MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Dec. 2001, p. 8184–8188 Vol. 21, No. 23
The Mouse Snail Gene Encodes a Key Regulator of the
ETHAN A. CARVER, RULANG JIANG,† YU LAN,† KATHLEEN F. ORAM, AND THOMAS GRIDLEY*
The Jackson Laboratory, Bar Harbor, Maine 04609
Received 2 August 2001/Accepted 28 August 2001
Snail family genes encode DNA binding zinc finger proteins that act as transcriptional repressors. Mouse
embryos deficient for the Snail (Sna) gene exhibit defects in the formation of the mesoderm germ layer. In
Sna?/?mutant embryos, a mesoderm layer forms and mesodermal marker genes are induced but the mutant
mesoderm is morphologically abnormal. Lacunae form within the mesoderm layer of the mutant embryos, and
cells lining these lacunae retain epithelial characteristics. These cells resemble a columnar epithelium and
have apical-basal polarity, with microvilli along the apical surface and intercellular electron-dense adhesive
junctions that resemble adherens junctions. E-cadherin expression is retained in the mesoderm of the Sna?/?
embryos. These defects are strikingly similar to the gastrulation defects observed in snail-deficient Drosophila
embryos, suggesting that the mechanism of repression of E-cadherin transcription by Snail family proteins
may have been present in the metazoan ancestor of the arthropod and mammalian lineages.
Genes of the Snail family encode zinc finger proteins that
function as transcriptional repressors in a variety of experi-
mental systems (8, 10, 12, 16, 17, 21; reviewed in reference 11).
The first gene of this family studied was the Drosophila snail
gene, which is one of two genes required zygotically for meso-
derm formation during Drosophila embryogenesis (1, 5, 9, 13,
24; reviewed in reference 19). Embryos homozygous for null
mutations of snail exhibit defects in mesoderm formation, gas-
trulation movements, and germ band retraction (9, 24). The
snail protein is a transcriptional repressor which acts to main-
tain proper germ layer boundaries by repressing the expression
within the mesoderm of regulatory genes involved in ectoder-
mal development (18). Snail family genes are evolutionarily
conserved, and studies have implicated Snail family proteins in
the regulation of epithelial-mesenchymal transitions in tissue
culture systems and in both vertebrate and invertebrate em-
bryos (3, 7, 17, 19, 20, 23, 26, 27).
Two mouse homologs of snail, termed Sna and Slug, have
been cloned (15, 22, 28, 30). It has been previously demon-
strated that mice homozygous for a null mutation of the Slug
gene are viable, although they exhibit postnatal growth defi-
ciency (15). We describe here the construction and analysis of
a targeted mutation of the Sna gene. During gastrulation, Sna
is expressed in the primitive streak and the mesoderm germ
layer (22, 30). Sna-deficient mouse embryos die early in ges-
tation, exhibiting defects in gastrulation and mesoderm forma-
MATERIALS AND METHODS
Gene targeting. The Sna targeting vector was constructed from an 18-kb
genomic clone containing the entire Sna gene (14). The 5? arm was a 2.5-kb
SalI-NruI genomic fragment subcloned upstream of a PGK-neo expression cas-
sette. The 3? arm was a 1.2-kb XbaI-EcoRI fragment. This resulted in the
deletion of a 1.6-kb genomic fragment containing exons 1 and 2 of the Sna gene,
which deletes the translation initiation site and amino acids 1 to 203 of the Sna
protein, including degenerate zinc finger 1 and zinc fingers 2 and 3 of the DNA
binding domain. A herpes simplex virus (HSV)-tk cassette was introduced for
negative selection. Embryonic stem (ES) cell electroporation and selection and
blastocyst injections were performed as previously described (31). DNAs from
individual ES cell colonies were prescreened by PCR, and positive colonies were
then screened by Southern blotting, using a 0.5-kb EcoRI-SphI genomic frag-
ment as a probe on SphI-digested genomic DNA. Germ line transmission of the
Sna mutant allele was obtained for two independently targeted clones. The
official nomenclature for this mutant allele is Snatm1Grid.
