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First published online 18 March 2009
doi: 10.1242/dev.028472

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C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV

¹ Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Toronto, M5G 1X5, Canada.
² Genome Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
³ RIKEN Center for Developmental Biology, Kobe, 650-0047, Japan.
⁴ Department of Molecular Genetics, University of Toronto, Toronto, M5S 1AB, Canada.

View larger version (30K): [in this window] [in a new window]   Fig. 1. mig-6 class-l mutations reduce the rate of DTC migration. (A) Schematic of an adult C. elegans hermaphrodite: DTCs (green), distal gonad arms with no eggs (gray), proximal arms with eggs (gray ovals), spermathecae (red) and fertilized eggs (white ovals) are shown. (B) Images of gonad arms and DTC (arrows) relative to the vulva (triangle) of N2 (left panels) (in two focal planes for the adult) and class-l mig-6(oz113) (right panels) at mid-L3, mid-L4 and adult stages. Asterisks indicate posterior. Scale bars: 50 µm. Black bar applies to top four panels. (C) Lengths of anterior and posterior gonad arms of individual animals (from a partially synchronized population) are plotted against developmental stage, as determined by vulva shape. Rates of movement for mutants (grouped) and controls were obtained by the slope of the line, as determined by the least squares method.

View larger version (132K): [in this window] [in a new window]   Fig. 2. mig-6 class-s mutations cause phase 2 DTC migration defects. Broken and unbroken arrows trace phase 1 and subsequent paths of DTC migration, respectively. DTCs in class-s mig-6(ev701) heterozygous hermaphrodites show (A) phase 2D, (B) phase 2V and (C) phase 2M (meandering - see Results) defects. The vulva (triangles) and the posterior of the animal (asterisks) are also indicated. Scale bars: 20 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Cloning and characterization of the mig-6/papilin gene. (A) mig-6 was mapped to the region between two deficiencies, sDf35 and sDf20, on LG V. (B) Schematic drawing of cosmid clones (white bars with single black region denoting the mig-6 gene) and subclones (unbroken lines representing the KpnI and FspI fragments), with rescue results with these DNA fragments shown on the right and exon/intron organization of mig-6 shown below (exons and 3' UTRs are black and white boxes, respectively). Rescue of mig-6 class-s (ev701) lethality and mig-6(e1931) sterility were determined as described in the Materials and methods. These experiments were internally controlled by scoring transgenic Dpy animals and their non-transgenic Dpy siblings. The number of rescuing transgenic lines over the number of lines tested is indicated in parentheses. DTC defects, which in mig-6(s) and mig-6(l) alleles manifest as clear patches in the body, were also rescued (denoted `+'). (C) Northern blot analysis of 3 µg of mRNA from a mixed-stage population of wild-type C. elegans. A probe predicted to hybridize to both mig-6S and mig-6L detected two bands (lane 1). Longer exposure with a mig-6L-specific probe (exon 14-17) shows a single band at 7.7 kb (lane 2). (D) Schematic diagrams of predicted protein structures of MIG-6S and MIG-6L with sites of molecular lesions found in class-l (blue) and class-s (red) alleles. The extent of the ev788 deletion is indicated. The region corresponding to the peptide used for raising an antibody is indicated by an underlying red bar.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Expression of mig-6 encoded mRNAs and proteins. (A,B) In situ hybridization using a mig-6L-specific probe showing DTC (d) specific expression (A), and using a probe common for mig-6S and mig-6L showing expression in the body wall muscles and other unidentified cells (B). (C-E) From L2 to adult stages, a mig-6 promoter::gfp transcriptional reporter construct is expressed in DTCs (d) (C), in body wall muscles (m), in the CAN neuron (can) (D), in the head mesodermal cell (hmc), in GLR cells (glr) and in coelomocytes (cc) (E). (F,G) Anti-MIG-6 antibody staining of late embryos (released from their shells) shows MIG-6 protein is expressed by body wall muscle primordium (green) in the wild type (F) but not in mig-6(ev788) (G), whereas control antibody, anti-SAD-1 (orange) stains nerve ring and ventral nerve cord in both strains. (H) In early larval stages, anti-MIG-6 antibody stains basement membranes of the gonad primordium (g), pharynx (ph) and intestine (i). This staining persists throughout larval and adult stages. (I,J) Anti-MIG-6 antibody stains basement membrane of the gonad and migrating DTCs (d) of L3 (I) and L4 (J) stages. Enlargement in I shows staining on the DTC. (K,L) The same basement membranes and DTCs are stained by anti-MIG-6 antibody in class-s mig-6(ev700) (K) and in mig-6(k177), which displays a phase 2M DTC defect (L) (see also Fig. S2 in the supplementary material). Vulvae are indicated by white triangles. Asterisks indicate posterior. Dorsal is upwards in all panels. Scale bars: 20 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effect of mig-6s mutations on MIG-17::GFP distribution. (A) Double heterozygotes of mig-6 and mig-17 display enhancement of class-s DTC defects. Black and gray bars represent percentages of anterior and posterior DTCs, respectively, with defective migrations (total of phase 2D, 2V and 2M defects - see Fig. 2). Lines on the end of the bars indicate standard errors of the expected mean. Numbers of animals scored are indicated towards the right. Asterisks indicate comparisons in which P<0.001 by the chi-square test. (B-E) Localization of MIG-17::GFP is visualized by anti-GFP antibody staining of whole-mount animals (B,D) and frozen cross-sections (green in C,E). Lower panels are Nomarski images of the same animal in the upper panel. MIG-17::GFP in a control genetic background displays uniform distribution on the gonad surface in lateral view (B) and on the gonad, intestine and body wall muscle periphery in cross-section (arrowheads in C). MIG-17::GFP in class-s mig-6(ev700) displays patchy staining along the phase 2 DTC path of a gonad arm (D) and outside of gonad membranes (arrows) in cross-section (E). Staining on the remaining gonad and intestine surfaces is weaker in the mutant genetic background (arrowhead in E) than in the wild type (C) (equal exposure times were used). Eight out of 15 animals in the mutant background showed accumulation of MIG-17::GFP away from the gonad and gut surfaces (arrow), whereas only one of 28 animals in the wild-type background showed this pattern. Phalloidin staining of body wall muscle actin filaments is orange in cross-sections. Positions of intestine (i) and gonad arms (g) are indicated. Scale bars: 20 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Effect of collagen IV mutations. (A,B) Temperature-sensitive collagen IV mutant emb-9(g23) cultured at non-permissive temperature from L2 to L4 larval stages showed slow gonad elongation (B), which is similar to class-l defect of mig-6(e1931) (A). Scale bars: 20 µm. (C) Reduction of collagen IV alpha1 by emb-9-specific RNAi (by feeding) or by combining with a heterozygote for the emb-9 null allele suppressed the class-s DTC phenotype of ev701 heterozygotes, as did a mutation of let-2/collagen IV alpha2. Balancer eT1 (aka unc-36) and eT1* (equals eT1 with a let-500 mutation in the balancing translocation) significantly enhances the penetrance of DTC migration defect in class-s heterozygote for unknown reasons. Black and gray bars represent percentages of anterior and posterior DTCs, respectively, that show defective migrations (total of phase 2D, 2V and 2M defects). Numbers of animals scored are indicated towards the right. Asterisks indicate comparisons in which P<0.001 by the Chi-square test.

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