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Molecular and Cellular Biology, April 2001, p. 2880-2890, Vol. 21, No. 8
Department of Genetics, University of
Washington, Seattle, Washington
Received 27 July 2000/Returned for modification 1 September
2000/Accepted 3 January 2001
The transition from preimplantation to postimplantation development
leads to the initiation of complex cellular differentiation and
morphogenetic movements, a dramatic decrease in cell cycle length, and
a commensurate increase in the size of the embryo. Accompanying these
changes is the need for the transfer of nutrients from the mother to
the embryo and the elaboration of sophisticated genetic networks that
monitor genomic integrity and the homeostatic control of cellular
growth, differentiation, and programmed cell death. To determine the
function of the murine zinc finger protein ZFR in these events, we
generated mice carrying a null mutation in the gene encoding it.
Homozygous mutant embryos form normal-appearing blastocysts that
implant and initiate the process of gastrulation. Mutant embryos form
mesoderm but they are delayed in their development and fail to form
normal anterior embryonic structures. Loss of ZFR function leads to
both an increase in programmed cell death and a decrease in mitotic
index, especially in the region of the distal tip of the embryonic
ectoderm. Mutant embryos also have an apparent reduction in apical
vacuoles in the columnar visceral endoderm cells in the extraembryonic
region. Together, these cellular phenotypes lead to a dramatic
development delay and embryonic death by 8 to 9 days of gestation,
which are independent of p53 function.
Gastrulation of the mammalian
embryo initiates shortly after implantation into the maternal uterine
tissue. The growing population of epiblast cells becomes the source for
the generation of the definitive embryonic tissues: the mesoderm,
endoderm, and ectoderm. Cell proliferation, differentiation, and
morphogenetic movements all contribute to the establishment of the
major body axes and the patterning of the definitive tissues
(7).
Prior to implantation, embryonic cells divide very slowly, with little
increase in mass. After implantation, as gastrulation initiates, a
massive increase in growth occurs, accompanied by a decrease in cell
cycle length (18). The number of ectodermal cells in
particular rises from an estimated 120 cells at embryonic day 5.5 (E5.5) to 660 cells at E6.5 and 8,060 cells at E7.5. To accommodate
this proliferation, the cell cycle length drops from approximately
11.5 h at E5.5 to 4.4 h at E6.5. In contrast, the newly
formed mesodermal cells, first produced at E6.5, divide every 8 to
10 h.
The accelerated growth during gastrulation imposes increased
biosynthetic and energy requirements on the embryo. A null mutation in
the dihydrolipoamide dehydrogenase gene (Dld), which encodes an enzyme functioning in mitochondrial oxidative metabolism, results in
developmental delay and lethality at this stage (8). The high proliferation rate of the epiblast also renders the cells more
sensitive to metabolic inhibitors (12, 15). Furthermore, in comparison to trophoblasts, the epiblast cells are more vulnerable to damaging X-ray or UV irradiation (12).
The rapid mitotic division at gastrulation also places a
stringent requirement on DNA surveillance systems. Several DNA repair and genome surveillance genes are expressed at gastrulation and are
required for embryonic survival. In the absence of these regulators, instability in the genome accumulates and progression through the cell
cycle slows or arrests, bringing development to a halt. This course of
degeneration is observed in mouse embryos deficient for the DNA repair
genes Xrcc1 (20) and Rad51
(9) and the tumor suppressor genes Brca1 and
Brca2 (6, 10, 17, 19). Thus, the Rad51,
Brca1, Brca2 and Xrcc1 genes together constitute a
class of genes that functions at the cellular level in the maintenance of the integrity of the genome, and they are required for the progression of early postimplantation development.
The murine gene Zfr (zinc finger RNA binding) encodes a
novel zinc finger protein that is expressed at high levels during gametogenesis (13). Conserved in Drosophila
melanogaster, Caenorhabditis elegans, and Homo sapiens,
Zfr encodes three widely spaced C2H2 zinc
fingers followed by a 316-amino-acid domain, which is conserved among a
small class of double-stranded RNA-binding proteins. This common
feature in amino acid composition, together with the means by which
Zfr was identified, suggests the gene encodes a zinc finger
RNA-binding protein. However, results from Northwestern and
Southwestern binding assays showed that recombinant ZFR protein has the
capacity to bind both DNA and RNA in vitro (13). At this
time, the nature of the ZFR substrates in vivo has not been identified.
