Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution
Figure 5
Dorsalization of the S. kowalevskii Embryo by Application of Exogenous Bmp4 Protein
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.
(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.
(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.
(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.
(D) Expression of bmp2/4 following treatment with Bmp4 protein showing activation of endogenous expression throughout the ectoderm.
(E) Stereomicrographs of uncleared embryos showing the expression of chordin in control embryos, with broad ventral expression, and (F) embryos following treatment with Bmp4 protein at 100 ng/ml.
(G) Stereomicrographs of uncleared embryos showing the expression of elv 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 elv expression; it is not repressed by Bmp4.
(I) Ubiquitous ectodermal expression of dlx following treatment with 100 ng/ml of Bmp4.
(J) Expression of tbx2/3 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.
(K) Expression of pitx expands from a spot to a ring around the base of the prosome in both the ectoderm and underlying mesenchyme, after Bmp4 protein treatment.
(L) Control expression of hex at day 4 of development, and (M) following treatment with Bmp4 at 100ng/ml.
(N) Pax1/9 expression expands from a dorsolateral spot to a circumferential ring in the endoderm following Bmp4 treatment at 100 ng/ml.
(O) Like pax1/9, the nk2–3/2–5 domain expands from a short dorsal stripe to a ring in the endoderm, after Bmp4 treatment.
(P) Expression of admp 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.