Version Changes
Revised. Amendments from Version 1
The methods subsection - generation of the list of zebrafish jogging genes section: edited to make it clearer how the list of zebrafish jogging genes was generated and the limitation of the approach taken to create a list of ‘heart jogging genes’.
In Discussion- paragraph 4: edited to create more positive statements about the role of the 'jogging ortholog' genes, in human heart development.
Abstract
For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer’s vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer’s vesicle determine asymmetry in the developing heart, the direction of ‘heart jogging’ and the direction of ‘heart looping’. ‘Heart jogging’ is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward ‘jog’. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.
We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging. We found that the human, mouse and zebrafish ‘heart jogging orthologs’ are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.
This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach.
Introduction
An understanding of heart development is important for the treatment of both congenital and acquired heart disease. The majority of heart development studies use model organisms for ethical and practical reasons. Transparent fish embryos, as well chick embryos, enable the developing heart to be studied in real time 1, and the mouse continues to be a key model organism used to investigate mammalian heart development 2. Although there is substantial evolutionary conservation in the development of left-right axis asymmetry, there is divergence between species 3. The earliest events in mammalian heart development are of great interest, but are poorly understood relative to externally developing organs, due to practical constraints.
For the majority of developing vertebrate embryos left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer’s vesicle in the zebrafish 4, 5. In the zebrafish, laterality cues from the Kuppfer’s vesicle determine asymmetry in the developing heart, and consequently the direction of heart jogging and heart looping. At 24 hours post-fertilization (hpf) the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward ‘jog’. At 36hpf the heart tube then loops to the right to create the asymmetric heart 5, 6. Cilia within the Kuppfer’s vesicle are known to be instrumental in establishing left-right asymmetry and consequently play a significant role in determining the direction of heart jogging 7 and heart looping 8. However, a failure of heart jogging does not necessarily imply that there will be a failure in heart looping, and vice versa. In addition, asymmetric cell migration has been implicated as a key factor in the process of heart jogging 9– 13. Several of the genes involved in zebrafish heart jogging have been identified from mutation, morpholino and functional complementation studies 6, 10, 13– 26.
We sought to determine whether the use of Gene Ontology (GO) annotation could offer mechanistic clues to early mammalian heart development. GO is a controlled vocabulary that is used to describe gene product function 27. GO describes three aspects of a gene product’s biology: the biological process that the gene product is involved in, the specific molecular function of the gene product and the cellular component that the gene product is located in. GO terms are associated in a directed acyclic graph (DAG), and thus have defined relationships to each other.
The process of heart looping has been described in a variety of higher eukaryotes 2, 28, 29, and the occurrence of dextral-looping, the early phase of heart looping, appears to be conserved from zebrafish to chicken to humans. In addition, many congenital heart abnormalities, such as dextrocardia and isomerisms are thought to be due to abnormal heart looping 2, 30 and ciliary dysfunction has been associated with 50% of patients with congenital heart disease and heterotaxy 31. However, the process of heart jogging has only been described in zebrafish 6. Biben and Harvey describe a leftward shift of the developing mouse caudal heart prior to looping, which may be analogous to heart jogging in zebrafish 28, but to our knowledge this has not been investigated further, and heart jogging is not considered to occur in mammals. Consequently, when the ontology describing heart development was expanded 32, limitations were included to prevent the association of the GO term ‘heart jogging’ to mammalian gene products 33. However, an absence of evidence is not evidence of absence, hence it remains a possibility that heart jogging also occurs in mammalian systems.
Although there has been substantial progress in heart development research 1, 3, 4, 29, there are clearly gaps in our understanding of early heart development, particularly in the mammal. Functional enrichment analysis of genes known to be involved in zebrafish heart jogging, and also of the human and mouse orthologs of these zebrafish heart jogging genes, identifies many conserved biological processes, functions and cellular locations across these three species. The results of these analyses support the role of cilia in symmetry breaking and the importance of cell signalling in early heart development.
Methods
Generation of the list of zebrafish heart jogging genes
A list of 30 zebrafish genes that affect heart jogging was compiled using a variety of approaches. Twelve zebrafish proteins were identified as they were already annotated to the ‘heart jogging’ GO terms, the remaining 18 proteins were then identified using the ZFIN ( http://zfin.org/) Site Search, with the search phrase 'heart jogging', and filtering using the 'Expression/Phenotypes' category. This search retrieves figures from papers that have ‘heart jogging’ in the figure legend, and thus are likely to be describing specific zebrafish genes (and proteins) involved in this process. Many of these genes had not yet been curated with GO terms. Each of the papers identified in this way were reviewed; of the 23 zebrafish genes identified in these papers five (Bmpr1aa, Tbx1, unm_hu119, unm_hu202, unm_hu304) were eliminated, as none of these papers provided experimental evidence for the involvement of these genes in heart jogging. This left 30 zebrafish proteins with strong evidence for a role in the heart jogging process ( Table 1). The experimental evidence describing the association of each gene to the process of heart jogging was manually reviewed, to ensure consistent criteria were applied.
Table 1. Proteins included in zebrafish ‘jogging’ gene list and the human and mouse ‘jogging ortholog’ gene lists.
The evidence for these 30 zebrafish proteins having a role in heart jogging comes from mutant, morpholino or functional complementation studies, as described in the associated publications.
