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

First published online 29 September 2004
doi: 10.1242/dev.01404

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 Kato, K. Articles by Hayashi, S. Search for Related Content PubMed PubMed Citation Articles by Kato, K. Articles by Hayashi, S.

Hedgehog and Decapentaplegic instruct polarized growth of cell extensions in the Drosophila trachea

Kagayaki Kato^1,2, Takahiro Chihara^2,* and Shigeo Hayashi^1,2,3,

¹ Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku Kobe 650-0047, Japan
² National Institute of Genetics and the Graduate School for Advanced Studies, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan
³ Department of Life Science, Kobe University Graduate School of Science and Technology, 1-1 Rokkodai-cho, Nada-ku, Kobe-shi, Hyogo, 657-8501, Japan

View larger version (77K): [in a new window]   Fig. 1. Extension of the dorsal branch (DB) is coupled to dorsal closure of the epidermis. (A-I) Images of time-lapse recordings. White arrowheads indicate the fusion point of the DB (A-C,I,J) or dorsal epidermis (D-F). Green arrowheads indicate the terminal branch. Anterior is leftwards (A-F). (A-C) Dorsal view of DB8 and DB9 labeled with btl>>gfp-moe (UAS-gfp-moe driven by btl-gal4) taken from Movie 1. (D-F) Simultaneous labeling of the trachea and the P compartment of the epidermis with gfp-moe, expressed by btl-gal4 and en-gal4. Tracheal cells are mostly hidden beneath the en stripe. Fusion cells are indicated by red dots. (G-I) Transverse optical sections obtained by y-z scanning of a btl>>gfp-TT-ras embryo. This combination of insertions caused fortuitous GFP expression in most epidermal cells. Trachea (green), epidermis (blue) and amnioserosa (red) were identified by their location and cell shape and were pseudo-colored. The contact point of the epidermis and the DB tip is indicated by an arrow. Dorsal is rightwards. (A,D,G) Time=0 minutes (early stage 15: the dorsal epidermis meets at the midline). The tip of the DB adheres to epidermal cells located two or three cells behind the leading edge (G), and follows dorsal closure prior to 0 minutes. (B,E,H) Time=60 minutes: contact of fusion cells. Fusion cells from opposing sides contact each other with numerous filopodia (B, white arrowhead). The terminal branch has begun ventral extension (B, green arrowhead). (C,F,I) Time=130 minutes (early stage 16): completion of branch fusion. Terminal branch extension is still in progress. (J) Dorsal (upper) and transverse (lower) views of a DB fusion point. The terminal branch follows the curved shape of the epidermis (green arrowheads), whereas the stalk does not associate closely with the epidermis (open arrowhead). (K) Schematic drawing of the DB. T, terminal cell; F, fusion cell.

View larger version (23K): [in a new window]   Fig. 2. Kinetics of terminal branch extension. (A) The tip of the DB is labeled with GFP-moesin (green) and Dlg (purple) labels the cell boundaries. The broken line indicates the signal of Dlg that accumulates in the boundary between the terminal cell (T) and the fusion cell (F). The junction between the fusion cell and terminal cell was identified by the high concentration of GFP-moe, and was designated as a reference point (red dot). The tips of filopodia are indicated by yellow crosses. (B) Time-lapse images of tracheal cells labeled with GFP-moe that were used for measurements. Elapsed time in minutes is indicated in the top right-hand corner of each image. In the 60 minute frame, the actin core in the terminal cell and stalk cell is indicated by a red arrowhead and arrow, respectively. (C) Plot of filopodial movement. The asterisk indicates an example of a terminal branch that transiently extended in the dorsal direction.

View larger version (68K): [in a new window]   Fig. 3. Relationship between the tip of the DB and the expression domains of dpp, en and bnl. Anterior is leftwards. Broken lines in A,B,D indicate the dorsal midline. Tracheal cells are labeled with lacZ marker of 1-eve-1 tracheal reporter (brown in A, green in B) or with GFP-moe driven by btl-gal4 (green in C-E). (A) Dorsal view of two consecutive segments in a stage 15 embryo double-labeled for bnl mRNA in the epidermis (purple dot) and trachea (brown, outlined in one segment). Strong expression of bnl mRNA overlaps the tip of the DB, and weaker expression extends ventrally. (B) Activated MAP kinase expression (red, arrowhead) detected in the terminal cell. Lateral view. (C) During stage 14, the tip of the DB (white arrowhead) has entered the en stripe from behind and localizes immediately ventral to dpp-lacZ expression. (D) dpp-lacZ expression in dorsal midline at the completion of dorsal closure. Late stage 15. (E) pMAD expression (purple) in the DB (green) of a stage 14 embryo (white arrowhead). A grayscale image of pMAD expression is shown on the right.

