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First published online 4 October 2006
doi: 10.1242/dev.02588

Development 133, 4257-4267 (2006)
Published by The Company of Biologists 2006
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Compartmentalisation of Rho regulators directs cell invagination during tissue morphogenesis

Sérgio Simões1,2, Barry Denholm3, Dulce Azevedo1, Sol Sotillos4, Paul Martin5, Helen Skaer3, James Castelli-Gair Hombría4 and António Jacinto1,2,*

1 Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Portugal.
2 Instituto Gulbenkian de Ciência, Oeiras, Portugal.
3 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.
4 Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Sevilla, Spain.
5 Department of Physiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.


Figure 1
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  		Fig. 1. Posterior spiracle cell invagination involves apical constriction and basolateral membrane elongation. (A) Stills of a movie showing the A8 segment (bracket) of a DE-Cad-GFP-expressing embryo. At stage 11 (t=0 minutes), spiracular chamber cells (dashed line) are superficial and localise posteriorly to the A8 tracheal pit (arrow). Later, notice apical constriction and inward cell movement to form a lumen (arrow). (B) Spiracle cells are expressing {alpha}-Catenin-GFP (green), using the ems-GAL4 driver (spiracle-specific); Armadillo (ß-Catenin) is in red. All images are top views. (i,ii) Initial lumen formation (stage 11) involves rearrangement of the most anterior cells (arrowheads) around the tracheal opening (arrow). (iii) the more posterior cells (left from white outline) constrict apically and invaginate later (iv), to form the complete chamber (v). (C) Stills of a live embryo expressing GFP-Actin in the spiracle invaginating cells. t=0 minutes corresponds to end of stage 12. Notice apical enrichment of GFP-Actin and elongation of the basolateral cell domain, with the cell nuclei (black) remaining basally localised. (D) Phalloidin staining (red) in the spiracular chamber cells expressing GFP (green). Notice the accumulation of F-Actin at the apical/luminal side of the spiracular chamber cells (lateral view). (E) Diagram showing invagination of the posterior spiracle cells (lateral view). The cells belonging to the spiracle primordium (red and green) constrict apically (i,ii); the more anterior cells (red) rearrange around the A8 tracheal dorsal branch (DB) (iii) and invaginate first, occupying deeper positions in the spiracle. These are followed by the most posterior cells (green), which then occupy more superficial positions. The invaginated cells form a multicellular tube (spiracular chamber) in continuity with the tracheal dorsal trunk. During invagination, cells also elongate their basolateral membrane, acquiring a bottle shape (iv). Anterior is to the left in all panels; note that in the extended germ band stage (stage 11) the anterior cells are on the right side because the embryo is folded. Scale bars: 20 µm in A; 10 µm in B-D. DT, tracheal dorsal trunk; SC, spiracular chamber.

 

Figure 2
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  		Fig. 2. Myosin II is apically enriched and essential for spiracle invagination. (A) Myosin II distribution is visualised in the spiracular chamber cells using embryos expressing Myosin II regulatory light chain (Spaghetti squash) fused to GFP (Sqh-GFP). Discs large (Dlg) (red) labels the basolateral membrane at stage 11/12 and only the lateral membrane at stage 13. The images are sagittal views and brackets indicate the position of the invaginating spiracle cells. Apical is up. (B) Spiracular chamber defects in sqh1GLC embryos (i,ii) compared with wild type; spiracular chamber cells are labelled with GFP-Actin, using the ems-GAL4 driver. In i, spiracle cells remain on the surface of the embryo; the arrowhead indicates two spiracle cells detached from the main cluster; notice that some cells fail apical constriction (cell labelled with bracket in i', which is a magnification of the rectangle in i), as opposed to other cells with a wedge shape and high accumulation of apical Actin (e.g. cell labelled with an asterisk). (ii) The arrow indicates the disrupted pattern of apical Actin in the mild class of sqh1GLC spiracles. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Rho1 is essential for spiracle invagination. (A) Wild-type Filzkörpers. (B) Cuticle of a null mutant for Rho1 (Rho11B) showing irregular Filzkörpers (arrows). (C) Severe class of Rho11B embryos, with one uninvaginated Filzkörper. (D) GFP-Actin (green) distribution in the spiracular chamber of wild type and Rho11B/Rho172R mutants (stage 17); red, Filzkörper autofluorescence obtained with the 488 nm laser. In wild-type spiracles, a continuous line of Actin surrounds the Filzkörper, while in late Rho1 mutants this pattern is partially lost. (E,F) Expression of the dominant negative form of Rho1, RhoN19, impairs Filzkörper secretion (E) and the invagination of spiracle cells (F, arrow); green, GFP-Actin. (G,H) Wild-type spiracle and a spiracle in which RhoN19 has been expressed visualised with GFP-Actin. Expression of RhoN19 disrupts the accumulation of apical Actin (arrowhead), while elongation of the basolateral membrane is still observed (arrow). Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. Rho1 activity is apically restricted during spiracular chamber formation. (A) Immunofluorescence against Rho1 protein showing its apical accumulation around the lumen of the spiracular chamber (arrows); (i) dorsal view and (ii) lateral view. (ii) The PKNG58AeGFP probe overlaps with apical Rho1, reflecting the local activation of this RhoGTPase. (B) Rho1 activity during formation of the spiracular chamber. Spiracle cells co-expressing PKNG58AeGFP and mRFP-Actin. early st11 - low levels of PKNG58AeGFP are detected throughout the spiracle primodium. The asterisk indicates the A8 tracheal pit position. Anterior is to the right; late st11 - the onset of Rho1 activity is detected in the first invaginating cells (arrow) localised posteriorly to the last tracheal pit. Notice also the higher accumulation of mRFP-Actin in these cells; b.c. - single invaginating bottle-shaped cell showing apical activation of Rho1 (arrowhead), which overlaps with apical accumulation of mRFP-Actin (apical is up); stage 13 and stage 17 - transverse and lateral views, respectively, of spiracular chambers showing accumulation of Rho1-GTP and mRFP-Actin at the luminal/apical surface (arrows). (C) Ectopic Rho1 activation blocks basolateral elongation and impairs cell invagination. The left panel shows active Rho1 (PKNG58AeGFP fluorescence) around the lumen of a wild-type spiracle (lateral view, stage 14); the dashed line outlines a single invaginated cell. The two middle panels represent spiracle cells co-expressing RhoV14 and PKNG58AeGFP (i) or GFP-Actin (ii). The right panel (iii) shows the Filzkorper defects (arrows) caused by the expression of RhoV14 (cuticle). In i, ectopic Rho1 activation is detectable on the cell membranes, as opposed to the apically restricted pattern in the wild type (same confocal settings as the WT control). ii shows a cluster of three spiracle cells with a mini-bottle shape, due to inhibition of basolateral elongation (compare with wild-type control, Fig. 3G). Scale bars: 10 µm, except B b.c, 3 µm.

