snail


EVOLUTIONARY HOMOLOGS

Snail homologs in invertebrates

Podocoryne carnea is a typical representative of the class Hydrozoa (jellyfish). With few exceptions, hydrozoans are marine and exhibit a life cycle that consists of the free swimming planula larva, the sessile polyp and the sexual stage, the medusam which is formed from polyps through budding. In some hydrozoans, the medusa generation has become secondarily reduced. It is generally assumed that all cnidarians are formed of an outer and an inner layer of multifunctional myoepithelial cells. The other cell types, interstitial cells, nerve cells or nematocytes are interspersed in either of the two layers. Therefore, Cnidaria are classified as diploblasts or simple bilayered animals. While this classification accurately describes the basic tissue organization of the planula larva and the polyp, the anatomy of the medusa is more complex. Most of the differences are found in the medusa bell, which not only can carry complicated sense organs such as lens eyes, statocysts, and nerve rings but also consists of two nonmyoepithelial cell layers and additionally a third layer of epithelial mononucleated striated muscle cells. In the entire phylum, the planula larva and the polyp lack these medusa-specific cell types and sense organs (Spring, 2002 and references therein).

In bilaterians, the striated and smooth muscle tissues are in general a derivative of the third or middle germ layer, the mesoderm. In the hydrozoan jellyfish, the striated muscle is a derivative of the entocodon, a tissue layer that separates from the ectodermal layer early in medusa development. The entocodon is located between the distal ectodermal and the endodermal tissue, and is separated from both layers by an extracellular matrix. Entocodon cells in early bud stages are embryonic in appearance and highly proliferative. Later the entocodon forms a cavity, which finally connects to the outside by the developing velar opening. In older bud stages mitotic activity in the bell gradually stops and the outer wall of the entocodon differentiates into the striated muscle while the inner wall forms the smooth muscle of the feeding and sex organ of the animal (Spring, 2002 and references therein).

The histology and developmental pattern of muscle formation in medusa development has led to the idea that the entocodon could be a mesoderm-like layer. Striated myofilaments are usually found in cells derived from the mesoderm, however, exceptions are known. Tentacles of entoprocts contain flagellated ectodermal epithelia that contain striated myofilaments. This indicates that the appropriate structural genes can be activated independent of the germ layer and that the analysis of the striated muscle-specific structural genes alone would not be sufficient. The molecular analysis of muscle development in Podocoryne has demonstrated that the structural genes for a tropomyosin and a myosin heavy chain are structurally and functionally conserved and specific for the striated muscle tissue. Furthermore, the presence of the homeobox gene Otx, a head and gastrulation regulator in bilaterians, in jellyfish striated muscle and the basic helix-loop-helix (bHLH) factor Twist during the formation of the entocodon in medusa development, indicates that genes with specific roles in mesoderm patterning of bilaterians are already present in the common ancestor with bilaterians. Next to homeodomain and bHLH transcription factors, the best-studied regulatory genes are members of the T-box, MADS-box and zinc finger families, such as Brachyury, Mef2, and Snail, respectively. These three gene families are involved at different levels in the specification of the mesodermal and myogenic lineage of bilaterian animals from Drosophila to vertebrates (Spring, 2002 and references therein).

To investigate the hypothesis that the entocodon of jellyfish is homologous to the mesoderm of bilaterians, a Podocoryne homolog of each of the three gene families was isolated and structure and expression patterns were studied throughout the life cycle and specifically during muscle development. The results demonstrate that all three genes are expressed during myogenic differentiation. Additionally, as is true for their bilataterian cognates, they appear to have other functions as well. The sequence and expression data demonstrate that the genes are structurally and functionally conserved and even more similar to humans or other deuterostomes than to protostome model organisms such as Drosophila or Caenorhabditis elegans. The data further strengthen the hypothesis that the common ancestor of cnidarians and bilaterians already used the same regulatory and structural genes and comparable developmental patterns to build muscle systems (Spring, 2002).

The winged helix transcription factor Forkhead and the zinc finger transcription factor Snail are crucially involved in germ layer formation in Bilateria. A homolog of forkhead/HNF3 (FoxA/group 1) and of snail was isolated and characterized from a diploblast, the sea anemone Nematostella vectensis. Nematostella forkhead expression starts during late Blastula stage in a ring of cells that demarcate the blastopore margin during early gastrulation, thereby marking the boundary between ectodermal and endodermal tissue. snail, by contrast, is expressed in a complementary pattern in the center of forkhead-expressing cells marking the presumptive endodermal cells fated to ingress during gastrulation. In a significant portion of early gastrulating embryos, forkhead is expressed asymmetrically around the blastopore. While snail-expressing cells form the endodermal cell mass, forkhead marks the pharynx anlage throughout embryonic and larval development. In the primary polyp, forkhead remains expressed in the pharynx. The detailed analysis of forkhead and snail expression during Nematostella embryonic and larval development further suggests that endoderm formation results from epithelial invagination, mesenchymal immigration, and reorganization of the endodermal epithelial layer, that is, by epithelial-mesenchymal transitions (EMT) in combination with extensive morphogenetic movements. snail also governs EMT at different processes during embryonic development in Bilateria. These data indicate that the function of snail in Diploblasts is to regulate motility and cell adhesion, supporting the notion that the triggering of changes in cell behavior is the ancestral role of snail in Metazoa (Fritzenwanker, 2004).

In most Bilateria, the formation of the two inner germ layers, endoderm and mesoderm, is intimately linked during the process of gastrulation. In vertebrates, endodermal and mesodermal cells immigrate or invaginate together as endomesoderm and become separated morphologically only later during gastrulation. The evolutionary origin of the mesoderm is currently a matter of intense investigation, but still not clear. Some evidence from the two major diploblastic phyla, Cnidaria (also known as coelenterates) and Ctenophora, support the view of an endodermal origin of the mesoderm. However, other molecular data suggest that the third germ layer arose from the blastopore region with contributions from both ectoderm and endoderm (Fritzenwanker, 2004).

The evolution of the bilaterian foregut is also debated. Textbook knowledge postulates that foregut (and mouth) formation in Protostomes and Deuterostomes is fundamentally different and evolved convergently. However, similar expression of conserved transcription factors in the foregut anlage of basal deuterostome and protostome ciliary larva challenged this view and suggested a conserved molecular regulation of mouth development and homology of the foregut in Bilateria. The foregut is of great interest because it is the boundary between ectoderm and endoderm. In insects, both foregut (stomodeum) and hindgut (proctodeum) are regarded as an ectodermal derivative because in the adult these structures have a chitinized cuticula (Fritzenwanker, 2004).

One of the crucial conserved genes for mesoderm formation in Bilateria codes for the zinc finger transcription factor Snail. In insects, snail has been shown to repress the expression of neuroectodermal genes thereby marking the boundary between mesodermal and neurogenic region in the Drosophila embryo. In vertebrates, snail function has been implicated in epithelial-mesenchymal transitions of migrating cells of the developing mesoderm and of the neural crest. Snail has also been isolated from Podocoryne carnea, a hydrozoan cnidarian and from the coral Acropora millepora. Podocoryne snail is expressed in the entocodon of the developing medusa bud, suggesting a role in muscle development of the medusa, while Acropora snail is expressed in the endoderm during embryogenesis indicating a role in germ layer specification (Fritzenwanker, 2004).

A conserved marker gene for the foregut in Bilateria codes for the winged helix transcription factor Forkhead. The founder member forkhead is expressed in the foregut and hindgut anlage in Drosophila. Forkhead belongs to the group 1/HNF3/FoxA subfamily. In vertebrates, three highly related HNF3 genes, alpha, beta, and gamma, exist that differ by the timing and location of expression. In particular, HNF-3beta plays a crucial role during early vertebrate development. In mice and frogs, HNF-3beta is expressed in the organizer (node) and in the derivatives, the notochord but also the floor plate. HNF-3beta is involved in formation of the dorsoventral axis; HNF-3beta −/− mutant mice have defects in the DV patterning of the neural tube and of the dorsal mesoderm. This gene also has a conserved role in mesoderm formation in a dose-dependent manner and acts synergistically with brachyury to specify axial mesoderm in chordates (Fritzenwanker, 2004).

In insects, forkhead plays a conserved role in terminal patterning and formation of the foregut and hindgut anlage. The first forkhead homolog, from a diploblast, budhead, was isolated from the hydrozoan Hydra. budhead is expressed in the hypostome, the polyps' mouth and appears to have a role in axial patterning. The role of forkhead during cnidarian embryogenesis, however, is unknown. Since Hydra embryogenesis is highly derived and not easily accessible at all stages, a new model organism, the anthozoan Nematostella vectensis, was examined. Anthozoa are regarded as the basal group among Cnidaria and embryogenesis in Nematostella is inducible and readily accessible. This study reports the isolation and characterization of forkhead and snail homologs from Nematostella vectensis. The analysis shows that forkhead is expressed at the blastopore margin, that is, the boundary between ectoderm and endoderm and it marks the presumptive pharynx of the primary polyp. By contrast, snail has a virtually complementary expression pattern and marks all ingressing endodermal cells. The detailed analysis of forkhead and snail expression highlights that endoderm formation in this basal cnidarian is characterized by a relatively complex cellular behavior involving epithelial-mesenchymal transitions and morphogenetic movements (Fritzenwanker, 2004).

The evolution of the bilaterian gut has been studied by comparing the expression pattern of specific marker genes from a variety of organisms. It appears that a conserved cassette of developmental genes, mostly transcription factors, is expressed in fore- and hindgut primordia in all or most bilateria. These include the transcription factors caudal, brachyury, and forkhead and the signalling molecule wingless. All four genes are expressed in the blastopore in vertebrates and many insects, where they specify the derivatives of the blastopore, the foregut, and the hindgut (in Drosophila, the amnioproctodeal and the stomodeal invagination). While comparative data on caudal expression in different organisms are still scarce, at least brachyury, forkhead and wingless appear to have overlapping expression domains in most animals studied, hence they form an evolutionarily conserved synexpression group, suggesting that they might act in concert to specify a homologous structure in a wide range of animals. For instance, in the cnidarian Hydra, homologs of brachyury, Wnt3a, and forkhead have overlapping spatio-temporal expression domains in the hypostome, which correspond to the organizer of the polyp. Similarly, in Nematostella embryos, brachyury and forkhead are coexpressed at the ectodermal margin of the blastopore during gastrulation. A comparative analysis of expression patterns of these two genes shows that they are co-expressed in all animals analyzed. This suggests a close functional relationship of these two genes during animal development throughout metazoan evolution. Although a direct interaction of the two proteins has not been demonstrated to date, in Xenopus, they act synergistically to form dorsal mesoderm, in particular the notochord. Thus, forkhead and brachyury are an evolutionarily ancient synexpression group in Eumetazoa (Fritzenwanker, 2004).

Forkhead expression in particular is surprisingly conserved among metazoans. Together with brachyury, it marks the future blastopore and its derivatives, that is, foregut and hindgut. For instance, in hemichordates and echinoderms, forkhead is expressed in the vegetal plate cells before gastrulation, later in the involuting endoderm, and finally most strongly in the stomodeum anlage and the proctodeum of the Tornaria and Pluteus larva, respectively. Hence, in these lower deuterostomes, expression appears ectodermally restricted (if proctodeum and stomodeum are defined as ectodermal structures). Yet, at least in chordates, forkhead expression is not germ layer specific, but rather region- and organ-specific. In mice, the forkhead homolog HNF-3beta is expressed in the visceral endoderm, the node, (which gives rise to axial mesoderm, the notochord) and the floor plate . In the urochordates and protochordates (Amphioxus and Ascidians), the forkhead homolog is also expressed in gastrulating endoderm, the notochord and the floor plate. This suggests a close association of endoderm and the dorsal mesoderm (the notochord). In line with this, the notochord has been proposed to be a derivative of the archenteron roof in lower vertebrates, based on classical embryology (Fritzenwanker, 2004).