View larger version (97K): [in a new window]   Fig. 5. Hh signaling restricts terminal branching to the P compartment. Broken lines indicate the dorsal midline, and white arrowheads indicate the terminal branch. (A,E,H,K) Stage 16 embryos labeled with anti-EN (purple), with the trachea shown in green (GFP-moe). Anterior is leftwards. (B) Double-labeling of en-lacZ (purple) and the trachea (green; GFP-moe). The terminal branch runs along the anterior border of the en-lacZ stripe. (C,F,I) Images taken from Movies 1 (C), 3 (F) and 4 (I) showing migration patterns of the terminal branch. The length (in µm) of one terminal branch (colored purple) is indicated in C. (D,G,J) Expression of SRF (purple). (A-D) Wild-type embryos. Terminal branches are aligned along the AP compartment borders. In C, terminal cells are pseudo-colored with purple to highlight the shape of terminal branches. (E-G) When Hh signaling is hyperactivated by CiAct in the trachea, terminal branches are not confined within the P compartment and cell extensions pointing in the AP direction are observed (arrowhead). (H-J) Downregulation of Hh signaling in the trachea by CiR expression results in the formation of secondary terminal branches that extend outside the P compartment (red arrowheads). Compare this with terminal branches that formed first (white arrowheads). An example of a single terminal cell extending two terminal branch (colored green) is labeled with asterisk in J. (K,K') Hh expressed in a broad longitudinal band in the DE (using the ush-gal4 driver) caused terminal branch misguidance. Tracheal cells were visualized with btl-gfp-moe. (L) Expression pattern of ush;gfpNLS. (M-O) Expression of ptc-lacZ. Expression in terminal cells (yellow circle) was low in control embryos (M) but was elevated when CiAct was ectopically expressed in the trachea using btl-Gal4 (N) and was reduced by CiR (O). The yellow arrowhead indicates an epidermal cell expressing a high level of ptc-lacZ. Broken green line in M indicates the outline of the DB detected by GFP (for clarity, the GFP signal is not shown).

View larger version (73K): [in a new window]   Fig. 4. Preferential association of terminal cells with the P compartment. Ectopic formation of a terminal cell was induced by expression of the Drosophila homolog of Axin. (A) Duplicated terminal cells associated with the P compartment (EN, purple). (B) Frequency of terminal cells located in the EN stripe. (C) Relocation of terminal cells from the anterior to the P compartment (Movie 2). A terminal cell located in the A compartment (white arrowhead) elongated along the AP axis and had fewer filopodia than cells located in the P compartment (red arrowhead). This cell moved to the P compartment and started to extend a terminal branch dorsally.

View larger version (100K): [in a new window]   Fig. 6. DPP signaling inhibits terminal branch extension. (A) When a high level of Dpp signaling was maintained by expressing dpp, elongating terminal branches were misoriented and were stalled (white arrowheads). Compare this with the control terminal branch shown in Fig. 5C. Overexpression of Dpp promoted excess recruitment of tracheal cells into DB (Vincent et al., 1997), but the number of terminal cells expressing SRF remained one per DB (data not shown). (B-F) Premature reduction of Dpp signaling by overexpression of Dad causes misdirection of the terminal branch. (B) Time-lapse images of a terminal branch that crossed the dorsal midline (arrowhead). (C-F) Phenotypes of Dad expression in terminal branches were classified as either class 1 (C, normal), class 2 (D, drifting along the AP axis) or class 3 (E, midline crossing). Class 2 and 3 phenotypes included another subcategory, the class 4 phenotype (F, bipolar elongation). The asterisk indicates that this phenotype is a subset included within the class 2 and 3 phenotypes. A total of 103 terminal branches that reached dorsal midline (broken line) were scored.

View larger version (29K): [in a new window]   Fig. 7. A model of the terminal branch guidance mechanism. Hh is expressed in the P compartment and forms a concentration gradient that diminishes toward the anterior and posterior directions. Terminal cells adhere to the basal surface of the epidermis and receive Hh, which promotes cell adhesiveness and thereby maintains terminal cell localization at the top of the gradient. Dpp, which is expressed at the dorsal edge of the epidermis, acts as a barrier against terminal branching to ensure monopolar growth.