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First published online 21 April 2004
doi: 10.1242/dev.01110

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A regulatory network of T-box genes and the even-skipped homologue vab-7 controls patterning and morphogenesis in C. elegans

¹ Genetics Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
² Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (82K): [in a new window]   Fig. 8. T-box mediated anterior repression of vab-7. (A) Upper line, schematic of vab-7 5' regulatory region of vab-7::GFP construct showing putative T-box binding sites A and B. Lower line, sequence of mutated sites A and B, following site directed mutagenesis. These mutated sites would not be expected to bind T-box proteins (Sinha et al., 2000). (B,D,F) Expression of vab-7::GFP in transgenic animals carrying this mutated construct. The normal posterior pattern of vab-7::GFP expression can be seen, together with ectopic expression at the anterior. (B) Ectopic anterior expression can be seen at around the 100-cell stage (arrows). The inset panel in (C) shows WT vab-7::GFP expression in four posterior Cxxp cells at the same stage, for comparison. Note there is no anterior expression in this case. (D) Ectopic anterior expression around the 400-cell stage (arrows) together with the usual posterior muscle expression of vab-7::GFP seen at this stage. (F) Ectopic expression at the anterior at the 1.5-fold stage (arrows) together with the usual posterior muscle and hypodermal VAB-7::GFP observable at this stage. (C,E,G) expression of WT, non-mutated vab-7::GFP in tbx-30(RNAi) embryos. Ectopic expression of vab-7 is observable in the same anterior cells at comparable stages to the T-box binding site-mutated vab-7 reporter, suggesting that TBX-30 normally represses vab-7 expression at the anterior of embryos by binding to these sites. (C) Ectopic expression of vab-7 around the 100-cell stage in the same cells as in (B) (arrows). The inset panel shows WT vab-7::GFP expression at the same stage, for comparison. (E) Ectopic anterior expression around the 400-cell stage (arrows), comparable to that seen in (D). (G) Ectopic anterior expression at the 1.5-fold stage, comparable to that seen in (F). The usual posterior vab-7 expression pattern is seen in all cases (slightly out of focus in (G)) Posterior is to the right, dorsal is up in panels (B-G). Scale bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 1. Phylogenetic analysis of T-box genes. (A) Phylogenetic tree. T-box domain amino acid sequences of all C. elegans T-box genes and representative members of the defined T-box subfamilies found in other organisms (Papaioannou, 2001) were aligned using ClustalW accessed via the European Bioinformatics Institute (EBI, http://www.ebi.ac.uk/). The alignment was subjected to Phylip analysis using the same EBI interface and Phylip outputs were interpreted using the tree drawing programme TreeView. Non-C. elegans genes are in red. Species abbreviations are as follows: As, ascidian; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus. New C. elegans gene names (tbx-30-41) have been approved by the Caenorhabditis Genetics Centre (CGC). (B) Genomic organisation of tbx-8 and tbx-9 (and neighbouring genes) in C. elegans and C. briggsae. Ce-tbx-8 and Ce-tbx-9 are transcribed in opposing directions (arrows), whereas Cb-tbx-8 and Cb-tbx-9 are transcribed in the same direction (arrows). The thick dotted line represents the intergenic region. Ce-tbx-8 and Cb-tbx-8 are 73% identical throughout their T-box domains. Ce-tbx-9 and Cb-tbx-9 are 56% identical. There is evidence of a local chromosome inversion in C. briggsae compared with C. elegans, which extends for several genes to the right of Cb-tbx-9 (neighbouring region not drawn to scale). Red bar denotes the T-box domain of Ce-tbx-8 and Ce-tbx-9 (59% identical).

View larger version (100K): [in a new window]   Fig. 2. RNA interference (RNAi) phenotypes of tbx-8 and tbx-9. (A) Wild-type adult hermaphrodite tail whip. (B) Tail region of adult hermaphrodite progeny of wild-type worm injected with dsRNA corresponding to tbx-8. These worms are indistinguishable from wild type. (C) Tail region of tbx-9(RNAi) worms. The adult hermaphrodite tail is thickened (arrow) and often has a `bobbed tail' appearance. (D) Tail region of adult hermaphrodite vab-7(e1562) animal. The tail is similar in appearance to that in (C). (E) L1 larvae from hermaphrodite mother co-injected with tbx-8 and tbx-9 dsRNA. In addition to thickening of the tail, these animals display gross midbody and posterior morphological abnormalities, often with large dorsal bulges (arrow). Most die as unelongated embryos or short L1s. (F) Wild-type L1. Scale bars, 10 µm. Posterior is to the right and dorsal is up in all panels. (G) RT-PCR analysis of tbx-8 and tbx-9 transcripts in wild-type worms and those subjected to tbx-8 and tbx-9 RNAi. ama-1 was chosen as a suitable control, as mRNA would be expected to be constant throughout. For each gene, primers were chosen to specifically amplify mRNA of approximately 200 bp (see Materials and methods). Results were identical for three separate experiments.

