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Evolutionary Scenarios of Notch Proteins

Evolutionary Scenarios of Notch Proteins Abstract Notch is a highly conserved family of transmembrane receptors and transcription factors that are key players in several developmental processes. In this study, we identified novel Notch sequences from various species covering from worm to human and conducted a comprehensive phylogenetic analysis in order to confirm and extend the evolutionary history of Notch. Our findings confirm an independent duplication event in Caenorhabditis elegans resulting in two Notch genes and show that the vertebrate Notch genes resulted from two duplication events, both of which occurred before the divergence of teleosts and tetrapoda. Furthermore, we demonstrate that the vertebrate Notch2 group is phylogenetically closer to Notch3 and that Notch2 appeared at the first round of vertebrate duplication events. Moreover, there is evidence that the two Notch1 genes in fish, appeared by a recent duplication of Notch1 in teleost after the divergence of teleost and tetrapoda. Whether this is from ancient whole genome duplication (WGD) or gene duplication remains to be elucidated. The fourth group of Notch (Notch4) was found only in mammals. We suggest two possible scenarios for the origin of the Notch4 subfamily: 1) Notch4 appeared at the time of the two WGDs in the early chordate but has been maintained only in the mammalian lineage and was lost in the other lineages, 2) a recent independent duplication event took place in the mammalian lineage. The increase of the sequencing data from Xenopus tropicalis, Gallus gallus genome projects and of other avian and reptile genomes will shed more light on this event. Nevertheless, the great divergence of Notch4, from the other three Notch genes, suggests a rapid divergence raising questions about the functional implication of this event. In addition, comparison of the organization of Notch syntenic genes among species supports the coordinated rearrangements during evolution for Nοtch, PBX, and BRD families that may lead to possible functional relationships. Notch, homologs, evolution, synteny Introduction Notch genes found both in vertebrate and invertebrate species represent a highly studied family of receptors that act also as transcription coactivators. They represent the principal molecules of the Notch signaling pathway that regulates cellular interactions, proliferation, differentiation, cell fate, apoptosis, and embryonic development (Weinmaster et al. 1991; Chitnis 1995; de la Pompa et al. 1997). The Notch gene was named after an irregular structure of the wing blades in Drosophila melanogaster (Mohr 1919), and the genomic locus responsible for this phenotype was reported to be involved in the embryonic development (Poulson 1940). The first Notch gene was cloned in 1983 (Artavanis-Tsakonas et al. 1983) and was shown to encode a cell-surface receptor (Wharton et al. 1985). Genomic analysis in this area revealed that Notch is important for the cell fate during development of D. melanogaster (Artavanis-Tsakonas et al. 1991; Artavanis-Tsakonas and Simpson 1991). Moreover, two Notch genes (glp-1 and lin-12) have been identified in Caenorhabditis elegans (Greenwald et al. 1983; Austin and Kimble 1987; Priess et al. 1987; Schnabel 1994). In the vertebrates, an increased number of Notch genes were identified (Ellisen et al. 1991; Weinmaster et al. 1992; Lardelli et al. 1994; Uyttendaele et al. 1996). Table 1 summarizes the species in which a Notch representative was reported with sequence analysis or functional studies. Table 1 Notch Genes Identified from Sequence and Functional Studies Species  Na  SA  SA-Referencesb  FS  FS-Referencesb  Drosophila melanogaster  N  +  Wharton et al. (1985)  +  Wharton et al. (1985)  Aedes aegypti  N  +  Nene et al. (2007)  —  —  Caenorhabditis elegans  glp-1  +  Yochem and Greenwald (1989)  +  Lambie and Kimble (1991)  lin-12  +  Yochem et al. (1988)  +  Lambie and Kimble (1991)  Ciona intestinalis  N  +  Satou et al. (2002)  —  —  Lytechinus variegatus  N  +  Sherwood and McClay (1997)  +  Sherwood and McClay (1997)  Branchiostoma floridae  N  +  Holland et al. (2001)  +  Holland et al. (2001)  Danio rerio  N1a  +  Bierkamp and Campos-Ortega (1993)  +  Bierkamp and Campos-Ortega (1993)  N1b  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N2  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N3  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  Xenopus tropicalis  N1  +  Klein et al. (2002)  +  Ogino et al. (2008)  Xenopus laevis  N1  +  Coffman et al. (1990)  +  Coffman et al. (1990)  Mus musculus  N1  +  del Amo et al. (1993)  +  Swiatek et al. (1994)  N2  +  Lardelli and Lendahl (1993)  +  Lardelli and Lendahl (1993)  N3  +  Lardelli et al. (1994)  +  Lardelli et al. (1994)  N4  +  Robbins et al. (1992)    Robbins et al. 1992)  Rattus norvegicus  N1  +  Weinmaster et al. (1991)  +  Weinmaster et al. (1991)  N2  +  Weinmaster et al. (1992)  +  Weinmaster et al. (1992)  N3  +  Tanigaki et al. (2001)  +  Lindsell et al. (1996)  N4  +  Uyttendaele et al. (2000)  +  Uyttendaele et al. (2000)  Homo sapiens  N1  +  Ellisen et al. (1991)  +  Aster et al. (1994)  N2  +  Stifani et al. (1992)  +  Hsieh et al. (1997)  N3  +  Tournier-Lasserve et al. (1993)  +  Felli et al. (1999)  N4  +  Sugaya et al. (1994)  +  Uyttendaele et al. (1998)  Macaca fascicularis  N1  +  Wang et al. (2007)  —  —  N2  +  Wang et al. (2007)  —  —  Species  Na  SA  SA-Referencesb  FS  FS-Referencesb  Drosophila melanogaster  N  +  Wharton et al. (1985)  +  Wharton et al. (1985)  Aedes aegypti  N  +  Nene et al. (2007)  —  —  Caenorhabditis elegans  glp-1  +  Yochem and Greenwald (1989)  +  Lambie and Kimble (1991)  lin-12  +  Yochem et al. (1988)  +  Lambie and Kimble (1991)  Ciona intestinalis  N  +  Satou et al. (2002)  —  —  Lytechinus variegatus  N  +  Sherwood and McClay (1997)  +  Sherwood and McClay (1997)  Branchiostoma floridae  N  +  Holland et al. (2001)  +  Holland et al. (2001)  Danio rerio  N1a  +  Bierkamp and Campos-Ortega (1993)  +  Bierkamp and Campos-Ortega (1993)  N1b  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N2  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N3  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  Xenopus tropicalis  N1  +  Klein et al. (2002)  +  Ogino et al. (2008)  Xenopus laevis  N1  +  Coffman et al. (1990)  +  Coffman et al. (1990)  Mus musculus  N1  +  del Amo et al. (1993)  +  Swiatek et al. (1994)  N2  +  Lardelli and Lendahl (1993)  +  Lardelli and Lendahl (1993)  N3  +  Lardelli et al. (1994)  +  Lardelli et al. (1994)  N4  +  Robbins et al. (1992)    Robbins et al. 1992)  Rattus norvegicus  N1  +  Weinmaster et al. (1991)  +  Weinmaster et al. (1991)  N2  +  Weinmaster et al. (1992)  +  Weinmaster et al. (1992)  N3  +  Tanigaki et al. (2001)  +  Lindsell et al. (1996)  N4  +  Uyttendaele et al. (2000)  +  Uyttendaele et al. (2000)  Homo sapiens  N1  +  Ellisen et al. (1991)  +  Aster et al. (1994)  N2  +  Stifani et al. (1992)  +  Hsieh et al. (1997)  N3  +  Tournier-Lasserve et al. (1993)  +  Felli et al. (1999)  N4  +  Sugaya et al. (1994)  +  Uyttendaele et al. (1998)  Macaca fascicularis  N1  +  Wang et al. (2007)  —  —  N2  +  Wang et al. (2007)  —  —  SA—Sequence Analysis and FS—Functional studies. a  Notch genes. b  For some species, there are too many references; therefore, we apologize to the authors that their work was not referenced here. View Large As in all multigene vertebrate families, the increased number of Notch homologs in vertebrates is believed to be the result of duplication events (Westin and Lardelli 1997). Furthermore human Notch1, 2, and 3 genes were found in syntenic regions (Larsson et al. 1994) giving evidence that they appeared from two rounds of genome duplication during metazoan evolution. Nevertheless, the increased number of Notch signaling pathways in vertebrate species was suggested to be necessary for the additional complexity of the body plan (Kortschak et al. 2001). However, in C. elegans, which is an organism with lower functional and structural complexity than Drosophila, two Notch genes were found and are believed to be the result of an independent duplication event (Maine et al. 1995). Phylogenetic analysis of vertebrate Notch proteins suggested that Notch1a and Notch1b resulted from a duplication near teleost/mammalian divergence (Kortschak et al. 2001). The same study reports evidence that Notch4 in mammals is the result of a rapid divergence from Notch3. A limiting factor in previous phylogenetic studies on Notch genes (Maine et al. 1995; Kortschak et al. 2001) was the low number of examined species, due to the lack of fully sequenced genomes. The availability of sequenced genomes of various species may allow a more comprehensive investigation on the evolution of Notch. In addition, the availability of amphioxus, chicken and reptile Notch genes may allow us to fill the gaps of the previously described scenarios. Amphioxus was reported to be the closest living invertebrate to the vertebrates (Simmen et al. 1998; Furlong and Holland 2002) although this has its opponents (Delsuc et al. 2006). Nevertheless, its draft assembly genome has been available recently (Putnam et al. 2008) and is a valuable source for further studies. Therefore, how many Notch genes can we identify on its genome? Does the topology of previously described Notch trees change with the inclusion of this lineage? Previous analyses on Notch genes were focused only on model fish organisms like zebrafish (Westin and Lardelli 1997; Kortschak et al. 2001). Do all fish have two Notch1 genes? It has been previously reported that this duplication was around the time of teleost/mammalian divergence (Kortschak et al. 2001). Can we specify if the duplication occurred before or after the divergence? Can we specify if the two copies were the result of genome duplication or simple gene duplication? Do avian and reptile species have all four Notch duplicates like mammals or do they have two copies of Notch1 and no Notch4 like fish? Do all vertebrates have a Notch3 gene? Which species do possess a Notch4 gene and what is its origin? A final question to be looked into is if there are relationships between Notch and genes found syntenic to Notch from worm to human. This will help us identify potential mechanisms concerning their expansion and possibly predict coevolutionary events with functional implications. Materials and Methods Homolog Identification Notch protein homologs were identified with the bidirectional best hit method (Hirsh and Fraser 2001; Jordan et al. 2002), where orthologs are determined if two