Germline Sex Determination in C. elegans (nematode)

Caenorhabditis elegans is sexually dimorphic organism– hermaphrodite (XX), male (XO).

Caenorhabditis elegans sex determination is a highly regulated process during which animals with two sets of autosomes and two X chromosomes (AA XX) develop as self-fertile hermaphrodites, while those with one X chromosome (AA XO) develop as males (Kasturi et al, 2010).

The core sex determination pathway in germline cells involves a series of negative regulations among proteins that ultimately specify male or female cell fates.

Stage-specific regulation of tra-2 and fem-3 in the hermaphrodite germline facilitates production of sperm in the L4 larval stage and oocytes in the adult (Gilbert, 2010).

How does the regulation of tra-2 and fem-3 expression affect spermatogenesis and oogenesis in C. elegans?

Video 1: Young Hermaphrodite C. elegans

Translational Regulation of Germline Sex Determination in C. elegans:


Figure 1: Germline sex determination in the nematode (Gilbert, 2010).

  • The fem-2 mRNA is regulated at the level of translation. The product of the laf-1 gene is thought to bind to the 3′ UTR of fem-2 mRNA and inhibit its translation.
  • In XX nematodes, the product of the tra-3 gene is present at embryonic stages and allows the female body plan to be constructed. At the second larval stage, it is turned off, permitting spermatogenesis. At the fourth larval stage, it is reactivated, causing egg production. In XO individuals, the activation of the her-1 gene inhibits Tra-2, allowing for the male phenotype and sperm production only. The regulation by multiple inhibitions forces one to think in quintuple negatives here (Gilbert, 2010).

Figure 2: The transition from spermatogenesis to oogenesis during the fourth instar larva of C. elegans is regulated by translation of the tra-2 and fem-3 messages. In both cases, the blocking of translation occurs through the binding of an inhibitory protein to the respective 3' UTR (Gilbert, 2010).

Laf-1:

  • Mutations of laf-1 affect sex determination, and genetic analysis suggests that LAF-1 promotes male cell fates (Hubert et al, 2009).
  • Laf-1 mutations were isolated as dominant suppressors of fem-3 gain-of-function (gf) alleles. The germlines of fem-3(gf) XX animals are masculinized, containing excess sperm and no oocytes.
  • Laf-1/+; fem-3(gf) animals produce both sperm and oocytes and are self-fertile.
  • 10–30% of laf-1/+ XX heterozygotes are feminized, producing oocytes but no sperm. A similar percentage of XO animals are partially feminized in both the germline and soma. These data suggest that wild-type LAF-1 promotes male fates in all tissues (Hubert et al, 2009).

Figure 3: Laf-1 mRNA expression. Northern blot comparing laf-1 expression in wildtype adult males, L4 hermaphrodites, and young adult hermaphrodites. The same blot was probed for rpl-6 as a loading control and for vit-2, which is expressed only in hermaphrodites (Hubert et al, 2009).

Results:

  • The phenotypes of laf-1/+ animals are similar to those of strong tra-2(gf) mutants. Both of these genotypes cause XX animals to develop as females and partially feminize the XO soma and germline (Hubert et al, 2009).
  • Additionally, both tra-2(gf) mutations and laf-1/+ heterozygosity suppress the germline masculinization of fem-3(gf) mutants (Mason et al, 2008).
  • Reporter transgenes carrying the tra-2 3′UTR are misregulated in laf-1/+ mutants. Transgenes carrying the wild-type tra-2 3′UTR are repressed in wild type but are derepressed in laf-1/+ mutants (Hubert et al, 2009).
  • Transgenes that lack the TGEs fail to be repressed in wild type and exhibit no additional derepression in laf-1/+ heterozygotes, indicating that laf-1 acts through the TGEs (Hubert et al, 2009).
  • These data suggest that laf-1 acts upstream of tra-2 and inhibits its expression. Feminization of laf-1/+ heterozygotes may thus result from overexpression of TRA-2.

