The Snail gene family

Aixa V. Morales and M. Angela Nieto*

Instituto Cajal, CSIC. Doctor Arce, 37, 28002- Madrid, Spain

* Author for correspondence

Tel: 34-91-5854723

Fax: 34-91-5854754

e-mail: anieto@cajal.csic.es

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Introduction

The conversion of an epithelial monolayer into a multilayered structure is a conserved strategy

during the early stages of animal development. This process is accomplished by well-orchestrated

morphogenetic movements that occur during gastrulation, and serves not only to generate the three

germ layers in tripoblastic animals (ectoderm, mesoderm and endoderm), but also to shape the

embryo and to ensure that cells receive the right signals at the right time.

The Snail superfamily plays a central role in different morphogenetic processes during

embryonic development and, in particular, during gastrulation (Nieto, 2002). The first family member

described, snail, was identified in Drosophila as a mesoderm determinant essential for embryonic

development (Grau et al., 1984; Nusslein-Volhard et al., 1984). Embryos functionally-deficient for

snail show defects in the invagination of the presumptive mesoderm and retraction of the germ band.

Since then, a plethora of homologues have been described in different species, ranging from jellyfish

to humans (Spring et al., 2002; Nieto, 2002), many of which share a role in mesoderm development.

Snail genes also participate in other processes that involve long-range movements, such as the

development of the neural crest and the acquisition of the invasive and migratory phenotype during

tumor progression. In addition, different family members have been assigned roles in neural

differentiation, the development of appendages, cell fate and cell survival and the establishment of

left-right asymmetry.

In this chapter, we will discuss the role of the different family members during mesoderm

formation, a complex process that involves the coordination of cell fate determination, cell shape

changes and the control of cell cycle. All these cellular processes occur concomitant with large-scale

morphogenetic movements, the hallmark of gastrulation.

The Snail superfamily of transcriptional repressors

The Snail gene superfamily encompasses the closely related Snail and Scratch families

(Manzanares et al., 2001; Nieto, 2002). They encode transcription factors of the zinc-finger type,

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where the number of fingers varies from four to six, although it is thought that a minimum of four

fingers are required for them to be functional (Fuse et al., 1994; Grimes et al., 1996, Nakayama et

al., 1998). The fingers correspond to the C2H2 type, where C and H are the cysteine and hystidine

residues that form the zinc-binding site and they are located at the carboxi-terminus of the protein

(Fig. 1).

The zinc-finger domain is the most conserved structural feature of the Snail superfamily.

Within this domain, the third and fourth fingers contain a consensus sequence that is conserved in in

all Snail Superfamily members described to date (>40). The sequence of the second and fifth fingers

serve to unambiguously assign proteins to the Snail or the Scratch families, while the first finger and

the Scratch and Slug domains in the N-terminal region are diagnostic clues to identify Scratch genes

and vertebrate Slug subfamily members, respectively (Fig. 1 and Manzanares et al., 2001).

Like most of the zinc-fingers in other proteins, the Snail zinc-fingers are involved in sequence-

specific DNA binding. They recognize a six-base sequence, CAGGTG, a type of E-box that is also

the binding site for basic helix-loop-helix (bHLH) transcription factors (Mauhin et al., 1993; Fuse et al,

1994; Inukai et al., 1999; Batlle et al., 2000; Cano et al., 2000). Interestingly, in vitro studies have

shown that Snail proteins compete with bHLH proteins for the same binding sequences suggesting

that in vivo, competition may occur for these sites (Fuse et al., 1994; Nakayama et al., 1998;

Kataoka et al., 2000; Pérez-Moreno et al., 2001).

Upon binding to the specific E-box consensus site, Snail proteins function as transcriptional

repressors (reviewed in Hemavathy et al., 2000a and Nieto, 2002). Nevertheless, under certain

circumstances, human Slug and Drosophila snail seem to contain a transcriptional activation domain

(Hemavathy et al, 2000b), and thus, the possibility that they act as transcriptional activators cannot

be excluded.

Transcriptional repression is mediated by the N-terminal region in both vertebrates and

invertebrates (Gray and Levine, 1996; LaBonne and Bronner-Fraser, 2000; Mayor et al., 2000).

Within the N-terminus domain, two different sequences seem to be responsible for repression

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depending on the species. Snail proteins in jellyfish (Spring et al., 2002), echinoderms (Manzanares

et al., 2001), cephalochordates (Langeland et al., 1998), one in the limpet (Lespinet et al., 2002),

Drosophila scratch and all vertebrate Snail and Scratch proteins (Manzanares et al., 2001) contain

and likely utilize the SNAG (Snail/Gfi-1) domain (Fig. 1). This domain was initially described in the

zinc-finger protein Gfi-1 as necessary and sufficient for repression (Grimes et al., 1996).