By Northern analysis, Zfr appears to be expressed at varying
levels within all tissues. Within the testis and ovary, ZFR
immunostaining is particularly strong in the nuclei of cells actively
engaged in meiotic recombination. ZFR localizes to the meiotic
chromosomes and is absent from interchromosomal regions. ZFR is also
expressed in a subset of somatic cells within the gonads but is not
detected in others.
The discovery of the unique motif organization within the ZFR
amino acid sequence, in conjunction with the observed ZFR chromosome localization, led us to learn more about the functional properties of
Zfr in the mouse. Through gene targeting in embryonic stem (ES) cells, a loss-of-function mutation in Zfr was created
to investigate the requirement for Zfr in cell function and
tissue development.
Gene targeting Zfr locus in embryonic stem
cells.
A 129X1/SvJ mouse genomic bacteriophage library was
screened with the SacI-PstI fragment (nucleotides
1215 to 1731) from cDNA Zfr.1. A 15-kb genomic clone was
isolated, mapped, and used to generate a gene targeting construct (Fig.
1A). A 1.35-kb
HpaI-PstI genomic fragment, containing exonic
sequence at the 3' end, was fused upstream of and in frame with a
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.8.2880-2890.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Requirement for the Murine Zinc Finger Protein ZFR
in Perigastrulation Growth and Survival
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
geo-polyadenylation signal cassette that lacked the
splice acceptor sequence. A 6-kb region from the 3' end of the genomic
clone constituted the right homologous arm. A cassette consisting of
the polyglycerate kinase (Pgk) promoter, the diphtheria
toxin (dt) gene, and a polyadenylation signal was cloned
downstream of the right arm in the same orientation. The targeting
construct DNA (25 µg) was linearized with SacII and electroporated into 107 129X1/SvJ ES cells (Bio-Rad Gene
Pulsar II, 500 µF, 0.24 kV). ES cells were cultured on a feeder layer
of SNL cells in Dulbecco's modified Eagle's medium supplemented with
15% fetal bovine serum, glutamine, antibiotics, 0.1 mM
-mercaptomethanol, and 300 µg of G418 (Gibco BRL) per ml for 10 days (14). The plasmid bearing the geo and
dt cassettes, the AK7 ES cells, and the SNL cells were
generously provided by P. Soriano. A fraction of each G418-resistant colony was picked with a drawn capillary tube for PCR analysis. PCR was
performed using a Zfr genomic primer that maps 5' of the targeting construct's left arm (primer b, MM115: 5' TCC ATA TGG TGG ACT CTC AG 3'), a primer to the 5' end of geo
(primer a, 5geo2: 5' GGG ATC CGC CAT GTC ACA G 3'), and
Gittschier buffer PCR conditions (7). Twenty of 37 clones
were positive for the 2.2-kb PCR product. DNA samples from six clones
were prepared and digested with EcoRV,
HindIII, or StuI, electrophoresed on a 1%
agarose gel, blotted, and probed three times (16). The template for the 5' probe was the genomic
EcoRV-StuI fragment located 5' of the left arm in
the targeting construct. The template for the 3' probe was PCR
amplified with the Zfr cDNA sequence corresponding to the
three exons in the right arm of the targeting construct. The
Neo probe was a 660-bp PCR product from the
geo plasmid (primer e, neol: 5' GAT TGC ACG CAG GTT
CTC C 3'; and primer f, neo2: 5' GTA GCC AAC GCT ATG TCC TG
3'). Five of the six lines were heterozygous at the
Zfr locus and had no additional random integrations.
View larger version (28K):
[in a new window]
FIG. 1.
