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, 8 (11), e1000537

Genetic Control of Organ Shape and Tissue Polarity

Affiliations

Genetic Control of Organ Shape and Tissue Polarity

Amelia A Green et al. PLoS Biol.

Abstract

The mechanisms by which genes control organ shape are poorly understood. In principle, genes may control shape by modifying local rates and/or orientations of deformation. Distinguishing between these possibilities has been difficult because of interactions between patterns, orientations, and mechanical constraints during growth. Here we show how a combination of growth analysis, molecular genetics, and modelling can be used to dissect the factors contributing to shape. Using the Snapdragon (Antirrhinum) flower as an example, we show how shape development reflects local rates and orientations of tissue growth that vary spatially and temporally to form a dynamic growth field. This growth field is under the control of several dorsoventral genes that influence flower shape. The action of these genes can be modelled by assuming they modulate specified growth rates parallel or perpendicular to local orientations, established by a few key organisers of tissue polarity. Models in which dorsoventral genes only influence specified growth rates do not fully account for the observed growth fields and shapes. However, the data can be readily explained by a model in which dorsoventral genes also modify organisers of tissue polarity. In particular, genetic control of tissue polarity organisers at ventral petal junctions and distal boundaries allows both the shape and growth field of the flower to be accounted for in wild type and mutants. The results suggest that genetic control of tissue polarity organisers has played a key role in the development and evolution of shape.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Shape of the wild-type Antirrhinum flower.
(A) Face view. Dorsal lobes are coloured red, lateral lobes orange, ventral lobe yellow. Lip region is shaded darker. (B) Side view. Dorsal tube is dark red, lateral tube dark orange, ventral tube dark yellow. The rim is traced in green. (C) Dissected, flattened petal parts: dorsal (D), lateral (L), and ventral (V) lobes and half of the tube (T) cut along the dorsal and ventral midlines of the flower. Petal parts coloured as in (A) and (B). Palate region of the tube is shaded darker. Numbered dots show connecting positions in the un-dissected corolla.
Figure 2
Figure 2. Antirrhinum corolla morphogenesis.
Images and sections from OPT scans of Antirrhinum flowers (with sepals removed) at a series of developmental stages (day 10–20 after flower initiation). A mature flower was also sectioned (day 24). Each flower is shown in face view (Top). Dorsal lobes are coloured bright red, dorsal tube dark red, lateral lobes bright orange, lateral tube dark orange, ventral lobe bright yellow, and ventral tube dark yellow. Grey lines mark planes of virtual sections, shown below (D–V). The white arrow indicates the ventral furrow and the green arrow indicates a ventral fold in the lobes (day 14–24). Initially (day 10) the corolla is almost radially symmetrical. The petals arch over to form a near spherical shell (day 12). The ventral furrow then forms (day 14). At later stages the ventral tube stretches out and straightens (day 17–24). Eventually the dorsal and ventral parts of the rim meet to form an enclosed chamber and the lobes unfold to give an open flower (day 24). Scale bar is 0.5 mm for day 10–14 and 2 mm for day 17–24.
Figure 3
Figure 3. Clonal analysis of wild-type corolla.
(A) Clones on dissected, flattened petals from 6 flowers induced at day 12.5 (i), day 16 (ii), and day 19 (iii), warped to a mean shape for each petal section (D, L, V, T—see Figure 1C) and overlaid, using a different colour for each flower. (B) Regional growth parameters based on clonal analysis (using grids in Figure S12). (i) Maximal growth rates, Kmax, for the period day 16–17.5. Rates are highest (red/yellow) along the lobe edges. (ii) Principal orientations of growth for the period day 12.5–14, shown as short lines scaled according to the value of Kmax. (iii) Principal orientations of growth for the day 16–17.5 interval. Cyan spots indicate foci at the ventral lobe junctions (numbered 2 in Figure 1C).
Figure 4
Figure 4. The cyc dich div ground state.
