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First published online 10 July 2006
doi: 10.1242/dev.02475

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Specification of the C. elegans MS blastomere by the T-box factor TBX-35

Gina Broitman-Maduro¹, Katy Tan-Hui Lin^1,2,*, Wendy W. K. Hung^1,2,* and Morris F. Maduro^1,

¹ Department of Biology, University of California, Riverside, Riverside, CA 92521, USA.
² Graduate Program in Cell, Molecular and Developmental Biology, University of California, Riverside, Riverside, CA 92521, USA.

View larger version (31K): [in a new window]   Fig. 1. Positions and fates of eight-cell stage C. elegans embryonic blastomeres in wild-type and mutant backgrounds. A diagram showing the relative positions of the wild-type AB granddaughters (AB4) and the P1 granddaughters MS, E, C and P3 is given at the top. Depletion of mex-1 and pie-1 individually and together results in the fate transformations shown at the bottom of the figure (Mello et al., 1992). The table summarizing the major embryonic cell types made by descendants of AB, MS, D and C was adapted from lineage data (Sulston et al., 1983). In embryo diagrams, anterior is towards the left, and dorsal is upwards.

View larger version (70K): [in a new window]   Fig. 2. The tbx-35 gene encodes a T-box factor. (A) Gene structure of tbx-35 on LGII. The left end of the gene is preceded by the start codon of ZK177.1. The deleted regions in tm1789 and tm618 are shown above the gene. The predicted mRNA is shown below. The coding region is shaded, with the T-box emphasized. The locations of MED-1 binding sites are shown as grey circles for the RAGTATAC site defined by MED-1 footprinting of the end-1 and end-3 promoters (Broitman-Maduro et al., 2005), and white circles for three lower-affinity RGGTATAC sites based on in vitro competition assays (G.B.-M., K.L., W.H. and M.M., unpublished). No additional MED sites are found elsewhere in the gene. We were unable to amplify the 5' end of the transcript using primers to detect SL1 or SL2, suggesting that the tbx-35 mRNA is not trans-spliced (Conrad et al., 1993); hence, the bona fide 5' end of the transcript is not known. A polyadenylation sequence (AATAAA) is found 115 bp downstream of the predicted stop codon, but we have not confirmed the precise 3' end of the transcript. (B) Alignment of the conserved T-box region of TBX-35 with those of other Tbx genes. TBX-35 is 25-28% identical (35-39% similar) to vertebrate eomesodermin, mouse brachyury, Drosophila Dorsocross2 and its closest homolog in C. elegans, TBX-37. The TBX-37 T-box is more closely related to the other T-box regions shown (e.g. 37% identical and 50% similar to brachyury). This alignment was generated with AlignX (within Vector NTI 6) using the default settings. An asterisk indicates a conserved glutamic acid residue that was mutated in a heat shock fusion construct (see text). Xl, Xenopus laevis; Hs, Homo sapiens; Mm, Mus musculus; Dm, Drosophila melanogaster. Accession numbers: eomesodermin, P79944 (Xenopus) and NP005433 (human); brachyury, CAA35985; Dorsocross2, AAM11545. (C) MED-1 binds the tbx-35 promoter. A 190 bp fragment of the tbx-35 promoter containing the proximal MED site cluster was incubated with increasing concentrations of recombinant DNA-binding domain of MED-1 as described (Broitman-Maduro et al., 2005). Double-stranded competitor oligonucleotides: wild type, 5'-TCATTTGTATACTTTATCTACAATAT; mutant, 5'-TCATTTGTTATCATTATCTACAATAT-3'. Underlined nucleotides represent the wild-type and mutated core MED-1 binding sites, respectively.