(A) Gene targeting strategy, showing maps of the
targeting construct and the wild-type and targeted alleles. Exons are
shown as black boxes. Exons encoding zinc fingers 1, 2, or 3 are
labeled ZF1, ZF2, or ZF3. The 5' probe for Southern blotting is
represented by the open box on the left. The 3' probe sequences are
represented by the three open boxes on the right. The PCR primers used
for genotyping are designated a through f. A subset of restriction
enzyme sites are shown. RV, EcoRV; S, StuI; H,
HpaI; HIII, HindIII; P, PstI. (B)
Southern blots of ES cell DNA from clone 25 and clone 32 cut with
EcoRV, StuI, or HindIII. The 5'
probe detects the wild-type 7.0-kb and the mutant 3.8-kb
EcoRV alleles. The 3' probe detects the wild-type 11.5-kb
and mutant 17.3-kb StuI alleles and the wild-type 8.2-kb and
mutant 13.0-kb HindIII alleles. (C) PCR products from
E7.5 embryos, which were derived from a Zfr heterozygote
intercross, electrophoresed on a 3% agarose gel. First lane, 1-kb DNA
ladder (Life Technologies Gibco-BRL). PCR primers c and d detect the
wild-type allele and generate a 377-bp band. Neo primers e and f
generate a 660-bp band.
Generation of Zfr mutant mice. Cells from lines 25 and 32 were injected into day 3.5 C57BL/6 (Jackson Laboratories) blastocysts and implanted in DBA/BL6 pseudopregnant females. Germline-transmitting chimeric males, obtained from both lines, were backcrossed to C57BL/6 and 129S4/SvJae females. Progeny from all crosses were PCR genotyped using a wild-type allele-specific primer (primer d, MM105: 5' CAG CTG ATC TTA CAA ACA TCA C 3') with a primer to the left arm (primer c, MM112GR: 5' CAA CAC CTG ACT GTC AAG TAA 3') and targeted allele-specific neo primers e and f (listed above).
X-Gal staining of preimplantation embryos.
Preimplantation
embryos were flushed from the oviduct or uterine horns of pregnant
females. Embryos were placed in 50-µl microdrops under oil. Embryos
were washed in phosphate-buffered saline (PBS) plus 10 mg of bovine
serum albumin/ml for 5 min, fixed in PBS plus 10 mg of bovine serum
albumin/ml, 2% formaldehyde, and 0.2% glutaraldehyde for 10 min, and
subsequently washed three times for 10 min. Embryos were stained in
wash plus 2 mM MgCl2, 5 mM ferricyanide, 5 mM ferrocyanide,
and 1 mg of
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
per ml.
Embryo dissections and histological analysis. Timed matings were conducted with F1 Zfr heterozygous hybrid (129S4/SvJae × C57BL/6) mice. Females with copulation plugs the next day were considered to be at day 0.5 of gestation. Pregnant females were sacrificed at different time points of gestation, and the embryos were dissected free of maternal tissue, examined, photographed, and genotyped by PCR (see above). For histological preparations, embryos in decidua were fixed in methacarns (60% methanol, 30% chloroform, 10% glacial acetic acid) overnight at 4°C. Tissues were processed three times for 15 to 30 min in methanol, once for 45 to 60 min in methylbenzoate, twice for 20 to 30 min in xylenes, and twice for 20 to 45 min in paraffin. Incubation times depended on the sizes of the samples. Five-micrometer sections of tissues were cut from paraffin blocks. Sections were stained with hematoxylin and eosin.
Immunocytochemistry. Slides with paraffin sections were deparaffinized in xylenes and rehydrated. Slides were blocked in PBS plus 3% goat serum for 20 to 60 min. Anti-ZFR antibodies were diluted 1:500 in PBS with goat serum and incubated with sections at 4°C overnight. Sections were then washed in PBS three times for 5 min, incubated with 1:200 biotinylated goat anti-rabbit immunoglobulin G (Zymed, San Francisco, Calif.) for 30 min, washed, incubated with streptavidin-HRP (Zymed) for 15 min, and washed again. The aminoethylcarbazole color substrate was prepared according to instructions from Zymed and incubated for 5 to 15 min, followed by a 5-min rinse in tap water. Sections were mounted with Aquamount (Lerner Laboratories, Pittsburgh, Pa.).
