E) Expression of <i>msx</i> in the late gastrula, and (F) at day 3. Expression occurs exclusively in the ectoderm of the metasome and is down-regulated in the telotroch, the cilated band, as marked (white arrowhead).</p></div