Much less information is available from Protostomes. However, in several species the expression domains are strikingly similar: in the Ecdysozoa, such as Drosophila, and Tribolium and in C. elegans, forkhead marks and is essential for the developing fore- and hind-gut before and during gastrulation. Among the Lophotrochozoa, expression of forkhead has been studied in the mollusc Patella vulgata. Strikingly, forkhead is expressed in the endoderm and anterior mesoderm, deriving from the anterior edge of the blastopore. At larval stages, forkhead is most strongly expressed in the stomodeum and somewhat weaker in the endoderm, reminiscent of the situation of vertebrates, where forkhead is also expressed in the prechordal plate. Based on expression data of forkhead in Nematostella vectensis, it is proposed that forkhead has an ancestral role in defining the blastopore and one derivative, the ectodermally derived pharynx. The evolutionary conservation of the synexpression group of brachyury, forkhead and several other genes suggest an establishment and coevolution of a cassette of conserved transcription factors in the blastopore during early metazoan evolution. Since forkhead and other node-specific genes also play important roles in dorsal-ventral axis formation in vertebrates, it is proposed that the blastopore evolved as an organizer for axis formation and mesoderm formation during early metazoan evolution (Fritzenwanker, 2004).

The ces-1 and ces-2 genes of C. elegans control the programmed deaths of specific neurons. Genetic evidence suggests that ces-2 functions to kill these neurons by negatively regulating the protective activity of ces-1; ces-2 encodes a protein closely related to the vertebrate PAR family of bZIP transcription factors, and a ces-2/ces-1-like pathway may play a role in regulating programmed cell death in mammalian lymphocytes. ces-1 encodes a Snail family zinc finger protein, most similar in sequence to the Drosophila neuronal differentiation protein Scratch. An element important for ces-1 regulation has been defined and evidence is provided that CES-2 can bind to a site within this element and thus may directly repress ces-1 transcription. These results suggest that a transcriptional cascade controls the deaths of specific cells in C. elegans (Metzstein, 1999).

The NSM cells of the nematode Caenorhabditis elegans differentiate into serotonergic neurons, while their sisters, the NSM sister cells, undergo programmed cell death during embryogenesis. The programmed death of the NSM sister cells is dependent on the cell-death activator EGL-1, a BH3-only protein required for programmed cell death in C. elegans, and can be prevented by a gain-of-function (gf) mutation in the cell-death specification gene ces-1, which encodes a Snail-like DNA-binding protein. The genes hlh-2 and hlh-3, which encode a Daughterless-like and an Achaete-scute-like bHLH protein, respectively, are required to kill the NSM sister cells. A heterodimer composed of HLH-2 and HLH-3, HLH-2/HLH-3, binds to Snail-binding sites/E-boxes in a cis-regulatory region of the egl-1 locus in vitro that is required for the death of the NSM sister cells in vivo. Hence, it is proposed that HLH-2/HLH-3 is a direct, cell-type specific activator of egl-1 transcription. Furthermore, the Snail-like CES-1 protein can block the death of the NSM sister cells by acting through the same Snail-binding sites/E-boxes in the egl-1 locus. In ces-1(gf) animals, CES-1 might therefore prevent the death of the NSM sister cells by successfully competing with HLH-2/HLH-3 for binding to the egl-1 locus (Thellmann, 2003).

Coordination of cell proliferation and cell fate determination by CES-1 snail

The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. This study presents evidence that the Snail-related transcription factor CES-1 of Caenorhabditis elegans coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene egl-1, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. CES-1 also affects cell cycle progression in this lineage. Specifically, it was found that CES-1 can repress the transcription of the cdc-25.2 gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of cya-1, which encodes Cyclin A. The results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the ces-1 gene leads to incorrect regulation of this functional 'module'. Finally, evidence is provided that dnj-11 MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. These findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination (Yan, 2013).

Homologs of the Drosophila genes dorsal and snail have been cloned from the glossiphoniid leech Helobdella robusta (Phylum Annelida). Sequences from one dorsal-class gene (Hro-dl) and two snail-class genes (Hro-sna1 and Hro-sna2) were identified. Polyclonal antibodies were raised against the most conserved domains of HRO-DL and HRO-SNA1. Nuclear staining appeared for both proteins in mid-embryogenesis, in mesodermal and ectodermal precursors. During segmentation, segmentally iterated stripes of cells with strong HRO-DL staining appeared. The stripes of HRO-DL staining were first concentrated in the cytoplasm of cells, and later in the nuclei. Around this time, HRO-SNA levels also appeared in nuclei in segmentally iterated stripes. The localization of HRO-DL and HRO-SNA proteins raise the possibility that these genes are part of a conserved genetic pathway that, instead of specifying the dorsoventral axis and the mesoderm as in flies, might play a role in the diversification of cell types within segment primordia during leech development (Goldstein, 2001).

It is unlikely that the HRO-DL and HRO-SNA proteins act in dorso-ventral axis specification, since neither show patterns of localization consistent with this possibility; for example, neither appear to be localized in a pattern expected for a spiralian dorsoventral determinant. It remains possible that they function in mesoderm specification, although since the proteins are expressed in both mesodermal and ectodermal precursors, this seems unlikely. The results suggest that the role of Dorsal-class proteins in specification of the dorsoventral axis and the mesoderm might have been present in the common ancestor to leeches and flies and was lost in the lineage leading to leeches, or it might not have been present in the common ancestor and was gained in the lineage leading to flies. Preliminary tests of an innate immunity function in leeches -- determining whether bacterial infection can cause cytoplasmic HRO-DL to become nuclear, as Dorsal does in flies -- have produced negative results. Specific HRO-SNA expression in the nervous system has not been observed. It is concluded therefore that none of the developmental functions known for these genes in flies have been found yet in leeches. Widespread expression appears transiently in leech development, in all the micromeres and teloblast derivatives between stage 6b and late stage 8. How these genes might function in these cells, or whether they have a function during this period, remains unclear. The most suggestive parts of the leech expression patterns are the stripes of nuclear staining that arise during segmentation: this pattern raises the possibility that these genes are part of a genetic pathway which, instead of specifying the dorsoventral axis and the mesoderm as in flies, might play a role in the diversification of cell types within segment primordia during leech development. Segmentally iterated stripes of staining have also been seen in Xenopus embryos for the dorsal homolog Xrel1. With the caveat in mind that it is not know if or how these striped patterns of Dorsal-class proteins might function in either leech or Xenopus embryos, these results at least raise the possibility that this may be a feature of a Dorsal pattern that was present in the ancient urbilaterians but was lost in the lineage leading to flies. Alternatively, Dorsal-class proteins might not have been expressed in iterated stripes in the common ancestor of these organisms, and might have independently acquired at least superficially similar patterns in the lineages leading to the leeches and frogs. Snail-class genes have been proposed to have an ancestral function in cell migration; whether HRO-DL activates snail in a segmentally iterated population of cells which will later migrate remains to be determined (Goldstein, 2001).

Snail genes have been found to play a role in mesoderm formation in two of the three clades of bilaterians, deuterostomes (comprising the chordates) and ecdysozoans (comprising the arthropods). No clear data are available on the role these genes play in development of the mesoderm in the third clade, that of lophotrochozoans (comprising annelids and molluscs). Two new members of the snail gene family have been identified in the gastropod mollusc Patella vulgata. Phylogenetic analysis has shown that the two genes clearly belong to the snail sub-family. Their expression patterns do not indicate a role during early mesoderm formation. In fact, contrary to expectations, the snail genes of Patella are mostly expressed in the ectoderm. In view of the location of their expression sites, it is suggested that these genes could be involved in regulating epithelial-mesenchymal transitions (EMT) and cell motility, as has recently been demonstrated for snail genes in vertebrates. This may well correspond to the ancestral function of these genes. The results are discussed in the light of the evolutionary origin of the mesoderm (Lespinet, 2002).

To date, about 35 members of the snail family have been isolated and sequenced. In order to compare these proteins all together and to avoid comparison biases due to different protein lengths, SSPA score identity was used instead of classical percentage identity. This analysis shows that protein pairwise identity scores of the sequences outside of the snail motive are quite low (below 40%) except for the vertebrate slug proteins (above 84%). The conservation of the snail-box is much higher and even much higher in vertebrates (score identity >77%) than in ecdysozoans (score identity >51%) (Lespinet, 2002).

These results led to the use of only the snail box in order to try to resolve the orthology relationships between members of the snail family. By distance analysis [through the Neighbour-Joining (NJ) algorithm] the sequences distribute themselves into two broad groups, respectively the snail/slug and the scratch sub-families. Since species belonging to the whole range of Bilateria are found in each of these two sub-families, this is indicative of a pre-bilaterian duplication of the ancestral snail gene to yield the two main paralogy groups. The NJ tree also shows that the slug genes originate within the snail sub-family. However, the tree also provides strong suspicions of a number of artifacts related to the fact that the various sequences appear to evolve at different rates. Within the snail/slug portion of the tree, the three Drosophila genes (snail, escargot and worniu) form a monophyletic group suggesting that they result from a relatively recent set of duplications that could be specific to arthropods. All the deuterostomes snail/slug genes, with the notable exception of the sea urchin and amphioxus snail, now form a monophyletic group. Surprisingly, however, two vertebrate snail sequences, those of chick and Xenopus, do not emerge with the other snail genes. Instead they form an independent clade also comprising the slug genes. The latter point clearly shows that slug genes originate from a duplication that occurred early on in the vertebrate lineage (Lespinet, 2002).

The Patella mesoderm is induced between the 32- and 40-cell stage but no expression of either Patella snail gene was found at this stage. During gastrulation, the mesoderm moves to the inside of the embryo; it was therefore expected that the snail orthologs would be expressed internally. Only four Pv-sna1-expressing cells were found located inside the larva but if they are probably not of ectodermal origin, these cells cannot be asigned to any germ layers. Since they do not fit with any well known structures, the fate of these four Pv-sna1-expressing cells remains unknown (Lespinet, 2002).

All other sites of expression found here for the two snail genes are ectodermal. The ectodermal Pv-sna1-expressing cells are located asymmetrically at the right side of the foot. Although the significance of this staining remains cryptic it is very tentatively suggested that, as in the chick and mouse, this expression could be related to the control of left-right asymmetry in this region. The ectodermal Pv-sna2 expression in the apical plate is at the base of the apical tuft. The tuft develops into a neural sensory structure suggesting that Pv-sna2 could be involved in the neural differentiation of this structure. This situation can be readily compared to the neural expression and role in neural differentiation of many of the snail family members. The other site of Pv-sna2 expression might correspond to the base of the mantle folds. Cells in this region are forming pockets that will expand inward and become the epithelial covering of the cavity enclosed by the shell. Although there is no direct evidence, an EMT process could be involved in this pocketing of the ectoderm. A function in EMT and cell-motility control has been found for vertebrate members of the snail gene family (Lespinet, 2002).

