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First published online 29 June 2005
doi: 10.1242/dev.01903

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Drosophila WntD is a target and an inhibitor of the Dorsal/Twist/Snail network in the gastrulating embryo

¹ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
² Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
³ Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
⁴ Program in Cell Dynamics, University of Massachusetts Medical School, Worcester, MA 01605, USA
⁵ Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

View larger version (80K): [in a new window]   Fig. 1. (A) Alignment of Wnt8/WntD protein sequences. Sequences of Wnt proteins from Drosophila melanogaster (WntD), Branchiostoma floridae (cephalochordate, BfWnt8), Gallus gallus (chicken, CWnt8), and Danio rerio (zebrafish, ZWnt8) are shown. The lightly shaded boxes highlight the conserved amino acid residues. WntD has fewer conserved residues when compared with other members of this subfamily. However, 20/22 of the characteristic cysteine residues are conserved (asterisks). (B) Degree of conservation among WntD and Wnt8 family members. The percent identity/percent similarity is shown in the table. WntD is more distally related to other members in the Wnt8 subfamily. (C-J) Expression pattern of Drosophila wntD in wild-type embryos. In situ hybridization was performed using an antisense probe generated from a wntD cDNA clone. The embryos are oriented with the anterior to the left. For sagittal views, the dorsal side is up (C,D); for other embryos, the ventral views are shown (E-J). The embryo in C is a pre-cellular blastoderm (stage 4), D is a cellular blastoderm (stage 5), E is an early gastrula-stage embryo (stage 6), and F is a gastrula-stage embryo with a ventral furrow, indicated by the arrow (stage 6). The embryos in G-J are at various stages of germ-band extension (stages 7, 8, 9 and 10). During gastrulation, the cephalic furrow (arrowhead in panels E,F) is formed at approximately the same time as the ventral furrow. The expression of wntD appears first in the anterior and posterior regions of the pre-cellular blastoderm (C), and then in the ventral cells and mesectoderm (D-F). Expression continues in the ventral mesectoderm (G), and de novo expression appears in the ventral neuroectoderm (G-J).

View larger version (99K): [in a new window]   Fig. 2. Genetic regulation of wntD expression. (A-F) The expression of wntD and sim in wild-type (WT) embryos. Both genes have lateral stripes of expression. Only wntD shows a lower level of expression in ventral cells, and only sim has a characteristic stripe in the posterior region (arrows in C and E). The embryo in E and F showed both characters, indicating that it contained both wntD and sim probes. Panels B, D and F are higher magnifications of the regions indicated by the brackets in A, C and E, respectively. The double in situ hybridization (E,F) shows that the wntD and sim patterns overlap in the mesectoderm. (G) In embryos laid by dorsal–/– mothers, no wntD staining was observed at any stage of embryogenesis. (H) In embryos laid by Toll10b mothers, an expansion of wntD expression into the dorsal side was observed. (I) Heterozygous snail embryos had increased expression in ventral cells but the mesectodermal expression was unchanged. (J) In the snail homozygous background, the mesectodermal expression of wntD disappeared, and the ventral staining became stronger. (K) In twist mutant embryos, the wntD pattern was narrower but overall was similar to that observed in wild-type embryos. (L) In twist snail double-mutant embryos, the mesectodermal staining disappeared, while a weaker ventral staining remained. (M) Sagittal view of a wild-type embryo during germ-band extension, showing wntD expression in the neuroectoderm. (N) In embryos that were zygotically mutant for Delta, the wntD expression in the neuroectoderm disappeared.

View larger version (84K): [in a new window]   Fig. 3. Increased WntD expression blocks ventral invagination by interfering with twist and snail expression. The panels in the left column, except panel I, are wild-type embryos. The panels in the right column are embryos expressing WntD by the nanos-Gal4-UAS system. The in situ hybridization probes used are indicated on the left. Panels C and D are cross-sections with dorsal side up, and all other panels are ventral views with anterior to the left. Arrow indicates ventral furrow; arrowhead indicates cephalic furrow. (A,B) The overall expression level of wntD was higher in nanos-Gal4-UAS-wntD embryos than in wild-type embryos and the expression was ubiquitous. The pictures were underexposed to show the cell morphology. The embryo in panel B had no ventral furrow, whereas the cephalic furrow appeared normal. (C,D) Cross-sections of gastrulating embryos showing that no mesoderm was formed during gastrulation in embryos overexpressing WntD. (E,F) The twist expression pattern was much reduced in embryos overexpressing WntD. In wild-type embryos, twist expression is approximately 22 cells wide along the dorsoventral axis at the onset of gastrulation. The embryo shown in E already had some of the cells invaginated. (G) A wild-type embryo showing the normal snail pattern. (H-J) The panels show the reduced snail pattern with increasing severity in embryos overexpressing WntD. Some embryos showed narrower patterns of expression whereas others showed no expression in the anterior regions. (K-N) WntD overexpression also causes sim and rhomboid to show abnormal expression patterns. In wild-type embryos, the expression of sim and rhomboid in the ventral cells is repressed by Snail. Moreover, the positioning of sim also requires Snail. Thus, the abnormal patterns of sim and rhomboid in panels L and N correlate well with the reduced snail pattern.