proteins in a different proteome find each other as the best hit in the other proteome. Searches were made through the NCBI (www.ncbi.nlm.nih.gov) and Ensembl (www.ensembl.org; release 46) protein databases. The four human proteins, (Nocth1: ENSP00000277541; Notch2: ENSP00000256646; Notch3: ENSP00000263388; and Notch4: ENSP00000364163) were first retrieved and afterward used as reference for the query of protein databases. Threshold of searching was set to 50% identity. Species searched were (in bold species where there are no published reports describing analysis of Notch genes): Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Bos taurus,Canis familiaris, Felis catus, Cavia porcellus, Loxodonta africana, Erinaceus europeus, Monodelphis domestica,Dasypus novemcinctus, Ornithorynhus anatinus, Oryctolagus cuniculus, Otolemur garnettii, Spermophilus tridecemlineatus, Tupaia belangeri, Myotis lucigucus, Sorex araneus, Echinops telfairi, Gallus gallus, Anolis carolinensis, Xenopus tropicalis, Danio rerio, Gasterosteus aculeatus, Tetraodon nigroviridis, Oryzias latipes, Takifugu rubripes, Branchiostoma floridae, Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, Ciona savignyi, Ciona intestinalis, Lytechinus variegatus, and Caenorhabditis elegans. Extensive genomic repositories including mRNA, cDNAs, and expressed sequence tags (ESTs) were searched using BlastN and TBlastN algorithm, as well as species-specific genomic databases when appropriate (see supplementary table ST1, Supplementary Material online, for details in genomic searches). In addition, in silico proteins were obtained using CAP3 program (Huang and Madan 1999) or Wise2 (Birney et al. 2004) and when this was not possible, ab initio cDNAs were retrieved from Ensembl. Multiple Sequence Alignments and Phylogenetic Analysis The Notch protein sequences accession numbers and identifiers are shown in supplementary table ST2, Supplementary Material online. After the exclusion of the in silico predicted, partial proteins, and those annotated as “novel projection” in Ensembl, we obtained a data set of 87 sequences (shown in bold in supplementary table ST2, Supplementary Material online). Full-length alignments were constructed using the MUSCLE program (version 3.7) (Edgar 2004) available at European Bioinformatics Institute (http://www.ebi.ac.uk/; version 3.6). The alignments were manually edited with BioEdit (version 7.0.9.0) (Tippmann 2004) for optimization. Evolutionary relationships were inferred using the Neighbor-Joining (NJ) and maximum parsimony (MP) methods implemented in the PHYLIP package (http://evolution.gs.washington.edu/phylip.html; release 3.67) (Felsenstein 1996) and maximum likelihood (ML) method implemented in PHYML (version 3.0) (Guindon et al. 2005). For the NJ method, a distance matrix was first calculated by Protdist program based on the multiple sequence alignment. The matrix was transformed to an unrooted tree under the evolutionary model Jones–Taylor–Thornton using the Neighbor program. For the character-based MP method, the unrooted tree was created with Protpars, whereas for the ML method, as in NJ, the tree was generated under the evolutionary model of Jones–Taylor–Thornton. The phylogenetic trees were viewed with the TreeExplorer program in MEGA 4 package (Kumar et al. 2008), whereas the robustness of the inferred trees of NJ and MP was tested by bootstrapping. The consensus trees were deduced from 100 trials. For PHYML method, approximate likelihood ratio test was used for branch support. Additionally, to examine the evolution and occurrence of Notch4 group and to check which of the groups Notch2 and Notch3 is closer to the clade Notch1, individual multiple alignments were constructed with MUSCLE for whole length, Notch intracellular region coding domain (NICD) and ankyrin repeat region (ANK). Phylogenetic analysis was conducted with PHYML using human proteins Notch1, Notch2, Notch3, Notch4, and Drosophila Notch protein as an outgroup. Genomic Positions of Notch and Neighboring Genes For the identification of the chromosomal location of the Notch genes, and their neighboring genes in H. sapiens, C. intestinalis, D. melanogaster, and C. elegans a text-based query was conducted using NCBI Map Viewer and GeneView of Ensembl. Neighboring genes were chosen from the syntenic regions surrounding the Notch genes of human and mouse, and the corresponding homologous neighboring genes in other species were identified using BlastP and TBlastN. Results Notch Orthologs and Paralogs One hundred and four (104) Notch homologous sequences were identified for 37 eukaryotic species (see supplementary table ST2, Supplementary Material online). In the mammalian species H. sapiens, M. mulatta, P. troglodytes, M. musculus, R. norvegicus, C. familiaris, and M. domestica, four copies of Notch homologs were identified. Nevertheless, for other mammals, characterized by a lower coverage of sequenced genome (1.5–2×), we could not identify all full-length copies (for details see supplementary table ST2, Supplementary Material online) and for some of them ESTs or cDNAs were identified (supplementary table ST1, Supplementary Material online). The G. gallus genome is the only sequenced genome of birds today. Besides the absence of Notch4, Notch3 was not detected either. In addition, even if the chicken genome has been sequenced to a great extent (7.1×), several genes from the Notch3 syntenic area of human appear to be absent as well. However, the detection of this gene in frog, reptile, and fish species, the still draft genome assembly of G. gallus, along with its unsequenced microchromosomes (Costantini et al. 2007), do not allow us to exclude Notch3 from G. gallus. In the genome of frog, X. tropicalis, the homologous sequences of Notch1, 2, and 3 were detected, whereas Notch4 sequence was once more missing. The accuracy of this finding is quite reliable if we consider the high coverage of sequenced genome (7.65×). Furthermore, in the recent draft assembly (6.3×) of A. carolinensis, it was possible to identify the Notch1 and Notch3 genes, which were previously not reported. In the fish genomes, included in this analysis (D. rerio, T. nigroviridis, T. rubripes, O. latipes, and G. aculeatus), two genes for Notch1 (Notch1a and Notch1b), one for Notch2 and Notch3 were detected, whereas Notch4 was once again absent. Finally, concerning those species distantly related to vertebrates, like insects, Ciona, sea urchin, and amphioxus, only one Notch gene was identified, whereas in C. elegans two were identified as previously described (Lambie and Kimble 1991). Table 2 shows the protein identity among species from representative Notch sequences. Table 2 Identities of Notch Proteins Based on Similarity Searches   N2_H.sa  N3_H.sa  N4_H.sa  B.flo  L.va  A.ae  A.ga  D.me  C.int  C.ele_lin12  C.ele_glp1  N1_H.sa  55  45  37  51  43  49  49  48  46  29  29  N2_H.sa    45  35  46  41  48  49  48  46  29  25  N3_H.sa      35  45  39  44  44  43  40  28  28  N4_H.sa        34  37  34  35  39  34  31  27  B.flo          44  49  49  47  45  25  29  L.va            45  45  44  39  29  30  A.ae              80  64  43  27  28  A.ga                69  44  29  27  D.me                  41  29  29  C.int                    30  32  C.ele_lin12                      46    N2_H.sa  N3_H.sa  N4_H.sa  B.flo  L.va  A.ae  A.ga  D.me  C.int  C.ele_lin12  C.ele_glp1  N1_H.sa  55  45  37  51  43  49  49  48  46  29  29  N2_H.sa    45  35  46  41  48  49  48  46  29  25  N3_H.sa      35  45  39  44  44  43  40  28  28  N4_H.sa        34  37  34  35  39  34  31  27  B.flo          44  49  49  47  45  25  29  L.va            45  45  44  39  29  30  A.ae              80  64  43  27  28  A.ga                69  44  29  27  D.me                  41  29  29  C.int                    30  32  C.ele_lin12                      46  Only representative sequences are used. NOTE.—Identity numbers were extracted from a Blast output after an all-to-all comparison of Notch sequences. Abbreviations: N1_H.sa, N1_Homo sapiens; N2_H.sa, N2_Homo sapiens; N3_H.sa, N3_Homo sapiens; N4_H.sa, N4_Homo sapiens; B.flo, Branchiostoma floridae; L.va, Lytechinus variegatus; A.ae, Aedes aegypti; A.ga Anopheles gambiae; D.me, Drosophila melanogaster; C.in, Ciona intestinalis; C.ele_lin12, Caenorhabditis elegans_lin12; C.ele_glp-1, C.elegans_glp1. View Large Evolution of Notch Family The evolutionary relationships of the Notch protein family were inferred through distance-NJ, MP, and ML methods. The unrooted phylogenetic trees generated from each method, are shown in figure 1, supplementary figures S1 and Supplementary Data (Supplementary Material online), respectively. NJ and ML methods generated overall congruent trees, whereas the tree generated using MP was different. The principle of the MP method is to minimize the number of substitutions, irrespective of the branch lengths on the tree. Therefore, a substitution on a long branch, counts as much as a substitution on a short one, consequently rapidly evolving lineages are inferred as closely related ones. Therefore, we took into consideration only the two congruent NJ-based and ML-based trees. FIG. 1.— View large Download slide Phylogenetic tree of 87 Notch proteins. The unrooted tree was generated using the distance-based method Neighbor-Joining, with Caenorhabditis elegans (lin-12) protein as outgroup. The tree was visualized and edited with TreeExplorer in MEGA 4.0 (Kumar et al. 2008). The branch lengths reflect evolutionary divergence. Some of the branch lengths are indicated on the tree (numbers with decimals). The integer numbers presented in the tree nodes represent the bootstrap values obtained by 100 resamples. Trees obtained with MP (S1) and ML (S2) methods are available as Supplementary Material online. The corresponding accession numbers for the protein identifiers are shown in the third column of supplementary table ST2, Supplementary Material online. FIG. 1.