Elongation of the head-to-tail body axis by convergent extension is a conserved developmental process throughout metazoans. In Drosophila, patterns of transcription factor expression provide spatial cues that induce systematically oriented cell movements and promote tissue elongation. However, the mechanisms by which patterned transcriptional inputs control cell polarity and behaviour have long been elusive. We demonstrate that three Toll family receptors, Toll-2, Toll-6 and Toll-8, are expressed in overlapping transverse stripes along the anterior–posterior axis and act in combination to direct planar polarity and polarized cell rearrangements during convergent extension. Simultaneous disruption of all three receptors strongly reduces actomyosin-driven junctional remodelling and axis elongation, and an ectopic stripe of Toll receptor expression is sufficient to induce planar polarized actomyosin contractility. These results demonstrate that tissue-level patterns of Toll receptor expression provide spatial signals that link positional information from the anterior–posterior patterning system to the essential cell behaviours that drive convergent extension.
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We thank K. Anderson, K. Kasza, W. Razzell, G. Sabio, M. Shirasu-Hiza, A. Spencer, M. Tamada and R. Zallen for comments on the manuscript, B. Glick for the fast-folding YFP, M. Buszczak for pUASp-w-attB, and the BAC-Recombineering Core Facility at the University of Chicago for Toll-8–YFP. This work was funded by NIH/NIGMS grants GM079340 and GM102803 to J.A.Z. J.A.Z. is an Early Career Scientist of the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Extended data figures and tables
a–c, Control and dsRNA-injected embryos stained for Runt (red, middle) and Wingless (Wg) (green, bottom) proteins. a, In uninjected wild-type embryos, Runt is expressed in seven broad stripes and Wg is expressed in 14 narrow stripes. b, In embryos injected with eve dsRNA alone, Runt is more uniformly expressed, and the Wg expression pattern collapses into fewer, broader stripes, similar to eve mutants (data not shown). c, In embryos co-injected with eve and runt dsRNAs, Runt protein is undetectable, indicating that the runt dsRNA effectively inhibits Runt expression, and the Wg expression pattern collapses into fewer, broader stripes, similar to eve and runt mutants (data not shown). Anterior left, ventral down. Scale bars, 100 μm. d–f, Quantitative RT–PCR analysis of Toll-2 (2), Toll-6 (6), Toll-7 (7) and Toll-8 (8) expression in late stage 6 embryos before axis elongation. CT values were normalized to the internal control gene RpL32. d, Relative transcript levels in WT embryos were calculated using the 2−ΔCT method. Toll-2, Toll-6 and Toll-8 were expressed at comparable levels, whereas Toll-7 was expressed at much lower levels. e, Toll-8 expression in Toll-859/145 embryos was reduced 76-fold compared with WT embryos. f, Gene expression was specifically reduced in embryos injected with single dsRNAs targeting Toll-2, Toll-6 or Toll-8 compared with embryos injected with a control Toll-3 dsRNA, as determined using the 2−ΔΔCT method.
a–f, Toll-2, Toll-6 and Toll-8 transcripts (green top, white bottom) and Runt protein (magenta) in wild-type (WT) embryos during early (stage 7, a–c) and mid-elongation (stage 8, d–f). The embryos are the same as in Fig. 1a–f. Coloured bars indicate the position of the Toll-2, Toll-6 and Toll-8 stripes (green) relative to Runt (magenta). Anterior left, ventral down. Scale bars, 100 μm.
Extended Data Figure 3 Time-lapse imaging of embryos defective for combinations of Toll-2, Toll-6 and Toll-8.
