Functional identity of the gamma tropomyosin gene: Implications for embryonic development, reproduction and cell viability

Abstract

The actin filament system is fundamental to cellular functions including regulation of shape, motility, cytokinesis, intracellular trafficking and tissue organization. Tropomyosins (Tm) are highly conserved components of actin filaments which differentially regulate filament stability and function. The mammalian Tm family consists of four genes; αTm, βTm, γTm and δTm. Multiple Tm isoforms (>40) are generated by alternative splicing and expression of these isoforms is highly regulated during development. In order to further identify the role of Tm isoforms during development, we tested the specificity of function of products from the γTm gene family in mice using a series of gene knockouts. Ablation of all γTm gene cytoskeletal products results in embryonic lethality. Elimination of just two cytoskeletal products from the γTm gene (NM1,2) resulted in a 50% reduction in embryo viability. It was also not possible to generate homozygous knockout ES cells for the targets which eliminated or reduced embryo viability in mice. In contrast, homozygous knockout ES cells were generated for a different set of isoforms (NM3,5,6,8,9,11) which were not required for embryogenesis. We also observed that males hemizygous for the knockout of all cytoskeletal products from the γTm gene preferentially transmitted the minus allele with 80-100% transmission. Since all four Tm genes are expressed in early embryos, ES cells and sperm, we conclude that isoforms of the γTm gene are functionally unique in their role in embryogenesis, ES cell viability and sperm function.

Figure 1

Schematic representation of the γTm gene and targeted deletions of both amino- and carboxy-terminal exons. (A) The entire γTm gene showing alternatively spliced variants., All cytoskeletal products contain the exon 1b promoter, with choice of either exon 6a or 6b, and variations of the carboxy-terminal exon 9. Open boxes represent UTRs, lines represent introns and A represents poly A tail. (B) The γTm gene structure showing targeted deletions of exons 1b, 9a + 9b, 9c and 9d summarized as −1b, −9ab, −9c and −9d. Arrow-heads (in red) represent the LoxP sites remaining following exon deletions.

Figure 2

Western blot analysis of mouse sperm. Total mouse sperm protein (10 µg) from WT 129/SvJ and C57BL/6 mouse lines and primary mouse embryonic fibroblasts (MEF) control protein (10 µg for Part A; 1 µg for Parts B–D) were analyzed with antibodies for the presence of high and low molecular weight Tm gene products. Blots were probed with (A) α/9d, (B) γ/9d and (C) δ/9d which recognize products from α, β, γ and δ Tm genes. (D) β actin control blot.

Figure 3

Design and screening of −9d ES cells. (A) The −9d^+/− homologously recombined (parental) allele containing the Neomycin cassette. An oligonucleotide primer set designed to the Neomycin cassette is also shown. (B) Following Cre-mediated recombination to remove the Neomycin cassette the expected −9d KO allele is shown with oligonucleotide primer sets common to both WT and −9d KO alleles. (C) Genomic DNA isolated from ES cell clones screened by PCR analysis. PCR analysis of outcomes from the three primer set F, R1 and R2 is shown in the upper part, with outcomes from the Neomycin primer set shown in the lower part. -ve, no template control; +/+, WT C57BL/6 ES cell DNA; −/−, genomic DNA from Δ9d deleted mouse line; Parental, homologously recombined −9d^+/− clone containing the NEO cassette; Clone#1, clone containing the NEO cassette; Clone#18, clone with NEO cassette removed. (D) Following re-targeting of the −9d^+/− ES clone (#18) with the −9d targeting construct, the possible outcomes from this re-targeting are WT (due to random integration), re-targeting back to the original 9d KO locus and targeting of the alternative allele. Oligonucleotide primer sets designed to detect the presence of exon 9d are shown. (E) Genomic DNA isolated from ES cell clones screened by PCR analysis using 9dF1 and 9dR1 primers. -ve, no template control; WT, C57BL/6 ES cell DNA; #1-#5 sample of the 330 clones. The predicted 527 bp fragment for exon 9d is shown. (F) The 330 ES cell clones were also analyzed by PCR for random and original locus re-targeting using F and R2 primers (upper part) and 9dF1 and 9dR1 primers (bottom part). -ve, no template control; WT, C57BL/6 ES cell DNA, −9d^+/− ES Clone#18; re-target, clone where −9d targeting construct has re-inserted into the original targeted locus; random, clone where −9d targeting construct has re-inserted randomly. The 900 bp fragment for knockout of exon 9d and the 527 bp fragment for presence of exon 9d is shown.

Figure 4

Design and screening of −9ab ES cells. (A) The γTm gene showing the subcloned 6.7 kb SpeI fragment containing exons 9a and 9b with flanking intron sequence. The targeting construct with a LoxP sequence and a cassette containing LoxP—pGK Thymidine Kinase—pGK Neomycin^R—LoxP. A 1.2 kb probe located outside the construct region is shown. (B) Southern blot analysis of C57BL/6 ES cell DNA following EcoRV digestion shows the expected 5.5 kb band for the WT allele and a 3.1 kb band for the recombined allele (Recomb) following probing with the 1.2 kb probe. (C) Following Cre recombinase and Gancyclovir selection the resulting alleles expected are WT, a floxed exon 9a + 9b or a full 9ab KO allele. Oligonucleotide primer sets common to the WT and both altered alleles are shown. (D) Genomic DNA isolated from ES cell clones was screened by PCR analysis. WT: +/+, Parental: homologously recombined clone containing exons 9a + b and the TKNEO cassette, −9ab^+/−: clone lacking exons 9a + 9b and −9abFlox: clone containing a floxed exon 9a + 9b allele. PCR analysis outcomes from the primer set 9723F and 9b3′R is shown in the upper part and primer set 9724F and 9b3′R lower part. (E) Following re-targeting of the −9ab^+/− clone with the −9ab construct the expected outcomes are a 9ab KO and homologously recombined alleles. (F) Genomic DNA isolated from ES cell clones was screened by PCR analysis using primer sets 9724F and 9b3−R to yield a 400 bp band and 9723F and 9b3′R to yield a 315 bp band. (G) Following the second round of Cre recombination and selection the expected outcome will be two −9ab^−/− alleles. An oligonucleotide primer set common to both WT allele and the fully deleted exon 9a + 9b allele was used in order to confirm any −9ab^−/− ES cell clones. (H) Genomic DNA isolated from ES cell clones were screened by PCR analysis. A single −9ab^−/− ES cell clone was determined by long range PCR against both WT^+/+ and −9ab^+/− controls. The expected 2.6 kb fragment for the WT allele and a 400 bp fragment for the deleted exons 9a and 9b is shown.