A Thoc1 null allele was generated by targeted homologous recombination in murine ES cells. The targeting vector was designed to delete 11.5 kbp of genomic DNA containing exons 2 through 8 of Thoc1 (Fig. ). The deleted exons include codons 19 to 173, which span a region of Thoc1 protein (pThoc1) that is highly conserved throughout evolution. The deletion also creates a reading frame shift, causing premature termination of potential readthrough translation. Hence, the targeted allele does not express pThoc1. Two of 128 G418-resistant ES cell clones isolated upon transfection of the targeting vector are heterozygous for the correctly targeted allele as assessed by Southern blotting using 5′ and 3′ flanking probes (Fig. ). These two ES cell clones were used to generate germ line-transmitting chimeras that served as strain founders. The phenotypes observed are completely penetrant in each strain, so data from the two strains are not further distinguished. Routine genotyping was performed using a PCR assay designed to detect the wild-type and mutant alleles (Fig. ).
FIG. 1. Generation of a Thoc1 null allele in the mouse. (A) A representation of the exon/intron structure of the targeted region of the Thoc1 gene, the targeting vector, and the expected structure of the successfully targeted mutant allele. Exons are numbered (more ...)
Adult Thoc1 heterozygous (Thoc1+/−) mice are born at the expected Mendelian frequency and are overtly normal. In contrast, no homozygous null (Thoc1−/−) mice were recovered from more than 121 genotyped offspring from intermating of Thoc1+/− mice (Table ). The lack of Thoc1−/− mice indicates that Thoc1 is required for embryonic development. We investigated the timing of presumptive embryonic mortality by genotyping embryos from heterozygote intercrosses at various stages of gestation. No Thoc1−/− postimplantation embryos were detected among those analyzed at E8.5 to E11.5. However, empty deciduae were often observed. While the empty deciduae may account for the missing Thoc1−/− embryos, pure embryonic tissue sufficient for PCR genotyping could not be recovered to confirm this possibility. In contrast, preimplantation Thoc1−/− blastocysts genotyped at E3.5 were recovered at the expected Mendelian ratio, suggesting that embryonic development ceases around the time of implantation.
Genotypes of neonates and embryos from Thoc1+/− intercrossesa
To determine whether preimplantation Thoc1−/− embryos exhibit developmental defects, embryos were flushed from the oviducts or uteri and cultured in vitro. Freshly isolated E3.5 Thoc1−/− embryos had a morphology similar to that of wild-type (Thoc1+/+) embryos but generally failed to hatch from the zona pellucida or form blastocyst outgrowths in culture (Fig. ). A small fraction (<5%) of freshly isolated E3.5 Thoc1 null embryos were able to hatch upon in vitro culture, and a few cells with trophoblast morphology were able to attach to the culture dish. However, such embryos never produced viable blastocyst outgrowths, as cells of the inner cell mass (ICM) were lacking (data not shown). While E3.5 Thoc1−/− embryos cultured in vitro failed to hatch normally, the frequency of empty deciduae detected in vivo suggests that development may proceed sufficiently to induce a decidual reaction.
FIG. 2. Developmental defects in Thoc1 nullizygous embryos cultured in vitro. (A) E3.5 embryos produced by intermating Thoc1+/− mice were collected and cultured in vitro for up to 3 days. Representative phase-contrast images of embryos of the (more ...)
Freshly isolated E1.5 Thoc1−/−
embryos cultured in vitro compacted normally after 1 day in culture (E2.5) and developed into normal-appearing blastocysts by E3.5 (Fig. ). In contrast to Thoc1+/+
embryos, however, these Thoc1−/−
embryos typically failed to reach the fully expanded blastocyst stage and did not form blastocyst outgrowths even after experimental removal of the zona pellucida (Fig. ). These data suggest that Thoc1−/−
embryos suffer from developmental defects at the late blastocyst stage, which compromises hatching, implantation, and subsequent development. Consistent with this possibility, freshly isolated Thoc1−/−
E2.5 embryos cultured in vitro for 1.5 days had significantly fewer cells, on average, than Thoc1+/+
embryos (25 versus 36 cells per embryo, respectively; P
< 0.0005). Similarly cultured Thoc1−/−
embryos exhibited an increase in the number of apoptotic cells as measured by immunostaining for the activated form of caspase 3 (Fig. ). Condensed nuclear fragments characteristic of the remnants of apoptotic cells were also apparent in Thoc1−/−
embryos as visualized by DNA staining. However, there was no significant difference in the mitotic indexes of Thoc1+/+
E2.5 embryos cultured in vitro for 1.5 days as assayed by immunostaining for the phosphorylated form of histone H3 (Fig. ). Further, E2.5 Thoc1−/−
embryos cultured in vitro for only 1 day showed no difference in total cell numbers (24 versus 25; P
> 0.8). Hence, cell numbers in Thoc1−/−
embryos are relatively normal up to around E3.5 but thereafter fail to accumulate due to a loss of cell viability. This loss of cell viability is the likely cause of the embryonic lethality observed around the time of implantation.