In contrast, Drosophila Snail family members (snail, escargot and worniu) lack the SNAG

domain, and mediate repression by interacting with the co-repressor CtBP (C-terminal Binding

Protein). This co-repressor binds to a conserved sequence P-DLS-K/R that is present in two copies

in the fly snail genes (Nibu et al., 1998; Ashraf et al., 1999; Fig. 1). Interestingly, Snail ascidian

proteins also lack the SNAG motif and contain CtBP consensus sites (Corbo et al, 1997; Wada et al.,

1999). Therefore, transcriptional repression via Snail proteins seems to be an activity that has been

conserved throughout evolution, either through the use of the SNAG domain or by co-repression with

CtBP. Indeed, vertebrate Slug proteins may use both the SNAG domain and CtBP binding to induce

repression (Fig. 1).

At the N-termini of the Drosophila Snail family members another conserved motif exists that is

known as the NT box. Due to the presence of several basic residues in this motif, it has been

proposed that it is responsible for nuclear localization (Hemavathy et al., 2000b).

Evolutionary history of the Snail gene Superfamily

The phylogenetic analysis of the Snail gene superfamily has shed some light on the evolution of the

family from ancestral genes (Manzanares et al., 2001). The appearance of this gene family is closely

associated with the origins of the metazoans and not a eukaryote character, as no Snail-like genes

have been found in yeast or plants. Members of the family have been identified from jellyfish to

humans, and an early duplication of a Snail gene in the metazoan ancestor appears to have given

rise to two highly related genes, Snail and Scratch. Since the Scratch genes have been implicated in

neural differentiation and do not seem to play roles in mesoderm formation (see Nieto, 2002, for a

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discussion on the roles assigned to the two different families), the rest of the chapter we will

concentrate on the Snail family.

After the early duplication, independent duplication events in different groups gave rise to a

different number of Snail genes. In Drosophila there are three members of the family -snail, escargot

and worniu- that are linked together in an interval of about 150 kb on the second chromosome. This

fact together with functional complementation of the three genes in neural development (Ashraf et

al., 1999) suggest that they arose through an intrachromosomal tandem duplication event (Fig. 2).

Only one Snail gene has been found in jellyfish (Springs et al., 2002), sea urchin and non-vertebrate

chordates such as ascidians (Corbo et al., 1997; Wada and Saiga, 1999) and amphioxus (Langeland

et al., 1998). However, independent duplications occurred in vertebrates and in lophotrochozoans,

since two Snail genes have been found both in the limpet and the leech (Lespinet et al., 2002;

Goldstein et al., 2001; Fig. 2).

With respect to the duplications in the vertebrate lineage, at least two Snail family members

can be found in each species analyzed. Thus, it seems likely that either the duplication of the whole-

genome (Holland et al., 1994), or a large-scale gene duplication event (Wolfe, 2001) at the base of

the vertebrates led to the generation of Snail and Slug (Fig. 2). The presence of two very closely

related Snail genes (snail1 and snail2) in zebrafish and pufferfish (Hammerschmidt and Nusslein-

Volhard, 1993; Thisse et al., 1993; Thisse et al., 1995; Smith et al., 2000) is in agreement with the

proposed tetraploidization in the teleost lineage (Postlethwait et al., 1998). It is interesting to note

here that a new vertebrate Snail homologue -Smuc- has been characterized in the mouse (Kataoka

et al., 2000) although it occupies an unusual position in the phylogenetic tree of the Snail family

(Manzanares et al., 2001) and it is not expressed during early mesodermal development. However, it

seems to participate in myogenesis (Kataoka et al., 2000).

Snail family members share an evolutionary conserved role in mesoderm formation in insects,

ascidian, amphioxus, and in vertebrates (reviewed in Nieto, 2002). These functional aspects will be

discussed in the next coming sections.

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Establishment of the mesodermal fate

In Drosophila, gastrulation is first visible when a longitudinal fold appears of on the ventral side of the

blastoderm. The cells of the ventral furrow then invaginate and give rise to the mesoderm and part of

the anterior gut. The morphogenetic movements that occur at gastrulation are discussed in detail in

Leptin/Weischaus. A prerequisite for gastrulation is the establishment of the territory that will

invaginate from the ventral side of the blastoderm and that represents the mesoderm primordium.

The maternal gradients established by the pathway of Toll and Dorsal define the ventral fate. Dorsal

is a transcription factor that at high concentrations directly binds to the promoters of the zygotic

genes twist and snail activating their transcription in the ventral cells (Jiang et al., 1991, and Fig. 3).

Twist acts as a transcriptional activator for mesodermal genes (N-cadherin, PS2α-integrin and

myosin; Leptin, 1991) and collaborates with Dorsal to establish high levels of snail expression and a

sharp boundary in the presumptive mesoderm (Ip et al., 1992, and Fig. 4). Snail functions as a

repressor of non-mesodermal genes binding to the promoters of at least two genes: single-minded

(Kasai et al., 1992) and rhomboid (Ip et al., 1992). Other relevant genes in this process that are de-

repressed in snail mutants include lethal of scute, crumbs, short gastrulation, Delta and genes of the

Enhancer of Split complex (reviewed in Ip and Gridley, 2002). However, whether they are direct

targets of snail remains to be determined since promoter analyses have not been carried out.

