PMC

Bar graph showing the number of CEH-22::GFP⁺ cells in WT and mutant embryos at the bean stage, comma stage, 1.5-fold stage, and 2-fold stage. WT n = 12, mutant n = 7. error bars are s.d.

LIN-26 is not expressed in the pharynx of wild-type (A) or mutant (B) embryos, as detected by αLIN-26 antibody, but the epidermis stains (edges). WT and mutant embryos were discerned by pharyngeal adherens junction morphology with MH27 (C, D) Each embryo is ~50 μm long.

Notch signaling and lag-1 are required to activate the organ selector gene pha-4 in cells destined to become pharynx.. lag-1 also activates the ref-1 family of transcriptional repressors, and these limit tbx-2 expression to ABa descendants around the 8E stage, directly or indirectly. TBX-2 and PHA-4 enter a positive feedback loop, which is required for commitment and developmental progression of anterior pharyngeal muscles by the 1.5 fold/comma stage.

PHA-4::GFP (A, green) but not TBX-2::GFP (C, green) in both ABa and MS cell descendants at the 4E stage (nuclei, blue). (B) PHA-4::GFP in cells of the pharynx and intestine at the 8-10 E stage. (D) TBX-2::GFP is first detected in 11–12 posteriorly-located nuclei at the 8-10 E stage. (E) TBX-2::GFP in an L1 pharynx is present in pm3, pm4, and pm5 muscle groups (arrows). (F) At the 8-10E stage, TBX-2 is nuclear. (G) At the 1.5-fold stage, TBX-2::GFP is both cytoplasmic and nuclear in pm3 and pm5. Note the filamentous appearance in G. Each embryo is ~50 μm long.

(A) tbx-2::GFP 5.2kb translational reporter is de-repressed at the 8E stage when the ref-1 family is inactivated by feeding RNAi, but activated normally with glp-1 and lag-1 inactivation. TBX-2::GFP expression in 8E cell embryos (B, D, F H, J) and 16E cell embryos (C, E, G, I, K). WT embryos (B, C), compared to feeding RNAi of glp-1 (D,E), lag-1 (F,G), and ref-1members (H,I) shows de-repression of tbx-2::GFP at the 16E cell stage. pha-4(RNAi) reduces the number of TBX-2::GFP expressing cells (J,K). Error bars indicate SD. P values of tbx-2::GFP cell counts of RNAi embryos compared to wild type are glp-1 p= 0 .32, refs p = 0.001, lag-1 p = 0.0003, pha-4 p = 0.0000003.

(A) Eight tbx-2::GFP translational reporter transgenes (top) and one transcriptional reporter (bottom) in wild-type worms (blue triangles). Orange arrows depict LAG-1 consensus binding sequences, red dots show potential bHLH (REF-1) binding sites, and green lines indicate PHA-4 consensus binding sequences. (B-E) TBX-2::GFP at the 8E stage for 5.2 kb (B), 2.4 kb (C), 1.0 kb (D) translational and 5.2 kb tbx-2::GFP transcriptional (E) reporters. (F-G) GFP at the comma stage for 5.2 kb (F), 2.4 kb (G), 1.0 kb (H) translational and 5.2 kb (I) transcriptional reporters. (J-M) TBX-2::GFP in an L1 larva for 5.2 kb (J), 2.4 kb (K), 1.0 kb (L) translational and 5.2 kb (M) transcriptional reporters. Embryos are ~50 μm long; an L1 pharynx is ~45 μm long.

(A,B) TBX-38 (red) is expressed normally in all embryos from tbx-2(ok529)/+ mothers (DAPI, blue). (C) Wild-type comma-stage embryo with 94 PHA-4::GFP⁺ cells in the pharynx resembles (D) a tbx-2(ok529) comma-stage embryo with 93 PHA-4::GFP⁺ cells. (E) Embryo C viewed 30 minutes later with 94 PHA-4::GFP⁺ cells while embryo D had only 81 PHA-4::GFP⁺ cells (F). (G) WT and (H) tbx-2(ok529) embryos had approximately the same number of ceh-22::GFP expressing cells at the bean stage (arrows), when CEH-22::GFP is first detectable. Following the same embryos in panels G and H until the two-fold stage, wild-type embryos consistently have more ceh-22::GFP expressing cells (I, arrows) compared to tbx-2(ok529) embryos (J, arrows). (K) tbx-2::GFP WT 8E embryo and (L, arrows) tbx-2::GFP pha-4(RNAi) embryo with TBX-2::GFP expressing cells (M) Differential interference contrast micrograph (DIC) of a pha-4(q490) larva lacking a pharynx and (N) the same larva showing no TBX-2::GFP in the head other than in two non-pharyngeal cells (arrows). Each embryo is ~50 μm long.

(A) tbx-2 consists of five exons based on EST alignments, Genefinder, and ORFeome 3.1 (). We constructed a GFP reporter with 5.2 kb of sequence upstream of the ATG and the open reading frame fused to GFP at the predicted carboxyl terminus. This construct uses the unc-54 3′UTR and polyadenylation site (). The ok529 deletion spans exon four (red). (B) TBX-2 has sequences similar to the amino-terminal repressor domain of vertebrate Tbx2 and Tbx3 (yellow) (), the conserved T-box (underlined, ), and nuclear localization signal (orange; (; ). The ok529 mutation results in an in-frame deletion of exon 4 (red), compromising the conserved T-box DNA binding domain. (C) Alignment of the amino terminus of C. elegans TBX-2 with sequences from the amino terminal repression domain of vertebrate Tbx 2/3 ().

(A). Diagram of a pharynx showing the eight groups of pharyngeal muscles (pm; adapted from ). Cells affected by tbx-2 mutations are shaded. (B,C,D) Differential interference contrast micrographs of first stage (L1) larvae. (B) The pharynx of a tbx-2(RNAi) L1 with a wild-type posterior bulb (asterisk), but abnormal anterior bulb (arrow). (C, E, G, I, K, M, O) wild-type larvae, (D, F, H, J, L, N, P) tbx-2(ok529) larvae. (C) Wild-type larva with anterior and posterior bulbs (arrow, asterisk). (D) tbx-2(ok529) L1 with a relatively normal posterior bulb (asterisk), but morphologically aberrant procorpus, anterior bulb, and isthmus (arrows). Muscle marker 3NB12 is lost from anterior cells in tbx-2 mutants compared to wild type (E,F; (; ). Pharyngeal myosin is absent from anterior muscles in tbx-2(ok529) larvae (). CEH-22::GFP () is expressed in fewer pm3, 4, and 5 cells (arrows) while pm7 expression appears WT (asterisk) in tbx-2(ok529) (I, J). ITR-1::GFP, () is lost from pm4 and pm5 (arrows) while expression is still detected in pm6 (asterisk) and the intestine of tbx-2(ok529) worms (K, L). AVR-15::GFP expression in wild type (M) and tbx-2(ok529) (N) larvae () reveals only a subset of pm4 and pm5 cells express in mutant larvae. Wild-type (O) and tbx-2(ok529) (P) larvae stained with an antibody that recognizes intermediate filaments to reveal pharyngeal marginal cells (). All three groups of marginal cells are present and morphologically wild-type (arrows). Wild-type L1 pharynx is ~45 μm long.