a–e, Axis elongation (tissue AP length relative to t = 0) (first row), total cell rearrangements (average number of neighbours lost per cell) (second row), T1 processes resulting from the contraction of single edges7 (third row), and rosette rearrangements resulting from the contraction of multiple connected edges8 (fourth row) over time in wild-type embryos (a) and embryos defective for one (b), two (c), or three (d, e) Toll receptors. Images were acquired every 15 s. f–i, Axis elongation (f), average number of cell rearrangements (g), T1 processes (h), and rosettes (i) per cell at t = 30 min. j, Edge contraction rate for AP edges (oriented 75–90° relative to the AP axis) at mid-stage 7 (averaged from t = 5–8 min after the onset of elongation). k, The orientation of shrinking edges relative to the AP axis (0°) was similar for all conditions. Single average values were obtained for each embryo, and plots show the mean ± s.e.m. across embryos. *P = 0.01–0.03, **P < 0.005 (unpaired t-test). n = 3–8 embryos per genotype, 164–365 cells per embryo (see Supplementary Table 2 for full list of n values). l, Cross sections of the ventrolateral epithelium in wild-type and Toll-2Δ76, Toll-61B, Toll-714F, Toll-859 mutant (Toll-2,6,7,8) embryos, showing that apical–basal polarity is unaffected in quadruple mutants. Myosin II (green) and Par-3 (red, white) are enriched at apical adherens junctions. Apical up, basal down. Scale bars, 10 μm. WT (Spider–GFP in a, e–k; Resille–GFP in a, f–k; and Resille–GFP + Toll-3 dsRNA in a–d and f–k); Toll-2 (Resille–GFP + Toll-2 dsRNA); Toll-6 (Resille–GFP + Toll-6 dsRNA); Toll-8 (Resille–GFP; Toll-859/145); Toll-2,6 (Resille–GFP + Toll-2/Toll-6 dsRNAs); Toll-2,8 (Resille–GFP; Toll-859/145+ Toll-2 dsRNA); Toll-6,8 (Toll-2Δ76/CyO; Toll-859, Toll-65A, Spider–GFP); Toll-2,6,8 (Toll-2Δ76; Toll-859, Toll-65A, Spider–GFP), Toll-2,6,8 i1 (Resille–GFP; Toll-859/145+ Toll-2/Toll-6 dsRNAs set 1); Toll-2,6,8 i2 (Resille–GFP; Toll-859/145+ Toll-2/Toll-6 dsRNAs set 2); runt (runtLB5; Spider–GFP/+); and eve (eveR13; Spider–GFP/+).
a, The crossing strategy used to generate Toll-2,6,8 triple mutants and Toll-2,6,7,8 quadruple mutants. Toll-7 and Toll-2 are 285 kb apart on the right arm of chromosome II and Toll-8 and Toll-6 are 94 kb apart on the left arm of chromosome III. b, Three unique Toll-6 null alleles (Toll-61B, Toll-64B and Toll-65A) were generated on the Toll-859 chromosome using TALEN-mediated mutagenesis to create Toll-8, Toll-6 double-mutant chromosomes. c, Six unique Toll-7 null alleles (Toll-71C, Toll-74D, Toll-75A, Toll-75F, Toll-714F and Toll-716A) were generated on the Toll-2Δ76 chromosome using TALEN-mediated mutagenesis to create Toll-7, Toll-2 double-mutant chromosomes. Each Toll-6 and Toll-7 allele is a frame-shift mutation leading to premature translational termination. TALENs were designed to induce double-stranded breaks immediately downstream of the ATG translational start sites. Orange letters indicate the TALEN binding sites, and the spacer regions are shown in bold. The AvaII and AvaI restriction sites used for screening are indicated with dotted boxes. Shown below are the predicted amino acid sequences of the mutant proteins compared with the wild-type sequence. Residues that are identical in the mutant and wild-type proteins are shown in green.
a–l, Planar polarity distributions for myosin II (left panels) and Par-3 (right panels) in Toll-2 single mutants (a, b), Toll-6,8 double mutants (c, d), Toll-2,6,8 triple mutants (e, f), Toll-2,6,7,8 quadruple mutants (g, h), runt mutants (i, j) and eve mutants (k, l). Vertical lines indicate the means of the distributions. Error bars indicate s.e.m. between embryos. Mean planar polarity was shifted towards 1 (absolute ratio; 0 on the log2 scale) in Toll-2 single mutants (P = 0.005 for myosin and P < 0.00005 for Par-3), Toll-2,6,8 triple mutants (P < 0.00002 for myosin and Par-3), Toll-2,6,7,8 quadruple mutants (P < 0.00001 for myosin and Par-3), runt mutants (P = 0.002 for myosin and P < 0.00001 for Par-3) and eve mutants (P < 0.00001 for myosin and Par-3), indicating reduced planar polarity (unpaired t-test with the means of the distributions used as the test statistic). Planar polarity in Toll-2,6,7,8 quadruple mutants was not significantly enhanced relative to triple mutants. Single values were obtained for each embryo, and plots show the mean ± s.e.m. across embryos. n = 2,166–4,909 cells in 7–20 embryos per genotype (Supplementary Table 2).
