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The role of Notch in patterning the human vertebral column
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The role of Notch in patterning the human vertebral column Sally L Dunwoodie1,2 The components of the Notch signaling pathway and the mechanics of signal transduction have largely been established in Drosophila. Although essential for many developmental processes in invertebrates and vertebrates, this review focuses on Notch signaling in the vertebrate-specific process of somitogenesis. More specifically it describes that mutations in genes encoding Notch pathway components (DLL3, MESP2, LFNG and HES7) cause severe congenital vertebral defects in humans. Importantly, this review highlights studies demonstrating that Dll3 is unique amongst DSL ligands acting as an inhibitor and not an activator of Notch signaling. Addresses 1 Developmental Biology Division, Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW 2010 Sydney, Australia 2 St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia
How the spatial and temporal aspects of Notch signaling, which are essential for proper somitogenesis, are controlled is the subject of considerable research. Recent findings demonstrate that Dll3 is required for the correct spatio-temporal activation of Notch1 during somitogenesis. Moreover, cell culture assays and a gene replacement strategy in mouse show that Dll3 is unique amongst DSL ligands; this mammalian-specific DSL ligand inhibits Notch signaling and unlike other DSL ligands, cannot activate signaling.
The Notch signaling pathway in mammals
The backbone is a segmented structure that provides both rigidity and flexibility to the body, and protection for the spinal cord. Its segmentation allows for a wide range of movements, and at the same time facilitates the repeated distribution of spinal axons along its length. Many and varied congenital vertebral disorders exist [1] and in 2000, mutations in the DLL3 gene linked one of these (spondylocostal dysostosis) to the Notch signaling pathway. More recently three additional components of the Notch signaling pathway have been connected to spondylocostal dysostosis. These findings are significant as they establish the genetic etiology of a complex congenital malformation, and they inform patient diagnosis, treatment and counseling.
The Notch signaling pathway is evolutionarily conserved and critical to invertebrate and vertebrate development and human disease [3–7]. Since this review focuses on the role of Notch1 signaling in somitogenesis, the Notch components most relevant to somite formation and pattering are discussed (Figure 1). In mammals there are four Notch receptors (Notch1–4), all single pass transmembrane proteins. Signaling is juxtacrine with ligand and receptor present on separate cells. Notch is synthesized as a single polypeptide and specific epidermal growth factor (EGF)-like repeats are modified by O-fucosylation in the endoplasmic reticulum by protein O-fucosyltransferase 1 (Pofut1). As Notch1 traffics through the Golgi, it may be further modified by elongation of the O-linked fucose with an N-acetylglucosamine; this is catalyzed by a fucose-specific b1,3-N-acetyl glucosaminyltransferase, encoded by the Lunatic fringe (Lfng) gene [8]. In the trans-Golgi network, Notch1 undergoes S1-cleavage into noncovalently linked extracellular Notch (ECN) and transmembrane and intracellular (TMIC) subunits. ECN and TMIC traffic to the cell surface and present as a heterodimer. Ligand binding to Notch1 in trans triggers proteolytic S2-cleavage producing the transmembrane bound fragment with the extracellular truncation (EXT) [9]. Subsequent constitutive S3-cleavage of Notch1 EXT releases the Notch1 intracellular domain (ICD) that translocates to the nucleus. Notch1 ICD forms a complex with the DNA-binding protein CSL/Rbpj converting it from a transcriptional repressor to an activator of transcription. Direct targets of Notch, relevant to somitogenesis include Lfng, Mesoderm posterior 2 (Mesp2), and hairy/enhancer-of-split 7 (Hes7). The stability of Notch1 ICD is tightly regulated by phosphorylation and ubiquitylation, making it a substrate for proteasomal degradation.
The vertebral column is derived from somites, and Notch signaling through studies in Xenopus, zebrafish, chicken and mouse is well established as a critical component of somite formation and vertebral column development [2].
There are five DSL (Delta and Serrate in Drosophila, Lag2 in Caenorhabtitis elegans) ligands in mammals; these are grouped as delta-like (Dll1, Dll3, Dll4) or serrate-like (Jagged1 and Jagged2). These ligands are also single-pass
Corresponding author: Dunwoodie, Sally L ([email protected])
Current Opinion in Genetics & Development 2009, 19:329–337 This review comes from a themed issue on Pattern formation and developmental mechanisms Edited by Kathryn Anderson and Kenneth Irvine Available online 14th July 2009 0959-437X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2009.06.005
Introduction
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Figure 1
Notch1 signaling in mammalian somitogenesis. (a) Notch1: N-terminal to the transmembrane domain there are 36 epidermal growth factor (EGF)-like repeats that are required for ligand binding, three LIN12/Notch repeats (LNR) that prevent ligand-independent signaling, and a heterodimerization domain (overlapping parallel lines) which contains the S2-cleavage site. C-terminal to the transmembrane domain there is an Rbpj/CSL-associated molecule (RAM) domain that binds Rbpj/CSL, six ankyrin (ANK) repeats which generally moderate protein interactions, two nuclear localization sequence (NLS), a transcription activation domain (TAD), and a C-terminal polypeptide enriched in proline, glutamine, serine and threonine residues (PEST) that are associated with protein degradation. Dll1: N-terminal to the transmembrane domain there is a DSL (Delta/Serrate/Lag) domain that interacts with EGF-like repeats of Notch, and eight EGF-like repeats. C-terminal to the transmembrane domain the protein is unstructured and a PDZ ligand-binding domain (PDZLDB) is located at the C-terminus. Dll3: N-terminal to the transmembrane domain there are six EGF-like repeats and Cterminal to the transmembrane domain, and the protein is unstructured. Membrane (M). (b) Notch1 is synthesized as a single polypeptide: (1) specific EGF-like repeats are modified by O-fucosylation in the endoplasmic reticulum (ER) by protein O-fucosyltransferase 1 (Pofut1). As Notch1 passes through the Golgi, (2) N-glycosylation occurs on O-fucose by Lunatic fringe (Lfng) and (3) Notch1 is S1-cleavaged by a Furin-like convertase. The ligand Dll3 is localized to the cis-Golgi. The Notch1 heterodimer consisting of N-terminal extracellular truncation (TMIC) and C-terminal transmembrane
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Notch in patterning the human vertebral column Dunwoodie 331
transmembrane proteins; generally speaking their binding of Notch in trans initiates intercellular signaling, which begins with S2-cleavage of Notch. S2-cleavage leads to shedding of the Notch ECN, and enables the DSL ligand together with the bound ECN to undergo endocytosis in the ligand-presenting, signal-sending cell [9]. Ubiquitylation, and endocytosis of DSL ligands are necessary for the efficient activation of Notch processing following trans interaction of ligand and receptor. In addition to activation of the Notch receptors, DSL ligands are capable of inhibiting signal transduction when expressed in the same cell as the receptor. This phenomenon, referred to as cis-inhibition, was first described in Drosophila where cells expressing ligand do not undergo Notch signaling but rather, they induce signaling in neighboring cells [10]. There is currently very little mechanistic understanding of how cis-inhibition occurs and there is controversy over whether the ligand and receptor interact at the cell surface (Drosophila; [11]) or inside the cell (mammalian cultured cells; [12]). The importance of cis-inhibition in vertebrates has not been investigated in vivo as it has been in Drosophila; in vertebrates it is likely to be an important means of modifying the signaling output of Notch receptors. Some recent advances in our understanding of Notch signaling with reference to somite formation concern the DSL ligand Dll3. Dll3, like Dll1, is expressed in the presomitic mesoderm and the absence of either ligand in mouse leads to severe, but distinct, defects in somitogenesis [13–15]. The different phenotypes generated in Dll1 and Dll3 null mice indicated that these proteins performed different function, but it was also possible that the two genes generated functionally equivalent ligands of Notch and that the phenotypic differences were because of the fact that Dll1 and Dll3 are differentially expressed in the presomitic mesoderm [13]. Geffers et al. [16] generated mice in which Dll1 is replaced by the Dll3 cDNA. Here, since Dll3 expression was unable to compensate for the loss of Dll1, they concluded that these two ligands are functionally distinct. This conclusion is supported by in vitro studies using a coculture system that recapitulates ligand-dependent activation of Notch signaling; ligand-expressing cells are added to Notchexpressing cells that also express a CSL-promoter reporter of Notch signaling activity [17]. Ladi and colleagues made a number of key findings; importantly they showed that Dll3, unlike Dll1 and other DSL ligands, could not activate the signaling of Notch1 or Notch2, when pre-
