The forkhead transcription factor UNC-130/FOXD integrates both BMP and Notch signaling to regulate dorsoventral patterning of the C. elegans postembryonic mesoderm

Abstract

The proper development of a multicellular organism requires precise spatial and temporal coordination of cell intrinsic and cell extrinsic regulatory mechanisms. Both Notch signaling and bone morphogenetic protein (BMP) signaling function to regulate the proper development of the C. elegans postembryonic mesoderm. We have identified the C. elegans FOXD transcription factor UNC-130 as a major target functioning downstream of both BMP signaling and Notch signaling to regulate dorsoventral patterning of the postembryonic mesoderm. We showed that unc-130 expression in the postembryonic M lineage is asymmetric: its absence of expression in the dorsal side of the M lineage requires the antagonism of BMP signaling by the zinc finger transcription factor SMA-9, while its expression in the ventral side of the M lineage is activated by LIN-12/Notch signaling. We further showed that the regulation of UNC-130 expression by BMP signaling and Notch signaling is specific to the M lineage, as the ventral expression of UNC-130 in the embryonically-derived bodywall muscles was not affected in either BMP pathway or Notch pathway mutants. Finally, we showed that the function of UNC-130 in the M lineage is independent of UNC-129, a gene previously shown to function downstream of and be repressed by UNC-130 for axon guidance. Our studies uncovered a new function of UNC-130/FOXD in the C. elegans postembryonic mesoderm, and identify UNC-130 as a critical factor that integrates two independent spatial cues for the proper patterning and fate specification of the C. elegans postembryonic mesoderm.

Keywords: BMP; Coelomocyte (CC); Dorsoventral patterning; FOXD; LIN-12; M lineage; Mesoderm; Notch; SMA-9; Schnurri; Sex myoblast (SM); unc-129; unc-130.

Figure 1. SMA-9, BMP signaling and LIN-12/Notch signaling regulate M lineage dorsoventral patterning

Diagrams showing the M lineage (A-D) and the locations of CCs (E-H) in wild-type or sma-9(0); BMP pathway(0) (A, E), sma-9(0) (B, F), lin-12(0) (C, G), and lin-12(0); sma-9(0) (D, H) animals. Blue arrowheads point to the two M lineage-derived CCs. BWM: body-wall muscle, CC: coelomocyte, SM: sex myoblast. d: dorsal, v: ventral, l: left, r: right, a: anterior, p: posterior.

Figure 2. unc-130 is asymmetrically expressed in the M lineage on the ventral side

(A-G) UNC-130∷GFP(evIs120) expression (B, E) in the undifferentiated M lineage cells marked by hlh-8∷NLS∷mCherry∷lacZ (A, D) at the 2-M stage (A-C), and the 16-M stage (D-F). C and F show the corresponding merged images. UNC-130∷GFP is not detectable in the M lineage at the 2-M stage (A-C), but detectable in all the ventral M lineage cells (arrowheads in E) at the 16-M stage (D-F). Note that the posterior two cells, M.vlpa and M.vlpp, show stronger GFP signal in panel E. The arrows in panels B and E point to embryonically-derived BWMs that express UNC-130∷GFP. (G) Summary of UNC-130∷GFP expression in the early M lineage, based on both the integrated UNC-130(evIs120) translational reporter and the endogenously tagged and functional UNC-130∷GFP generated via CRISPR, unc-130(gv45). UNC-130∷GFP-expressing cells are in green. Darker green means stronger UNC-130∷GFP expression. UNC-130∷GFP expression can be detected in the ventral M lineage starting at the 8-M stage. (H-N) unc-129p∷GFP (I, L) expression pattern at the 2-M stage (H-J) and the 14-M stage (K-M). The undifferentiated M lineage cells are marked by hlh-8∷NLS∷mCherry∷lacZ (H, K). J and M show the corresponding merged images. unc-129p∷GFP is not expressed in the M lineage, but is expressed in the dorsal embryonically-derived BWMs (open arrowheads in I and L). (N) Summary of lack of unc-129p∷GFP expression in the M lineage. All images in this figure and subsequent figures are shown with anterior to the left and dorsal up. Only images of the left side of the animal are shown in panels A-F and H-M. Similar expression pattern is also seen in the equivalent cells on the right side (not shown).