 

Figure 5
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  		Fig. 5. RhoGEF64C is a positive regulator of Rho1 and its mRNA and protein are apically localised. (A) In situ hybridisation for RhoGEF64C showing expression in the posterior spiracle primordium (black arrows) and hindgut (white arrowhead), during retraction of the germ band. (B) Staining for the apical RhoGEF64C (red) in GFP-Actin expressing spiracle cells (stage 15). (C,D) RhoGEF64C mRNA is apically localised, surrounding the lumen of the posterior spiracles (C, lateral view) and hindgut (D, dorsal view) (stage 15). (E) Deletion of the Dbl plus PH domain (GEF64C{Delta}Dbl) abrogates apical localisation of RhoGEF64C mRNA in the posterior spiracles, as opposed to truncations of the UTR regions (GEF64C FL ({Delta}5'UTR) and GEF64C {Delta}5'3'UTR); CDS - coding sequence. Dorsal views. (F) Expression of RhoGEF64C FL rescues the RhoN19-induced phenotype, as opposed to the truncated form RhoGEF64C{Delta}Dbl. (G) RhoGEF64C RNAi downregulates the expression of this gene, as assessed by in situ hybridisation. Notice the formation of irregular Filzkörpers with partially disrupted cortical Actin. Green, GFP-Actin. Scale bars: 50 µm in A; 10 µm in B-D,F,G; and 20 µm in E.

 

Figure 6
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  		Fig. 6. RhoGAP Cv-c is localised basolaterally and controls Rho1 activity. (A) Cuticles and distribution of GFP-Actin (green) in the spiracles of wild-type and cv-c7 mutant embryos. Notice the partially uninvaginated Filzkörpers in cv-c7 mutants (arrows) accompanied by disruption of the apical Actin (inset). (B) Expression of Venus-Cv-c (green) in spiracle cells using the ems-GAL4 driver, co-stained with the basolateral marker {alpha}-Spectrin (red) (i-iii) and with RhoGEF2 (red) (i'-iii'). (C) Cv-c gain of function (using emsGAL4 and UAS-Cv-c) leads to invagination failure of the most distal cells of the spiracular chamber (arrowheads) correlating with a disruption of their apical Actin (arrow). Green, GFP-Actin; red, Armadillo. (D) PKNG58AeGFP (i) and mRFP-Actin (i') profiles in spiracles overexpressing RhoGAP Cv-c. The cell cluster on the right (bracket) failed invagination and shows weaker apical Rho1 activity (yellow arrowhead) than the remaining invaginated cells (white arrowhead). (E) PKNG58AeGFP expression in a cv-c7 mutant spiracle with a severe phenotype. Apical and basal sections (dorsal view) and probe distribution along the xz axis. Notice the apical restriction of active Rho1, non-uniformly associated with the apical junctions. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. Model for Rho1 activity during spiracle cell invagination and tube formation. Rho1 is exclusively active at the apical domain of the invaginating cells that form the spiracular chamber, by the action of RhoGEF2 and RhoGEF64C. At the apical side Rho1-GTP promotes Myosin II and F-Actin assembly/activity, being essential for correct cell invagination and lumen maintenance. Rho1 function is excluded from the basolateral domain both by the absence of RhoGEF activity and by the presence of the RhoGAP Cv-c. Inactivation of Rho1 at the basolateral domain is also required to maintain the steady state levels of apical Rho1-GTP.

 




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