sented to the receptors in trans. Theoretically there are a number of possible explanations for this, but many were dispelled when they demonstrated that Dll3 does not bind Notch1 in a trans conformation. These binding studies were performed with equal amounts of soluble ligand (Dll1 or Dll3) interacting with Notch1 on the surface of cultured cells. The reason for a lack of interaction between Dll3 and Notch1 in trans was not clear as HA-tagged Dll3 was detected on the cell surface to the same extent as HA-tagged Dll1 and so its localization was not an impediment to Dll3 being able to interact with Notch in trans. They also showed that Dll3, like Dll1, when expressed in the same cells as Notch1 or Notch2 inhibited Notch signaling. This demonstrated that Dll3 is unique amongst DSL ligands, as it is unable to activate Notch signaling in trans and functions solely to inhibit Notch in cis. One reason for this uniqueness may stem from the finding that the endogenous Dll3 protein, unlike endogenous Dll1, is not localized to the cell surface in presomitic mesoderm of mouse embryos [16]; this indicates that Dll3 is not normally in a location to interact with Notch in trans. This finding also suggests that overexpression of Dll3 [17] may force the tagged protein to the cell surface, and that this is not what normally occurs. Moreover, Geffers et al. demonstrated that Dll3 is predominantly localized to the cis-Golgi [16], and it is possible that this locale is significant with respect to its function as a cis-inhibitor of Notch signaling (Figure 1b). There is clearly much to be done in order to establish how DSL ligand-dependent cis-inhibition of Notch1 occurs. One could imagine that Dll3 residing in the cis-Golgi might interfere with S1-cleavage or fucosylation of Notch1, which occurs as the receptor passes through the Golgi network. The inference here is that Notch1 would be rendered incapable of signaling as a result of altered processing or modification. This mechanism may not apply to cis-inhibition mediated by Dll1, as virtually all Dll1 is localized to the cell surface; however, it should be noted that some Dll1 localizes to the cis-Golgi [16]. A recent report by Cordle et al. has revealed that the disulfide-rich DSL domain, which defines DSL ligands, is required for both the activation of Notch in trans, and for cis-inhibition of receptor signaling in Drosophila [18]. Moreover, they identify using human peptides the interaction sites of Notch1 (EGF-like repeats 11–13) and the DSL ligand Jagged1 (DSL domain and EGF-like repeat 3). Importantly, these interaction sites are necessary for both the activation of Notch in trans and for cis-inhibition
( Figure 1 Legend continued ) and intracellular domain (ECN) traffics to the cell surface. The DSL ligand Dll1, ubiquitylated (Ub) by Mindbomb 1 (Mib1), is on the surface of the signal-sending cell. Dll1 binds Notch1 ECN in trans, and this activates S2-cleavage by the ADAM protease TACE (4). (5) Dll1 and Notch1 ECN are released and endocytosed into the signal-sending cell. (6) The membrane anchored Notch1 extracellular truncation (EXT) may undergo ubiquitylation (Ub) facilitating endocytosis. (7) Notch1 EXT undergoes S3-cleavage in the transmembrane domain. This is mediated by the gammasecretase complex and it releases Notch1 ICD. (8) Notch1 ICD enters the nucleus and binds the DNA-binding protein CSL/Rbpj, and this triggers the release of corepressor (CoR) proteins and histone deacetylases, and facilities the binding of Mastermind and coactivators (CoA) such as histone acetylases, which facilitate transcription of target genes such as Lfng, Mesp2 and Hes7. This figure was assembled using the following: ExPASy http:// www.expasy.org/sprot/, PESTfind Analysis Webtool https://emb1.bcc.univie.ac.at/toolbox/pestfind/pestfind-analysis-webtool.htm [7,47–50]. www.sciencedirect.com
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of Notch signaling in Drosophila. This finding indicates that the same surfaces of both Notch and the DSL ligand, form structurally distinct complexes, which mediate opposite effects on Notch signaling. Indeed molecular modeling supports this finding as the two protein structures can form anti-parallel and parallel conformations, mimicking receptor-ligand interaction in trans and in cis. Dll3 is highly divergent and the cysteine-rich region Nterminal to EGF-like repeat 1 lacks significant homology to the DSL domain of other DSL ligands of Notch [13] (Figure 1a); therefore it is possible that structurally this region can only support interaction with Notch in cis. Alternatively, other regions of the Dll3 protein might mediate cis-inhibition of Notch.
or a few vertebrae are affected; the other 10% of cases are manifested by multiple, contiguous vertebral anomalies. There are numerous disorders and syndromes that include AVS as a feature, and for some of these the genetic etiology has been established [1]. One of these is spondylocostal dysostosis (SCD); it is characterized by AVS throughout much of the spine and remarkably, unlike cases with AVS occurring once or twice along the vertebral column, the thoracic cage is symmetric and scoliosis is limited and nonprogressive (Figure 2b). SCD is also characterized by irregularly aligned ribs with variable points of fusion along their length [1].
Abnormal vertebral segmentation in humans
The breakthrough aligning aberrant Notch signaling with SCD came when Turnpenny et al. identified a region on chromosome 19q13.1–q13.3, which segregated with SCD in a large consanguineous kindred with seven affected individuals, using autozygosity mapping [25]. The stark similarity between the vertebral defects present in individuals with SCD and the mouse null for the DSL ligand Dll3 [26], and the fact that the syntenic region on mouse chromosome 7 contains the Dll3 gene, were the impetus for sequencing DLL3 in these individuals with SCD. Fittingly, mutations in DLL3 were identified in three pedigrees; these were homozygous in those with SCD and heterozygous or absent in unaffected family members [27]. Since then 24 cases of SCD have been attributed to the mutation of DLL3 (SCDO1: OMIM 277300) [1]. The majority of these introduce a premature stop codon. There are also five missense mutations in the EGF-like repeats and one in the transmembrane domain. These mutations have not been functionally compared with the wildtype DLL3, but are considered to be null, given the similarity in vertebral disruption observed in the Dll3 null mice [15]. Considerable progress has been made in recent years using a combination of autozygosity mapping and candidate gene sequencing, in consanguineous families; mutations of three additional genes have been identified as causative of SCD. The link to Notch was strengthened when a mutation in MESP2 was identified in an individual with SCD (SCDO2; OMIM 608681) [28]. Again the similarities between the phenotypes of Mesp2 null mice and SCD individuals were instructive [29]. The Mesp2 gene is a direct target of Notch signaling and it encodes for a bHLH transcription factor that activates the expression of at least two genes involved in somitogenesis, Lfng and Ripply2 [22,29–31] (Figure 3a). An in vitro transcription assay showed that the mutant MESP2 protein is unable to activate transcription of the mouse Lfng enhancer to the same extent as wildtype MESP2 [32].
Abnormal vertebral segmentation (AVS) occurs when somite formation is disrupted, and can be defined as altered vertebral integrity, size, shape or position. AVS is a common congenital abnormality that occurs with a prevalence of about 2-3/1000 births [24]. AVS can be divided into two main groups based on the number of vertebrae affected (Figure 2a). In some 90% of cases one
The past two years has additional more Notch-associated genes, LFNG and HES7, added to the list of those that cause SCD. Mutation of LFNG causes SCDO3 (OMIM 609813), here only a single case has been reported; the patient is homozygous for a missense mutation in a conserved amino acid (F188L) [33]. Lfng was selected
Somitogenesis and vertebral column formation The subdivided nature of the vertebral column offers postural support and flexibility; it is duly derived from the segmental arrangement of its progenitor tissues, the somites. These are paired blocks of mesoderm located on either side of the neural tube that form in a repeated manner at the rostral end of the presomitic mesoderm. This reiterative segmentation is controlled by an oscillator (segmentation clock) that generates pulses of Notch, Wnt and FGF (Fibroblast Growth Factor) signals; these are converted into the periodic formation of somite boundaries [19]. During their formation each somite is patterned into rostral and caudal halves, and it is this identity that dictates which cells of the sclerotome combine to form a vertebra [2]. Each vertebra is formed from the caudal half of one somite combining with the rostral half of the following somite; therefore, if the anteroposterior identity of somitic cells is not correctly defined, abnormal vertebra will result. Notch signaling also plays a crucial role in establishing this somite polarity and its target gene Mesp2 is central to this process [20]. Indeed, the analysis of mouse mutants has played a critical step in demonstrating the importance of Notch signaling in somite formation and patterning. To date, the following Notch pathway components (Dll1, Dll3, Lfng, Mib1, Notch1, Pofut1, Psen1, CSL/Rbpj), Notch1 ICD target genes (Hes7, Lfng, Mesp2) and target genes of Mesp2 (Tbx6, Ripply2), have been identified for their crucial role in somiogenesis [21–23]. Moreover, many of these mouse mutants have been critical in discovering gene mutations that cause congenital defects of the vertebral column in humans.