Recent results on mouse and human have shown that snail is able to bind the E-cadherin promoter and thus to repress the expression of this molecule that plays a central role in the control of cell adhesiveness and migration. Also, snail genes have repeatedly been linked to EMT, e.g., in neural crest formation and heart development in vertebrates, and in tumor formation. This has led to the suggestion that EMT and the control of cell motility are ancient functions of this gene family (Lespinet, 2002).

The Snail repressor is required for PMC ingression in the sea urchin embryo

In metazoans, the epithelial-mesenchymal transition (EMT) is a crucial process for placing the mesoderm beneath the ectoderm. Primary mesenchyme cells (PMCs) at the vegetal pole of the sea urchin embryo (Echinodermata) ingress into the floor of the blastocoele from the blastula epithelium and later become the skeletogenic mesenchyme. This ingression movement is a classic EMT during which the PMCs penetrate the basal lamina, lose adherens junctions and migrate into the blastocoele. Later, secondary mesenchyme cells (SMCs) also enter the blastocoele via an EMT, but they accompany the invagination of the archenteron initially, in much the same way vertebrate mesenchyme enters the embryo along with endoderm. This study identified a sea urchin ortholog of the Snail transcription factor, and focus was placed on its roles regulating EMT during PMC ingression. Functional knockdown analyses of Snail in whole embryos and chimeras demonstrate that Snail is required in micromeres for PMC ingression. Snail represses the transcription of cadherin, a repression that appears evolutionarily conserved throughout the animal kingdom. Furthermore, Snail expression is required for endocytosis of cadherin, a cellular activity that accompanies PMC ingression. Perturbation studies position Snail in the sea urchin micromere-PMC gene regulatory network (GRN), downstream of Pmar1 and Alx1, and upstream of several PMC-expressed proteins. Taken together, these findings indicate that Snail plays an essential role in PMCs to control the EMT process, in part through its repression of cadherin expression during PMC ingression, and in part through its role in the endocytosis that helps convert an epithelial cell to a mesenchyme cell (Wu, 2007).

Patterning of brain precursors in ascidian embryos

In terms of their embryonic origins, the anterior and posterior parts of the ascidian central nervous system (CNS) are associated with distinct germ layers. The anterior part of the sensory vesicle, or brain, originates from ectoderm lineages following a neuro-epidermal binary fate decision. In contrast, a large part of the remaining posterior CNS is generated following neuro-mesodermal binary fate decisions. This study addresses the mechanisms that pattern the anterior brain precursors along the medial-lateral axis (future ventral-dorsal) at neural plate stages. Functional studies show that Nodal signals are required for induction of lateral genes, including Delta-like, Snail, Msxb and Trp (see Drosophila Delta, Snail, Msx and Trp). Delta-like/Notch signalling induces intermediate (Gsx; see Drosophila Ind) over medial (Meis; see Drosophila Hth) gene expression in intermediate cells, whereas the combinatorial action of Snail and Msxb prevents the expression of Gsx in lateral cells. It is concluded that despite the distinct embryonic lineage origins within the larval CNS, the mechanisms that pattern neural precursors are remarkably similar (Esposito, 2017).

Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A well-characterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT (Saunders, 2014).

Snail homologs in Urochordates

Studies on ascidians (phylum Urochordata) provided the first evidence for localized determinants in animal development. The destruction of particular blastomeres leads to the specific loss of muscle derivatives. Lineage studies have established a tight correlation between the distribution of yellow crescent and muscle differentiation in Styela. The yellow crescent becomes localized to the vegetal cytoplasm shortly after fertilization and is ultimately inherited by the two B4.1 blastomeres that form most of the tail muscles in the ascidian tadpole. A new T-box gene resembling Drosophila Optomotor blind, CiVegTR, that fulfils the criteria of the classic muscle determinant, has been isolated in the ascidian Ciona intestinalis. CiVegTR maternal RNAs become localized to the vegetal cytoplasm of fertilized eggs and are incorporated into muscle lineages derived from the B4.1 blastomere. The CiVegTR protein binds to specific sequences within a minimal, 262-bp enhancer that mediates Ci-snail expression in the tail muscles. Mutations in these binding sites abolish expression from an otherwise normal lacZ reporter gene in electroporated embryos. In addition to the previously identified AC-core E-box sequences, T-box recognition sequences are conserved in the promoter regions of many genes expressed in B4.1 lineages in both Ciona and the distantly related ascidian Halocynthia. These results suggest that CiVegTR encodes a component of the classical muscle determinant that was first identified in ascidians nearly 100 years ago (Erives, 2000).

A Ciona ortholog of the Drosophila Snail repressor (Ci-sna) was found to repress a 434-bp notochord-specific enhancer in the promoter region of the Ciona Brachyury gene. Ci-sna is expressed in the developing tail muscles, where it is important for restricting Ci-Bra expression to the developing notochord. Ci-sna is activated early during muscle specification (32-cell stage), at the time when maternal determinants first activate zygotic genes. The present study identifies a 262-bp enhancer from the Ci-sna 5' flanking region that is sufficient to mediate expression in derivatives of the B4.1 blastomeres. This enhancer contains two conserved sequence motifs that are also present in the regulatory regions of muscle-specific genes in the distantly related ascidian Halocynthia. One of the motifs corresponds to a specialized E-box sequence (CAACTG), whereas the other contains conserved residues recognized by different T-box DNA binding proteins (GT-GNNA). Mutations in either motif diminish or abolish the expression driven by otherwise normal Ci-sna/lacZ transgenes (Erives, 2000).

Two axial structures, a neural tube and a notochord, are key structures in the chordate body plan, and a closer look at these structures furthers understanding of the origin of chordates. The neural tube of ascidian larvae is composed of about 340 cells, and is divided into three regions along the anteroposterior axis, which are, from anterior to posterior, the sensory vesicle, the visceral ganglion and the caudal neural tube. The sensory vesicle is composed solely of the a-line (anterior-animal) cells. The visceral ganglion present at the junction between the trunk and tail consists of the A-line (anterior-vegetal) cells. The caudal neural tube running along the length of the tail consists of four (dorsal, ventral and two lateral) rows of ependymal cells: the lateral and ventral cells are of A-line origin and the dorsal cells are of b-line (posterior-animal) origin. Beneath the neural tube, a stack of exactly 40 notochord cells runs along the tail. The anterior 32 cells (primary notochord) and the posterior 8 cells (secondary notochord) are derived from A-line and b-line cells, respectively. To expand knowledge on mechanisms of development of the neural tube in lower chordates, isolation and characterization of HrzicN, a new member of the Zic family gene of the ascidian, Halocynthia roretzi, was undertaken. HrzicN expression is detected by whole-mount in situ hybridization in all neural tube precursors, all notochord precursors, anterior mesenchyme precursors and a part of the primary muscle precursors. Embryos injected with HrzicN morpholino ('HrzicN knockdown embryos') exhibit failure of neurulation and tail elongation, and develop into larvae without a neural tube and notochord. Analysis of mesodermal marker gene expression in HrzicN knockdown embryos revealed unexpected roles for this gene in the development of mesodermal tissues. HrzicN knockdown leads to loss of HrBra (Halocynthia roretzi Brachyury) expression in all of the notochord precursors: this may be the cause for notochord deficiency. Hrsna (Halocynthia roretzi snail) expression is also lost from all the notochord and anterior mesenchyme precursors. By contrast, expression of Hrsna and the actin gene is unchanged in the primary muscle precursors. These results suggest that HrzicN is responsible for specification of the notochord and anterior mesenchyme (Wada, 2002).

The notochord and dorsal ectoderm induce dorsoventral compartmentalization of the vertebrate neural tube through the differential regulation of genes such as HNF-3beta, Pax3, Pax6 and snail. The expression of HNF-3beta (Drosophila homolog: forkhead) and snail (Drosophila homolog: snail) homologs were examined in the ascidian, Ciona intestinalis, a member of the subphylum Urochordata, the earliest branch in the chordate phylum. Ciona Snail is composed of 584 amino acids; the terminal stretch of 153 amino acids has five zinc fingers. The Ci-fkh homolog is has 587 amino acids and is highly conserved. The Ciona HNF-3beta homolog is expressed in the ventralmost ependymal cells of the neural tube. It is likely that Ci-fkh has a conserved function in the specification of an ascidian floor plate. Ciona snail homolog is expressed at the junction between the invaginating neuroepithelium and dorsal ectoderm, similar to the patterns seen in vertebrates. The snail expressing lateral border of the neural plate forms the neural crest and the dorsal roof of the neural tube. Ci-sna is also apparent in muscle/trunk mesenchyme precursors. These findings provide evidence that dorsoventral compartmentalization of the chordate neural tube is not an innovation of the vertebrates. It is propose that precursors of the floor plate and neural crest were present in a common ancestor of both vertebrates and ascidians (Corbo, 1997).

A minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra) is sufficient to direct a notochord-specific pattern of gene expression. Evidence is presented that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays have identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages. Repression appears to depend on tight linkage between sna1 and sna2 sites and Su(H) activator sites, located on the minimal enhancer. The insertion of spacer sequences between sna1 and Su(H)1 or between Su(H)2 and sna2 results in a severe derepression of Ci-Bra/lacZ transgenes in the tail muscles. Intact sna1 and sna2 sites appear to be required for the repression of Ci-Bra in the tail muscles. The function of Ci-Snail in creating a boundary between notochord and muscle is likened to the function of Snail in Drosophila in creating a boundary between neuroectoderm and mesoderm (Fujiwara, 1998).

Genomic cis-regulatory networks in the early Ciona intestinalis embryo

Precise spatiotemporal gene expression during animal development is achieved through gene regulatory networks, in which sequence-specific transcription factors (TFs) bind to cis-regulatory elements of target genes. Although numerous cis-regulatory elements have been identified in a variety of systems, their global architecture in the gene networks that regulate animal development is not well understood. This determined the structure of the core networks at the cis-regulatory level in early embryos of the chordate Ciona intestinalis by chromatin immunoprecipitation (ChIP) of 11 TFs. The regulatory systems of the 11 TF genes examined were tightly interconnected with one another. By combining analysis of the ChIP data with the results of previous comprehensive analyses of expression profiles and knockdown of regulatory genes, it was found that most of the previously determined interactions are direct. Focus was placed on cis-regulatory networks responsible for the Ciona mesodermal tissues by examining how the networks specify these tissues at the level of their cis-regulatory architecture. Many interactions were found that had not been predicted by simple gene knockdown experiments, and a significant fraction of TF-DNA interactions were found to make major contributions to the regulatory control of target gene expression (Kubo, 2010).

The developmental fates of blastomeres in the Ciona embryo have been determined by the gastrula stage. A comprehensive study has revealed that 53 TF genes are zygotically expressed and regulate one another in complex networks before gastrulation begins. To dissect the architecture of these networks at the level of protein-DNA interactions, focus was placed on 11 TF genes that play core roles in gene regulatory networks for endomesoderm specification: Brachyury, FoxA-a, FoxD, MyoD, Neurogenin, Otx, Snail, SoxC, Tbx6b, Twist-like1 and ZicL. Because the Ciona genome contains multiple copies of FoxD, Tbx6b and ZicL as gene clusters and their precise copy numbers have not yet been determined, these genes are collectively referred to FoxD, Tbx6b and ZicL in this paper. Likewise, there are two copies of Twist-like1, which are highly similar to each other, and these are collectively referred to as Twist-like1 (Kubo, 2010).

