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First published online 6 July 2005
doi: 10.1242/dev.01926

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Tec29 controls actin remodeling and endoreplication during invagination of the Drosophila embryonic salivary glands

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA

View larger version (93K): [in a new window]   Fig. 1. Tec29 is expressed in embryonic salivary gland and has a salivary gland phenotype. In all the figures, anterior is towards the left. (A-D) Tec29 mRNA is expressed in the salivary placodes at stage 11 and in the invaginating salivary glands (arrowhead). Tec29 is also expressed in the salivary ducts from stage 12 onwards (D, arrow). (E,F) The shorter isoform of Tec29, type 1, is expressed in the salivary primordium (E), while the longer isoform, type 2, is expressed only in the nervous system (F). (G,H) Immunostaining for TEC29 protein shows that it is expressed in the salivary placodes and is apically localized in the invaginating gland (white arrowhead). (I-L) Immunostaining with anti-CREB antibody showed that multiple alleles of Tec29, Tec29K00206, Tec29K00206/Tec29K05610 and Tec29e482 have long salivary glands compared with w1118 embryos (I). (M,N) The salivary ducts in Tec29K00206, as visualized by dead ringer (dri), do not go through normal morphogenesis (arrows). (O,P) Double labeling for FKH and EN show that the salivary placodes at stage 11 are normal and respect their AP boundary in Tec29K00206 embryos.

View larger version (31K): [in a new window]   Fig. 2. Tec29 embryos show delays in invagination of the salivary placodes. (A) Changes in the length of the salivary gland (µm) as the placode cells invaginate during stage 12 of embryogenesis and at stages 13 through 16. Progression through stage 12 was monitored by the position of the tip of the germ band (as percent embryo length measured from the posterior end of the embryo). The bars show the mean length of the salivary glands in embryos whose germ band tip lies in the indicated intervals. Seventy-five wild-type salivary glands and 46 Tec29K00206 salivary glands from embryos at different stages were measured. As the germ band has finished retraction by the end of stage 12, the later stages are indicated only by stage numbers, not position. In w1118 embryos (blue bars), the length of the salivary glands gradually increases during stage 12 and then decreases slightly at stages 13-16. In Tec29K00206 embryos (red bars), there is no increase in length of gland observed at mid stage 12 (*); however, the length of the gland increases and exceeds w1118 at stage 15 (**). (B) This graph shows the area of the salivary placodes on the surface measured in the same embryos used for measurement in A. Although there is a decrease in the surface area of the placodes in w1118 embryos (blue bars), in Tec29K00206 embryos (red bars) the area of the placodes does not decrease such that at stage 13-14, one-third of the placode is still on the surface. Uninvaginated cells remain on the surface of Tec29K00206 embryos at stage 15-16; however, these cells have moved more anteriorly in the head and are hard to quantitate.

View larger version (97K): [in a new window]   Fig. 3. The salivary glands at stage 15 are stretched in Tec29 mutant embryos. (A-D) SCRIBBLE and FKH staining in the salivary glands show that the cells are isodiametric in w1118 but are stretched along the axis of the glands in Tec29K00206 embryos (arrowheads). (E,F) Staining with CRUMBS and FKH also shows that the proximal cells of the salivary glands are stretched in Tec29 embryos (arrows). Because the salivary gland cells are wedge shaped with their apical surface facing the lumen, CRUMBS, being more apical, produces a smaller outline of the cells than does SCRIBBLE.

View larger version (144K): [in a new window]   Fig. 4. Actin is disorganized in the salivary placodes of Tec29K00206 embryos. All the panels show optical sections of embryos focused on the apical surface of the salivary placodes. In all the panels, the circle outlines the salivary placodes. (A,B) The apical staining for -spectrin looks normal in Tec29K00206 embryos. (C,D) Actin disorganization on the apical surface of the cells in Tec29K00206 embryos is evident at early stage 12 when the embryos are stained with a monoclonal antibody against ACTIN, which detects both G-actin and F-actin.

View larger version (97K): [in a new window]   Fig. 5. Tec29 phenotype in the salivary glands is enhanced by chickadee and suppressed by twinstar. (A-C) More cells in the salivary glands of Tec29K00206 chic221 double mutants (B) are on the surface at stage 14 compared with Tec29K00206 alone (A). However, Tec29K00206 tsrK05633 double mutants can rescue the Tec29K00206 phenotype in the salivary glands (C). (D-F) The actin disorganization in Tec29K00206 embryos is increased by chic221 and rescued by tsrK05633 (circle). (G) The graphical representation of the enhancement and suppression by chic221 and tsrK05633, respectively. At stage 14, a third of the salivary placodes is left on the surface in Tec29K00206 embryos (759±36 µm2, n=30), whereas it is almost doubled in Tec29K00206chic221 embryos (1304±45 µm2, n=30) as well as in Tec29K00206Src42AE1 embryos (1278±43 µm2, n=30) and it is halved in Tec29K00206tsrK05633 (330±59 µm2, n=30).

View larger version (59K): [in a new window]   Fig. 6. Tec29 is necessary to delay endoreplication in the salivary glands. There are more BrdU-labeled nuclei in the invaginated gland labeled with FKH antibody (green) at early stage 12 in Tec29K00206 compared with w1118 (A,B, arrowhead) and also ventrally in the salivary placodes at mid stage 12 (arrowhead, compare E with F). In addition, there are more BrdU labeled nuclei in Tec29K00206chic221 embryos compared with Tec29K00206 alone at early stage 12 (C) and at mid stage 12 (G). BrdU labeling in Tec29K00206tsrK05633 is similar to Tec29K00206 embryos (D,H). (I,J) Delaying endoreplication by driving UAS-CycE using prd-GAL4 in Tec29K00206 embryos rescues the long salivary gland phenotype of Tec29K00206 embryos (arrows).

View larger version (67K): [in a new window]   Fig. 7. Tec29 interacts with Src42 and Src64 in the salivary glands. (A-D) The invagination defects in Tec29K00206 embryos (A) are enhanced by Src42AE1 (B). In addition, phenotypes of both homozygous and heterozygous Tec29K00206 embryos are enhanced by Src64PI (C,D, arrow). (E-H) Disorganization of actin observed in Tec29K00206 Src42AE1 double mutants is not obviously more severe than Tec29K00206 alone (E,F, circle); however, there are more cells labeled with BrdU in the Tec29K00206Src42AE1 compared with Tec29K00206 (arrowhead, compare H with G).

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