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Notch in patterning the human vertebral column Dunwoodie 333
Figure 2
Classification algorithm for abnormal vertebral segmentation. (a) A classification algorithm has been proposed by the international consortium for vertebral anomalies and scoliosis (ICVAS; http://www.icvas.org/ index.html). Abnormal vertebral segmentation (AVS), spondylocostal dysostosis (SCD), spondylothoracic dysostosis (STD); Alagille syndrome (OMIM 118450); VACTERL association (OMIM 192350); CHARGE syndrome (OMIM 214800). (b) Line diagrams (not to scale) derived from X-ray radiographs and magnetic resonance imaging (MRI) of individuals with vertebral anomalies. From the left: single AVS X-ray; SCD type 1 (DLL3) MRI; SCD type 2 (MESP2) MRI; SCD type 3 (LFNG) MRI; SCD type 4 (HES7) X-ray. Vertical line indicates thoracic vertebrae.
for sequencing on the basis that it is a target of Notch signaling, its expression is deregulated in the presomitic mesoderm of Dll3 null embryos, and Lfng null mice have SCD-like vertebral defects [15,34–36]. Lfng catalyzes the addition of N-acetylglucosamine to O-fucose on the EGFlike repeats of Notch in the Golgi [37,38]. In this capacity, Lfng enhances the Dll1-activated signaling of Notch1 in the coculture assay [39,40]. Confusingly Lfng functions to inhibit Notch1 signaling in the presomitic mesoderm; overexpression of Lfng in chicks leads to suppression of Notch signaling [41] and in mouse Notch1 activity is increased in the absence of Lfng [30] (Figure 3a). The identified human mutant protein (LFNG F188L), unlike the wildtype protein, is not localized to the Golgi and lacks enzymatic activity [33]. A mutation in HES7 was identified when autozygosity mapping in a consanguiwww.sciencedirect.com
neous family identified a 10.1 Mb run of 1081 homozygous SNPs on chromosome 17 that was present in the proband, but absent from parents and unaffected siblings. HES7 maps to this region and was a strong candidate for causing SCD in this individual as Hes7 is expressed in the presomitic mesoderm in mouse, it is a target gene of Notch signaling, and Hes7 null mice have SCD-like vertebral defects [42]. Sequencing revealed that the patient is homozygous for a missense mutation that changed a conserved amino acid [43]. The Hes7 gene encodes for a bHLH-Orange domain transcriptional repressor that represses its own transcription and that of Lfng [44,45] (Figure 3a). The mutant protein HES7 R25W, unlike the wildtype protein, is unable to repress transcription from promoters containing either N-boxes or E-boxes [43]. Current Opinion in Genetics & Development 2009, 19:329–337
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Figure 3
SCD-associated genes refine Notch1 signaling in the anterior presomitic mesoderm in mammalian somitogenesis. (a) Interaction between Notch pathway components. Dll1 activates Notch1 signaling producing N1ICD, and Dll3 inhibits Notch1 signaling [17]. N1ICD activates transcription of Mesp2 [31], Lfng [51,52] and Hes7 [44]. Hes7 protein inhibits its own transcription [44] and that of Lfng [45]. The effect of Lfng on Notch1 signaling is contradictory; it can potentiate Notch1 signaling in cultured mammalian cells [40], and inhibit signaling in the embryo [30,41]. Mesp2 protein activates the transcription of Lfng [30], Ripply2 [22] and Epha4 [53]. Ripply2 inhibits the transcription of Mesp2 [22] and Epha4 is implicated in somite border formation in zebrafish [54] but is not required for this in mouse [55,56]. (b) Upper: localization of activated Notch1 (N1ICD), proteins (Mesp2, Hes7) and transcripts (Lfng) central in mouse somitogenesis. In wildtype mouse embryos, at the anterior of the presomitic mesoderm, Notch1 signaling is active (N1ICD; green) in a narrow domain of cells. Mesp2 (blue) and Lfng (yellow) expression is coincident and occurs posterior to this domain of N1ICD [30,57]. Hes7 (red) is expressed posterior to and nonoverlappping with Mesp2 and Lfng [58]. The next somite boundary (dashed line) will form posterior to the N1ICD domain [30]. Uncx4.1 transcripts (purple) define somitic cells with caudal identity, rostral cells are defined by no Uncx4.1 expression (white). Lower: In mice null for Dll3 or Mesp2, the N1ICD domain of activated Notch1 remains broad and does not narrow [16,30]. In mice null for Lfng, Notch1 is activated (N1ICD) throughout much of the presomitic mesoderm [30]. In Hes7 null embryos, although N1ICD has not been measured directly, elevated Lfng expression throughout the presomitic mesoderm implies that Notch1 activity is absent [42]. Uncx4.1 expression, and caudal identity, is disrupted in the mouse mutants. Formed somites (S1, S2) with rostral (R) and caudal (C) identity, presomitic mesoderm with somites next to form (S0, S-1, S-2). Dashed line shows the position of the forming somite boundary. Anterior to the left.
Recently, mutation of MESP2 was also shown to cause spondylothoracic dysostosis (STD) [32]. STD, like SCD, is also an autosomal recessive disorder defined by multiple contiguous AVS (Figure 2a); STD individuals have greater truncal shortening than those with SCD. Regularly positioned ribs that are fused posteriorly at their vertebral origins distinguish STD, from SCD [1]. Cornier et al. discuss how distinct mutations in MESP2 might cause STD and SCD [32]. Not all cases of SCD are associated with mutation in DLL3, MESP2, LFNG or HES7 and so it is likely that additional genes associated with the Notch signaling Current Opinion in Genetics & Development 2009, 19:329–337
pathway might also cause SCD. This list of candidates consists of over 100 genes and so in order to prioritize, one needs to garner information about the four SCD-associated genes. Apart from the fact that these four genes are expressed in the presomitic mesoderm and are associated with Notch signaling, each has a distinct function in the signaling pathway (Figure 1). Dll3 is a ligand of Notch that inhibits receptor signaling, and Lfng is a glycosyltransferase that modifies the Notch receptor and in doing so alters its ability to be activated by ligand. Mesp2 and Hes7 are bHLH transcription factors, with Mesp2 activating gene transcription (Lfng, Ripply2 and Epha4 are direct targets), while Hes7 represses the transcription of www.sciencedirect.com
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Lfng, and through the inhibition of its own transcription sets up the oscillatory nature of Notch1 signaling in the presomitic mesoderm. Although this looks like a disparate group of proteins with distinct function, null mutation in mouse indicates that they each are required for the normal pattern of Notch1 activity in the anterior presomitic mesoderm. Normally at the anterior of the presomitic mesoderm the domain of cells that are undergoing Notch1 signaling, is broad occurring in cells of both S0 and S-1. This domain of Notch1 signaling narrows to a band of a few cells wide in S0, residing just rostral to where the next somite boundary will form [30] (Figure 3b). In Dll3 and Mesp2 null embryos the domain of cells that are undergoing Notch1 signaling remains broad and does not narrow [16,30]. In Lfng null embryos, Notch1 signaling occurs throughout the presomitic mesoderm [30], again Notch1 signaling does not get restricted in the anterior presomitic mesoderm. Although Notch1 signaling has not been directly determined in Hes7 null embryos, Lfng expression is derepressed as transcript are throughout the presomitic mesoderm [42,46]. Since constant Lfng expression in chick presomitic mesoderm phenocopies loss of Notch signaling [41], the inference here is that Notch signaling is largely lost in Hes7 null embryos. In sum, Notch1 signaling is disrupted in the presomitic mesoderm in each case and the crucial, narrow band of cells actively undergoing Notch1 signaling is absent. This impacts on both somite formation and patterning; epithelial somites fail to form adjacent to the presomitic mesoderm and cells with caudal somite identity are not localized to the caudal part of the somite. Another feature that these genes have in common is that they each in mouse are required for somitogenesis but have no other function critical for survival. This is not the case for other Notch pathway-associated genes required for somitogenesis such as Dll1, Mib1, Notch1, Pofut1 and CSL/Rbpj, as null embryos die during gestation and therefore it is unlikely that the mutation in these genes would be identified in those born with SCD. It is also possible that the mutation of genes associated with FGF and Wnt signaling could cause SCD, making the candidate list even more extensive. The same set of criteria should apply to these: expressed in presomitic mesoderm; cause somite defects in mutant mouse models; required for correct spatial activation of Notch1 signaling in the presomitic mesoderm; and not required for embryo viability.
