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First published online March 1, 2004
doi: 10.1242/10.1242/dev.01012

Development 131, 1425-1436 (2004)
Published by The Company of Biologists 2004
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A forkhead protein controls sexual identity of the C. elegans male somatic gonad

Weiru Chang1,2, Christopher Tilmann3,4, Kara Thoemke1,2, Finn-Hugo Markussen3,4, Laura D. Mathies3,4,*, Judith Kimble3,4 and David Zarkower1,2,{dagger}

1 Department of Genetics, Cell Biology and Development, University of Minnesota, 321 Church Street SE, Minneapolis, MN 55455, USA
2 Developmental Biology Center, University of Minnesota, 321 Church Street SE, Minneapolis, MN 55455, USA
3 Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706-1544, USA
4 Howard Hughes Medical Institute, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706-1544, USA



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  		Fig. 1. fkh-6 is required for gonadal development. (A) Molecular basis of fkh-6 mutations. Boxes indicate exons; lines indicate introns. The forkhead domain (black) is encoded by two exons. Arrows indicate locations of mutations. The ez16 and q641 alleles are both single base changes, indicated in red: ez16 affects the ATG start codon and q641 affects the predicted splice donor site of the second intron. The q641 transcript is spliced aberrantly, resulting in a two amino acid insertion in the forkhead domain (see text). (B,C) Adult XO males stained with DAPI to highlight gonadal nuclei. (B) Wild-type adult male gonad with elongate, J-shaped gonad. (C) fkh-6(ez16) adult male with disorganized gonad that has failed to elongate. Sperm are indicated by arrow. (D-F) Male gonads, differential interference contrast (DIC) optics. (D) Wild-type adult male with germline (gl), mature sperm, seminal vesicle (sv) and vas deferens (vd) indicated. (E) fkh-6(ez16) adult male with vulva indicated (arrowhead). (F) fkh-6(ez16) male at higher magnification, with mature sperm indicated (arrow). (G) Wild-type young adult XX hermaphrodite showing posterior gonad arm, with spermatheca (spth), oocytes (ooc) and embryo (emb) indicated. (H) Young adult fkh-6(ez16) hermaphrodite; embryo is lodged in spermatheca and fertilized eggs, and oocytes can be seen backing up behind embryo. (I,J) Adult hermaphrodites stained with DAPI. (I) Wild-type adult hermaphrodite with two gonad arms indicated by brackets. (J) fkh-6 adult hermaphrodite showing the one-armed gonad phenotype that occurs in ~10% of XX mutants. (K,L) Early L3 wild-type and fkh-6 hermaphrodites at ten-cell somatic gonad primordium (SPh) stage. Dorsal and ventral uterine precursor cells are indicated by triangles; larger sheath/spermathecal precursor cells (SS) are indicated by arrow. (K) Wild-type early L3 hermaphrodite. Three of the six dorsal and ventral uterine precursor cells are visible, as is one of the four SS cells (other SPh cells are out of focal plane or obscured by intestine). (L) fkh-6 early L3 hermaphrodite showing normal position of SPh cells. Three of six dorsal and ventral uterine precursors are visible, including differentiating anchor cell (AC). Two of four SS cells are in focal plane. (M) Wild-type adult male tail, with sensory rays and other copulatory structures. (N) fkh-6 adult male tail, showing normal morphology. (O) Ventrolateral view of wild-type adult male showing egl-15::gfp reporter expression in sex muscles in the tail (Harfe et al., 1998Go). (P) Ventral view of fkh-6 adult male expressing egl-15::gfp in sex muscles with normal morphology.

 


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  		Fig. 2. Sex reversal of cell fates in the fkh-6 male gonad. Left column (A,D,G,J,M): wild type XX hermaphrodites. Middle column (B,E,H,K,N): wild type XO males. Right column (C,F,I,L,O): fkh-6(ez16) mutant XO males. All panels are overlaid DIC and fluorescence images, except those showing leIs8, which are DIC images of ß-gal stained animals. For each reporter, fluorescence images are identical exposures for all genotypes. Cell types expressing each reporter in wild-type hermaphrodites are as follows. cdh-3::gfp is expressed in the hermaphrodite anchor cell (AC, arrow), but not in wild-type males. lim-7::gfp is expressed in hermaphrodite sheath cells. leIs8 is a lacZ reporter expressed in hermaphrodite spermathecal cells and some uterine cells. leEx780 is a gfp reporter expressed in spermathecal cells. ezIS1 is a gfp reporter not expressed in hermaphrodites, but expressed in male seminal vesicle and vas deferens (see Materials and methods).

