Early Midline Signaling of Bmp and Chordin

<div><p>All embryos are cleared (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040291#s4" target="_blank">Materials and Methods</a>) otherwise specified. All embryos have a similar orientation: anterior in the top left and dorsal in the top right of each panel, unless otherwise specified.</p> <p>(A) Expression of <i>bmp2/4</i> at late gastrula stage and (B) at day 3 of development.</p> <p>(C) Hatched juvenile oriented with dorsal toward the viewer. White arrowhead indicates the position of the collar where the <i>bmp2/4</i> expression domain follows the submerged ectodermal track of the dorsal cord.</p> <p>(D) Section of a juvenile of a similar stage to (C). Dorsal is at the top of the panel. Note the submerged <i>bmp</i> domain, stained purple.</p> <p>(E) Dorsal view of <i>bmp5/8</i> expression at day 3 of development with dorsal toward the viewer, and (F) in a hatching juvenile at day 5 of development. White arrowhead indicates the submerged domain in the collar (mesosome) similar to that of <i>bmp2/4</i> in panel D.</p> <p>(G) Expression of <i>bmp5/8</i> in the far posterior of a late juvenile at day 13 of development. White arrow indicates the position of the anus, before which the <i>bmp5/8</i> domain stops. Posterior to the anus is the post anal tail.</p> <p>(H) Expression of <i>tolloid</i>/<i>xolloid</i> at day 3 of development.</p> <p>(I) Expression of <i>bambi</i> at the late gastrula stage. (J) Expression of <i>crossveinless</i> at the late gastrula stage and (K) at day 3 of development, with the dorsal midline oriented toward the viewer.</p> <p>(L) <i>Tsg</i> expression at day two of development.</p> <p>(M–P) <i>Chordin</i> expression from early to late developmental stages.</p> <p>(M) Expression at mid gastrula.</p> <p>(N) Sagittal section an embryo at day 2 of development just anterior to the telotroch; ventral is at the bottom of the panel.</p> <p>(O) Day 3 of development, a surface view of the lateral ectoderm, (P) and day 4 of development, in sagittal section.</p> <p>(Q) <i>Admp</i> and <i>bmp2/4</i> double in situ hybridization at day 2 of development; brown is <i>admp</i> and blue is <i>bmp2/4</i>, and (R) a cross section of the same stage just anterior to the telotroch.</p> <p>(S) <i>Admp</i> expression at day 5 of development, and (T) surface view of an uncleared embryo, ventral midline toward the viewer.</p></div

The Anatomy of S. kowalevskii to Illustrate Differentiations along the Dorsoventral Axis

<div><p>The ventral side, assigned because of the mouth location, is down in this schematic figure. Anterior is to the left. 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). Ectodermal derivatives are colored blue, mesodermal red, and endodermal yellow. Prominent structures of the dorsoventral axis include the dorsal and ventral axon tracts, the dorsal and ventral blood vessels, the dorsal stomochord with the heart/kidney complex, the dorsal gill slits, the ventral mouth, and the ventral tail (only in the juvenile, not shown).</p> <p>Redrawn with modification from Benito and Pardos [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040291#pbio-0040291-b105" target="_blank">105</a>].</p></div

Ventralization of the S. kowalevskii Embryo following Injection of the Egg with Anti-<i>bmp</i> siRNAs

<div><p>Eggs were injected immediately following fertilization with <i>bmp</i> siRNA and development was allowed to continue.</p> <p>(A) Low magnification micrograph of a group of embryos at the beginning of day 3 of development from injected eggs, showing the consistency of the phenotype. The dorsoventral orientation cannot be determined since the embryos are cylindrically symmetric. Note the deep indentation between the prosome and mesosome, and the short posterior end beyond the telotroch (ciliated band).</p> <p>(B) Optical section of an embryo at the same stage of development from an injected egg.</p> <p>(C) In the siRNA treated embryo, the expression of <i>dlx</i> continues in scattered cells in the anterior ectoderm of the prosome but disappears from the dorsal midline.</p> <p>(D) Expression of <i>pitx</i> at day 4 of development in a siRNA treated embryo, showing expanded expression throughout the majority of the metasome anterior to the telotroch, whereas the prosome dorsal spot is absent. The prosome has detached from the mesosome at this stage, as the mouth indentation encircles the embryo. The same developmental stage is shown in panel E and F.</p> <p>(E) Expression of <i>admp</i> expands strongly throughout the ectoderm of the detached prosome.</p> <p>(F) Low magnification micrograph of day 4 embryos showing expansion of expression of <i>netrin</i> from a ventral midline stripe to the entire ectoderm including that of the detached prosome. White arrowhead shows the position of the telotroch.</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

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

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

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

Summary of Wnts, Wnt antagonists, and Fzs receptor expression.

