The t-complex-encoded guanine nucleotide exchange factor Fgd2 reveals that two opposing signaling pathways promote transmission ratio distortion in the mouse

Abstract

Transmission ratio distortion (TRD), the preferential inheritance of the t haplotype from t/+ males, is caused by the cooperative effect of four t-complex distorters (Tcd1-4) and the single t-complex responder (Tcr) on sperm motility. Here we show that Fgd2, encoding a Rho guanine nucleotide exchange factor, maps to the Tcd2 region. The t allele of Fgd2 is overexpressed in testis compared with wild type. A loss-of-function allele of Fgd2 generated by gene targeting reduces the transmission ratio of the t haplotype t(h49), directly demonstrating the role of Fgd2 as Distorter. Fgd2 identifies a second Rho G protein signaling pathway promoting TRD.

Figure 1.

Fgd2 maps to the Tcd2 region. (A) Structure of a complete (t^w5) and various partial (t^x) t haplotypes used for mapping Fgd2. The Tcd2 region is defined as the segment of t chromatin that is present in t^h18 and exchanged for wild-type chromatin in t^w18. The centromere is shown as a filled circle at the left, wild-type chromatin is symbolized by filled bars, t chromatin is indicated by open bars, and inversions (In1–In4) are indicated by arrows. Markers and t haplotypes have been described (Lyon 1984); only three Distorters are indicated. (wt) Wild type. (B) Southern blot analysis of genomic DNA digested with PstI, using a full-length Fgd2 cDNA clone as probe, reveals a t-specific band of 4.5 kb that occurs in t haplotypes carrying Tcd2—but not in t^w18 or t^h49—thus mapping Fgd2 to the Tcd2 region; only polymorphic fragments are shown.

Figure 2.

Fgd2 expression from the t-haplotype allele is strongly enhanced as compared with wild-type alleles. (A) Domain structure of Fgd2 proteins encoded by the long and short transcript variants derived from this gene. (GEF) Catalytical domain of guanosine nucleotide exchange factors; (PH) Pleckstrin homology domain; (FYVE) FYVE domain. (B) Temporal expression profile of the long Fgd2 transcript in postnatal testes representing the first cycle of spermatogenesis. (Top panels) Northern blot. (Bottom panels) RT–PCR analysis. (p.p.) Post-partum, after birth. (C) In situ hybridization of Fgd2 antisense (left) or sense (right) control transcripts to testis cryosections showing expression of Fgd2 in early stages of spermatogenesis. (Middle) Schematic view of a transversal section through a seminiferous tubule. (se) Sertoli cell; (sg) spermatogonia; (sc) spermatocytes; (rs) round spermatid; (sz) spermatozoa. Bar, 20 μm. (D) Northern blot analysis of Fgd2 expression in testis derived from wild-type (+/+) and t⁶/t^w5 males demonstrates strongly enhanced expression of the long (L)—and simultaneous strongly reduced expression of the short (S)—Fgd2 transcript in t haplotypes. (Gapdh) Gapdh loading control. (E) Quantitative RT–PCR analysis of the long Fgd2 testis transcript in various strains and t haplotypes, demonstrating up to sixfold higher expression of Fgd2 in t haplotypes compared with wild-type strains, which show considerable differences. (+) Wild-type strain BTBR/TF.

Figure 3.

Gene targeting of Fgd2 by homologous recombination. (A) Scheme of gene targeting. A neo selection cassette was introduced into the Fgd2 locus replacing exons 3–6 and part of exon 7. Probe locations and predicted diagnostic restriction fragments are indicated. (B) Southern blot analysis of DNA derived from targeted and control embryonic stem (ES) cells (left) and of mice (right) carrying the mutant allele (−), with the 5′ and 3′ probes. (C) Northern blot analysis of testis RNA derived from wild-type and mutant animals. The long Fgd2 transcript is not detected in homozygous mutant animals. (Gapdh) Gapdh loading control; (ko) band derived from targeted allele; (wt) wild-type fragment; (+) wild type; (−) mutant.

Figure 4.

Model of TRD: The t haplotype encodes several Distorters (only two are shown), which are expressed in all sperm cells derived from a t/+ male and act on two opposing Rho signaling pathways regulating Smok1. Smok1 is thought to be involved in sperm motility control. Tagap1^Tcd1a and Fgd2^Tcd2 represent hypermorphic alleles expressing strongly elevated gene activity as compared with the wild type. Enhanced down-regulation of the inhibitory pathway by Tagap1 and stronger up-regulation of the activating pathway by Fgd2 additively induce hyperactivation of Smok1 in all sperm, resulting in abnormal flagellar function and low fertilization probability. This harmful effect of the Distorters is rescued by the dominant-negative action of Tcr, which is restricted to t sperm, giving the latter an advantage in fertilizing the egg cells. Neither the Rho switch molecules nor their target effector proteins (X, Y) are known. Arrows symbolize activation, bars represent inhibition, green arrow indicates normal signaling, and purple arrow indicates impaired signaling.