Eleven gene-fusion constructs were prepared that encode GFP-tagged TFs expressed under the control of their own promoters (e.g. a fusion gene that encodes GFP-tagged Brachyury driven by the Brachyury promoter). When these constructs were introduced into eggs, the resultant embryos expressed the fusion genes at the same time and in the same blastomeres as the endogenous genes. Exceptions were the Twist-like1 and the Snail constructs. Twist-like1 is normally expressed in three cell lineages (A7.6, B7.7 and B8.5), but the construct drove Twist-like1-GFP expression only in the B7.7 and B8.5 lines. Snail expression in the notochord lineage is normally very weak. The Snail construct did not recapitulate this expression in the notochord lineage but did drive Snail-GFP expression in the remaining lineages (Kubo, 2010).

Expression of these genes did not affect embryonic morphology at the stage when the embryos were fixed. The fixed embryos were subjected to ChIP using anti-GFP antibodies, and subsequently to microarray analysis. To define significant regions, two programs were used employing totally different algorithms. DNA segments regarded as positive by both programs were defined as significant. To confirm that this approach successfully identified TF binding sites, the sequences of ZicL and Tbx6b binding regions defined with three different false discovery rates (FDRs) were analyzed, as the consensus binding motifs of these two TFs are known. The frequencies of matches to the consensus binding sequences for ZicL and Tbx6b around peaks in 0.1% FDR were generally better than in 0.01% and 1% FDRs. As expected, the frequencies of the consensus binding sequences for ZicL and Tbx6b were markedly higher around peaks in the identified regions, suggesting that the method was able to successfully identify the TF binding regions (Kubo, 2010).

Brachyury and Ci-tropomyosin-like are the only known direct targets of ZicL and Brachyury, respectively. As an independent confirmation, the TF binding sites of these genes was expected. The ZicL ChIP profile showed a sharp peak around two known strong ZicL binding sites. The Brachyury ChIP profile also showed a peak around the known Brachyury binding site in the Ci-tropomyosin-like promoter. These peaks were included in significant regions identified with all the FDRs described above. ChIP-qPCRs were performed for these two known interactions. The ChIP-qPCR results showed excellent agreement with the ChIP-chip results (Kubo, 2010).

Next, the promoters were examined of genes that were identified in previous studies as likely direct targets of one of the 11 TFs on the basis of expression assays and gene knockdown assays. Among 29 interactions that had been found in the gene knockdown assays and for which both the source and target genes are expressed in the same cells, 28, 23 and 19 interactions were indicated to be direct under the FDRs of 1%, 0.1% and 0.01%, respectively. The remainder of the interactions were not regarded as direct. Otx expression in the A-line lineage requires a cis-regulatory module that includes Fox binding sites and is suppressed in FoxA-a morphants. The FoxA-a binding to this cis-regulatory element was counted with FDRs of 1% and 0.1%, but not with the most stringent FDR (0.01%). Similarly, several lines of evidence have suggested that MyoD is directly regulated by ZicL. First, MyoD expression is suppressed in ZicL morphants. Second, MyoD and ZicL are both expressed in presumptive muscle cells and the time windows of their expression overlap. Lastly, there is a putative ZicL binding site near to the peaks found in the MyoD upstream region. This putative binding was observed under the FDRs of 1% and 0.1%, but not under the most stringent FDR of 0.01%. On the basis of the above observations, in the following sections the results obtained at an FDR of 0.1% are generally described (Kubo, 2010).

The frequencies of the consensus sequences for ZicL and Tbx6b binding were markedly higher around peaks in the identified regions. Since the consensus binding motifs of the other nine TFs had not been determined previously, similar analyses was performed with motifs of homologs in other animals. The frequencies of the consensus binding motifs for six of the TFs, but not FoxD, SoxC or Twist-like1, were markedly higher around peaks. Because the position weight matrices (PWMs) for FoxD, SoxC and Twist-like1 gave higher background, no significant changes were seen. However, the number of matches to the motifs was markedly higher around peaks than in flanking regions and the background. These observations suggested that the method was able to successfully identify the TF binding regions (Kubo, 2010).

As has been reported in other animals, it was found that the regions bound by Brachyury, MyoD, Neurogenin, Snail, Tbx6b, Twist-like1 and ZicL, especially around the peaks, showed a marked GC bias. This bias is likely to be related to the consensus sequences, because the consensus sequences for these TFs are generally more GC-rich than those of the remaining TFs. The observed enrichment of recognition sequences was unlikely to be an artifact of GC bias because even if background sequences were picked with a base composition comparable to the averaged GC content of the bound regions (the difference between the average GC content of the bound and background regions was less than 0.8%), matches to the PWMs were enriched around peaks versus each of the GC-adjusted backgrounds (Kubo, 2010).

Next, attempts were made to discover overrepresented motifs in the regions (360 bp) around the peaks identified by each ChIP experiment using the Trawler program. It was found that overrepresented motifs were similar to the PWMs that were determined experimentally (Tbx6b and ZicL) or to those of homologs in other animals (the remaining nine TFs). This further supported the conclusion that the results of the ChIP experiments were of high quality (Kubo, 2010).

It is generally believed that TFs tend to bind near promoters, although many examples are known in which TFs bind to enhancers far from promoters. The distributions of peaks in all experiments, except Snail ChIP, were higher around transcription start sites. The reason why Snail binding sites were not enriched around transcription start sites is unclear, but this does not necessarily indicate that the results of the Snail ChIP were of low quality. Altogether, these observations support the conclusion that all of the ChIP experiments revealed in vivo occupancies of the TFs (Kubo, 2010).

TF genes were significantly enriched among the target genes of the 11 TFs. Among 670 potential TF genes in the Ciona genome, at least 607 encode proteins with known TF motifs or proteins with two or more zinc-finger motifs that potentially bind to DNA. A significantly greater number of TF genes were found among the targets than would be expected from random sampling. This enrichment indicates that the TFs examined bind targets selectively and not randomly (Kubo, 2010).

The ChIP data was compared with the results of the comprehensive gene knockdown experiments of a previously study. Among 76 interactions previously found in the early embryo, the ChIP assays indicated that 58 are direct. In addition, 251 novel interconnections were found. Among 121 (11×11) possible interconnections, 84 were observed in the present study. The data indicate that these genes are tightly interconnected with one another (Kubo, 2010).

Because the gene regulatory network model previously constructed from comprehensive expression profiles and comprehensive knockdowns of regulatory genes is of single-cell resolution, the ChIP data was interpred into this network by assuming that the examined TFs bind to the targets wherever their mRNAs are expressed. The reconstructed networks had a complex architecture (Kubo, 2010).

The reconstructed regulatory networks allow tracing of development at the single-cell level. Figs S8 and S9 in the supplementary material show the interconnections among the core 11 TFs in A-line and B-line blastomeres, which give rise to endomesodermal tissues, from the 8-cell to the early gastrula stage. Two of the three mesenchymal lineages (B-line mesenchymal cells) and 28 out of 36 muscle cells (B-line muscle cells) in the tadpole larvae are derived from B4.1 blastomeres at the 8-cell stage. Thirty-two and eight notochord cells are derived from A4.1 and B4.1 blastomeres, respectively. Previous studies demonstrated that Twist-like1, MyoD and Brachyury are essential for specification of the mesenchyme, muscle and notochord, respectively (Kubo, 2010).

Twist-like1 is expressed exclusively in the mesenchymal lineage and is regulated by FoxA-a, Otx and ZicL, as indicated by the fact that knockdown of any of these three genes results in loss or reduction of Twist-like1 expression. No direct binding was detected of FoxA-a to the Twist-like1 promoter, but it was found that FoxA-a binds to the upstream regions of Otx and ZicL, and that ZicL and Otx bind to the promoter of Twist-like1. Therefore, it is highly likely that FoxA-a mainly activates Twist-like1 indirectly through activating Otx and ZicL (Kubo, 2010).

Twist-like1 expression begins in B7.7 (the posterior B-line mesenchyme) at the 64-cell stage and in B8.5 (the anterior B-line mesenchyme) at the early gastrula stage. These two mesenchymal lines contribute to distinct adult tissues after metamorphosis. ZicL might be associated with the differences between these two lineages because the contribution of ZicL to Twist-like1 activation is weaker than that of Otx. To confirm this idea, a mutant Twist-like1 promoter was tested, from which a 150 bp segment containing the identified ZicL binding region was deleted. Because the Otx ChIP result indicated that the Otx binding region is distinct from the ZicL binding region, Otx was expected to bind to this mutant promoter. When introduced into fertilized eggs by electroporation, the wild-type promoter (1550 bp) drove reporter expression in 65% of the embryos, whereas the mutant promoter drove reporter expression in 36% of the embryos. In addition to the significant decrease in the number of embryos expressing the reporter, the overall fluorescence was weaker and the posterior B-line mesenchyme did not appear to express the reporter in the mutant construct. To confirm this observation, the experimental embryos were cleavage-arrested at the 110-cell stage. Cells in the arrested embryos cannot divide further, but the developmental programs proceed as in normal embryos. The mutant construct failed to drive reporter expression in the posterior B-line mesenchyme. These results suggest that ZicL contributes to the difference between these two lineages (Kubo, 2010).

A previous study showed that nine mesenchyme-specific non-regulatory genes are under the control of Twist-like1. None of these genes was identified as a direct target in the present study. Even when applied with an FDR of 1%, only one gene was identified as a direct target. Therefore, it is likely that Twist-like1 regulates the expression of mesenchyme-specific genes through its downstream regulatory gene circuit, although there is a possibility that Twist-like1 binds to the regulatory elements of these genes at later stages (Kubo, 2010).

The B6.2 and B6.4 cell pairs in the 32-cell embryo have the potential to give rise to mesenchyme and muscle. At the 64-cell stage, these cells divide, and one of the daughter cells becomes specified to give rise to the muscle cells. Previous functional assays showed that ZicL, Tbx6b and MyoD are essential for specification of muscle cells. Tbx6b begins to be expressed at the 16-cell stage, and cells expressing Tbx6b give rise not only to muscle cells but also to mesenchyme cells. Tbx6b expression declines to undetectable levels before the tailbud stage. ZicL starts to be expressed at the 32-cell stage in a variety of cells, including those with developmental fates of muscle, mesenchyme, notochord and neurons. ZicL expression in the muscle lineage disappears before the late gastrula stage. MyoD expression begins at the 44-cell stage exclusively in the muscle lineage under the control of Tbx6b and ZicL. The present study showed that ZicL, Tbx6b and MyoD constituted a tightly interconnected gene circuit that is responsible for this specification: (1) ZicL bound to the promoters of MyoD and Tbx6b; (2) Tbx6b bound to the promoters of MyoD and ZicL; and (3) MyoD bound to the promoter of Tbx6b and to its own promoter. All of these interactions, except MyoD binding to the Tbx6b promoter, have been confirmed by functional assays (Kubo, 2010).

To understand how this gene circuit regulates downstream muscle-specific genes, the promoters were examined of 13 muscle structural genes that are well annotated and known to be expressed in the larval tail muscle. Of these, ten were directly bound by MyoD and Tbx6, one by MyoD and ZicL, one by Tbx6b and ZicL, and one by MyoD alone (Kubo, 2010).

