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

First published online 31 March 2004
doi: 10.1242/dev.01097

This Article Summary Full Text Full Text (PDF) 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 Szafranski, P. Articles by Goode, S. Search for Related Content PubMed PubMed Citation Articles by Szafranski, P. Articles by Goode, S.

A Fasciclin 2 morphogenetic switch organizes epithelial cell cluster polarity and motility

¹ Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
² Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
³ Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
⁴ Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (80K): [in a new window]   Fig. 3. BC motility assay. (A1-A3) Wild-type stage 9 egg chambers of increasing maturity. As the border cells (BCs, arrow) move to the oocyte, oocyte area increases relative to the rest of the egg chamber (B,C,E). This oocyte growth can thus be used as a clock to measure the rate of BC migration. The % oocyte area (=oocyte area divided by the total area of the egg chamber; 23%) was plotted against the % migration (migration distance divided by the path distance; i.e. 66%). The results of from 400 wild-type egg chambers are shown in E. For all genotypes: 0.75r0.88, P<0.001. (F) Graphs for wild-type, rosy and Fas2null BC clusters are shown. Delamination time is the point at which the BCs completely leave the epithelium (0% migration; green arrows). Migration time is the difference between the completion time (100% migration; red arrow) and the delamination time (green arrows). (D) BA3-Gal4 expression pattern. Slbo-Gal4 expression pattern is shown in Fig. 2D.

View larger version (80K): [in a new window]   Fig. 1. Fas2, Dlg and Slbo expression during oogenesis. (A) Transmembrane Fas2 binds to PDZ1 and PDZ2 domains of cortical Dlg. (B) Dlg is expressed in egg chamber germ cells (GCs) and follicle cells throughout oogenesis (g, germarium; s, stage of oogenesis; e, early; l, late). Border cell (BC) differentiation at stage 8 is marked by Slbo expression. At stage 9, the BCs migrate between the nurse cells (NCs) to the front edge of the oocyte (OO) (arrow). (C) Same egg chambers as (B). Fas2 is expressed in all follicle cells through stage 7 (see also D'). At stage 8, Fas2 expression is lost in anterior follicle cells that include the BCs (bracket), precisely at the time when the BCs differentiate. (D-D'') At stage 7, just preceding BC differentiation, Fas2 and Dlg are uniformly distributed around the circumference of the polar cells (PCs, inset). Fas2 and Dlg predominantly colocalize in all follicle cells (arrowheads), but at late stage 7, Fas2 starts becoming localized more apically (completes apical localization at stage 8, see E'), while Dlg remains uniformly distributed on the lateral membrane. (E-E''') Anterior of a stage 8 egg chamber. (E) When the BCs differentiate (Slbo expression), Dlg redistributes in the PCs toward the leading edge of the cluster (inset, arrows). (E') Fas2 expression is lost in stretch cells and BCs, but continues in PCs at the center of the cluster, where it becomes precisely colocalized with Dlg in a graded leading-to-trailing pattern (inset, arrows). BC extensions that express Dlg on their surface encapsulate the PCs at the leading edge of the cluster (E''', arrowheads). These extensions pioneer BCs invasion at stage 9 (F,F', arrow). Fas2 is expressed in PC, but not BC membranes (F', inset, arrows). (G) Fas2 and Dlg (inset) continue to be polarized in PCs toward the leading edge of the BCs as the cluster migrates (arrows). (H,I) Fas2 enhancer-trap expression during oogenesis. The Fas2 transcription pattern (H) resembles Fas2 protein pattern (C). At stage 9, Fas2 is expressed in PCs but not BCs (I).

View larger version (79K): [in a new window]   Fig. 2. Fas2 polarization and signaling. (A-A'') Wild-type BC cluster. Fas2, Dlg and Lgl are polarized to the leading half of the PCs (arrowheads; leading-to-trailing Fas2 polarity ratio: 8.6±1.6; see also Fig. 6A-A''). Fas2 is localized at the highest level in the interface between the PCs (A, arrow). (B-B'') Targeting Fas2 to BCs using Slbo-Gal4 (Slbo-Gal4 is not expressed in PCs; D) causes Fas2 to become localized around the circumference of the PCs (leading-to-trailing Fas2 polarity ratio: 1.1±0.2), and dramatically reduced at the interface between the PCs (arrow, compare with A). Dlg and Lgl colocalize in a similar pattern. (C) Targeting chimeric CD8-Fas2 to BCs does not change Fas2 polarity (leading-to-trailing polarity ratio: 6.1±2.0; anti-Fas2 antibody partially recognizes also CD8-Fas2). (E-E') Fas2 mosaic, in which one PC has no Fas2. The PCs and cluster are turned perpendicular to the normal orientation for initiating movement, with the Fas2+ PC oriented in the direction of migration (arrow in E', compare with A'). High Fas2 level between PCs is lost (E, arrow, compare with A). Leading-to-trailing Fas2 polarity ratio is 6.8. (E') Dlg has cortical localization in BCs that contact the Fas2+ PC, but is mislocalized in the BC in contact with the Fas2null PC (outline). Lgl shows the same distribution (inset, E), indicating that Fas2 signals maintenance and localization of Dlg and Lgl in BCs. (F-F'') Fas2 mosaic epithelium (at the time of BC delamination). Dlg and Lgl levels, and localization are similar in both Fas2+ and Fas2null follicle cells. In Fas2+ follicle cells, Fas2 is lost from the membranes that contact Fas2null cells (arrowheads), but remains at the site where they contact Fas2+ cells (arrows). (G) A late stage 7 Fas2 mosaic, in which loss of Fas2 from the presumptive BC epithelial cell (pBCs) causes loss of Fas2 from the membrane of the adjacent Fas2+ PC (arrowheads). (H) In an early stage 8 Fas2 mosaic, Fas2 is present in the PC membrane (arrowheads) adjacent to a Fas2null BC. This suggests the presence of a Fas2 receptor in stage 8 BCs (see text).