View larger version (59K): [in a new window]   Fig. 4. Muscle and intestinal defects in tbx-8/tbx-9(RNAi) animals. (A-D) Transgenic embryos carrying an hlh-1::GFP body wall muscle reporter. Body wall muscle nuclei are green. (A-B) Embryos around the 400-cell stage. (A) Wild type, dorsal view. Two rows of muscle cells (one on the right and one on the left side of the embryo) are visible at this stage. (B) tbx-8/tbx-9(RNAi) embryo. Some muscle cells are not in regular rows (arrow). (C-D) Embryos at the 1.5-fold stage. (C) Wild type, left lateral view. Muscles have split into dorsal and ventral rows on each side of the embryo. One dorsal and one ventral row can be seen in this focal plane. (D) tbx-8/tbx-9(RNAi) embryo. There is no clear separation of dorsal and ventral rows, especially at the posterior (arrows). (E-F) transgenic animals carrying a myo-3::GFP body wall muscle reporter. (E) Dorsal view of wild-type L1, showing two of the four rows of muscle nuclei; two ventral rows are not visible in this focal plane. (F) Muscle nuclei in tbx-8/tbx-9(RNAi) animals. Instead of lying in straight rows, nuclei are often bunched (arrows), and there are gaps in the row (arrowheads). (G-J) Intestinal defects in tbx-8/tbx-9(RNAi) animals. (G-H) tbx-8/tbx-9(RNAi) animals that have survived to hatching. The intestine has severe morphological defects with the gut lumen being highly distended (arrows). In comparison, a wild-type gut lumen is visible in (E) (arrow). (I-J) Transgenic worms carrying an elt-2::GFP intestinal cell reporter. Intestinal nuclei are green. (I) Wild-type L1 showing the positions of 16 of the 20 intestinal nuclei (the other four are out of focus). (J) tbx-8/tbx-9(RNAi) L1 worm. The intestinal nuclei are out of position, often being bunched together at either end of the intestine (arrows) and missing from the middle (arrowheads). Counting reveals that all nuclei are usually present, however. Posterior is to the right in all panels. Scale bars, 10 µm.

View larger version (76K): [in a new window]   Fig. 3. Hypodermal defects in tbx-8/tbx-9(RNAi) animals. The arrangement of hypodermal cells in embryos and L1 larvae has been visualised using the ajm-1::GFP reporter. (A-D) Dorsal view. (A) Wild-type embryo around 300 minutes following first cleavage. The two rows of dorsal hypodermal cells born 1 hour previously have intercalated into a single row and their advancing edges have come into contact with the opposing lateral hypodermal cells (arrows). (B-D) tbx-8/tbx-9(RNAi) embryos at approximately the same stage. Dorsal hypodermal cells are clearly disorganised. In most embryos dorsal intercalation begins relatively normally at the anterior (B-D, arrow) but does not proceed normally in the midregion or posterior (B-D, arrowheads). In these embryos, dorsal intercalating cells that are not the appropriate wedge-shape can be seen (B-D, asterisks), and many cells do not extend contralaterally in the correct way. Dorsal intercalation often arrests at this point, the same misarranged cells being observable at least 1 hour later. (E-H) Arrangement of lateral hypodermal (seam) cells in tbx-8/tbx-9(RNAi) embryos and L1 larvae. (E) Wild-type embryo beginning elongation, lateral view. A linear row of seam cells can be seen. (F) tbx-8/tbx-9(RNAi) embryo, same stage and view. The lateral row of cells is interrupted, with some cells being pinched out of line (asterisks). (G) tbx-8/tbx-9(RNAi) animal that has survived to hatching; some seam cells are pinched out of line (asterisks) and are misshapen at the site of the dorsal bulge (arrow). Animals are much shorter than they should be in L1 (compare with (H) wild type, posterior half only of animal, same scale). Posterior is to the right in all panels. Scale bars, 10 µm.

View larger version (81K): [in a new window]   Fig. 5. Embryonic expression of tbx-8 and tbx-9 reporters. The earliest detectable tbx-8::GFP and tbx-9::GFP expression is seen in the nuclei of the gut cell precursors Ea and Ep at the onset of gastrulation, around 100 minutes following first cleavage (A-B). Ea and Ep are moving dorsally, into the interior. (A) TBX-8::GFP (Ea, arrowhead, Ep, arrow), (B) TBX-9::GFP (Ea, arrowhead, Ep, arrow). tbx-8 and tbx-9 are subsequently expressed in muscle and hypodermal cells. (C) Expression of tbx-8::GFP in dorsal muscle cells (arrows) and lateral hypodermal cells (arrowheads) at the 1.5-fold stage. To confirm these cells types, co-staining was performed with muscle and hypodermal specific markers. (D) TBX-9 stained green with a GFP antibody. The outlines of muscles cells are stained red with the muscle antibody NE8/4C6.3 and dorsal muscle cells expressing tbx-9 are indicated (arrows). Similar results were obtained for TBX-8 (data not shown). (E-I) Hypodermal expression of tbx-8::GFP and tbx-9::GFP. TBX-8::GFP (E) and TBX-9::GFP (F) can be seen in the nuclei of dorsal hypodermal cells undergoing intercalation (around 300 minutes following first cleavage). The expression is particularly strong in cells that are just starting to intercalate (E,F, arrows). Outlines of cells undergoing the early stages of intercalation are shown with a black dotted line. Weaker expression can also be seen in lateral hypodermal cells (E,F, arrowheads). (G-I) Embryo co-stained with antibodies to show TBX-9::GFP in green and LIN-26 (expressed in all hypodermal cells) in red. (G) Green channel only showing cells expressing TBX-9::GFP stained with a GFP antibody. (H) Red channel only showing cells expressing LIN-26. (I) Merged image. Yellow nuclei strongly expressing both LIN-26 and TBX-9 can be seen in dorsal hypodermal cells that are beginning to intercalate (white solid arrow). Once cells are intercalated at the anterior TBX-9 expression is weaker and nuclei appear orange-yellow (dashed arrow). Cells at the posterior that have yet to intercalate express TBX-9 very weakly in this embryo and the nuclei appear orange-red (dotted arrow). Weak TBX-9 expression can also be seen in lateral hypodermal cells, in which the nuclei appear orange (arrowhead). Ventral hypodermal cells (not visible in this focal plane), which express LIN-26 but not TBX-9, stained red (not shown). Underlying muscle cell nuclei, which express TBX-9 but not LIN-26, appear green (blue arrow). Similar results were obtained with a tbx-8::GFP reporter. Posterior is to the right in all panels. Scale bar, 10 µm.

