Fig. 8. Ectopic expression of LvTbx2/3 mRNA causes profound morphological defects in embryonic development but does not prevent expression of markers of ectoderm, mesoderm and endoderm specification. Nomarski (A) and polarized light (B) images of 24 hour control, glycerol-injected embryos. These embryos exhibit the morphology, skeletal pattern and tripartite gut characteristic of the pluteus stage of development. Embryos that ectopically express LvTbx2/3 mRNA (0.75-1.0 pg/pl; three to five times the endogenous copy number per nucleus, but in all nuclei) and imaged under Nomarski (C,E,G) or polarized light (D,F,H) optics are positioned to show a vegetal view. Twenty-four hours after injection, ectopic LvTbx2/3-expressing embryos appear radialized with multiple spicule clusters forming around the circumference of the embryo (D). They are, in many cases, delayed in gastrulation compared with control embryos as the archenteron has not yet reached the animal pole. The embryos contain derivatives of all germ layers, including pigment and blastocoelar cells derived from the nonskeletogenic mesenchyme, indicating that early specification events have not been eliminated. (E-H) embryos ectopically expressing LvTbx2/3 for 48 hours. These embryos exhibit severe morphological defects in tissues derived from all three germ layers. They lack the typical pluteus form, have drastically mispatterned skeletons and have archenterons composed of multiple chambers rather than the normal three. Two embryos (F,H) display the variability in the skeletal phenotypes. No two embryos that ectopically express LvTbx2/3 display identical defects in their skeletons, although all are severely mispatterned. When stained for terminal markers of pattern formation in these tissues, these embryos express markers for each known cell lineage. In the endoderm, the mid/hindgut marker, Endo1, is expressed and, in many cases, is localized to several of the additional chambers that have formed (I). LvBrac is normally expressed in two domains, a blastopore/hindgut domain and an oral/stomodael domain. Within the endoderm of injected embryos, LvBrac expression remains around the blastopore (J). EctoV is normally expressed in a refined domain corresponding to the oral ectoderm. In injected embryos, EctoV expression is still refined, indicating an oral axis has formed (K). (L) LvBrac is also expressed normally in the stomodael domain, indicating that substructures have been specified in the oral ectoderm and that domain is not `aboralized'. (M) mAb 295, which recognizes the ciliated band, a structure at the boundary between oral and aboral cells, is also expressed in these embryos. However, instead of being a tight band, in many cases the ciliary band is broadly dispersed, indicating a loss of a refined O/A boundary.

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