QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 January 2007
doi: 10.1242/dev.02748

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fox, D. T. Articles by Peifer, M. Search for Related Content PubMed PubMed Citation Articles by Fox, D. T. Articles by Peifer, M.

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

¹ Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.

View larger version (91K): [in this window] [in a new window]   Fig. 1. Abl is required for coordinated apical constriction in the VF. Embryos, genotypes and antigens are indicated. (A-D') Germband extension. (A,B) Dorsal view, anterior left; arrows indicate the ventral midline, bracket indicates the germband. (C-D') Living embryos. Moesin::GFP prior to (C,D) and after (C',D') cell intercalation. White dots highlight the rearrangement of cell contacts. (E-H) Grazing-sections, ventral. (E,F) Apical view of the VF at early gastrulation. (E) Arrow indicates uniform constriction in wild type. (F) abl mutant. Constricted (arrow) and unconstricted (arrowhead) cells are shown. (G,H) Subapical view at mid-gastrulation. (G) Arrow indicates lateral mesoderm orienting parallel to the VF. (H) abl mutant. Lateral mesoderm are pulled towards the VF at varying angles. (I-L) VF cross-sections. (I,J) Early gastrulation. (I) Wild type. Central cells constrict and point towards the furrow. Arrow indicates the outermost cells stretch over their neighbors. (J) abl mutant. Arrows indicate constricted cells; arrowhead indicates an unconstricted cell. (K,L) Late gastrulation. (K) Wild type. Arrow indicates wedge-shaped constricted cells; arrowhead indicates unconstricted lateral cells. (L) abl mutant. (M,N) Cad (red) and Twist (green) expression in wild-type (M) and abl-mutant (N) embryos in the late VF. (O-P') Midline morphogenesis. (Q,R) Stills of living embryos expressing Moesin::GFP. (Q) Wild-type central cells constrict (t=10 minutes, arrow); lateral cells internalize without constriction (t=15 minutes, arrow). (R) abl mutant. At t=0 minutes, ectopic F-actin before VF formation (arrow) can be seen. At t=10 minutes, constricting cells (arrows) are interspersed with unconstricted cells (arrowhead). Inset in Q,R shows asymmetry in cell shape. At t=15, 20 and 25 minutes, mesodermal cells remain at the surface (arrows). Cad, DE-Cadherin; Nrt, Neurotactin; Sim, Single-minded; Moe::GFP, Moesin::GFP. Scale bar: 20 µm.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Abl localization and activity. Wild-type or Abl::GFP embryos are shown. Antigens are indicated. (A) abl::GFP fusion construct. (B-D) Syncytial blastoderm. (B) Grazing-section. (C,D) Cross-sections showing apical Abl::GFP (C, arrow) and apical phosphorylated Abl (P-Abl; D, arrow). (E-H'') Cellularization. (E) Grazing-section. (F-H'') Cross-sections. (F) Abl::GFP localizes apically throughout (arrow). Nrt marks basolateral membranes. (G) Mid-cellularization; Abl::GFP is at high levels apically (arrow) and is at low levels in furrow canals (arrowhead). (H-H'') Late cellularization. Apical Abl::GFP (arrow) partially overlaps AJs (Cad). (I-J) Grazing-sections of the VF. Abl::GFP is elevated in the VF (arrows), overlapping constricting AJs (Cad). (J) P-Abl is elevated in the VF (arrow). (K-N') Cross-sections of the VF. Abl::GFP (K,L) and P-Abl (M,N) concentrate in mesoderm in the early (K-K') and late (L-L') VF (arrows), and persist apically in non-mesoderm tissue (arrowheads; L,N). (O) Apical Abl::GFP in the posterior midgut (arrow). (P) Basal Abl::GFP localization (arrow) in the late VF. (Q,R) Germband-extension. Abl::GFP (Q) and P-Abl (R) at the cell cortex. (S-S'') Abl::GFP (S, green in S'') overlaps ß-integrin (S', red in S'') in ectoderm (arrows). P-Abl, Phospho-Abl; ß-Int, ß-PS-Integrin. Scale bar: 20 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 3. Myosin and RhoGEF2 fail to uniformly assemble a contractile network in abl mutants. Embryos, antigens and genotypes are indicated. (A-F'') Cross-sections. (A-D) Low magnification. (A,B) Myosin in the basal furrow canals (arrows) in wild type (A) and abl mutants (B) at late cellularization. (C,D) Mesoderm in the early VF. Myosin disappears basally (arrowheads) and accumulates apically (arrows) in wild type (C) and abl mutants (D). (E-F'') Close-ups of the VF. (G-H'') Grazing-sections. (E-E'',G-G'') Myosin accumulates apically in wild-type constricting cells (arrow). (F-F'',H-H') Myosin concentrates in constricting cells (arrows) in abl mutants, but is diffuse in unconstricting cells (arrowheads). (I-N'') Cross-sections taken at late cellularization-gastrulation onset. (I-K) Wild type. RhoGEF2 is lost basally in mesoderm (I, arrow vs arrowhead), and then accumulates apically (J,K, arrows). (L) abl mutant. RhoGEF2 accumulates apically (arrow). (M-N'') Cross-sections of the VF. (O,P) Grazing-sections of the VF. (M,O) In wild-type embryos, RhoGEF2 localizes strongly to all cells (arrows). (N,P) In abl