Histology, in situ hybridization, and immunofluorescence. Embryos were dis-
sected at embryonic day 7.5 (E7.5) from timed matings of Sna?/?heterozygotes.
Mutant homozygotes were identified by allele-specific PCR or by their charac-
teristic morphology. A strict correlation was observed between genotype and the
characteristic Sna?/?mutant phenotype. Some embryos were sectioned in their
decidua for histological analysis. Decidua and isolated embryos were fixed in
Bouin’s fixative for histological analysis. Fixed embryos were dehydrated through
graded alcohols, embedded in paraffin, sectioned, and stained with hematoxylin
and eosin. Embryos for in situ hybridization were fixed overnight at 4°C in 4%
paraformaldehyde in phosphate-buffered saline. Whole-mount in situ hybridiza-
tion was performed as previously described (15). At least three Sna?/?mutant
embryos were analyzed for each of the probes. For analysis of E-cadherin RNA
expression, embryos were embedded in plastic resin after whole-mount in situ
hybridization and sectioned.
Sna?/?mutant embryos and control littermates were stained at E7.5 in whole
mount with a monoclonal anti-E-cadherin antibody (Zymed). Antibody staining
was detected with fluorescein-conjugated anti-rat immunoglobulin G (Jackson
ImmunoResearch). Whole-mount embryos were cut into thick sections using
electrolytically sharpened tungsten needles, mounted in Vectashield mounting
medium (Vector Laboratories), and examined with a fluorescent microscope.
Transmission electron microscopy. Embryos were fixed overnight in 2.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). After being washed, embryos
were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer. Embryos were
washed, dehydrated in an ethanol series, and treated with propylene oxide.
Embryos were infiltrated with Epon-araldite, and ultrathin sections were cut.
Specimens were imaged on a JEOL 100CXII transmission electron microscope.
Disruption of the mouse Sna gene. To analyze the role of the
Sna gene during embryogenesis in mice, we used gene target-
ing to construct a mutant allele from which exons 1 and 2 of the
Sna gene had been deleted (Fig. 1A). This deletion removes
the exons encoding the translation initiation site and amino
* Corresponding author. Mailing address: The Jackson Laboratory,
600 Main St., Bar Harbor, ME 04609. Phone: (207) 288-6237. Fax:
(207) 288-6077. E-mail: email@example.com.
† Present address: Center for Oral Biology and Department of Bi-
ology, University of Rochester, Rochester, NY 14642.
acids 1 to 203 of the 264-amino-acid Sna protein, including
degenerate zinc finger 1 and zinc fingers 2 and 3 of the DNA
binding domain. Germ line transmission of the Sna mutant
allele was obtained for two independently targeted clones (Fig.
1B). Heterozygous Sna?/?mice appeared normal.
No homozygous Sna?/?mice were found among the prog-
eny of the intercross of heterozygous Sna?/?mice, indicating
that our Sna mutant allele is a recessive lethal mutation. To
determine when homozygous mutant embryos were dying, em-
bryos were isolated from timed matings. At E6.5, Sna?/?mu-
tant embryos were not distinguishable from the embryos of
heterozygous and wild-type littermates. At E7.5, however, the
homozygous mutant embryos were smaller than the embryos of
their littermates (Fig. 1C). By E8.5, the Sna?/?embryos were
severely retarded compared to those of littermates and were
being resorbed (data not shown).
Sna?/?mutant embryos form a mesoderm cell layer. Histo-
logical analysis of Sna?/?mutant embryos at E7.5 demon-
strated the presence of three germ layers (see below), indicat-
ing that a mesoderm layer had formed in the mutants. This was
confirmed by analysis of the expression of several marker genes
(Fig. 2). In wild-type embryos, the Brachyury (T) gene is ex-
pressed in the primitive streak and in the rostral axial meso-
derm (32). In the Sna?/?embryos, expression levels of the T
gene were reduced compared to those of the controls and
expression did not extend as far rostrally in the embryo (Fig.
