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doi: 10.1242/10.1242/dev.00409

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LvTbx2/3: a T-box family transcription factor involved in formation of the oral/aboral axis of the sea urchin embryo

Jeffrey M. Gross^*, Robert E. Peterson, Shu-Yu Wu and David R. McClay

Development, Cell and Molecular Biology Group, Box 91000 LSRC, Duke University, Durham, NC 27710, USA
^* Present address: Department of Molecular and Cellular Biology, Harvard University, BioLabs 2094, Cambridge, MA 02138, USA

View larger version (48K): [in a new window]   Fig. 1. (A) Nucleotide and predicted amino acid sequences of LvTbx2/3. (B) Phylogenetic tree of LvTbx2/3, Tbx2, Tbx3 and Tbx2/3 orthologs from other organisms generated by the neighbour-joining method. Bootstrap values indicated on nodes.

View larger version (53K): [in a new window]   Fig. 2. Developmental northern blot of LvTbx2/3 expression. Poly(A)+ RNA (3 µg per lane) was loaded (calculated by OD260). Loading was verified by probing the blot with a ubiquitin fragment from L. pictus (data not shown). Egg; 60 cell stage; MB, mesenchyme blastula; EG, early gastrula; LG, late gastrula; PR, prism; PL, pluteus larva.

View larger version (110K): [in a new window]   Fig. 3. Tbx2/3 polyclonal and preimmune sera controls. Western-blot analysis of protein extracts from late gastrula (1500 embryos) using polyclonal LvTbx2/3 and preimmune serum. In blots probed with LvTbx2/3, two immunoreactive bands appear, one of 70 kDa and one of 35 kDa. A 35 kDa band was also recognized by preimmune serum, indicating the presence of a nonspecific antigen.

View larger version (109K): [in a new window]   Fig. 4. Aboral distribution of LvTbx2/3 protein (red) in prism-stage embryos, demonstrated by co-staining with 5a7 (EctoV; green). The EctoV antigen is expressed from late gastrula stages onward, solely in the oral ectoderm and foregut. Prism-stage embryos viewed aborally (A) and in a vegetal cross-section (B). Complementary expression is observed, indicating that LvTbx2/3 is distributed asymmetrically about the oral/aboral axis, and localized in aboral territories of the endoderm, ectoderm and mesoderm. In many prism and pluteus-stage embryos, a patch of cilia on the oral surface crossreacts with Cy3 secondary antibodies nonspecifically, as observed in the LvTbx2/3 (red) images. (C) Whole-mount, immunofluorescent analysis of fixed embryos probed with preimmune serum. No nuclear staining is observed at any stage (early prism stage shown). (D) Whole-mount, immunofluorescent analysis of fixed embryos probed with with polyclonal LvTbx2/3 serum that had been preincubated with recombinant fusion protein. No nuclear staining is observed at any stage examined (early pluteus stage shown).

View larger version (91K): [in a new window]   Fig. 5. LvTbx2/3 protein is asymmetric throughout embryonic development. Cross-section images (A,C,E,G,I,K) and surface projections (B,D,F,H,J). LvTbx2/3 expression first appears at the mesenchyme blastula stage and is distributed asymmetrically in the presumptive endoderm and ectoderm, as viewed in cross-section (A) and in a vegetal-surface view (B). Early-gastrula stage embryos in cross-section (C) and a vegetal view (D) maintain asymmetric distribution of LvTbx2/3 in the presumptive endoderm and ectoderm, whereas the endoderm and mesoderm that have invaginated into the blastocoel do not express protein. (E,F) Mid-gastrula stage embryo (cross-section and surface projection of the same embryo). LvTbx2/3 expression is maintained asymmetrically in the presumptive endoderm and ectoderm and is not present in invaginated endoderm or mesoderm. (G,H) Late gastrula distribution of LvTbx2/3 protein (cross-section and surface projection of the same embryo). Asymmetric expression is observed in the invaginated endoderm, the ectoderm and in all of the skeletogenic mesenchyme cells at this stage (also see Fig. 6). (I) Animal view of early pluteus embryo optically sectioned to remove the most superficial layers of ectoderm and expose the archenteron and stomodaeum. LvTbx2/3 is distributed asymmetrically in the surface ectoderm and the length of the archenteron. (J) Vegetal surface view. Distribution of LvTbx2/3 protein is asymmetric in the aboral ectoderm nuclei. High concentrations of LvTbx2/3 are also observed in the distal most nuclei of the extending pluteus arms. (K) Vegetal cross-section of a late pluteus embryo. Asymmetric distribution is maintained in cells of the ectoderm, endoderm and skeletogenic mesoderm.

View larger version (78K): [in a new window]   Fig. 6. Dynamic expression of LvTbx2/3 in the skeletogenic mesenchyme cells (PMCs). (A) Expression in PMCs begins at mid-late gastrula stage in all PMCs. (B,C) Two views of the same early-prism stage embryo. (B) Superficial view of the PMCs under the ectoderm. At this stage, asymmetric distribution of LvTbx2/3 is observed in the PMC lineage. (C) Deeper, cross-sectional view of the embryo in B co-stained for LvTbx2/3 (red) and EctoV (green). LvTbx2/3 localization in PMCs is in the aboral territory of the embryo. Note the clear, asymmetric distribution in the endoderm. (D) An oblique view of a late prism-stage embryo stained for LvTbx2/3 (red) and 5a7 (green). Distribution of LvTbx2/3 protein is limited to the aboral PMCs and is not present in the ventrolateral clusters that have begun to form triradiate spicules. Animal (E) and vegetal (F) cross-sectional views of early-pluteus stage embryos. LvTbx2/3 protein persists in the aboral PMCs and is not observed in the oral PMCs.

View larger version (46K): [in a new window]   Fig. 7. Perturbation of the oral/aboral axis and the consequences for LvTbx2/3 expression. (A,B) Control embryos depicting normal expression of EctoV and LvBrac. (A) Cross-sectional view of EctoV oral ectoderm distribution in the late gastrula. (B) Mid-gastrula surface view of normal blastopore and somodael LvBrac expression. (C,D) Injection of LvG-cadherin mRNA animalizes the embryo by binding to endogenous ß-catenin and preventing its nuclear localization. These embryos lack endoderm and mesoderm and, as previously reported, express the EctoV antigen uniformly (C). They do not express aboral LvTbx2/3 (D). (E,F) NiCl2 ventralization. (E) LvBrac expression expands to all ectoderm cells after ventralization with NiCl2. (F) LvTbx2/3 is not expressed in any germ layer of these embryos. (G,H) Ectopic expression of BMP2/4 radializes the ectoderm of the embryo, as indicated by the formation of multiple triradiate spicules (Angerer et al., 2000). Such embryos express normal levels of vegetal LvBrac around the blastopore but do not express oral LvBrac (G) and aboral LvTbx2/3 in the tissue of any germ layer (H), indicating that ectopic expression of BMP2/4 antagonizes normal specification events along the A/V axis and in the O/A axis in all germ layers. (I,J) Disruption of the extracellular matrix with ßAPN, a drug that prevents collagen crosslinking, results in the failure to express oral LvBrac, but vegetal expression of LvBrac is, apparently, unaffected (I). (J) ßAPN also prevents LvTbx2/3 expression.

View larger version (82K): [in a new window]   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.