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A molecular analysis of ascidian metamorphosis reveals activation of an innate immune response

¹ Box 351800, Zoology Department and Center for Developmental Biology, University of Washington, Seattle, WA 98195-1800, USA
² Friday Harbor Laboratories, 620 University Road, University of Washington, Friday Harbor, WA 98250-9299, USA

View larger version (44K): [in a new window]   Fig. 1. Ascidian metamorphosis (Cloney, 1990; Hirano and Nishida, 1997; Satoh, 1994). (A) Pre-competent ascidian larvae, showing the chordate dorsal nerve cord and notochord. The three cell lineages within the trunk mesenchyme form distinct mesodermal structures, as in B. (B) An adult ascidian showing the chordate endostyle and pharyngeal slits. The trunk lateral cells form structures (colored in red) including the blood cells, parts of the body wall musculature and the pharyngeal gill slit endothelia. The mesenchyme cells (colored green) migrate into the tunic. The trunk ventral cells form structures (colored in blue) including the heart and parts of the body wall musculature. (C-D) Upon detection of the appropriate settlement cue, competent larvae adhere to the substrate through adhesives secreted by the papillae. (E) The papillae retract, pulling the larval head against the substrate. (F) Over the next 20 minutes the tail is resorbed. (G) Within an hour, the outer larval tunic is molted. (H,I) Close up diagrams of the newly settled juvenile during the first few hours of metamorphosis. During this time (i) the cerebral vesicle is resorbed; (ii) the viscera rotates 90 degrees; (iii) ampullae extend from the anterior epidermis, pushing the juvenile tunic along the substrate; and (iv) there is extensive migration of trunk mesenchyme cells both within the body, across the epidermis into the tunic and through a tube to the exterior.

View larger version (101K): [in a new window]   Fig. 2. Temporal expression of immune-related transcripts visualized through RT-PCR analysis. Results for nine transcripts over 10 developmental stages are shown. Stages include four embryonic stages (in hours after fertilization), two larval stages (pre-competent larvae two hours after hatching and competent larvae 10 hours after hatching), and four juvenile stages (time after settlement). The 16S mtl rRNA bands at the bottom were used as a loading control. Transcripts are grouped according to the screens by which they were isolated. The title of each screen is labeled above the relevant set of transcripts and the timing of the subtractive hybridization is indicated by an arrow.

View larger version (102K): [in a new window]   Fig. 3. Expression patterns of isolated transcripts visualized through in situ hybridization. Results for five immune-related transcripts and one control transcript over the four developmental stages included in our screens are displayed. Transcripts are identified to the left of each row of images displaying their expression pattern over time. (A-E) Pre-competent larva, 1-2 hours after hatching. (F-U) Competent larvae, 10-12 hours after hatching. For all larval pictures we have chosen to display only the head, because this allows the details of expression to be discerned and there was no significant expression in the tail region for any of the genes examined. (K-V) Juveniles 1 hour after settlement. (P-W) Juveniles two days after settlement.

View larger version (105K): [in a new window]   Fig. 4. Close up views of expression patterns. (A) Bv-Crn (Coronin) expression in a pre-competent larva. Note expression in the mesenchymal cells at the base of the larval trunk. (B) Bv-Crn expression in a competent larva. Note expression in scattered mesenchyme cells that have migrated anteriorly along the trunk; also note the lack of strong staining in the papillae region, characteristic of the immune-related transcripts. (C) Bv-Ptx (Pentraxin) expression in newly settled juveniles. Note expression in the area of the resorbing cerebral vesicle (cv). (D) Bv-Tenc (Tenascin) expression in a juvenile shortly after settlement. Note expression in the papillae region (p), in the resorbing cerebral vesicle, in the tail and in the muscle granules (mg) which are undergoing phagocytosis. (E) Bv-Sccp2 (Selectin) expression in a juvenile two days after settlement. Note expression in the epidermis, particularly in the ampullae (amp), as well as in blood cells (bc) in and around the ampullae. (F) Bv-Ccp3 (Complement receptor) expression in a juvenile two days after settlement. This is a close up view of a single ampullae. Note expression in the ampullae epidermis and in the cytoplasm of blood cells around the ampullae.

View larger version (98K): [in a new window]   Fig. 5. Propidium iodide staining reveals timing of trans-epidermal migrations. (A,B,D,E) Projected compilations of confocal z-series, C is a DIC image (anterior is to the right). (A) Pre-competent larvae two hours after hatching; note the lack of any cells outside of the epidermis. Pre-competent larva at five hours after hatching also lack cells within the juvenile tunic (data not shown). (B) A competent larva, seven hours after hatching. Cells that have migrated into the tunic are labeled with an asterisk. (C) Close up DIC image of a cell that has migrated into the tunic in a 7-hour old larva (indicated in B by the white box). Note that the cell clearly lies on the outside of the epidermis but still within the juvenile tunic. The migrated cell is marked by a black dot, an asterisk indicates the epidermis, a black arrowhead indicates the juvenile tunic and a white arrowhead indicates the larval tunic. (D) A competent larva 15 hours after hatching; a regular array of cells can clearly be seen within the juvenile tunic, closely apposed to the outside of the epidermis. (E) Newly settled juvenile, 17 hours after settlement; migrated cells are now embedded within the expanding juvenile tunic, away from the epidermis. The juvenile tunic is indicated by the white arrowheads (at this point the larval tunic has been molted).

View larger version (204K): [in a new window]   Fig. 6. Observations of PAT cell migrations (anterior is downward). (A) A competent larva viewed ventrally. Note extended papillae (pap), two layers of tunic (lt, larval tunic; jt, juvenile tunic), rounded blood cells (*) within the hemocoel (h). (B) 15 minutes after induction of settlement with 50 mM KCl filtered sea water. Note retraction of the papillae, an anterior cone of PAT cells has extended into the tunic space (black arrowhead), the anterior region has flattened and the tunic has begun to expand anteriorly. (C) 25 minutes after induction, note the anterior tunnel through the tunic and the PAT cell clearly migrating through the tunnel (black arrowhead). (D) 45 minutes after induction, the migrating PAT cell is now outside of the juvenile tunic. Under natural conditions the larval tunic is molted, leaving the migrating PAT completely exposed to the external environment. Also note the continued migration of rounded blood cells across the epidermis in the anterior (black arrowhead).

View larger version (26K): [in a new window]   Fig. 7. Diagram of blood cell extravasation across the endothelia during vertebrate inflammation. Modified from Alper (Alper, J., 2001). Our results indicate that a similar process may underlie cell migrations across the epidermis and ampullae during ascidian metamorphosis.

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