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First published online 31 March 2004
doi: 10.1242/dev.01088

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The T-box transcription factors TBX-37 and TBX-38 link GLP-1/Notch signaling to mesoderm induction in C. elegans embryos

Kathryn Good^1,2,*, Rafal Ciosk^1,2,*, Jeremy Nance^1,2,*, Alexandre Neves^1,2, Russell J. Hill^1,2,3 and James R. Priess^1,2,4,

¹ Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
² Howard Hughes Medical Institute, Seattle, WA 98109, USA
³ Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA
⁴ Department of Zoology, University of Washington, Seattle, WA 98195, USA

View larger version (27K): [in a new window]   Fig. 1. Cell interactions that diversify AB descendants. (A) Four-cell and 12-cell embryos showing cells described in the text. ABa, ABp and their descendants each express GLP-1/Notch (green); ABp granddaughters at the 12-cell stage are outlined in bold. (B). Lineage diagram showing the first few divisions of ABa and ABp. Cell fates are shown in the absence of Notch signaling (– Notch) and when both the first (four cell) and second (12 cell) Notch interactions occur (+ Notch). The fates of the ABp descendants called ABpla and ABplp are further modified by the third and fourth Notch interactions, respectively (see Table 1). Signaling from MS leads to PHA-4 expression by the descendants of ABalp and ABara, and in addition represses LAG-2 in ABara descendants.

View larger version (46K): [in a new window]   Fig. 2. tbx-37 and tbx-38 map positions and T-box DNA-binding domains. (A) Region from the right arm of chromosome III deleted by both zuDf1 and zuDf3. Bold lines indicate DNA lacking in the various deficiency strains used. Terminal arrowheads indicate that the deficiency extends further than shown, black and white circles indicate defined and approximate deficiency breakpoints, respectively. The physical positions of listed genes are indicated in Mb according to the Wormbase web site (http://www.wormbase.org, release WS100, 2003). (B) The highly conserved T-box DNA-binding domain from mouse Tbx6 is shown with the corresponding domains from the C. elegans TBX-37, TBX-38 and TBX-8 proteins. The DNA-binding domains of TBX-37 and TBX-38 are nearly identical; residues shared between either of these proteins and TBX-8 or Tbx6 are shown in bold. Mutations in tbx-37 are as follows with base changes underlined: zu464 [glycine to glutamic acid change in DNA-binding domain (asterisk); GCCTCCGAATTC], zu466 (premature stop in exon 4; GGAGCTTAAAAT) and zu467 (5' splice site in intron 3; CAGATTGGGATT). Mutations in tbx-38 are as follows: zu463 [3' splice site in intron 2 (double asterisk); TTTCCAAAAC], zu460 [deletion beginning at 5' splice site in intron 2 (double asterisk); AGGTTT//GACATA]; and zuDf5 (deletion that removes tbx-38 and the flanking genes).

View larger version (139K): [in a new window]   Fig. 3. Comparison of wild-type and tbx-37 tbx-38 embryos. Left and right columns compare wild-type embryos and tbx-37 tbx-38 embryos at identical stages with the same orientations. (A,B) Embryos undergoing morphogenesis, lateral view. Embryos are bent within the eggshell, head to the left and tail wrapped below the head. Anterior and posterior halves of the pharynx in A are indicated with brackets; the arrowhead indicates the rectum near the end of the tail. (C,D) Internal view of embryos immunostained for adherens junctions; labeling as above. (E,F) Surface view of embryos immunostained for adherens junctions. Note additional lateral cells (arrows) in the tbx-37 tbx-38 embryo. (G,H) Dorsal view of embryos prior to morphogenesis. Clones of cells derived from each of the four ABarp granddaughters are colored as follows: ABarpaa(magenta), ABarpap (green), ABarppa (yellow), ABarppp (blue). Descendants of the C cell (white) are indicated for reference. White arrow in H indicates surface gap created by the abnormal ingressions of some ABarpaa and ABarpap descendants. All embryos are approximately 50 µm in length.

