Endocytic and recycling endosomes modulate cell shape changes and tissue behaviour during morphogenesis in Drosophila

Abstract

During development tissue deformations are essential for the generation of organs and to provide the final form of an organism. These deformations rely on the coordination of individual cell behaviours which have their origin in the modulation of subcellular activities. Here we explore the role endocytosis and recycling on tissue deformations that occur during dorsal closure of the Drosophila embryo. During this process the AS contracts and the epidermis elongates in a coordinated fashion, leading to the closure of a discontinuity in the dorsal epidermis of the Drosophila embryo. We used dominant negative forms of Rab5 and Rab11 to monitor the impact on tissue morphogenesis of altering endocytosis and recycling at the level of single cells. We found different requirements for endocytosis (Rab5) and recycling (Rab11) in dorsal closure, furthermore we found that the two processes are differentially used in the two tissues. Endocytosis is required in the AS to remove membrane during apical constriction, but is not essential in the epidermis. Recycling is required in the AS at early stages and in the epidermis for cell elongation, suggesting a role in membrane addition during these processes. We propose that the modulation of the balance between endocytosis and recycling can regulate cellular morphology and tissue deformations during morphogenesis.

Figure 1. Rab5 and Rab11 are planar polarized in the epidermis.

(A–F″) Confocal sections from wild type Drosophila embryos immunostained with DE-Cadherin, Rab5 (A–C) and Rab11 (D–F) antibodies, before epidermal cell elongation (first column of panels), after epidermal cell elongation (second column of panels) and during zippering (third column of panels). Rab5 vesicles accumulate at the LE, especially after epidermal cell elongation (B″, arrowheads), and this is maintained during zippering (C″, arrowheads). There is an enrichment of Rab11 labelled vesicles in the DME cells after epidermal cell elongation (E″) that is also maintained during zippering (F″, yellow dots indicate the borders of DME cells). Quantification of fluorescence levels from boxes B'', E'' (G). Vesicles labelled for Rab11 are more abundant in epidermal cells than in the AS cells, but not Rab5 (G). Yellow arrow indicates the LE, grey box the approximated location of the DME cells.

Figure 2. Blocking endocytosis affects actin organization, but not DE-Cadherin levels.

Confocal sections from Drosophila embryos immunostained with DE-Cadherin (A–L) and phalloidin to visualize F-actin (A'–L'). (A–C'). Wild type embryos during different DC stages show the dynamic distribution of DE-Cadherin and F-actin. Arrowheads in inset B highlight the clearance of DE-Cadherin from the LE and accumulation at the ANCs. In this and all subsequent genotypes three different stages of DC are shown (D–F'). Early stages of DC embryos expressing Rab5^DN ubiquitously show irregular AS cell shapes (D, green contours) and big lamellipodia (D', yellow arrowhead). Later during DC, these embryos exhibit impaired epidermal elongation and puckering (E, yellow arrow) and a strong actin cable with bigger and more filopodia (E', green arrowheads). Actin projections are also observed throughout the epidermis (E', red arrowheads). Zippering is impaired in these embryos (F, F'). (G–I') Embryos expressing Rab5^DN in the AS results in changes in cell morphology, particularly evident at early and late stages of DC (G,I), but epidermal cell elongation is not compromised (H). Zippering is also affected (I). Abnormal filopodia and lamellipodia are observed in the AS (G', I', yellow arrowheads) throughout DC. Embryos expressing Rab5^DN in the engrailed domain do not exhibit defects in epidermal cell elongation nor in zippering. DE-Cadherin shows a wild type distribution (J–L), but stronger levels of Actin are observed in the engrailed domain (J', K'). Rab5^DN domain of expression is enclosed in the yellow dashed line (J–L').

Figure 3. AS and Epidermal dynamics upon expression of Rab5^DN or Rab11^DN.

Still images from a time-lapse movie of a wild type embryo (A and Movie S1, D and Movie S4), an AS>Rab5^DN (B and Movie S2), an AS>Rab11^DN (C and Movie S6), an Epidermal>Rab5^DN (E and Movie S5), and an Epidermal>Rab11^DN (F and Movie S7) embryos, expressing ubi-DE-CadherinGFP, at different stages after the start of DC (indicated by the time stamp). Notice the irregular shapes of AS cells in B and C. No defects are observed in Epidermal>Rab5^DN embryos (E). Epidermal>Rab11^DN embryos exhibit a delay in the advancement of epidermal cells (F-0' arrows, HistoneYFP shows Rab11^DN domain of expression) and AS cells are very elongated in the D/V direction, in particular the ones facing the engrailed domain (F-60' yellow arrowheads). Ultimately the epidermis detaches from the AS (F-120', arrowhead). (A'–C') Measurement of the distance between the two opposing epithelia for a wild type (A'), an AS>Rab5^DN (B') and an AS>Rab11^DN (C') embryo. Closure is slower in AS>Rab5^DN embryos than in wild type ones (B'). Epidermal>Rab11^DN embryos exhibit a narrower AS (C') due to cell shape defects (see Fig. 5).

Figure 4. Rab11^DN expression impairs epidermal advancement and mislocalizes DE-Cadherin.