(A) Mature (day 24) cyc dich div flower: all petals are identical and bilaterally symmetrical. (B) Representation of a flattened cyc dich div petal, divided into its key regions (see [11]). Patterning along the mediolateral axis of the petal is shown below as distributions of the identity factors LAT (highest at lateral edges) and MED (highest medially). (C) Observed clone pattern on the flattened div mutant ventral lobe (induced at ∼ day 14; clones for several lobes overlaid, see Figure S13 for clones induced at other times). (D) Initial canvas (day 10) used for modelling the cyc dich div triple mutant corolla. Identity factors are established during the setup phase in domains along the proximodistal and mediolateral axes of each petal. Polarity organisers PROXORG and DISTORG are located proximally and distally. Black arrows indicate gradient of POL. (E) PRN summarising the influences of the polarity organisers PROXORG and DISTORG on polarity through POL (POLARISER). See main text for explanation, and see Model 1 in Text S1B for details of implementation. (F) KRN. Sections shaded grey are active only during the early or late phases (unshaded regions are active throughout early and late phases). See main text for explanation, and see Model 1 in Text S1B for detailed implementation. (G) Shape generated by growing the initial canvas (D) to maturity (day 24). A subset of identity factors and polarity organisers are coloured as in (D). Black arrows indicate gradient of POL. (H) One petal from (G), computationally flattened for comparison with (B). Regions with non-zero Gaussian curvature cannot be flattened without distortion or crushing. We used a heuristic to minimise the distortion . (I) Ellipse pattern on a computationally flattened lobe from (G) (ellipses originated as circles on day 14). Compare with (C). (J) Shape generated by growing the initial canvas (D) to maturity using a variant model with isotropic specified growth (no POL gradient). For detailed implementation, see Model 1, mutant 1 in Text S1B. (K) Shape generated through growing the initial canvas (D) to maturity using a variant model with no inhibition of specified growth rates in medial region of lobes. For detailed implementation, see Model 1, mutant 2 in Text S1B. All scale bars are 5 mm unless otherwise marked.
Figure 5
Figure 5. Introducing DIV: The cyc dich corolla.
(A) Mature (day 24) cyc dich flower: all petals are identical, bilaterally symmetrical, and fold back at tube rim. Arrow marks projection at a petal junction. (B) Representation of a flattened cyc dich petal, divided into its key regions . Patterning along the mediolateral axis of the petal is shown below as distributions of the identity factors LAT (highest at lateral edges) and MED (highest medially), plus DIV (present everywhere). Compare with Figure 4B. (C) Observed clone pattern on the flattened cyc dich lobe (induced at ∼ day 14; clones for several lobes overlaid, see Figure S13 for clones induced at other times). (D) Initial canvas used for modelling the cyc dich double mutant corolla. Identity factors are present in domains along the proximodistal and mediolateral axes of each petal. The same identity factors and polarity organisers are present as in Figure 4D, with the addition of DIV (yellow; present everywhere). (E) KRN. Sections shaded grey are active only during the early or late phases (unshaded regions are active throughout early and late phases). Sections coloured blue are influenced by DIV. For detailed implementation, see Model 2 in Text S1B. (F) Shape generated by growing the initial canvas (D) to maturity. A subset of identity factors and polarity organisers are coloured as in (D). (G) One petal from (F) computationally flattened for comparison with (B). (H) Face view of the mature canvas (oblique view shown in (F)). (I) Ellipse pattern on a computationally flattened lobe from (F) (ellipses originated as circles on day 14). Compare with (C). All scale bars are 5 mm unless otherwise marked.
Figure 6
Figure 6. Introducing CYC, DICH, and RAD: The wild-type corolla.