BrdU labeling of embryos. Pregnant females at E6.5 were injected intraperitoneally with 100 µg of bromodeoxyuridine (BrdU) per gram of body weight and were sacrificed 1 h later. Decidua were removed, fixed in 4% paraformaldehyde overnight at 4°C, and processed as described previously (1). Five-micrometer sections were processed for anti-BrdU labeling. After deparaffinization, sections were incubated in 50% formamide-1× SSC-0.1% Tween (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 70°C for 30 min, microwaved in 10 mM citric acid (pH 6.0) for 10 min to expose the BrdU antigen, and incubated with a 1:50 dilution of anti-BrdU antibody overnight at 4°C (Becton Dickinson, San Jose, Calif.). Biotinylated anti-mouse antibodies were used as secondary antibodies. Sections were counterstained with hematoxylin, mounted, and photographed. Cells from the embryonic, extraembryonic, and ectoplacental cones of each embryo were counted and scored for BrdU staining.
TUNEL analysis of embryos. E6.5 embryos in decidua were fixed in 4% paraformaldehyde and processed as described above. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were performed on sections by using the Apoptag assay (Intergen, Purchase, N.Y.). Sections were counterstained briefly with methyl green, mounted with Permount, and photographed.
In vitro culture of E3.5 embryos. F1 Zfr heterozygotes were allowed to mate for 2 h and checked for copulation plugs. E3.5 blastocysts were flushed from the uteri of plugged females and placed in gelatinized 24-well dishes. Embryos were cultured in ES cell media without the addition of leukemia inhibitory factor and then were photographed every 24 h. After 7 days in culture, the outgrowths were picked with drawn Pasteur pipettes and genotyped by PCR.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Targeted disruption of Zfr gene.
To disrupt the
Zfr gene, a targeting vector was designed in which a 6-kb
region was deleted and replaced with a promoterless lacZ-neomycin resistance fusion gene (geo)
(Fig. 1A). Homologous recombination at the Zfr locus would
result in the deletion of three exons, including the sequence encoding
zinc fingers 1 and 2, disruption in the expression of the remaining
downstream exons, and fusion of the Zfr transcription unit
to geo. ES cells from two homologous recombinant lines
were identified and injected into C57BL/6 blastocysts (Fig. 1B and C).
Chimeras were backcrossed to C57BL/6 and 129S4/SvJae mice to study the
mutant phenotype on hybrid as well as pure genetic backgrounds. The
phenotypes described below were consistently seen in both inbred
strains and in the hybrid 129S4/SvJae × C57BL/6 mutants.
ZFR expression in early embryo.
Immunolocalization experiments
in the ovary had revealed that ZFR staining is strong in primary and
growing oocytes (13). To determine whether maternal ZFR
protein localizes to the newly formed pronuclei in the one-cell embryo,
anti-ZFR immunofluorescence and 4',6'-diamidino-2-phenylindole
(DAPI) staining were performed with E0.5 embryos. Anti-ZFR staining was
found in the male and the female pronucleus but not in the polar bodies
(Fig. 2A). Thus, ZFR is present at the
beginning of embryogenesis.
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Zfr mutation results in early embryonic lethality.
Heterozygous Zfr F1 hybrids (C57BL/6 × 129S4/SvJae) displayed no overt phenotype and were fertile.
Intercrosses between heterozygotes were established to generate
homozygous mutant offspring. No viable Zfr homozygotes were
identified among the 105 born F2 progeny, indicating that
the homozygous mutation causes embryonic lethality (Table
1).
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Histological analysis of Zfr homozygotes.