— View large Download slide Phylogenetic tree of 87 Notch proteins. The unrooted tree was generated using the distance-based method Neighbor-Joining, with Caenorhabditis elegans (lin-12) protein as outgroup. The tree was visualized and edited with TreeExplorer in MEGA 4.0 (Kumar et al. 2008). The branch lengths reflect evolutionary divergence. Some of the branch lengths are indicated on the tree (numbers with decimals). The integer numbers presented in the tree nodes represent the bootstrap values obtained by 100 resamples. Trees obtained with MP (S1) and ML (S2) methods are available as Supplementary Material online. The corresponding accession numbers for the protein identifiers are shown in the third column of supplementary table ST2, Supplementary Material online. Based on the trees generated with NJ (fig. 1) and ML (supplementary fig. S2, Supplementary Material online) methods, we observe a classification in six distinct groups. The first group clusters the two Notch copies of C. elegans, which shows a great differentiation from the other taxa. The group of mammalian Notch4 proteins forms its own second branch. This branch is expected to lie within the vertebrates. However its great divergence from the other groups, indicated by the longer branch length (fig. 1), shows that this paralog group has diverged more rapidly than any of the other groups. Furthermore, we observe the third clade of invertebrate species carrying only one Notch. This includes the species of insects, Ciona, sea urchin, and amphioxus. Amphioxus Notch protein is clustered at the top of the invertebrate clade, phylogenetically closer to the vertebrates. The remaining Notch proteins on the tree belong to vertebrates and are classified in three groups. The main branch is divided into the Notch1 group and the Notch2–Notch3 group. In the Notch1 clade, we observe that the fish possess two Notch1 proteins, whereas in the other clades (Notch2 and Notch3), fish have only one member. In addition, Notch2 is evolutionary closer to Notch3. The branch lengths in figure 1 indicate that Notch2 diverged before Notch3 possibly at the first round of duplication events in vertebrates. From the phylogenetic tree of the Notch family, we tried to map the existence and to date the various duplication events that are part of this family history. We confirm previous observations and claim that: Notch in C. elegans resulted from an independent duplication event within its linage and that the three Notch (Notch1, Notch2, and Notch3) are paralogs that originated prior to the divergence of mammals, birds, reptiles, amphibians, and teleost. From our analysis, we report that in fish, the two copies of Notch1 gene are due to a duplication event that took place after the differentiation of teleost and tetrapods. This appeared probably from whole genome duplication (WGD) or from independent duplication within the lineage. Furthermore, branch lengths (fig. 1) indicate that Notch2 differentiated from Notch1 before Notch3 leading to the conclusion that Notch2 arose from the first duplication, whereas Notch3 from the second. Based on the simpler analysis to detect the origin of Notch4, we did not detect a correlation when whole length proteins, NICD, or ANK domains were used. It was not possible to detect Notch4 origin due to its rapid differentiation since duplication. Syntenies We first chose gene families that were found in syntenic positions between Notch (1–4) in H. sapiens and M. musculus. The genes chosen for the genomic analysis were the following: pre-B cell leukemia homebox (PBX), Lim homeobox 3 (LHX3), Notch regulated ankyrin repeat protein (NRARP), bromodomain (BRD), and CAMSAP1 (calmodulin regulated spectrin-associated protein 1). Their chromosomal locations and homologous positions were retrieved in C. elegans, D. melanogaster, C. intestinalis, and H. sapiens using Mapviewer, Geneview, and the BlastP and TBlastN algorithms. Schematic representation of their chromosomal positions for each species is given in figure 2 and their corresponding coordinates in table 3. The recent sequencing of the Florida lancelet, B. floridae (Putnam et al. 2008), provides a critical point in the research of evolution and syntenies, because the amphioxus species are considered primitive chordates. The syntenic genes were mapped on different scaffolds of those available for amphioxus as well (data not shown). However, because the relative locations of scaffolds in the genome are still unknown, they do not allow drawing any conclusions. Therefore, the imminent improvement of amphioxus genome annotation might reveal the exact location of syntenic genes and consequently allow the study of coordinate arrangements in this species. Table 3 Chromosomal Positions of Notch, PBX, NRARP, LHX3-4, CAMSAP1, and BRD Gene Families Species  Notch  PBX  NRARP  LHX3-4  CAMSAP1  BRD  Caenorhabditis elegans  III (9,092,224–9,099,594)  III (6,979,820–6,981,724)  IV (410,949–415,603)  X (7,528,561–7,532,677)  X (17,021,735–17,030,133.)  I (4,526,428–4,534,799)  III (9,060,153–9,071,299)  X (12,318,161–12,320,621)          Drosophila melanogaster  X (2,991,028–3,028,418)  X (15,886,520–15,890,040)  X (19,578,370–19,579,684)  2L (19,079,234–19,108,108)  2R (13,034,543–13,050,512)  X (7,944,180–7,949,460)  Ciona intestinalis  9q (2,333,255–2,357,750)  13q (2,032,218–2,043,568)  13q (2,090,301–2,091,974)  13q (1,991,460–2,001,042.)  13q (661,596–672,320)  9p (359,085–366,460)  Homo sapiens  9 (138,508,717–138,560,135)  9 (127,548,372–127,769,477)  9 (139,219,277–139,219,687)  9 (138,227,919–138,236,776)  9 (137,840,154–137,938,891)  9 (135,886,477–135,922,913)  1 (120,255,701–120,413,799)  1 (162,795,684–163,082,933)    1 (178,466,065–178,510,811)  1 (198,975,309–199,096,452)  1 (92,187,516–92,252,573)  19 (15,131,445–15,172,792)  19 (19,533,524–19,590,462)      19 (7,566,788–7,588,981)  19 (15,209,301–15,252,262)  6 (32,270,599–32,299,822)  6 (32,260,488–32,265,941)        6 (33,005,845–33,018,696)                  Species  Notch  PBX  NRARP  LHX3-4  CAMSAP1  BRD  Caenorhabditis elegans  III (9,092,224–9,099,594)  III (6,979,820–6,981,724)  IV (410,949–415,603)  X (7,528,561–7,532,677)  X (17,021,735–17,030,133.)  I (4,526,428–4,534,799)  III (9,060,153–9,071,299)  X (12,318,161–12,320,621)          Drosophila melanogaster  X (2,991,028–3,028,418)  X (15,886,520–15,890,040)  X (19,578,370–19,579,684)  2L (19,079,234–19,108,108)  2R (13,034,543–13,050,512)  X (7,944,180–7,949,460)  Ciona intestinalis  9q (2,333,255–2,357,750)  13q (2,032,218–2,043,568)  13q (2,090,301–2,091,974)  13q (1,991,460–2,001,042.)  13q (661,596–672,320)  9p (359,085–366,460)  Homo sapiens  9 (138,508,717–138,560,135)  9 (127,548,372–127,769,477)  9 (139,219,277–139,219,687)  9 (138,227,919–138,236,776)  9 (137,840,154–137,938,891)  9 (135,886,477–135,922,913)  1 (120,255,701–120,413,799)  1 (162,795,684–163,082,933)    1 (178,466,065–178,510,811)  1 (198,975,309–199,096,452)  1 (92,187,516–92,252,573)  19 (15,131,445–15,172,792)  19 (19,533,524–19,590,462)      19 (7,566,788–7,588,981)  19 (15,209,301–15,252,262)  6 (32,270,599–32,299,822)  6 (32,260,488–32,265,941)        6 (33,005,845–33,018,696)                  Data are based on the genome annotations available from Ensembl. View Large FIG. 2.— View largeDownload slide Schematic representation of the chromosomal position of Notch and of their neighboring genes in Caenorhabditis elegans, Drosophila. melanogaster, Ciona intestinalis, and Homo sapiens. Notch homologous genes are indicated by gray boxes, whereas the remaining ones are shown in plain white boxes. FIG. 2.— View largeDownload slide Schematic representation of the chromosomal position of Notch and of their neighboring genes in Caenorhabditis elegans, Drosophila. melanogaster, Ciona intestinalis, and Homo sapiens. Notch homologous genes are indicated by gray boxes, whereas the remaining ones are shown in plain white boxes. The two Notch genes of C. elegans (lin-12 and glp-1) were found on chromosome III. One of the copies of PBX was also found on chromosome (III) along with the Notch genes. The LHX3 homolog LHX, PBX, and CAMSAP1 were located on chromosome X, NRARP on chromosome IV and the homolog for BRD on chromosome I. In D. melanogaster, the Notch, BRD, PBX, and NRARP genes were found on chromosome X, whereas the CAMSAP1 and LHX on chromosomes, 2R and 2L, respectively. In C. intestinalis, most of the genes (LHX, PBX, CAMSAP1, and NRARP) were clustered on the chromosome 13q, whereas BRD and Notch in 9p and 9q, respectively. In C. intestinalis, only one copy from the genes under investigation was identified, whereas in vertebrates, most of them are duplicated. We have noted that in H. sapiens, there was an increase in number for most of the genes studied here. Notch1, BRD3, PBX3, LHX3, NRARP, and the one copy of CAMSAP1 were identified on chromosome 9, whereas Notch2, BRDT, PBX1, LHX4, and the second homolog of CAMSAP1 were located on chromosome 1. The Notch3, BRD4, PBX4, and the third homolog of CAMSAP1 were located on chromosome 19, and finally Notch4, BRD2, and PBX2 were found on chromosome 6. The chromosomal location of these genes examined here follows the distribution of Notch. It is obvious that in human there are coordinate arrangements between each of the paralog genes within BRD, PBX, and Notch families (table 4). In addition, the two LHX genes are found next to Notch1 and Notch2, whereas CAMSAP1 paralogs lie next to Notch1, Notch2, and Notch3 genes. Table 4 Correlation of Notch, BRD, and PBX Genes—in Human—Based on Chromosomal Arrangement Notch1  Notch2  Notch3  Notch4  BRD3  BRDT  BRD4  BRD2  PBX3  PBX1  PBX4  PBX2  Notch1  Notch2  Notch3  Notch4  BRD3  BRDT  BRD4  BRD2  PBX3  PBX1  PBX4  PBX2  In rat and mouse, the correlation is the same only for the groups Notch1–BRD3–PBX3 and Notch4–BRD2–PBX2. View Large Discussion In this work, an extended analysis on the evolution of Notch sequences was conducted using a great number of species not presented beforehand to this extent. We confirm that several independent duplications occurred in the family as well as duplications that involved genome duplications as indicated in previous studies. Caenorhabditis elegans has two Notch genes, whereas in insects, Ciona species, sea urchin, and amphioxus, there is only one copy of Notch. Besides this difference, a direct comparison of Notch protein sequences between nematodes and vertebrates did not reveal one to one correlations with other Notch genes (table 2). Therefore, our results confirm that the two Notch genes in C. elegans emerged from an independent duplication event, as suggested by Maine et al. (1995). The high bootstrap values in the phylogenetic tree, using NJ method (fig. 1), for the Notch1, Notch2, and Notch3 groups in vertebrates, suggest two duplication events that occurred prior to the split of tetrapoda and teleost. This is also supported by the existence of the three groups in teleost. In addition, figure 1 suggests that from the first duplication event, Notch1 led to the emergence of Notch2, and the second duplication event led to the appearance of Notch3 from Notch2. Nevertheless, the evolutionary picture regarding events of Notch family in birds and reptiles is still unclear. We did not identify any Notch3 or Notch4 in chicken. Moreover, Notch4 was not identified in the genome of the reptile A. carolinensis. The sequencing of more avian genomes, and the better coverage of the