In snail mutant embryos the expression domains of genes normally confined to the ectoderm

invade the mesoderm anlagen and, as in the twist mutant embryos, the ventral invagination is largely

abolished (Fig. 5). In this case, no mesodermal differentiation occurs and fully developed mutant

embryos lack all mesoderm derivatives (Grau et al., 1984). Rescue of the ventral-cell invagination

can be obtained by inducing snail expression in the absence of twist but not vice versa, indicating

that snail has a more direct role in regulating the cellular events that drive gastrulation.

As in Drosophila, snail is expressed in the mesodermal precursors of non-vertebrate

chordates –ascidian and amphioxus- (Corbo et al., 1997; Wada and Siaga, 1999; Langeland et al.,

1998). With respect to anamniote vertebrates, Slug expression is observed in the dorsal marginal

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zone in Xenopus embryos (Mayor et al., 2000) and snail-1 is expressed in the involuting cells of the

germ ring in the zebrafish (Hammerschmidt and Nusslein-Volhard, 1993; Thisse et al., 1993). Thus,

it appears that they may also be involved in the determination of the mesodermal fate in these

species.

In amniotes, the early ectodermal cells are recruited to the primitive streak, a transient

structure that forms along the posterior midline of the embryo (reviewed in Tam and Behringer,

1997). At the primitive streak, cells undergo an epithelial to mesenchymal transition (EMT, see

below) and then ingress between the primitive ectoderm and endoderm to give rise to both the

mesoderm and the definitive endoderm. Fate-mapping studies have demonstrated that the order and

the site of progenitor cell ingression through the primitive streak determine both the spatial

distribution and the different mesodemal fates (Kinder et al., 1999; Nicolet, 1971; Psychoyos and

Stern, 1996). Snail genes are indeed expressed in the primitive streak, although the family member -

either Snail or Slug- depends on the corresponding species (Sefton et al., 1998, and Fig. 4).

In spite of their expression pattern, there are not definitive evidences for the role of Snail family

members in the specification of the mesoderm in vertebrates. Indeed, mouse embryos homozygous

for a null mutation in the snail gene still form the three embryonic tissue layers. However, their

mesodermal cells express low levels of the mesodermal marker Brachyury and the expression of the

epiblast marker Otx-2 is extended (Carver et al., 2001). The main defect observed in these mutants

is that these “mesodermal” cells exhibit an epithelial character, since they fail to undergo the EMT

(Fig. 5).

In support of Snail having an ancestral function in mesoderm specification is its expression in

the entocodon, a tissue that some authors regard as the equivalent of mesoderm in diploblastic

animals and that gives rise to the smooth muscle tissue of the jellyfish at the medusa stage (Springs

et al., 2002). Nevertheless, the expression pattern of snail genes in the leech and limpet embryos is

not compatible with this role in mesoderm specification in Lophotrochozoans (Goldstein et al., 2001;

Lespinet et al., 2002).

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Intracellular morphogenetic changes prior to migration

In the Drosophila blastoderm, once the mesoderm territory has been determined and prior to

invagination, the ventral cells undergo coordinated changes in shape that moves the mesoderm

anlagen towards the interior of the embryo. After flattening their apical (outer) surface, the nuclei of

the ventral cells migrate to a basal position and they progressively constrict their apical sides until

they adopt a wedge shape that probably helps the cells to move into the interior (discussed in

Leptin/Weischaus).

In snail mutants, the mis-specified ventral epithelium becomes very thin, suggesting that a

shortening of the cells occurs. However, no apical constrictions can be seen at all and, as a

consequence, the furrow does not form (Leptin, 1991; Fig. 5). Furthermore, in mutant alleles with a

reduced snail function, the ventral cells express both neuroectodermal and mesodermal markers and

in this case, are able to invaginate ventrally (Hemavathy et al., 1997). This suggest that Snail,

together with Twist, not only controls the specification of the mesoderm but also regulates a separate

set of molecules involved in cell-shape changes in the ventral furrow (Fig. 3). One of these

molecules is Folded Gastrulation (fog), a putative secreted ligand that controls normal concerted

apical constrictions during invagination and that is aberrantly expressed in snail mutant embryos

(Morize et al., 1998). It has been postulated that fog can exert its function by binding to a receptor

associated to the Gα-like protein concertina (Costa et al., 1994) and it interacts with the exchange

factor RhoGEF2. This reveals the importance of the small GTPases in the changes in cell shape that

occur during invagination (Barrett et al., 1997).

In a similar manner to mesoderm invagination in Drosophila, the amphibian and fish

prospective mesoderm, together with the endoderm first involutes at the blastopore lip, prior to

converging medially and extending longitudinally. In both these morphogenetic movements the cells

move as a single multilayered sheet (discussed by Keller and by Locascio and Nieto, 2001). The

convergent extension movements are triggered by signaling cascades involving the mesoderm-

inducing factors activin and FGF, which induce the expression of T-box transcription factors. One of

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these, no tail (Brachyury), has been shown to enhance snail1 expression in zebrafish (Thisse et al.,

1993; Hammerschmidt and Nusslein-Volhard, 1993).

