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First published online 4 August 2004
doi: 10.1242/dev.01288

Development 131, 4333-4343 (2004)
Published by The Company of Biologists 2004
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TRA-1/GLI controls development of somatic gonadal precursors in C. elegans

Laura D. Mathies1,2, Mara Schvarzstein3, Kristin M. Morphy2, Robert Blelloch4, Andrew M. Spence3 and Judith Kimble1,*

1 Department of Biochemistry and Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706-1544, USA
2 Department of Genetics, North Carolina State University, Raleigh, NC 27695-7614, USA
3 Department of Molecular and Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada
4 Whitehead Institute, Cambridge, MA 02142-1479, USA



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  		Fig. 1. tra-1 controls gonadal symmetry. (A-D) Left, DIC images; right, immunofluorescence of same gonad in which (A,B) lag-2::GFP marks SGPs (Blelloch et al., 1999Go) and (C,D) GFP::POP-1 marks SGPs and their daughters. (A) Gonadal primordium in wild-type XO male with SGPs (Z1 and Z4) at poles and PGCs located centrally. (B) Gonadal primordium in tra-1(e1099) XX pseudomale, Z4 is dorsal and more anterior than normal. (C) Wild-type XO male, Z1 divides asymmetrically to produce Z1.a, which is smaller and has less nuclear GFP::POP-1 (small arrowhead); and Z1.p, which is larger and has more GFP::POP-1 (large arrowhead), a phenomenon also seen in hermaphrodites (Siegfried et al., 2004Go). In this gonad, Z4 has not divided. (D) tra-1(RNAi) XX pseudomale, polarity of Z1 division is reversed: Z1.a is larger and has more nuclear GFP::POP-1 (large arrowhead), while Z1.p, which is smaller, has less GFP::POP-1 (small arrowhead). In this gonad, Z4 has not divided and is located more dorsal and anterior than normal. (E,F) Z1/Z4 asymmetric divisions. (E) In wild-type males, central daughters (Z1.p/Z4.a, dark) are larger than polar daughters (Z1.a/Z4.p, light). (F) In tra-1 mutants, the polarity of Z1 division is reversed, so its polar daughter is larger (Z1.a, dark) than its central daughter (Z1.p, light), but the Z4 division has its normal polarity. (G,H) Laser ablation experiments test the fate of SGP daughters. In each experiment, three out of four SGP daughters were ablated (X), leaving one daughter to be assessed for fate. (G) In wild-type males, isolated Z1.p generated a linker cell (LC, n=1), while isolated Z1.a generated a distal tip cell (DTC, n=1). (H) In tra-1 mutants, isolated Z1.p generated a DTC (n=2), while isolated Z1.a generated a LC (n=6).

 


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  		Fig. 2. TRA-1 expressed in embryonic and larval SGPs. (A-F) Embryos or larvae stained with {alpha}-TRA-1 antibodies; (A-C,E,F) {alpha}-TRA-1(AS) polyclonal antibody; (D) {alpha}-TRA-1(DZ) peptide antibody. (A,B) Wild-type XX embryo at ~320 minutes, when gonadal primordium has just formed; this embryo carries a hnd-1::GFP reporter to identify SGPs. (A) TRA-1 is present in SGPs (only one is visible) of the newly formed gonadal primordium and appears to be predominantly nuclear. SGP staining is reproducible. PGC staining with {alpha}-TRA-1 is variable and may be non-specific. (B) hnd-1::GFP identifies SGP shown in A. (C) Wild-type XX threefold embryo with strong nuclear TRA-1 staining. (D) Wild-type XX L1 gonadal primordium. TRA-1 (red) in SGP nuclei (only one is visible); PGCs are identified by {alpha}-NOS-3 (green). (E) Wild-type XX L1 gonadal primordium; TRA-1 in SGPs is predominantly nuclear. (F) tra-2; xol-1 XX pseudo-male L1 gonadal primordium. TRA-1 in masculinized SGPs is expressed at a lower level and is distributed between nucleus and cytoplasm; a similar pattern is observed in wild-type XO males (not shown). (G-J) Larvae expressing a TRA-1 translational reporter (GFP::TRA-1). (G,I) DIC images; (H,J) immunofluorescence. (G,H) Wild-type L1 XX hermaphrodite. GFP::TRA-1 is expressed in SGPs (only one is visible) and intestinal cells (int). This rescuing fusion protein is predominantly nuclear. (I,J) Wild-type L1 XO male; GFP::TRA-1 is expressed at a lower level in male SGPs than in hermaphrodite SGPs, and it is distributed through both nucleus and cytoplasm. The exposure in J is longer than that of H to highlight SGP cytoplasmic expression. Expression of GFP::TRA-1 in intestinal cells is not regulated in a sex-specific manner; it therefore serves as a measure of relative exposure between H and J.

