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First published online 20 April 2005
doi: 10.1242/dev.01829

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crossveinless-c is a RhoGAP required for actin reorganisation during morphogenesis

¹ Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
² Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK
³ School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
⁴ Facultad de Ciencias, Centro de Biologia Molecular, `Severo Ochoa', CSIC-UAM, Cantoblanco, Madrid 28049, Spain
⁵ Centro Andaluz de Biología del Desarrollo,Universidad Pablo de Olavide, Carretera de Utrera, Km 1, Sevilla 41013, Spain

View larger version (60K): [in a new window]   Fig. 1. New alleles of cv-c. (A) The wing of a wild-type adult fly has five longitudinal veins, and an anterior and posterior crossvein (ACV and PCV). (B) A wing from a cv-c1/cv-c1 homozygous fly. The PCV is completely absent (a small piece of vein tissue running parallel to the fifth longitudinal vein is often observed) and the ACV is detached from the fourth longitudinal vein. (C) A wing from a cv-c1/cv-cM62 fly exhibiting a PCV phenotype identical to cv-c1/cv-c1. (D) A wing in which the UAS-RhoGAP88C-dsRNA construct has been expressed throughout the developing wing disc using Bx-Gal4. The phenotypes produced are identical to cv-c1 wings; the PCV is significantly reduced and the ACV is detached from longitudinal vein 4.

View larger version (103K): [in a new window]   Fig. 4. Embryonic phenotypes of cv-c mutants. Cuticle preparations of late embryos viewed under phase contrast showing the head skeleton (A,B), the posterior spiracles (C,D) and the dorsal cuticle (E,F). Wild-type (A,C,E), cv-cM62 homozygote (B,D) and cv-c7 homozygote (F) embryos. (A) In wild-type embryos, the medial tooth occupies an internal position (arrowhead in A), whereas in 80% of cv-cM62 embryos (B) the medial tooth is located on the exterior (arrowhead); the mouth hooks, which would normally be in this position, are displaced laterally (mouth hooks in A and B are indicated with asterisks). (C) A wild-type embryo with normal posterior spiracle morphology. The Filzkörper (black arrowhead), a filter formed in the internal tube connecting the spiracle to the tracheal system, is located inside the dome-like stigmatophore (white arrowhead). (D) In 28% of cv-cM62 posterior spiracles analysed, the cells that form the Filzkörper do not invaginate and instead form a `lawn' on the exterior (black arrowhead). In 67% of posterior spiracles analysed, the Filzkörper cells invaginate but do so aberrantly such that the final Filzkörper is branched (white arrowhead). (E) A wild-type embryo with normal dorsal cuticle morphology. (F) A cv-c7 embryo showing puckering of the dorsal cuticle (arrowhead). (G,H) Stage 15 embryos stained with anti-SAS showing the latter stages of dorsal closure. (G) The leading edge cells of the lateral epidermis zip up along the dorsal midline (arrows) during stage 15 in wild-type embryos. (H) In cv-c mutant embryos, this process is delayed and less orderly. Arrows and arrowheads indicate the dorsal midline and regions of delayed closure. (I,J) Midgut morphology of stage 16 embryos visualised with the Fas3 antibody (which marks the visceral muscle overlying the midgut) in wild-type (I) or cv-cM62 (J) embryos. The anterior-most constriction (1) does not occur in cv-cM62 embryos, and the posterior-most constriction (3) is variably affected. (K-P) MpT morphology is disrupted in cv-c embryos. (K-N) MpT development visualised by staining with the Cut antibody, in wild-type (K,L) and cv-cM62 embryos (M,N). Defects in cv-cM62 embryos become obvious by stage 13 as the MpTs start their convergent extension movements (compare K with M). By stage 16, the wild-type MpTs have formed four long, thin tubules, positioned invariantly within the body cavity (L), whereas the cv-cM62 MpTs have coalesced into a large cyst-like ball (N). Anterior (arrowhead) and posterior (asterisk) tubules are marked where they can be distinguished. (O,P) Nitrogenous waste products are excreted as urates that precipitate to form uric acid crystals in the acidic environment of the tubule lumen; these can be visualised in stage 17 embryos using polarised light. (O) A wild-type embryo, with normal posterior tubule morphology. (P) A cv-cM62 homozygous embryo in which the MpTs have formed a cyst-like ball; urates are excreted into a large central lumen. (Q) A stage 16 cv-c mutant embryo in which wild-type cv-c has been expressed in the MpTs. The MpT phenotype is partially rescued. Embryos are shown from lateral (A-E,I,J,O,P) or dorsal (F-H,K-N,Q) perspectives.