Both MyoD and Tbx6 bound to the promoters of more than three-quarters of the muscle genes examined. To test the action of this feed-forward loop comprising MyoD and Tbx6b in the regulation of muscle-specific gene expression, the expression patterns of genes under the control of this circuit were examined. Of the 155 genes under the direct control of MyoD and Tbx6b, 50 (including the above ten) were already known to be expressed in muscle cells. From the remaining 105 genes, 20 were randomly chosen, and 15 were found to be expressed in muscle cells, suggesting that this circuit is widely used for the regulation of genes expressed in muscle cells, and also that this circuit might not necessarily be sufficient for driving expression of the target (Kubo, 2010).

Brachyury is activated at the 64-cell stage exclusively in the notochord lineage, and this expression specifies the notochord fate. ZicL directly binds to the Brachyury promoter and activates its expression. It has also been shown that FoxD and FoxA-a are required for Brachyury expression, probably through regulating ZicL expression, and that FGF signaling is also required for Brachyury expression. The present assays showed that not only ZicL, but also FoxD binds to the Brachyury promoter. Although FoxD mRNA is not present in the notochord lineage at the 32-cell and 64-cell stages, when ZicL and Brachyury are activated, respectively (FoxD is expressed in the ancestors of cells in which ZicL and Brachyury are expressed), the ChIP assay indicated that FoxD binds to the promoters of ZicL and Brachyury. Because knockdown of FoxD eliminates ZicL and Brachyury expression and because the FoxD-GFP fusion protein exists in the notochord lineage at the 32-cell stage, it is likely that FoxD protein exists in these cells and binds to the promoters of ZicL and Brachyury when these two genes begin to be expressed (Kubo, 2010).

FoxA-a binding to the Brachyury promoter was not identified under 0.1% FDR. There was, however, a small peak that was counted as significant under 1% FDR. The possibility could not be ruled out that FoxA-a binds weakly to the Brachyury promoter. It is also possible that FoxA-a could bind weakly to a FoxD binding site because the FoxA-a binding peak coincided with that of FoxD. Even if this weak binding occurs in vivo, the regulation of Brachyury by FoxA-a would largely be achieved indirectly through FoxD and ZicL, since strong binding was found of FoxA-a to the promoters of FoxD and ZicL (Kubo, 2010).

Next, 14 non-regulatory genes were examined that are known to be expressed in the notochord under the control of Brachyury. Among them, 11 were identified here as direct targets of Brachyury. The present results suggest that the remaining three genes are regulated indirectly through a gene circuit under the control of Brachyury, although it cannot be ruled out that Brachyury binds to the regulatory elements of these three genes at later stages (Kubo, 2010).

The present study found many interactions between TFs and genomic DNA that were unexpected from preceding gene knockdown assays. Similar observations were also reported in preceding ChIP studies. To estimate what proportion of the binding makes a major contribution to gene regulation in Ciona embryos, MyoD mRNA or an MO against MyoD was injected into eggs, and their effects were analyzed on the expression of the same targets that were analyzed at the gastrula stage or at the tailbud stage, respectively. The mRNA levels of 14 targets, ten of which were expressed in muscle, were significantly increased (>2-fold) in embryos injected with MyoD mRNA, and MyoD MO injection significantly reduced the mRNA levels of three of these targets. The mRNA level of one target (KH.C12.38), which was weakly expressed in muscle at the tailbud stage, was significantly decreased in embryos injected with MyoD mRNA, whereas the mRNA level of one target (KH.C9.27), which was expressed in muscle at the gastrula stage, was significantly increased in embryos injected with the MyoD MO. In total, the mRNA levels of 16 targets were significantly altered by MyoD mRNA overexpression or gene suppression. The remaining four were not significantly affected, although three of these were expressed in muscle, implying that MyoD binding makes a relatively small contribution to activating these target genes. It was also found that eight of 15 Brachyury targets and seven of 12 Twist-like1 targets were significantly affected in the embryos by overexpression or knockdown of Brachyury or Twist-like1, respectively. Therefore, it is estimated that more than half of TF binding makes a major contribution to the regulatory control of gene expression (Kubo, 2010).

Snail homologs in vertebrates

The members of the Snail family of zinc-finger transcription factors have been implicated in the formation of distinct tissues within the developing vertebrate and invertebrate embryo. Two members of this family have been described in higher vertebrates, Snail (Sna) and Slug (Slu), where they have been implicated in the formation of tissues such as the mesoderm and the neural crest. The mouse homolog of the Slug gene has been isolated, enabling an analysis and comparison of the amino acid sequences and the patterns of expression of both Sna and Slu in the chick and mouse. Features in the sequences have been detected that allow the unequivocal ascription of any family member to the Sna or Slu subfamilies. A stretch of 29 amino acids immediately preceding the zinc-finger domain is exclusive to and highly conserved in vertebrate Slug proteins. The vertebrate Snail proteins appear to contain several amino acid stretches of variable size at conserved positions that are absent in the Slug protein sequences. The identity between each of these short sequences is not sufficiently conserved so as to enable these stretches of amino acids to be considered as diagnostic for Snail proteins. When compared to the three members of the Snail family in Drosophila (snail, escargot and scratch) it appears that all vertebrate members of the family show a slightly greater degree of identity to the product of escargot (between 50 and 59% identity) than to that of snail (between 44 and 55% identity), scratch being the most distant relative of the Drosophila genes. The region 5' to the zinc-finger domains in each of the Drosophila proteins is notably larger than that of the vertebrate homologs, as is the case for the only Snail family homologs described in sea urchin and ascidians. The vertebrate proteins of the Sna and Slu subfamilies contain sequences in this 5' region that are specific to both Esg and Sna. It has been observed that during early stages of development many of the sites of Slu and Sna expression in the mouse and chick embryo are swapped. Thus, an inversion in the expression of Snail family members appears to have occurred between the chick and mouse in the premigratory neural crest, early mesoderm and during early somite formation. These swaps appear to have taken place within the avian lineage. Later in development, the sites of expression of Slu and Sna are conserved between these two species. These data, together with the data available in other species, lead to a proposal that Slu and Sna arose as a duplication of an ancestor gene and that an extra duplication in the fish lineage has given rise to two Sna genes. This analysis of the Snail family may also shed new light on the origin of the neural crest. The neural crest first appears at the edges of the neural folds, precisely the region where the ascidian snail homolog is expressed. This raises the possiblity that these cells, that also appear in the cephalochordata, are the evolutionary precursors of the neural crest (Sefton, 1998).

Xsna, the Xenopus homolog of Drosophila snail, is expressed in both mesoderm and ectoderm. Expression occurs initially in all mesoderm but is down regulated in a tissue-specific fashion at the end of gastrulation in a way that reveals the subdivision of the mesoderm before its derivatives are overtly differentiated. Xsna is also expressed in the ectoderm of the prospective neural fold from stage 11; it is found here in a distinct band of cells surrounding the prospective neural plate designated as the neural plate border. The deep and superficial ectoderm compartments labelled by Xsna represent the prospective neural crest and the prospective roof of the neural tube, respectively. Xsna expression persists in neural crest cells during their subsequent migration (Mayor, 1993).

Expression of the Xsna gene during Xenopus embryogenesis has been analysed by in situ hybridization. Like its homolog snail in Drosophila, Xsna is expressed zygotically in all early mesoderm. Expression starts during stage 9 in the dorsal marginal zone and spreads to the ventral side by stage 10. During gastrulation, each cell begins to express as it involutes so that cells newly expressing Xsna are added to the forming mesoderm mantle in an anterior-to-posterior progression. Xsna expression is then down-regulated in a tissue-specific fashion that reveals the subdivision of the mesoderm before its derivatives are overtly differentiated; e.g., the appearance of the notochord, myotomes, and pronephroi are preceded by the disappearance of Xsna mRNA, while undifferentiated mesoderm remains labelled, even into tadpole stages. Xsna is expressed in the suprablastoporal endoderm during gastrulation and in its derivatives, the prechordal and sub-notochordal endoderm, during neurulation. Xsna is also expressed in the prospective neural fold ectoderm from stage 11 in a low arc above the dorsal marginal zone, precisely identifying a distinct band of cells that surrounds the prospective neural plate that is designated the neural plate border. In the anterior transverse neural fold, which becomes forebrain,Xsna expression ceases during neurulation. Both the deep and the superficial ectoderm compartments labelled by Xsna expression in the longitudinal neural folds, are respectively, the prospective neural crest and prospective roof of the neural tube. Xsna expression persists in the neural crest during migration and in some derivatives at least until metamorphosis but ceases in the roof of the neural tube soon after neurulation (Essex, 1993).

A mouse gene (Sna) is closely related to snail, xsnail and another Drosophila gene, escargot, which also encodes a zinc-finger protein. Mouse Sna encodes a 264 amino acid protein that contains four zinc fingers. Developmental RNA blot analysis shows that Sna transcripts are expressed throughout postimplantation development. Analysis of the spatial and temporal localization of Sna transcripts by in situ hybridization to both whole-mount and sectioned embryos reveals that in the gastrulating embryo, Sna is expressed throughout the primitive streak and in the entire mesodermal germ layer. By 9.5 days post coitum Sna is expressed at high levels in cephalic neural crest and limb bud mesenchyme. In fact, by 10.5 days, Sna expression is observed in most mesenchymal cells, whether of neural crest or mesodermal origin (Smith, 1992).

A gene encoding a zinc finger protein of the Snail family, cSnR, is expressed in the right-hand lateral mesoderm during normal chick development. Antisense disruption of cSnR function during the hours immediately preceding heart formation randomizes the normally reliable direction of heart looping and subsequent embryo torsion. Implanted ectopic sources of intercellular signal proteins that are involved in establishing normal left-right information randomize the handedness of heart development and also alter the asymmetry of cSnR expression. cSnR thus appears to act downstream of these signals, or perhaps acts in parallel with the latest expressed of them, the Nodal protein, in controlling the anatomical asymmetry (Isaac, 1997).

Slug, a zinc-finger-containing gene of the snail family, is expressed in a dynamic fashion that is regulated by axial signalling systems. Expression can be correlated with three regions: the zone of polarizing activity, the progress zone and the interdigital areas. Slug expression is first detected in the ectoderm covering the limb bud and flank at stage 18. It is interesting to note that snail expression in the development of the wing imaginal disc in Drosophila is also ectodermal (Fuse, 1996). Soon after Slug ectodermal expression is established, Slug is expressed in the mesenchyme, a tissue of mesodermal origin. The maintenance of slug expression is dependent on signals from the apical ectodermal ridge and is independent of Sonic Hedgehog. The mesodermal domain of Slug expression extends distally under the apical ectodermal ridge into the progress zone, an area of undifferentiated, rapidly proliferating mesenchymal cells located under the ridge, whose maintenance depends of the presence of the ridge. FGF-2, presumably expressed in the apical ectodermal ridge, can function to maintain Slug mesenchymal expression. In the interdigit areas, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at various stages of tissue differentiation is consistent with its roles in early development: maintaining the mesenchymal phenotype and repressing the differentiation processes. It is suggested that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone (Ros, 1997).

An epithelial-mesenchymal cell transformation occurs during the development of the endocardial cushions in the atrioventricular (AV) canal of the heart. It was hypothesized that the transcription factor Slug is required for this epithelial-mesenchymal cell transformation since Slug is required for similar transformations during gastrulation and neural crest differentiation in chicken embryos. The temporal and spatial localization of Slug in the embryonic chicken heart is consistent with a role for Slug in endocardial cushion formation. Moreover, Slug expression by AV canal endothelial cells is induced by a signal provided by AV canal myocardium. Slug appears to be required for epithelial-mesenchymal cell transformation in the chicken heart, since treatment of AV canal explants with antisense Slug oligodeoxynucleotides inhibits mesenchymal cell formation in vitro. Antisense Slug oligodeoxynucleotides prevents endothelial cell-cell separation, suggesting that Slug acts early in the transformation pathway (Romano, 1999).