genomes of G. gallus and A. carolinensis, could allow elucidating the evolutionary gap of Notch family, between teleost fish and mammals. Three WGD events have been proposed in ancient vertebrate history, two of them are described at the origin of this group and the third is described in fishes (Meyer and Van de Peer 2005). The first two rounds of WGDs are still controversial (Dehal and Boore 2005), but for the WGD in fish, the picture is clearer. The occurrence of the second Notch1 copy only in fish suggests that it was generated from an independent duplication event after the differentiation of tetrapoda and teleost and may have occurred from the WGD event in fish. As previously described by Westin and Lardelli (1997) for zebrafish, we extend the scenario for all fish that the existence of multiple Notch in fish agrees with the assumption that a single Notch was duplicated twice in chordate lineage in the two rounds of genome duplication. The existence of the second Notch1 gene though, agrees with an independent duplication in teleost because no second Notch1 was identified in any other group of species. Mammals is the only lineage in which Notch4 genes were identified, and as shown in figure 1, they are classified in a separate branch and the latter is separated before even Drosophila Notch. Based on the fact that Notch4 is found only in mammals, we cannot claim that this group was differentiated before mammals and insects. The fact that Notch4 group is present only in the mammalian lineage allows us to present two possible scenarios: 1) a recent independent duplication of a Notch gene took place in the mammalian lineage giving rise to Notch4 or 2) this gene arose by the two WGDs in the early chordate and has been maintained by evolution only in the mammalian lineage but lost in all the others. Nevertheless, the increase of sequencing data, from avian and reptile genomes, will ultimately shed more light to our suggestions. Furthermore, it has been previously claimed that Notch4 diverged from Notch3 (Kortschak et al. 2001). However, from our observations, we cannot say with confidence from which Notch gene, Notch4 evolved, but it is clear that it evolved more rapidly than the other Notch genes. Figure 3 summarizes schematically the proposed evolutionary scenarios in representative taxa of this study, where two-gene duplications occurred in early chordate and an independent duplication event in mammals. An alternative scenario is schematically shown in figure 4, where two WGD events occurred in the early chordate and four Notch genes emerged but Notch4 has been maintained only in the mammalian lineage. FIG. 3.— View largeDownload slide Evolutionary scenario of Notch duplication events. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two duplication events prior to the differentiation of teleostei and tetrapoda. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. Light gray: Proposed independent duplication event that gave rise to Notch4 in mammalian lineage. FIG. 3.— View largeDownload slide Evolutionary scenario of Notch duplication events. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two duplication events prior to the differentiation of teleostei and tetrapoda. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. Light gray: Proposed independent duplication event that gave rise to Notch4 in mammalian lineage. FIG. 4.— View largeDownload slide Alternative evolutionary scenario of Notch duplication events predicted in this study. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two WGD events prior to the differentiation of teleostei and tetrapoda in the early chordate. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. FIG. 4.— View largeDownload slide Alternative evolutionary scenario of Notch duplication events predicted in this study. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two WGD events prior to the differentiation of teleostei and tetrapoda in the early chordate. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. Previous phylogenetic analyses have provided evidence of large segmental duplication of vertebrate genomes (Pebusque et al. 1998; Abi-Rached et al. 2002; McLysaght et al. 2002; Robinson-Rechavi et al. 2004) and of coordinated arrangements of genes that code for proteins known to be functionally related (Martin 2001; Pennisi 2001). In this work, we investigated whether Notch family resulted from genome duplication events. If this is the case, then there should be coordinate rearrangements with other gene families, functionally related to Notch genes, on the same chromosomes. The gene families of PBX, LHX3, NRARP, and BRD, along with Notch, are found in syntenic regions and were chosen for this investigation. The family of PBX (pre-B cell leukemia homebox) genes code for the TALE group of transcription factors. The gene LHX3 codes for a protein that belongs to a family in which proteins have two LIM domains. These proteins function as transcription factors and interact with other transcription factors as coactivators or corepressors (Bach et al. 1999). The NRARP (Notch regulated ankyrin repeat protein) gene, was found to be expressed with Delta and Notch proteins (Gawantka et al. 1998) and was shown to be an evolutionary conserved transcriptional target of Notch signaling pathway (Krebs et al. 2001; Lamar et al. 2001; Lahaye et al. 2002; Topczewska et al. 2003). The BRD proteins, which are members of BET subclass, have an ET domain in the carboxylic end and two bromodomain repeats which bind to acetyloluicine at the N-terminal of histones. In mammals, the BRD family consists of the BRD2, BRD3, BRD4, and BRDT families, and it is believed to have a critical role in the organization of chromosomes and spermatogenesis (Denis et al. 2000; Pivot-Pajot et al. 2003; Kanno et al. 2004). Finally, the gene CAMSAP1 encodes for a protein regulated by calmodulin and it is associated with spectrin. However, an unknown function is assigned to this protein. Summarizing, we show that the members of the PBX and BRD families most probably, coevolved with Notch genes, and segmental duplication events preserved these genes in close positions. There is evidence that these genes code for proteins that function together. Indeed, from studies in C. elegans, it seems that PBX is essential for the Notch signaling pathway (Takacs-Vellai et al. 2007), and further studies might also confirm that this functional relation exists also in higher species. BRD homolog in Drosophila, named fs(1)h, has been described as a multifunctional agent, regulated by the signaling pathway of Ras, the latter correlated many times with Notch (Sundaram 2005). Other syntenic analyses have shown a similar evolutionary history of BRD with Notch genes (Paillisson et al. 2007). In human, there is a coordinated arrangement of the homologs BRD, PBX, and Notch on the chromosomes (table 4), and this is probably translated as a functional link with both the PBX and BRD genes. The gene NRARP, which presents a Notch target and is regulated by Notch signaling, is found in close vicinity to Notch1 in human and to Notch in the other distantly related species. The NRARP gene, has not been duplicated, and only one copy of it is found in human, supporting the hypothesis that functionally related genes can be found in close vicinity on the genome (Martin 2001). Unlike Notch, BRD, and PBX, gene families of CAMSAP1 and LHX3 do not have four copies in mammals. CAMSAP1 has three homologs that are coordinately arranged on chromosomes with Notch1, Notch2, and Notch3, suggesting that they followed the two duplication events in vertebrates that gave rise to Notch1, Notch2, and Notch3, whereas LHX3 genes seem to have appeared during the first duplication event that gave rise to Notch1 and Notch2 genes. Conclusion In this work, we present a comprehensive phylogenetic analysis that confirms the high conservation among Notch proteins, in vertebrates and invertebrates. These genes were duplicated several times during evolution, leading to four genes in mammals. However, the ancient origin of Notch still remains unclear. There is still no evidence of the existence of Notch genes in any group besides metazoan phyla, suggesting that Notch appeared as a necessity for complex cellular communication and organization. Furthermore, the evolutionary picture in birds and reptiles is still unclear, and the sequencing of more avian and reptile genomes will shed light on various hypotheses. Additionally, the Notch4 family, present only in mammals, has diverged in a way that it was not possible to predict the origin of this duplication. Nevertheless, for this gene, we make two hypotheses. First, that this gene was duplicated from one of the other Notch genes in the mammalian lineage and second that it arose by the two WGD in the early chordate and has been maintained by evolution only in the mammalian lineage. The coordinate arrangement of other neighboring gene families, in close proximity to Notch genes, gives evidence of segmental duplication events. Their distribution does not seem to be random because a close proximity to Notch gene is also found in distantly related species. These results suggest that the molecular evolution of the Notch family involved ancestral segmental duplications in vertebrates and the genes found to be conserved in proximal positions may have a functional relation with Notch. These findings may be the foundation for further structural, functional, and evolutionary investigations, as the number of sequenced genomes increases. We thank Athanasia Pavlopoulou and Manolis Ladoukakis for their valuable comments concerning the manuscript. Prof Marc Baumann and Dr Sophia Kossida acknowledge the Sigrid Juselius Foundation for the Fellowship awarded to the latter, which enabled part of this work. Athina Theodosiou is thankful to CIMO (Centre of International Mobility) for the travel grant that enabled her to spend some fruitful time in Prof Baumann's group. Finally, we are indebted for the comments and suggestions of the anonymous referees in an early version of this manuscript. Comments: During the process of review of this manuscript, it has been published that Pbx1 is a Notch3 target gene in humans (Park et al. 2008). 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Author 2009. 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Evolutionary Scenarios of Notch Proteins

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Oxford University Press
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© The Author 2009. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
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10.1093/molbev/msp075
