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Caudal regulates the spatiotemporal dynamics of pair-rule waves in Tribolium - PubMed

  • ️Wed Jan 01 2014

Caudal regulates the spatiotemporal dynamics of pair-rule waves in Tribolium

Ezzat El-Sherif et al. PLoS Genet. 2014.

Abstract

In the short-germ beetle Tribolium castaneum, waves of pair-rule gene expression propagate from the posterior end of the embryo towards the anterior and eventually freeze into stable stripes, partitioning the anterior-posterior axis into segments. Similar waves in vertebrates are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. However, neither a molecular candidate nor a functional role has been identified to date for such a frequency gradient, either in vertebrates or elsewhere. Here we provide evidence that the posterior gradient of Tc-caudal expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. We show this by analyzing the spatiotemporal dynamics of Tc-even-skipped expression in strong and mild knockdown of Tc-caudal, and by correlating the extension, level and slope of the Tc-caudal expression gradient to the spatiotemporal dynamics of Tc-even-skipped expression in wild type as well as in different RNAi knockdowns of Tc-caudal regulators. Further, we show that besides its absolute importance for stripe generation in the static phase of the Tribolium blastoderm, a frequency gradient might serve as a buffer against noise during axis elongation phase in Tribolium as well as vertebrates. Our results highlight the role of frequency gradients in pattern formation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Tc-cad expression in Tribolium.

(A–D) Concurrent Tc-cad in situ hybridization (red; first column) and Tc-EVE antibody staining (green; second column) were merged (third column) to show that Tc-cad expression overlaps with the emerging first two stripes of Tc-Eve in the blastoderm (A, B), and retreats to the posterior while the third stripe emerges (C). Tc-cad expression is confined in the growth zone during the germband stage to overlap with emerging stripes (fourth stripe in D). (E, F) Measuring Tc-cad expression across AP axis of the blastoderm (E, Text S3) and fitting raw measurements (thin blue line in F) to a linear-with-plateau curve (thick blue line in F) and calculating its three descriptors (F, Text S3). (G, H) As revealed by the change in the three descriptors of Tc-cad gradient over time (G), Tc-cad expression gradient builds up during 14–17 hours AEL but does not shift. Tc-cad dynamics are summarized in H; dashed curve: early, solid curve: late expression. Anterior to left. Error bars represent 95% confidence intervals.

Figure 2
Figure 2. Characterization of Tc-cad gradient in WT and RNAi knockdowns.

(A, A′) Tc-cad gradient in WT. (B) A model for Tc-cad regulation in the Tribolium blastoderm. (C–D″) Tc-cad gradient expression in a Tc-lgs RNAi embryo (C, C′), and the average of its three descriptors normalized to WT values (Text S3) in 14–17 AEL (D) and 17–20 AEL (D′). As inferred from (D, D′), a comparison between the spatial distribution of Tc-cad gradient in Tc-lgs RNAi embryos and that of WT is summarized in D″ (not to scale). The same was performed for Tc-pan (E–F″), Tc-apc1 (G–H″; in H″: dashed curve for 14–17 AEL and solid curve for 17–20 AEL), Tc-zen1 (I–J″), and Tc-lgs;Tc-zen1 (K–L″) RNAi embryos. (M–M″) the average of the three descriptors of the Tc-cad expression gradient in Tc-pan RNAi normalized to Tc-lgs RNAi values (Text S3). (N–N″) the average of the three descriptors of the Tc-cad expression gradient in Tc-lgs;Tc-zen1 RNAi normalized to Tc-lgs RNAi values. Anterior to the left. Error bars represent 95% confidence intervals. Asterisk (*) represents p-value<0.05.

Figure 3
Figure 3. Tc-eve expression in WT and RNAi knockdowns.

