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First published online 21 November 2007
doi: 10.1242/dev.010892

Development 135, 33-41 (2008)
Published by The Company of Biologists 2008
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chongmague reveals an essential role for laminin-mediated boundary formation in chordate convergence and extension movements

Michael T. Veeman, Yuki Nakatani*, Carolyn Hendrickson, Vivian Ericson, Clarissa Lin and William C. Smith{dagger}

Department of Molecular, Cell and Developmental Biology, University of California Santa Barbara, Santa Barbara CA, 93106, USA.


Figure 1
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  		Fig. 1. Notochord morphology in chongmague. All images show the notochord-specific bra:GFP transgene (white in A-C, green in D-I). Although not fused to any localization signal, the GFP is consistently brighter in the nucleus and cell cortex, and also brighter in the eight secondary lineage notochord cells at the posterior tip of the tail. Panels D-I also show the actin cytoskeleton labeled with phallacidin in red. (A-C) Maximum intensity projections of confocal stacks through identically staged wild-type (A), chm/chm (B) and aim/aim (C) embryos. Anterior to the top. Green arrowheads in B show notochord cells at the edges of the notochord that have their long axes inappropriately oriented along the anteroposterior and not the mediolateral axis. (D,F) Mid-tailbud-stage wild-type embryo. Anterior is to the left. (D) A volume rendering of the entire confocal stack and (F) a single slice at the level indicated by the blue line (inset). The inset shows a cross section along the orange line in the main panel. (E,G-I) chm/chm sibling. Anterior is to the left. (E) A volume rendering and (G-I) single slices at the levels indicated on the cross-section inset in G as indicated by the coloured lines to the left of each panel. The blue arrowheads mark an isolated notochord cell at the periphery of the tail. The red arrowhead marks a notochord cell interdigitated between two muscle cells. The yellow bracket marks the characteristic epidermal protrusion at the tip of the chm tail. Scale bar: 50 µm.

 

Figure 2
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  		Fig. 2. Time-lapse analysis of ascidian notochord boundary formation. (A-C) Nomarski time-lapses of notochord boundary formation in the highly transparent ascidian Ascidiella aspersa. (A) Emergence of a morphologically distinct notochord boundary (marked with orange dots). Frames are 100 minutes apart. (B) Cell behaviors at the notochord boundary late in intercalation. Note the rapid spreading of the yellow cell as it contacts the boundary (indicated by the yellow arrowhead), while the adjacent green cell becomes temporarily displaced. Frames are 3 minutes apart. (C) Gradual shearing of muscle cells (one marked in yellow) versus notochord cells along the notochord boundary. Frames are 75 minutes apart. (D) Confocal time-lapse of bra:GFP fluorescence in a chm/chm Ciona savignyi embryo. Anterior is to the top. Note the small group of cells in the first frame (blue arrow) that break away from the main mass of the notochord and migrate toward the tip of the tail (red arrow in the final frame). There is also considerable movement of cells at the notochord boundary, especially on the bottom right side of the image. Frames shown are 16 minutes apart. (E) A single frame of bra:GFP fluorescence in a wild-type sibling imaged just after the time-lapse in D was completed.

 

Figure 3
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  		Fig. 3. Chm is Cs-lam{alpha}3/4/5. (A) Schematic of AFLP and SSCP mapping showing a predicted alpha laminin gene on paired scaffolds 312 and 44 in C. savignyi. (B-D) Whole-mount in situ hybridization for Cs-lam{alpha}3/4/5 message showing notochord-specific expression in (B) late neurula, (C) mid-tailbud and (D) late tail extension stages. (E) Sense control and (F) decreased expression in a chm/chm embryo. (G) Embryo injected with control morpholino. (H,I) Cs-lam{alpha}3/4/5 morphant embryos showing the range of phenotypes observed. (J) bra:GFP expressing embryo co-injected with Cs-lam{alpha}3/4/5 morpholino and red fluorescent dextran as an injection marker. Note the disorganized notochord boundary, the dispersed bra:GFP-expressing cells in the tail (blue arrowheads) and the characteristic epidermal protrusion (yellow bracket). (K) Schematic of Cs-lam{alpha}3/4/5 domain structure showing a 9 amino acid insertion in chm and a frameshift predicted to cause to premature termination in chm35. The antigen used to make the Cs-lam{alpha}3/4/5 antibody is indicated. (L) Chromatograms showing the chm35 frameshift in genomic DNA from wt/wt, chm35/chm35 and chm35/wt individuals. wt, wild type.