Figure 3. SMA-9 antagonizes the BMP pathway to repress unc-130 expression in the early M lineage and in dorsal M lineage cells

UNC-130∷GFP(evIs120) expression (B, E, I, L, P, S) in the undifferentiated M lineage cells marked by hlh-8∷NLS∷mCherry∷lacZ (A, D, H, K, O, R) in wild-type (A-G), sma-9(cc604) (H-N) and sma-3(jj3); sma-9(cc604) (O-U) animals at the 2-M stage (A-C, H-J, O-Q), and the 16-M stage (D-F, K-M, R-T). The corresponding merged images are shown in C, F, J, M, Q and T. Panels G, N and U show summaries of the M lineage expression pattern of UNC-130∷GFP in the different genetic background. The arrows in panels B, E, I, L, P and S point to embryonically-derived BWMs that expresses UNC-130∷GFP. The arrowheads in panels E, I, L and S point to M lineage cells that express UNC-130∷GFP. Only images of the left side of the animal are shown in panels D-F, K-M and R-T. Similar expression pattern is also seen in the equivalent cells on the right side (not shown). Note that unlike wild-type or sma-3(jj3); sma-9(cc604) animals which only have UNC-130∷GFP expression in the ventral M lineage starting at the 8-M stage (G, U), UNC-130∷GFP expression can be detected in the early M lineage cells, and both ventral and dorsal M lineage cells in sma-9(cc604) mutants (N).

Figure 4. UNC-130 functions in parallel to the BMP pathway to regulate body size

(A) Growth curves of wild-type (N2), unc-130(ev505) and sma-3(jj3) worms at 20°C. Sixty to 120 animals were examined for each genotype at each time point. The body lengths of unc-130(ev505) animals are significantly different (P<0.0001, unpaired two-tailed Student's t-test) from the body lengths of wild-type and sma-3(jj3) animals at 48, 72 and 96 hours, respectively. (B) Relative body length of worms with various genotypes at 96 hours post-plating (see Materials and methods). The body length of unc-130(ev505) worms was normalized to 1.0. Similar results were obtained using worms at 72 hours post-plating (data not shown). *** P<0.0001 (unpaired two-tailed Student's t-test). Error bars represent standard deviation.

Figure 5. LIN-12/Notch signaling promotes unc-130 expression in the ventral M lineage cells

UNC-130∷GFP expression (B, F, J, N) in the undifferentiated M lineage cells marked by hlh-8∷NLS∷mCherry∷lacZ (A, E, I, M) in wild-type (A-D), lin-12(n941) (E-H), lin-12(n941)/+; sma-9(cc604) (I-L) and lin-12(n941); sma-9(cc604) (M-P) animals at the 16-M stage. The corresponding merged images are shown in C, G, K and O. Panels D, H, L and P show summaries of the M lineage expression pattern of UNC-130∷GFP in the different genetic background. UNC-130∷superGFP (gv45) was used to assay for UNC-130∷GFP expression in lin-12(n941) (E-G), lin-12(n941)/+; sma-9(cc604) (I-K) and lin-12(n941); sma-9(cc604) animals (M-O), because the UNC-130∷GFP(OE) transgene evIs120 (shown in A-C) appeared to map to the same chromosome as lin-12. The arrows in panels B, F, J and N point to embryonically-derived BWMs that expresses UNC-130∷GFP. The arrowheads in panels B, J and N point to M lineage cells that express UNC-130∷GFP. Only images of the left side of the animal are shown. Similar expression pattern is also seen in the equivalent cells on the right side (not shown). Note the lack of M lineage expression of UNC-130∷GFP in lin-12(n941) animals (F), and the dorsal, but relatively fainter, M lineage expression of UNC-130∷GFP in lin-12(n941); sma-9(cc604) animals (N).

Figure 6. A model for how UNC-130 integrates both BMP and Notch signaling to regulate dorsoventral patterning of the M lineage

We propose that in the dorsal side of the M lineage, SMA-9 represses DBL-1/BMP-dependent expression of UNC-130, while in the ventral side of the M lineage, UNC-130 expression is activated by LIN-12/Notch signaling. When expressed, UNC-130 acts as a transcriptional repressor to repress the expression of UNC-130 target genes, which promote the CC fate and repress the SM fate. The double negative gate in the dorsal side is sufficient to allow for proper CC specification. In the ventral side of the M lineage, UNC-130 likely functions together with additional LIN-12/Notch target genes to allow for the proper specification of the SMs. Solid lines do not imply direct regulatory relationship. Grey color is used to indicate lack of expression of a specific gene or fate. X refers to additional LIN-12 target(s) involved in SM fate specification.