(A) Mature (day 24) wild-type flower. (B) Representation of a flattened dorsal petal, divided into its key regions . Patterning along the mediolateral axis of the petal is shown below as distributions of the identity factors LAT (highest at lateral edges), MED (highest medially), DICH (present in the dorsal half), plus CYC and RAD (present everywhere). (C) Representation of a flattened lateral petal, divided into its key regions. Patterning along the mediolateral axis of the petal is shown below as distributions of the identity factors LAT and MED, plus DIV (highest at the most ventral edge) and non-autonomous RAD (highest at the most dorsal edge). (D) Initial canvas used for modelling the wild-type corolla. Identity factors are present in domains along the proximodistal and mediolateral axes of each petal. The same identity factors and polarity organisers are present as in Figure 5D, with the addition of CYC (bright red; present throughout the dorsal petals), RAD (not shown—same distribution as CYC), and DICH (red-brown: present in the dorsal halves of the dorsal petals). (E) Gene Regulatory Network (GRN). Section shaded grey is active only during the late phase. These interactions are supported by genetic data as described in the main text. For detailed implementation, see Model 3 in Text S1B. (F) KRN incorporating CYC, DICH, and RAD (blue sections). CYC promotes Kper, enhanced by LIP, leading to broadening of the dorsal petals. CYC and DICH promote Kpar in combination with PLT, leading to increased palate length. CYC promotes Kpar in combination with DTL in the absence of DICH leading to enhanced growth of the non-medial region of the dorsal lobe. DICH promotes Kpar in combination with PLT leading to extension of the dorsal palate. RAD inhibits Kpar in combination with LIP or PLT (in the absence of CYC or DICH modulated by LAT), reducing the length of the lip and palate regions. We also assume that LTS inhibits Kper at early stages, consistent with observations on reduced lateral petal width observed with OPT, and that CYC and DICH modulate relative growth on the two surfaces of the canvas, leading to bending back of the dorsal lobes. For detailed implementation, see Model 3 in Text S1B. (G) Face view of the shape generated by growing the initial canvas (D) to maturity (day 24). (H) Oblique view of the shape generated by growing the initial canvas (D) to maturity (day 24). (I) Dorsal petal from (H) computationally flattened for comparison with (B). Numbers refer to positions as shown in Figure 1C. (J) Lateral petal from (H) computationally flattened for comparison with (C). Numbers refer to positions as shown in Figure 1C. (K) Ellipse pattern on a computationally flattened lateral lobe from (H) (ellipses originated as circles on day 14). (L) Observed clone pattern on the flattened lateral lobe (induced at ∼ day 14; clones for several lobes overlaid). Dot marks point towards which clones tend to be oriented. Compare with (K). (M) Longitudinal section view of canvas at day 14 when ventral petal has arched over (compare with Figure 2). (N) Longitudinal section view of canvas at maturity (day 24). Note that the ventral tube bulges out, in contrast to what is observed experimentally (Figure 2). All scale bars are 5 mm unless otherwise marked.
Figure 7
Figure 7. Correction of clonal patterns by modification of the wild-type model.
(A) Part of the KRN (rest as in Figure 6F) showing modification of specified growth in the medial region of the lateral petal. For detailed implementation, see Model 4 in Text S1B. (B) Oblique view of mature wild-type canvas grown with the modifications in (A). Black arrows indicate gradient of POL. (C) Longitudinal section (clipped dorsoventrally) of the modified wild-type canvas shown in (B). Note the ventral petal is still bulging outwards. The distal petal lobes grow through the tube as there is no collision detection in the model. (D) Ellipse pattern on a computationally flattened lateral lobe from (B) (ellipses originated as circles on day 14). Compare with Figure 6K and L. Numbers refer to positions shown in Figure 1C. (E) PRN showing addition of a new polarity organiser: CENORG. For detailed implementation, see Model 5 in Text S1B. (F) Part of the KRN (rest as in Figure 6F) showing additional modification in the medial lip and palate of lateral petals. For detailed implementation, see Model 5 in Text S1B. (G) Wild-type canvas before (i) and after (ii) reorientation of growth through activation of CENORG (cyan at the ventral rim indicates location of CENORG). (H) Ellipse pattern on a computationally flattened lateral lobe from the mature canvas grown according to modifications in (E) and (F) (ellipses originated as circles on day 14). Compare with (D) and Figure 6K and L. Numbers refer to positions shown in Figure 1C. (I) Oblique view of mature (day 24) wild-type canvas grown with the modifications described in (E) and (F). (J) Longitudinal section (clipped dorsoventrally) of the modified mature (day 24) wild-type canvas shown in (I). (K) Ellipse pattern on a computationally flattened ventral lobe from the mature canvas grown according to modifications in (E) and (F) (ellipses originated as circles on day 14). All scale bars are 5 mm unless otherwise marked.