Figure 4: Phenotypes of laf-1(RNAi) and vbh-1(RNAi). Fed wild-type L4 hermaphrodites bacteria expressing laf-1 dsRNA, vbh-1 dsRNA, both laf-1 and vbh-1 dsRNA, or empty vector, and scored the phenotypes of their offspring over a period of 3 days. The percentage of offspring displaying each phenotype was calculated for each of n mothers, and the values reported are the mean percentage±one standard deviation. Results that are significantly different from empty vector (unpaired t-test, pb0.01) are indicated in boldface. “Other defects” includes sterility, slow growth, larval arrest, bursting, and post-embryonic death (Hubert et al, 2009).

Regulation of TRA-2 Expression:

  • The function of LAF-1 involves repression of tra-2 expression. TRA-2 promotes female fates, and regulation of its expression is critical for normal sexual development in all tissues.
  • Translation of tra-2 mRNA is repressed in the hermaphrodite germline to allow spermatogenesis.
  • This repression requires two 28-nucleotide elements termed TGEs (tra-2 and GLI elements) located in the tra-2 3′ untranslated region (3′UTR). Disruption or deletion of these elements, such as in tra-2(gf) mutants, causes excess tra-2 activity and feminizes the hermaphrodite germline (Gilbert, 2010).
  • The STAR protein GLD-1 binds to the TGEs and mediates tra-2 repression.
  • Regulation of tra-2 also requires FOG-2, an F-box protein that physically interacts with GLD-1 (Kalis et al, 2010).

Regulation of Spermatogenesis:

  • The initiation of sperm formation is achieved by the repression of the tra-2 message. The Tra-2 protein is essential for the development of eggs and female body cells, and repression of tra-2 mRNA translation in germ cells causes them to become sperm (Sasagawa et al, 2009).
  • If the 3′ UTR of this message that contains two regions of 28 nucleotides are mutated, the translation of the tra-2 mRNA is not repressed, no sperm is made, and the nematode is functionally female instead of hermaphroditic (Mason et al, 2008).
  • Tra-3, appears to promote female development by freeing the tra-2 message from Laf-1 suppression.
  • During the larval stages L2 and L3, the Tra-3 protein is absent. At this time, Laf-1 can inhibit Tra-2 message, and sperm can be formed.

Evalution:

The strength of the paper by Hubert is that the result section is clear and easy to understand; results are demonstrated with multiple figures. Moreover, experimental methods were listed and described in detail. But the paper did not indicate any possible future works. It would have been better if the paper analyzed the role of laf-1 gene in other organisms as well.

Additional Links for Nematodes:

1. Worm Atlas http://wormatlas.psc.edu/ver1/index.htm

2. Worm Book http://www.wormbook.org/

References:

1. Gilbert, Scott. “Translational Regulation of Germline Sex Determination in C. elegans.” Developmental Biology. (2010): 712-14. Print.

2. Hubert, Amy, and Philip Anderson. “The C. Elegans Sex Determination Gene Laf-1 Encodes a Putative DEAD-box RNA Helicase.” Developmental Biology, 330.2 (2009): 358-367.

3. Kalis, Andrea, Mary Kroetz, Kathleen Larson, and David Zarkower. “Functional Genomic Identification of Genes Required for Male Gonadal Differentiation in Caenorhabditis Elegans.” Genetics, 185.2 (2010): 523-521.

4. Kasturi, Prasad, Simone Zanetti, Myriam Passannante, Zarifja Saudan, Fritz Muller, and Alessandro Puoti. “The C. Elegans Sex Determination Protein MOG-3 Functions in Meiosis and Binds to the CSL Co-repressor CIR-1.” Developmental Biology, 344.2 (2010): 593-602.

5. Mason, D. Adam, Jeremy Rabinowitz, and Douglas Portman. “Dmd-3, a Doublesex-related Gene Regulated by Tra-1, Governs Sex-specific Morphogenesis in C. Elegans.” Development (09501991), 135.14 (2008): 2.

6. Sasagawa, Yohei, Mieko Otani, Nahoko Higashitani, Atsushi Higashitani, Ken Sato, Teru Ogura, and Kunitoshi Yamanaka. “Caenorhabditis Elegans P97 Controls Germline-specific Sex Determination by Controlling the TRA-1 Level in a CUL-2-dependent Manner.” Journal of Cell Science, 122.20 (2009): 9.

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