In amniotes, the morphogenetic movement of convergent extension only influences the

development of axial mesoderm and the accumulation of the epiblast cells in the primitive streak

prior to the onset of gastrulation. Once formed, the cells of the primitive streak acquire a bottle-like

shape, broader at the basal or leading edge. These changes in cell shape share characteristics with

those described in Drosophila: blebbing of the dorsal and ventral sides; disruption of cell junctions;

breakdown of actin filaments; reorientation of microtubules; loss of basal lamina and decrease in

adhesion molecules (Bellairs, 1987). In snail mutant mouse embryos, the primitive streak and

mesoderm-like layer that is formed contain lacunae and the cells retain epithelial characteristics, with

microvilli in the apical surface and maintenance of E-cadherin expression and adherens junctions

between the cells (Carver et al., 2001; see Fig. 5). Once again, these changes are compatible with a

failure in undergoing the EMT and cannot be separated from the changes in cell shape that occur

before delamination.

Many of the cellular processes involved in gastrulation are also common to those that occur

during neural crest delamination (Bellairs, 1987). In chicken embryos, Slug gain of function in the

neural tube causes an upregulation of RhoB, an small GTPase involved in actin rearrangements (Del

Barrio and Nieto, 2002). This upregulation is accompanied by an increase in neural-crest migration

only in the cranial region, indicating that in the trunk, Slug plays a role in the specification of neural

crest cell progenitors and also in the changes in cell shape involving a rho pathway. In the light of

these results and given the connection between snail and RhoGEF2 in Drosophila, is seems that a

Rho-mediated signaling cascade is crucial for the cellular changes that occur during gastrulation in

invertebrates and vertebrates (Figs. 3 and 7).

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Coordination of cell division and migration

Morphogenetic movements and cell division both involve the reorganization of the cytoskeleton. This

reorganization can be a cause of incompatibility between the two processes especially during rapid

developmental events such as gastrulation. As such, this might explain the mitotic arrest in the

ventral cells of the gastrulating fly embryo, that only enter mitosis after mesoderm invagination is

complete (Foe, 1989). The cells of the ventral furrow start expressing string, a cdc25 homologue

essential for entry into mitosis, at the same time as the rest of the cells in the embryo. However,

string function is inhibited by tribbles and frühstart (Groβhans and Wieschaus, 2000; Seher and

Leptin, 2000; Mata et al., 2000) and this inhibition is dependent on snail function (Groβhans and

Wieschaus, 2000). Thus, snail seems to act as a mitotic inhibitor during gastrulation of the fly

embryo.

Interestingly, mammalian epithelial cells transfected with Snail undergo dramatic changes in

cell shape compatible with EMT and reduce their rate of proliferation (S. Vega and M. A. Nieto, in

preparation). Similarly, the invasive areas of tumors show lower rate of proliferation when compared

to non-invasive areas of the same tumor (Jung et al., 2001). Significantly, Snail is expressed in the

invasive front of tumors induced in the skin of the mouse (Cano et al., 2000) and of biopsies obtained

from patients with breast carcinoma (Blanco et al., 2002).

The triggering of the epithelial-mesenchymal transition

Once inside the Drosophila embryo, the mesodermal cells must disperse into single cells that divide,

attach and migrate along the ectoderm to form a single cell layer. The mechanism by which cells

become migratory is not yet fully understood, but it is known that it implies a switch of adhesion

molecules (Oda et al., 1998). E-cadherin is supplied maternally but its expression is also activated in

the embryo when zygotic transcription begins although it is repressed in the mesoderm (Oda et al.,

1994). In snail mutants the ventral cells of the embryo fail to downregulate E-cadherin expression

(Oda et al., 1998), consistent with the fact that Snail is a strong repressor of E-cadherin transcription

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in epithelial cells (Cano et al., 2000; Batlle et al., 2000). Snail is also required in Drosophila to

increase N-cadherin expression, indicating that it is involved in the switch from E-cadherin

(implicated in the maintenance of stable junctions) to N-cadherin expression (a weak intercellular

adhesion system), which is important for the movement of cells after invagination (Oda et al., 1998).

As mentioned before, in amniotes, cells that undergo EMT and delaminate give rise to the

mesoderm, cells migrating as individual cells through the extracellular matrix. The EMT process

implies the loss of epithelial markers and the gain or redistribution of mesenchymal markers, as well

as the acquisition of a fibroblastic-like phenotype concomitant with that of a migratory and invasive

phenotype (Hay, 1995; Thiery, 2002).

