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Morphogenetic pattern formation during ascidian notochord formation is regulative and highly robust

¹ Department of Zoology, University of Washington, Seattle, WA 98195, USA
² Friday Harbor Labs, Friday Harbor, WA 98250, USA

View larger version (28K): [in a new window]   Fig. 1. An overview of the cut and paste experiments reported here. (A) Isolation of primary notochord precursors. (B) Endoderm ablation. (C) Ablation of neural plate precursors. (D) Isolation of anterior half embryos. (E) Isolation of dorsal anterior quadrants. (F) Co-isolation of notochord/notoplate. (G) Muscle/notochord recombination. Crosses in B and C indicate which blastomeres were ablated. The dashed lines 1-3 represent the positions of specific cuts made to obtain D-F.

View larger version (11K): [in a new window]   Fig. 2. Summary of morphogenetic behaviors expressed by primary notochord rudiments after their isolation at the 64-cell stage (see text for details).

View larger version (68K): [in a new window]   Fig. 3. Expression of F-actin-rich protrusions in isolated A-line notochord rudiments. Confocal sections were taken at 0.3 µm intervals through entire notochord rudiments and then made 3D reconstructions using NIH image. Each stereo pair was produced by combining two projections through the same confocal stack, in which the angle of the axis of projection differs by 6 degrees. (A) A 16-cell rudiment at the mid-gastrula stage. No localized protrusions can be seen. (B) By approximately 1 hour PFD, many cells bear detectable protrusions along interior edges. (C) At 3 hours PFD, protrusions are larger and more numerous, but no planar bias is evident.

View larger version (47K): [in a new window]   Fig. 4. Comparison of tadpole stages of (A) anterior half embryos, (B) dorsal anterior quadrants, and (C) notochord/neural plate coisolates. Posterior is bottom right in all three panels. e, endoderm; br, anterior neural tissue that normally gives rise to the brain; arrows in A indicate epidermis. Arrows in B indicate disorganized notochord cells that lost contact with an external substrate. In all three panels, the extended neural plate lies out of focus beneath the notochord. Scale bar, 10 µm.

View larger version (97K): [in a new window]   Fig. 5. Intermediate stages of notochord formation in dorsal anterior quadrants. (A-D) A dorsal anterior quadrant stained with Bodipy and imaged at (A)10 minutes, (B) 30 minutes, (C) 50 minutes, and (D) 70 minutes after completion of the final notochord cell division. Posterior is left in all panels. The larger cells to the right are anterior endoderm. The darker cells lining the edge are epidermal cells. The posterior-most notochord cells constrict their apices and many enter the interior of the notochord plate as they do during blastopore closure in whole embryos (arrows). But those that remain at the boundary never elongate or intercalate perpendicular to the AP axis. Scale bar, 20 µm. (E,F) Posterior (E) and anterior (F) cross-sectional views of dorsal anterior quadrants at approximately 3 hours PFD, when control embryos have completed formation of a cylindrical rod. Dorsal is up in both views. Arrows indicate the boundary between the neural plate and lateral epidermal cells. Note the downward curvature of the neural plate, which has normally closed to form a tube by this stage.

View larger version (16K): [in a new window]   Fig. 6. Emergence of cell polarity in anterior half embryos. (A) Average cell orientation relative to the mediolateral axis of the notochord rudiment. Note the clear anterior-posterior progression in the emergence of a mediolateral orientation in rows 1-3, while cells in the posteriormost row do not become mediolaterally oriented. (B) The average length/width ratio of individual cells increases with a time course comparable to whole embryos. The initial decrease in L/W ratios for rows 2 and 3 reflect the fact that these cells are initially elongated along the AP axis of the embryoid and subsequently change their orientation by decreasing their cross-sectional extent along the AP axis and increasing it perpendicular to the AP axis in the same way that cells within intact embryos do (data not shown). Cells in the posterior-most row remain extended along the AP axis.

View larger version (19K): [in a new window]   Fig. 7. Formation of a normal notochord in embryos lacking endoderm. Organization of the tadpole is essentially normal except for the absence of anterior gut structures. Scale bar, 10 µm.

View larger version (38K): [in a new window]   Fig. 8. Formation of normal notochords in embryos lacking the posterior neural plate (notoplate). (A) Control embryos; the axial neural tube can be seen in cross section as a set of four cells lying above the notochord and just below the dorsal epidermis. (B) Embryo lacking posterior neural plate. The axial neural tube is gone and instead a pair of epidermal cells contact the fully formed notochord along the dorsal midline.

View larger version (82K): [in a new window]   Fig. 9. Intermediate stages of notochord formation in embryos lacking the posterior neural plate. Posterior is left in all top and side views. Dorsal is up in all cross sections. (A-C) Approximately 1 hour PFD. (A) Top view. (B) Side view. (C) Cross section through the AP midline. Note that the anterior and lateral portions of the notochord remain a monolayer plate, but one that is oriented along the dorsal-ventral axis. The lateral epidermis and anterior endoderm now bound the basal surface of the notochord instead of the neural plate. (D-G) Approximately 2 hours PFD. (D) Dorsal top view. (E) Ventral top view. (F) Lateral section through one lateral arm of the notochord rudiment. (G) Cross section through the AP midline. Note the fusion of the two lateral notochord arms posteriorly (D) and the gradual posterior extension of the dorsal anterior boundary of the notochord plate (arrow in D) which is matched by a posteriorwards extension of the overlying epidermis (not shown). Note also the elongation of individual notochord cells perpendicular to the AP axis within the dorsal plate (D) and its lateral arms (F), and the constriction of cell apices within the lateral arms (arrows in G). (H-J) Approximately 3 hours PFD. (H) Top view. (I) Posterior cross section. (J) Anterior cross section. By this stage, the formation of a cylindrical intermediate is nearly complete. The organization of posterior muscle is essentially normal and the epidermis now bounds the notochord rod dorsally along its entire extent. Scale bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 10. Notochord fails to form a cylindrical intermediate in embryos lacking both neural plate and muscle. (A) Late neurula stage. The lateral notochord arms have met at the posterior (compare with D and E in Fig. 9). (B) Late tailbud stage. Individual notochord cells have begun to swell as they do at later stages in control embryos, but the overall shape and organization of the notochord rudiment remains similar to that seen at late neurula stage.

View larger version (54K): [in a new window]   Fig. 11. Notochord extension in notochord/posterior cap recombinants. (A) Lateral view of a posterior cap cultured in isolation until the tailbud stage. Posterior muscle cells have rearranged to form (approximately) three rows of six cells each arrayed along either side of the axial midline. But individual muscle cells do not extend along the AP axis. (B) Notochord/posterior cap recombinant at the same stage. The notochord has extended with respect to the AP axis of the posterior cap and individual muscle cells are considerably elongated along the AP axis. Scale, 10 µm.

View larger version (16K): [in a new window]   Fig. 12. Different modes of ascidian notochord formation. In notochord/neural plate co-isolates (and dorsal anterior quadrants) (A), basal contact with neural plate (and planar contact with anterior endoderm) specify convergent extension along and invagination about the embryonic AP axis. In embryos lacking neural plates (B), basal contact with anterior endoderm and/or lateral mesenchyme and/or epidermis specifies invagination/extension along a perpendicular axis. The extending lateral arms subsequently meet at the posterior and then re-anneal along the embryonic midline. In notochord/posterior cap recombinants (C), contact with posterior muscle specifies formation and extension of a cylindrical rod along the AP axis of the posterior cap, regardless of the initial orientation of the notochord rudiment. In normal embryos (D) multiple tissue interactions redundantly specify and support the normal mode of convergent extension and invagination.