|
Zebrafish gene symbol
(protein ID) |
Human gene symbol
(protein ID) |
Mouse gene symbol
(protein ID) |
|---|---|---|
| acvr1l 6 (Q9DGI6) | ACVRL1 (P37023) | Acvrl1 (Q61288) |
| apc 57 (F1QN37) | APC (P25054) | Apc (Q61315) |
| bmp4 6, 13 (O57574) | BMP4 (P12644) | Bmp4 (P21275) |
| bmp7a 6 (Q9PTF9) | BMP7 (P18075) | Bmp7 (P23359) |
| vbmpr2a 20 (Q288P3) | BMPR2 (Q13873) | Bmpr2 (O35607) |
| bmpr2b 20 (Q288P2) | ||
| camk2a 14 (Q32PV2) | CAMK2A (Q9UQM7) | Camk2a (P11798) |
| camk2b2 14 (E7F012) | CAMK2B (Q13554) | Camk2b (P28652) |
| camk2g1 14 (Q4V9P8) | CAMK2G (Q13555) | Camk2g (Q923T9) |
| ccdc103 6 (Q6DGB6) | CCDC103 (Q8IW40) | Ccdc103 (Q9D9P2) |
| ccdc40 6 (Q56A40) | CCDC40 (Q4G0X9) | Ccdc40 (Q8BI79) |
| cobl 23 (I1X3U9) | COBL (O75128) | Cobl (Q5NBX1) |
| dand5 15 (Q76C29) | DAND5 (Q8N907) | Dand5 (Q76LW6) |
| dnaaf1 6, 10 (Q7ZV84) | DNAAF1 (Q8NEP3) | Dnaaf1 (Q9D2H9) |
| dub 22 (Q0P484) | RCSD1 (Q6JBY9) | Rcsd1 (Q3UZA1) |
| fgfr2 18 (Q8JG38) | FGFR2 (P21802) | Fgfr2 (P21803) |
| foxh1 6, 58 (Q9I9E1) | FOXH1 (O75593) | Foxh1 (O88621) |
| foxj1a 25 (Q08CI2) | FOXJ1 (Q92949) | Foxj1 (Q61660) |
| foxj1b 25 (F1R8Z9) | ||
| fzd2 22 (Q90YL7) | FZD2 (Q14332) | Fzd2 (Q9JIP6) |
| gsk3b 17 (Q9IBD2) | GSK3B (P49841) | Gsk3b (Q9WV60) |
| has2 13 (Q9DG41) | HAS2 (Q92819) | Has2 (P70312) |
| lrrc6 6 (B3DH20) | LRRC6 (Q86X45) | Lrrc6 (O88978) |
| nipbla 21 (F5HSE3) | NIPBL (Q6KC79) | Nipbl (Q6KCD5) |
| Niplblb 21 (F1QBY1) | ||
| nkd1 24 (Q2TJA6) | NKD1 (Q969G9) | Nkd1 (Q99MH6) |
| nphp3 26 (P0CI65) | NPHP3 (Q7Z494) | Nphp3 (Q7TNH6) |
| pkd2 6 (Q6IVV8) | PKD2 (Q13563) | Pkd2 (O35245) |
| ptpn11a 16 (Q7ZW17) | PTPN11 (Q06124) | Ptpn11 (P35235) |
| southpaw 10, 19 (Q7ZZT5) | no mammalian orthologs | |
Generation of the list of human and mouse ‘jogging ortholog’ genes
The HUGO Gene Nomenclature Committee Comparison of Orthology Predictions (HCOP) search tool ( http://www.genenames.org/cgi-bin/hcop.pl) was used to identify the closest possible human and mouse ortholog for each of the 30 zebrafish genes. HCOP displays predictions from 11 homology prediction tools, including EnsemblCompara, Homologene and Inparanoid 34. For all but one gene, southpaw, HCOP returned human or mouse homologs for the zebrafish genes. The lack of a close mammalian ortholog of southpaw was confirmed with a UCSC BLAT analysis against the human and mouse genomes 35. BLAST analysis 36 showed that the closest possible human and mouse homolog for the zebrafish southpaw gene was Nodal (33% identity). Indeed, both southpaw and nodal are specifically expressed in the left lateral plate mesoderm 5, 37 and knockdown of murine Nodal in this region leads to a disruption of cardiac asymmetry, as does injection of southpaw morpholinos, suggesting a functional orthology between southpaw and Nodal 5, 37. However a reciprocal HCOP search showed that the zebrafish genes nodal-related 1 and 2 are the closest orthologs of human NODAL. Hence we have not included a human or mouse ortholog for zebrafish southpaw ( Table 1). Three pairs of zebrafish paralogs ( bmpr2a/ bmpr2b; foxj1a/ foxj1b; nipbla/ nipblb) have a single corresponding ortholog in human and mouse. Therefore, there are 26 human and 26 mouse orthologs to the 30 zebrafish genes identified as relevant to zebrafish heart jogging ( Table 1).
Gene ontology annotation
The human ‘jogging ortholog’ genes were fully manually annotated, by an experienced GO curator 38. Individual PubMed queries were run for each gene using the approved human gene symbol and filtering on ‘human’. To achieve full annotation, all of the relevant publications (a total of 232) containing unique functional data for each gene were annotated, regardless of the specific biology described in each paper. This approach enabled consistent annotation of all experimental data relating to each gene, thus ensuring an unbiased overview of any common processes associated with these genes. In addition, the GO term ‘heart looping’ was associated with a ‘jogging ortholog’ human gene if dextrocardia or situs inversus totalis phenotypes had been associated with a mutation in the gene, in order to follow the generally agreed view that leftward heart looping will have resulted in these phenotypes 2.
Functional enrichment analysis
The Mouse Genome Informatics functional enrichment tool VLAD (VisuaL Annotation Display; http://proto.informatics.jax.org/prototypes/vlad-1.0.3/) was used to look for overrepresentation of GO terms in each gene list relative to the whole genome of the organism. The annotation datasets used for the analysis were zfin (4th March 2013), goa_human (5th March 2013) and mgi (7th March 2013) for the zebrafish, human and mouse analyses respectively, and the ontology dataset used was dated 10th March 2013. The query gene lists (as UniProt IDs) were pasted into the ‘Query Set’ field, the ‘Universe Set’ field was left blank (to specify all genes in species specific annotation file) and the ‘Display Settings’ options selected were ‘pruning threshold’:3 and ‘collapsing threshold’:6. No evidence codes were excluded from the analyses. For this analysis the total number of genes (universe set size) having annotations in the biological process ontology were 14,577, 30,441 and 24,813 for zebrafish, human and mouse respectively. In line with common practice, when using functional analysis tools, enriched GO terms with 1 or 2 associated query genes were excluded from the final results table.
Creation of an ‘early heart development’ mouse gene list
A list of 103 mouse genes likely to play a role in early heart development was created by combining gene lists derived from three sources: The Mouse Genome Informatics Mammalian Phenotype Ontology browser http://www.informatics.jax.org/searches/MP_form.shtml 39, the QuickGO browser http://www.ebi.ac.uk/QuickGO/ 40 and the ‘jogging ortholog’ gene list described above (see Mousegenelist.csv in Data File). The Mammalian Phenotype Ontology browser was queried for genotypes annotated with the terms ‘abnormal direction of heart looping’, ‘ situs inversus totalis’, ‘dextrocardia’ and ‘mesocardia’, creating a list of 180 genotypes with an associated gene. Due to the multiple phenotypes associated with each of these genotypes only 58 genes were identified through this approach, and of these only 5 overlap with the 26 ‘jogging ortholog’ genes. Thirty-five genes were identified by filtering on the GO term ‘determination of heart left/right asymmetry’ and its child terms, the evidence code IMP (Inferred by Mutant Phenotype), and the mouse taxon. Of these only two are also present in the ‘jogging ortholog’ gene lists and 11 are present in the phenotype gene list. Twenty-six mouse ‘jogging ortholog’ genes were added to this combined gene list, and any duplicated genes were removed.
Results
Annotation of the zebrafish heart jogging genes and the human ‘jogging ortholog’ genes
Thirty zebrafish genes were annotated to the GO term ‘heart jogging’ or one of its child terms based on experimental data from the literature ( Table 1). Human and mouse orthologs of these genes were identified, as described in the Methods section, resulting in a list of 26 mammalian ‘jogging orthologs’.
