van der Zee et al 10.1073/pnas.0605154103 |
Fig. 7. The Tribolium Short gastrulation protein (Sog). (A) Domain structure of Drosophila melanogaster Sog (Dm-sog), Anopheles gambiae Sog (Ag-sog), and Xenopus laevis Chordin (Xl-chrd). Black boxes indicate the cystein-rich (CR) domains and display the percentage of amino acid identity compared with Tribolium castaneum Sog (Tc-sog). CR domains bind BMPs. No other sog-like genes were found in the available Tribolium genome sequence. (B) Neighbor-joining distance tree by using the entire Chordin/Sog protein sequences from metazoan species. Sequences were loaded in MEGA 3.1 software. One thousand replicates were used for bootstrap analysis. Species name and GenBank/ensembl accession nos. are shown in parentheses. Chordin-Gg (Gallus gallus, NP_990311), Chordin-Xl (X. laevis, Q91713), Chordin-Hs (Homo sapiens, AAG35767), Chordin-Mm (Mus musculus, AAD19895), Chordin-Ci (Ciona intestinalis, BAE06347), Chordin-Hr (Urochordata, Halocynthia roretzi, AAK83138), Chordin-Nv (Cnidaria, Nematostella vectensis DQ358700.1), Sog-Am (Apis mellifera, XP_393520.1), Sog-Tc (T. castaneum), Sog-Dm (D. melanogaster, Q24025), and Sog-Ag (An. gambiae ENSF00000008622).
Fig. 8. Tc-sog RNAi reduces the head, Tc-dpp RNAi enlarges the head. Tc-mlpt in situ hybridizations. (A-C) Differentiated blastoderm stage (D-F) DAPI images of the embryos shown in A-C, respectively. (G-I) Extending germ-band embryos. (A) Tc-sog RNAi. Tc-mlpt was detected in a narrow stripe in the anterior of the germ rudiment and in the primitive pit. (B) Wild-type. Tc-mlpt is detected in an anterior triangle and in the primitive pit. (C) Tc-dpp RNAi embryo. Tc-mlpt is detected in a broad anterior band and in the primitive pit. (D) After Tc-sog RNAi, the serosa/germ rudiment border is straight and located at a position corresponding to the WT dorsal border (white line). (E) In WT, the germ rudiment/serosa border is oblique and runs from a dorsal, more posterior point to a ventral, more anterior point (white lines). (F) After Tc-dpp RNAi, the germ rudiment/serosa border is straight and located at a position corresponding to the WT ventral position (white line). (G) After Tc-sog RNAi, Tc-mlpt is detected only in a posterior domain. (H) WT. Tc-mlpt is detected in a posterior domain and in three stripes in the head (arrowheads). (I) After Tc-dpp RNAi, Tc-mlpt is detected in a posterior domain and in three stripes in an enlarged head (arrowheads).
Fig. 9. Hox gene expression after Tc-sog RNAi. In situ hybridization with Tc-Deformed (Tc-Dfd) (A and B), Tc-Sex combs reduced (Tc-Scr) (C and D), and Tc-Antennapedia (Tc-Antp) (E and F). (A) WT. Tc-Dfd is expressed in the mandibular (Md) and maxillary (Mx) segment (1). (B) Tc-sog RNAi. No Tc-Dfd expression was detected, indicateing that the Md and Mx segment are absent. (C) WT. Tc-Scr is expressed in the labial (Lb) segment (2). (D) Tc-sog RNAi. The anteriormost (reduced) segment expresses Tc-Scr, indicating that this reduced segment has a labial identity. (E) WT. Tc-Antp is expressed in all thoracic and abdominal segments (3). (F) Tc-sog RNAi. Except for the anteriormost (reduced) segment, all segments express Tc-Antp. Thus, except for the anteriormost, reduced segment, the embryo only consists of thorax and abdomen.
1. Brown S, Hotzman S, Kaufman T, Denell R (1999) Dev Genes Evol 209:389-398.
2. Curtis CD, Brisson JA, DeCamillis MA, Shippy TD, Brown SJ, Denell RE (2001) Genesis 30:12-20.
3. Stuart JJ, Brown SJ, Beeman RW, Denell RE (1993) Development (Cambridge, UK) 117:233-243.
Fig. 10. Cuticles after Tc-sog RNAi. (A-C) WT cuticles. (D-F) Cuticles after Tc-sog RNAi. (A) Lateral view. Thoracic segments are labeled. (B) Ventral pattern of four bristles (asterisks at their base) of abdominal segment 7. (C) Dorsal pattern of eight bristles (thoracic segment 2). Two outer bristles carry a typical scale at their base (arrows). (D) After Tc-sog RNAi, limb buds are present at the thoracic segments. (E) Thoracic region after Tc-sog RNAi. The limbs probably lost their normal polarity, as revealed by their symmetrical appearance. Anterior to T1, only one segment is present (arrow). (F) Bristle pattern found between the legs (first thoracic segment) of Tc-sog RNAi embryos. Dorsal hairs with a scale (arrows) are present. The mirror image polarity in the ectoderm makes the limb buds more symmetrical.