2A and B). The Lim1 gene in wild-type embryos is expressed in
the primitive streak and the mesodermal wings (2). Lim1 was
expressed in both of these structures in the Sna?/?embryos
(Fig. 2C and D). In wild-type embryos at the prestreak and
early streak stages, the Otx2 gene is expressed throughout the
epiblast but its expression gradually becomes restricted to the
anterior neuroectoderm (29). In Sna?/?mutants, Otx2 expres-
sion did not become anteriorly restricted (Fig. 2E and F). The
Cer1 gene is expressed in wild-type embryos in the anterior
visceral endoderm and the definitive endoderm (4). Cer1 was
expressed in these tissues in Sna?/?embryos, but expression
levels were reduced compared to those of the controls (Fig. 2G
and H). These marker studies confirm that the mesoderm,
ectoderm, and endoderm all differentiate in Sna?/?mutant
embryos, although some differences in expression levels or
details of the expression patterns were detected in the mutants.
The Sna?/?mutant mesoderm retains epithelial character-
istics. While histological and marker analyses clearly indicated
that a mesoderm cell layer differentiated in the Sna?/?mutant
embryos, morphological abnormalities were apparent in the
mutant mesoderm. In wild-type and heterozygous embryos by
E7.5, the mesoderm cell layer had delaminated from the prim-
itive streak and had migrated anteriorly between the embry-
onic ectoderm and the visceral endoderm to form the meso-
dermal wings (Fig. 3A, C, and E). Cells in the mesoderm layer
of these embryos had a morphology characteristic of that of
mesenchymal cells. In Sna?/?mutant embryos, a primitive
streak and a mesoderm layer formed and the cells migrated
anteriorly to form the mesodermal wings. However, many of
the mutant mesoderm cells did not have a characteristic mes-
enchymal morphology (Fig. 3B, D, and F). In most Sna?/?
mutant embryos, cavities or lacunae formed in the mesoderm
layer (Fig. 3D and F) and the mesoderm cells abutting these
lacunae exhibited an epithelial morphology. The cells lining
these lacunae had the appearance of a columnar epithelium
(Fig. 3F and 4B). Transmission electron microscopic analysis
revealed that the mutant mesoderm exhibited apical-basal po-
larity, which is typically observed in an epithelial cell layer.
These cells contained microvilli along the apical surface (i.e.,
the surface facing into the lacunae) (Fig. 4C) and contained
electron-dense adhesive junctions that resembled adherens
junctions (Fig. 4C and D).
E-cadherin expression is not downregulated in the Sna?/?
mutant mesoderm. Recent work has shown that Sna expres-
sion represses E-cadherin transcription in cultured epithelial
cell lines by binding to E boxes present in the E-cadherin
promoter region and that Sna overexpression causes epithelial
cell lines to adopt a fibroblast-like morphology and to acquire
tumorigenic and invasive properties (3, 7). E-cadherin protein
is a component of adherens junctions, and downregulation of
E-cadherin expression in cells in the primitive streak is be-
lieved to be important for gastrulation in vertebrates (6). We
therefore analyzed expression of the E-cadherin gene by in situ
hybridization of Sna?/?mutant embryos and littermate con-
FIG. 1. Targeted disruption of the mouse Sna gene. (A) Targeting
scheme. The upper line shows the genomic organization of the Sna
gene (14). The three exons are indicated by boxes. The region encod-
ing the amino terminus of the Sna protein is indicated by gray boxes,
the region encoding the zinc fingers is indicated by black boxes, and the
3? untranslated region is indicated by a white box. The middle line
represents the structure of the targeting vector. The lower line repre-
sents the predicted structure of the Sna locus following homologous
recombination of the targeting vector. The probe used for Southern
blot analysis is indicated. N, NruI; R, EcoRI; S, SalI; Sp, SphI; X, XbaI;
TK, thymidine kinase. (B) DNAs isolated from targeted ES cells were
digested with SphI, blotted, and hybridized with the indicated probe.
Wild-type (wt) and mutant hybridization bands are indicated. Three
independently targeted ES cell clones are shown. (C) Whole-mount
morphology of a Sna?/?embryo (right) and a control littermate em-
bryo (left) at E7.5. In all figures, normal littermate embryos were
either Sna?/?or Sna?/?and are indicated with a plus sign.