Tc-eve expression waves in WT (A), mild Tc-cad (B), Tc-lgs (C), Tc-pan (D), Tc-apc1 (E), Tc-zen1 (F) and Tc-lgs;Tc-zen1 (G) RNAi embryos (First cycle/wave/stripe in red, second in gold, and third in green). Tc-eve expression patterns were classified according to the cycle of Tc-eve oscillation in the posterior end of the embryo (roman numerals) and the phase of the cycle (1 for high phase, and 0 for low; e.g. I.1: high phase of the first cycle). Embryos were mapped on the time axis according to timing data (see text). Arrows indicate the position of the anterior border of Tc-eve expression at 20–23 hours AEL in WT (black arrow) and in different knockdowns (white arrows). Shown also are snapshots of computer simulations of a Tc-eve oscillator the frequency of which is modulated by the Tc-cad gradient of WT (A′; see Movie S1, upper panel), mild Tc-cad and Tc-lgs RNAi (C′; see Movie S2, lower panel), Tc-pan RNAi (D′; see Movie S3, lower panel), Tc-apc1 (E′; see Movie S4, lower panel), Tc-zen1 (F′; see Movie S5, lower panel), and Tc-lgs;Tc-zen1 (G′; see Movie S6, lower panel) RNAi embryos; blue: Tc-eve expression, red curve: Tc-cad expression gradient. Snapshots were taken at the end of the corresponding simulations. Anterior to the left. Simulations were performed using Matlab (code is available in Text S1).

Figure 4
Figure 4. Spatial characteristics of Tc-eve waves over time in WT and RNAi knockdowns.

(A, B, C, D, E, F) average position of the anterior border of Tc-eve expression over time in mild Tc-cad (A), Tc-lgs (B), Tc-pan (C), Tc-apc1 (D), Tc-zen1 (E) and Tc-lgs;Tc-zen1 (F) RNAi embryos (red) compared to WT (blue; along with Tc-lgs RNAi in case of Tc-lgs;Tc-zen1, green). Same comparisons were performed for average width of first (A′, B′, C′, D′, E′, F′) and second (A″, B″, C″, D″, E″, F″) Tc-eve stripes. At top is a depiction of Tc-eve expression (black stripes) in a WT Tribolium embryo at late blastoderm stage; anterior to the left. All measurements were normalized to AP axis lengths (Text S3 and Figure S5). A missing data point for a certain stripe indicates that stripe has not formed yet; a stripe proper should have both anterior and posterior borders. Error bars represent 95% confidence intervals.

Figure 5
Figure 5. Temporal dynamics of Tc-eve expression at the posterior end of the embryo in WT and RNAi knockdowns.

(A, B, C, D, E, F) percentage distributions of Tc-eve expression classes (classification was based on Tc-eve oscillation cycle in the posterior end, see Figure 3) in different timed egg collections in multiple RNAi knockdowns (red bars) in comparison with WT (blue bars): mild Tc-cad (A), Tc-lgs (B), Tc-pan(C), Tc-apc1(D), Tc-zen1(E), and Tc-lgs;Tc-zen1(F) RNAi embryos. Cycle I embryos are those going from high (phase I.1) to low (phase I.0) Tc-eve expression levels at the posterior end to from the first Tc-eve stripe (Figure 3); cycle II embryos are those going from high (phase II.1) to low (phase II.0) Tc-eve expression levels to form the second Tc-eve stripe (and so on). Class distributions were used to estimate the duration of different Tc-eve oscillation cycles (A′, B′, C′, D′, E′, and F′; see Text S3). Error bars represent 95% confidence intervals.

Figure 6
Figure 6. Frequency profile and robustness of the clock-and-wavefront model.

A computer simulation of a two-dimensional (2D) lattice of oscillators (horizontal and vertical axes of the lattice represent the AP and lateral axes of the embryo, respectively; posterior to the right). Each oscillator runs independently with a frequency determined by a smooth spatial gradient that retracts from anterior to posterior, with or without applying a threhold. (A) one-dimensional (1D) lateral cross section of the 2D smooth gradient (see Movies S7 for the 2D version; direction of movement is shown in blue arrow). (B) stripes generated if the smooth gradient shown in (A) is directly applied to the oscillators lattice (high phase of oscillation is shown in white and low phase in black); see Movie S7. (C) a 2D thresholded version of the smooth gradient shown in (A). (D) stripes generated if the thresholded gradient shown in (C) is applied to oscillators lattice; see Movie S8. (E–H) are the same as (A–D) after adding noise to frequency gradient intensity; see Movies S9 and S10. Simulations were performed using Matlab (code is available in Text S2).

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