interdependencies that exist between Notch signaling components, and also their absolute requirement in normal somitogenesis. Recent years have seen the genetic etiology of AVS disorders in humans linked to the Notch signaling pathway; impaired activities of any of four components of Notch signaling cause this debilitating birth defect. The combination of clinical genetics with molecular genetics of development in mouse has here proven to be important. Now that many critical components that refine the domain of Notch1 signaling at the anterior of the presomitic mesoderm have been defined, the onus is on developing an understanding of the molecular mechanisms that control the extent of Notch signaling. Ligand-dependent cis-inhibition of Notch signaling will prove to be central to our understanding of the control of Notch signaling, and understanding how Notch signaling is modified is of therapeutic importance in pathologies characterized by increased Notch signaling. Dll3 is likely to be crucial here as it holds the unique position among DSL ligands of Notch, as an inhibitor and not an activator of signaling.
Acknowledgements I apologize to those who have made contributions to our understanding of this research area and who were not cited in this review. I acknowledge the support of the National Health and Medical Research Council (NHMRC) via project grant 404805 and a Senior Research Fellowship.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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Conclusions The Notch signaling pathway is central to numerous developmental processes. Somite boundary formation and rostro-caudal patterning of somites relies on the tightly controlled spatio-temporal pattern of Notch1 activity. The analysis of mice lacking individual components of the Notch signaling pathway, in combination with cell culture studies, has demonstrated the complex www.sciencedirect.com
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11. Glittenberg M, Pitsouli C, Garvey C, Delidakis C, Bray S: Role of conserved intracellular motifs in Serrate signalling, cisinhibition and endocytosis. EMBO J 2006, 25:4697-4706. 12. Sakamoto K, Ohara O, Takagi M, Takeda S, Katsube K: Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev Biol 2002, 241:313-326. 13. Dunwoodie SL, Henrique D, Harrison SM, Beddington RS: Mouse Dll3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo. Development 1997, 124:3065-3076. 14. Hrabe de Angelis M, McIntyre J, 2nd, Gossler A: Maintenance of somite borders in mice requires the Delta homologue DII1. Nature 1997, 386:717-721. 15. Dunwoodie SL, Clements M, Sparrow DB, Sa X, Conlon RA, Beddington RS: Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm. Development 2002, 129:1795-1806. 16. Geffers I, Serth K, Chapman G, Jaekel R, Schuster-Gossler K, Cordes R, Sparrow DB, Kremmer E, Dunwoodie SL, Klein T et al.: Divergent functions and distinct localization of the Notch ligands DLL1 and DLL3 in vivo. J Cell Biol 2007, 178:465-476. This study shows that the Dll3 ligand of Notch is distinct from the Dll1 ligand. The Dll3 cDNA, expressed from the Dll1 locus in mouse was unable to rescue the somite defect in Dll1 null embryos. Using unique antibodies to Dll1 and Dll3, it was revealed in the mouse embryonic presomitic mesoderm that Dll1 is localized to the cell surface as expected, and Dll3 is localized to the Golgi, which was an unexpected finding. 17. Ladi E, Nichols JT, Ge W, Miyamoto A, Yao C, Yang LT, Boulter J, Sun YE, Kintner C, Weinmaster G: The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands. J Cell Biol 2005, 170:983-992. Using mammalian cells in culture it was shown that Dll3, unlike Dll1, could not bind Notch in trans and activate signaling. Instead, Dll3 acts solely as a cis-inhibitor of Notch signaling. 18. Cordle J, Johnson S, Tay JZ, Roversi P, Wilkin MB, de Madrid BH, Shimizu H, Jensen S, Whiteman P, Jin B et al.: A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition. Nat Struct Mol Biol 2008, 15:849-857. X-ray structure identifies the sites of interaction between Notch1 and Jagged1. The DSL domain and EGF-like repeat 3 of Jagged1 interact with EGF-like repeats 11–13 of Notch1. Studies in Drosophila demonstrate that this surface of Jagged1 is required to activate Notch in trans and inhibit signaling in cis. 19. Dequeant ML, Pourquie O: Segmental patterning of the vertebrate embryonic axis. Nat Rev Genet 2008, 9:370-382. 20. Saga Y: Segmental border is defined by the key transcription factor Mesp2, by means of the suppression of Notch activity. Dev Dyn 2007, 236:1450-1455. 21. Chan T, Kondow A, Hosoya A, Hitachi K, Yukita A, Okabayashi K, Nakamura H, Ozawa H, Kiyonari H, Michiue T et al.: Ripply2 is essential for precise somite formation during mouse early development. FEBS Lett 2007, 581:2691-2696. 22. Morimoto M, Sasaki N, Oginuma M, Kiso M, Igarashi K, Aizaki K, Kanno J, Saga Y: The negative regulation of Mesp2 by mouse Ripply2 is required to establish the rostro-caudal patterning within a somite. Development 2007, 134:1561-1569. 23. Kusumi K, Sewell W, O’Brien M: Mouse Mutations DIsrupting Somitogenesis and Vertebral Patterning. In Somitogenesis, vol. 638. Edited by Moroto M, Whittock N. Springer; 2009:140-156. 24. Alexander PG, Tuan RS: Carbon monoxide-induced axial skeletal dysmorphogenesis in the chick embryo. Birth Defects Res A Clin Mol Teratol 2003, 67:219-230. 25. Turnpenny PD, Bulman MP, Frayling TM, Abu-Nasra TK, Garrett C, Hattersley AT, Ellard S: A gene for autosomal recessive spondylocostal dysostosis maps to 19q13.1–q13.3. Am J Hum Genet 1999, 65:175-182. Current Opinion in Genetics & Development 2009, 19:329–337
26. Kusumi K, Sun ES, Kerrebrock AW, Bronson RT, Chi DC, Bulotsky MS, Spencer JB, Birren BW, Frankel WN, Lander ES: The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries. Nat Genet 1998, 19:274-278. 27. Bulman MP, Kusumi K, Frayling TM, McKeown C, Garrett C, Lander ES, Krumlauf R, Hattersley AT, Ellard S, Turnpenny PD: Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. Nat Genet 2000, 24:438-441. 28. Whittock NV, Sparrow DB, Wouters MA, Sillence D, Ellard S, Dunwoodie SL, Turnpenny PD: Mutated MESP2 causes spondylocostal dysostosis in humans. Am J Hum Genet 2004, 74:1249-1254. 29. Saga Y, Hata N, Koseki H, Taketo MM: Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev 1997, 11:1827-1839. 30. Morimoto M, Takahashi Y, Endo M, Saga Y: The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity. Nature 2005, 435:354-359. 31. Yasuhiko Y, Haraguchi S, Kitajima S, Takahashi Y, Kanno J, Saga Y: Tbx6-mediated Notch signaling controls somitespecific Mesp2 expression. Proc Natl Acad Sci U S A 2006, 103:3651-3656. 32. Cornier AS, Staehling-Hampton K, Delventhal KM, Saga Y, Caubet JF, Sasaki N, Ellard S, Young E, Ramirez N, Carlo SE et al.: Mutations in the MESP2 gene cause spondylothoracic dysostosis/Jarcho-Levin syndrome. Am J Hum Genet 2008, 82:1334-1341. This study shows that the mutation in MESP2 causes the congenital vertebral disorder, spondylothoracic dysostosis. This is interesting as the mutation in MESP2 also causes the related, but distinct, spondylocostal dysostosis. It is unclear, how what appear to be equal deficiencies in MESP2 transcriptional activity in vitro, can cause these distinct vertebral anomalies in humans. 33. Sparrow DB, Chapman G, Wouters MA, Whittock NV, Ellard S, Fatkin D, Turnpenny PD, Kusumi K, Sillence D, Dunwoodie SL: Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype. Am J Hum Genet 2006, 78:28-37. This study shows that mutation of LFNG, a component of the Notch signalling pathway, causes the congenital vertebral disorder, spondylocostal dysostosis. It also shows that the single amino acid change in LFNG leads to its loss of activity in Notch signalling, and that this is because of the abrogation of its glycosyltransferase activity. 34. Evrard YA, Lun Y, Aulehla A, Gan L, Johnson RL: lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 1998, 394:377-381. 35. Zhang N, Gridley T: Defects in somite formation in lunatic fringe-deficient mice. Nature 1998, 394:374-377. 36. Zhang N, Norton CR, Gridley T: Segmentation defects of Notch pathway mutants and absence of a synergistic phenotype in lunatic fringe/radical fringe double mutant mice. Genesis 2002, 33:21-28. 37. Bruckner K, Perez L, Clausen H, Cohen S: Glycosyltransferase activity of fringe modulates Notch-Delta interactions. Nature 2000, 406:411-415. 38. Moloney DJ, Panin VM, Johnston SH, Chen J, Shao L, Wilson R, Wang Y, Stanley P, Irvine KD, Haltiwanger RS et al.: Fringe is a glycosyltransferase that modifies Notch. Nature 2000, 406:369-375. 39. Hicks C, Johnston SH, diSibio G, Collazo A, Vogt TF, Weinmaster G: Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat Cell Biol 2000, 2:515-520. 40. Yang LT, Nichols JT, Yao C, Manilay JO, Robey EA, Weinmaster G: Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1. Mol Biol Cell 2005, 16:927-942. 41. Dale JK, Maroto M, Dequeant ML, Malapert P, McGrew M, Pourquie O: Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock. Nature 2003, 421:275-278. www.sciencedirect.com
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42. Bessho Y, Sakata R, Komatsu S, Shiota K, Yamada S, Kageyama R: Dynamic expression and essential functions of Hes7 in somite segmentation. Genes Dev 2001, 15:2642-2647.
