Linda L. Runft

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.

Echinoderm eggs and embryos: Procurement and culture

The protocols outlined here hopefully will provide researchers with healthy, beautiful echinoderm oocytes, eggs, and embryos for experimental use. The large size of echinoderm oocytes and eggs, the ease with which they can be manipulated, and (in many species) their optical clarity, make them an ideal model system for studying not only the events specific to oocyte maturation and fertilization, but also for investigating more general questions regarding cell cycle regulation in an in vivo system. The quick rate at which development proceeds after fertilization to produce transparent embryos and larva makes the echinoderm an advantageous organism for studying deuterostome embryogenesis. Continued use of the echinoderms as model systems will undoubtedly uncover exciting answers to questions regarding fertilization, cell cycle regulation, morphogenesis, and how developmental events are controlled.

Two-stage dependence for 1-methyladenine induced reinitiation of meiotic maturation in starfish oocytes

The maturation hormone 1-methyladenine (1-MA) causes meiotic resumption of prophase arrested immature starfish oocytes. Continuous exposure to > or = 0.5 microM 1-MA causes germinal vesicle breakdown (GVBD) in approximately 20 min, but oocytes pretreated for > 30 min with a subthreshold dose of 1-MA undergo GVBD much faster (approximately 10 min) when they are exposed to 1 microM 1-MA. Furthermore, a very low subthreshold 1-MA suffices to start the maturation process: oocytes exposed to 0.005 microM 1-MA for up to 10 min followed by 1 microM 1-MA is equivalent to continuous exposure to 1 microM 1-MA. These dose and timing relationships indicate that there is a two-stage dependence on 1-MA. A possible explanation for this dependence is that there are two processes involved: an initial process that is triggered by a low dose of 1-MA, and a second process that cannot start until the first process is completed and is stimulated by a higher dose of 1-MA. These subthreshold 1-MA effects on GVBD timing are not directly coupled to changes in calcium physiology that also occur during maturation. Subthreshold 1-MA was found to cause a transient accumulation of Cdc2/cyclin B into the nucleus. The two-stage dependence indicates that there are unsuspected features in this well-studied pathway leading to GVBD. In the animal, this hormone dependence may help to synchronize maturation throughout all parts of the ovary.

Correction: Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution.

The in situ staining patterns of Fig 4C and 4D were incorrectly assigned to the nkx2.2 gene of Saccoglossus kowalevskii. Subsequent sequencing and analysis has shown that these images were actually expression patterns for the foxA gene. Fig 4 has been updated with images of the in situ staining patterns of the nkx2.2 gene of Saccoglossus kowalevskii and the figure legend has been amended to reflect this. Fig 4 Expression in S. kowalevskii of Orthologs of Chordate Genes Important in Dorsoventral Patterning of the Neural Tube. The corrected legend for Fig 4 appears below. Additionally, the authors would like to amend the text description of nk2-2 expression on p.1610 (right hand column), which incorrectly concerned foxa; and restate the conclusion about nk2-2 expression in the Discussion on p.1615 (left column). The authors confirm this conclusion is unchanged, but the old foxa data were irrelevant to the cross-phylum comparison of nk2-2 expression among S. kowalevskii, chordates, and Drosophila. The corrected text appears below. Page 1610: In S. kowalevskii, we find that the expression profiles of the hh, nk2.2, and msx orthologs do not at all resemble the expression domains of vertebrates or Drosophila, as shown in Fig 4. We could isolate only one kind of hedgehog ortholog from S. kowalevskii, but this may exhaust the hemichordate repertoire since basal deuterostomes such as amphioxus and sea urchins have but one gene [86,87], whereas vertebrates have up to four paralogs of hh [86]. The expression of hh in S. kowalevskii begins at day 2, in a small patch at the apical tip of the prosome ectoderm (Fig 4A), and it continues in the same domain throughout all stages examined (Fig 4B). Low level expression also occurs in the anterior gut (unpublished data). At no time is hh expressed in a dorsal or ventral midline domain, for example, close to the netrin ventral domain. The nk2-2 gene (also called nk2.2 and nkx2.2) of S. kowalevskii was cloned by low stringency hybridization. During gastrulation, it is expressed in the archenteron endoderm (Fig 4C) rather than ectoderm, as has also been found in amphioxus, though not other chordates [88]. Expression in S. kowalevskii is excluded from the anterior mesendoderm that becomes the prosome mesoderm, and the level of expression is reduced in the posterior endoderm. During days 2 and 3 of development, nk2-2 expression is further down-regulated in in the posterior and midline endoderm, leaving by day 3.5 (Fig 4D) a left and right patch of expression in the pharyngeal endoderm, ventral to the site of gill pore formation. At no stage was nk2-2 expressed in an ectodermal dorsal or ventral midline domain. Thus, like hh of S. kowalevksii, the nk2-2 gene, which in chordates depends on Hh signaling for expression, is not expressed in a chordate-like ectodermal midline domain. Page 1615: Nonetheless hemichordates use the Bmp-Chordin axis for a second phase, the “morphogenetic phase” for patterning the three germ layers. The patterning of neuronal cell types within the diffuse nervous system is included in the patterning, for example, the giant neurons near the dorsal midline (the Bmp pole); a nerve tract rich in motor neuron axons at the ventral midline, a specialized patch of sensory neurons near the mouth, and a line of poxN expressing cells, perhaps specialized neurons, on the posterior dorsal midline. However, chordates appear to achieve much more patterning of neuronal cell types than do hemichordates. Indeed, the entire Bmp-Hh double gradient used by chordates to select and place ten gene expression domains in the dorsoventral axis of the neural tube [96] is absent from hemichordates. Hh is localized to the apical ectoderm and anterior gut in hemichordates; it has no dorsoventral expression. Whereas the neural tube domains of chordates include those of pax6, dbx, en, irx, nk2.2, and msx, none of these genes shows a dorsoventral ectodermal midline domain in S. kowalevskii (see Results here and in [45]). Yet all of these are expressed in the hemichordate embryo in anteroposterior domains, as they are in chordates in domains additional to the dorsoventral domains. Particularly noteworthy are the nk2.2 and msx domains since these are thought to have similar expression in Drosophila neurectoderm and the chordate neural ectoderm of the neural tube [15], with roles in neural patterning. Yet neither gene has a dorsal or ventral ectodermal domain in S. kowalevskii. 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. 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.