The human ‘jogging ortholog’ genes were then fully annotated with GO terms based on published experimental data. All manual annotations to the human, mouse and zebrafish genes can be visualized with the QuickGO Gene Ontology browser http://tinyurl.com/humanortholog, http://tinyurl.com/mouseortholog and http://tinyurl.com/zebrafishgenes.
Functional enrichment analysis
The zebrafish heart jogging gene list and the human and mouse ‘jogging ortholog’ gene lists were analysed using the VLAD enrichment tool. This identified 155 biological process GO terms that were significantly enriched in the zebrafish (see Human_data.csv in Data File), 431 in the human (see Human_data.csv in Data File) and 402 in the mouse (see Mouse_data.csv in Data File) gene lists. The enriched GO terms from all three species were grouped into five biological areas: Development, Patterning, Cellular Process, Signalling and Movement. The relative enrichment of key GO terms from each area was compared across all three species (see Biological_process_summary.csv in Data File; summarized in Table 2).
Table 2. Comparison of enriched Gene Ontology terms across orthologous gene lists from zebrafish, human and mouse.
The enriched GO terms were grouped into specific ontology areas, with a selection of more specific child term (preceded with a dash) also included. The full list of grouped GO terms can be found in Table S4, which also shows the genes annotated to each term from each of the three species. k: the number of genes in each gene list annotated to the GO term; M: the number of genes in the species proteome annotated to the GO term.
| Gene Ontology terms | Zebrafish | Human | Mouse | ||||||
|---|---|---|---|---|---|---|---|---|---|
| k | M | k/M as % | k | M | k/M as % | k | M | k/M as % | |
| DEVELOPMENT | |||||||||
| GO:0032502 developmental process | 30 | 2357 | 1.3% | 24 | 6803 | 0.4% | 22 | 3945 | 0.6% |
| - GO:0009888 tissue development | 30 | 673 | 4.5% | 17 | 1849 | 0.9% | 14 | 1138 | 1.2% |
| - GO:0072358 cardiovascular system development | 30 | 487 | 6.2% | 15 | 1095 | 1.4% | 10 | 679 | 1.5% |
| - GO:0001944 vasculature development | - | - | - | 7 | 666 | 1.1% | 7 | 436 | 1.6% |
| - GO:0007507 heart development | 30 | 268 | 11.2% | 13 | 634 | 2.1% | 8 | 390 | 2.1% |
| - GO:0001947 heart looping | 22 | 83 | 26.5% | 7 | 68 | 10.3% | - | - | - |
| - GO:0007399 nervous system development | 8 | 700 | 1.1% | 15 | 2802 | 0.5% | 12 | 1486 | 0.8% |
| - GO:0072001 renal system development | 6 | 100 | 6.0% | 9 | 386 | 2.3% | 6 | 200 | 3.0% |
| - GO:0007423 sensory organ development | - | - | - | 10 | 738 | 1.4% | 8 | 480 | 1.7% |
| - GO:0048736 appendage development | 4 | 123 | 3.3% | 4 | 237 | 1.7% | 4 | 159 | 2.5% |
| - GO:0050793 regulation of developmental process | 6 | 267 | 2.2% | 14 | 2383 | 0.6% | 14 | 1542 | 0.9% |
| PATTERNING | |||||||||
| GO:0007389 pattern specification process | 30 | 435 | 6.9% | 15 | 644 | 2.3% | 12 | 414 | 2.9% |
| - GO:0009799 specification of symmetry | 30 | 177 | 16.9% | 10 | 147 | 6.8% | 7 | 90 | 7.8% |
| - GO:0061371 determination of heart left/right
asymmetry |
30 | 97 | 30.9% | 8 | 71 | 11.3% | 4 | 48 | 8.3% |
| CELLULAR PROCESS | |||||||||
| GO:0071840 cellular component organization or
biogenesis |
12 | 1124 | 1.1% | 17 | 5991 | 0.3% | 15 | 3306 | 0.5% |
| - GO:0030030 cell projection organization | 10 | 274 | 3.6% | 12 | 1311 | 0.9% | 11 | 644 | 1.7% |
| - GO:0051128 regulation of cellular component
organization |
- | - | - | 10 | 1894 | 0.5% | 10 | 1293 | 0.8% |
| - GO:0031344 regulation of cell projection organization | - | - | - | 5 | 452 | 1.1% | 5 | 311 | 1.6% |
| GO:0006468 protein phosphorylation | 8 | 696 | 1.1% | 8 | 842 | 1.0% | 9 | 748 | 1.2% |
| - GO:0001932 regulation of protein phosphorylation | - | - | - | 8 | 1124 | 0.7% | 8 | 722 | 1.1% |
| GO:0006357 regulation of transcription from RNA
polymerase II promoter |
8 | 166 | 4.8% | 10 | 1939 | 0.5% | 12 | 1268 | 0.9% |
| GO:0042127 regulation of cell proliferation | 5 | 70 | 7.1% | 9 | 1769 | 0.5% | 9 | 1123 | 0.8% |
| GO:0007049 cell cycle | - | - | - | 9 | 1644 | 0.5% | 8 | 915 | 0.9% |
| - GO:0051726 regulation of cell cycle | - | - | - | 6 | 934 | 0.6% | 6 | 538 | 1.1% |
| SIGNALLING | |||||||||
| GO:0023052 signaling | 12 | 2131 | 0.6% | 16 | 6682 | 0.2% | 16 | 4568 | 0.4% |
| - GO:0023051 regulation of signaling | 7 | 521 | 1.3% | 16 | 3227 | 0.5% | 16 | 1931 | 0.8% |
| GO:0050896 response to stimulus | - | - | - | 20 | 10767 | 0.2% | 21 | 6679 | 0.3% |
| - GO:0048583 regulation of response to stimulus | 7 | 557 | 1.3% | 15 | 3799 | 0.4% | 15 | 2140 | 0.7% |
| MOVEMENT | |||||||||
| GO:0007017 microtubule-based process | 6 | 188 | 3.2% | 6 | 873 | 0.7% | 4 | 358 | 1.1% |
| GO:0040011 locomotion | 10 | 289 | 3.5% | 10 | 1448 | 0.7% | 8 | 753 | 1.1% |
| - GO:0040012 regulation of locomotion | - | - | - | 6 | 657 | 0.9% | 5 | 473 | 1.1% |
| - GO:2000145 regulation of cell motility | - | - | - | 6 | 608 | 1.0% | 5 | 439 | 1.1% |
| - GO:0016477 cell migration | 8 | 200 | 4.0% | 6 | 931 | 3.6% | 5 | 504 | 1.0% |
Enrichment of heart development terms. As expected there was a significant enrichment of developmental process terms in all three gene lists, including an enrichment of the GO term ‘heart development’. However, there was also enrichment of terms such as ‘renal system development’ and ‘nervous system development’, indicating the role of these proteins in regulating the development of a range of organ systems and tissues. These data analyses also show an enrichment of terms describing specific, but universal, cellular processes, such as signalling and regulation of transcription ( Table 2). These terms represent essential aspects of development, but are grouped discretely due to their roles in many other biological processes.