51. Cole SE, Levorse JM, Tilghman SM, Vogt TF: Clock regulatory elements control cyclic expression of Lunatic fringe during somitogenesis. Dev Cell 2002, 3:75-84.
43. Sparrow DB, Guillen-Navarro E, Fatkin D, Dunwoodie SL: Mutation of Hairy-and-Enhancer-of-Split-7 in humans causes spondylocostal dysostosis. Hum Mol Genet 2008, 17:3761-3766. This study shows that mutation of HES7, a target of the Notch signalling pathway, causes the congenital vertebral disorder spondylocostal dysostosis. It also shows that the single amino acid change in HES7 leads to a loss of its transcriptional repressive activity.
52. Morales AV, Yasuda Y, Ish-Horowicz D: Periodic Lunatic fringe expression is controlled during segmentation by a cyclic transcriptional enhancer responsive to notch signaling. Dev Cell 2002, 3:63-74.
44. Bessho Y, Miyoshi G, Sakata R, Kageyama R: Hes7: a bHLH-type repressor gene regulated by Notch and expressed in the presomitic mesoderm. Genes Cells 2001, 6:175-185. 45. Chen J, Kang L, Zhang N: Negative feedback loop formed by Lunatic fringe and Hes7 controls their oscillatory expression during somitogenesis. Genesis 2005, 43:196-204. 46. Hirata H, Bessho Y, Kokubu H, Masamizu Y, Yamada S, Lewis J, Kageyama R: Instability of Hes7 protein is crucial for the somite segmentation clock. Nat Genet 2004, 36:750-754. 47. Kurooka H, Kuroda K, Honjo T: Roles of the ankyrin repeats and C-terminal region of the mouse notch1 intracellular region. Nucleic Acids Res 1998, 26:5448-5455. 48. Oberg C, Li J, Pauley A, Wolf E, Gurney M, Lendahl U: The Notch intracellular domain is ubiquitinated and negatively regulated by the mammalian Sel-10 homolog. J Biol Chem 2001, 276:35847-35853. 49. Tamura K, Taniguchi Y, Minoguchi S, Sakai T, Tun T, Furukawa T, Honjo T: Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-J kappa/ Su(H). Curr Biol 1995, 5:1416-1423. 50. Ilagan MX, Kopan R: SnapShot: notch signaling pathway. Cell 2007, 128:1246.
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53. Nakajima Y, Morimoto M, Takahashi Y, Koseki H, Saga Y: Identification of Epha4 enhancer required for segmental expression and the regulation by Mesp2. Development 2006, 133:2517-2525. 54. Barrios A, Poole RJ, Durbin L, Brennan C, Holder N, Wilson SW: Eph/Ephrin signaling regulates the mesenchymal-to-epithelial transition of the paraxial mesoderm during somite morphogenesis. Curr Biol 2003, 13:1571-1582. 55. Dottori M, Hartley L, Galea M, Paxinos G, Polizzotto M, Kilpatrick T, Bartlett PF, Murphy M, Kontgen F, Boyd AW: EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract. Proc Natl Acad Sci U S A 1998, 95:13248-13253. 56. Kullander K, Croll SD, Zimmer M, Pan L, McClain J, Hughes V, Zabski S, DeChiara TM, Klein R, Yancopoulos GD et al.: Ephrin-B3 is the midline barrier that prevents corticospinal tract axons from recrossing, allowing for unilateral motor control. Genes Dev 2001, 15:877-888. 57. Oginuma M, Niwa Y, Chapman DL, Saga Y: Mesp2 and Tbx6 cooperatively create periodic patterns coupled with the clock machinery during mouse somitogenesis. Development 2008, 135:2555-2562. 58. Bessho Y, Hirata H, Masamizu Y, Kageyama R: Periodic repression by the bHLH factor Hes7 is an essential mechanism for the somite segmentation clock. Genes Dev 2003, 17:1451-1456.
Current Opinion in Genetics & Development 2009, 19:329–337
The role of Notch in patterning the human vertebral column Sally L Dunwoodie1,2 The components of the Notch signaling pathway and the mechanics of signal transduction have largely been established in Drosophila. Although essential for many developmental processes in invertebrates and vertebrates, this review focuses on Notch signaling in the vertebrate-specific process of somitogenesis. More specifically it describes that mutations in genes encoding Notch pathway components (DLL3, MESP2, LFNG and HES7) cause severe congenital vertebral defects in humans. Importantly, this review highlights studies demonstrating that Dll3 is unique amongst DSL ligands acting as an inhibitor and not an activator of Notch signaling. Addresses 1 Developmental Biology Division, Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW 2010 Sydney, Australia 2 St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia
How the spatial and temporal aspects of Notch signaling, which are essential for proper somitogenesis, are controlled is the subject of considerable research. Recent findings demonstrate that Dll3 is required for the correct spatio-temporal activation of Notch1 during somitogenesis. Moreover, cell culture assays and a gene replacement strategy in mouse show that Dll3 is unique amongst DSL ligands; this mammalian-specific DSL ligand inhibits Notch signaling and unlike other DSL ligands, cannot activate signaling.
The Notch signaling pathway in mammals
The backbone is a segmented structure that provides both rigidity and flexibility to the body, and protection for the spinal cord. Its segmentation allows for a wide range of movements, and at the same time facilitates the repeated distribution of spinal axons along its length. Many and varied congenital vertebral disorders exist [1] and in 2000, mutations in the DLL3 gene linked one of these (spondylocostal dysostosis) to the Notch signaling pathway. More recently three additional components of the Notch signaling pathway have been connected to spondylocostal dysostosis. These findings are significant as they establish the genetic etiology of a complex congenital malformation, and they inform patient diagnosis, treatment and counseling.