a, Single-cell analysis of Par-3 planar polarity in wild-type (WT) (left) and Toll-2 mutant (right) embryos. Toll-8-expressing cells were identified by fluorescence in situ hybridization. Cyan lines, boundaries between stripes; Toll-8+, Toll-8-expressing cells. AP enriched (red), DV enriched (blue). Cells without at least one AP and one DV edge were not scored (grey). b, c, Myosin II (cyan, white) and Toll-2 mRNA (red) in stage 7 WT (b) and eve mutant (c) embryos. Arrows, residual myosin cables in eve embryos. Anterior left, ventral down. Scale bars, 20 μm.
This file contains toll receptor expression levels and significantly misregulated genes in eve/runt dsRNA-injected embryos. The transcriptomes of embryos injected with both eve and runt dsRNAs were compared with water-injected controls using RNA sequencing (Gene Expression Omnibus accession number GSE61689). Embryos were hand-selected at the late blastoderm stage, 15-30 min prior to the start of axis elongation (late stage 5/early stage 6). (Top) Toll-8 (Tollo) and Toll-2 (18w) were strongly expressed and Toll-6 and Toll-7 were weakly expressed at this stage. Toll (Tl) is maternally and zygotically expressed at this stage51. Expression of the other Drosophila Toll family genes was not detected. (Middle) Transcripts that were significantly upregulated in eve/runt double RNAi embryos (p<0.01). (Bottom) Transcripts that were significantly downregulated in eve/runt double RNAi embryos (p<0.01). (XLSX 16 kb)
This file contains a summary of genotypes used and the number of cells and embryos in each experiment. (XLSX 15 kb)
Time-lapse videos of a wild-type control-injected embryo (WT) (top) and a Toll-8 mutant embryo injected with Toll-2/Toll-6 dsRNAs (Toll-2,6,8) (bottom). Cell membranes were labeled with Resille:GFP. Cells are labeled according to number of neighbors lost (purple=0, blue=1, green=2, yellow=3, red=4). Anterior left, ventral down; t = 0 is the onset of elongation. Images were acquired at 40× magnification with 15 s intervals between time points. (MOV 9109 kb)
Time-lapse videos of a wild-type embryo (WT) (top) and a Toll-2,6,8 triple mutant embryo (bottom). Cell membranes were labeled with Spider:GFP. Cells are labeled according to number of neighbors lost (purple=0, blue=1, green=2, yellow=3, red=4). Anterior left, ventral down; t = 0 is the onset of elongation. Images were acquired at 40× magnification with 15 s intervals between time points. (MOV 14150 kb)
Time-lapse video of myosin:GFP in a wild-type stage 15 embryo. Yellow arrows indicate the anterior boundaries of two engrailed stripes, anterior to the denticle field (white dots). Anterior left, ventral down; t = 0 is the beginning of stage 15 (time in h:min:sec). Images were acquired at 40× magnification with 30 s intervals between time points. (MOV 7160 kb)
Time-lapse video of myosin:GFP in a stage 15 embryo expressing Toll-2:HA in ectopic stripes under the control of engrailed-Gal4. Yellow arrows indicate the anterior boundaries of two engrailed stripes, which form an ectopic myosin cable. Anterior left, ventral down; t = 0 is the beginning of stage 15 (time in h:min:sec). Images were acquired at 40× magnification with 30 s intervals between time points. (MOV 6513 kb)
Time-lapse video of myosin:GFP in a stage 15 embryo expressing Toll-8:HA in ectopic stripes under the control of engrailed-Gal4. Yellow arrows indicate the anterior boundaries of two engrailed stripes, which form an ectopic myosin cable. Anterior left, ventral down; t = 0 is the beginning of stage 15 (time in h:min:sec). Images were acquired at 40× magnification with 30 s intervals between time points. (MOV 7256 kb)
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Paré, A., Vichas, A., Fincher, C. et al. A positional Toll receptor code directs convergent extension in Drosophila. Nature 515, 523–527 (2014). https://doi.org/10.1038/nature13953
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