View larger version (116K): [in a new window]   Fig. 4. WntD regulates Dorsal nuclear localization. All the panels show immunofluorescence staining using an anti-Dorsal antibody. A,D,G,J and L are side views; M and P are ventral views of whole embryos. B,E,H and K are sagittal views, after 2D deconvolution, of the regions indicated by the brackets in A,D,G and J, respectively. C,F and I are ventral views of gastrulae. N and Q are higher magnification views of the posterior regions of the embryos shown in M and P, respectively. O and R are sagittal views, after 2D deconvolution, of cellular blastoderms at the posterior region, including the pole cells. The genotype of each embryo is shown at the bottom right-hand corner. gd, gastrulation-defective; Toll10b is a gain of function Toll. (A-C) In wild-type blastoderm and gastrula, Dorsal protein is localized in the ventral nuclei. (D-F) In gastrulation-defective mutant embryos, the Dorsal protein remains cytoplasmic. (G-I) In many WntD-overexpression embryos, Dorsal is also cytoplasmic. (J,K) In Toll10b embryos, the nuclear Dorsal staining extended into the dorsal side of the embryo. (L) Essentially no signal was detected in a Dorsal protein null embryo. (M,N) Ventral view of a wild-type gastrula showing high levels of nuclear Dorsal around the ventral furrow. The higher magnification in N shows that the posterior region (right side) had less staining. (O) Sagittal view of a wild-type cellular blastoderm at the posterior end, showing the staining of Dorsal changing from nuclear to cytoplasmic in cells ventral to the pole cells. (P-R) Embryos derived from the Df(3R)l26c strain, which has many genes, including wntD, uncovered, showed increased Dorsal nuclear staining in the posterior region, as indicated by the arrow. (R) In a cellular blastoderm before germ-band extension, the nuclear staining of Dorsal already extents further to the dorsal side, using pole cells as a reference.

View larger version (111K): [in a new window]   Fig. 5. Loss of WntD function leads to expansion of a Dorsal target gene. The blue staining in all the panels is RNA in situ staining using an antisense snail probe. The brown staining in E and F is in situ staining using an antisense huckebein probe. (A) Sagittal view of a wild-type blastoderm. The bracket at the posterior end indicates the retracted expression from the pole. (B) Ventral view of a wild-type gastrula, showing the sharp pattern of snail in the lateral and posterior regions. (C) Sagittal view of a Df(3R)l26c blastoderm. The bracket and the arrow indicate the expanded staining in the posterior and anterior regions, respectively. (D) Ventral view of a Df(3R)l26c gastrula; the expanded staining is similarly indicated by the bracket and the arrow. (E,F) Double staining of snail and huckebein, showing their complementary patterns in the posterior region of a wild-type embryo but overlapping pattern in a Df(3R)l26c embryo. (G) Ventrolateral view of an embryo derived from Df(3R)l26c strain that also contained a transgenic wntD genomic construct. All embryos from this rescued strain showed snail expression identical to that observed in wild-type embryos. (H) Ventral view of another wild-type blastoderm, with a retracted posterior pattern. (I) Sagittal view of a wild-type blastoderm previously injected with wntD dsRNA, showing a slightly expanded posterior expression. (J) Ventral view of a wild-type blastoderm previously injected with wntD dsRNA. The posterior sharpening is not as obvious as in wild-type embryos injected with buffer alone.

View larger version (104K): [in a new window]   Fig. 6. Feedback regulation of snail expression by de-repressed WntD expression. snail in situ probe, brown; wntD probe, blue. The embryos shown in E,K and M were from an experiment using the snail probe only; other embryos were from experiments using the snail and wntD probes together. (A,B) Wild-type embryos showing the patterns of snail and wntD expression. (A) Ventral view of an early gastrula-stage embryo; (B) Sagittal view of a mid-gastrula-stage embryo. (C) A snail mutant at germ-band extension stage showing the de-repressed wntD and the reduced snail mRNA expression. (D) A Df(3R)l26c embryo double stained for wntD and snail. The snail pattern expanded into the anterior and posterior regions, and the staining of wntD was absent, demonstrating that it was a homozygous deficiency embryo. (E) A gastrulating snail mutant embryo stained for snail mRNA alone. The mutant embryo did not have a ventral furrow, although the cephalic furrow had already formed. The lateral border of the snail expression (arrow) was fuzzy in contrast to the sharp pattern observed in wild-type embryos. (F) A double-mutant embryo stained for both snail and wntD. The lateral borders of the snail pattern were sharp (arrow). (G,H) Higher magnifications of the embryo shown in E, showing the cephalic furrow (arrowhead) and the cellularization in the sagittal view (G), and the slightly dorsally moved pole cells (arrow, H). (I,J) Higher magnification of the embryo shown in F, showing that it was at a similar stage to the embryo shown in E. (K) Ventral view of a snail mutant during early germ-band extension showing the disappearing snail mRNA. (L) Ventral view of a similar stage double-mutant embryo showing a higher level of the snail mRNA staining. (M,N) Sagittal views of gastrulating embryos of the genotype indicated. The snail staining disappeared more slowly in the double-mutant background.

View larger version (14K): [in a new window]   Fig. 7. A model of WntD and Dorsal/Twist/Snail interaction in the Drosophila embryo. Dorsal and Twist cooperate to activate the expression of snail, wntD, and other genes in the ventral and lateral regions. Snail represses wntD and other neuroectodermal genes in the ventral region, thereby restricting their expression to the lateral regions and allowing ventral invagination to proceed normally. WntD in turn can negatively regulate Dorsal, probably at a step upstream of Dorsal nuclear localization.

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