View larger version (78K): [in a new window]   Fig. 3. Expression of tbx-35 is specific for the early MS lineage. (A,B) A reporter tbx-35::GFP transcriptional fusion shows GFP fluorescence in the two daughters of MS (MSa and MSp) and their descendants for several divisions. (C) Depletion of mex-1 by RNAi results in ectopic expression (arrows) of tbx-35 in AB descendants, consistent with a transformation of the AB granddaughters to MS (Mello et al., 1992). (D) Expression of tbx-35::GFP (arrows) in early C descendants, consistent with a C to MS transformation in pie-1(RNAi) embryos (Mello et al., 1992). (E) Depletion of the divergent ß-catenin wrm-1 by RNAi results in an E to MS transformation (Rocheleau et al., 1997), and concomitant expression of tbx-35::GFP in both the E and MS lineages. (F) Although MS adopts an E-like fate in pop-1(-) embryos (Lin et al., 1995), expression of tbx-35::GFP occurred in the early MS lineage of all mutant embryos examined (n>30). (G) tbx-35 mRNA accumulates in MS as detected by in situ hybridization. Seventy-eight percent of embryos at this stage (n=50) showed MS expression and 22% of embryos did not stain. (H) Ectopic tbx-35 mRNA in a mex-1(RNAi) embryo. Seventy percent of embryos at this stage (n=54) showed ectopic expression, 9% showed normal MS-specific expression and 20% did not stain. (I) Ectopic activation of tbx-35 in E in a lit-1(t1512) embryo grown at 25°C (Kaletta et al., 1997). Sixty-five percent of embryos (n=55) showed expression in MS and E, 22% showed MS-specific expression and 13% did not stain. (J) Normal expression of tbx-35 mRNA in a pop-1(RNAi) embryo. Seventy-eight percent of embryos (n=65) showed staining in MS, while 22% did not stain. For these and subsequent embryo images, anterior is towards the left, and dorsal is upwards, and the eggshell (seen by Nomarski optics) is shown with a broken blue line. Scale bar: 10 µm.

View larger version (89K): [in a new window]   Fig. 4. Deficiency of MS-derived cell types in tbx-35(tm1789) embryos. (A) Differential interference contrast (DIC) image showing the grinder (gr), indicative of MS-derived pharynx, in a wild-type threefold stage embryo. (B) A ceh-22::GFP reporter (pseudocolored yellow) shows the fully elongated pharynx with MS- and ABa-derived halves (Okkema and Fire, 1994). (C) Terminal glp-1(or178) embryo showing ceh-22 expression in only MS-derived pharynx. (D) Terminal tbx-35(tm1789) embryo arrested at 1.5-fold elongation, similar in appearance to med-1,2(-) embryos (Coroian et al., 2005). The grinder is absent, and internal cavities (arrows) are observed in 35% of such embryos, similar to the hypodermis-lined voids in skn-1(-) embryos (Bowerman et al., 1992). (E) ceh-22 expression in only ABa-derived pharynx in tbx-35(-). (F) Absence of pharynx in a glp-1(-); tbx-35(-) embryo. (G) Wild-type embryo at the 1.5-fold stage. (H) Expression of hlh-1::GFP in the embryo shown in G indicates muscle precursors (Chen et al., 1994). In wild type, an average of 46.8±1.4 cells (n=10) were scored as expressing at this stage. (I) In terminal pal-1(RNAi) embryos, muscles made by C and D are not made, resulting in a loss of posterior hlh-1 expression (arrows; compare arrows in H). Expression was seen in an average of 20±0.6 cells (n=33). (J) A 1.5-fold stage tbx-35(tm1789) embryo. (K) hlh-1::GFP expression in the embryo shown in J shows lack of signal in the anterior (arrowheads; compare H), while posterior expression persists (arrows) and a large cluster of hlh-1-expressing cells is seen near the center (*). An average of 34.8±2.4 cells (n=10) was scored in these embryos. (L) Terminal tbx-35(-); pal-1(RNAi) embryos express hlh-1::GFP in only a small number of cells, with an average of 5.7±0.5 cells (n=40).

View larger version (54K): [in a new window]   Fig. 5. MS lineage defects in tbx-35(-) embryos confirmed by in situ hybridization. (A) pha-4 transcripts in a wild-type onefold embryo have an intense pharynx component (with ABa- and MS-derived regions) and a weaker endoderm-specific component. All stained embryos (n=37) demonstrated this type of staining. (B) Expression of the pharyngeal myosin gene myo-2 in a wild-type late-stage embryo. All (n=37) stained embryos displayed a pattern similar to the one shown. (C) Expression of the body muscle myosin gene myo-3 mRNA in a twofold embryo. Anterior muscles (descendants of MS) are shown by arrows. (D) Zygotic activation of pal-1 in the early C lineage. Among 31 embryos at this stage, four showed no staining, while the remaining 27 (87%) showed expression only in the C lineage, as shown here. (E) Eighty-eight percent (n=25) of stained tbx-35(-) embryos displayed a reduced anterior region of high pha-4 expression, similar to the embryo shown here. Twelve percent of embryos displayed apparent wild-type staining (not shown). (F) Detection of myo-2 in only the anterior half of the pharynx in tbx-35(-). (G) Absence of anterior MS-derived muscle (arrows) in a tbx-35(-) embryo stained for myo-3. Additional staining is present in the center of the embryo (*). (H) Detection of zygotic pal-1 mRNA in both the MS and C lineages in a tbx-35(-) embryo, evidence of an early MS to C transformation. Among 36 embryos from the MS516 strain, three showed no staining and 33 showed strong expression in the C lineage. Of these, six also showed strong signal in the MS lineage, and two showed weak MS signal. As the rescuing array in MS516 is inherited by 60% of embryos (n=94), we estimate that 30% of tbx-35(-) embryos express pal-1 in the MS lineage.

