1. RNA-Regulated TRA-1 Nuclear Export Controls Sexual Fate
S.P. Segal, L.E. Graves, J. Verheyden, E.B. Goodwin 
Developmental Cell 
Volume 1, Issue 4, Pages (October 2001)
DOI: /S (01)

2. Figure 1 Western Analysis of TRA-1 Proteins
(A) Genetic control of sex determination in C. elegans. Genes that act to control both sex determination and dosage compensation are omitted and only the genes involved in somatic sex determination are indicated (for a review, see Cline and Meyer, 1996). Eight genes determine somatic sexual fates: her-1, three tra genes, three fem genes, and laf-1. In XO animals, her-1 and laf-1 inhibit tra-2, the fem genes inhibit tra-1, and male development ensues. In XX animals, her-1 is inactive and tra-3 promotes tra-2 activity; hence, tra-2 represses the fem genes and tra-1 promotes female fate.
(B) Cartoon of tra-2 3′ UTR showing the location of the TRE (solid bar), the TRA-1 binding site (hatched box), the TGEs (arrows), and the 108 nt deletion present in the tra-2(e2020gf) 3′ UTR.
(C) Western analysis with peptide TRA-1 antibody of TRA-1 proteins in wild-type and mutant animals. Four bands are detected from whole-worm XX extracts (lane 1) and XO (lane 3) but not from tra-1(null) (lane 2) extracts. Wild-type XO (lane 4) and tra-2(e2020gf) XO (lane 5) make similar amounts of TRA-1. Wild-type XX (lane 6) and tra-2(e2020gf) XX (lane 7) animals make similar amounts of TRA-1. Blots were probed for myosin as a loading control.
(D) Western analysis with Myc-antibody of Myc-TRA-1A expression in wild-type hermaphrodites (lane 1), wild-type males (lane 2), and tra-2(e2020gf) males (lane 3), all of which make similar amounts of Myc-TRA-1A. Blots were probed for SMG-3 as a loading control
Developmental Cell 2001 1, DOI: ( /S (01) )

3. Figure 2 TRA-1 Has Sex-Specific Localization in the Intestine
Samples were costained with TRA-1 (left) and Mab414 (right; nuclear rim marker) antibodies.
(A) In wild-type XX hermaphrodites, TRA-1 is nuclear and cytoplasmic with higher nuclear levels.
(B) In wild-type XO males, TRA-1 is evenly distributed between the nucleus and cytoplasm.
(C) tra-1(e1834null) animals do not stain for TRA-1.
(D and E) Wild-type XX and XO animals expressing myc-tra-1A transgene stained with Myc antibody. In XX animals, Myc-TRA-1A is higher in the nucleus than in the cytoplasm (D). In XO animals, Myc-TRA-1A is evenly distributed between the nucleus and cytoplasm (E).
(F) Animals not carrying a myc-tra-1A transgene do not stain for Myc-TRA-1A.
(G and H) In wild-type XX L4 hermaphrodites (G) and tra-2(lf) animals (H), TRA-1 is evenly distributed between the nucleus and cytoplasm.
(I) In tra-2(lf); fem-1(lf) XX females, TRA-1 is nuclear and cytoplasmic with higher nuclear levels
Developmental Cell 2001 1, DOI: ( /S (01) )

4. Figure 3 TRA-1 Has Sex-Specific Localization in the Germline
(A and B) Cartoon of XX and XO germlines showing the mitotic (1), transition (2), and meiotic (3) zones.
(C) In wild-type hermaphrodites, TRA-1 is nuclear and cytoplasmic with strong nuclear staining in the distal mitotic region through the transition zone. TRA-1 is evenly distributed between the nucleus and cytoplasm in the meiotic pachytene region.
(D) GLP-1 staining showing the mitotic region.
(E–G) Higher magnification of TRA-1 staining in the hermaphrodite mitotic (E), transition (F), and meiotic pachytene (G) zones.
(H) In wild-type males, TRA-1 is nuclear and cytoplasmic, with strong nuclear staining confined to the mitotic region.
(I) GLP-1 staining of the same germline.
(J–L) Higher magnification of TRA-1 staining in the male mitotic (J), transition (K), and meiotic pachytene (L) zones.
(M) In L4 XX animals, TRA-1 is strongly nuclear only in the mitotic region and is evenly distributed throughout the rest of the germline.
(N) In tra-2(lf); fem-1(lf) XX germlines, TRA-1 is strongly nuclear throughout the germline. Lines indicate the mitotic (1), transition (2), and meiotic pachytene (3) zones
Developmental Cell 2001 1, DOI: ( /S (01) )