Fig. 11. orthodenticle is expressed in dorsalized Drosophila embryos. (A-C) Cellular blastoderm and early gastrulating embryos (lateral views, anterior faces to the left and dorsal faces up). (A and B) Dm-orthodenticle (Dm-otd) and LacZ in situ hybridizations. (A) brkM68 sogYSO6/FM7c ftz-LacZ. These embryos develop like WT (1). (B) brkM68 sogYSO6/Y. The absence of ftzLacZ expression proves that this embryo lacks brk and sog function. The double mutant results in a complete loss of the neuroectoderm because of the ventral expansion of Dpp activity (1). This absence of brk and sog does not result in a loss of Dm-otd expression. (C) Dm-otd in situ hybridizations. Maternal genotype: gd9/gd9. Loss-of-function mutations in gastrulation defective (gd) result in completely dorsalized embryos with uniform Dpp activity along the DV circumference (2). Dm-otd is not significantly altered compared to WT embryos.
1. Jazwinska A, Ruslow C, Roth S (1999) Development (Cambridge, UK) 126:3323-3334.
2. Ponomareff G, Giordano H, DeLotto Y, DeLotto R (2001) Genetics 159:635-645.
Fig. 12. Morphogenetic movements after changes in Dpp signaling. (A-C) Tc-pnr in situ hybridizations at early gastrulation. DAPI images from the surface of the embryos have been superimposed. (A) In WT, the amnion expresses Tc-pnr and folds over the germ rudiment at the ventral side (arrowhead). DAPI staining reveals the serosal nuclei at the surface. (B) Tc-dpp RNAi embryo. No Tc-pnr expression could be detected. DAPI staining reveals that a part of the germ rudiment remains at the posterior surface. The germ band is more or less symmetrically folded inward at the primitive pit and extends toward the anterior. (C) Tc-sog RNAi embryo. Tc-pnr is detected along the anterior margin of the germ rudiment and in the primitive pit, but not at the dorsal side, where the amnion should form (arrowhead). DAPI staining reveals the serosal nuclei at the surface. (D-F) Schematic drawings of the embryos shown in A-C with Tc-pnr staining in black. Dashed lines indicate the serosa. Solid lines indicate germ rudiment. GZ, growth zone. The arrow in E indicates the direction of germ-band extension.
Fig. 13. Schematic drawings of the Tc-sog and Tc-dpp RNAi phenotypes. (A and B) Tc-sog RNAi. (A) The neurogenic ectoderm (green) is absent. Dorsal cell fates occupy domains along the anteroposterior (AP) axis: dorsal serosal cells (red) are present in a broad band anterior to the germ rudiment, amniotic cells (dark blue) are present along the anterior margin of the germ rudiment and in the primitive pit. Dorsal amnion is absent. The serosa/germ rudiment border is straight and is located at the position of the dorsal border in WT. The presumptive mesoderm (dotted line with arrow) is correspondingly shorter along the AP axis. (B) The enlarged serosa and remaining amniotic cells allow for a rather normal gastrulation, but the amniotic cavity (ac) never closes. The mesoderm invaginates normally. (C and D) Wild-type. (C) Dorsal serosal cells and amniotic cells are localized to the dorsal side. Neurogenic ectoderm and mesoderm are present. The germ rudiment/serosa border is oblique and runs from a dorsal, posterior position to a ventral, anterior position. (D) The amnion folds over the embryo at the ventral side forming the amniotic cavity (ac). The serosal window (sw) closes. (E and F) Tc-dpp RNAi. (E) Dorsal serosa and amnion are absent. Mesoderm and an anterior serosa (orange) are present. The germ rudiment/serosa border is straight and located at the position of the WT ventral border. The rest of the embryo consists of neurogenic ectoderm. (F) The embryo folds symmetrically inwards at the primitive pit and extends with the growth zone toward the anterior (arrow). The small serosa covers only two-thirds of the yolk; the anterior regions of the embryo proper remain at the posterior surface of the egg (big arrow). The mesoderm invaginates normally toward the yolk, i.e., toward the exterior of the tube-like embryo (small arrows in E and F, see also G). (G) Cross-section of the tubular Tc-dpp RNAi embryo. The invaginated mesodermal cells, stained in brown with a Twist antibody, migrate and cover the whole outer circumference of the tube at extending germ-band stages. pp, primitive pit.