<p>Wnts are expressed in nested domains posteriorly, whereas <i>sfrps</i> are expressed in the anteriormost ectoderm at the blastula and gastrula stages. <i>dkk1/2/4</i> is expressed in three discrete domains of gastrula ectoderm. At juvenile stages, Sfrps are expressed in the very anterior ectoderm (apical tuft), and <i>sfrp1/5</i> is also expressed in the entire proboscis mesoderm, whereas <i>dkk1/2/4</i> is broadly expressed in the anterior ectoderm. Wnts are expressed in three discrete ectodermal domains: the base of the proboscis, the anterior trunk (over the first gill slit), and the posterior-most ectoderm. In addition, <i>wnt9</i> and <i>wntA</i> are expressed in posterior internal tissues. Fz genes are expressed in nested domains along the ectoderm. Territories are color coded: endomesoderm (grey), posterior ectoderm (dark blue), intermediate ectoderm (medium blue) and anterior ectoderm (light blue). Fz, frizzled; Sfrp, secreted frizzled-related protein.</p

Expression of Wnt genes with anterior ectodermal localizations.

<p>Whole mount in situ hybridization of Wnt genes with ectodermal localization in the more anterior domain of the embryo. All data are presented as optical sagittal or frontal sections following clearing in Murray Clear. Developmental staging is from blastula to 72 h of development. All embryos are oriented with anterior, or animal (in the case of blastula), to the top left of the panel and posterior, or vegetal, to the bottom right of the panel. Right column, ventral is to the bottom left. Unless otherwise noted, expression is ectodermal. (Ai-Av), Expression of <i>wnt8</i> at blastula stage (Ai), midgastrula stage (Aii), 30 h (Aiii), 50 h, side view (Aiv), and 72 h of development, side view (Av). (Bi-Bv), Expression of <i>wnt 2</i>. Blastula stage (Bi), at late gastrula (Bii), at 36 h (Biii), at 48 h, frontal view (Biv), and at 72 h of development, side view (Bv). (Ci-Cv), Expression of <i>wnt11</i> at blastula stage (Ci), at gastrula stage (Cii), at 48 h, side view (Ciii), 60 h, side view (Civ), and 72 h of development, side view. (Di-Dv), Expression of <i>wnt7</i> at blastula stage (Di), gastrula stage (Dii), 36 h (Diii), 48 h, showing a dorsal view with focal plane through the dorsal ectoderm (Div), and 72 h of development, side view (Dv). (Ei-Ev), Expression of <i>wnt5</i> at blastula stage (Ei), at midgastrula stage (Eii), at 48 h, dorsal view (Eiii), at 60 h, side view (Eiv), and at 72 h of development in side view (Ev). (Fi-Fv), Expression of <i>wntA</i>, no expression for the first 48 h at blastula stage (Fi), gastrula stage (Fii), and at 40 h of development (Fiii). Expression begins at 48 h shown as a frontal section from a dorsal view (Fiv), and at 72 h of development with a side view (Fv). (Gi-Gv), Expression of <i>wnt9</i> at blastula stage (Gi), at midgastrula stage (Gii), at 48 h, showing a ventral view with optical section through the ventral ectoderm and posterior mesoderm (Giii), at 60 h of development again in ventral view (Giv), and at the same stage in side view (Gv).</p

Blocking cWnt pathway anteriorizes the embryo.

<p>(i), Overexpression of <i>sfrp1/5</i> and <i>dkk1/2/4</i> produces embryos with enlarged proboscis and reduced trunk. Control embryos (A, C, E, G, I and K). Embryos injected with <i>sfrp1/5</i> mRNA (B and D), <i>dkk1/2/4</i> mRNA (F and H), and <i>fz5/8</i> siRNA (J and L). In situ hybridization for the most apical marker <i>foxQ2-1</i> (E, F, I and J), the proboscis markers <i>six3</i> (A, B, G and G) and <i>rx</i> (K and L), and the anterior trunk marker <i>engrailed</i> (C and D). 48 h embryos (C, D, G-L). Seventy-two h embryos (A, B, E and F). Anterior to the top left, ventral to the bottom left. Numbers indicate embryos with the displayed phenotyped over the number of analyzed embryos. (ii), Knock down of β-catenin extends anterior-most fate only into anterior ectoderm. siRNA against β-catenin was injected at 4-cell stage into single blastomeres with a rhodamine tracer (red cell in diagrams, which then develop into a full quadrant of the embryo at gastrula, also shown in the model in red). Expression by in situ hybridization at 28 h of the apical marker <i>foxQ2-1</i> (A-C), the midtrunk marker <i>msx</i> (D-F), and the posterior marker <i>hox9/10</i> (G-I). Control embryos are shown in (A,D and G), and G represents an injection control. Panels B, E, and H all show the expression of markers genes following injection of β-catenin siRNA. Panels C, F, and I also show the fluorescent tracer showing the lineage of the injected blastomeres at gastrulation. siRNA, short interfering RNA.</p