To
investigate the nature of the phenotype in greater detail, histological
sections of mutant embryos from different gestation time points were
stained with hematoxylin and eosin and analyzed. In general, the
development and organization of the germ layers in E6.5 mutants
resembled those of wild-type embryos (Fig. 4A and
B). However, two deficiencies were
detected in mutants at this stage. Numerous pyknotic cells were
consistently found in the proamniotic cavity and in the epiblast
(embryonic ectoderm) (Fig. 4C). Furthermore, the apical vacuoles in the
extraembryonic endoderm cells appeared to be diminished, as greater
eosin staining was observed in this region.
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Increased cell death in ectoderm of Zfr mutant embryos. Pyknotic cells were commonly observed in histological sections of Zfr mutant embryos. Especially prominent in the embryonic ectoderm, these abnormal cells were often the first indication of the mutant genotype at E6.5. To determine whether the frequent pyknosis and growth failure of Zfr mutant embryos were associated with increased programmed cell death, a TUNEL assay was performed on sections of E6.5 embryos.
The TUNEL assay was performed on sagittal sections near the midline of 7 Zfr mutants and 22 normal E6.5 embryos (Fig. 7A and B). Apoptotic cells were expected to be observed in sections of Zfr wild-type and heterozygous embryos, as programmed cell death naturally occurs during early embryogenesis (11). Among the normal embryo sections, an average of 13.2 TUNEL-positive cells per section were counted. In contrast, mutant embryos displayed a prominent and statistically significant increase in the number of TUNEL-positive cells, averaging 43.7 TUNEL-positive cells per section (Student's t test, P < 0.0005). The apoptotic bodies were especially common in the embryonic ectoderm tissue and were often clustered near the distal end of the mutant embryos (Fig. 7B).
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Decreased proliferation rates in Zfr mutant embryos. A second possibility that could contribute to the small size and narrow cavities in Zfr homozygous mutant embryos is a defect in embryonic cell proliferation. To examine the mitotic index of the Zfr mutants, pregnant females from Zfr heterozygote intercrosses carrying E6.5 embryos were injected with BrdU 1 h prior to dissection. Sections of embryos in decidual swellings were processed for anti-BrdU immunohistochemistry. Sagittal sections close to the midline of five mutant and four normal embryos were selected and examined (Fig. 7C and D). BrdU-positive and -negative cells were counted and classified as belonging to the embryonic, extraembryonic, or ectoplacental region. Calculation of the number of BrdU-positive cells divided by the total number of cells generated the mitotic index value. When cells from all regions of the embryos were counted, an average mitotic index of 0.78 was found for the normal class of embryos. A significantly lower average mitotic index of 0.69 was found for the mutant embryos (Student's t test, P < 0.025).
In transverse sections of E7.5 Zfr mutants, a more pronounced developmental delay was observed in the embryo proper versus the extraembryonic region. Similarly, the TUNEL assay showed localized programmed cell death in the embryonic ectoderm. It therefore was possible that, in mutant embryos, proliferation rates in embryonic tissues were more severely affected than those in extraembryonic tissues. When calculated for the embryonic regions exclusively, the average mitotic indices were 0.76 for normal embryos and 0.61 for mutant embryos (Student's t test, P < 0.005). In contrast, the average mitotic index for the combined extraembryonic and ectoplacental regions was 0.76 for normal embryos and 0.73 for mutant embryos. The fraction of BrdU incorporation in mutants was therefore especially low in the embryo proper but not significantly different in the extraembryonic tissues.The Zfr mutation causes degeneration of in vitro-cultured blastocyst outgrowths. To assay the intrinsic growth and survival potential of the Zfr mutant embryos, E3.5 embryos obtained from timed Zfr heterozygous intercross matings were cultured in vitro for 7 days. Grown in ES cell media without supplemental LIF, the blastocyst outgrowths were monitored daily and ultimately genotyped by PCR on the last day. Eleven Zfr heterozygous, five Zfr homozygous, and six wild-type embryos were assayed. As blastocysts, the Zfr mutants displayed no overt anomalies, suggesting that the Zfr mutation does not lead to a preimplantation phenotype. All of the blastocysts successfully attached to the culture dish, hatched from the zona pellucida, and initiated growth. Proliferating and differentiating inner cell masses, surrounded by trophoblasts, were observed in wild-type and Zfr heterozygous outgrowths. Cavitation of the inner cell mass was observed by the seventh day of culture for 10 of the 17 wild types and heterozygotes.