‘Pattern specification’, described in GO as a ‘developmental process that results in the creation of defined areas or spaces within an organism to which cells respond and eventually are instructed to differentiate’ and several of its more specific child terms (such as ‘specification of symmetry’), were also enriched in all three gene lists. Within the symmetry ontology, the GO term ‘determination of heart left/right asymmetry’ is annotated to all 30 genes in the zebrafish jogging gene list, however, it is only associated with 8 and 4 jogging ortholog genes in the human and mouse respectively. Of the 97 zebrafish genes associated with ‘determination of heart left/right asymmetry’ 31% are also present in the zebrafish jogging gene list. In contrast, only 11% of the human and 8% of the mouse genes associated with this term are also ‘jogging orthologs’. These results confirm an overlap in the functional role of the zebrafish jogging genes and the human and mouse orthologs in the determination of heart left/right symmetry. However, this relatively low level of overlap may reflect the limitations of model organism and human research in this area, rather than a lack of functional conservation of these genes.
In addition, there were some differences in the developmental terms that were enriched between species. For example, the GO terms ‘vasculature development’ and ‘sensory organ development’ are enriched in both the human and mouse ‘jogging ortholog’ gene lists ( Table 2), but neither of these processes are enriched in the zebrafish jogging ortholog genes. This difference may reflect the type of experiments zebrafish are used for, rather than reflecting a difference between zebrafish and mammals in the genes required for these developmental processes.
Enrichment of cilia terms. Terms in the cellular component organization or biogenesis ontology were enriched across all three gene lists ( Table 2 and Biological_process_summary.csv in Data File). Specifically there was an enrichment of terms describing ‘cilium morphogenesis’ and ‘protein complex assembly’ ( Figure 1). Within each of these, some more specific terms were enriched, for example the human and mouse ‘jogging ortholog’ gene lists were enriched for the term ‘axonemal dynein complex assembly’, whilst the zebrafish and human gene lists showed an enrichment of the term ‘cilium assembly’.
Figure 1. View of relationships between enriched terms from the cellular component organization or biogenesis ontology.
The GONUTs view of relationships between enriched terms from the cellular component organization or biogenesis ontology 84. The grey arrows are used where a term has an ‘is a’ relationship to its parent term, the blue arrows indicate a ‘part of’ relationship. The bars below each GO term indicates which of these terms are enriched in the zebrafish ‘jogging’ gene list, and the human and mouse ‘jogging ortholog’ gene lists.
Terms such as ‘regulation of cell projection organization’ were also enriched in the human and mouse ‘jogging ortholog’ gene lists. ‘Regulation’ terms have a ‘regulates’ relationship with the relevant processes; for example the term ‘positive regulation of cell projection organization’ has a ‘positively_regulates’ relationship to the term ‘cell projection organization’. In GO an important benefit of building a DAG, rather than a flat-list of controlled vocabulary terms, is that relationships can be used to make inferences from one term to another. However, the VLAD enrichment tool does not automatically create a transitive relationship between ‘regulation’ terms and the processes or functions they regulate. Consequently genes annotated to a ‘regulation’ term will not be associated with the regulated process term (unless there is an independent annotation to the process term). It is also important to recognise that it can be difficult for a curator to choose between annotating to the biological process itself, or to the term describing the regulation of that biological process, based on the published experimental data. Therefore, to get a full picture of the genes involved in a process, including the genes that regulate the process, it is necessary to combine the genes annotated to GO terms describing both the ‘process’ and the ‘regulation of the process’. For example, 10 zebrafish, 12 human and 14 mouse genes within the ‘jogging ortholog’ gene lists are annotated to either ‘cell projection organization’ or ‘regulation of cell projection organization’ (or children of these terms). This represents 33%, 40% and 47% of these zebrafish, human and mouse ‘jogging’ gene lists respectively, indicating that the process of cell projection organization is an important function for this group of genes. In addition, many of the ‘jogging’ and ‘jogging ortholog’ genes annotated to ‘cell projection organization’ terms have also been annotated to the cellular component term ‘cell projection’ (7, 11, 13, genes in zebrafish, human and mouse, respectively, see Data File). The enrichment of the biological process term ‘cell projection organization’ and cellular component term ‘cell projection’ within these gene lists is consistent with the key role of cilia located in the node/Kuppfer’s vesicle to determine heart left/right asymmetry in all three species.
Enrichment of cell migration terms. Cell migration also plays a key role in the establishment of the heart cone, heart jogging and heart looping 9, 10 and enrichment of the GO term ‘cell migration’ is seen in the ‘jogging’ gene lists of all three species ( Table 2 and Biological_process_summary.csv in Data File). Lenhart et al. (2013) 9 identified FoxH1, spaw, Bmp4, Lefty2 and Has2 as essential to the asymmetric cell migration that leads to heart jogging. However, our literature review suggests that some genes may have functions in both cilia assembly, within the Kuppfer’s vesicle, and cell migration. For example, thymocytes from Foxj1 transgenic mice display defective migration 41, whereas Foxj1-null mice are defective in ciliogenesis 42. Similarly, in zebrafish, Fzd2 has been shown to play a role in cilium assembly 22 as well as pancreatic insulin-cell migration 43. Consequently, further investigations into the role of these genes in heart jogging cell migration may provide further insight into this process.
Co-annotation of heart development associated genes
In order to investigate the contribution of individual genes in the multiple processes associated with early heart development we created human and mouse heart development gene lists and examined the associated GO biological processes terms. A list of 103 mouse genes with roles in early heart developmental processes was created by merging the three gene lists created using the Mouse Genome Informatics phenotype browser, the QuickGO browser as well as the ‘jogging ortholog’ gene list (Mousegenelist.csv in Data File).
GO captures a range of biological processes that a single gene is involved in. By comparing the overlap between the GO terms associated with specific gene lists it is possible to see what cellular mechanisms are likely to be contributing to the various heart developmental processes. Using the QuickGO browser, genes in the zebrafish ‘heart jogging’ gene list, which were associated with the GO terms ‘heart looping’, ‘signal transduction’, ‘cell migration’ and ‘cell projection organization’ (and all child terms, including ‘regulation’ terms), were downloaded, as well as the genes associated with these terms that were also present in the mouse ‘early heart development’ gene list (Mousegenelist.csv in Data File).