An epithelial-mesenchymal cell transformation (EMT) occurs during the development of endocardial cushions in the atrioventricular (AV) canal of the heart. This is a complex developmental process regulated by multiple extracellular signals and signal transduction pathways. It has recently been shown that the transcription factor Slug is expressed in the AV canal and is required for initial steps of EMT. Treatment of AV canal explants with either antisense oligodeoxynucleotides toward Slug or anti-TGFbeta2 antibody inhibits the initial steps of EMT. Other studies have identified roles for HGF and BMP during EMT in the heart. Both HGF and BMP are known to regulate Slug in other cell types. To determine whether TGFbeta2 or other signaling factors regulate Slug expression during EMT in the heart, AV canal explants were cultured in the presence of anti-TGFbeta2 antibody, anti-TGFbeta3 antibody, pertussis toxin, retinoic acid, noggin, or anti-HGF antibody. Only treatment with anti-TGFbeta2 antibody or retinoic acid inhibits Slug expression in AV canal explants. Consistent with these data, it was found that retinoic acid disrupts initial steps of EMT, while antagonists of BMP and HGF signaling disrupt later steps of EMT. Transfection of AV canal explants with Slug rescues the inhibitory effect of anti-TGFbeta2 antibody but not retinoic acid on EMT. Slug is thus an essential target of TGFbeta2 signaling during EMT in the developing chicken heart (Romano, 2000).

Based primarily on studies in the chick, it has been assumed that the zinc finger transcription factor Slug is required for neural crest migration. In the mouse, however, Slug is not expressed in the premigratory neural crest, which forms normally in Slug -/- animals. To study the role of Slug in Xenopus laevis, the injection of XSlug antisense RNA and tissue transplantation were used. Injection of Slug antisense RNA does not suppress the early expression of the related gene XSnail, but leads to reduced expression of both XSlug and XSnail in later stage embryos, whereas the expression of another neural crest marker, XTwist, is not affected. Down-regulation of XSlug and XSnail is associated with the inhibition of neural crest cell migration and the reduction or loss of many neural crest derivatives. In particular, the formation of rostral cartilages is often highly aberrant, whereas the posterior cartilages are less frequently affected. The effects of Slug antisense RNA on neural crest migration and cartilage formation are rescued by the injection of either XSlug or XSnail mRNA. These studies indicate that XSlug is required for neural crest migration, that XSlug and XSnail may be functionally redundant, and that both genes are required to maintain one another's expression in the neural crest development of Xenopus laevis (Carl, 1999).

The Snail family of genes comprises a group of transcription factors with characteristic zinc finger motifs. One of the members of this family is the Slug gene. Slug has been implicated in the development of neural crest in chick and Xenopus by antisense loss of function experiments. Functional derivatives of Xslug have been generated by constructing cDNAs that encode the Xslug protein fused with the transactivation domain of the virus-derived VP16 activator or with the repressor domain of the Drosophila Engrailed protein. The results suggest that Xslug normally functions as a transcriptional repressor and that Xslug-VP16 behaves as a dominant negative of Xslug. Xslug functions by controlling its own transcription. Xslug is expressed in the dorsal mesendoderm at the beginning of gastrulation, where is it able to upregulate the expression of dorsal genes. When Xslug is expressed outside of the organizer it represses the expression of ventral genes. These results indicate that this effect on mesodermal patterning depends on BMP activity, showing that Xslug can directly control the transcription of BMP-4 (Mayor, 2000).

The most novel finding of this study is the early function that Xslug has in mesendoderm development. Xslug is expressed in the organizer region and is able to control the expression of organizer genes such as chordin, cerberus and other dorsal mesodermal genes such as goosecoid, pintallavis and Frzb. That Xslug is required for the normal development of the embryo is shown by the injection of Xslug-VP16; these embryos lack a head. This phenotype is probably explained by the inhibition of the expression of some dorsal genes, particularly cerberus, which is known to be involved in head development. The lack of ectopic head structures and a proper secondary axis after Xslug injection could be explained by the fact that the level of cerberus induced ectopically by Xslug overexpression is not sufficient to activate the cascade required by the organizer activity (Mayor, 2000).

When Xslug is expressed in ventral mesoderm it induces dorsal genes and represses ventral genes, BMP-4 being one of these ventral genes. However, when Xslug-VP16 RNA is injected in dorsal blastomeres, dorsal genes are inhibited while no expression of ventral mesodermal genes is observed. This can be explained by the requirement of BMP-4 for the expression of ventral markers (Xvent-1, Xwnt-8), and as BMP-4 is not expressed in the dorsal mesoderm no ventral marker can be induced there. Interestingly, these results show that Xslug is able to block BMP transcription, and as a consequence, Xvent-1, a gene downstream of BMP, is blocked by Xslug mRNA injection. In addition, the effect of Xslug-VP16 can be rescued only by co-injection of BMP mRNA but not with Xwnt-8. The inhibition of Xwnt-8 caused by Xslug overexpression could also be a consequence of the interference with BMP-4 transcription, since a dominant negative BMP-4 receptor suppresses Xwnt-8 expression. Taken together these results indicate that Xslug could be a repressor of BMP in dorsal mesoderm. Thus, when it is ectopically expressed in ventral mesoderm the downregulation of BMP leads to an upregulation of dorsal genes such as chordin or cerberus (Mayor, 2000).

Epithelial-mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. EMT can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in an NBT-II cell model for EMT. The zinc-finger protein Slug is expressed in chicken neural crest cells just before they emerge from the neural tube and later during their migration phase. Interestingly, Slug is also expressed by epiblast cells lining the primitive streak during gastrulation, just before the emergence of mesenchymal cells. Treatment of developing embryos with antisense slug interferes with these two processes, suggesting a potential causal role for Slug in the EMT precess in vivo. Slug mRNA and protein levels increase transiently in FGF-1-treated NBT-II cells. Transient or stable transfection of Slug cDNA in NBT-II cells results in a striking disappearance of the desmosomal markers desmoplakin and desmoglein from cell-cell contact areas, mimicking the initial steps of FGF-1 or HGF/SF- induced EMT. Stable transfectant cells express Slug protein and are less epithelial, with increased cell spreading and cell-cell separation in subconfluent cultures. Interestingly, NBT-II cells transfected with antisense Slug cDNA are able to resist EMT induction by FGF-1 or even HGF/SF. This antisense effect is suppressed by retransfection with Slug sense cDNA. These results indicate that Slug induces the first phase of growth factor-induced EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Moreover, the antisense inhibition experiments suggest that Slug is also necessary for EMT (Savagner, 1997).

The expression pattern of the zinc-finger protein slug during rat and mouse embryonic development is described. Expression is mostly confined to migratory neural crest cells and several mesodermal derivatives. slug expression could not be detected in premigratory rodent neural crest cells, unlike previously studied vertebrates; the earliest substantial expression of slug is found in migratory cranial neural crest cells invading the first branchial arch. Their derivatives, comprising most of the craniofacial region, continue to express slug. Concomitantly, slug is expressed in sclerotome precursor cells prior to their separation from the differentiating somites. During organogenesis, slug is expressed in mesenchymal components of lung, digestive tract, and meso- and meta-nephros until late stages. Slug is also found in mesenchymal cells undergoing cartilage and bone differentiation. Expression is down-regulated in parallel with chondrocyte phenotypic differentiation. Overall, slug appears to be expressed by mesenchymal cells at predifferentiation stages involving cell migration and phenotype modulation. Expression is generally down-regulated afterwards. However, residual slug mRNA is found in several adult tissues, including liver and lung (Savagner 1998).

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, evidence is provided that the attenuation of Wnt3a signaling observed in Fgfr1-/- embryos can be rescued by lowering E-cadherin levels. It is proposed that modulation of cytoplasmic ß-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak (Ciruna, 2001).

FGFR1 regulates the morphogenesis and migration of mesodermal cells by differentially regulating intercellular adhesion properties of progenitor populations in the primitive streak. More specifically, FGFR1 signaling is required for the expression of mSnail, a key mediator of epithelial to mesenchymal transitions in development. Furthermore, it is proposed that mSnail expression downstream of FGFR1 is required for the normal downregulation of E-cadherin. Given the morphoregulatory roles for differential cell adhesion during embryogenesis, ectopic E-cadherin expression at the primitive streak of Fgfr1 mutants provides a molecular explanation for the observed defects in epithelial to mesenchymal transition (EMT), progenitor cell migration, and the sorting of Fgfr1-/- from WT cells during gastrulation (Ciruna, 2001).

The mechanisms by which FGFR1 signaling regulates both the morphogenesis and patterning of mesoderm at gastrulation are intricately entwined. Gene dosage and chimeric analyses of Brachyury function have demonstrated that the level of T expression in progenitor cell populations influences the timing and pattern of ingression through the primitive streak. Furthermore, T box genes may also regulate cell adhesion and EMT at gastrulation. In zebrafish, the Brachyury homolog no tail, and the T box gene spadetail have both been implicated as positive regulators of Snail expression. Although regulation of mouse Snail by T has yet to be determined, it is intriguing that in late gastrula-staged Fgfr1 -/- embryos, the only observed domain of mSnail expression overlaps with an Fgfr1-independent domain of T expression at the base of the allantois. Therefore, T may positively regulate Snail expression at the primitive streak, providing another link between Brachyury expression, intercellular adhesion, and the morphogenesis of the mesodermal germ layer (Ciruna, 2001).

In addition, it is proposed that FGFR1 signaling indirectly regulates Wnt signal transduction at the primitive streak. In Fgfr1 -/- embryos, although Wnt3a is expressed in the late primitive streak, direct targets of Wnt signaling (i.e., Brachyury and the T-lacZ reporter transgene) are not activated. It is suggested that ectopic E-cadherin expression in Fgfr1 mutants attenuates Wnt3a signaling by sequestering free ß-catenin from its intracellular signaling pool, and demonstrates that forced downregulation of E-cadherin in Fgfr1 -/- explants can rescue endogenous Wnt signaling at the primitive streak. Evidence that cadherins act as regulators of ß-catenin signaling is well documented. E-Cadherin and LEF-1 bind to partially overlapping sites in the central region of ß-catenin; consequently, LEF-1 and E-cadherin form mutually exclusive complexes with ß-catenin and compete for the same intracellular signaling pool. Furthermore, overexpression of cadherins during Drosophila and Xenopus embryogenesis has been shown to phenocopy Wnt/ß-catenin signaling mutants (Ciruna, 2001).

The Snail genes are implicated in processes that involve cell movement, both during embryonic development and tumour progression. In teleosts, the vertebrate Snail1 gene is represented by two distinct genes, snail1a and snail1b (previously snail1 and snail2). These genes are expressed in complementary mesodermal domains and their combined expression matches that of their mammalian counterpart. By analysing their loss and gain of function, it was found that the most-anterior axial mesendodermal cells, the precursors of the polster, move in a cohesive manner directed by the activity of snail1a- and snail1b-expressing cells surrounding these precursors. The cell-autonomous function of Snail1 proteins regulates cell motility and influences the behaviour of Snail-negative neighbouring cells. Snail1a is required by the prechordal plate for it to reach its normal position, whereas Snail1b controls the acquisition of its normal shape. These non-redundant functions of Snail1a and Snail1b in controlling axial mesendoderm migration comply with the duplication-degeneration-complementation model, and indicate that Snail genes not only act as inducers of epithelial-to-mesenchymal transition, but also as more general regulators of cell adhesion and movement (Blanco, 2007).

Regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition

The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. This study found that Snail is highly unstable, with a short half-life about 25 min. GSK-3beta binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its beta-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3beta results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3beta together function as a molecular switch for many signalling pathways that lead to EMT (Zhou, 2004).

Transcriptional regulation of Snail

The E2A-HLF fusion gene transforms human pro-B lymphocytes by interfering with an early step in apoptotic signaling. A search for E2A-HLF-responsive genes identified a zinc finger transcription factor, SLUG, whose product belongs to the Snail family of developmental regulatory proteins. Importantly, SLUG bears close homology to the CES-1 protein of C. elegans, which acts downstream of CES-2 in a neuron-specific cell death pathway. Consistent with the postulated role of CES-1 as an antiapoptotic transcription factor, SLUG is nearly as active as Bcl-2 or Bcl-xL in promoting the survival of IL-3-dependent murine pro-B cells deprived of the cytokine. It is concluded that SLUG is an evolutionarily conserved transcriptional repressor whose activation by E2A-HLF promotes the aberrant survival and eventual malignant transformation of mammalian pro-B cells otherwise slated for apoptotic death (Inukai, 1999).

Expression of Snail transcriptional factor is a determinant in the acquisition of a mesenchymal phenotype by epithelial tumor cells. However, the regulation of the transcription of this gene is still unknown. This study describes the characterization of a human SNAIL promoter that contains the initiation of transcription and regulates the expression of this gene in tumor cells. This promoter was activated in cell lines in response to agents that induce Snail transcription and the mesenchymal phenotype, as addition of the phorbol ester PMA or overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or v-Akt. Although other regions of the promoter were required for a complete stimulation by Akt or ILK, a minimal fragment (-78/+59) was sufficient to maintain the mesenchymal specificity. Activity of this minimal promoter and SNAIL RNA levels are dependent on ERK signaling pathway. NFkappaB/p65 also stimulates SNAIL transcription through a region located immediately upstream the minimal promoter, between -194 and -78. These results indicate that Snail transcription is driven by signaling pathways known to induce epithelial to mesenchymal transition, reinforcing the role of Snail in this process (Barbera, 2004).

The activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial-mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. NFkappaB was identified as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition (Bachelder, 2005).

Adipose Snail1 regulates lipolysis and lipid partitioning by suppressing Adipose Triacylglycerol Lipase expression

Lipolysis provides metabolic fuel; however, aberrant adipose lipolysis results in ectopic lipid accumulation and lipotoxicity. While adipose triacylglycerol lipase (ATGL) (see Drosophila Brummer) catalyzes the first step of lipolysis, its regulation is not fully understood. This study demonstrated that adipocyte Snail1 (see Drosophila Snail) suppresses both ATGL expression and lipolysis. Adipose Snail1 levels are higher in fed mice than in fasted mice and higher in obese mice as opposed to lean mice. Insulin increases Snail1 levels in both murine and human adipocytes, wherein Snail1 binds to the ATGL promoter to repress its expression. Importantly, adipocyte-specific deletion of Snail1 increases adipose ATGL expression and lipolysis, resulting in decreased fat mass and increased liver fat content in mice fed either a normal chow diet or a high-fat diet. Thus, this study has identified a Snail1-ATGL axis that regulates adipose lipolysis and fatty acid release, thereby governing lipid partitioning between adipose and non-adipose tissues (Sun, 2016).

Snail family members and the neural crest

The Slug gene encodes a zinc finger protein, homologous to the product of the Drosophila Snail gene, that is implicated in the generation and migration of both mesoderm and neural crest cells in several vertebrate species. The mouse Slug (Slugh) gene encodes a 269-amino-acid protein that shares 92% amino acid identity with the product of the chicken Slug gene. Slugh expression is first detected in extraembryonic mesoderm and is later detected in many mesodermal subsets, although it is not detected in the primitive streak. In contrast to many other vertebrates, the mouse Slug gene is not expressed in premigratory neural crest cells but is expressed in migratory neural crest cells. Analysis of a targeted null mutation that deletes all Slugh coding sequences reveals that Slugh is not required for mesoderm formation or for neural crest generation, migration, or development in mice. These results indicate that neither the expression pattern nor the biological function of the Slug gene is conserved among all vertebrates. These data also raise interesting questions about the regulation of neural crest generation, which is one of the distinguishing characteristics of the vertebrate subphylum (Jiang, 1998).

The neural crest is a transient population of precursor cells that arises at the border between the neural plate and prospective epidermis in vertebrate embryos. The earliest known response to neural-crest-inducing signals is the expression of the zinc-finger transcription factors slug and snail. Although it is widely believed that these transcription factors play an essential role in neural crest development, relatively little is understood about their mechanism of action during this process. Overexpression of XSlug leads to expanded expression of neural crest markers and an excess of at least one neural crest derivative, melanocytes. In order to further investigate XSlug function, mutant constructs, in which the DNA-binding domain is fused to either the activation domain from Gal4 or the repressor domain from Drosophila Engrailed, were overexpressed. The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. In contrast, overexpression of either the activation domain fusion or the DNA-binding domain alone was found to inhibit XSlug function. Using a hormone-inducible inhibitory mutant, it has been shown that inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration, demonstrating for the first time that this transcriptional repressor is required during multiple stages of neural crest development (LaBonne, 2000).

The markers Xslug, Xsnail, and Xtwist all are expressed in the presumptive neural folds and are thought to delineate the presumptive neural crest. However, their interrelationship and relative spatiotemporal distributions are not well understood. A detailed in situ hybridization analysis of the relative patterns of expression of these transcription factors from gastrulation through neurulation and post-neural crest migration is presented. The three genes mark the prospective neural crest and roof plate, coming on sequentially, with Xsnail preceding Xslug preceding Xtwist. By combining gene expression analysis with a fate map of the same region using DiI labeling, the correspondence between early and late domains of gene expression has been determined. At the beginning of gastrulation, Xsnail is present in a unique domain of expression in a lateral region of the embryo in both superficial and deep layers of the ectoderm, as are Xslug and Xtwist. During gastrulation and neurulation, the superficial layer moves faster toward the dorsal midline than the deep layer, producing a relative shift in these cell populations. By early neurula stage, the Xsnail domain is split into a medial domain in the superficial ectoderm (fated to become the roof plate) and a lateral domain in the deep layer of the ectoderm (fated to become neural crest). Xsnail is down-regulated in the most anterior neural plate and up-regulated in the posterior neural plate. These results show that changes in the expression of Xsnail, Xslug, and Xtwist are a consequence of active cell movement in some regions coupled with dynamic changes in gene expression in other regions (Linker, 2000).

Homologs of the Drosophila snail gene have been characterized in several vertebrates. In addition to being expressed in mesoderm during gastrulation, vertebrate snail genes are also expressed in presumptive neural crest and/or its derivatives. Given that neural crest is unique to vertebrates and is considered to be of fundamental importance in their evolution, the expression of a snail gene from amphioxus, a cephalochordate widely accepted as the sister group of the vertebrates, has been cloned and characterized. At the amino acid sequence level, the amphioxus snail gene is a clear phylogenetic outlier to all the characterized vertebrate snail genes. During embryogenesis, snail expression initially becomes restricted to the paraxial or presomitic mesoderm of amphioxus. Later, snail is expressed at high levels in the lateral neural plate, where it persists during neurulation. It is suggested that a population of cells homolgous to the pre-migratory neural crest may have been present in the common ancestor of cephalochordates and vertebrates. These results indicate that an ancestral function of snail genes in the lineage leading to vertebrates is to define the paraxial mesoderm. Furthermore, these results indicate that a cell population homologous to the vertebrate neural crest may be present in amphioxus, thus providing an important link in the evolution of this key vertebrate tissue. It must be considered that precursors of neural crest in the vertebrate ancestor may have resided within the neural tube itself and that neural crest does not arise from epidermal cells that overgrow the invaginating neural tube. It is tempting to speculate that early in the chordate lineage, while the mesodermal role of snail was conserved, this morphogenetic role of ventral furrow formation, as found in Drosophila, was co-opted into the process of neural plate invagination (Langland, 1999).

The Snail gene family of transcription factors plays crucial roles in different morphogenetic processes during the development of vertebrate and invertebrate embryos. Slug is involved in neural crest formation in the chick embryo. A series of gain-of-function experiments has been carried out that show that Slug overexpression in the neural tube of the chick embryo induces an increase in neural crest production. The analysis of electroporated embryos shows that Slug can induce the expression of the small GTPase rhoB and an increase in the number of HNK-1-positive migratory cells, indicating that it lies upstream of these cells in the genetic cascade of neural crest development. The increase in neural crest production after Slug overexpression is confined to the cranial region, indicating that the mechanisms of crest induction somehow differ between head and trunk. The expression of the two vertebrate family members, Slug and Snail, is peculiar with respect to the neural crest. Slug is not expressed in the premigratory crest in the mouse, whereas it is expressed in this cell population in the chick and the opposite is true for Snail. This raises the question of whether they can be functionally equivalent. To test this hypothesis both intra- and interspecies, a series of ectopic expression experiments were performed by electroporating chick and mouse Snail in the chick embryo hindbrain. Both genes elicit the same responses in the neural tube. These results indicate that they can be functionally equivalent, although the embryos show a higher response to the endogenous gene, chick Slug (del Barrio, 2002).

The transcription factors of the Sox family play important roles in diverse developmental processes. A number of genetic studies have established that Sox10 is a major regulator of neural crest formation. This study reports the cloning and functional analysis of the Xenopus Sox10 gene. Sox10 mRNA accumulates during gastrulation at the lateral edges of the neural plate, in the neural crest-forming region. In this tissue, Sox10 expression is regulated by Wnt signaling and colocalizes with two major regulators of neural crest formation, Slug and Sox9. While initially expressed in neural crest cells from all axial levels, at the tailbud stage, Sox10 is downregulated in the cranial neural crest and persists mostly in neural crest cells from the trunk region. Overexpression of Sox10 causes a dramatic expansion of the Slug expression domain. The C-terminal portion of Sox10 is sufficient to mediate this activity. Later during embryogenesis, Sox10-injected embryos show a massive increase in pigment cells (Trp-2-expressing cells). The responsiveness of the embryo to Sox10 overexpression by expansion of the Slug expression domain and ectopic production of Trp-2-positive cells and differentiated melanocytes is lost during gastrulation, as revealed by a hormone-inducible Sox10 construct. These results suggest that Sox10 is involved in the specification of neural crest progenitors fated to form the pigment cell lineage (Aoki, 2003).