 


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  		Fig. 3. Feminization of early cellular events in the fkh-6 male gonad. (A-D) Left, Z1/Z4 cell lineages; right, schematics of early gonadogenesis; L1, first larval stage; L2, second larval stage; a, anterior daughter; p, posterior daughter; AC, anchor cell; LC, linker cell; DTC, distal tip cell. Open arrows, size asymmetry of Z1/Z4 divisions in the wild-type males. In all schematics, Z1 and Z4 are black, and their daughters are either light grey (Z1.a, Z4.p) or dark grey (Z1.p, Z4.a). (A) Wild-type hermaphrodite. Z1 and Z4 produce daughter cells of similar size. Z1.a and Z4.p generate DTCs, while either Z1.p or Z4.a generates an AC. (B) fkh-6 hermaphrodite. Z1/Z4 early lineage appears normal, but is delayed relative to wild type (n=2). (C) Wild-type male. Z1 and Z4 divide asymmetrically to generate daughter cells with a pronounced size asymmetry (open arrows). Z1.p and Z4.a migrate anteriorly, and one or the other generates a LC. (D) fkh-6 male. Z1 and Z4 do not produce daughters with a dramatic size difference (n=8), Z1.p and Z4.a do not migrate anteriorly (n=8), and Z1.a/Z4.p were able to divide (broken lines, 3/6 cells examined). In animals with Z1.a/Z4.p divisions, extra DTCs were observed. (E,F) DIC micrographs of L1 developing gonad. (E) Wild-type male. Z1.a is smaller than Z1.p. Z4.p (out of focal plane) is similarly smaller than Z4.a. (F) fkh-6 (q641) male. Z4.a and Z4.p are about the same size; Z1.a and Z1.p (out of focal plane) also are similar in size.

 


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  		Fig. 4. fkh-6::gfp is expressed in the early somatic gonad and the spermatheca. (A-H) Expression of integrated fkh-6(pro)::gfp array. (A,B) DIC and fluorescence micrographs in four-cell gonad of wild-type L1 male, with fkh-6::gfp expression in Z1 and Z4 (arrows). (C,D) Four-cell gonad of wild-type L1 hermaphrodite showing expression in Z1 (arrow). Z4 is out of focal plane. (E,F) Wild-type male gonad in late L1, with expression of fkh-6::gfp reporter in four daughters of Z1 and Z4, which have moved to the anterior (bracket). Reporter expression is no longer detectable in hermaphrodites by this stage (not shown). (G,H) Wild-type L4 hermaphrodite expressing fkh-6::gfp reporter in spermatheca (spth) and weakly in sheath (sh). Fluorescence outside the gonad in these images is intestinal autofluorescence. Reporter expression is not detected in males at this stage (not shown).

 


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  		Fig. 5. fkh-6 acts downstream of tra-1 in the early gonad. (A) Simplified version of C. elegans sex-determination pathway (Hodgkin et al., 1986Go). The ratio of X chromosomes to autosomes (Madl and Herman, 1979Go) signals via a regulatory cascade and regulates tra-1, such that high tra-1 activity promotes female somatic development and represses male development. Early genes in the pathway and some genetic interactions are not shown. (B) Gonad is severely underdeveloped in fkh-6;tra-1 XX adult (broken line indicates gonadal boundary). (C-E) Comparison of Z4 daughters in fkh-6, tra-1, and fkh-6; tra-1 mutants. Z4.a and Z4.p are similar in size in fkh-6(q641) XO L1 male, whereas Z4.a is larger than Z4.p in tra-1(e1099) XX L1 pseudomale. In fkh-6; tra-1 double mutant XX L1 pseudomales, Z4.a and Z4.p are similar in size. Z1 daughters (out of focal plane) have the same size polarities. (F) Summary of early events of gonadogenesis in fkh-6 and tra-1 single mutants and fkh-6; tra-1 double mutants. Relative sizes of Z1 and Z4 daughters and male-specific anterior migration of Z1.p and Z4.a were scored. Eight Z1/Z4 divisions were scored for each mutant class (fkh-6 XO, tra-1 XX, and fkh-6; tra-1 XX). (G,H) DIC and fluorescence images showing tra-1::gfp expression in the gonadal primordium of fkh-6(q641) XO L1 male. Arrowhead indicates Z4. Reporter also is expressed in Z1, which is out of focal plane. Other brightly expressing nuclei are intestinal. (I,J) DIC and fluorescence images showing fkh-6(pro)::gfp expression in the gonadal primordium of tra-1(e1099) XX L1 pseudomale. Z1 (arrow) and Z4 daughters (arrowheads) express fkh-6::gfp. Z4 daughters are displaced to the dorsal side of the gonad, a common tra-1 defect (L.M. and J.K., unpublished). (K) Summary of fkh-6::gfp and tra-1::gfp expression. Wild-type timing of Z1/Z4 division and anterior migration of Z1.p/Z4.a is indicated at top (unbroken lines). Periods of GFP reporter expression in wild-type and mutant transgenic animals are indicated (broken-ended lines indicate imprecision in timing of expression).

 

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© The Company of Biologists Ltd 2004