Outgrowths from four Zfr homozygous mutant blastocysts grew aberrantly in culture. Initially, mutant blastocyst outgrowths were indistinguishable from the remaining outgrowths. Comparable growth of the inner cell mass and trophoblasts could be seen in a wild-type and in a mutant blastocyst after 2 and 4 days in culture (Fig. 8). Shortly thereafter, by day 5, obviously undersized inner cell masses were observed in the four mutants. In three cases, the inner cell mass subsequently deteriorated, leaving mostly trophoblast cells behind (Fig. 8). The fourth mutant outgrowth did not degenerate and instead grew slightly through day 7 despite its reduced size. These observations thus confirm that the Zfr mutation can detrimentally affect the growth of blastocysts when cultured outside the maternal environment. The impaired growth of the mutant embryos could be a manifestation of increased programmed cell death, decreased cellular proliferation, or both. Among the Zfr mutant blastocysts, one exception to the course of development described above was found. A fifth mutant outgrowth grew as well as the normal embryos and developed a substantial, differentiated, but not cavitated, inner cell mass by day 7.
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Analysis of Zfr-deficient embryos in
Trp53 null background.
The tumor suppressor gene
Trp53 (encoding p53) plays a central role in the control of
apoptosis and cell cycle progression. To determine whether the
phenotype seen in Zfr mutant embryos would be altered by the
loss of p53 function, Zfr heterozygous females were mated to
Trp53 heterozygous males to obtain compound heterozygotes.
The Zfr+/
Trp53+/
animals were intercrossed, and litters were dissected at E8.5. The
rationale for choosing this time point was based on the Zfr phenotype and results from genetic crosses of Trp53 with
other mutations. A Trp53-deficient genetic background was
found to partially rescue the developmental life span of embryos
deficient for Brca1, Brca2, Rad51, or Xrcc1,
presumably because cell cycle checkpoints, apoptosis, or both pathways,
were no longer activated (9, 10, 20). At E8.5, the
Zfr null embryos could still be dissected and accurately
genotyped but were tiny and deformed. Thus, any rescue of the
Zfr phenotype by the Trp53 mutation would most
likely be morphologically distinct at this stage. Among 126 embryos
dissected, only one Zfr/
Trp53/ double mutant embryo was isolated,
strongly suggesting that loss of p53 in Zfr mutants does not
rescue the Zfr embryos from demise at E8.5.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The Zfr mutation results in embryonic cell death, growth impairment, and dysmorphogenesis. Elimination of the Zfr gene has severe consequences for postimplantation and gastrulation stage development. Early indications of embryonic failure can be detected prior to primitive streak formation at E6.5. At this stage, Zfr mutants display increased cell death in the epiblast and decreased vacuolization in the visceral endoderm. Cellular proliferation slows, and consequently, primitive streak embryos are growth retarded. As gastrulation proceeds, a loose amnion develops to cover the constricted amniotic cavity. At their most advanced state, the tiny headfold-stage Zfr mutants are morphologically anomalous and are degenerating. The embryonic tissues appear to be particularly blighted in comparison with the extraembryonic region, which exhibits a grossly normal chorion and allantois. Despite the growth deficiency and abnormal levels of programmed cell death at E6.5, Zfr mutants are not impaired in primitive streak formation and mesoderm differentiation and migration. Extraembryonic mesodermal structures, including proamniotic folds, chorion, and allantois, develop, and attachment of the allantois to the chorion was observed. Thus, the Zfr mutation is distinct from mutations that interrupt the gastrulation process directly.
