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First published online 11 June 2008
doi: 10.1242/dev.017046

Development 135, 2373-2382 (2008)
Published by The Company of Biologists 2008
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dmd-3, a doublesex-related gene regulated by tra-1, governs sex-specific morphogenesis in C. elegans

D. Adam Mason1, Jeremy S. Rabinowitz2 and Douglas S. Portman1,2,3,*

1 Center for Neural Development and Disease, University of Rochester, Rochester, NY 14642, USA.
2 Department of Biology, University of Rochester, Rochester, NY 14642, USA.
3 Department of Biomedical Genetics, University of Rochester, Rochester, NY 14642, USA.


Figure 1
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  		Fig. 1. dmd-3 acts with mab-3 to direct cell fusion and retraction of the male tail. (A) The dmd-3 locus and dmd-3 reporter genes. Thin gray lines indicate the dmd-3 promoter and 3' UTR; gray boxes mark exons. The two DM domains, the E(ht) enhancer element and the deletions in the dmd-3(tm2863) and dmd-3(ok1327) alleles are indicated. (B) DIC images of the tails of a wild-type adult male (i), wild-type adult hermaphrodite (ii), dmd-3(ok1327) adult male (iii,iv), mab-3(e1240) adult male (v) and mab-3(e1240); dmd-3(ok1327) adult male (vi). Representative examples of the two classes (short and long) of Lep tails observed in dmd-3 males are shown (iii,iv). (C) Individual wild type (i-iii), dmd-3 mutant (iv-vi) and mab-3; dmd-3 double mutant (vii-ix) L4 males were observed periodically by DIC microscopy. Tail-tip retraction is indicated by a bold white arrow; anterior tail retraction is indicated by a bold black arrow. Thin white and black arrows indicate the partial tail tip retraction and partial anterior tail retraction, respectively, seen in dmd-3 larvae. (D) Confocal images of wild-type (i,ii), dmd-3 mutant (iii,iv) and mab-3; dmd-3 mutant L4 males (v,vi) expressing the apical junction marker AJM-1::GFP. The broken line indicates the larval cuticle. Solid arrowheads indicate intact cell boundaries; broken arrowheads indicate cell fusions. (E) AJM-1::GFP/DIC images of the unretracted tail tips of an adult hermaphrodite (i), an adult dmd-3 male (ii) and an adult mab-3; dmd-3 male (iii). Arrowheads indicate intact cell boundaries.

 

Figure 2
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  		Fig. 2. dmd-3 and mab-3 reporters are sex-specifically expressed in the tail tip hypodermis. (A) dmd-3::YFP (fsIs2) expression in an L4 male (i) demonstrating expression in the hindgut, B lineage, tail tip (hyp8-11) and hyp13. The future position of the dmd-3-expressing RnA neurons is marked with a broken white line. An L4 hermaphrodite carrying dmd-3::YFP (ii) demonstrates the male-specificity of expression. In late-L3 (iii) and L4 males, dmd-3::YFP is expressed in the linker cell of the developing male gonad. (B) Schematic diagrams of the progression of tail tip morphogenesis. Circles indicate tail tip hypodermal cells; green shading reflects the intensity of dmd-3 expression. Solid lines between hypodermal cells indicate intact boundaries and broken lines indicate cell fusion events. The thick black line represents the L4 cuticle, whereas the thick gray line indicates the newly formed adult cuticle. See Materials and methods for a description of L4 staging criteria. (C,D) Confocal microscopy images of L4 males (i-vi) and hermaphrodites (vii) carrying dmd-3::YFP (fsIs2) (C) or MAB-3::GFP (fsIs10) (D). Numbers indicate tail tip hypodermal cells. hg and RnA indicate the hindgut and ray RnA neurons, respectively. The larval cuticle is outlined with dashed lines; dotted lines indicate the developing adult cuticle.

 