The Notch signaling pathway is evolutionarily conserved and critical to invertebrate and vertebrate development and human disease [3–7]. Since this review focuses on the role of Notch1 signaling in somitogenesis, the Notch components most relevant to somite formation and pattering are discussed (Figure 1). In mammals there are four Notch receptors (Notch1–4), all single pass transmembrane proteins. Signaling is juxtacrine with ligand and receptor present on separate cells. Notch is synthesized as a single polypeptide and specific epidermal growth factor (EGF)-like repeats are modified by O-fucosylation in the endoplasmic reticulum by protein O-fucosyltransferase 1 (Pofut1). As Notch1 traffics through the Golgi, it may be further modified by elongation of the O-linked fucose with an N-acetylglucosamine; this is catalyzed by a fucose-specific b1,3-N-acetyl glucosaminyltransferase, encoded by the Lunatic fringe (Lfng) gene [8]. In the trans-Golgi network, Notch1 undergoes S1-cleavage into noncovalently linked extracellular Notch (ECN) and transmembrane and intracellular (TMIC) subunits. ECN and TMIC traffic to the cell surface and present as a heterodimer. Ligand binding to Notch1 in trans triggers proteolytic S2-cleavage producing the transmembrane bound fragment with the extracellular truncation (EXT) [9]. Subsequent constitutive S3-cleavage of Notch1 EXT releases the Notch1 intracellular domain (ICD) that translocates to the nucleus. Notch1 ICD forms a complex with the DNA-binding protein CSL/Rbpj converting it from a transcriptional repressor to an activator of transcription. Direct targets of Notch, relevant to somitogenesis include Lfng, Mesoderm posterior 2 (Mesp2), and hairy/enhancer-of-split 7 (Hes7). The stability of Notch1 ICD is tightly regulated by phosphorylation and ubiquitylation, making it a substrate for proteasomal degradation.
The vertebral column is derived from somites, and Notch signaling through studies in Xenopus, zebrafish, chicken and mouse is well established as a critical component of somite formation and vertebral column development [2].
There are five DSL (Delta and Serrate in Drosophila, Lag2 in Caenorhabtitis elegans) ligands in mammals; these are grouped as delta-like (Dll1, Dll3, Dll4) or serrate-like (Jagged1 and Jagged2). These ligands are also single-pass
Corresponding author: Dunwoodie, Sally L ([email protected])
Current Opinion in Genetics & Development 2009, 19:329–337 This review comes from a themed issue on Pattern formation and developmental mechanisms Edited by Kathryn Anderson and Kenneth Irvine Available online 14th July 2009 0959-437X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2009.06.005
Introduction
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Figure 1
Notch1 signaling in mammalian somitogenesis. (a) Notch1: N-terminal to the transmembrane domain there are 36 epidermal growth factor (EGF)-like repeats that are required for ligand binding, three LIN12/Notch repeats (LNR) that prevent ligand-independent signaling, and a heterodimerization domain (overlapping parallel lines) which contains the S2-cleavage site. C-terminal to the transmembrane domain there is an Rbpj/CSL-associated molecule (RAM) domain that binds Rbpj/CSL, six ankyrin (ANK) repeats which generally moderate protein interactions, two nuclear localization sequence (NLS), a transcription activation domain (TAD), and a C-terminal polypeptide enriched in proline, glutamine, serine and threonine residues (PEST) that are associated with protein degradation. Dll1: N-terminal to the transmembrane domain there is a DSL (Delta/Serrate/Lag) domain that interacts with EGF-like repeats of Notch, and eight EGF-like repeats. C-terminal to the transmembrane domain the protein is unstructured and a PDZ ligand-binding domain (PDZLDB) is located at the C-terminus. Dll3: N-terminal to the transmembrane domain there are six EGF-like repeats and Cterminal to the transmembrane domain, and the protein is unstructured. Membrane (M). (b) Notch1 is synthesized as a single polypeptide: (1) specific EGF-like repeats are modified by O-fucosylation in the endoplasmic reticulum (ER) by protein O-fucosyltransferase 1 (Pofut1). As Notch1 passes through the Golgi, (2) N-glycosylation occurs on O-fucose by Lunatic fringe (Lfng) and (3) Notch1 is S1-cleavaged by a Furin-like convertase. The ligand Dll3 is localized to the cis-Golgi. The Notch1 heterodimer consisting of N-terminal extracellular truncation (TMIC) and C-terminal transmembrane
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Notch in patterning the human vertebral column Dunwoodie 331
transmembrane proteins; generally speaking their binding of Notch in trans initiates intercellular signaling, which begins with S2-cleavage of Notch. S2-cleavage leads to shedding of the Notch ECN, and enables the DSL ligand together with the bound ECN to undergo endocytosis in the ligand-presenting, signal-sending cell [9]. Ubiquitylation, and endocytosis of DSL ligands are necessary for the efficient activation of Notch processing following trans interaction of ligand and receptor. In addition to activation of the Notch receptors, DSL ligands are capable of inhibiting signal transduction when expressed in the same cell as the receptor. This phenomenon, referred to as cis-inhibition, was first described in Drosophila where cells expressing ligand do not undergo Notch signaling but rather, they induce signaling in neighboring cells [10]. There is currently very little mechanistic understanding of how cis-inhibition occurs and there is controversy over whether the ligand and receptor interact at the cell surface (Drosophila; [11]) or inside the cell (mammalian cultured cells; [12]). The importance of cis-inhibition in vertebrates has not been investigated in vivo as it has been in Drosophila; in vertebrates it is likely to be an important means of modifying the signaling output of Notch receptors. Some recent advances in our understanding of Notch signaling with reference to somite formation concern the DSL ligand Dll3. Dll3, like Dll1, is expressed in the presomitic mesoderm and the absence of either ligand in mouse leads to severe, but distinct, defects in somitogenesis [13–15]. The different phenotypes generated in Dll1 and Dll3 null mice indicated that these proteins performed different function, but it was also possible that the two genes generated functionally equivalent ligands of Notch and that the phenotypic differences were because of the fact that Dll1 and Dll3 are differentially expressed in the presomitic mesoderm [13]. Geffers et al. [16] generated mice in which Dll1 is replaced by the Dll3 cDNA. Here, since Dll3 expression was unable to compensate for the loss of Dll1, they concluded that these two ligands are functionally distinct. This conclusion is supported by in vitro studies using a coculture system that recapitulates ligand-dependent activation of Notch signaling; ligand-expressing cells are added to Notchexpressing cells that also express a CSL-promoter reporter of Notch signaling activity [17]. Ladi and colleagues made a number of key findings; importantly they showed that Dll3, unlike Dll1 and other DSL ligands, could not activate the signaling of Notch1 or Notch2, when pre-
sented to the receptors in trans. Theoretically there are a number of possible explanations for this, but many were dispelled when they demonstrated that Dll3 does not bind Notch1 in a trans conformation. These binding studies were performed with equal amounts of soluble ligand (Dll1 or Dll3) interacting with Notch1 on the surface of cultured cells. The reason for a lack of interaction between Dll3 and Notch1 in trans was not clear as HA-tagged Dll3 was detected on the cell surface to the same extent as HA-tagged Dll1 and so its localization was not an impediment to Dll3 being able to interact with Notch in trans. They also showed that Dll3, like Dll1, when expressed in the same cells as Notch1 or Notch2 inhibited Notch signaling. This demonstrated that Dll3 is unique amongst DSL ligands, as it is unable to activate Notch signaling in trans and functions solely to inhibit Notch in cis. One reason for this uniqueness may stem from the finding that the endogenous Dll3 protein, unlike endogenous Dll1, is not localized to the cell surface in presomitic mesoderm of mouse embryos [16]; this indicates that Dll3 is not normally in a location to interact with Notch in trans. This finding also suggests that overexpression of Dll3 [17] may force the tagged protein to the cell surface, and that this is not what normally occurs. Moreover, Geffers et al. demonstrated that Dll3 is predominantly localized to the cis-Golgi [16], and it is possible that this locale is significant with respect to its function as a cis-inhibitor of Notch signaling (Figure 1b). There is clearly much to be done in order to establish how DSL ligand-dependent cis-inhibition of Notch1 occurs. One could imagine that Dll3 residing in the cis-Golgi might interfere with S1-cleavage or fucosylation of Notch1, which occurs as the receptor passes through the Golgi network. The inference here is that Notch1 would be rendered incapable of signaling as a result of altered processing or modification. This mechanism may not apply to cis-inhibition mediated by Dll1, as virtually all Dll1 is localized to the cell surface; however, it should be noted that some Dll1 localizes to the cis-Golgi [16]. A recent report by Cordle et al. has revealed that the disulfide-rich DSL domain, which defines DSL ligands, is required for both the activation of Notch in trans, and for cis-inhibition of receptor signaling in Drosophila [18]. Moreover, they identify using human peptides the interaction sites of Notch1 (EGF-like repeats 11–13) and the DSL ligand Jagged1 (DSL domain and EGF-like repeat 3). Importantly, these interaction sites are necessary for both the activation of Notch in trans and for cis-inhibition