 

Figure 4
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  		Fig. 4. Immunolocalization of Cs-lam{alpha}3/4/5. (A-C) Single confocal sections through the notochords of wild-type ascidian embryos stained for Cs-lam{alpha}3/4/5 at (A) late neurula, (B) early tailbud and (C) late tail stages. Anterior is to the left. Green arrowheads indicate the lateral edges of the notochord or notochord primordium. (D) Tangential section grazing the perinotochordal surface of a wild-type late-tail-stage embryo. (E) Reconstructed z-section across the notochord of a mid-tail wild-type embryo. Red 'm's indicate the lateral blocks of muscle cells. (F) Single confocal section through the notochord of a mid-tail-stage chm/chm embryo. (G-I) Single confocal sections through the notochords of (G) mid-tail, (H) mid-late tail and (I) late tail stage aim/aim embryos. Red arrowheads show ectopic laminin localization on intranotochordal cell surfaces. (J) Reconstructed z-section across the notochord of an aim/aim mid-late-tail-stage embryo. Red 'm's indicate the lateral blocks of muscle cells, and the red arrowhead shows ectopic laminin. (K) Representative images from multiple wild-type embryos stained under standard conditions with the Cs-lam{alpha}3/4/5 antibody, the Cs-lam{alpha}3/4/5 antibody blocked by prebinding to Cs-lam{alpha}3/4/5 peptide, or with control non-immune normal rabbit serum. (L,M) Cs-lam{alpha}3/4/5 antibody staining (red) and GFP staining (green) in C. intestinalis embryos electroporated with (L) notochord-specific expression plasmids for both Cs-lam{alpha}3/4/5 and GFP, or (M) GFP plasmid alone.

 

Figure 5
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  		Fig. 5. Cs-lam{alpha}3/4/5 can drive considerable tail extension in the absence of a functional PCP pathway in C. savignyi. (A,B) Confocal section through (A) late neurula stage wild-type embryo and (B) mid-tail-stage aim/aim embryo. Anterior to the left. bra:GFP in green, phallacidin in red. White arrowheads indicate medially polarized actin-rich structures. (C) Brightfield images of hatched larvae of the indicated genotypes. (D) Widefield bra:GFP fluorescence in an aim/aim;chm/chm embryo. Wild-type sibs (not shown) are at late tailbud stage. (E) Neurula-stage wild-type embryo, with phallacidin staining in white and the notochord primordium pseudocolored in red.

 

Figure 6
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  		Fig. 6. A model of ascidian notochord morphogenesis. In wild-type embryos, the PCP pathway mediates mediolateral intercalation behavior and a perinotochordal laminin-containing ECM mediates boundary formation. When the PCP pathway is disrupted in aim/aim embryos, mediolaterally-biased notochord cell intercalation is perturbed but considerable convergence and extension still slowly occur. This may be the result of randomly moving notochord cells being `trapped' where they stochastically contact the notochord boundary. The PCP pathway is also required to maintain perinotochordal/intranotochordal Cs-lam{alpha}3/4/5 polarity. The failure of aim/aim notochord cells to complete the final stages of intercalation may reflect this deposition of laminin on intranotochordal surfaces. In chm/chm embryos there is a defect in perinotochordal boundary formation and notochord cells become dispersed in the tail. Despite this morphogenetic defect, there is still a moderate degree of convergence and extension, which is probably due to the PCP pathway given that the aim/aim;chm/chm double mutant shows a near complete failure in convergence and extension. Although the lack of epistasis suggests that the two processes act at least partly in parallel, the true relationship is clearly complex, given the role of aim in maintaining Cs-lam{alpha}3/4/5 polarity and the role of Cs-lam{alpha}3/4/5 in maintaining polarized cell behaviors. wt, wild type.

 

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© The Company of Biologists Ltd 2008