View larger version (56K): [in a new window]   Fig. 4. PHA-4 expression in wild-type and tbx-37 tbx-38 embryos. Wild-type and tbx-37 tbx-38 embryos are shown in the top and bottom rows, respectively. (A,E) PHA-4::GFP expression at morphogenesis stages as shown and labeled in Fig. 3A,B. PHA-4 is present at high levels in all of the pharyngeal nuclei (brackets) and rectal nuclei (left of arrowhead in each panel), and present at lower levels in the intestinal nuclei. (B,F) Light micrographs of living embryos just prior to morphogenesis; the daughters of the MS cell were killed during early embryogenesis. (C,G) PHA-4::GFP expression in the embryos shown in B and F, respectively. In the wild-type embryo (B,C), cells in the anterior pharyngeal primordium (ant) and the intestinal cells express GFP, but the posterior pharyngeal primordium (pos) has not formed. In the tbx-37 tbx-38 embryo (F,G), no pharyngeal cells are present. (D,H) PHA-4::GFP expression in 44-cell embryos. Both embryos show PHA-4::GFP in the MS granddaughters (top 4 GFP-positive nuclei) but only the wild-type embryo expresses PHA-4::GFP in ABa descendants (arrows indicate ABaraaa and ABarapa).

View larger version (77K): [in a new window]   Fig. 5. LAG-2 and TBX-37 expression. (A,B) LAG-2::GFP expression in 44-cell tbx-37 tbx-38 embryos with or without killing MS. Arrows indicate positions of the ABara granddaughters in both panels. (C,D) Wild-type 24-cell embryo stained with mAbT38 (C) and with DAPI to visualize nuclei (D). This dorsal view shows four ABa descendants (white numbers; 1, ABalap; 2, ABarpa; 3, ABarpp; 4, ABalpp) and two ABp descendants (magenta; 1, ABplaa; 2, ABplap). (E,F) Wild-type 26-cell embryo showing TBX-37::GFP expression (E) and viewed with Nomarski optics (F). This ventral view shows five ABa descendants (white; 4, ABalpp; 5, ABalpa; 6, ABalaa; 7, ABaraa; 8, ABarap) and five ABp descendants (magenta; 3, ABplpa; 4, ABplpp; 5, ABprpa; 6, ABprpp; 7, ABprap); additional cells from other lineages are labeled in black. (G,H) 26-cell wild-type (G) and apx-1 mutant (H) embryos with the same dorsolateral orientation showing TBX-37::GFP expression. Two ABp descendants are indicated by arrows in both panels.

View larger version (102K): [in a new window]   Fig. 6. ABp development. (A) apx-1 mutant embryo showing the lack of body morphogenesis and the massive pharynx (outlined by arrows) caused by the hyperinduction of both ABa and ABp descendants to become mesodermal cells. (B) apx-1;tbx-37 tbx-38 embryo with a small, half-pharynx (small arrows), a tail, a rectum (large arrow) and a tail spike (inset). (C,D) Ventral views of wild-type and apx-1; tbx-37 tbx-38 embryos at the onset of morphogenesis showing ventral hypodermal cells (white arrows) and the nascent rectum (black arrow). Scale bar: 5 µm.

View larger version (18K): [in a new window]   Fig. 7. TBX-37, TBX-38 expression and the first and second Notch interactions. Schematic diagrams of ABa and ABp development in wild-type embryos, apx-1 embryos, and apx-1; tbx-37 tbx-38 mutant embryos. (A) Arrows extending from MS indicate ABa and ABp descendants that contact MS or its sister, E (not shown). (B) When ABa or ABp descendents express TBX-37, TBX-38 and are signaled by MS or E, they adopt the tertiary fates shown and become mesodermal precursors. (C) The ABp descendants are assumed here to adopt secondary fates in the apx-1; tbx-37 tbx-38 mutant embryos because these embryos form tails. Fates of ABa descendants presumably are the same as in tbx-37 tbx-38 mutant embryos, but were not examined directly (indicated by?).