View larger version (42K): [in a new window]   Fig. 6. tbx-35 is required to specify ectopic MS-like cells. (A) Ectopic MS-type muscle (hlh-1::GFP) in tbx-35(+); pie-1(RNAi); pal-1(RNAi) embryos. (B) Ectopic pharynx cells (ceh-22::GFP, pseudocolored yellow) in a tbx-35(+); mex-1(RNAi); pie-1(RNAi) embryo. (C) Very few hypodermal cells (lin-26::GFP, pseudocolored cyan) are made in a tbx-35(+); mex-1(RNAi); pie-1(RNAi) embryo (an average of 6.5±2.1 cells, n=16). (D) Dramatic reduction in number of muscle cells in a tbx-35(-); pie-1(RNAi); pal-1(RNAi) embryo. (E) Near absence of pharynx in a tbx-35(-); mex-1(RNAi); pie-1(RNAi) embryo. (F) Production of hypodermal cells in a tbx-35(-); mex-1(RNAi); pie-1(RNAi) embryo (an average of 26.7±3.9 nuclei, n=11). Simultaneous depletion of pal-1 greatly reduces the production of these extra hypodermal cells (Table 1).

View larger version (27K): [in a new window]   Fig. 7. Overexpression of TBX-35 is sufficient to specify pharynx and muscle fates. Images depict terminal embryos that received a 30-minute heat shock at 33°C before the 100-cell stage. (A) Absence of pharynx muscle (ceh-22::GFP) in a skn-1(RNAi) embryo (0.0±0.0 cells, n=36). (B) Small numbers of muscle cells (hlh-1::GFP) made in a skn-1(RNAi); pal-1(RNAi) background (6.7±0.5 cells, n=47). (C) Pharynx muscle cells are made when tbx-35 is overexpressed in a skn-1(RNAi) background. Thirty-two percent of embryos (45/141) had an average of 13.8±1.8 cells per embryo, with 10 embryos containing at least 25 cells as shown here, or an overall average of 4.4±0.8 (n=141). (D) An increase in body muscle cells in a skn-1(RNAi); pal-1(RNAi) background following ectopic activation of tbx-35 (an average of 13.2±1.7 cells, n=87). Fifteen percent of embryos (13/87) had at least 30 muscle cells, similar the embryo shown here. (E) Numbers of pharynx muscle cells (ceh-22::GFP) in a skn-1(RNAi) background. (F) Numbers of body muscle cells (hlh-1::GFP) in a skn-1(RNAi); pal-1(RNAi) background.

View larger version (21K): [in a new window]   Fig. 8. A model for specification of the MS blastomere. A gene cascade that begins with maternal SKN-1 progresses through activation of med-1,2 in EMS, which in turn activate tbx-35 in MS. POP-1 represses endoderm specification in MS. In E, the sister of MS, a Wnt/MAPK-dependent mechanism represses tbx-35 through an unknown effector (not shown). TBX-35 acts upstream of genes that specify pharynx and muscle fates, through regulators such as pha-4 and hlh-1, respectively (Gaudet and Mango, 2002; Krause et al., 1990). TBX-35 also represses targets of maternal PAL-1, blocking C fates. Activation of med-1,2 by SKN-1 is direct (Maduro et al., 2001), as is activation of tbx-35 by MED-1,2 (this work). In addition to med-1,2, SKN-1 also activates the expression of one or more Delta/Serrate/Lag (DSL) proteins that enable the MS cell to signal the AB lineage to make anterior pharynx (Priess et al., 1987).