View larger version (112K): [in a new window]   Fig. 6. vab-7 expression in tbx-8/tbx-9(RNAi) embryos. (A-C) Similarity of expression domains of tbx-8 (A), tbx-9 (B) and vab-7 (C) GFP reporter constructs around the 400-cell stage. In all three cases, expression is seen in posterior muscle cells, although the domain of tbx-8 expression is slightly wider, with some non-vab-7-expressing nuclei showing up (A, arrowheads). (D) vab-7::GFP expressing nuclei at the 1.5-fold stage. Expression is seen in posterior muscle cells (arrowheads), the majority of which are dorsal, and in the posterior hypodermal cells hyp 8-11 (arrow, 4-5 nuclei). The vab-7::GFP construct gives a similar expression pattern to the vab-7::lacZ construct and antibody staining previously reported (Esmaeili et al., 2002; Ahringer, 1996). (E) vab-7::GFP expression in tbx-8/tbx-9(RNAi) embryos at the same stage. No muscle-specific expression of vab-7 is observed, even though the same number of muscle cells are present (see text). vab-7 expression is still seen in one or two posterior hypodermal cells (arrow), which appear to have shifted slightly anteriorly, presumably due to the general disorganisation of tbx-8/tbx-9(RNAi) embryos. Posterior is to the right. Scale bar, 10 µm.

View larger version (109K): [in a new window]   Fig. 7. Expression of vab-7 in embryos overexpressing tbx-8 or tbx-9. Expression of vab-7::GFP was monitored in worms that also carried integrated hsp16-2::tbx-8 + hsp16-41::tbx-8 or hsp16-2::tbx-9 + hsp16-41::tbx-9 constructs in which high level expression of tbx-8 or tbx-9 could be ubiquitously induced by heat treatment. (A-B) Following heat-shock induction of tbx-8 at 33°C for 45 minutes and a subsequent incubation at 20°C for 2 hours, ectopic vab-7::GFP expression can be seen up to the 400-cell stage. (A) Early embryos; (B) later embryos around the 400-cell stage. (C-D) Similar results were obtained following heat shock induction of tbx-9, using the same heat treatment conditions. (E-F) vab-7::GFP embryos that do not contain the hsp16::tbx-8 or hsp16::tbx-9 constructs, subject to the same heat treatment regime. VAB-7 is seen in very few posterior cells around the 200-cell stage (E), and in muscle cells around the 400-cell stage, as has been previously reported (Ahringer, 1996) (this report). Posterior is to the right in all panels. Scale bar, 10 µm.

View larger version (97K): [in a new window]   Fig. 9. MAB-9 localisation in vab-7 mutants. (A,B) Embryo at the 1.5-fold stage carrying an integrated mab-9::GFP translational reporter construct (nomarski image on the left, (A), corresponding fluorescence image on the right, (B)). MAB-9 can be seen in three nuclei at this stage around the presumptive rectum, B, F and hyp 7, as has been previously reported (Woollard and Hodgkin, 2000). (C,D) vab-7(e1562) embryo also carrying an integrated mab-9::GFP reporter at the same stage (nomarski image on the left, (C), fluorescence image on the right, (D)). Ectopic MAB-9 can be seen in more posterior nuclei (arrows). Posterior is to the right in all panels. Scale bar, 10 µm.

View larger version (16K): [in a new window]   Fig. 10. Interactions between T-box genes and vab-7 in C. elegans. vab-7 expression is activated (probably indirectly) at the posterior of embryos by TBX-8 and TBX-9 working together. VAB-7, in turn, functions to repress mab-9 expression in posterior cells. Meanwhile, at the anterior of embryos, vab-7 is itself repressed by the action of TBX-30. This repression is likely to be direct, acting through T-box binding sites in the vab-7 5' regulatory region.

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