In the zebrafish ‘heart jogging’ gene list a similar proportion of the genes have the potential to play a role in cell projection organisation (10 genes), cell migration (8 genes) and signal transduction (13 genes) ( Figure 2A). In the list of 103 mouse genes that are associated with early heart development, either by phenotype, annotation or homology to the zebrafish ‘heart jogging’ gene list, 82 have been annotated to the GO term heart looping. In contrast to the zebrafish ‘jogging’ gene list, signal transduction appears to play a major role in the mouse early heart development, with 27 genes associated with both signal transduction and heart looping, whereas only 18 and 9 genes, respectively, are associated with cell migration and cell projection organization ( Figure 2B). These results fit well with what is known about these gene lists. The zebrafish ‘jogging’ gene list defines a group of genes whose functions are required very early in heart development, when the role of cilia in symmetry breaking initiates the heart jogging process. Whereas, in the mouse ‘early heart development’ gene list the genes included have roles in heart looping, which is developmentally later event than heart jogging. Therefore, although the initial events associated with breaking of left-right symmetry are represented within this gene list, the genes involved in the later process of ensuring the complex looping of the heart tube, through controlled signalling and cell migration, contribute to a large proportion of this list.
Figure 2. Venn diagrams describing the multiple roles of genes associated with heart development.
Venn diagrams showing the overlap between the GO terms associated with A) the zebrafish ‘heart jogging’ gene list (30 genes) and B) the mouse combined heart development gene list (103 genes).
Human disease phenotypes associated with the ‘jogging ortholog’ genes
While annotating the 26 human ‘jogging ortholog’ genes we noticed that almost half of these genes have not been associated with a specific disease phenotype ( Table 3). However, of the 26 genes examined, mutations in 14 had been associated with a disease phenotype, a fifth of which were ciliopathies. Dextrocardia or situs inversus totalis (reversal or mirroring of the major visceral organs) was associated with 6 of the human ‘jogging ortholog’ genes. Location of the heart on the right side (rather than the left) is generally agreed to be the result of left-handed, instead of right-handed looping of the heart tube in early embryogenesis 2. The association of these ‘jogging ortholog’ genes with heart looping defects confirm that there is conserved functional homology between at least some of these orthologous zebrafish and human genes in the very early stages of heart development, which lead to the initial heart asymmetry. All four of ciliopathy-associated ‘jogging orthologs’ were also described as associated with situs inversus totalis, confirming the conserved role of these genes in the cilia within the symmetry determining left-right organizer.
Table 3. Diseases associated with the human ‘jogging ortholog’ genes.
The associated diseases are described in the listed publications.
|
Human gene symbol
(protein ID) |
Heart relevant phenotype | Other associated phenotypes |
|---|---|---|
| ACVRL1 (P37023) | - | Hereditary haemorrhagic telangiectasia type 2
(HHT2) 59, HHT2 with pulmonary hypertension 60 |
| APC (P25054) | - | Familial adenomatous polyposis coli-1 61 |
| BMP4 (P12644) | - | Microphthalmia, syndromic 6 62, orofacial cleft 11 63 |
| BMP7 (P18075) | - | - |
| BMPR2 (Q13873) | - | Pulmonary hypertension 64 |
| CAMK2A (Q9UQM7) | - | - |
| CAMK2B (Q13554) | - | - |
| CAMK2G (Q13555) | - | - |
| CCDC103 (Q8IW40) | Dextrocardia, situs inversus totalis 65 | Ciliary dyskinesia, primary, 17 65 |
| CCDC40 (Q4G0X9) | Situs inversus totalis 66 | Ciliary dyskinesia, primary, 15
67, Kartagener’s
Syndrome 66 |
| COBL (O75128) | - | - |
| DAND5 (Q8N907) | - | - |
| DNAAF1 (Q8NEP3) | Situs inversus totalis 68, 69 | Ciliary dyskinesia, primary, 13 68, 69 |
| FGFR2 (P21802) | - | Several craniosynostosis
70,
71, see OMIM for more
information |
| FOXH1 (O75593) | Ventricular septal defect
72, transposition
of the great arteries 54 |
- |
| FOXJ1 (Q92949) | - | - |
| FZD2 (Q14332) | - | - |
| GSK3B (P49841) | - | - |
| HAS2 (Q92819) | - | - |
| LRRC6 (Q86X45) | Situs inversus totalis 73 | Ciliary dyskinesia, primary, 19, Kartagener’s
Syndrome 73 |
| NIPBL (Q6KC79) | Cardiac septal defects (not confirmed as
associated with NIPBL mutations) 74 |
Cornelia de Lange syndrome 1 74, 75 |
| NKD1 (Q969G9) | - | Colorectal adenocarcinoma 76 |
| NPHP3 (Q7Z494) | Situs inversus totalis 77 | nephronophthisis type 3
78, Meckel syndrome type
7 77, renal-hepatic-pancreatic dysplasia 77, 79 |
| PKD2 (Q13563) | Dextrocardia, situs inversus totalis 53 | Polycystic kidney disease 2 53, 80 |
| PTPN11 (Q06124) | atrioventricular canal defects 81 | juvenile myelomonocytic leukemia
82, LEOPARD
syndrome 81, Noonan syndrome 81, 83 |
| RCSD1 (Q6JBY9) | - | - |
Zebrafish data: GO terms significantly enriched in zebrafish ‘heart jogging’ gene list. The GO biological process, molecular function and cellular component terms that are highly enriched in the zebrafish ‘jogging’ gene list. The GO terms are listed in order of decreasing p-values and the ‘universe set’ size included in the functional analysis using VLAD is 14,577.
Human data: GO terms significantly enriched in human orthologs of zebrafish ‘heart jogging’ genes. The GO biological process, molecular function and cellular component terms that are highly enriched in the human ‘jogging ortholog’ gene list. The GO terms are listed in order of decreasing p-values and the ‘universe set’ size included in the functional analysis using VLAD is 30,441.
Mouse data: GO terms significantly enriched in mouse orthologs of zebrafish ‘heart jogging’ genes. The GO biological process, molecular function and cellular component terms that are highly enriched in the mouse ‘jogging ortholog’ gene list. The GO terms are listed in order of decreasing p-values and the ‘universe set’ size included in the functional analysis using VLAD is 24,813.
Biological process summary: Summary of all enriched biological process GO terms across all three species. The enriched biological process GO terms are grouped into general biological areas (‘development’, ‘patterning’, ‘cellular process’, ‘signaling’, ‘movement’ and ‘high level terms’, text in upper case). Each area is subdivided into more specific ontologies, based on the Gene Ontology structure. A subset of the biological process GO terms and data are reported in Table 2 in the main article. High-level terms were not included in any further discussion, as these terms are not specific enough to be informative. k: the number of genes in each gene list annotated to the GO term; M: the number of genes in the species proteome annotated to the GO term. Starred genes are annotated directly to the enriched term. Non-starred genes are annotated to a ‘child’ of the enriched term. DAR: zebrafish, HUM: human, MUS: mouse.
Mouse gene list:‘Early heart development’ mouse gene list. The evidence that these 103 mouse genes have a role in the early stages of heart development comes from their orthology to the zebrafish ‘heart jogging’ gene list, from phenotypes associated with specific genotypes, or annotation to the GO term ‘determination of heart left/right asymmetry’ or it’s child terms (see method section in main article for full details).