Confocal sections from Drosophila embryos expressing Rab11^DN immunostained with DE-Cadherin (A–Q, R–S') and phalloidin to visualize F-actin (A'–F'). Three different stages of DC are shown (A–F'). At early stages, AS cells expressing Rab11^DN do not exhibit wavy membranes as in the wild type (A). Expression of Rab11^DN does not impair epidermal cell elongation (B), but zippering is deffective (C). DE-Cadherin levels (A–C) and Actin distribution (A'–C') in the AS are not affected. Embryos expressing Rab11^DN in the engrailed domain show a delay in the advancement of the dorsal epidermal cells and are less elongated (D, arrowheads). Later embryos exhibit ectopic grooves in the Rab11^DN domain of expression (E) and zippering does not occur (F). Actin accumulates at the LE in the DME cells expressing Rab11^DN (E'). Epidermal cell elongation in Rab11^DN expressing cells and wild type cells (H-yellow dashed line outline Rab11^DN domain of expression) and quantification of cell elongation of DME cells in the D/V direction in wild type cells (green) and Rab11^DN expressing cells (red) in Epidermal > Rab11^DN embryos (G - Magnification from D). Cell elongation is significantly reduced in Rab11^DN expressing cells [p<0.01 (N = 234 and 145 (G)]. In the mutant cells the levels of DE-Cadherin are higher at the cell membrane and in the cytoplasm of epidermal cells (H - Magnification from D, gradient represents fluorescence intensity values). DE-Cadherin is dispersed along the A/P axis in epidermal cells where Rab11^DN is expressed (I-P). Projection of sections I-P (Q). Overexpression and reduction of DE-Cadherin in embryos expressing Rab11^DN in epidermal stripes (R-S'). Epidermal cells expressing Rab11^DN and CadherinGFP exhibit a delay in epidermal advancement and impaired cell elongation similar to embryos expressing Rab11^DN alone in epidermal stripes [R, R', G (N = 129 and 69)]. In shg^R64 mutants (reduction of DE-Cadherin levels) epidermal cells expressing Rab11^DN do not elongate and detach from the AS (S, red dashed line outlines the holes). The levels of DE-Cadherin at the cell membrane are higher in the region where Rab11^DN is expressed (R', S').

Figure 5. Quantification of AS apical membrane content, AS apical constriction and cell elongation.

(A) Schematic representation of the three parameters measured in this study: apical surface area, elongation and membrane content. (A') Examples of AS cells followed in embryos of the indicated genotypes expressing Ubi-DE-CadherinGFP. (B–D) Quantification of apical surface area (B), cell anisotropy (C) and membrane content (D) for 5 cells of a representative embryo of each of the indicated genotypes. (E–F) Automated analyses of apical cell areas of the whole AS tissue in embryos at the beginning of DC. Cell area is shown with a colour code (E) and the proportion of cells in each cell area class is depicted in the bar graphs using the same colour code. In Rab5^DN expressing embryos, cells are more evenly distributed among the different cell area classes when compared with wild type (F). In Rab11^DN expressing embryos, the cell area is mostly concentrated in two cell area classes with small areas (F).

Figure 6. Balance between endocytosis and recycling modulates cellular morphology.

(A-C) Confocal sections from Drosophila embryos expressing Rab5^DN and Rab11^DN simultaneously in the AS immunostained with an anti-DE-Cadherin antibody, showing cell shapes similar to the wild types. Still images from a time-lapse video of an embryo co-expressing Rab5^DN and Rab11^DN in the AS (D) and ubi-DE-CadherinGFP at indicated stages after the start of DC (Movie S8). (E) Measurement of the distance between the two opposing epithelia in these embryos. (F) Quantification of apical surface area, cell anisotropy and membrane content of 5 cells of 1 embryo over time. This embryo show features more similar to the wild type for the parameters analyzed, however zippering is defective and DC fails (D-240’). (G) Automated analysis of apical cell areas at the beginning of DC, as shown in Figure 5E–F. We found two different types of AS > [Rab11^DN + Rab5^DN] embryos. In G1, an example embryo is depicted in which values for cell areas, direction of elongation and gradient of cell areas are close to wild type embryos. In G2 type embryos cells are smaller, the gradient is not as defined and cell elongation is random. The proportion of cells in each of the cell area classes is very similar to wild type (G3). (H) Model of the balance between endocytosis and recycling in cell morphology. When Rab5 mediated endocytosis is blocked there is less removal of membrane, which results in more membrane in AS cells and when Rab11 function is impaired, the insertion of membrane through Rab11 endosomes is blocked, and the apical membrane content is reduced.

Figure 7. Different temporal requirements of endocytosis and recycling in the epidermis and AS.

In wild type embryos AS cells are initially wiggly, then they reduce their apical surface area by contracting in the D/V direction, and this leads to reduction of AS tissue area. Epidermal cells elongate in the D/V direction which results in tissue spreading. If endocytosis is blocked, in the AS there is an increase in the apical membrane content and defects in apical surface reduction, indicating that Rab5 is required in the AS to remove membrane. In the epidermis it seems that expression of Rab5^DN does not have effects on epidermal cell shape changes. When recycling is blocked, AS cells exhibit straight boundaries at the onset of DC suggesting that Rab11 is required initially to maintain the membrane content of AS cells. In addition, Rab11 is required for epidermal cell elongation, which together with the observation in the AS cells, suggests that Rab11 is required for membrane growth.