The FoxF/FoxC factor LET-381 directly regulates both cell fate specification and cell differentiation in C. elegans mesoderm development

Abstract

Forkhead transcription factors play crucial and diverse roles in mesoderm development. In particular, FoxF and FoxC genes are, respectively, involved in the development of visceral/splanchnic mesoderm and non-visceral mesoderm in coelomate animals. Here, we show at single-cell resolution that, in the pseudocoelomate nematode C. elegans, the single FoxF/FoxC transcription factor LET-381 functions in a feed-forward mechanism in the specification and differentiation of the non-muscle mesodermal cells, the coelomocytes (CCs). LET-381/FoxF directly activates the CC specification factor, the Six2 homeodomain protein CEH-34, and functions cooperatively with CEH-34/Six2 to directly activate genes required for CC differentiation. Our results unify a diverse set of studies on the functions of FoxF/FoxC factors and provide a model for how FoxF/FoxC factors function during mesoderm development.

Fig. 1.

CEH-34/Six2 regulates the specification of non-muscle coelomocyte fate in the mesoderm. (A) Schematic of the early M lineage in a wild-type hermaphrodite. M, M mesoblast; d, dorsal; v, ventral; l, left; r, right; a, anterior; p, posterior; CC, coelomocyte; BWM, body wall muscle; SM, sex myoblast. (B) Model for the specification of dorsal M lineage fates (see Amin et al., 2009). CC fates are specified by CEH-34 and EYA-1, which require a number of known upstream factors, and at least one unknown factor, X.

Fig. 2.

The ceh-34 promoter contains a 348-bp enhancer element necessary and sufficient for M lineage expression. (A) Schematic of deletion constructs of the ceh-34 promoter, with the wild-type reference (pNMA94) at the top. Nucleotide positions relative to the ATG of the ceh-34 ORF are indicated above the constructs. The expression of each gfp::ceh-34 transgene is indicated to the right: +++, wild-type expression; +, expression in fewer cells than in wild-type; −, no expression. (B-D) A representative lateral image of a wild-type animal expressing E1::pes-10Δp::gfp (B), labeled with anti-FOZI-1 antibodies (C). (D) Merged image of B and C. (E) Alignment of the C. elegans 348-bp E1 element shown in A with corresponding sequences from C. briggsae, C. remanei and C. brenneri. Putative transcription factor (TF) binding sites are designated by black bars above each of the sites. FoxF sites are highlighted in yellow.

Fig. 3.

LET-381/FoxF directly regulates ceh-34 expression to regulate CC fate specification. (A) Plasmids and oligonucleotides used for mutagenesis of the ceh-34 promoter. Wild-type and mutant sequences from –2210 bp to –2170 bp of the ceh-34 promoter are shown. M lineage expression of the gfp::ceh-34 reporter is indicated to the right of each oligonucleotide sequence. We observed rare cases in which mutating FoxF site 1 resulted in the transient ectopic expression of gfp::ceh-34 in the posterior sister cells of M-derived CCs (asterisks; 3.7%, n=109). (B) ceh-34(tm3733) L1 larvae lack functional CCs, as a myo-3p::secreted CC::GFP reporter is diffused throughout the pseudocoelom. (C,D) Transgenic ceh-34(tm3733) adults expressing pNMA94(C) or pNMA116(D). pNMA94 restores the presence of M-derived CCs and the functionality of the embryonic CCs (C), whereas pNMA116 rescues neither (D). (E-G) let-381(RNAi) results in the loss of gfp::ceh-34 expression in the M lineage (E). (F) M lineage cells labeled by anti-FOZI-1. (G) Corresponding merged image of E and F. (H) EMSA using end-labeled NMA-269/270, purified LET-381 protein, LET-381 antibody and unlabeled NMA-269/270 competitor oligonucleotides. Lanes 5 and 6 have competitors in 200- and 2000–fold excess, respectively. (I) Gel-shift using end-labeled mutant oligonucleotides as indicated above each lane. (J) Gel shift using end-labeled NMA-269/270 and different unlabeled competitior oligonucleotides. An excess of competitors relative to the labeled oligos was used: 500-fold (lanes 3, 8), 1000-fold (lanes 4, 9), 2000-fold (lanes 5, 10), 5000-fold (lanes 6, 11) and 10,000-fold (lanes 7, 12-15). Asterisks (**) in H-J denote binding by degraded LET-381 proteins.

Fig. 4.

LET-381 is required for dorsal M lineage CC fates. (A,B) Early M lineage in wild-type (A) and let-381(RNAi) (B) animals. (C,D) L4440-RNAi treated control (C) and let-381(RNAi) (D) adults. CCs are visualized using intrinsic CC::gfp, with embryonic CCs labeled by arrowheads and M-derived CCs by arrows. Type I vulval muscles are visualized using egl-15::gfp, denoted by asterisks. M-derived CCs are missing and extra vulval muscles are present in let-381(RNAi) animals (D). (E,F) Sterile let-381 deletion mutants, gk302 (E) and tm288 (F). let-381 mutants have extra type I vulval muscles (E) and lack functional CCs, as the myo-3p secreted CC::GFP is diffused throughout the pseudocoelom (F).