Discussion
We have used GO to annotate the key genes involved in zebrafish heart jogging and their human and mouse orthologs. Heart jogging is not a process that is thought to occur in mammals. However, these genes are conserved between species and play essential roles in many developmental processes. The information available about these genes in several diverse species can be used to shed light on the roles of these genes and possible mechanisms in heart jogging and other heart developmental processes. Our analyses are in agreement with the well described essential role of cilia in early development 4, 5, 31, with a third of the zebrafish ‘heart jogging’ genes associated with the biological process ‘cell projection organization’ ( Table 2).
However, it is also important to recognise that although there is considerable evidence for conserved mechanisms of heart development across vertebrates there are also areas of divergence 44. For example, in the mouse, zebrafish and Xenopus the rotation of cilia is responsible for the early asymmetric gene expression pattern around the left-right organizer, whereas cilia do not play a role in symmetry breaking in the chicken or pig 44.
The early phases of heart development are particularly difficult to study in mammals, however various approaches are enabling progress in this area 2, 29, 45, 46 and using phenotype, annotation and orthology data we have created a list of 103 genes with a putative role in early mouse heart developmental processes. Furthermore, the phenotypes associated with experimentally generated mutant mice provide further clues to the likely role of these genes in human heart development; the genes associated with situs inversus totalis phenotypes are most likely to have functional roles within the node. Conversely, genes not associated with situs inversus totalis but associated with an abnormal direction of heart looping, dextrocardia or mesocardia are likely to be involved in the response of the embryonic heart tube to the left/right asymmetry signals. This is not a completely reliable interpretation, for example mutations in the transcription factor Pitx2 lead to mice with situs inversus totalis, however, Pitx2 is expressed in the left lateral plate and its continued asymmetric expression is necessary for asymmetric morphogenesis of most visceral organs 44. The mouse knockout consortia data 47 will continue to help with the identification of additional early heart development genes, and informed interpretation of these phenotypes will make it possible to separate those genes likely to be associated with the node from those with functions within the heart tube.
In humans, defects in early heart development are likely to result in spontaneous abortion and therefore many genes required for early heart development will go undetected 48. Consequently, human embryos with heart defects, which develop to full term, represent the less severe end of the spectrum. Mutations in several human genes have now been identified as causative of abnormal heart looping, such as ACVR2B, LEFTY2, GJA1 and ZIC3 49– 52, and some of the ‘jogging ortholog’ genes ( CCDC103, CCDC40, DNAAF1, LRRC6, NPHP3 and PKD2) are also associated with heart looping defects. Thus providing evidence to support an involvement of these genes in left-right asymmetry determination in the heart. Furthermore, mutations in some of the ‘jogging ortholog’ human genes, FOXH1 and PTPN11, are associated with heart septal defects in humans, which seems to imply that in individuals with these mutations early heart developmental processes have proceeded normally, suggesting that, contrary to their role in zebrafish, these genes may not be involved in the early stages of human heart development. However, there are other possible reasons why there is a poor association of heart defects with the ‘jogging ortholog’ gene list. This may simply be due to the lack of detection of situs inversus totalis 53, or reflect a redundancy in gene function, or it may be that the majority of mutations in these genes are simply not detected in humans because they are masked by first trimester spontaneous abortions, which are known to have a high level of heart defects 48.
The impact of lethal mutations on detection of genes associated with heart development would suggest that mutations in these genes would only be detected in individuals with mutations with relatively minor impact on gene function. This idea is supported by the recent identification of multiple ‘minor’ heterozygous mutations within a functional network in three patients with transposition of the great arteries. All of these genes either participate or cooperate within the Nodal signaling pathway 54 and the carriers of single mutations exhibit no heart or laterality defects. The impact of ‘minor’ mutations, such as these, may explain the contribution of ‘genetic modifiers’ to congenital heart defects with variable penetrance within a family 55, or may suggest a polygenic basis for some of these diseases 56. This is supported by model organism data, which provides evidence of multigenic origins for congenital heart disease 56. However, model organisms are rarely used to examine the impact of genetic modifiers on heart development, as the majority of model organisms are inbred and examination of mutations leading to ‘minor’ phenotypic variations is often not viewed with the same level of interest as the more extreme heart development defects.
Next Generation Sequencing (NGS) has the potential to identity many more instances of multiple mutations in genes which are functionally linked through a specific pathway. However, teasing out which gene mutations are contributing to a disease, as a genetic modifier or as the causative gene variant, and which are not involved in the disease, is likely to take considerable time. Gene Ontology, KEGG and Reactome pathways, along with protein interaction networks have the potential to inform the process of identifying genetic variants associated with heart defect risk through the identification of pathways and networks which are common to the genes associated with the risk gene variants. Consequently, interpretation of NGS data will be greatly improved with full annotation of the candidate genes involved. The identification of these risk gene variants is likely to be of considerable value to those patients seeking prenatal diagnosis. In addition, the identification of more genes associated with heart defects will also help clarify the conserved and divergent heart development pathways that exist between humans and key model organisms.
Conclusions
This study demonstrates that full annotation, using GO, of a set of genes known to be associated with early stages of heart development in zebrafish can be used to confirm functional conservation of the role of these genes in a variety of developmental processes. While this study supports the assertion of gene function based on orthology between genes, it also identifies that for some genes there is no direct evidence for their conserved involvement in specific developmental processes through evolution. Consequently, for evolutionary studies, manual annotation of the genome of individual species will be necessary to enable a bioinformatics approach to investigating the evolution of developmental processes.
Acknowledgements
Many thanks to Dr. Jim Hu and Dr Mary Dolan for their help with the creation of Figure 1 and Dr Fotios Drenos for creating the Venn diagrams in Figure 2.