The first indication that the Snail family was involved in EMT during gastrulation came from

studies in the chick embryo. The incubation of early chicken embryos with antisense oligonucleotides

to inhibit Slug function led to the formation of an abnormally cell dense, wrinkled tissue at the

primitive streak region. This tissue was generated due to the incapacity of the cells to convert to

mesenchyme and migrate as mesodermal precursors (Nieto et al., 1994; Fig. 6). Since Snail rather

than Slug is expressed in the primitive streak of the mouse embryo (Fig. 4), it was proposed that

Snail might be responsible for triggering EMT in mammals (Sefton et al., 1998). Indeed, mouse Snail

is able to induce a complete EMT when expressed in mammalian epithelial cells (Cano et al., 2000;

Batlle et al., 2000) and the in vivo demonstration comes from the fact that Snail mutant mice die at

gastrulation due to a failure of the mesodermal cells to undergo EMT (Carver et al., 2001; Fig. 5) . As

in Drosophila, loss of E-cadherin expression is essential for the mesodermal cells to ingress at

gastrulation in mouse embryos (Burdsal et al., 1993). Indeed, as already mentioned, in Snail

knockout mouse embryos the mesodermal layer forms but cells maintain their epithelial character,

retaining an apical-basal polarity, microvilli and adherens junctions (Carver et al., 2001, and Fig. 5).

While they establish a low level of expression of some mesodermal markers, as in Drosophila snail

mutants, they fail to downregulate E-cadherin expression (Carver et al., 2001; Oda et al., 1998),

further evidence that Snail acts as a repressor of E-cadherin transcription (Cano et al., 2000; Batlle

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et al., 2000). Thus, the repression of E-cadherin expression by Snail appears to have been

conserved through evolution from insects to mammals. However, the levels of E-cadherin in the

mesoderm layer of Snail mutant mouse embryos are lower than those of the embryonic ectoderm in

the same embryos, indicating that other E-cadherin repressors might be present. Candidates include

HLH-type transcription factors such as SIP1 and E12/E47 (Comjijn et al., 2001, Pérez-Moreno et al.,

2001), which although weaker repressors, are both expressed in the mesoderm.

Although covergent-extension is the main feature of gastrulation in anamniote vertebrates,

examples of individual cell migration have been reported. In Xenopus embryos, the first mesodermal

cells to ingress during gastrulation transiently express snail, they migrate as individual cells and they

give rise to the most anterior mesoderm (R. Mayor, personal communication; Fig. 8). In a similar

way, in zebrafish embryos, snail2 mRNA appears in a reticular staining formed by single cells in the

anterior cephalic paraxial mesoderm (Thisse et al., 1995; Fig. 8). Interestingly, the formation of the

mesendoderm layer in zebrafish has been recently shown to involve a combined process of

ingression of individual cells and the involution-like movement observed in Xenopus gastrulation

(Carmany-Rampey and Schier, 2001). Thus, these examples of individual cell migration are

accompanied by Snail expression and the triggering of an epithelial mesenchymal transition. Even in

a gastropod mollusc, Patella vulgata, snail-2 is expressed in the involuting cells of the mantle tip,

compatible with it controlling EMT and cell motility at this site. Altogether, these results have led to

the suggestion that EMT and the control of cell motility are ancient functions associated with the

Snail gene family (Lespinet et al., 2001; Manzanares et al., 2001). In keeping with this idea, Snail

genes have been implicated in the different EMTs that occur at different sites and times during

vertebrate embryonic development. This includes processes such as the formation of the parietal

endoderm (Velmaat et al., 2000), the delamination of the neural crest (Nieto et al., 1994; Carl et al.,

1998; LaBonne and Bronner-Fraser, 2000; Mayor et al., 2000; Del Barrio and Nieto, 2002), the

formation of the heart cushions (Romano and Runyan, 2000), the decondensation of the somites

(Sefton et al., 1998) and the closure of the palate (Martínez-Alvarez et al., Submitted). There is also

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good evidence that Snail is involved in the triggering of EMTs in pathological situations, such as

when single carcinoma cells dissociate from the site of primary tumors (Blanco et al., 2002) and that

associated with the transformation of mesothelial cells after continuous peritoneal dialysis (Yáñez-

Mo et al., 2003).

Signaling pathways in vertebrates

It is clear that different members of the TFG-beta superfamily are involved in mesoderm

induction and are associated with the activation of different Snail family members in vertebrates

(reviewed in Beddington and Robertson, 1999 and Nieto, 2002). Additionally, other signaling

pathways such as those involving Wnt and FGF have also been implicated in both mesoderm

induction and Snail expression (Smith, 1993; Kimelman and Griffin, 2000; Nieto, 2002). With respect

to the involvement of Wnt signaling, the analysis of the promoter of Xenopus Slug genes has

revealed a functional binding site for the transcription factor Lef1, which regulates gene expression

following activation of the Wnt pathway (Vallin et al., 2001). However, this may be dependent on the

cellular context, as Snail genes are not upregulated in epithelial cells or colon carcinoma cells

overexpressing LEF (Kim et al., 2002; Tan et al., 2001).