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19369596
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Abstract

Abstract Notch is a highly conserved family of transmembrane receptors and transcription factors that are key players in several developmental processes. In this study, we identified novel Notch sequences from various species covering from worm to human and conducted a comprehensive phylogenetic analysis in order to confirm and extend the evolutionary history of Notch. Our findings confirm an independent duplication event in Caenorhabditis elegans resulting in two Notch genes and show that the vertebrate Notch genes resulted from two duplication events, both of which occurred before the divergence of teleosts and tetrapoda. Furthermore, we demonstrate that the vertebrate Notch2 group is phylogenetically closer to Notch3 and that Notch2 appeared at the first round of vertebrate duplication events. Moreover, there is evidence that the two Notch1 genes in fish, appeared by a recent duplication of Notch1 in teleost after the divergence of teleost and tetrapoda. Whether this is from ancient whole genome duplication (WGD) or gene duplication remains to be elucidated. The fourth group of Notch (Notch4) was found only in mammals. We suggest two possible scenarios for the origin of the Notch4 subfamily: 1) Notch4 appeared at the time of the two WGDs in the early chordate but has been maintained only in the mammalian lineage and was lost in the other lineages, 2) a recent independent duplication event took place in the mammalian lineage. The increase of the sequencing data from Xenopus tropicalis, Gallus gallus genome projects and of other avian and reptile genomes will shed more light on this event. Nevertheless, the great divergence of Notch4, from the other three Notch genes, suggests a rapid divergence raising questions about the functional implication of this event. In addition, comparison of the organization of Notch syntenic genes among species supports the coordinated rearrangements during evolution for Nοtch, PBX, and BRD families that may lead to possible functional relationships. Notch, homologs, evolution, synteny Introduction Notch genes found both in vertebrate and invertebrate species represent a highly studied family of receptors that act also as transcription coactivators. They represent the principal molecules of the Notch signaling pathway that regulates cellular interactions, proliferation, differentiation, cell fate, apoptosis, and embryonic development (Weinmaster et al. 1991; Chitnis 1995; de la Pompa et al. 1997). The Notch gene was named after an irregular structure of the wing blades in Drosophila melanogaster (Mohr 1919), and the genomic locus responsible for this phenotype was reported to be involved in the embryonic development (Poulson 1940). The first Notch gene was cloned in 1983 (Artavanis-Tsakonas et al. 1983) and was shown to encode a cell-surface receptor (Wharton et al. 1985). Genomic analysis in this area revealed that Notch is important for the cell fate during development of D. melanogaster (Artavanis-Tsakonas et al. 1991; Artavanis-Tsakonas and Simpson 1991). Moreover, two Notch genes (glp-1 and lin-12) have been identified in Caenorhabditis elegans (Greenwald et al. 1983; Austin and Kimble 1987; Priess et al. 1987; Schnabel 1994). In the vertebrates, an increased number of Notch genes were identified (Ellisen et al. 1991; Weinmaster et al. 1992; Lardelli et al. 1994; Uyttendaele et al. 1996). Table 1 summarizes the species in which a Notch representative was reported with sequence analysis or functional studies. Table 1 Notch Genes Identified from Sequence and Functional Studies Species  Na  SA  SA-Referencesb  FS  FS-Referencesb  Drosophila melanogaster  N  +  Wharton et al. (1985)  +  Wharton et al. (1985)  Aedes aegypti  N  +  Nene et al. (2007)  —  —  Caenorhabditis elegans  glp-1  +  Yochem and Greenwald (1989)  +  Lambie and Kimble (1991)  lin-12  +  Yochem et al. (1988)  +  Lambie and Kimble (1991)  Ciona intestinalis  N  +  Satou et al. (2002)  —  —  Lytechinus variegatus  N  +  Sherwood and McClay (1997)  +  Sherwood and McClay (1997)  Branchiostoma floridae  N  +  Holland et al. (2001)  +  Holland et al. (2001)  Danio rerio  N1a  +  Bierkamp and Campos-Ortega (1993)  +  Bierkamp and Campos-Ortega (1993)  N1b  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N2  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N3  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  Xenopus tropicalis  N1  +  Klein et al. (2002)  +  Ogino et al. (2008)  Xenopus laevis  N1  +  Coffman et al. (1990)  +  Coffman et al. (1990)  Mus musculus  N1  +  del Amo et al. (1993)  +  Swiatek et al. (1994)  N2  +  Lardelli and Lendahl (1993)  +  Lardelli and Lendahl (1993)  N3  +  Lardelli et al. (1994)  +  Lardelli et al. (1994)  N4  +  Robbins et al. (1992)    Robbins et al. 1992)  Rattus norvegicus  N1  +  Weinmaster et al. (1991)  +  Weinmaster et al. (1991)  N2  +  Weinmaster et al. (1992)  +  Weinmaster et al. (1992)  N3  +  Tanigaki et al. (2001)  +  Lindsell et al. (1996)  N4  +  Uyttendaele et al. (2000)  +  Uyttendaele et al. (2000)  Homo sapiens  N1  +  Ellisen et al. (1991)  +  Aster et al. (1994)  N2  +  Stifani et al. (1992)  +  Hsieh et al. (1997)  N3  +  Tournier-Lasserve et al. (1993)  +  Felli et al. (1999)  N4  +  Sugaya et al. (1994)  +  Uyttendaele et al. (1998)  Macaca fascicularis  N1  +  Wang et al. (2007)  —  —  N2  +  Wang et al. (2007)  —  —  Species  Na  SA  SA-Referencesb  FS  FS-Referencesb  Drosophila melanogaster  N  +  Wharton et al. (1985)  +  Wharton et al. (1985)  Aedes aegypti  N  +  Nene et al. (2007)  —  —  Caenorhabditis elegans  glp-1  +  Yochem and Greenwald (1989)  +  Lambie and Kimble (1991)  lin-12  +  Yochem et al. (1988)  +  Lambie and Kimble (1991)  Ciona intestinalis  N  +  Satou et al. (2002)  —  —  Lytechinus variegatus  N  +  Sherwood and McClay (1997)  +  Sherwood and McClay (1997)  Branchiostoma floridae  N  +  Holland et al. (2001)  +  Holland et al. (2001)  Danio rerio  N1a  +  Bierkamp and Campos-Ortega (1993)  +  Bierkamp and Campos-Ortega (1993)  N1b  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N2  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  N3  +  Westin and Lardelli (1997)  +  Westin and Lardelli (1997)  Xenopus tropicalis  N1  +  Klein et al. (2002)  +  Ogino et al. (2008)  Xenopus laevis  N1  +  Coffman et al. (1990)  +  Coffman et al. (1990)  Mus musculus  N1  +  del Amo et al. (1993)  +  Swiatek et al. (1994)  N2  +  Lardelli and Lendahl (1993)  +  Lardelli and Lendahl (1993)  N3  +  Lardelli et al. (1994)  +  Lardelli et al. (1994)  N4  +  Robbins et al. (1992)    Robbins et al. 1992)  Rattus norvegicus  N1  +  Weinmaster et al. (1991)  +  Weinmaster et al. (1991)  N2  +  Weinmaster et al. (1992)  +  Weinmaster et al. (1992)  N3  +  Tanigaki et al. (2001)  +  Lindsell et al. (1996)  N4  +  Uyttendaele et al. (2000)  +  Uyttendaele et al. (2000)  Homo sapiens  N1  +  Ellisen et al. (1991)  +  Aster et al. (1994)  N2  +  Stifani et al. (1992)  +  Hsieh et al. (1997)  N3  +  Tournier-Lasserve et al. (1993)  +  Felli et al. (1999)  N4  +  Sugaya et al. (1994)  +  Uyttendaele et al. (1998)  Macaca fascicularis  N1  +  Wang et al. (2007)  —  —  N2  +  Wang et al. (2007)  —  —  SA—Sequence Analysis and FS—Functional studies. a  Notch genes. b  For some species, there are too many references; therefore, we apologize to the authors that their work was not referenced here. View Large As in all multigene vertebrate families, the increased number of Notch homologs in vertebrates is believed to be the result of duplication events (Westin and Lardelli 1997). Furthermore human Notch1, 2, and 3 genes were found in syntenic regions (Larsson et al. 1994) giving evidence that they appeared from two rounds of genome duplication during metazoan evolution. Nevertheless, the increased number of Notch signaling pathways in vertebrate species was suggested to be necessary for the additional complexity of the body plan (Kortschak et al. 2001). However, in C. elegans, which is an organism with lower functional and structural complexity than Drosophila, two Notch genes were found and are believed to be the result of an independent duplication event (Maine et al. 1995). Phylogenetic analysis of vertebrate Notch proteins suggested that Notch1a and Notch1b resulted from a duplication near teleost/mammalian divergence (Kortschak et al. 2001). The same study reports evidence that Notch4 in mammals is the result of a rapid divergence from Notch3. A limiting factor in previous phylogenetic studies on Notch genes (Maine et al. 1995; Kortschak et al. 2001) was the low number of examined species, due to the lack of fully sequenced genomes. The availability of sequenced genomes of various species may allow a more comprehensive investigation on the evolution of Notch. In addition, the availability of amphioxus, chicken and reptile Notch genes may allow us to fill the gaps of the previously described scenarios. Amphioxus was reported to be the closest living invertebrate to the vertebrates (Simmen et al. 1998; Furlong and Holland 2002) although this has its opponents (Delsuc et al. 2006). Nevertheless, its draft assembly genome has been available recently (Putnam et al. 2008) and is a valuable source for further studies. Therefore, how many Notch genes can we identify on its genome? Does the topology of previously described Notch trees change with the inclusion of this lineage? Previous analyses on Notch genes were focused only on model fish organisms like zebrafish (Westin and Lardelli 1997; Kortschak et al. 2001). Do all fish have two Notch1 genes? It has been previously reported that this duplication was around the time of teleost/mammalian divergence (Kortschak et al. 2001). Can we specify if the duplication occurred before or after the divergence? Can we specify if the two copies were the result of genome duplication or simple gene duplication? Do avian and reptile species have all four Notch duplicates like mammals or do they have two copies of Notch1 and no Notch4 like fish? Do all vertebrates have a Notch3 gene? Which species do possess a Notch4 gene and what is its origin? A final question to be looked into is if there are relationships between Notch and genes found syntenic to Notch from worm to human. This will help us identify potential mechanisms concerning their expansion and possibly predict coevolutionary events with functional implications. Materials and Methods Homolog Identification Notch protein homologs were identified with the bidirectional best hit method (Hirsh and Fraser 2001; Jordan et al. 2002), where orthologs are determined if two proteins in a different