A missense mutation in the transcription factor ETV5 leads to sterility, increased embryonic and perinatal death, postnatal growth restriction, renal asymmetry and polydactyly in the mouse

Abstract

ETV5 (Ets variant gene 5) is a transcription factor that is required for fertility. In this study, we demonstrate that ETV5 plays additional roles in embryonic and postnatal developmental processes in the mouse. Through a genome-wide mouse mutagenesis approach, we generated a sterile mouse line that carried a nonsense mutation in exon 12 of the Etv5 gene. The mutation led to the conversion of lysine at position 412 into a premature termination codon (PTC) within the ETS DNA binding domain of the protein. We showed that the PTC-containing allele produced a highly unstable mRNA, which in turn resulted in an undetectable level of ETV5 protein. The Etv5 mutation resulted in male and female sterility as determined by breeding experiments. Mutant males were sterile due to a progressive loss of spermatogonia, which ultimately resulted in a Sertoli cell only phenotype by 8 week-of-age. Further, the ETV5 target genes Cxcr4 and Ccl9 were significantly down-regulated in mutant neonate testes. CXCR4 and CCL9 have been implicated in the maintenance and migration of spermatogonia, respectively. Moreover, the Etv5 mutation resulted in several developmental abnormalities including an increased incidence of embryonic and perinatal lethality, postnatal growth restriction, polydactyly and renal asymmetry. Thus, our data define a physiological role for ETV5 in many aspects of development including embryonic and perinatal survival, postnatal growth, limb patterning, kidney development and fertility.

Figure 1. The SCO mouse line carries a missense mutation within the Etv5 gene.

(A) Schematic of the mouse Etv5 gene and the location of the identified mutation. Exon positions are based on the ENSMUST00000079601 transcript. (B) ETV5 protein and the location of the missense amino acid.

Figure 2. Etv5 mutant (Etv5^sco/sco) males are sterile due to the progressive loss of germ cells.

(A) Testis of 8 weeks-old Etv5^sco/sco and a wild-type (WT) littermate. (B) Testis weight to body weight ratio of Etv5^sco/sco mice compared to WT littermates. n = 12 per group. *p<0.05 (unpaired t-test, two-tailed). (C–F) PAS staining of 4–8 weeks-old testes of Etv5^sco/sco and WT mice.

Figure 3. Levels of Etv5 mRNA in postnatal day 3 testes (A), kidney (B) and spleen (C) in Etv5^sco/sco and wild-type (WT) mice.

(D) ETV5 immunoblotting showed an absence of full length and truncated proteins in the mutant spleen. HPRT was used as a loading control. Levels of Cxcr4 (E) and Ccl9 (F) mRNAs in Etv5^sco/sco and WT postnatal day 3 testes. A–C, E–F, WT values were set as 100%. n = 3 mice per group, **p<0.01, ***p<0.001 (unpaired t-test, two-tailed).

Figure 4. Etv5^sco/sco mice exhibit several developmental abnormalities.

(A) Increased number of re-absorbed embryos (indicated by arrows) at E16.5 from a heterozygous breeding pair. (B) Body weight of Etv5^sco/sco and WT mice at 3 weeks and 3 months. Data are shown as mean ± standard deviation (S.D.), n = 6 per genotype, *p<0.05, **p<0.01 (unpaired t-test, two-tailed). (C) Renal asymmetry (D) and polydactyly in adult (12 weeks-old) Etv5^sco/sco mice.

Figure 5. Allelic series of Etv5 mouse models.

(A) The Etv5^tmKmm mouse line carried a targeted deletion of exons 2–5. (B) Etv5^tm1.2Xsun allele mouse line carried a targeted deletion of exons 10–11. (C) The Etv5^tm1Hass mouse line carried a targeted deletion of exons 11–12 which were replaced with a LacZ-PGK-Neo cassette. (D) Etv5^sco/sco mouse line contained an ENU-induced PTC-mutation in exon 12.