mutants, RhoGEF2 localizes apically in constricting cells (arrows), but is diffuse in non-constricting cells (arrowheads). (Q,R) Cross-sections of the VF showing the expression of P-Myo by constant-level imaging. Similar levels of Myo-P (P-Myo) were observed at apical constriction sites (arrows) in wild type (Q) versus abl mutants (R). Myo, Myosin; GEF, RhoGEF2; Arm, Armadillo; P-Myo, Phospho-Myosin. Scale bar: 20 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 4. cta mutants resemble abl mutants. Embryos, antigens and genotypes are indicated. (A-F) VFs from cta mutants. (A,B,E,F) Grazing-sections, (C,D,G-J') Cross-sections. (A,C) Constricted (arrows) and unconstricted (arrowheads) cells at early gastrulation. (B,D) Late gastrulation. (B, arrow) Mesoderm internalized at varying angles. (D, arrow) Asymmetric cell shape. (E,F) During furrow ingression, apical myosin and RhoGEF2 are concentrated in constricting VF cells (arrows) and are diffusely localized elsewhere (arrowheads). (G-J') Myosin and RhoGEF2 levels are comparable at sites of apical constriction (arrows) in wild type (G,I) and cta mutants (H,J), but, in the mutants, they are not concentrated in unconstricted cells (arrowheads). Scale bar: 20 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 5. In S2 cells, Abl promotes actin accumulation while Cta promotes myosin accumulation. S2 cells are shown, and the antigens, transfections and RNAi treatments are indicated. (A) UAS-Abl::GFP fusion construct. (B-E''') Transfected cells. Arrows indicate UAS-Abl overexpressing cells in B,C; UAS-activated Cta in D; and UAS-activated Rho in E. Arrowheads indicate untransfected controls. Abl-transfected cells display elevated phosphorylated Tyr (P-Tyr; B') and concentrated actin, but show no change in Myo-P (P-Myo) expression (C). (D) Cta-transfected cells display concentrated P-Myo, but not actin. (E) Rho-transfected cells exhibit concentrated actin and P-Myo, which are organized into a central ring. (F-H') abl RNAi does not block Rho gain-of-function. (F) Control RNAi. (G,G') Control RNAi+active Rho. Notice increased Rho and concentrated actin relative to F,F'. (H,H') abl RNAi+active Rho. Rho and actin localization is similar to active Rho alone (G,G'). (I) Immunoblot showing knockdown of the Abl protein from the S2 cells shown in G-H'. Samples were non-adjacent on the same gel. Pnut, loading control; PTyr, Phospho-Tyrosine. Scale bar: 30 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 6. Actin localization is altered in abl and RhoGEF2, but not in cta, mutants. Embryos and antigens are indicated. (A-D) Wild type; (E-H) abl mutant; (I-L) cta mutant; (M-P) RhoGEF2 mutant. (A,E,I,M) Late cellularization. Actin is localized basally in all genotypes (arrows), but also accumulates apically (arrowheads) in abl (E) and RhoGEF2 (M) mutants. Ena is diffusely localized in wild-type (A'), and in cta (I') and RhoGEF2 (M') mutants, but accumulates apically in abl mutants (E', arrowheads). (B,F,J,N) VF initiation. Ectopic apicolateral actin localization (arrowheads) occurs in abl (F) and RhoGEF2 (N) mutants, but not in wild-type (B) or cta mutants (J). (F) Red arrowhead shows accumulated actin in constricting cells. (C,G,K,O) Close-up of the VF. (C) Wild-type. Actin is at apical AJs in most cells (white arrowhead). In the mesoderm, actin accumulates apically in constricting cells (yellow arrowhead), and disappears basally (arrow). (G) abl mutant. Ectopic actin in apicallyconstricting cells (yellow arrowhead) and non-constricting cells (white arrowhead). Arrow indicates the disappearance of normal basal actin. (K) cta mutant. Actin localization is normal (arrow, arrowheads as in C). (O) RhoGEF2 mutant. Actin defects resemble those in abl mutants. Arrows and arrowheads are as in G. (D,H,L,P) Close-up of the VF. Arrows indicate non-mesodermal Ena. (D) Wild type. Ena concentrates at AJs in non-mesodermal cells, but remains diffuse in mesodermal cells (arrowhead). (H) abl mutant. Ena accumulates apicolaterally in non-mesodermal (arrow) and mesodermal (arrowhead) cells. (L,P) Neither cta (L) nor RhoGEF2 (P) mutants accumulate ectopic mesodermal Ena (arrowhead). (Q-R'') RhoGEF2 mutant at late gastrulation. In non-mesodermal cells, excess apical actin colocalizes with Ena in ectopic structures (arrows). (R) Close-up of Q. (S,T) ena heterozygosity suppresses the VF phenotype of abl mutants. (S) abl mutant. (T) ena/+;abl embryo. Scale bar: 20 µm.

View larger version (103K): [in this window] [in a new window]   Fig. 7. cta;abl double mutants resemble RhoGEF2 mutants. Moesin::GFP. (A) Wild type; (B) abl mutant; (C) cta mutant. (A-C) Mesoderm is internalized and midline cells meet (arrows). (D) RhoGEF2 mutant; (E,F) cta;abl double mutant. Mesoderm is not internalized (arrowhead) and midline cells do not meet (arrows). Scale bar: 20 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 8. A mechanistic model of ventral furrow formation.