proteome find each other as the best hit in the other proteome. Searches were made through the NCBI (www.ncbi.nlm.nih.gov) and Ensembl (www.ensembl.org; release 46) protein databases. The four human proteins, (Nocth1: ENSP00000277541; Notch2: ENSP00000256646; Notch3: ENSP00000263388; and Notch4: ENSP00000364163) were first retrieved and afterward used as reference for the query of protein databases. Threshold of searching was set to 50% identity. Species searched were (in bold species where there are no published reports describing analysis of Notch genes): Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Bos taurus,Canis familiaris, Felis catus, Cavia porcellus, Loxodonta africana, Erinaceus europeus, Monodelphis domestica,Dasypus novemcinctus, Ornithorynhus anatinus, Oryctolagus cuniculus, Otolemur garnettii, Spermophilus tridecemlineatus, Tupaia belangeri, Myotis lucigucus, Sorex araneus, Echinops telfairi, Gallus gallus, Anolis carolinensis, Xenopus tropicalis, Danio rerio, Gasterosteus aculeatus, Tetraodon nigroviridis, Oryzias latipes, Takifugu rubripes, Branchiostoma floridae, Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, Ciona savignyi, Ciona intestinalis, Lytechinus variegatus, and Caenorhabditis elegans. Extensive genomic repositories including mRNA, cDNAs, and expressed sequence tags (ESTs) were searched using BlastN and TBlastN algorithm, as well as species-specific genomic databases when appropriate (see supplementary table ST1, Supplementary Material online, for details in genomic searches). In addition, in silico proteins were obtained using CAP3 program (Huang and Madan 1999) or Wise2 (Birney et al. 2004) and when this was not possible, ab initio cDNAs were retrieved from Ensembl. Multiple Sequence Alignments and Phylogenetic Analysis The Notch protein sequences accession numbers and identifiers are shown in supplementary table ST2, Supplementary Material online. After the exclusion of the in silico predicted, partial proteins, and those annotated as “novel projection” in Ensembl, we obtained a data set of 87 sequences (shown in bold in supplementary table ST2, Supplementary Material online). Full-length alignments were constructed using the MUSCLE program (version 3.7) (Edgar 2004) available at European Bioinformatics Institute (http://www.ebi.ac.uk/; version 3.6). The alignments were manually edited with BioEdit (version 7.0.9.0) (Tippmann 2004) for optimization. Evolutionary relationships were inferred using the Neighbor-Joining (NJ) and maximum parsimony (MP) methods implemented in the PHYLIP package (http://evolution.gs.washington.edu/phylip.html; release 3.67) (Felsenstein 1996) and maximum likelihood (ML) method implemented in PHYML (version 3.0) (Guindon et al. 2005). For the NJ method, a distance matrix was first calculated by Protdist program based on the multiple sequence alignment. The matrix was transformed to an unrooted tree under the evolutionary model Jones–Taylor–Thornton using the Neighbor program. For the character-based MP method, the unrooted tree was created with Protpars, whereas for the ML method, as in NJ, the tree was generated under the evolutionary model of Jones–Taylor–Thornton. The phylogenetic trees were viewed with the TreeExplorer program in MEGA 4 package (Kumar et al. 2008), whereas the robustness of the inferred trees of NJ and MP was tested by bootstrapping. The consensus trees were deduced from 100 trials. For PHYML method, approximate likelihood ratio test was used for branch support. Additionally, to examine the evolution and occurrence of Notch4 group and to check which of the groups Notch2 and Notch3 is closer to the clade Notch1, individual multiple alignments were constructed with MUSCLE for whole length, Notch intracellular region coding domain (NICD) and ankyrin repeat region (ANK). Phylogenetic analysis was conducted with PHYML using human proteins Notch1, Notch2, Notch3, Notch4, and Drosophila Notch protein as an outgroup. Genomic Positions of Notch and Neighboring Genes For the identification of the chromosomal location of the Notch genes, and their neighboring genes in H. sapiens, C. intestinalis, D. melanogaster, and C. elegans a text-based query was conducted using NCBI Map Viewer and GeneView of Ensembl. Neighboring genes were chosen from the syntenic regions surrounding the Notch genes of human and mouse, and the corresponding homologous neighboring genes in other species were identified using BlastP and TBlastN. Results Notch Orthologs and Paralogs One hundred and four (104) Notch homologous sequences were identified for 37 eukaryotic species (see supplementary table ST2, Supplementary Material online). In the mammalian species H. sapiens, M. mulatta, P. troglodytes, M. musculus, R. norvegicus, C. familiaris, and M. domestica, four copies of Notch homologs were identified. Nevertheless, for other mammals, characterized by a lower coverage of sequenced genome (1.5–2×), we could not identify all full-length copies (for details see supplementary table ST2, Supplementary Material online) and for some of them ESTs or cDNAs were identified (supplementary table ST1, Supplementary Material online). The G. gallus genome is the only sequenced genome of birds today. Besides the absence of Notch4, Notch3 was not detected either. In addition, even if the chicken genome has been sequenced to a great extent (7.1×), several genes from the Notch3 syntenic area of human appear to be absent as well. However, the detection of this gene in frog, reptile, and fish species, the still draft genome assembly of G. gallus, along with its unsequenced microchromosomes (Costantini et al. 2007), do not allow us to exclude Notch3 from G. gallus. In the genome of frog, X. tropicalis, the homologous sequences of Notch1, 2, and 3 were detected, whereas Notch4 sequence was once more missing. The accuracy of this finding is quite reliable if we consider the high coverage of sequenced genome (7.65×). Furthermore, in the recent draft assembly (6.3×) of A. carolinensis, it was possible to identify the Notch1 and Notch3 genes, which were previously not reported. In the fish genomes, included in this analysis (D. rerio, T. nigroviridis, T. rubripes, O. latipes, and G. aculeatus), two genes for Notch1 (Notch1a and Notch1b), one for Notch2 and Notch3 were detected, whereas Notch4 was once again absent. Finally, concerning those species distantly related to vertebrates, like insects, Ciona, sea urchin, and amphioxus, only one Notch gene was identified, whereas in C. elegans two were identified as previously described (Lambie and Kimble 1991). Table 2 shows the protein identity among species from representative Notch sequences. Table 2 Identities of Notch Proteins Based on Similarity Searches   N2_H.sa  N3_H.sa  N4_H.sa  B.flo  L.va  A.ae  A.ga  D.me  C.int  C.ele_lin12  C.ele_glp1  N1_H.sa  55  45  37  51  43  49  49  48  46  29  29  N2_H.sa    45  35  46  41  48  49  48  46  29  25  N3_H.sa      35  45  39  44  44  43  40  28  28  N4_H.sa        34  37  34  35  39  34  31  27  B.flo          44  49  49  47  45  25  29  L.va            45  45  44  39  29  30  A.ae              80  64  43  27  28  A.ga                69  44  29  27  D.me                  41  29  29  C.int                    30  32  C.ele_lin12                      46    N2_H.sa  N3_H.sa  N4_H.sa  B.flo  L.va  A.ae  A.ga  D.me  C.int  C.ele_lin12  C.ele_glp1  N1_H.sa  55  45  37  51  43  49  49  48  46  29  29  N2_H.sa    45  35  46  41  48  49  48  46  29  25  N3_H.sa      35  45  39  44  44  43  40  28  28  N4_H.sa        34  37  34  35  39  34  31  27  B.flo          44  49  49  47  45  25  29  L.va            45  45  44  39  29  30  A.ae              80  64  43  27  28  A.ga                69  44  29  27  D.me                  41  29  29  C.int                    30  32  C.ele_lin12                      46  Only representative sequences are used. NOTE.—Identity numbers were extracted from a Blast output after an all-to-all comparison of Notch sequences. Abbreviations: N1_H.sa, N1_Homo sapiens; N2_H.sa, N2_Homo sapiens; N3_H.sa, N3_Homo sapiens; N4_H.sa, N4_Homo sapiens; B.flo, Branchiostoma floridae; L.va, Lytechinus variegatus; A.ae, Aedes aegypti; A.ga Anopheles gambiae; D.me, Drosophila melanogaster; C.in, Ciona intestinalis; C.ele_lin12, Caenorhabditis elegans_lin12; C.ele_glp-1, C.elegans_glp1. View Large Evolution of Notch Family The evolutionary relationships of the Notch protein family were inferred through distance-NJ, MP, and ML methods. The unrooted phylogenetic trees generated from each method, are shown in figure 1, supplementary figures S1 and Supplementary Data (Supplementary Material online), respectively. NJ and ML methods generated overall congruent trees, whereas the tree generated using MP was different. The principle of the MP method is to minimize the number of substitutions, irrespective of the branch lengths on the tree. Therefore, a substitution on a long branch, counts as much as a substitution on a short one, consequently rapidly evolving lineages are inferred as closely related ones. Therefore, we took into consideration only the two congruent NJ-based and ML-based trees. FIG. 1.— View large Download slide Phylogenetic tree of 87 Notch proteins. The unrooted tree was generated using the distance-based method Neighbor-Joining, with Caenorhabditis elegans (lin-12) protein as outgroup. The tree was visualized and edited with TreeExplorer in MEGA 4.0 (Kumar et al. 2008). The branch lengths reflect evolutionary divergence. Some of the branch lengths are indicated on the tree (numbers with decimals). The integer numbers presented in the tree nodes represent the bootstrap values obtained by 100 resamples. Trees obtained with MP (S1) and ML (S2) methods are available as Supplementary Material online. The corresponding accession numbers for the protein identifiers are shown in the third column of supplementary table ST2, Supplementary Material online. FIG. 1.