Consistent with the interrogation of gene expression databases, we find that Thoc1
RNA is present in the fertilized oocyte and throughout preimplantation embryonic development (Fig. ). Thoc1
protein is also widely expressed at later stages of development. Sagittal sections of E12.5 wild-type embryos have been immunostained for pThoc1, and nuclear pThoc1 staining is observed in all tissues and cell types that are detectable in such sections (Fig. ). Similar results are observed in E13.5 embryos (data not shown). We conclude that Thoc1
is widely expressed during embryonic development. Thoc1
expression has previously been detected in a wide range of adult tissues (5
FIG. 3. Thoc1 expression during embryonic development. (A) Total RNA was isolated from wild-type embryos at the indicated developmental stages (C, cell number stage; M, morula; B, blastocyst). RNA was reverse transcribed, and Thoc1 cDNA was amplified by PCR using (more ...)
Since pThoc1 may be present in early Thoc1−/− embryos due to translation of maternally supplied mRNA, we determined whether the loss of cell viability observed in Thoc1 null embryos coincided with the disappearance of pThoc1. Immunostaining of E2.5 embryos showed that nuclear pThoc1 staining was barely detectable in E2.5 Thoc1−/− embryos while it was readily apparent in heterozygous embryos (Fig. ). By E3.5, pThoc1 nuclear staining was undetectable in Thoc1−/− embryos while strong nuclear pThoc1 staining remained in Thoc1+/− embryos. Hence, pThoc1 levels were declining by E2.5 and undetectable by E3.5 in Thoc1−/− embryos, coinciding with the timing of loss of cell and embryo viability.
The blastocyst stage marks segregation into the first two cell lineages in the mammalian embryo, the ICM, comprised of undifferentiated embryonic stem cells that ultimately give rise to the embryo proper, and the differentiating trophoectoderm (TE), which contributes to extraembryonic tissues, like the placenta. We immunostained blastocysts for the differentiation markers Oct4 (ICM) and Cdx2 (TE) to determine if cellular differentiation occurs in Thoc1−/− blastocysts and to identify which cell type fails to accumulate. As expected, wild-type late-stage blastocysts showed a well-organized inner cell mass comprised of cells expressing Oct4, but not Cdx2 (Fig. ). Cells comprising the presumptive TE of Thoc1-expressing blastocysts express Cdx2, but not Oct4. In contrast to wild-type blastocysts, there were very few Oct4-positive cells in age-matched Thoc1−/− embryos, and a well-organized inner cell mass was not apparent (Fig. ). However, the few Oct4-positive cells observed were Cdx2 negative. The presumptive TE of Thoc1−/− blastocysts contained approximately normal numbers of Cdx2-positive and Oct4-negative cells in the appropriate spatial organization (Fig. ). Proper cellular differentiation of the TE in Thoc1−/− blastocysts is also supported by the appearance of the blastocoel cavity, which requires formation of adherens junctions and tight junctions, the apparent initiation of a decidual reaction (see above), and normal-appearing E-cadherin staining (data not shown). These data suggest that cellular differentiation is properly initiated in Thoc1−/− embryos but that Oct4-positive Cdx2-negative cells of the ICM fail to survive. We conclude that Thoc1 deficiency causes peri-implantation embryonic lethality due initially to the failure of Oct4-positive cells of the ICM to survive.
FIG. 4. Thoc1−/− blastocyst stage embryos lack Oct4-positive cells of the inner cell mass. (A) Freshly isolated E2.5 embryos of the indicated genotypes were cultured in vitro for 1.5 days and then immunostained for pThoc1 and Oct4 protein. DNA (more ...)
The widespread expression of pThoc1 in developing embryos and adults suggests the possibility that pThoc1 may also be required for later stages of embryonic development, as well as for normal homeostasis of adult tissue. Due to the early embryonic lethality observed in Thoc1−/− mice, however, it is currently unclear whether this is the case. Alternatively, undifferentiated stem or progenitor cells may be uniquely dependent on pThoc1. This requirement would block early embryonic development but might not influence later stages of embryonic or adult development. We currently favor the latter hypothesis based on the preliminary observation that conditional ablation of mouse Thoc1 in differentiating mammary epithelial cells or embryonic fibroblasts has little effect on cell viability (X. Wang, Y. Li, and D. Goodrich, unpublished data). Phenotypic characterization of mice containing such conditionally null or hypomorphic alleles of Thoc1 will be necessary to definitively test this hypothesis.
The mammalian TREX component pThoc1 is essential for the viability of at least some cell types in mice. Given that murine pThoc1 has a high degree of primary amino acid sequence similarity to pThoc1 from other metazoan species, the requirement for pThoc1 is likely to extend to other multicellular organisms. For example, Drosophila melanogaster
and human cancer cell lines both exhibit reduced viability upon depletion of their respective orthologous Thoc1
). In contrast, HPR1
is not essential for the viability of the unicellular yeast S. cerevisiae
, although Hpr1p-deficient yeasts grow more slowly, are temperature sensitive for growth, and have a reduced cellular life span (14
). The differences in the physiological requirements for HPR1/Thoc1
may reflect TREX-independent functions of pThoc1 or may be due to differences in the functions of the yeast and metazoan TREX complexes. While all detectable pThoc1 is resident within TREX complexes (13
), we currently cannot exclude the possibility that TREX independent functions are responsible for the pThoc1 requirement for cell viability. Since the yeast and metazoan TREX complexes differ in subunit composition, we favor the second hypothesis, that metazoan TREX complexes may have functions and regulatory inputs distinct from their yeast counterpart and that these differences account for the different physiological requirements for HPR1/Thoc1
. Resolution of this issue will require further comparison of the molecular, cellular, and physiological functions of the yeast and metazoan TREX complexes.