Figure 3
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  		Fig. 3. dmd-3 is likely to be a direct target of TRA-1A and is sufficient to trigger hermaphrodite tail tip retraction. (A) DIC images of an adult wild type X0 male (i), an adult tra-1(e1099) XX pseudomale (ii), an adult dmd-3(ok1327) X0 male (iii) and an adult tra-1(e1099); dmd-3(ok1327) XX pseudomale (iv). Overlaid dmd-3::YFP/DIC images of a wild-type late mid-L4 X0 male (v) and a tra-1(e1099) late mid-L4 XX pseudomale (vi) expressing dmd-3::YFP (fsIs2). (B) Sequences of the TRA-1A consensus binding site (Zarkower and Hodgkin, 1993Go; Yi et al., 2000Go), the putative TRA-1A binding site in the dmd-3 promoter and the {Delta}TRA-1 mutation. The single nucleotide difference between the consensus site and the dmd-3 site is shown in green; nucleotides mutated in {Delta}TRA-1 are shown in red. (C) DMD-3::GFP/DIC (i, ii, iv, v, vii, viii, xi, xii), DMD-3::GFP/MH27 antibody staining (red) (ix, xiii) and DIC (iii, vi, x, xiv) images of late L3 (i, iv, vii and xi), mid-L4 (ii, v, viii, ix, xii, and xiii) and adult (iii, vi, x, xiv) males (i-vi) and hermaphrodites (vii-xiv) carrying E(ht)::DMD-3::GFP (fsIs9) (i-iii and vii-x) or E(ht){Delta}TRA-1::DMD-3::GFP (fsIs7) (iv-vi and xi-xiv). Expression of E(ht){Delta}TRA-1::DMD-3::GFP in hermaphrodite tail tip hypodermal cells (numbers in xii) and hindgut (hg) (white arrowheads in xi and xii) is indicated. Gray arrowheads (vii and viii) indicate expression in phasmid neurons (ph). Solid red arrowheads (ix and xiii) mark intact hyp8-11 boundaries; the dashed red arrowhead (xiii) indicates cell fusion. White arrows (ii, iv, v, xi and xiii) mark hyp8-11 retraction.

 

Figure 4
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  		Fig. 4. dmd-3 expression in the tail tip is regulated by lin-41, Wnt signaling and a positive-feedback loop. (A) dmd-3::YFP (fsIs3) expression in wild-type (i) and lin-41(bx42gf) (ii) mid-L4 males. The arrowhead in ii indicates the absence of dmd-3::YFP expression in hyp10 in lin-41(bx42) males. (B) dmd-3::YFP (fsIs3) expression in wild type (i) and lin-41(ma104lf) (ii) late L3 males. The arrow in ii highlights the precocious tail tip retraction in lin-41(ma104) males. (C) dmd-3::YFP expression in wild type (i-iii), lin-44(n1792) (iv-vi) and tlp-1(bx85) (vii-ix) L4 males of the indicated stages. (D) dmd-3::YFP (fsIs2) expression in wild type (i), dmd-3(ok1327) (ii), mab-3(e1240) (iii) and mab-3(e1240); dmd-3(ok1327) (iv) mid-L4 males. In A,C, the larval cuticle is shown with dashed lines. Dotted lines indicate the developing adult cuticle.

 

Figure 5
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  		Fig. 5. EFF-1 is regulated by dmd-3 and mab-3. (A) AJM-1::GFP (syIs78)/DIC and DIC images of wild-type and eff-1(ok1021) L4 (i,ii) and adult (iii,iv) males. The broken green arrowhead indicates cell fusion; the solid green arrowhead marks intact cell boundaries. The white arrows indicate tail tip retraction. (B) Tail tip expression of an EFF-1::GFP translational reporter (fsEx135) in male (i-v) and hermaphrodite (vi) larvae. (C) Expression of EFF-1::GFP in wild type (i), mab-3 (ii), dmd-3 (iii,iv) and dmd-3; mab-3 (v) mid-L4 males. The examples in iii and iv represent the range of EFF-1::GFP expression levels seen in dmd-3 mutants. (D) Categorization of EFF-1::GFP fluorescence intensity in wildtype (n=39), mab-3 (n=24), dmd-3 (n=61) and mab-3; dmd-3 (n=33) mid-L4 males and wild-type mid-L4 hermaphrodites (n=31) based on confocal images. (E) Expression of EFF-1::GFP::outron::mCherry in a wild-type mid-L4 male (i-iii) and hermaphrodite (iv-vi). GFP (i,iv), mCherry (ii,v) and GFP/mCherry (iii,vi) overlays are shown.

 

Figure 6
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  		Fig. 6. dmd-3 and mab-3 occupy the central node of the tail tip retraction network. Our results support a model in which multiple upstream regulatory pathways converge on dmd-3 and mab-3 to regulate the temporal (red), sexual (green) and cell-type (yellow) specificity of tail tip morphogenesis. Temporal specificity is imparted by the heterochronic pathway via let-7 and lin-41. Sexual specificity arises by the regulation of dmd-3 by tra-1, which is likely to be direct. At least two pathways can be thought of as cell-type determinants: the yellow `1' depicts the induction phase of dmd-3 expression in hyp8-11, while the yellow `2' indicates the maintenance and amplification phase. Downstream of dmd-3 and mab-3 lie multiple effectors of morphogenesis, including eff-1. The targets that mediate hyp8-11 retraction are unknown, as is the upstream regulatory pathway that initiates dmd-3 expression (1) in the tail tip. Black arrows and bars indicate regulatory events that are likely to be direct. The solid gray bar indicates indirect regulation. Broken gray and black arrows and bars indicate steps for which the molecular mechanism is unknown. Thin gray arrows indicate that the function of mab-3 in tail tip morphogenesis is secondary to that of dmd-3.

 

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© The Company of Biologists Ltd 2008