— View large Download slide Phylogenetic tree of 87 Notch proteins. The unrooted tree was generated using the distance-based method Neighbor-Joining, with Caenorhabditis elegans (lin-12) protein as outgroup. The tree was visualized and edited with TreeExplorer in MEGA 4.0 (Kumar et al. 2008). The branch lengths reflect evolutionary divergence. Some of the branch lengths are indicated on the tree (numbers with decimals). The integer numbers presented in the tree nodes represent the bootstrap values obtained by 100 resamples. Trees obtained with MP (S1) and ML (S2) methods are available as Supplementary Material online. The corresponding accession numbers for the protein identifiers are shown in the third column of supplementary table ST2, Supplementary Material online. Based on the trees generated with NJ (fig. 1) and ML (supplementary fig. S2, Supplementary Material online) methods, we observe a classification in six distinct groups. The first group clusters the two Notch copies of C. elegans, which shows a great differentiation from the other taxa. The group of mammalian Notch4 proteins forms its own second branch. This branch is expected to lie within the vertebrates. However its great divergence from the other groups, indicated by the longer branch length (fig. 1), shows that this paralog group has diverged more rapidly than any of the other groups. Furthermore, we observe the third clade of invertebrate species carrying only one Notch. This includes the species of insects, Ciona, sea urchin, and amphioxus. Amphioxus Notch protein is clustered at the top of the invertebrate clade, phylogenetically closer to the vertebrates. The remaining Notch proteins on the tree belong to vertebrates and are classified in three groups. The main branch is divided into the Notch1 group and the Notch2–Notch3 group. In the Notch1 clade, we observe that the fish possess two Notch1 proteins, whereas in the other clades (Notch2 and Notch3), fish have only one member. In addition, Notch2 is evolutionary closer to Notch3. The branch lengths in figure 1 indicate that Notch2 diverged before Notch3 possibly at the first round of duplication events in vertebrates. From the phylogenetic tree of the Notch family, we tried to map the existence and to date the various duplication events that are part of this family history. We confirm previous observations and claim that: Notch in C. elegans resulted from an independent duplication event within its linage and that the three Notch (Notch1, Notch2, and Notch3) are paralogs that originated prior to the divergence of mammals, birds, reptiles, amphibians, and teleost. From our analysis, we report that in fish, the two copies of Notch1 gene are due to a duplication event that took place after the differentiation of teleost and tetrapods. This appeared probably from whole genome duplication (WGD) or from independent duplication within the lineage. Furthermore, branch lengths (fig. 1) indicate that Notch2 differentiated from Notch1 before Notch3 leading to the conclusion that Notch2 arose from the first duplication, whereas Notch3 from the second. Based on the simpler analysis to detect the origin of Notch4, we did not detect a correlation when whole length proteins, NICD, or ANK domains were used. It was not possible to detect Notch4 origin due to its rapid differentiation since duplication. Syntenies We first chose gene families that were found in syntenic positions between Notch (1–4) in H. sapiens and M. musculus. The genes chosen for the genomic analysis were the following: pre-B cell leukemia homebox (PBX), Lim homeobox 3 (LHX3), Notch regulated ankyrin repeat protein (NRARP), bromodomain (BRD), and CAMSAP1 (calmodulin regulated spectrin-associated protein 1). Their chromosomal locations and homologous positions were retrieved in C. elegans, D. melanogaster, C. intestinalis, and H. sapiens using Mapviewer, Geneview, and the BlastP and TBlastN algorithms. Schematic representation of their chromosomal positions for each species is given in figure 2 and their corresponding coordinates in table 3. The recent sequencing of the Florida lancelet, B. floridae (Putnam et al. 2008), provides a critical point in the research of evolution and syntenies, because the amphioxus species are considered primitive chordates. The syntenic genes were mapped on different scaffolds of those available for amphioxus as well (data not shown). However, because the relative locations of scaffolds in the genome are still unknown, they do not allow drawing any conclusions. Therefore, the imminent improvement of amphioxus genome annotation might reveal the exact location of syntenic genes and consequently allow the study of coordinate arrangements in this species. Table 3 Chromosomal Positions of Notch, PBX, NRARP, LHX3-4, CAMSAP1, and BRD Gene Families Species  Notch  PBX  NRARP  LHX3-4  CAMSAP1  BRD  Caenorhabditis elegans  III (9,092,224–9,099,594)  III (6,979,820–6,981,724)  IV (410,949–415,603)  X (7,528,561–7,532,677)  X (17,021,735–17,030,133.)  I (4,526,428–4,534,799)  III (9,060,153–9,071,299)  X (12,318,161–12,320,621)          Drosophila melanogaster  X (2,991,028–3,028,418)  X (15,886,520–15,890,040)  X (19,578,370–19,579,684)  2L (19,079,234–19,108,108)  2R (13,034,543–13,050,512)  X (7,944,180–7,949,460)  Ciona intestinalis  9q (2,333,255–2,357,750)  13q (2,032,218–2,043,568)  13q (2,090,301–2,091,974)  13q (1,991,460–2,001,042.)  13q (661,596–672,320)  9p (359,085–366,460)  Homo sapiens  9 (138,508,717–138,560,135)  9 (127,548,372–127,769,477)  9 (139,219,277–139,219,687)  9 (138,227,919–138,236,776)  9 (137,840,154–137,938,891)  9 (135,886,477–135,922,913)  1 (120,255,701–120,413,799)  1 (162,795,684–163,082,933)    1 (178,466,065–178,510,811)  1 (198,975,309–199,096,452)  1 (92,187,516–92,252,573)  19 (15,131,445–15,172,792)  19 (19,533,524–19,590,462)      19 (7,566,788–7,588,981)  19 (15,209,301–15,252,262)  6 (32,270,599–32,299,822)  6 (32,260,488–32,265,941)        6 (33,005,845–33,018,696)                  Species  Notch  PBX  NRARP  LHX3-4  CAMSAP1  BRD  Caenorhabditis elegans  III (9,092,224–9,099,594)  III (6,979,820–6,981,724)  IV (410,949–415,603)  X (7,528,561–7,532,677)  X (17,021,735–17,030,133.)  I (4,526,428–4,534,799)  III (9,060,153–9,071,299)  X (12,318,161–12,320,621)          Drosophila melanogaster  X (2,991,028–3,028,418)  X (15,886,520–15,890,040)  X (19,578,370–19,579,684)  2L (19,079,234–19,108,108)  2R (13,034,543–13,050,512)  X (7,944,180–7,949,460)  Ciona intestinalis  9q (2,333,255–2,357,750)  13q (2,032,218–2,043,568)  13q (2,090,301–2,091,974)  13q (1,991,460–2,001,042.)  13q (661,596–672,320)  9p (359,085–366,460)  Homo sapiens  9 (138,508,717–138,560,135)  9 (127,548,372–127,769,477)  9 (139,219,277–139,219,687)  9 (138,227,919–138,236,776)  9 (137,840,154–137,938,891)  9 (135,886,477–135,922,913)  1 (120,255,701–120,413,799)  1 (162,795,684–163,082,933)    1 (178,466,065–178,510,811)  1 (198,975,309–199,096,452)  1 (92,187,516–92,252,573)  19 (15,131,445–15,172,792)  19 (19,533,524–19,590,462)      19 (7,566,788–7,588,981)  19 (15,209,301–15,252,262)  6 (32,270,599–32,299,822)  6 (32,260,488–32,265,941)        6 (33,005,845–33,018,696)                  Data are based on the genome annotations available from Ensembl. View Large FIG. 2.— View largeDownload slide Schematic representation of the chromosomal position of Notch and of their neighboring genes in Caenorhabditis elegans, Drosophila. melanogaster, Ciona intestinalis, and Homo sapiens. Notch homologous genes are indicated by gray boxes, whereas the remaining ones are shown in plain white boxes. FIG. 2.— View largeDownload slide Schematic representation of the chromosomal position of Notch and of their neighboring genes in Caenorhabditis elegans, Drosophila. melanogaster, Ciona intestinalis, and Homo sapiens. Notch homologous genes are indicated by gray boxes, whereas the remaining ones are shown in plain white boxes. The two Notch genes of C. elegans (lin-12 and glp-1) were found on chromosome III. One of the copies of PBX was also found on chromosome (III) along with the Notch genes. The LHX3 homolog LHX, PBX, and CAMSAP1 were located on chromosome X, NRARP on chromosome IV and the homolog for BRD on chromosome I. In D. melanogaster, the Notch, BRD, PBX, and NRARP genes were found on chromosome X, whereas the CAMSAP1 and LHX on chromosomes, 2R and 2L, respectively. In C. intestinalis, most of the genes (LHX, PBX, CAMSAP1, and NRARP) were clustered on the chromosome 13q, whereas BRD and Notch in 9p and 9q, respectively. In C. intestinalis, only one copy from the genes under investigation was identified, whereas in vertebrates, most of them are duplicated. We have noted that in H. sapiens, there was an increase in number for most of the genes studied here. Notch1, BRD3, PBX3, LHX3, NRARP, and the one copy of CAMSAP1 were identified on chromosome 9, whereas Notch2, BRDT, PBX1, LHX4, and the second homolog of CAMSAP1 were located on chromosome 1. The Notch3, BRD4, PBX4, and the third homolog of CAMSAP1 were located on chromosome 19, and finally Notch4, BRD2, and PBX2 were found on chromosome 6. The chromosomal location of these genes examined here follows the distribution of Notch. It is obvious that in human there are coordinate arrangements between each of the paralog genes within BRD, PBX, and Notch families (table 4). In addition, the two LHX genes are found next to Notch1 and Notch2, whereas CAMSAP1 paralogs lie next to Notch1, Notch2, and Notch3 genes. Table 4 Correlation of Notch, BRD, and PBX Genes—in Human—Based on Chromosomal Arrangement Notch1  Notch2  Notch3  Notch4  BRD3  BRDT  BRD4  BRD2  PBX3  PBX1  PBX4  PBX2  Notch1  Notch2  Notch3  Notch4  BRD3  BRDT  BRD4  BRD2  PBX3  PBX1  PBX4  PBX2  In rat and mouse, the correlation is the same only for the groups Notch1–BRD3–PBX3 and Notch4–BRD2–PBX2. View Large Discussion In this work, an extended analysis on the evolution of Notch sequences was conducted using a great number of species not presented beforehand to this extent. We confirm that several independent duplications occurred in the family as well as duplications that involved genome duplications as indicated in previous studies. Caenorhabditis elegans has two Notch genes, whereas in insects, Ciona species, sea urchin, and amphioxus, there is only one copy of Notch. Besides this difference, a direct comparison of Notch protein sequences between nematodes and vertebrates did not reveal one to one correlations with other Notch genes (table 2). Therefore, our results confirm that the two Notch genes in C. elegans emerged from an independent duplication event, as suggested by Maine et al. (1995). The high bootstrap values in the phylogenetic tree, using NJ method (fig. 1), for the Notch1, Notch2, and Notch3 groups in vertebrates, suggest two duplication events that occurred prior to the split of tetrapoda and teleost. This is also supported by the existence of the three groups in teleost. In addition, figure 1 suggests that from the first duplication event, Notch1 led to the emergence of Notch2, and the second duplication event led to the appearance of Notch3 from Notch2. Nevertheless, the evolutionary picture regarding events of Notch family in birds and reptiles is still unclear. We did not identify any Notch3 or Notch4 in chicken. Moreover, Notch4 was not identified in the genome of the reptile A. carolinensis. The sequencing of more avian genomes, and the better coverage of the genomes of G. gallus and