Fig. 5.

The expression of LET-381 in the M lineage precedes that of CEH-34. Images are lateral views with anterior to the left and dorsal up. (A) Schematic of the let-381 translational reporter. Regions deleted in gk302 and tm288 are shown and drawn to scale. (B-D) Representative images of live embryos (B,C) and fixed late L1 larva (D) expressing let-381::gfp. (E-N) The left side of three L1 larvae double-labeled with LET-381::GFP (E,I), anti-FOZI-1 antibody (F,J) and DAPI (H) at the 14-M (E-H) and 16-M stage (I-K). (G,K) Merged images of E and F and of I and J, respectively. (L-N) An L1 larva double-labeled with anti-LET-381 (L) and anti-FOZI-1 antibodies (M) at the 16-M stage. (N) A merged image of L and M. (O) Summary of LET-381 expression in the M lineage, with LET-381-positive cells in green circles and CEH-34-positive cells outlined in black. (P-S) sma-9(cc604) (P,Q) and lin-12(n676n930ts) (R,S) animals double-labeled with anti-LET-381 (P,R) and anti-FOZI-1(P,S) antibodies at the 16-M stage. LET-381 is not expressed in the M lineage of cc604 mutants (P,Q) but is ectopically expressed in ventral M lineage cells of n676n930ts animals (R,S).

Fig. 6.

LET-381 and CEH-34 might directly regulate CC specification and differentiation. (A-C) Animals with three (A) and six (B,C) M-derived CCs. (C) Magnification of five CCs from the animal in B. (D) Forced expression of ceh-34 and eya-1 using the hlh-8 promoter leads to an increase in the number of M-derived CCs in two independent lines. (E) Heat-shock-induced expression of let-381 in the same transgenic lines containing hlh-8p::ceh-34+hlh-8p::eya-1 leads to higher efficiency and even more M-derived CCs. (F-I) Schematic of CC-specific gene promoters with putative FoxF-, in blue, and Six2-, in red (Noyes et al., 2008) and purple (Hu et al., 2008), binding sites indicated in capital letters. Mutated nucleotides are in lower case letters. Expression in embryonic and M-derived CCs is indicated on the right of each construct: +++, high level of GFP expression; ++, 4-fold reduction in GFP expression; +, 6-fold reduction in GFP expression, –, no GFP expression. (J-M) Representative images of adults listed in G. (J) A wild-type cup-4p::gfp showing six CCs labeled with GFP; (K) FoxF-mutated (mFoxF) cup-4p::gfp (pNMA143) with no GFP expression in the M-derived CCs; (L) Six2-mutated (mSix2) cup-4p::gfp (pNMA146) with six CCs labeled and (M) mFoxF + mSix2 cup-4p::gfp (pNMA150) with no GFP expression in any CC. (N) EMSA using end-labeled wild-type and mutant oligonucleotides as indicated above each lane. Competitor indicates presence of 600-fold excess of unlabeled oligonucleotides, identical to the oligonucleotides used for the gel shift. (O) Gel shift using end-labeled NMA-325/326 and different unlabeled competitor oligonucleotides (U-NMA-). An excess of competitors relative to the labeled oligonucleotides was used: 60-fold (lanes 3, 8), 330-fold (lanes 4, 9, 13), 600-fold (lanes 5, 10, 14), 1000-fold (lanes 6, 11, 15) and 1500-fold (lanes 7, 12).

Fig. 7.

LET-381 and CEH-34 function in a feed-forward manner to regulate mesoderm fates. Gene regulatory networks for cell fate specification in the dorsal M lineage are shown for each left/right pair that will adopt either a CC or BWM fate. LET-381 directly regulates the expression of the CC specification factor CEH-34, and both genes function in a feed-forward manner to regulate a number of CC differentiation genes and to suppress the BWM fate. The expression of ceh-34 in M.d(l/r)pa requires both LET-381 and additional factors, either activators in M.d(l/r)pa and M.d(l/r)pp (shaded in yellow) or repressors in M.d(l/r)aa and M.d(l/r)ap (shaded in green). LET-381 also acts to inhibit the specification of SM-specific genes in the dorsal M lineage. let-381 is regulated by dorsoventral signaling mechanisms (SMA-9–Sma/Mab) and mesoderm intrinsic factors (HLH-1, FOZI-1 and MAB-5), which might also regulate ceh-34 expression independently of LET-381. Thick blue lines represent likely direct regulatory relationships, whereas black lines represent relationships based solely on genetic data, and do not distinguish between direct and indirect. Gray lines and text indicate a lack of expression in the indicated cell.