A sensitized genetic background reveals evolution near the terminus of the Caenorhabditis germline sex determination pathway

Abstract

Caenorhabditis elegans and Caenorhabditis briggsae are both self-fertile hermaphroditic nematodes that evolved independently from male/female ancestors. In C. elegans, FEM-1, FEM-2, and FEM-3 specify male fates by promoting proteolysis of the male-repressing transcription factor, TRA-1. Phenotypes of tra-1 and fem mutants are consistent with this simple linear model in the soma, but not in the germline. While both XX and XO tra-1(lf) mutants have functional male somas, they produce both sperm and oocytes. Further, all three tra-1; fem double mutants retain the expected male soma, but make only oocytes (the germline fem phenotype). Thus, a poorly characterized tra-1 activity is important for sustained male spermatogenesis, and the fem genes affect germline sexual fate independently of their role in regulating TRA-1. C. briggsae tra-1 mutants are phenotypically identical to their C. elegans counterparts, while the fem mutants differ in the germline: XX and XO C. elegans fem mutants are true females, but in C. briggsae they are self-fertile hermaphrodites. To further explore how C. briggsae hermaphrodites regulate germline sex, we analyzed Cb-tra-1/Cb-fem interactions. Cb-tra-1 is fully epistatic to Cb-fem-2 in the germline, unlike the orthologous C. elegans combination. In contrast, Cb-fem-3 shifts the Cb-tra-1(lf) germline phenotype to that of a nearly normal hermaphrodite in the context of a male somatic gonad. This suggests that Cb-fem-3 is epistatic to Cb-tra-1(lf) (as in C. elegans), and that the normal control of C. briggsae XX spermatogenesis targets Cb-tra-1-independent factors downstream of Cb-fem-3. The effect of Cb-fem-3(lf) on Cb-tra-1(lf) is not mediated by change in the expression of Cb-fog-3, a likely direct germline target of Cb-tra-1. As Cb-fem-2 and Cb-fem-3 have identical single mutant phenotypes, Cb-tra-1 provides a sensitized background that reveals differences in how they promote male germline development. These results represent another way in which C. briggsae germline sex determination is incongruent with that of the outwardly similar C. elegans.

Figure 1

Comparison of the somatic and germ line sex determination pathways of C. elegans and C. briggsae. The top panel shows the so-called core pathway, which controls somatic sexual dimorphism all known Caenorhabditis species. The middle panel depicts how the core pathway is modified in the C. elegans germ line, with germline-specific controls of the XX hermaphrodite shown in gray. Those targeting tra-2 are required for XX spermatogenesis, while those targeting fem-3 are required for the switch to oogenesis. While both fog-1 and fog-3 both lie downstream of tra-1 by genetic criteria, only fog-3 has been shown to be directly regulated by tra-1. The bottom panel shows what is known about C. briggsae germ line sex determination. The locus of the regulation of XX spermatogenesis proposed by Hill et al. (2006) is shown to target tra-1 or points downstream, with the unknown factors promoting spermatogenesis depicted above the core pathway, and those promoting the switch to oogenesis below. The potential existence of a Cb-tra-1-independent sperm-promoting function for one or more Cb-fem genes (examined in this paper) is indicated with a dashed line.

Figure 2

C. briggsae and C. elegans tra-1(lf) mutants produce endomitotic oocytes. A. Cb-tra-1(nm2) XX Tra males produce oocytes as early at the L4 larval stage. Inset shows the tail of the same animal, which is beginning tip retraction. g, gut; o, oocytes; sc, spermatocytes; sv, seminal vesicle; vd, vas deferens. B. Adult XX Cb-tra-1(nm2) animals often have a mixture of sperm (sp), oocytes, and acellular material (am). C, D. Matched pair of DIC and Hoechst 33258 stained Cb-tra-1(nm2) animals (D) shows that the acellular material in Cb-tra-1(nm2) gonads corresponds to endomitotic oocytes (eo) that have lost their plasma membrane integrity. In the upper animal, immature spermatocytes (sc) are seen in the Emo gonad, while the lower animal is producing only sperm. E, F. Matched pair of an XX C. elegans tra-1(e1781) Emo mutant. In all panels, anterior is to the right.

Figure 3

Summary of somatic gonad and germline phenotypes. All animals were scored by Hoechst 33258 staining in the first day of adulthood. “Spermatogenesis then oogenesis” includes both Emo and non-Emo animals. “Abnormal germline” includes a change in the location and number of sperm, spermatocytes, oocytes and germline stem cells. “Abnormal gonad” includes animals with abnormally shaped gonad arms and double-armed gonads. The phenotypic distributions of Cb-tra-1(nm2) and Cb-tra-1(nm2); Cb-fem-2(nm27) are not significantly different (chi-square, omitting abnormal gonad category, p=0.667, df=2), while both of these are significantly different from Cb-tra-1(nm2); Cb-fem-3(nm63) (Cb-tra-1(nm2) vs. Cb-tra-1(nm2); Cb-fem-3(nm63): p<.001, df =2. Cb-tra-1(nm2); Cb-fem-2(nm27) vs. Cb-tra-1(nm2); Cb-fem-3(nm63): p<.001, df=2). The number of animals scored is indicated in the upper right corner of each panel.

Figure 4

Cb-tra-1; Cb-fem double mutant phenotypes. A, B are a matched pair of a Hoechst 33258-stained Cb-tra-1(nm2); Cb-fem-2(nm27) Tra male under DIC (A) or epifluorescence (B). Both sperm (sp) and endomitotic oocytes (eo) can be observed. Asterisk indicates the male tail. C. DIC microscopy of a live adult Cb-tra-1(nm2); Cb-fem-3(nm63) mutant, showing well defined mature oocytes (mo), endomitotic oocytes proximal to them (eo), and mature sperm (sp) in the seminal vesicle. D. Two adult Cb-tra-1(nm2); Cb-fem-3(nm63) mutants under DIC. The more reflexed animal (above and to the right) has both a normal testis whose distal tip is indicated (dt), but also a smaller anterior gonad (ag) arm with oocytes in it. The less reflexed animal (below and to the left) has only a testis, although it is also making oocytes. E, F. Matched pair of Hoechst 33258-stained Cb-tra-1(nm2); Cb-fem-3(nm63) double mutant, showing the testis with its distal tip indicated in E and a large number of endomitotic oocytes.

Figure 5

Quantitative RT-PCR analysis of Cb-fog-3 transcript levels in various mutants. Cb-fog-3 expression was normalized to a multi-gene actin amplicon amplified from the same cDNA prep and run in the same microtiter plate. Error bars represent standard errors of the mean for 6 biological replicates. See text for more information on statistical tests.