The T-box factor TBX-2 and the SUMO conjugating enzyme UBC-9 are required for ABa-derived pharyngeal muscle in C. elegans

Abstract

The C. elegans pharynx is produced from the embryonic blastomeres ABa and MS. Pharyngeal fate in the ABa lineage is specified by the combined activities of GLP-1/Notch-mediated signals and the TBX-37 and TBX-38 T-box transcription factors. Here, we show another T-box factor TBX-2 also functions in ABa-derived pharyngeal development. tbx-2 mutants arrest as L1 larvae lacking most or all ABa-derived pharyngeal muscles. In comparison, tbx-2 mutants retain ABa-derived marginal cells and pharyngeal muscles derived from MS. A tbx-2Colon, two colonsgfp translational fusion is expressed in a dynamic pattern in C. elegans embryos beginning near the 100-cell stage. Early expression is limited to a small number of cells, which likely include the ABa-derived pharyngeal precursors, while later expression is observed in body wall muscles and a subset of pharyngeal neurons. TBX-2 contains 2 consensus sumoylation sites, and it interacts in a yeast two-hybrid assay with the UBC-9 and GEI-17 components of the C. elegans SUMO-conjugating pathway. ubc-9(RNAi) has been previously shown to cause variable embryonic and larval arrest, and we find that, like tbx-2 mutants, ubc-9(RNAi) animals lack ABa-derived pharyngeal muscles. ubc-9(RNAi) also alters the subnuclear distribution of TBX-2::GFP fusion protein, suggesting that UBC-9 and TBX-2 interact in C. elegans. Together, these results indicate that TBX-2 and SUMO-conjugating enzymes are necessary for ABa-derived pharyngeal muscle, and we hypothesize that TBX-2 function requires sumoylation. Sumoylation is increasingly recognized as an important mechanism controlling activity of many nuclear factors, and these results provide the first evidence that T-box factor activity may require sumoylation.

RNA recognition by the embryonic cell fate determinant and germline totipotency factor MEX-3

“…The genes include ubc-9, pha-4, and tbx-2, all of which are required to specify ABa-derived pharyngeal muscle (39,40). The 3ЈUTR of ubc-9 contains four MRE sites, whereas tbx-2 and pha-4 each have one.…”

The T-Box Gene tbx-2, the Homeobox Gene egl-5 and the Asymmetric Cell Division Gene ham-1 Specify Neural Fate in the HSN/PHB Lineage

“…The gene tbx-2 maps just to the right of dpy-17, and mutations in this gene cause an L1 arrest and pharyngeal phenotypes that were similar to those exhibited by gm111 animals (Roy Chowdhuri et al 2006). Both gm111 and the tbx-2 mutation caused an L1 arrest phenotype that results from the inability of the animals to feed: the pharynx was often detached from the mouth (Pun phenotype), and those pharynxes that were attached were thin and abnormally shaped (Figure 4).…”

“…The 10-aminoacid sequence flanking this Ala has been conserved across all T-box proteins (Bollag et al 1994;Papaioannou and Silver 1998), and analysis of vertebrate T-box protein/ DNA cocrystals shows that this region and in particular Ala238 interacts with target DNA (Muller and Herrmann 1997;Coll et al 2002). The deletion allele tbx-2(ok529) is a 1082-bp in-frame deletion that removes part of the conserved DNA binding domain, and genetic analysis suggests that the lesion eliminates tbx-2 function (Roy Chowdhuri et al 2006;Smith and Mango 2007). The similarity of the gm111 and ok529 phenotypes indicates that gm111 also results in a severe loss if not a total absence of TBX-2 function.…”

“…First, TBX-2 is normally expressed in the HSN/PHB lineage, but the TBX-2TGFP levels were too low to detect or were expressed transiently and were not detected in our experiments, Second, the TBX-2TGFP may not be expressed appropriately from this transgene, at least in cells required for the L1 arrest and HSN phenotypes. However, this transgene did rescue the PHB defects, suggesting that it should be expressed in cells involved in regulation of PHB fate (data not shown) (Roy Chowdhuri et al 2006;Smith and Mango 2007). Third, TBX-2 could function cell nonautonomously outside the lineage to regulate HSN and PHB differentiation.…”

“…To test whether a tbx-2Tgfp transgene is expressed within the HSN/PHB lineage, we carried out similar colabeling experiments as described above. Having established that the egl-5TlacZ transgene is expressed in the precursor cell, we costained a strain carrying tbx2Tgfp (Roy Chowdhuri et al 2006) and muIs13 with both anti-GFP and anti-b-galactosidase antibodies. We saw no colabeling of the HSN/PHB precursor, suggesting that TBX-2 may not be expressed at that time (Figure 8, A9-D9).…”