Interestingly, the phenotype of FGF receptor 1 (FGFR1) mutant mice is reminiscent of that of

Snail mutants (Ciruna and Rossant, 2001). In an elegant study, Ciruna and Rossant showed that

FGF signaling is not involved in Snail induction but rather in the maintenance of its expression.

Indeed, Snail is transiently expressed in these mutants, permitting the formation of the

extraembryonic mesoderm (the first to develop) to be formed. However, subsequently, Snail

expression disappears and the embryonic mesoderm fails to develop (see Fig. 6). In these cases, E-

cadherin is not downregulated and the cells remain trapped close to the primitive streak while

expressing low levels of mesodermal markers as it occurs in both Drosophila and mouse Snail

mutants. In relation to this, it is interesting to note that the Drosophila FGF-R2 mutants –heartless-

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show a similar phenotype: the invaginated mesodermal cells remain aggregated along the ventral

midline and fail to migrate (Beiman et al., 1996; Gisselbrecht et al., 1996).

There is an interesting connection between FGF and Wnt signaling that was also unveiled in

the FGFR1 mouse mutants. In these embryos Wnt signaling is attenuated in the primitive streak but

this can be reverted by disrupting the abnormal high levels of E-cadherin. This result indicates that

the E-cadherin expression attenuates Wnt signaling by sequestering cytoplasmic β-catenin, making

it unavailable for binding to the Tcf/Lef family transcription factors and subsequent activation of Wnt

target genes (Ciruna and Rossant, 2001). The model that emerges is that the members of the TGF-

beta/BMP families induce Snail genes and FGF maintains their levels allowing for Wnt signaling to

be active (Fig. 7).

The distribution of mesodermal territories

We have already mentioned that in Drosophila snail is expressed in the prospective mesoderm

and act as a repressor of the neuroectodermal genes rhomboid (rho) and single-minded (sim)

(reviewed in Ip and Gridley, 2002). Thus, Snail specifies the mesodermal anlagen by preventing

alternative fates from being specified. In addition, Snail controls the territory in which Notch signaling

can act in the fly at early gastrulation stages and limits the expression of single-minded (sim) to the

mesectoderm (Cowden and Levine, 2002; Morel et al., 2003). Interestingly, Snail seems to have a

conserved role in setting up boundaries between tissues even at later stages for the distribution of

territories. It establishes the muscle/notochord boundary in ascidians, by repressing Brachyury (T)

expression in the muscles but not in the notochord (Fijiwara et al., 1998). Furthermore, it excludes

Forkhead expression from the lateral ependymal cells restricting it to the floor plate (Di Gregorio et

al., 2001).

In vertebrates, there are indications to suggest that Snail genes may play a role in defining

tissue boundaries. As discussed above, the phenotype of Drosophila and mouse mutants for Snail

and FGF signaling include the accumulation of mesodermal cells in the midline. The net result is an

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enlargement of the axial mesoderm territory at the expense of the paraxial mesoderm anlagen,

something that it is also observed in zebrafish spadetail (VegT) mutants (Thisse et al., 1993; Thisse

et al., 1995). However, since there is a failure in migration from the streak, this putative role in

defining territories is somehow mixed here with its role in inducing the movement of mesodermal

cells after invagination or ingression. Thus, it is likely that cells acquire characteristics of the different

mesodermal territories (paraxial and lateral) not only according to the time they delaminate from the

primitive streak, but also upon receiving signals that do not reach them while trapped close to the

primitive streak. Considering that VegT (spadetail) lies upstream of Snail it is tempting to speculate

that the spadetail phenotype mentioned above can be at least partly explained by the defective Snail

function. Indeed, Snail1 transcripts are detected in the involuting cells of the germ ring in zebrafish

and, as gastrulation begins, becomes restricted to the paraxial mesoderm. Undoubtedly, Snail loss of

function experiments in zebrafish will help to clarify this issue.

In Xenopus embryos, Slug is expressed in the dorsal marginal zone above the blastopore lip,

a territory that includes the prospective dorsal mesoderm and endoderm. In this region, Slug controls

dorsal mesoderm genes such as chordin and cerberus by inhibiting BMP transcription, a signal that

normally induces ventral (lateral) mesodermal fates (Mayor et al., 2000).

In addition to zebrafish and Xenopus, the analysis of expression patterns of Snail genes in

other vertebrates is also compatible with their function in the subdivision of mesoderm territories.

Following the duplication at the base of the vertebrate lineage, the two duplicates -Snail and Slug-

seem to have distributed their expression so that they show complementary patterns in the different

mesodermal territories along the medio-lateral axis (dorso-ventral in Xenopus and fish). This is

summarized in Fig. 8 where the expression of Snail family members in chordates is shown in the

early mesoderm and in its subsequent derivatives: axial, paraxial and lateral mesodermal tissues.

Slug was initially expressed in the notochord and its expression was lost in birds and mammals.

More interestingly, there is an interchange in the expression pattern of Snail and Slug in the avian

embryo as far as the early and the paraxial and lateral mesoderm is concerned (Sefton et al., 1998).