( Figure 1 Legend continued ) and intracellular domain (ECN) traffics to the cell surface. The DSL ligand Dll1, ubiquitylated (Ub) by Mindbomb 1 (Mib1), is on the surface of the signal-sending cell. Dll1 binds Notch1 ECN in trans, and this activates S2-cleavage by the ADAM protease TACE (4). (5) Dll1 and Notch1 ECN are released and endocytosed into the signal-sending cell. (6) The membrane anchored Notch1 extracellular truncation (EXT) may undergo ubiquitylation (Ub) facilitating endocytosis. (7) Notch1 EXT undergoes S3-cleavage in the transmembrane domain. This is mediated by the gammasecretase complex and it releases Notch1 ICD. (8) Notch1 ICD enters the nucleus and binds the DNA-binding protein CSL/Rbpj, and this triggers the release of corepressor (CoR) proteins and histone deacetylases, and facilities the binding of Mastermind and coactivators (CoA) such as histone acetylases, which facilitate transcription of target genes such as Lfng, Mesp2 and Hes7. This figure was assembled using the following: ExPASy http:// www.expasy.org/sprot/, PESTfind Analysis Webtool https://emb1.bcc.univie.ac.at/toolbox/pestfind/pestfind-analysis-webtool.htm [7,47–50]. www.sciencedirect.com
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of Notch signaling in Drosophila. This finding indicates that the same surfaces of both Notch and the DSL ligand, form structurally distinct complexes, which mediate opposite effects on Notch signaling. Indeed molecular modeling supports this finding as the two protein structures can form anti-parallel and parallel conformations, mimicking receptor-ligand interaction in trans and in cis. Dll3 is highly divergent and the cysteine-rich region Nterminal to EGF-like repeat 1 lacks significant homology to the DSL domain of other DSL ligands of Notch [13] (Figure 1a); therefore it is possible that structurally this region can only support interaction with Notch in cis. Alternatively, other regions of the Dll3 protein might mediate cis-inhibition of Notch.
or a few vertebrae are affected; the other 10% of cases are manifested by multiple, contiguous vertebral anomalies. There are numerous disorders and syndromes that include AVS as a feature, and for some of these the genetic etiology has been established [1]. One of these is spondylocostal dysostosis (SCD); it is characterized by AVS throughout much of the spine and remarkably, unlike cases with AVS occurring once or twice along the vertebral column, the thoracic cage is symmetric and scoliosis is limited and nonprogressive (Figure 2b). SCD is also characterized by irregularly aligned ribs with variable points of fusion along their length [1].
Abnormal vertebral segmentation in humans
The breakthrough aligning aberrant Notch signaling with SCD came when Turnpenny et al. identified a region on chromosome 19q13.1–q13.3, which segregated with SCD in a large consanguineous kindred with seven affected individuals, using autozygosity mapping [25]. The stark similarity between the vertebral defects present in individuals with SCD and the mouse null for the DSL ligand Dll3 [26], and the fact that the syntenic region on mouse chromosome 7 contains the Dll3 gene, were the impetus for sequencing DLL3 in these individuals with SCD. Fittingly, mutations in DLL3 were identified in three pedigrees; these were homozygous in those with SCD and heterozygous or absent in unaffected family members [27]. Since then 24 cases of SCD have been attributed to the mutation of DLL3 (SCDO1: OMIM 277300) [1]. The majority of these introduce a premature stop codon. There are also five missense mutations in the EGF-like repeats and one in the transmembrane domain. These mutations have not been functionally compared with the wildtype DLL3, but are considered to be null, given the similarity in vertebral disruption observed in the Dll3 null mice [15]. Considerable progress has been made in recent years using a combination of autozygosity mapping and candidate gene sequencing, in consanguineous families; mutations of three additional genes have been identified as causative of SCD. The link to Notch was strengthened when a mutation in MESP2 was identified in an individual with SCD (SCDO2; OMIM 608681) [28]. Again the similarities between the phenotypes of Mesp2 null mice and SCD individuals were instructive [29]. The Mesp2 gene is a direct target of Notch signaling and it encodes for a bHLH transcription factor that activates the expression of at least two genes involved in somitogenesis, Lfng and Ripply2 [22,29–31] (Figure 3a). An in vitro transcription assay showed that the mutant MESP2 protein is unable to activate transcription of the mouse Lfng enhancer to the same extent as wildtype MESP2 [32].
Abnormal vertebral segmentation (AVS) occurs when somite formation is disrupted, and can be defined as altered vertebral integrity, size, shape or position. AVS is a common congenital abnormality that occurs with a prevalence of about 2-3/1000 births [24]. AVS can be divided into two main groups based on the number of vertebrae affected (Figure 2a). In some 90% of cases one
The past two years has additional more Notch-associated genes, LFNG and HES7, added to the list of those that cause SCD. Mutation of LFNG causes SCDO3 (OMIM 609813), here only a single case has been reported; the patient is homozygous for a missense mutation in a conserved amino acid (F188L) [33]. Lfng was selected
Somitogenesis and vertebral column formation The subdivided nature of the vertebral column offers postural support and flexibility; it is duly derived from the segmental arrangement of its progenitor tissues, the somites. These are paired blocks of mesoderm located on either side of the neural tube that form in a repeated manner at the rostral end of the presomitic mesoderm. This reiterative segmentation is controlled by an oscillator (segmentation clock) that generates pulses of Notch, Wnt and FGF (Fibroblast Growth Factor) signals; these are converted into the periodic formation of somite boundaries [19]. During their formation each somite is patterned into rostral and caudal halves, and it is this identity that dictates which cells of the sclerotome combine to form a vertebra [2]. Each vertebra is formed from the caudal half of one somite combining with the rostral half of the following somite; therefore, if the anteroposterior identity of somitic cells is not correctly defined, abnormal vertebra will result. Notch signaling also plays a crucial role in establishing this somite polarity and its target gene Mesp2 is central to this process [20]. Indeed, the analysis of mouse mutants has played a critical step in demonstrating the importance of Notch signaling in somite formation and patterning. To date, the following Notch pathway components (Dll1, Dll3, Lfng, Mib1, Notch1, Pofut1, Psen1, CSL/Rbpj), Notch1 ICD target genes (Hes7, Lfng, Mesp2) and target genes of Mesp2 (Tbx6, Ripply2), have been identified for their crucial role in somiogenesis [21–23]. Moreover, many of these mouse mutants have been critical in discovering gene mutations that cause congenital defects of the vertebral column in humans.
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Figure 2
Classification algorithm for abnormal vertebral segmentation. (a) A classification algorithm has been proposed by the international consortium for vertebral anomalies and scoliosis (ICVAS; http://www.icvas.org/ index.html). Abnormal vertebral segmentation (AVS), spondylocostal dysostosis (SCD), spondylothoracic dysostosis (STD); Alagille syndrome (OMIM 118450); VACTERL association (OMIM 192350); CHARGE syndrome (OMIM 214800). (b) Line diagrams (not to scale) derived from X-ray radiographs and magnetic resonance imaging (MRI) of individuals with vertebral anomalies. From the left: single AVS X-ray; SCD type 1 (DLL3) MRI; SCD type 2 (MESP2) MRI; SCD type 3 (LFNG) MRI; SCD type 4 (HES7) X-ray. Vertical line indicates thoracic vertebrae.