 


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  		Fig. 3. tra-1 and ehn-3 redundantly control SGP development. (A,B) Young adults, DAPI stained; male tail and pharynx are indicated. (A) tra-1(e1099) single mutant with a normally shaped male gonad (bracket). (B) tra-1(e1099); ehn-3(q766) double mutant with no visible gonad (arrow). (C,D) L4/young adults, stained with antibodies to germ cell marker PGL-1. (C) ehn-3(q766) single mutant with normal two-armed gonad (brackets). Actual genotype is tra-1/+; ehn-3. (D) tra-1(e1099); ehn-3(q766) double mutant with only a few germ cells. (EH) L1 larvae expressing pes-1::GFP, an SGP-marker (Molin et al., 2000Go). (E,G) DIC; (F,H) immunofluorescence of same animal shown in E and G, respectively. (E,F) tra-1(e1099) mutant L1. (E) SGP (Z4, white arrow) has typical morphology. (F) Z4 expresses pes-1::GFP; (G,H) tra-1(e1099); ehn-3(q766) double mutant L1. (G) A small cell is associated with PGCs (white arrow); (H) this small cell does not express pes-1::GFP.

 


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  		Fig. 4. Gonadal masculinization in tra-1/+; ehn-3. (A-D) Gonadal leader cells marked with lag-2::GFP (Blelloch et al., 1999Go). (A) Wild-type XX hermaphrodite leader cell (DTC) is crescent shaped. (B) Wild-type XO male leader cell (LC) is round. (C,D) tra-1/+; ehn-3(q689) XX leader cells. (C) Leader cell with typical DTC crescent shape (most common). (D) Leader cell with typical LC round shape. A similar low-penetrance gonadal masculinization was also observed in tra-1/+; ehn-3(RNAi). (E,F) tra-1/+; ehn-3(q689) XX larvae, DIC. (E) Gonadal arm with normal hermaphrodite organization: DTC (white arrow) is adjacent to germ cells (bracket) and separated from other somatic gonadal cells (out of photo). (F) Gonadal arm with male organization: LC (white arrow) is adjacent to somatic gonadal cells (black arrows) and separated from germ cells.

 


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  		Fig. 5. ehn-3 encodes an SGP-specific protein with paired zinc fingers. (A) The ehn-3 locus. cDNA analyses identify three transcripts, each predicted to encode a protein with multiple zinc fingers (black boxes). All cDNAs are transpliced to the SL1 leader. ehn-3A encodes six zinc fingers; ehn-3B1 and B2 encode four zinc fingers. The gene predicted by the sequence consortium, called ZK616.10, corresponds to ehn-3B. ehn-3(q689) is associated with an insertion (T) and a C->T transition in exon 2 of ehn-3A. ehn-3(q766) deletes a region including exon 2 from ehn-3A, the next intron and part of the first common exon, including coding sequence for the first 14 amino acids of EHN-3B1 and B2. (B) Semi-quantitative PCR used to assess transcript levels in ehn-3 mutants. (Left) ehn-3 PCR products were less abundant when amplified from ehn-3 mutants than from wild type (lower band in N2 is ehn-3B2); by contrast, hlh-2 PCR product was similar in wild-type and ehn-3 mutants. (Right) Ratio of ehn-3 to hlh-2 PCR products. (C) Alignment of EHN-3 putative zinc fingers. ZNF1-ZNF3 and ZNF6 were predicted by Pfam; ZNF4 and ZNF5 were identified by eye. (D-F) SGP-specific expression of EHN-3::GFP. (D) EHN-3::GFP transgene composition: ehn-3 genomic DNA, including 429 bp of 5' flanking sequence plus the entire EHN-3-coding region, was fused in frame to the GFP-coding sequence. (E) Wild-type embryo expressing EHN-3::GFP. SGP (arrow) has assembled into the gonadal primordium; PGC, asterisk. (F) EHN-3::GFP is expressed in SGP (arrowhead) shortly after gonadal primordium is formed.

 


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  		Fig. 6. Overlapping expression of ehn-3, tra-1 and hnd-1 in embryonic SGPs. hnd-1, tra-1 and ehn-3 are expressed in the SGPs at different, but overlapping, times during embryogenesis. hnd-1::GFP is first detected in SGPs migrating towards germ cells and disappears about the two-fold stage of embryogenesis. TRA-1 is detected in newly formed gonadal primordium and may be expressed earlier in migrating SGPs (M.S. and A.S., unpublished). TRA-1 continues expression in SGPs during embryogenesis and into the first larval stage, when its level and distribution are modulated in the two sexes. EHN-3::GFP is expressed exclusively in SGPs shortly after gonadal primordium forms; it disappears by the threefold stage of embryogenesis.

 

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© The Company of Biologists Ltd 2004