5. Figure 4
TRA-1 Nuclear Levels Rise when CRM-1-Mediated Nuclear Export Is Inhibited by Treatment with LMB
(A and B) TRA-1 staining of XO intestines; TRA-1 (left) and Mab414 (right) staining. Nuclear TRA-1 levels are lower in buffer-treated (A) than in LMB-treated (B) intestines.
(C and D) Nuclear Myc-TRA-1A levels are lower in buffer-treated (C) than in LMB-treated (D) intestines.
(E and F) LMB treatment resulted in transcriptional activation of the transgene vit:GFP in wild-type males (E) but not in tra-1(e1834null) animals (F).
(G–H) tra-2 mRNA accumulates in the nucleus of LMB-treated XO intestines (G) but not in buffer-treated controls (H) (left). DAPI stain of the same samples (right).
(I–J) Nuclear TRA-1 levels are lower in buffer-treated (I) than in LMB-treated (J) tra-2(e1095lf) intestines.
(K and L) Cytoplasmic tra-2 mRNA levels are lower in tra-2(e1095lf) (K) than in tra- 2(e1095lf); smg-3(r930) (L) double mutant intestines. Arrows indicate nuclei
Developmental Cell 2001 1, DOI: ( /S (01) )

6. Figure 5
tra-2(e2020gf) Male Germlines and Intestines Have Increased Nuclear TRA-1
(A) In tra-2(e2020gf) germlines, TRA-1 is nuclear throughout.
(B) In tra-2(e2020gf) germlines, GLP-1 is identical to that in wild-type (see Figure 3I).
(C) DAPI staining of germline nuclei. Lines delineate the mitotic (1), transition (2), and meiotic pachytene (3) zones.
(D–F) Analysis of TRA-1 and Myc-TRA-1 in tra-2(gf) animals. TRA-1 staining (top); Mab414 staining (bottom). In tra-2(e2020gf) male intestines, nuclear TRA-1 (D) and nuclear Myc-TRA-1 (E) levels are high. Nuclear TRA-1 is not increased in intestines of tra-2(q122gf) XO animals (F)
Developmental Cell 2001 1, DOI: ( /S (01) )

7. Figure 6
The Increased Levels of Nuclear TRA-1 in tra-2(e2020gf) Animals Are Due to Decreased TRA-1 Nuclear Export
dsRNAi was used to inhibit the activity of ima-3. (Top) Model of effect on TRA-1 localization to ima-3 in wild-type and tra-2(e2020gf) cells treated with dsRNA. If the increased nuclear TRA-1 in tra-2(e2020gf) animals results from decreased export, then inhibition of nuclear import would cause a redistribution of TRA-1 in wild-type cells but not tra-2(e2020gf) cells. (Bottom) Shown are the distal regions of XX germlines. As compared with untreated wild-type XX animals (A), strong nuclear TRA-1 localization is lost in wild-type animals (D) but not tra-2(e2020gf) animals (G) treated with dsRNA for ima-3. Mab414 staining indicates nuclei in (B), (E), and (H), and merged images of Mab414 and TRA-1 staining are shown in (C), (F), and (I). Loss of ima-3 activity causes nucleoporin aggregates of variable size (E and H), and hence aggregate size does not correlate with a different degree of loss in ima-1 activity
Developmental Cell 2001 1, DOI: ( /S (01) )

8. Figure 7
Expression of a tra-2 3′ UTR with the TRA-1 Binding Site Reduces Nuclear TRA-1 in Both the Germline and Intestine of tra-2(e2020gf) XO Animals
Nuclear TRA-1 levels are high throughout the germline of tra-2(e2020gf) animals that do not carry a transgene (A) or that overexpress transgenic mRNA lacking the TRA-1 binding site (D). Mab414 staining indicates nuclei (B, E, and H) and DAPI staining indicates nuclear morphology (C, F, and I). Lines indicate the mitotic (1), transition (2), and meiotic (3) zones.
(G) Nuclear TRA-1 levels are reduced in the meiotic pachytene region and part of the transition zone of animals overexpressing a transgene with the TRA-1 binding site (hatched box).
(J and K) Nuclear TRA-1 levels are high in tra-2(e2020gf) XO intestines expressing transgenic RNA lacking the TRA-1 binding site (J) but not in tra-2(e2020gf) intestines overexpressing transgenic RNA with the TRA-1 binding site (K). Arrows indicate nuclei. (Far left) Cartoons of 3′ UTRs carried by transgenes
Developmental Cell 2001 1, DOI: ( /S (01) )