Supporting Results
Analysis of the Cuticle Confirms the "Double Dorsal" Phenotype.
The double dorsal phenotype in Tc-sog RNAi embryos (Fig. 5) also can be seen in cuticle preparations. In WT (Fig. 10A), the ventral cuticle displays a typical pattern of two groups of two bristles per segment (Fig. 10B), whereas the dorsal side shows a pattern of eight bristles, of which two always carry a scale at their base (Fig. 10C). Because of a reduction of the amnion, Tc-sog RNAi embryos do not close entirely at the dorsal side, making it difficult to score the dorsal cuticular markers. However, along the ventral midline, bristles of the dorsal type are found (Fig. 10F). In the thorax area, these bristles are localized between enlarged and abnormally shaped legs, indicating the presence of lateral ectoderm. This arrangement is in accordance with the double dorsal phenotype in which lateral ectoderm flanks ectopic dorsal ectoderm present along the ventral midline.No cuticles were obtained after Tc-dpp RNAi. The aberrant development of Tc-dpp RNAi embryos apparently prevents the excretion of cuticle.
Morphogenetic Movements and Gastrulation After Changes in Bone Morphogenetic Protein (BMP) Signaling.
Changes in Dpp signaling severely affect the specification and, consequently, the morphogenetic movements of the extraembryonic membranes. In WT, Tc-pnr expression is believed to mark the presumptive amniotic cells at the blastoderm stage (Fig. 2E; ref. 1). During gastrulation, the dorsal Tc-pnr-expressing cells fold over the germ rudiment at the ventral side (Fig. 12 A and D) to cover the embryo as an inner extraembryonic layer (Fig. 12 C and D). The serosa follows the amnion and soon covers the yolk and embryo as an outer extraembryonic membrane (Figs. 12 A and D and 13 C and D). The dorsal serosa cells marked by Tc-doc (Fig. 2C) undergo the largest translocation in this process.After Tc-dpp RNAi, Tc-pnr expression and, consequently, the amnion is absent (Figs. 2J and 12E). Furthermore, the size of the serosa is severely reduced, and the dorsal serosal cells are lost, as shown by the absence of Tc-doc expression (Figs. 2H and 13E). Thus, the small serosa only slightly expands and will cover about two-thirds of the yolk, but never will cover the complete embryo (Figs. 12 B and E and 13 E and F). Therefore, the anterior region of the embryo remains at the surface of the yolk throughout development and is positioned at the posterior pole of the egg (Figs. 12 B and E and 13F). Because ventral ectodermal cell fates are present around the entire circumference at the posterior pole, posterior pit formation and the subsequent invagination are largely symmetric. This symmetric invagination leads to the formation of an ectodermal tube, which extends with the growth zone toward the anterior (Figs. 12E and 13F). As a result, the posterior growth zone will be located at the anterior of the egg, whereas the head remains at the posterior. Despite this inverted orientation, all older Tc-dpp RNAi embryos in this paper are shown for simplicity with their heads to the left.
After Tc-sog RNAi, the serosa is enlarged with many dorsal serosal cells (Fig. 2 M and N). Amniotic cells, marked by Tc-pnr, still are specified along the anterior border of the germ rudiment and in the primitive pit (Figs. 2O and 13A). These amniotic cells, together with the dorsal serosal cells allow a rather normal gastrulation (Figs. 12 C and F and 13 A and B). However, the dorsal side of the blastodermal germ rudiment does not express Tc-pnr (Fig. 2O, arrow) and remains free of Tc-pnr transcripts during gastrulation (Fig. 13 C and F, arrowhead). This part does not behave like amnion and prevents the remaining amnion from closing completely, leaving the anterior of the germ rudiment uncovered throughout development. Although the abdomen of many embryos is covered by a normal amnion arising from the growth zone, some embryos also lack the posterior part of the amnion. Thus, Tc-sog RNAi embryos at least lack the dorsal amnion derived from the blastoderm, reminiscent of Drosophila sog mutants lacking the amnioserosa. However, this absence of dorsal amnion has only minor effects on gastrulation.
In summary, the absence of BMP signaling (Tc-dpp RNAi) leads to the total absence of the dorsalmost cell fates (dorsal serosal cells and amnion) and severely affects gastrulation. Knockdown of the transport mechanism (Tc-sog RNAi) leads to a reduction of the amnion, but only to mild effects on gastrulation.
1. van der Zee M, Berns N, Roth S (2005) Curr Biol 15:624-636.