The Caenorhabditis elegans synthetic multivulva genes prevent ras pathway activation by tightly repressing global ectopic expression of lin-3 EGF

Abstract

The Caenorhabditis elegans class A and B synthetic multivulva (synMuv) genes redundantly antagonize an EGF/Ras pathway to prevent ectopic vulval induction. We identify a class A synMuv mutation in the promoter of the lin-3 EGF gene, establishing that lin-3 is the key biological target of the class A synMuv genes in vulval development and that the repressive activities of the class A and B synMuv pathways are integrated at the level of lin-3 expression. Using FISH with single mRNA molecule resolution, we find that lin-3 EGF expression is tightly restricted to only a few tissues in wild-type animals, including the germline. In synMuv double mutants, lin-3 EGF is ectopically expressed at low levels throughout the animal. Our findings reveal that the widespread ectopic expression of a growth factor mRNA at concentrations much lower than that in the normal domain of expression can abnormally activate the Ras pathway and alter cell fates. These results suggest hypotheses for the mechanistic basis of the functional redundancy between the tumor-suppressor-like class A and B synMuv genes: the class A synMuv genes either directly or indirectly specifically repress ectopic lin-3 expression; while the class B synMuv genes might function similarly, but alternatively might act to repress lin-3 as a consequence of their role in preventing cells from adopting a germline-like fate. Analogous genes in mammals might function as tumor suppressors by preventing broad ectopic expression of EGF-like ligands.

Figure 1. n4441 is an allele of lin-3.

(A) Genetic map showing n4441 on LGIV. n4441 was localized between dpy-13 and unc-30 by three-factor mapping. SNP mapping using polymorphisms present in the CB4856 strain further localized n4441 to a 661 kb region between the SNPs dbP6 at 10909553 and uCE4-1148 at 11570158 of LGIV (data not shown). (B) The lin-3 locus. The lin-3a isoform (Wormbase web site, http://www.wormbase.org, release WS200, Mar 20 2009) is shown. Solid boxes, exons; open boxes, UTRs. The start of the transcript is indicated by an arrow. Arrowheads indicate the locations of mutations. n4951 is a nonsense mutation that truncates LIN-3 after 26 amino acids, and n4929 is a missense mutation that converts an arginine to a lysine at amino acid 347. (C) n4441 is a G-to-A mutation at nucleotide 30904 of cosmid F36H1, 211 bp upstream of the lin-3 transcript. No other mutations were present in n4441 mutants in the region shown in (B). (D) A lin-3 loss-of-function mutation suppresses the n4441 synMuv phenotype in cis but not in trans. lin-3(n1059) is a nonsense mutation in lin-3 . lin-3(n4441 n4951) and lin-3(n4441 n4929) heterozygotes also carried the nT1[qIs51] translocation. All animals were grown at 20°C. Animals were scored as Muv if any ventral ectopic protrusions were observed. n, total number of animals scored.

Figure 2. The lin-3(n4441) mutation specifically prevents repression of lin-3.

(A) The lin-3(n4441) mutation is located 211 bp upstream of the lin-3 transcript. The gene F36H1.12 is upstream of lin-3 in the opposite orientation, and lin-3(n4441) is located 465 bp from the predicted F36H1.12 transcript. Solid boxes, exons; open boxes, UTRs. (B) lin-3 mRNA levels in lin-3(n4441) single and double mutants. As reported previously , lin-3 mRNA levels are substantially increased in lin-15AB double mutants but not in lin-15A or lin-15B single mutants. Like other class A synMuv mutations, lin-3(n4441) caused a substantial increase in lin-3 mRNA levels only in a class B synMuv mutant background. Realtime RT-PCR experiments were performed using RNA harvested at the late L2 or early L3 stage from each strain shown. Relative lin-3 mRNA levels were normalized to the levels of mRNA encoding the ribosomal protein subunit rpl-26 using the ΔΔCt method . The means and standard deviations of relative lin-3 mRNA levels from two independent trials are shown. The lin-15A(n767), lin-15B(n744) and lin-15AB(e1763) alleles were used in this experiment. (C) F36H1.12 mRNA levels in synMuv single and double mutants. No combination of lin-3(n4441), lin-15A, and lin-15B mutations affected F36H1.12 mRNA levels. Realtime RT-PCR experiments were performed using RNA harvested at the late L2 or early L3 stage from each strain shown. Relative F36H1.12 mRNA levels were normalized to the levels of mRNA encoding the ribosomal protein subunit rpl-26 using the ΔΔCt method. The means and standard deviations of relative F36H1.12 mRNA levels from two independent trials are shown.

Figure 3. The synMuv genes prevent widespread ectopic expression of lin-3 mRNA.

FISH of lin-3 mRNA in late L2 to early L3 animals. Each dot represents a single mRNA molecule . lin-3 mRNAs are shown in red, and 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclei is shown in blue. The images shown are maximum intensity projections of a z-stack of images. The anchor cell (AC) is indicated by an arrowhead in each panel. (A) Wild type. lin-3 mRNA is expressed in the pharynx, anchor cell, and germline and is tightly repressed elsewhere. (B) lin-15A(n767). There is a low level of ectopic lin-3 expression, with approximately 60 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline. (C) lin-15B(n744). There is a very low level of ectopic lin-3 expression, with approximately 10 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline. The large bright spot at the edge of the head is outside the animal. (D) lin-15AB(e1763). lin-3 is ectopically expressed throughout the animal, with approximately 900 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline.

Figure 4. Multiple class B synMuv mutations have similar effects on lin-3 mRNA expression.

FISH of lin-3 mRNA in late L2 to early L3 animals. Each dot represents a single mRNA molecule . lin-3 mRNAs are shown in red, and 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclei is shown in blue. The images shown are maximum intensity projections of a z-stack of images. The anchor cell (AC) is indicated by an arrowhead in each panel. Each mutant displayed ectopic lin-3 mRNA expression throughout the animal in most if not all tissues. (A) lin-36(n766); lin-15A(n767). (B) lin-52(n771); lin-15A(n767). (C) lin-53(n833); lin-15A(n767).

Figure 5. The lin-3(n4441) mutation causes widespread ectopic expression of lin-3 mRNA.

FISH of lin-3 mRNA in late L2 to early L3 animals. Each dot represents a single mRNA molecule . lin-3 mRNAs are shown in red, and 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclei is shown in blue. The images shown are maximum intensity projections of a z-stack of images. The anchor cell (AC) is indicated by an arrowhead in each panel. (A) lin-3(n4441). there is a low level of ectopic lin-3 expression, with approximately 45 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline. (B) lin-3(n4441); lin-15B(n744). lin-3 is ectopically expressed throughout the animal, with approximately 1000 ectopic copies of lin-3 mRNA seen outside the pharynx, anchor cell, and germline.