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This interchange explains why the loss of Slug function in the chick (Fig. 6) produces a similar

mesodermal phenotype to that of mouse Snail or FGFR mutants (Nieto et al., 1994; Carver et al.,

2001; Figs. 5 and 6) and why the mouse Slug mutant does not show mesodermal defects (Jiang et

al., 1998).

Recent data indicate that the expression of Snail in the early mesoderm is also conserved in

turtle and lizard embryos, as reflected by its expression in the tail bud (Locascio et al., 2002),

considered to be a continuation of the blastopore or the primitive streak (Gont et al., 1993). The lack

of Snail expression at the tip of the tail in ascidian embryos is consistent with this, as the ascidia tail

is formed by convergent-extension movements of the notochord (Nishida, 1987) and Snail genes do

not seem to play a primary role in convergent-extension (Locascio and Nieto, 2001).

Finally, and beyond the scope of this review, Snail genes have been also associated with

mesoderm differentiation at later stages. Their expression during the decondensation of the

sclerotome has been related to their role in triggering of the EMT (Nieto, 2002). Moreover, Snail

expression in the muscle lineages in ascidia (Fijiwara et al., 1998) is consistent with Slug being a

target of MyoD as has been described in the mouse (Zhao et al., 2002).

Concluding remarks and perspectives

The analysis of Snail genes in invertebrates and vertebrates has unveiled their role in mesoderm

specification, migration and subsequent distribution of mesodermal territories. However, an ancestral

function in the specification of the mesoderm is not supported after the recent data obtained in

lophotrochozoans. It seems that its prominent role in mesoderm development is related to the

establishment of tissue boundaries and the promotion of cell shape changes and cell movement.

In spite of the increase in our knowledge in the inductive signals and targets and the

similarities observed in different systems, further work is needed to understand the interaction

between the different signaling pathways that have been implicated in these processes in both

Drosophila and vertebrates.

17

Independent duplications have occurred in different groups in evolution, giving rise to a

different number of genes in lophotrochozoans, arthropods and vertebrates. The duplicated genes

have adopted different sites of expression and functions in the different species and as a result,

some unexpected interchanges of expression patterns have arisen, although the mechanisms that

originated these remain to be determined. Nevertheless, in general, either Snail or Slug is

expressed in the relevant mesodermal populations of different species. Indeed, in the primitive

streak, Slug induces EMT in the chick and Snail does it in the mouse. In addition, the two of them

can behave as functionally equivalent when ectopically expressed at the appropriate sites both

intra- and interspecies (Del Barrio and Nieto, 2002). These transcription factors are composed of

two main domains, a DNA binding domain that is essentially identical in all Snail and Slug

vertebrate family members and a much more divergent putative protein-protein interaction domain.

Thus, it will be a challenge to determine the mechanisms that these two proteins have used to fulfill

the same role during evolution with respect to their target genes, their regulatory partners and the

genetic cascades in which they are involved.

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Legends to Figures.

Fig.1. Conserved features in the Snail superfamily members. The zinc-finger domain is the most

conserved feature, with diagnostic sequences in the third and fourth fingers that allow the

identification of the whole superfamily (pink). The sequences of the second and fifth fingers are

specific for either the Snail (blue) or the Scratch families (green). Sequences in the first finger and in

a central domain of the protein define the Scratch family (green) or the Slug subfamily (red). The

SNAG domain (yellow) is absent from Drosophila Snail family members. CtBP binding sites are

present in the fly Snail genes and in the vertebrate Slug subfamily. The absence of the first finger in

some vertebrate Snail subfamily members is indicated by a dotted line.

Fig. 2. Evolutionary history of the Snail gene family. The early duplication of a unique Snail gene

present in the metazoan ancestor likely gave rise to two genes: Snail and Scratch. The subsequent

duplications undergone by Snail are shown in all groups where family members have been described

to date. Name sin bold indicate present genes. For a picture of the proposed duplications within the

Scratch family see Nieto, 2002.

Fig. 3. Genetic pathways involved in Drosophila gastrulation. The arrows indicate the flow of the

pathway, not direct transcriptional regulation. Only rhomboid and single-minded have been shown to

be directly repressed by Snail, which also directly represses E-cadherin transcription in vertebrates

and very likely in the fly embryo. To better follow the sequence, genes and cellular processes

repressed or inactive are shown in red and those active are shown in green.

Fig. 4. Expression of Snail genes in Drosophila, chicken and mouse embryos during gastrulation.

Snail expression is observed in the mesodermal precursors prior to and during invagination in

Drosophila embryos. In vertebrates, Snail genes are expressed in the primitive streak and the early

25

mesodermal cells. However, the family member present in these tissues depends on the species:

Snail in the mouse and Slug in the chick. This figure has been adapted from Nieto, 2002. Pictures of

Drosophila embryos were kindly provided by Maria Leptin.

Fig. 5. Schematic representation of the Snail mutant phenotype in Drosophila and mouse embryos.