for sequencing on the basis that it is a target of Notch signaling, its expression is deregulated in the presomitic mesoderm of Dll3 null embryos, and Lfng null mice have SCD-like vertebral defects [15,34–36]. Lfng catalyzes the addition of N-acetylglucosamine to O-fucose on the EGFlike repeats of Notch in the Golgi [37,38]. In this capacity, Lfng enhances the Dll1-activated signaling of Notch1 in the coculture assay [39,40]. Confusingly Lfng functions to inhibit Notch1 signaling in the presomitic mesoderm; overexpression of Lfng in chicks leads to suppression of Notch signaling [41] and in mouse Notch1 activity is increased in the absence of Lfng [30] (Figure 3a). The identified human mutant protein (LFNG F188L), unlike the wildtype protein, is not localized to the Golgi and lacks enzymatic activity [33]. A mutation in HES7 was identified when autozygosity mapping in a consanguiwww.sciencedirect.com
neous family identified a 10.1 Mb run of 1081 homozygous SNPs on chromosome 17 that was present in the proband, but absent from parents and unaffected siblings. HES7 maps to this region and was a strong candidate for causing SCD in this individual as Hes7 is expressed in the presomitic mesoderm in mouse, it is a target gene of Notch signaling, and Hes7 null mice have SCD-like vertebral defects [42]. Sequencing revealed that the patient is homozygous for a missense mutation that changed a conserved amino acid [43]. The Hes7 gene encodes for a bHLH-Orange domain transcriptional repressor that represses its own transcription and that of Lfng [44,45] (Figure 3a). The mutant protein HES7 R25W, unlike the wildtype protein, is unable to repress transcription from promoters containing either N-boxes or E-boxes [43]. Current Opinion in Genetics & Development 2009, 19:329–337
334 Pattern formation and developmental mechanisms
Figure 3
SCD-associated genes refine Notch1 signaling in the anterior presomitic mesoderm in mammalian somitogenesis. (a) Interaction between Notch pathway components. Dll1 activates Notch1 signaling producing N1ICD, and Dll3 inhibits Notch1 signaling [17]. N1ICD activates transcription of Mesp2 [31], Lfng [51,52] and Hes7 [44]. Hes7 protein inhibits its own transcription [44] and that of Lfng [45]. The effect of Lfng on Notch1 signaling is contradictory; it can potentiate Notch1 signaling in cultured mammalian cells [40], and inhibit signaling in the embryo [30,41]. Mesp2 protein activates the transcription of Lfng [30], Ripply2 [22] and Epha4 [53]. Ripply2 inhibits the transcription of Mesp2 [22] and Epha4 is implicated in somite border formation in zebrafish [54] but is not required for this in mouse [55,56]. (b) Upper: localization of activated Notch1 (N1ICD), proteins (Mesp2, Hes7) and transcripts (Lfng) central in mouse somitogenesis. In wildtype mouse embryos, at the anterior of the presomitic mesoderm, Notch1 signaling is active (N1ICD; green) in a narrow domain of cells. Mesp2 (blue) and Lfng (yellow) expression is coincident and occurs posterior to this domain of N1ICD [30,57]. Hes7 (red) is expressed posterior to and nonoverlappping with Mesp2 and Lfng [58]. The next somite boundary (dashed line) will form posterior to the N1ICD domain [30]. Uncx4.1 transcripts (purple) define somitic cells with caudal identity, rostral cells are defined by no Uncx4.1 expression (white). Lower: In mice null for Dll3 or Mesp2, the N1ICD domain of activated Notch1 remains broad and does not narrow [16,30]. In mice null for Lfng, Notch1 is activated (N1ICD) throughout much of the presomitic mesoderm [30]. In Hes7 null embryos, although N1ICD has not been measured directly, elevated Lfng expression throughout the presomitic mesoderm implies that Notch1 activity is absent [42]. Uncx4.1 expression, and caudal identity, is disrupted in the mouse mutants. Formed somites (S1, S2) with rostral (R) and caudal (C) identity, presomitic mesoderm with somites next to form (S0, S-1, S-2). Dashed line shows the position of the forming somite boundary. Anterior to the left.
Recently, mutation of MESP2 was also shown to cause spondylothoracic dysostosis (STD) [32]. STD, like SCD, is also an autosomal recessive disorder defined by multiple contiguous AVS (Figure 2a); STD individuals have greater truncal shortening than those with SCD. Regularly positioned ribs that are fused posteriorly at their vertebral origins distinguish STD, from SCD [1]. Cornier et al. discuss how distinct mutations in MESP2 might cause STD and SCD [32]. Not all cases of SCD are associated with mutation in DLL3, MESP2, LFNG or HES7 and so it is likely that additional genes associated with the Notch signaling Current Opinion in Genetics & Development 2009, 19:329–337
pathway might also cause SCD. This list of candidates consists of over 100 genes and so in order to prioritize, one needs to garner information about the four SCD-associated genes. Apart from the fact that these four genes are expressed in the presomitic mesoderm and are associated with Notch signaling, each has a distinct function in the signaling pathway (Figure 1). Dll3 is a ligand of Notch that inhibits receptor signaling, and Lfng is a glycosyltransferase that modifies the Notch receptor and in doing so alters its ability to be activated by ligand. Mesp2 and Hes7 are bHLH transcription factors, with Mesp2 activating gene transcription (Lfng, Ripply2 and Epha4 are direct targets), while Hes7 represses the transcription of www.sciencedirect.com
Notch in patterning the human vertebral column Dunwoodie 335
Lfng, and through the inhibition of its own transcription sets up the oscillatory nature of Notch1 signaling in the presomitic mesoderm. Although this looks like a disparate group of proteins with distinct function, null mutation in mouse indicates that they each are required for the normal pattern of Notch1 activity in the anterior presomitic mesoderm. Normally at the anterior of the presomitic mesoderm the domain of cells that are undergoing Notch1 signaling, is broad occurring in cells of both S0 and S-1. This domain of Notch1 signaling narrows to a band of a few cells wide in S0, residing just rostral to where the next somite boundary will form [30] (Figure 3b). In Dll3 and Mesp2 null embryos the domain of cells that are undergoing Notch1 signaling remains broad and does not narrow [16,30]. In Lfng null embryos, Notch1 signaling occurs throughout the presomitic mesoderm [30], again Notch1 signaling does not get restricted in the anterior presomitic mesoderm. Although Notch1 signaling has not been directly determined in Hes7 null embryos, Lfng expression is derepressed as transcript are throughout the presomitic mesoderm [42,46]. Since constant Lfng expression in chick presomitic mesoderm phenocopies loss of Notch signaling [41], the inference here is that Notch signaling is largely lost in Hes7 null embryos. In sum, Notch1 signaling is disrupted in the presomitic mesoderm in each case and the crucial, narrow band of cells actively undergoing Notch1 signaling is absent. This impacts on both somite formation and patterning; epithelial somites fail to form adjacent to the presomitic mesoderm and cells with caudal somite identity are not localized to the caudal part of the somite. Another feature that these genes have in common is that they each in mouse are required for somitogenesis but have no other function critical for survival. This is not the case for other Notch pathway-associated genes required for somitogenesis such as Dll1, Mib1, Notch1, Pofut1 and CSL/Rbpj, as null embryos die during gestation and therefore it is unlikely that the mutation in these genes would be identified in those born with SCD. It is also possible that the mutation of genes associated with FGF and Wnt signaling could cause SCD, making the candidate list even more extensive. The same set of criteria should apply to these: expressed in presomitic mesoderm; cause somite defects in mutant mouse models; required for correct spatial activation of Notch1 signaling in the presomitic mesoderm; and not required for embryo viability.
interdependencies that exist between Notch signaling components, and also their absolute requirement in normal somitogenesis. Recent years have seen the genetic etiology of AVS disorders in humans linked to the Notch signaling pathway; impaired activities of any of four components of Notch signaling cause this debilitating birth defect. The combination of clinical genetics with molecular genetics of development in mouse has here proven to be important. Now that many critical components that refine the domain of Notch1 signaling at the anterior of the presomitic mesoderm have been defined, the onus is on developing an understanding of the molecular mechanisms that control the extent of Notch signaling. Ligand-dependent cis-inhibition of Notch signaling will prove to be central to our understanding of the control of Notch signaling, and understanding how Notch signaling is modified is of therapeutic importance in pathologies characterized by increased Notch signaling. Dll3 is likely to be crucial here as it holds the unique position among DSL ligands of Notch, as an inhibitor and not an activator of signaling.
Acknowledgements I apologize to those who have made contributions to our understanding of this research area and who were not cited in this review. I acknowledge the support of the National Health and Medical Research Council (NHMRC) via project grant 404805 and a Senior Research Fellowship.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
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