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First published online 16 March 2005
doi: 10.1242/dev.01782

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The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo

L. Ryan Baugh^1,*, Andrew A. Hill², Julia M. Claggett¹, Kate Hill-Harfe^1,2,, Joanne C. Wen¹, Donna K. Slonim^2,, Eugene L. Brown² and Craig P. Hunter^1,

¹ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
² Department of Genomics, Wyeth Research, Cambridge, MA 02140, USA

View larger version (40K): [in a new window]   Fig. 8. Projection of three-dimensional images of reporter expression patterns for PAL-1 target genes likely to be involved in patterning. Dorsal and lateral views of chromosomally integrated transcriptional reporters 210 minutes after the four-cell stage (22°C; 200 cells, 16 C-lineage cells) are shown for 13 validated targets. Images are orthogonal perspectives following three-dimensional volume rendering of many optical sections. A broken gray line marks the approximate outline of each egg case. (A) Ordination: 8 C ectodermal nuclei in blue, 8 C muscle nuclei in red and 4 D muscle nuclei in yellow; (B) pal-1; (C) vab-7; (D) cwn-1; (E) C55C2.1; (F) unc-120; (G) hnd-1; (H) hlh-1; (I) elt-3; (J) mab-21; (K) nhr-25; (L) nob-1; (M) tbx-8; (N) tbx-9. Movies for each reporter are available in the supplementary material.

View larger version (43K): [in a new window]   Fig. 1. Use of homeotic mutants to deconvolve lineage-specific expression from transcript profiles. Wild-type and mutant embryos were staged in small cohorts by morphology at the four-cell stage and collected in replicate at the ten time points indicated by black dots in A. The complete lineage through the 190-cell stage is depicted for wild type in A, and hypothetical lineages are depicted for pie-1(zu154) in B and mex-3(zu155); skn-1(RNAi) in C. Lineages specified by SKN-1 and PAL-1 are blue and red, respectively, and the pattern of cell fates produced is indicated below the wild-type lineage in A. (D) Approximate fraction of the embryo with different lineage identities for each genotype, with expected changes in transcript abundance for lineage-specific genes in parentheses.

View larger version (18K): [in a new window]   Fig. 2. A skn-1-dependent transcriptional cascade. (A) Published expression patterns and regulatory relationships for four transcription factors and a differentiation gene involved in endoderm development. (B) Temporal expression patterns in wild type, pie-1(zu154) and mex-3(zu155); skn-1(RNAi) measured by microarray. med-1 and med-2 cannot be distinguished on the microarray.

View larger version (41K): [in a new window]   Fig. 3. Identification and rank of candidate PAL-1 target genes. (A) A log-scale histogram of the number of genes versus cumulative scores (1-7) from three distinct tests designed to capture a large fraction of candidate PAL-1 target genes (see text, and Materials and methods for details). (B) Hierarchical clustergram for 308 candidate genes; yellow indicates high and blue low relative expression. The three time courses were concatenated end to end for hierarchical clustering but are separated here for clarity.

View larger version (51K): [in a new window]   Fig. 4. Target validation. Embryonic expression of pal-1, assayed by YFP reporter, is dependent on maternal PAL-1 activity. (A-C) Expression of a zygotic pal-1 reporter is shown in a mid-gastrula embryo with corresponding Nomarski images for wild-type (A,D), pal-1(RNAi) (B,E) and mex-3(RNAi) (C,F).

View larger version (24K): [in a new window]   Fig. 5. Reporters for muscle-specific transcription factors are regulated in a lineage-specific fashion. Expression of hnd-1::GFP::lacZ (A,D), hlh-1::YFP (B,E) and unc-120::YFP (C,F) in wild-type (A-C) and pal-1(RNAi) (D-F) embryos. pal-1 RNAi eliminates expression in the C and D lineages (posterior). hnd-1::gfp::lacZ embryos were imaged 210 minutes after the four-cell stage and hlh-1::yfp and unc-120::yfp at 260 minutes (22°C). Because of the larger size of the reporter protein the signal for hnd-1::gfp::lacZ is better localized to the nucleus than in the other two strains.

View larger version (42K): [in a new window]   Fig. 7. PAL-1 target genes likely involved in patterning are expressed in four temporal phases. Wild-type temporal expression patterns are plotted for 13 validated target genes that encode 11 transcription factors, a wingless ligand (cwn-1) and a novel protein (mab-21) known to be involved in a cell fate switch in the male tail. Developmental stages are indicated as the number of C-lineage cells across the top of the graph, and the phase each gene belongs to is indicated by color (based on when zygotic expression is first detected). vab-7 is omitted from the graph because it is not detected in wild type by microarray, but we know it is a phase II gene based on lacZ reporter and in situ hybridization (Ahringer, 1996), as well as its detection in the mex-3(zu155); skn-1(RNAi) timecourse data.

View larger version (17K): [in a new window]   Fig. 9. Proposed structure of the regulatory network specified by pal-1. A graph of predicted regulatory relationships based on the best candidate upstream activator for each gene (Table 4) is presented. Temporal phase is indicated on the left. Lines with arrows represent cell-autonomous regulation by transcription factors and lines with dots represent regulation by signaling molecules.

View larger version (16K): [in a new window]   Fig. 6. Expression of the posterior HOX gene nob-1 is regulated by cell lineage. (A) Wild-type expression of a nob-1 YFP reporter in a 200-cell embryo (210 minutes after the four-cell stage at 22°C). Arrows indicate the two posterior-most C-lineage cells. Expression of the nob-1 reporter in similarly staged pal-1(RNAi) (B) and mex-3(RNAi) (C) embryos shows loss of and ectopic expression, respectively.