Figure 8
Figure 8. Further modification to the wild-type corolla model.
(A) Part of the KRN (rest as in Figures 6F; 7F) showing modifications to specified growth rates that improve the corolla shape. DICH and LAT with LPB promote Kpar on the B (abaxial) surface and inhibit on the A (adaxial) surface leading to the dorsal petal lobes bending forwards. During the early stage, MLOBE in combination with CYC or DIV (but in the absence of DICH) inhibits Kpar on the A surface, leading to the lobes bending forward. DIV in combination with LPB (but in the absence of MED) promotes Kpar and Kper on the A surface, while inhibiting Kper on the B surface, leading to bulging of the ventral petal junctions. During the late stage, LTS in combination with LAT inhibits Kpar in the lip and palate, preventing excessive growth of lateral boundaries. Growth of the ventral palate is modulated by DIV promoting Kpar in combination with PLT, while DIV in combination with CENORG inhibits Kpar in the absence of LPB and enhanced by PLT and MED, restricting growth of the distal palate. Inhibition of Kper by DIV is promoted by PLT, and by LPB in the LOBE, leading to a narrow ventral palate and lip. For detailed implementation, see Model 6 in Text S1B. (B) Initial (day 10) distribution of additional identity factors used in the model, MLOBE and LPB. (C–E) Mature canvas generated by model based on modifications in (A) shown in oblique view (C), longitudinal section (D), and face view (E). (F–G) Ellipse patterns on computationally flattened ventral (F) and lateral (G) lobes from the mature canvas of the wild-type model incorporating changes in (A) (ellipses originated as circles on day 14). All scale bars are 5 mm unless otherwise indicated.
Figure 9
Figure 9. Final version of wild-type corolla model and exploration of mutant forms.
(A) Modified PRN incorporating effect of CYC and DICH on DISTORG. For detailed implementation, see Model 7 in Text S1B. (B–D) Mature canvas generated by model incorporating changes in (A), shown in oblique view (B), longitudinal section (C), and face view (D). (E–G) Flattened ventral (E), lateral (F), and dorsal (G) petals from wild-type model shown in (B–D). (H–I) Mature (day 24) canvas from model shown in (B–D), in which CYC and DICH are inactive (H, compare to cyc dich mutant Figure 5A), or in which CYC, DICH, and DIV are inactive (I, compare to cyc dich div mutant in Figure 4A). (J) Mature (day 24) canvas from a genotype lacking CYC, DICH, and DIV based on a modified model in which CENORG has weak activity in the absence of DIV. Note that there are now invaginations at the petal junctions (cyan areas), similar to those seen in Figure 4A. For detailed implementation, see Model 8 in Text S1B. (K) Ellipse patterns on a computationally flattened petal lobe from (J) (ellipses originated as circles on day 14). All scale bars are 5 mm.
Figure 10
Figure 10. Snip-and-fill mechanism for cell wall growth.
Highly schematic model for plant cell wall growth along one axis. (A) Turgor pressure is counterbalanced by load-bearing components in the wall, indicated by stretched springs with cross-links. Loosening of the wall occurs by breaking of covalent or non-covalent bonds. (B) Wall stretches as the load is shifted to the remaining wall components. (C) Space created by expansion allows new cross-links to be inserted, seeding the synthesis or insertion of additional load-bearing components. (D) Following insertion of new material, the original properties of the wall have been restored. The net effect is an increase in the resting length of the wall. Because the insertion of new material in this example is limited by the space created through expansion, residual stresses do not accumulate.

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