View larger version (72K): [in a new window]   Fig. 6. Localization of Fas2, Dlg and Lgl in mutant, misexpression and rescue clusters. The delamination and migration times are shown in Fig. 4. Polarity values are ratios of Fas2 at the leading half of PC to Fas2 at the trailing half of PC, and are mean±s.d. from on average 10 measurements. (A-A'') Fas2 colocalizes with Dlg and Lgl at the leading half of the PCs. Dlg and Lgl colocalize in the cortex of BCs. (B-B'') In Fas2rd1 clusters, Dlg and Lgl are mislocalized in BCs. In PCs, Fas2 remains polarized, and Dlg and Lgl colocalize in a similar pattern (arrowheads). (C-C'') Complete loss of Fas2 in Fas2null PCs causes Dlg and Lgl fragmentation and loss of polarity (arrowheads). As in Fas2rd1 clusters, Dlg and Lgl are mislocalized in BCs. Fas2 is polarized in dlghf/+ and lgl4/+ clusters that contain functional Dlg or Lgl reduced by half (D,F). Further reduction of Dlg in dlghf/dlglv55 clusters (E'), and Lgl in lgl4/lglts3 clusters (G'') results in the loss of Fas2 polarity (E,G). (H-H'') Fas2, Dlg and Lgl colocalize in a circumferential pattern in PCs in UAS-Fas2/+; BA3-Gal4/+ clusters. Fas2, Dlg and Lgl colocalize in polarized pattern in PCs in UAS-Fas23/+; BA3-Gal4/+ clusters (I-I''), and in UAS CD8-Fas2/+; BA3 Gal4/+ clusters (J-J''). (K) Fas2 polarity in Fas2rd1 PCs is restored when Fas2 is targeted to PCs. (L) Polarity of Fas2rd1 PCs is abolished when Dlg is targeted to BCs. In all cases, decrease of Fas2 polarity correlates with the delay of BC delamination. Arrowheads indicate the accumulation of Fas2, Dlg or Lgl.

View larger version (41K): [in a new window]   Fig. 4. Quantitative analysis of the role of Fas2, Dlg and Lgl in BC motility. Bar graphs show time of delamination (green) and migration (black) of BCs. Migration is faster when the level of Fas2, Dlg or Lgl is lower. These defects are rescued when Fas2 is targeted to PCs in Fas2 clusters using BA3-Gal4, or when Dlg is targeted to BCs in Fas2 or dlg clusters using Slbo-Gal4. Slower delamination of BCs correlates with the decrease of Fas2 polarity (see Fig. 6). Asterisks indicate statistically significant differences (General Linear Model analysis, P<0.05) from control (loss-of-function and misexpression experiments), or from Fas2 or dlg mutants (rescue experiments).

View larger version (146K): [in a new window]   Fig. 5. Misexpression of Fas2 and Fas23 in PCs. (A) Misexpression of Fas2 in PCs causes BCs to initiate migration behind the trailing edge of the epithelium (TE) (see Fig. 3A1-3 for wild-type migration). Delamination is significantly delayed, but the rate of migration is normal (Fig. 4). (B) Misexpression of Fas23, which is unable to interact with Dlg, does not affect time of delamination, though it accelerates migration by 10% (Fig. 4).

View larger version (50K): [in a new window]   Fig. 7. Polarity of the BC cluster and proposed mechanism by which Fas2, Dlg and Lgl control movement. (A-C') The polarity of the BC cluster is demonstrated by the high levels of Amph in trailing BCs (tBCs) compared with leading BCs (lBCs). Amph polarity is established just preceding BC differentiation (stage 7-8; pBCs, presumptive BCs). (D) Model of a delaminating BC cluster. At stage 7, Fas2 (orange), and Dlg and Lgl (blue) are localized around the circumference of PCs and on lateral membranes of presumptive BC epithelial cells. At stage 8, the BCs differentiate when Slbo turns on. Fas2 is lost from BCs, and other anterior epithelial cells, except PCs. Fas2 loss from BCs is crucial for Fas2 polarization to the leading half of the PCs (morphogenetic switch, Fig. 2B). Dlg and Lgl become localized around the circumference of the BCs. The PC Fas2-Dlg-Lgl complex acts through a putative BC receptor to maintain cortical organization of Dlg and Lgl in BCs, which is crucial for inhibiting rate of BC movement. Polarized communication between PCs and front BCs assures timely delamination of the BC cluster. The polarized nature of the cluster suggests that the work of the extension-retraction-contraction cycle found in single cells may be distributed between multiple cells in the migrating cluster and coordinated through Fas2 intercellular communication (1). Another possibility is that polarized Fas2 signaling is required to polarize front BCs in an active pattern similar to individual migrating cells, with the back border cells playing a passive role (2).