Funding Statement
The Cardiovascular GO Annotation Initiative is funded by the British Heart Foundation (SP/07/007/23671 and RG/13/5/30112) and PJT is supported by the British Heart Foundation (RG08/014). The Zebrafish Model Organism Database is funded by the National Human Genome Research Institute (P41 HG002659) of the National Institutes of Health.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
v2; ref status: indexed
References
- 1.Bakkers J: Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovasc Res. 2011;91(2):279–288 10.1093/cvr/cvr098 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Manner J: The anatomy of cardiac looping: a step towards the understanding of the morphogenesis of several forms of congenital cardiac malformations. Clin Anat. 2009;22(1):21–35 10.1002/ca.20652 [DOI] [PubMed] [Google Scholar]
- 3.Schlueter J, Brand T: Left-right axis development: examples of similar and divergent strategies to generate asymmetric morphogenesis in chick and mouse embryos. Cytogenet Genome Res. 2007;117(1–4):256–267 10.1159/000103187 [DOI] [PubMed] [Google Scholar]
- 4.Shiraishi I, Ichikawa H: Human heterotaxy syndrome - from molecular genetics to clinical features, management, and prognosis. Circ J. 2012;76(9):2066–2075 10.1253/circj.CJ-12-0957 [DOI] [PubMed] [Google Scholar]
- 5.Ahmad N, Long S, Rebagliati M: A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry. Trends Cardiovasc Med. 2004;14(2):43–49 10.1016/j.tcm.2003.11.001 [DOI] [PubMed] [Google Scholar]
- 6.Chen JN, van Eeden FJ, Warren KS, et al. : Left-right pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish. Development. 1997;124(21):4373–4382 [DOI] [PubMed] [Google Scholar]
- 7.Ferrante MI, Romio L, Castro S, et al. : Convergent extension movements and ciliary function are mediated by ofd1, a zebrafish orthologue of the human oral-facial-digital type 1 syndrome gene. Hum Mol Genet. 2009;18(2):289–303 10.1093/hmg/ddn356 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Speder P, Petzoldt A, Suzanne M, et al. : Strategies to establish left/right asymmetry in vertebrates and invertebrates. Curr Opin Genet Dev. 2007;17(4):351–358 10.1016/j.gde.2007.05.008 [DOI] [PubMed] [Google Scholar]
- 9.Lenhart KF, Holtzman NG, Williams JR, et al. : Integration of Nodal and BMP Signals in the Heart Requires FoxH1 to Create Left-Right Differences in Cell Migration Rates That Direct Cardiac Asymmetry. PLoS Genet. 2013;9(1):e1003109 10.1371/journal.pgen.1003109 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Baker K, Holtzman NG, Burdine RD: Direct and indirect roles for Nodal signaling in two axis conversions during asymmetric morphogenesis of the zebrafish heart. Proc Natl Acad Sci U S A. 2008;105(37):13924–13929 10.1073/pnas.0802159105 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Rohr S, Otten C, Abdelilah-Seyfried S: Asymmetric involution of the myocardial field drives heart tube formation in zebrafish. Circ Res. 2008;102(2):e12–19 10.1161/CIRCRESAHA.107.165241 [DOI] [PubMed] [Google Scholar]
- 12.de Campos-Baptista MI, Holtzman NG, Yelon D, et al. : Nodal signaling promotes the speed and directional movement of cardiomyocytes in zebrafish. Dev Dyn. 2008;237(12):3624–3633 10.1002/dvdy.21777 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Smith KA, Chocron S, von der Hardt S, et al. : Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells. Dev Cell. 2008;14(2):287–297 10.1016/j.devcel.2007.11.015 [DOI] [PubMed] [Google Scholar]
- 14.Francescatto L, Rothschild SC, Myers AL, et al. : The activation of membrane targeted CaMK-II in the zebrafish Kupffer’s vesicle is required for left-right asymmetry. Development. 2010;137(16):2753–2762 10.1242/dev.049627 [DOI] [PubMed] [Google Scholar]
- 15.Hashimoto H, Rebagliati M, Ahmad N, et al. : The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish. Development. 2004;131(8):1741–1753 10.1242/dev.01070 [DOI] [PubMed] [Google Scholar]
- 16.Jopling C, van Geemen D, den Hertog J: Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects. PLoS Genet. 2007;3(12):e225 10.1371/journal.pgen.0030225 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Lee HC, Tsai JN, Liao PY, et al. : Glycogen synthase kinase 3 alpha and 3 beta have distinct functions during cardiogenesis of zebrafish embryo. BMC Dev Biol. 2007;7:93 10.1186/1471-213X-7-93 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Liu DW, Hsu CH, Tsai SM, et al. : A variant of fibroblast growth factor receptor 2 (Fgfr2) regulates left-right asymmetry in zebrafish. PLoS One. 2011;6(7):e21793 10.1371/journal.pone.0021793 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Long S, Ahmad N, Rebagliati M: The zebrafish nodal-related gene southpaw is required for visceral and diencephalic left-right asymmetry. Development. 2003;130(11):2303–2316 10.1242/dev.00436 [DOI] [PubMed] [Google Scholar]
- 20.Monteiro R, van Dinther M, Bakkers J, et al. : Two novel type II receptors mediate BMP signalling and are required to establish left-right asymmetry in zebrafish. Dev Biol. 2008;315(1):55–71 10.1016/j.ydbio.2007.11.038 [DOI] [PubMed] [Google Scholar]
- 21.Muto A, Calof AL, Lander AD, et al. : Multifactorial origins of heart and gut defects in nipbl-deficient zebrafish, a model of Cornelia de Lange Syndrome. PLoS Biol. 2011;9(10):e1001181 10.1371/journal.pbio.1001181 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Oishi I, Kawakami Y, Raya A, et al. : Regulation of primary cilia formation and left-right patterning in zebrafish by a noncanonical Wnt signaling mediator, duboraya. Nat Genet. 2006;38(11):1316–1322 10.1038/ng1892 [DOI] [PubMed] [Google Scholar]
- 23.Ravanelli AM, Klingensmith J: The actin nucleator Cordon-bleu is required for development of motile cilia in zebrafish. Dev Biol. 2011;350(1):101–111 10.1016/j.ydbio.2010.11.023 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Schneider I, Schneider PN, Derry SW, et al. : Zebrafish Nkd1 promotes Dvl degradation and is required for left-right patterning. Dev Biol. 2010;348(1):22–33 10.1016/j.ydbio.2010.08.040 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Tian T, Zhao L, Zhang M, et al. : Both foxj1a and foxj1b are implicated in left-right asymmetric development in zebrafish embryos. Biochem Biophys Res Commun. 2009;380(3):537–542 10.1016/j.bbrc.2009.01.111 [DOI] [PubMed] [Google Scholar]
- 26.Zhou W, Dai J, Attanasio M, et al. : Nephrocystin-3 is required for ciliary function in zebrafish embryos. Am J Physiol Renal Physiol. 2010;299(1):F55–62 10.1152/ajprenal.00043.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ashburner M, Ball CA, Blake JA, et al. : Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25(1):25–29 10.1038/75556 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Biben C, Harvey RP: Homeodomain factor Nkx2-5 controls left/right asymmetric expression of bHLH gene eHand during murine heart development. Genes Dev. 1997;11(11):1357–1369 10.1101/gad.11.11.1357 [DOI] [PubMed] [Google Scholar]