In Drosophila snail mutants, the ventral invagination is abolished and the mesoderm does not form.

In the mouse, cells expressing low levels of mesodermal markers are formed. However, as in

Drosophila, these cells fail to downregulate E-cadherin expression and show epithelial

characteristics, indicating that they are unable to undergo the epithelial-mesenchymal transition.

Fig. 6. Schematic representation of the result of Slug loss of function in the chick and that of FGF

signaling in mouse embryos. In Slug-antisense treated chicken embryos, the early mesodermal cells

accumulate in the region of the primitive streak and a low number of cells are able to migrate to the

locations of paraxial and lateral mesoderm. Similarly, mouse embryos mutant for FGFR1 show a

similar phenotype, also reminiscent to that of the Snail mouse mutant. However, in the FGFR1 null

mice, Snail is transiently expressed in the primitive streak, allowing for the first mesoderm that forms

(extraembryonic) to develop. Therefore, FGF signaling seems to be essential for the maintenance of

Snail expression and the corresponding formation of the embryonic mesoderm.

Fig. 7. Genetic pathways involved in vertebrate gastrulation. Members of the TGF-beta superfamily

has been proposed to activate Snail in different vertebrate embryos, and data from FGFR1 mutant

mice indicate that FGF signalling is necessary for its maintenance (Ciruna and Rossant, 2001). The

repression of E-cadherin expression maintains cytoplamic beta-catenin available to transduce the

Wnt-signaling pathaway. As in Fig. 3, the arrows indicate the flow of the pathway, not direct

transcriptional regulation. Only E-cadherin and Mucin-1 have been shown to be directly repressed by

26

Snail. Again, the genes and cellular processes repressed or inactive are shown in red and those

active are shown in green.

Fig. 8. Schematic representation of the mesodermal expression of Snail family members during

Chordate evolution. A single Snail gene has been identified in Ascidians and Amphioxus where it is

expressed in the mesodermal precursors and in the paraxial mesoderm. Among vertebrates, Snail

family members are also expressed in different mesodermal populations. Interestingly, the

expression of Snail and Slug in these populations is largely complementary in each species and

compatible with a function in the subdivision of mesodermal territories. Note that expression in the

chicken is interchanged with respect to that of other vertebrates. For a recent analysis of Snail and

Slug expression in vertebrates including reptiles see Locascio et al., 2002. The expression of Snail in

Xenopus and Snail2 at the tip of the axial mesoderm corresponds to the first cells that ingress and

will form the head mesenchyme (see text).

Snail superfamily

Scratch family

Snail family

Snail superfamily Snail family

Slug subfamily Scratch family

Snail

Vertebrates

Drosophila II III IV V

Zn Fingers

I

Slug

Snail

NT box CtBP interaction motif

SNAG domain

Figure 1

snail

Metazoan Ancestor

snail

scratch

Protostomes Deuterostomes

Lophotrochozoans

snail2 snail1

Leech and Limpet snail

Ecdysozoans

Drosophila C.elegans snail

worniu escargot snail snail-like snail

Non-vertebrate Chordates

Ascidian and amphioxus

Vertebrates

Snail

Slug Snail

snail1 snail2 (Teleosts)

Smuc

Cnidarians

Jellyfish

snail

Figure 2

Snail

single-minded, rhomboid,

other...

E-cadherin

Gastrulation

Cell adhesion

Tribbles

String/cdc25

Cell cycle progression

Folded gastrulation

Concertina

RhoGEF2

Neuroectodermal fates

Cytoeskeletal changes

Toll Dorsal

Twist

Figure 3

Mitotic Block

Non-adhesive cells

Specification of mesodermal

territory

Changes in cell shape

Cell movements

Drosophila

m

snail

chicken mouse

Snail

Slug

Figure 4

Drosophila Mouse

WT embryo

snail mutant

WT embryo WT mesodermal cells

Snail mutant Snail mutant cells

Ectoderm

Mesoderm Failed “mesoderm” (Dros) or “mesoderm” of epithelial phenotype

Figure 5

Chick

Slug antisense treated embryo

WT embryo

Mouse

WT embryo

FGFR1 mutant

Ectoderm

Mesoderm

Mesoderm of epithelial phenotype

Endoderm

Figure 6

Snail

E-cadherin

FGF signaling

Fibronectin Vimentin

TGF-beta/BMP

Increased Wnt signaling

Induction

Maintenance

RhoB

Changes in cell shape Cell motility

Muc-1 Desmoplakin Cytokeratin 18

Cytoplasmic beta-catenin

Loss of epithelial markers

Gain of mesenchymal/mesodermal

markers

Cytoeskeletal changes

Figure 7

Amphioxus

Chicken

Snail/Slug ancestor

Snail or zSnail1

Slug

zSnail 2

Early mesoderm

Lateral mesoderm

Paraxial mesoderm

Somite

Notochord Ascidian

Xenopus Mouse

Zebrafish

zSnail 2 +Slug

Figure 8