VOL. 21, 2001 Sna GENE KNOCKOUT 8185
trols (Fig. 5A to C). In the control embryos, E-cadherin ex-
pression was downregulated in the mesoderm (Fig. 5A and B).
In Sna?/?embryos, E-cadherin RNA expression was main-
tained in the mesoderm layer (Fig. 5A and C). However, the
levels of E-cadherin RNA observed in the mutant mesoderm
were lower than those observed in the embryonic ectoderm.
We also examined whether E-cadherin protein expression was
maintained in the mesoderm of Sna?/?embryos. This analysis
revealed that, as we observed with E-cadherin RNA, expres-
sion of E-cadherin protein was retained in the mesoderm of
the Sna?/?embryos but at lower levels than were observed in
the embryonic ectoderm (Fig. 5D to G).
FIG. 2. Analysis of marker gene expression in Sna?/?mutant em-
bryos at E7.5. (A and B) T expression. T is expressed in axial meso-
derm cells, and expression extends rostrally from the node (A). In the
Sna?/?embryo, T is expressed, but at lower levels than in the control
embryos, and does not extend as far rostrally in the embryo (B). (C and
D) Lim1 expression. Lim1 is expressed in the primitive streak and the
mesodermal wings (C). Lim1 is expressed in these tissues in the Sna?/?
embryo (D). (E and F) Otx2 expression. Otx2 is expressed in the
visceral endoderm and epiblast, and expression is gradually restricted
to the anterior third of the embryo as the primitive streak extends (E).
In the Sna?/?mutant, Otx2 expression is not restricted to the anterior
portion of the embryo (F). (G and H) Cer1 expression. Cer1 is ex-
pressed in the anterior visceral endoderm and the definitive endoderm
(G). In the Sna?/?embryos, Cer1 expression is reduced (H). All
embryos are oriented with the anterior side towards the left.
FIG. 3. Morphological abnormalities in the Sna?/?mutant meso-
derm. (A and B) Sagittal sections of embryos at E7.5. In the Sna?/?
mutant embryo (B), a posterior amniotic fold forms (arrow) but no
amnion or chorion is formed. (C to F) Transverse sections of embryos
at E7.5. In Sna?/?mutant embryos, lacunae form within the mesoderm
layers (arrows in D and F). Mesoderm cells lining these lacunae exhibit
an epithelial morphology. (A to D) Hematoxylin-and eosin-stained
paraffin sections. (E and F) Toluidine blue-stained plastic sections.
Abbreviations: am, amnion; ch, chorion; ee, embryonic ectoderm; m,
mesoderm; ps, primitive streak.
8186CARVER ET AL.MOL. CELL. BIOL.
We describe here the construction and analysis of a targeted
null mutation of the mouse Sna gene. Although previous work
had demonstrated that the related Slug gene is not essential for
embryogenesis in mice (15), Sna?/?mutant embryos die early
in gestation. The mutant embryos exhibit defects in gastrula-
tion and in the epithelial-mesenchymal transition required for
generation of the mesoderm cell layer. Our data indicate that
formation of the mesoderm cell layer can occur despite the
retention of E-cadherin expression. However, many cells in the
mesoderm of Sna?/?mutant embryos retain apical-basal po-
larity and an epithelial morphology, presumably due to the
retention of adherens junctions between the mesoderm cells in
the mutant embryos. The phenotypic defects we observe in the
Sna?/?mutant mouse embryos are strikingly similar to the
gastrulation defects observed in snail mutant Drosophila em-
bryos (25). The Drosophila E-cadherin gene is normally ex-
pressed in the epithelial cells of the cellular-blastoderm-stage
embryo but is then downregulated in mesoderm precursor cells
prior to invagination. In Drosophila embryos homozygous for a
snail null mutation, E-cadherin downregulation does not occur
and mesoderm precursors in the ventral region of the embryo
retain adherens junctions and apical-basal polarity (25). As
noted by Wolpert (33), the morphogenetic movements of gas-
trulation are more highly conserved than the establishment of
the body plan during evolution. The similarity of the gastrula-
tion defects in mutant Snail genes of both Drosophila and mice
indicates that the molecules regulating mesoderm formation
and gastrulation movements are conserved over an extremely
wide evolutionary distance. This observation suggests that re-
FIG. 4. Apical-basal polarity and adhesive junctions in the Sna?/?
mutant mesoderm. Transmission electron microscopic images for anal-
ysis of wild-type (A) and Sna?/?(B to D) embryos at E7.5. are shown.