The pattern of programmed cell death was studied in the neural crest and how it is controlled by the activity of the transcription factors Slug and msx1 was examined. The results indicate that apoptosis is more prevalent in the neural folds than in the rest of the neural ectoderm. Through gain- and loss-of-function experiments with inducible forms of both Slug and msx1 genes, it was shown that Slug acts as an anti-apoptotic factor whereas msx1 promotes cell death, either in the neural folds of the whole embryos, in isolated or induced neural crest and in animal cap assays. The protective effect of expressing Slug can be reversed by expressing the apoptotic factor Bax, while the apoptosis promoted by msx1 can be abolished by expressing the Xenopus homologue of Bcl2 (XR11). Furthermore, Slug and msx1 control the transcription of XR11 and several caspases required for programmed cell death. In addition, expression of Bax or Bcl2 produced similar effects on the survival of the neural crest and on the development of its derivatives as those produced by altering the activity of Slug or msx1. Finally, it was shown that in the neural crest, the region of the neural folds where Slug is expressed, cells undergo less apoptosis, than in the region where the msx1 gene is expressed; this region corresponds to cells adjacent to the neural crest. The expression of Slug and msx1 controls cell death in certain areas of the neural folds, and how this equilibrium is necessary to generate sharp boundaries in the neural crest territory and to precisely control cell number among neural crest derivatives is discussed (Tribulo, 2004).

FGF, WNT, and BMP signaling promote neural crest formation at the neural plate boundary in vertebrate embryos. To understand how these signals are integrated, the role of the transcription factors Msx1 and Pax3 was analyzed. Using a combination of overexpression and morpholino-mediated knockdown strategies in Xenopus, it was shown that Msx1 and Pax3 are both required for neural crest formation; they display overlapping but nonidentical activities, and Pax3 acts downstream of Msx1. In neuralized ectoderm, Msx1 is sufficient to induce multiple early neural crest genes. Msx1 induces Pax3 and ZicR1 cell autonomously, in turn, Pax3 combined with ZicR1 activates Slug in a WNT-dependent manner. Upstream of this, WNTs initiate Slug induction through Pax3 activity, whereas FGF8 induces neural crest through both Msx1 and Pax3 activities. Thus, WNT and FGF8 signals act in parallel at the neural border and converge on Pax3 activity during neural crest induction (Monsoro-Burq, 2005).

Palate development requires precise regulation of gene expression changes, morphogenetic movements and alterations in cell physiology. Defects in any of these processes can result in cleft palate, a common human birth defect. The Snail gene family encodes transcriptional repressors that play essential roles in the growth and patterning of vertebrate embryos. This study reports the functions of Snail (Snai1) and Slug (Snai2) genes during palate development in mice. Snai2-/- mice exhibit cleft palate, which is completely penetrant on a Snai1 heterozygous genetic background. Cleft palate in Snai1+/- Snai2-/- embryos is due to a failure of the elevated palatal shelves to fuse. Furthermore, while tissue-specific deletion of the Snai1 gene in neural crest cells does not cause any obvious defects, neural-crest-specific Snai1 deletion on a Snai2-/- genetic background results in multiple craniofacial defects, including a cleft palate phenotype distinct from that observed in Snai1+/- Snai2-/- embryos. In embryos with neural-crest-specific Snai1 deletion on a Snai2-/- background, palatal clefting results from a failure of Meckel's cartilage to extend the mandible and thereby allow the palatal shelves to elevate, defects similar to those seen in the Pierre Robin Sequence in humans (Murray, 2007).

The neural crest, a transient population of migratory cells, forms the craniofacial skeleton and peripheral nervous system, among other derivatives in vertebrate embryos. The transcriptional repressor Snail2 is thought to be crucial for the epithelial-to-mesenchymal transition (EMT) that promotes neural crest delamination from the neural tube; however, little is known about its downstream targets. To this end, avian Snail2 was depleted in the premigratory neural crest using morpholino antisense oligonucleotides and effects on potential targets were examined by quantitative PCR. Several dorsal neural tube genes were upregulated by alleviating Snail2 repression; moreover, the cell adhesion molecule cadherin6B was derepressed within 30 minutes of blocking Snail2 translation. Examination of the chick cadherin6B genomic sequence reveals that the regulatory region contains three pairs of clustered E boxes, representing putative Snail2 binding sites. The clustered E boxes containing the sequence CAGGTA surrounding the second exon of Cad6B, the first coding exon. In vivo and in vitro biochemical analyses demonstrate that Snail2 directly binds to these sites and regulates cadherin6B transcription. These results are the first to describe a direct target of Snail2 repression in vivo and in the context of the EMT that characterizes neural crest development (Taneyhill, 2007).

Snail family transcriptional repressors regulate epithelial mesenchymal transitions during physiological and pathological processes. A conserved SNAG repression domain present in all vertebrate Snail proteins is necessary for repressor complex assembly. This study identified the Ajuba family of LIM proteins as functional corepressors of the Snail family via an interaction with the SNAG domain. Ajuba LIM proteins interact with Snail in the nucleus on endogenous E-cadherin promoters and contribute to Snail-dependent repression of E-cadherin. Using Xenopus neural crest as a model of in vivo Snail- or Slug-induced EMT, it was demonstrate that Ajuba LIM proteins contribute to neural crest development as Snail/Slug corepressors and are required for in vivo Snail/Slug function. Because Ajuba LIM proteins are also components of adherens junctions and contribute to their assembly or stability, their functional interaction with Snail proteins in the nucleus suggests that Ajuba LIM proteins are important regulators of epithelia dynamics communicating surface events with nuclear responses (Langer, 2008).

The Snail transcription factor regulates the numbers of neural precursor cells and newborn neurons throughout mammalian life

The Snail transcription factor regulates diverse aspects of stem cell biology in organisms ranging from Drosophila to mammals. This study asked whether it regulates the biology of neural precursor cells (NPCs) in the forebrain of postnatal and adult mice, taking advantage of a mouse containing a floxed Snail allele (Snailfl/fl mice). Inducibly ablating Snail in the embryonic cortex has long-term consequences for cortical organization. In particular, when Snailfl/fl mice are crossed to Nestin-cre mice that express Cre recombinase in embryonic neural precursors, this causes inducible ablation of Snail expression throughout the postnatal cortex. This loss of Snail causes a decrease in proliferation of neonatal cortical neural precursors and mislocalization and misspecification of cortical neurons. Moreover, these precursor phenotypes persist into adulthood. Adult neural precursor cell proliferation is decreased in the forebrain subventricular zone and in the hippocampal dentate gyrus, coincident with a decrease in the number of adult-born olfactory and hippocampal neurons. Thus, Snail is a key regulator of the numbers of neural precursors and newborn neurons throughout life (Zander, 2014; PubMed).

Slug, a highly conserved zinc finger transcriptional repressor, protects hematopoietic progenitor cells from radiation-induced apoptosis

A zinc finger transcriptional repressor, Slug, which is aberrantly upregulated by the E2A-HLF oncoprotein in pro-B cell acute leukemia, functions as an antiapoptotic factor in normal hematopoietic progenitor cells. Slug-/- mice were much more radiosensitive than wild-type mice, dying earlier and showing accentuated decreases in peripheral blood cell counts, as well as abundant microhemorrhages and widely disseminated bacterial microabscesses throughout the body. Slug expression was detected in diverse subsets of hematopoietic progenitors, but not in more differentiated B and T lymphoid cells, and there was a significant increase in apoptotic (TUNEL-positive) bone marrow progenitor cells in irradiated Slug-/- mice compared to wild-type controls. These results implicate Slug in a novel survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage (Inoue, 2002).

Snail1 is a transcriptional effector of FGFR3 signaling during chondrogenesis and achondroplasias

Achondroplasias are the most common genetic forms of dwarfism in humans. They are associated with activating mutations in FGFR3, which signal through the Stat and MAPK pathways in a ligand-independent manner to impair chondrocyte proliferation and differentiation. Snail1 has been implicated in chondrocyte differentiation as it represses Collagen II and aggrecan transcription in vitro. Snail1 overexpression in the developing bone leads to achondroplasia in mice. Snail1 acts downstream of FGFR3 signaling in chondrocytes, regulating both Stat and MAPK pathways. Moreover, FGFR3 requires Snail1 during bone development and disease as the inhibition of Snail1 abolishes its signaling even through achondroplastic- and thanatophoric-activating FGFR3 forms. Significantly, Snail1 is aberrantly upregulated in thanatophoric versus normal cartilages from stillborns. Thus, Snail activity may likely be considered a target for achondroplasia therapies (de Frutos, 2007).

Snail2 and Zeb2 repress P-Cadherin to define embryonic territories in the chick embryo

Snail and Zeb (see Drosophila Snail and Zinc finger homeodomain 1) transcription factors induce epithelial to mesenchymal transition (EMT) in embryonic and adult tissues by direct repression of E-Cadherin (see Drosophila Shotgun) transcription. The repression of E-Cadherin transcription by the EMT inducers Snail1 and Zeb2 plays a fundamental role in defining embryonic territories in the mouse, as E-Cadherin needs to be downregulated in the primitive streak and in the epiblast concomitant with the formation of mesendodermal precursors and the neural plate, respectively. This study shows that in the chick embryo, E-Cadherin is weakly expressed in the epiblast at pre-primitive streak stages where it is substituted by P-Cadherin. Snail2 and Zeb2 were shown to repress P-Cadherin transcription in the primitive streak and the neural plate, respectively. This indicates that E- and P-Cadherin expression patterns evolved differently between chick and mouse. As such, the Snail1/E-Cadherin axis described in the early mouse embryo corresponds to Snail2/P-Cadherin in the chick, but both Snail factors and Zeb2 fulfill a similar role in chick and mouse in directly repressing ectodermal Cadherins to promote the delamination of mesendodermal precursors at gastrulation and the proper specification of the neural ectoderm during neural induction (Acloque, 2017).

Dynamic chromatin modification sustains epithelial-mesenchymal transition following inducible expression of Snail-1

Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT, were analyzed. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6-48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers (Javaid, 2013).

Snail and tumors

The adhesion protein E-cadherin plays a central part in the process of epithelial morphogenesis. Expression of Human Snail protein is downregulated during the acquisition of metastatic potential at late stages of epithelial tumor progression. There is evidence for a transcriptional blockage of E-cadherin gene expression in this process. Snail, which is expressed by fibroblasts and some E-cadherin-negative epithelial tumor cell lines, binds to three E-boxes present in the human E-cadherin promoter and represses transcription of E-cadherin. The repressor effect is directly associated with the presence of the Snail zinc-finger domain. The functionality of the amino-terminal SNAG domain, a short domain common to all members of the vertebrate Snail family, was examined. The SNAG domain has been shown to mediate transcriptional-repressor features of both mouse Snail and Gfi-1. Snail proteins with point mutations (Sna-P2A) or deletions of the SNAG domain do not repress E-cadherin promoter activity in reporter assays, showing that integrity of both the zinc-finger and the SNAG domains are required for Snail's repressive activity at the E-cadherin promoter. Inhibition of Snail function in epithelial cancer cell lines lacking E-cadherin protein restores the expression of the E-cadherin gene (Batlle, 2000).

The Snail zinc-finger transcription factors trigger epithelial-mesenchymal transitions (EMTs), endowing epithelial cells with migratory and invasive properties during both embryonic development and tumor progression. During EMT, Snail provokes the loss of epithelial markers, as well as changes in cell shape and the expression of mesenchymal markers. This study shows that in addition to inducing dramatic phenotypic alterations, Snail attenuates the cell cycle and confers resistance to cell death induced by the withdrawal of survival factors and by pro-apoptotic signals. Hence, Snail favors changes in cell shape versus cell division, indicating that with respect to oncogenesis, although a deregulation/increase in proliferation is crucial for tumor formation and growth, this may not be so for tumor malignization. Finally, the resistance to cell death conferred by Snail provides a selective advantage to embryonic cells to migrate and colonize distant territories, and to malignant cells to separate from the primary tumor, invade, and form metastasis (Vega, 2004).


snail: Biological Overview | Regulation | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation | References

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