A. carolinensis, could allow elucidating the evolutionary gap of Notch family, between teleost fish and mammals. Three WGD events have been proposed in ancient vertebrate history, two of them are described at the origin of this group and the third is described in fishes (Meyer and Van de Peer 2005). The first two rounds of WGDs are still controversial (Dehal and Boore 2005), but for the WGD in fish, the picture is clearer. The occurrence of the second Notch1 copy only in fish suggests that it was generated from an independent duplication event after the differentiation of tetrapoda and teleost and may have occurred from the WGD event in fish. As previously described by Westin and Lardelli (1997) for zebrafish, we extend the scenario for all fish that the existence of multiple Notch in fish agrees with the assumption that a single Notch was duplicated twice in chordate lineage in the two rounds of genome duplication. The existence of the second Notch1 gene though, agrees with an independent duplication in teleost because no second Notch1 was identified in any other group of species. Mammals is the only lineage in which Notch4 genes were identified, and as shown in figure 1, they are classified in a separate branch and the latter is separated before even Drosophila Notch. Based on the fact that Notch4 is found only in mammals, we cannot claim that this group was differentiated before mammals and insects. The fact that Notch4 group is present only in the mammalian lineage allows us to present two possible scenarios: 1) a recent independent duplication of a Notch gene took place in the mammalian lineage giving rise to Notch4 or 2) this gene arose by the two WGDs in the early chordate and has been maintained by evolution only in the mammalian lineage but lost in all the others. Nevertheless, the increase of sequencing data, from avian and reptile genomes, will ultimately shed more light to our suggestions. Furthermore, it has been previously claimed that Notch4 diverged from Notch3 (Kortschak et al. 2001). However, from our observations, we cannot say with confidence from which Notch gene, Notch4 evolved, but it is clear that it evolved more rapidly than the other Notch genes. Figure 3 summarizes schematically the proposed evolutionary scenarios in representative taxa of this study, where two-gene duplications occurred in early chordate and an independent duplication event in mammals. An alternative scenario is schematically shown in figure 4, where two WGD events occurred in the early chordate and four Notch genes emerged but Notch4 has been maintained only in the mammalian lineage. FIG. 3.— View largeDownload slide Evolutionary scenario of Notch duplication events. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two duplication events prior to the differentiation of teleostei and tetrapoda. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. Light gray: Proposed independent duplication event that gave rise to Notch4 in mammalian lineage. FIG. 3.— View largeDownload slide Evolutionary scenario of Notch duplication events. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two duplication events prior to the differentiation of teleostei and tetrapoda. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. Light gray: Proposed independent duplication event that gave rise to Notch4 in mammalian lineage. FIG. 4.— View largeDownload slide Alternative evolutionary scenario of Notch duplication events predicted in this study. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two WGD events prior to the differentiation of teleostei and tetrapoda in the early chordate. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. FIG. 4.— View largeDownload slide Alternative evolutionary scenario of Notch duplication events predicted in this study. Commonly accepted tree of the taxa presented here was extracted from NCBI taxonomy browser. Spots indicate duplication events reported here in the Notch family. Black spot: Two WGD events prior to the differentiation of teleostei and tetrapoda in the early chordate. Dark gray spots: Independent recent duplication events, one for Notch1 in teleostei and one for Notch in nematode. Previous phylogenetic analyses have provided evidence of large segmental duplication of vertebrate genomes (Pebusque et al. 1998; Abi-Rached et al. 2002; McLysaght et al. 2002; Robinson-Rechavi et al. 2004) and of coordinated arrangements of genes that code for proteins known to be functionally related (Martin 2001; Pennisi 2001). In this work, we investigated whether Notch family resulted from genome duplication events. If this is the case, then there should be coordinate rearrangements with other gene families, functionally related to Notch genes, on the same chromosomes. The gene families of PBX, LHX3, NRARP, and BRD, along with Notch, are found in syntenic regions and were chosen for this investigation. The family of PBX (pre-B cell leukemia homebox) genes code for the TALE group of transcription factors. The gene LHX3 codes for a protein that belongs to a family in which proteins have two LIM domains. These proteins function as transcription factors and interact with other transcription factors as coactivators or corepressors (Bach et al. 1999). The NRARP (Notch regulated ankyrin repeat protein) gene, was found to be expressed with Delta and Notch proteins (Gawantka et al. 1998) and was shown to be an evolutionary conserved transcriptional target of Notch signaling pathway (Krebs et al. 2001; Lamar et al. 2001; Lahaye et al. 2002; Topczewska et al. 2003). The BRD proteins, which are members of BET subclass, have an ET domain in the carboxylic end and two bromodomain repeats which bind to acetyloluicine at the N-terminal of histones. In mammals, the BRD family consists of the BRD2, BRD3, BRD4, and BRDT families, and it is believed to have a critical role in the organization of chromosomes and spermatogenesis (Denis et al. 2000; Pivot-Pajot et al. 2003; Kanno et al. 2004). Finally, the gene CAMSAP1 encodes for a protein regulated by calmodulin and it is associated with spectrin. However, an unknown function is assigned to this protein. Summarizing, we show that the members of the PBX and BRD families most probably, coevolved with Notch genes, and segmental duplication events preserved these genes in close positions. There is evidence that these genes code for proteins that function together. Indeed, from studies in C. elegans, it seems that PBX is essential for the Notch signaling pathway (Takacs-Vellai et al. 2007), and further studies might also confirm that this functional relation exists also in higher species. BRD homolog in Drosophila, named fs(1)h, has been described as a multifunctional agent, regulated by the signaling pathway of Ras, the latter correlated many times with Notch (Sundaram 2005). Other syntenic analyses have shown a similar evolutionary history of BRD with Notch genes (Paillisson et al. 2007). In human, there is a coordinated arrangement of the homologs BRD, PBX, and Notch on the chromosomes (table 4), and this is probably translated as a functional link with both the PBX and BRD genes. The gene NRARP, which presents a Notch target and is regulated by Notch signaling, is found in close vicinity to Notch1 in human and to Notch in the other distantly related species. The NRARP gene, has not been duplicated, and only one copy of it is found in human, supporting the hypothesis that functionally related genes can be found in close vicinity on the genome (Martin 2001). Unlike Notch, BRD, and PBX, gene families of CAMSAP1 and LHX3 do not have four copies in mammals. CAMSAP1 has three homologs that are coordinately arranged on chromosomes with Notch1, Notch2, and Notch3, suggesting that they followed the two duplication events in vertebrates that gave rise to Notch1, Notch2, and Notch3, whereas LHX3 genes seem to have appeared during the first duplication event that gave rise to Notch1 and Notch2 genes. Conclusion In this work, we present a comprehensive phylogenetic analysis that confirms the high conservation among Notch proteins, in vertebrates and invertebrates. These genes were duplicated several times during evolution, leading to four genes in mammals. However, the ancient origin of Notch still remains unclear. There is still no evidence of the existence of Notch genes in any group besides metazoan phyla, suggesting that Notch appeared as a necessity for complex cellular communication and organization. Furthermore, the evolutionary picture in birds and reptiles is still unclear, and the sequencing of more avian and reptile genomes will shed light on various hypotheses. Additionally, the Notch4 family, present only in mammals, has diverged in a way that it was not possible to predict the origin of this duplication. Nevertheless, for this gene, we make two hypotheses. First, that this gene was duplicated from one of the other Notch genes in the mammalian lineage and second that it arose by the two WGD in the early chordate and has been maintained by evolution only in the mammalian lineage. The coordinate arrangement of other neighboring gene families, in close proximity to Notch genes, gives evidence of segmental duplication events. Their distribution does not seem to be random because a close proximity to Notch gene is also found in distantly related species. These results suggest that the molecular evolution of the Notch family involved ancestral segmental duplications in vertebrates and the genes found to be conserved in proximal positions may have a functional relation with Notch. These findings may be the foundation for further structural, functional, and evolutionary investigations, as the number of sequenced genomes increases. We thank Athanasia Pavlopoulou and Manolis Ladoukakis for their valuable comments concerning the manuscript. Prof Marc Baumann and Dr Sophia Kossida acknowledge the Sigrid Juselius Foundation for the Fellowship awarded to the latter, which enabled part of this work. Athina Theodosiou is thankful to CIMO (Centre of International Mobility) for the travel grant that enabled her to spend some fruitful time in Prof Baumann's group. Finally, we are indebted for the comments and suggestions of the anonymous referees in an early version of this manuscript. Comments: During the process of review of this manuscript, it has been published that Pbx1 is a Notch3 target gene in humans (Park et al. 2008). 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Author 2009. 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Journal

Molecular Biology and Evolution – Oxford University Press

Published: Apr 15, 2009

Keywords: Notch homologs evolution synteny

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