View larger version (125K): [in a new window]   Fig. 5. Cellular basis of the cv-c phenotype. (A-D) Planar polarity of the dorsal epidermis is unaffected in cv-c embryos. In wild-type embryos, the Cno protein (red) is initially expressed around the entire cell cortex, but later clears from sites of apposition with the amnioserosa and refines to distinct puncta (arrowheads), where adjacent epidermal cells meet. This pattern is seen in cv-c embryos (B,D) as in wild type (A,C). (E-H) Apicobasal polarity in the MpTs is unaffected in cv-c embryos. Embryos in which UAS-CD8-GFP was driven in the MpTs to label the entire membrane (green) were also stained with Baz (red) to mark the apical membrane. Wild-type (E,F) and cv-c (G,H) at stages 13 and 16. In cv-c tubules, apicobasal polarity is established and maintained. (I,J) The adherens junctions appear unaffected in cv-c embryos. Wild-type (I) and cv-c (J) stage 14 embryos stained with Sas (green) and Ecad (red). The level of expression and the relative position of the two markers in cv-c MpTs is identical to wild type. Asterisk in J indicates the lumen. (K-P) The level and distribution of cortical F-actin is disrupted in the MpTs of cv-c embryos. (K) In wild-type stage 13 MpTs, F-actin is localised to both the lateral and, in particular, the apical cell cortices. (L,M) F-actin continues to accumulate at the lateral and apical cell cortices; at stage 14/15, it appears compact and in close apposition to the plasma membrane (inset in L). (N) In stage 13 cv-c mutant MpTs, F-actin fails to accumulate in the cortex, remaining diffuse in the cytoplasm. (O) In stage 14 cv-c mutant MpTs, F-actin remains perinuclear, showing little concentration in the subcortex (inset in O). (P) In a small number of cv-c mutant MpTs, the distal end of the tubule elongates (arrowhead); in these cases, cortical F-actin is similar to wild type (M) seen at stage 16.

View larger version (92K): [in a new window]   Fig. 6. Overexpression of cv-c produces long actin-filled cellular extensions. (A,B) Wild-type (A) and cv-cM62 (B) stage 16 embryos stained with anti-Cut (light brown) to label the MpT nuclei and 22C10 (dark brown) to label the tip cell. The tip cell is present and has a normal morphology in cv-cM62 homozygotes. (C,D) The tip cell stalk of a wild-type embryo is 7 µm (C) but increases dramatically in length in embryos in which UAS-cv-c has been expressed in the tubule using CtB-Gal4; in many cases, the length of the stalk exceeds 50 µm (D) (n=56). (E,F) Stage 16 embryos expressing UAS-GMA in the MpTs to label F-actin (green), either in the absence of cv-c overexpression (E) or when cv-c is overexpressed (F); the extended tip cell stalk is associated with F-actin. (G) CtB-Gal4>UAS-GMA;UAS-cv-c embryo co-stained for GFP (green) and the apical membrane marker Sas (red, overlap in yellow). The tip cell stalk does not stain for Sas, indicating that the extension is of basolateral membrane. (H) 22C10 staining of a stage 16 embryo in which a dominant-negative Rho1 construct (UAS-RhoN19) has been overexpressed in the tubules; the tip cell stalk is extended (n=22). In all figures, arrowheads indicate the extent of the tip cell. Scale bars: 10 µm.