(A) Mesoderm cells in wild-type embryos exhibit a typical mesenchy-
mal morphology. (B) In Sna?/?embryos, mesoderm cells lining the
lacunae exhibit an ordered, columnar morphology. (B to D) The lu-
mens of the lacunae are indicated with asterisks. (C) Mesoderm cells
in the mutant embryos have microvilli (arrowheads) at the apical
surface and exhibit electron-dense adhesive junctions (arrow) between
the cells. (D) Adhesive junctions are numerous in the Sna?/?mutant
mesoderm. The positions of intercellular adhesive junctions are indi-
cated by arrows. Approximate magnifications: (A and B) ?4,000, (C)
?20,000, (D) ?3,000.
FIG. 5. E-cadherin expression is retained in the mesoderm of
Sna?/?mutant embryos. (A) Whole-mount in situ hybridization with
E-cadherin antisense riboprobes of a control littermate (left) and a
Sna?/?embryo (right). (B and C) Plastic sections of embryos treated
as described for panel A. E-cadherin RNA expression is downregu-
lated in the mesoderm of the control littermate embryo (B), but ex-
pression is retained (arrows) in the mesoderm of the Sna?/?embryo
(C). (D to G) Immunofluorescence with anti-E-cadherin monoclonal
antibody. (D and E) Nomarski optics. (F and G) Fluorescence optics.
In the Sna?/?embryo (G), E-cadherin protein expression is retained in
the mesoderm layer (arrows). Abbreviations: ee, embryonic ectoderm;
m, mesoderm; ps, primitive streak.
VOL. 21, 2001Sna GENE KNOCKOUT8187
pression of E-cadherin transcription by Snail family proteins Download full-text
may have been an ancestral condition in the metazoan precur-
sor to the arthropod and mammalian lineages.
Our studies provide the first genetic evidence that the Sna
gene functions as a key regulator of the epithelial-mesenchy-
mal transition in mice. Our data show that, as was found in
cultured cells and in metastatic carcinomas (3, 7), the E-cad-
herin gene is a target for repression by the Sna protein. How-
ever, the level of expression of E-cadherin in the mesoderm of
Sna?/?mutant embryos is considerably less than the level of
E-cadherin RNA expression in the embryonic ectoderm of
these embryos. This finding suggests that other regulators of
E-cadherin transcription may not be maintained in the meso-
derm of Sna?/?mutant embryos. For example, the embryonic
ectoderm may express a positive regulator of E-cadherin tran-
scription and this positive regulator might not be expressed in
the mesoderm of the Sna?/?mutant embryos.
It is intriguing that despite the retention of E-cadherin ex-
pression and intercellular adherens junctions, a primitive
streak forms and the mesoderm layer delaminates in Sna?/?
mutant embryos. This may be due to the fact that these regions
express distinctly lower levels of E-cadherin RNA than are
expressed in the embryonic ectoderm. It would be interesting
to overexpress E-cadherin in the primitive streak and the me-
soderm to test whether higher levels of E-cadherin expression
would entirely prevent streak formation and mesoderm de-
We thank B. Holdener, J. Mercer, and M. Shen for helpful discus-
sions; L. Bechtold and P. Finger for transmission electron microscopic
analysis and plastic sectioning; G. Martin for help with fluorescent
microscopy; C. Norton for technical assistance; S. Ang, R. Behringer,
B. Hermann, and R. Kemler for in situ probes; and S. Ackerman and
T. O’Brien for reading the manuscript.
This work was supported by a grant (HD34883) from the NIH to
T.G. and a subcontract to T.G. under NIH Project Center grant
DE13078 from Johns Hopkins University. This work was also sup-
ported by a training grant (CA09217) (E.A.C. and Y.L.) and a Core
grant (CA34196) from the National Cancer Institute to the Jackson
E.A.C. and R.J. contributed equally to this work.
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