The visceral endoderm, an extraembryonic tissue, serves a number of functions critical for the proper development of the conceptus. In addition to its role in developmental patterning of the underlying ectodermal tissue (2), the visceral endoderm cells supply nutrients to the embryonic tissue by synthesizing serum components and endocytosing maternal macromolecules that have passed through Reichert's membrane into the parietal yolk sac cavity (3). The columnar visceral endoderm cells adjacent to the extraembryonic ectoderm are specialized for this task; polarized in organization, these cells contain many lysosomes and microvilli at the apical surface. The anomalous morphology noted in the Zfr visceral endoderm cells could be an indication of a requirement for ZFR in visceral endoderm function. One possible interpretation of the Zfr phenotype is that some of the nutritional links between the visceral endoderm and the adjacent ectoderm are disrupted, which in turn leads to survival and proliferation problems in the embryonic ectoderm. The combination of ectodermal cell death and anomalous visceral endoderm histology at gastrulation stages has been described with other mouse mutants, notably the huntingtin and hnf4 mutants (4, 21). At the start of gastrulation at E6.5, Zfr mutants display increased apoptosis in the embryonic ectoderm and decreased cell proliferation rates in vivo. When cultured in vitro, most E3.5 mutant embryos degenerate by 7 days of culture. Wild-type and Zfr heterozygote outgrowths, by comparison, show continual growth of the inner cell mass. The compromised growth of the Zfr mutant embryonic cells in vivo and in vitro suggests the hypothesis that the embryonic cells are intrinsically impaired by the Zfr mutation. The Zfr phenotype becomes more apparent when the embryonic growth rate increases and the length of the cell cycle decreases. These changes at gastrulation could be responsible for the developmental course observed in Zfr mutants. However, in an investigation of four genes involved in cell cycle control and DNA surveillance, Brca1, p21, p53, and mdm2 showed no difference in expression among wild-type, heterozygous, and mutant E6.5 littermate embryos (unpublished data).Making it to gastrulation: possibilities for cell lethality and
maternal rescue in Zfr embryos.
Experiments to address
the tissue distribution of Zfr transcripts revealed that
Zfr is most highly expressed in the testis, brain, and
ovary, suggesting that ZFR has an important activity in the function or
development of cells in these tissues (13). The low-level
expression of Zfr in other tissues as well pointed to the
possibility that Zfr is expressed in a variety of cells at
modulated levels. Our first observation of ZFR expression during embryogenesis came from investigating whether the ZFR expression in
oocyte nuclei was continued in the one-cell embryo. The observed localization of ZFR in the male and female pronuclei and the detection of -galactosidase expressed from the targeted Zfr allele
in blastocysts suggested that Zfr might be essential for
preimplantation development.
/ embryo might be accumulating subtle
damage that only later leads to an observable phenotype, as has been
hypothesized for the DNA repair and tumor suppressor mutants described
earlier. A second factor that could permit the survival of
Zfr mutant embryos to gastrulation is rescue by maternal
Zfr RNA or protein present in one-cell embryos. Maternal
gene products are generally not considered to play an important role in
mouse embryonic development beyond the two-cell stage, at which time
the zygotic genome becomes activated. Coincident with this activation,
specific degradation of maternal RNAs and rapid turnover of proteins
encoded by maternal messages have been observed (5, 7).
However, reports of targeted mutations in the mitochondrial oxidative
gene Dld and cyclin A2 gene have nevertheless invoked the
possibility of maternal rescue prolonging the lifespan of the
mutants (8). It is possible that small amounts of maternal
ZFR protein are retained beyond implantation and permit development to
proceed to gastrulation.
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ACKNOWLEDGMENTS |
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We thank Phil Soriano for many helpful discussions and for the generous gift of the AK47 ES cells, Kathy Kafer for expert technical assistance, and Bill Buaas for many critical discussions and advice.
This work was supported by a grant from NICHD (HD12629) through a cooperative agreement as part of the Specialized Cooperative Centers Program in Reproduction Research.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Genetics, Box 357360, 1959 NE Pacific, University of Washington, Seattle, WA 98195. Phone: (206) 543-1818. Fax: (206) 543-0754. E-mail: braun{at}u.washington.edu.
Present address: Corixa Corporation, Seattle, WA 98104.
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Discussion
References
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Molecular and Cellular Biology, April 2001, p. 2880-2890, Vol. 21, No. 8
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.8.2880-2890.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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