View larger version (34K): [in a new window]   Fig. 2. cv-c encodes RhoGAP88C. (A) Genomic map depicting part of the 88B-88C interval of the third chromosome (centromere is towards the left). All known and predicted genes in the region are shown; those drawn above the line are transcribed left to right, whereas those below are transcribed right to left. Small nucleotide polymorphic (SNP) loci used for mapping are indicated with orange lollipops. The P-element inset l(3)06951 is represented by the blue triangle. Two transcription units without predicted translation products (green), are indicated by their EST clot numbers. (B) The Cv-c protein is 1017 amino acids in length and contains three evolutionarily conserved domains: the N-terminal sterile motif (SAM) (green), the GAP domain (blue) and the C-terminal START domain (orange). (C) Comparison between fly Cv-c and its homologues: human deleted in liver cancer 1 and 2 (DLC1 and DLC2); rat p122; mouse seriologically defined in colon cancer antigen 13 (SDCCA13); and worm gut-on-exterior interacting protein 1 (GEI-1). Percentage of amino acids that are identical in fly Cv-c and in each of the homologues is shown for the SAM (green), GAP (blue) and START (orange) domains. Molecular lesions for cv-cC524 [R369stop (black asterisk)], cv-c7 [R601Q] and cv-cM62 [Q666stop (blue asterisk)] are indicated.

View larger version (102K): [in a new window]   Fig. 3. Embryonic expression of cv-c. cv-c RNA in situ hybridisation in wild-type (A-E) or cv-cJ17 (F) embryos. (A) Expression is not detected in blastoderm embryos, indicating a lack of maternal contribution. (B) Expression in stage 8 embryos is seen in the head (arrow) and head mesoderm, at the tip of the cephalic furrow, in the amnioserosa (arrowhead), and within distinct regions of the hindgut and midgut primordia. (C) At stage 11, cv-c transcripts are detected at high levels in the tracheal pits and in the epidermal cells that will later form the leading edge cells during dorsal closure (arrowhead). (D) At stage 13, cv-c is expressed in many tissues, including the MpTs (arrowhead), the longitudinal visceral muscle (LVM) of the midgut (black arrow), lymph glands, peripheral nervous system and regions of the head. (E) At stage 16, the highest level of cv-c is in the gut visceral musculature (arrowhead). (F) cv-c transcripts in cv-cJ17 mutant embryos are detected in the LVM of the midgut but are either absent or considerably reduced in other tissues (stage 13 embryo shown). In this, and all subsequent figures, embryos are orientated with anterior towards the left.

View larger version (113K): [in a new window]   Fig. 7. Genetic interaction with Rho-family GTPases. (A-B') Stage 16 embryos stained with an antibody against Cut (dark brown) or ß-galactosidase (light brown). (A) Rho172R/CyOwg-LacZ; cv-cM62/cv-cM62 embryo exhibiting the typical cv-cM62 MpT phenotype. (A') Higher magnification of MpTs shown in A. (B) A much less severe MpT phenotype is seen in Rho172R/Rho172R; cv-cM62/cv-cM62 embryos (n=48), in which normal tubulogenesis often takes place. Arrowheads in B indicate elongated anterior MpTs. (B') Higher magnification of MpTs shown in B. (C-F) Cuticle preparations of late embryos. (C) Rac1J11,Rac2 mutant embryos show abnormal head involution (black arrowhead), germband retraction and strong dorsal closure phenotypes in 82% of the embryos (n=46). (D) In Rac1J11,Rac2,cv-cM62 mutant embryos, head involution (arrowhead), germband retraction and dorsal closure phenotypes are less severe; the dorsal closure phenotype is rescued in 37% of embryos (n=44). (E,F) cv-cM62 posterior spiracle phenotypes are enhanced in a Rac1J11,Rac2 background. (E) cv-cM62 homozygote showing variable penetrance of the posterior spiracle phenotype (arrowheads). (F) Rac1J11,Rac2,cv-cM62 showing complete penetrance of the posterior spiracle phenotype (arrowheads). In 71% of Rac1,Rac2,cv-cM62 embryos, posterior spiracle invagination fails and the spiracles remain on the exterior (compared with 28% in cv-cM62 embryos).

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