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First published online 19 November 2008
doi: 10.1242/dev.030981

Development 136, 35-39 (2009)
Published by The Company of Biologists 2009
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brachyury null mutant-induced defects in juvenile ascidian endodermal organs

Shota Chiba1, Di Jiang1,*, Noriyuki Satoh2 and William C. Smith1,{dagger}

1 Department of Molecular, Cell and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106, USA.
2 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.


Figure 1
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  		Fig. 1. An ENU-induced short tailed mutant. (A) Sibling larvae, either wild type (top) or homozygous for the 411 mutation (bottom). Scale bar: 100 µm. (B) SNP-based linkage analysis. Examples of unlinked (chromosome 01q, left panel) and linked (chromosome 12p, right panel) loci are shown. Panels show sequence traces from pooled mutant (411-/-) or heterozygous larvae and homozygous wild-type mixed siblings (411+/- + 411+/+). The red arrows indicate SNPs. (C) The mutant 411 has a premature stop codon in the brachyury gene. (Top) The gene model of brachyury. (Middle) Partial sequence of brachyury on exon 2. The point mutation is shown in red. (Bottom) Predicted models of wild-type and mutant brachyury protein. (D-G) Disrupted notochord morphology in bra-/- embryos. (D,E) Maximum intensity projections of confocal stacks through wild-type (D) and bra-/- (E) early tailbud stage embryos (dorsal view). Notochords were visualized by staining embryos carrying a stable brachyury promoter:GFP transgene with an anti-GFP antibody. The smaller cells in the background are mesenchyme (Me). (F,G) Lateral view of wild-type (F) and bra-/- (G) early tailbud stage embryos stained with phallacidin (green) and DAPI (blue). Notochord lineage cells are indicated by arrows. Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Notochord cells express endoderm markers in bra-/- mutant. (A-C) Alkaline phosphatase (AP) activity in wild-type (A) and bra-/- (B,C) larvae. The two bra-/- larvae are representative of the range of ectopic AP activity observed. (D-F) Expression of the endoderm/endodermal strand marker Kyotograil2005.572.7.1. in wild-type (D) and bra-/- (E,F) larvae. The black arrowhead indicates ectopic expression in the mutant larva. Genotypes were confirmed by PCR. The red arrowheads in the lower panels indicate the mutation that results in a premature stop codon in the bra- mutant. (G,H) Ectopic AP activity in the notochord lineage of wild-type and mutant larvae. The notochord lineage is indicated by GFP fluorescence. (G) Merged image for a wild-type larva. No colocalization of AP and GFP expression was found in anterior (A-line) notochord cells. (H) Merged image for a bra-/- larva. Ectopic AP activity and GFP fluorescence are colocalized (arrow) in the mutant. Scale bars: 100 µm.

 

Figure 3
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  		Fig. 3. bra-/- juveniles have defects in tail resorption and digestive tract development. (A) Stage 4 wild-type juvenile. A small amount of tail debris is still evident (arrow). (B,C) Stage 4 bra-/- juveniles showing the range of defects observed, from moderate (B) to severe (C). Note the extensive tail debris remaining in B (arrow). (D) Higher magnification image of digestive tract of juvenile A. (E) Higher magnification image of digestive tract of juvenile B. Although the esophagus and the stomach can be distinguished in the bra-/- juvenile, they are malformed. E, esophagus; ST, stomach. Scale bars: 100 µm. (F-J) Fate of notochord lineage as indicated by GFP fluorescence during metamorphosis. At stage 3 of metamorphosis the GFP-expressing cells are found displaced further towards the stalk in bra-/- (G) than in wild-type (F) juveniles. By stage 4, the remaining notochord debris is greatly reduced in wild-type (H), but not in bra-/- (I,J) juveniles. In the stage 4 bra-/- juveniles, the GFP-expressing cells have incorporated into definitive endodermal structures, such as the stomach (arrow in I) or intestine (arrow in J). (K) RT-PCR shows the absence of brachyury transcript in larvae and in stage 3 juveniles. 110, 110-cell stage; eTB, early tailbud stage; SL, larva; Juv, stage 3 juvenile.

 

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