- 29.Chen CM, Norris D, Bhattacharya S: Transcriptional control of left-right patterning in cardiac development. Pediatr Cardiol. 2010;31(3):371–377 10.1007/s00246-009-9610-3 [DOI] [PubMed] [Google Scholar]
- 30.Ramsdell AF: Left-right asymmetry and congenital cardiac defects: getting to the heart of the matter in vertebrate left-right axis determination. Dev Biol. 2005;288(1):1–20 10.1016/j.ydbio.2005.07.038 [DOI] [PubMed] [Google Scholar]
- 31.Nakhleh N, Francis R, Giese RA, et al. : High prevalence of respiratory ciliary dysfunction in congenital heart disease patients with heterotaxy. Circulation. 2012;125(18):2232–2242 10.1161/CIRCULATIONAHA.111.079780 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Khodiyar VK, Hill DP, Howe D, et al. : The representation of heart development in the gene ontology. Dev Biol. 2011;354(1):9–17 10.1016/j.ydbio.2011.03.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Deegan née Clark JI, Dimmer EC, Mungall CJ: Formalization of taxon-based constraints to detect inconsistencies in annotation and ontology development. BMC Bioinformatics. 2010;11(1):530 10.1186/1471-2105-11-530 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Eyre TA, Wright MW, Lush MJ, et al. : HCOP: a searchable database of human orthology predictions. Brief Bioinform. 2007;8(1):2–5 10.1093/bib/bbl030 [DOI] [PubMed] [Google Scholar]
- 35.Kent WJ: BLAT--the BLAST-like alignment tool. Genome Res. 2002;12(4):656–664 10.1101/gr.229202 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Magrane M, Consortium U: UniProt Knowledgebase: a hub of integrated protein data. Database (Oxford). 2011;2011:bar009 10.1093/database/bar009 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Lowe LA, Yamada S, Kuehn MR: Genetic dissection of nodal function in patterning the mouse embryo. Development. 2001;128(10):1831–1843 [DOI] [PubMed] [Google Scholar]
- 38.Khodiyar VK, Dimmer EC, Huntley RP, et al. : Fundamentals of gene ontology functional annotation. In: Knowledge-based Bioinformatics Edited by Alterovitz G, Ramoni M. Boston, Massachusetts: Wiley;2010;171–208 10.1002/9780470669716.ch8 [DOI] [Google Scholar]
- 39.Eppig JT, Blake JA, Bult CJ, et al. : The Mouse Genome Database (MGD): comprehensive resource for genetics and genomics of the laboratory mouse. Nucleic Acids Res. 2012;40(Database issue):D881–886 10.1093/nar/gkr974 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Binns D, Dimmer E, Huntley R, et al. : QuickGO: a web-based tool for Gene Ontology searching. Bioinformatics. 2009;25(22):3045–3046 10.1093/bioinformatics/btp536 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Srivatsan S, Peng SL: Cutting edge: Foxj1 protects against autoimmunity and inhibits thymocyte egress. J Immunol. 2005;175(12):7805–7809 [DOI] [PubMed] [Google Scholar]
- 42.Pan J, You Y, Huang T, et al. : RhoA-mediated apical actin enrichment is required for ciliogenesis and promoted by Foxj1. J Cell Sci. 2007;120(Pt 11):1868–1876 10.1242/jcs.005306 [DOI] [PubMed] [Google Scholar]
- 43.Kim HJ, Schleiffarth JR, Jessurun J, et al. : Wnt5 signaling in vertebrate pancreas development. BMC Biol. 2005;3:23 10.1186/1741-7007-3-23 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Nakamura T, Hamada H: Left-right patterning: conserved and divergent mechanisms. Development. 2012;139(18):3257–3262 10.1242/dev.061606 [DOI] [PubMed] [Google Scholar]
- 45.MacGrogan D, Nus M, de la Pompa JL: Notch signaling in cardiac development and disease. Curr Top Dev Biol. 2010;92:333–365 10.1016/S0070-2153(10)92011-5 [DOI] [PubMed] [Google Scholar]
- 46.Abu-Issa R, Kirby ML: Heart field: from mesoderm to heart tube. Annu Rev Cell Dev Biol. 2007;23:45–68 10.1146/annurev.cellbio.23.090506.123331 [DOI] [PubMed] [Google Scholar]
- 47.Skarnes WC, Rosen B, West AP, et al. : A conditional knockout resource for the genome-wide study of mouse gene function. Nature. 2011;474(7351):337–342 10.1038/nature10163 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.van der Linde D, Konings EE, Slager MA, et al. : Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis. J Am Coll Cardiol. 2011;58(21):2241–2247 10.1016/j.jacc.2011.08.025 [DOI] [PubMed] [Google Scholar]
- 49.Kosaki K, Bassi MT, Kosaki R, et al. : Characterization and mutation analysis of human LEFTY A and LEFTY B homologues of murine genes implicated in left-right axis development. Am J Hum Genet. 1999;64(3):712–721 10.1086/302289 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Kosaki R, Gebbia M, Kosaki K, et al. : Left-right axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB. Am J Med Genet. 1999;82(1):70–76 [DOI] [PubMed] [Google Scholar]
- 51.Britz-Cunningham SH, Shah MM, Zuppan CW, et al. : Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality. N Engl J Med. 1995;332(20):1323–1329 10.1056/NEJM199505183322002 [DOI] [PubMed] [Google Scholar]
- 52.Gebbia M, Ferrero GB, Pilia G, et al. : X-linked situs abnormalities result from mutations in ZIC3. Nat Genet. 1997;17(3):305–308 10.1038/ng1197-305 [DOI] [PubMed] [Google Scholar]
- 53.Bataille S, Demoulin N, Devuyst O, et al. : Association of PKD2 (polycystin 2) mutations with left-right laterality defects. Am J Kidney Dis. 2011;58(3):456–460 10.1053/j.ajkd.2011.05.015 [DOI] [PubMed] [Google Scholar]
- 54.De Luca A, Sarkozy A, Consoli F, et al. : Familial transposition of the great arteries caused by multiple mutations in laterality genes. Heart. 2010;96(9):673–677 10.1136/hrt.2009.181685 [DOI] [PubMed] [Google Scholar]
- 55.Rankin J, Auer-Grumbach M, Bagg W, et al. : Extreme phenotypic diversity and nonpenetrance in families with the LMNA gene mutation R644C. Am J Med Genet A. 2008;146A(12):1530–1542 10.1002/ajmg.a.32331 [DOI] [PubMed] [Google Scholar]
- 56.Stankunas K, Shang C, Twu KY, et al. : Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease. Circ Res. 2008;103(7):702–709 10.1161/CIRCRESAHA.108.175489 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Lin X, Xu X: Distinct functions of Wnt/beta-catenin signaling in KV development and cardiac asymmetry. Development. 2009;136(2):207–217 10.1242/dev.029561 [DOI] [PubMed] [Google Scholar]
- 58.Slagle CE, Aoki T, Burdine RD: Nodal-dependent mesendoderm specification requires the combinatorial activities of FoxH1 and Eomesodermin. PLoS Genet. 2011;7(5):e1002072 10.1371/journal.pgen.1002072 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Johnson DW, Berg JN, Baldwin MA, et al. : Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nat Genet. 1996;13(2):189–195 10.1038/ng0696-189 [DOI] [PubMed] [Google Scholar]
- 60.Trembath RC, Thomson JR, Machado RD, et al. : Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med. 2001;345(5):325–334 10.1056/NEJM200108023450503 [DOI] [PubMed] [Google Scholar]
- 61.Groden J, Thliveris A, Samowitz W, et al. : Identification and characterization of the familial adenomatous polyposis coli gene. Cell. 1991;66(3):589–600 10.1016/0092-8674(81)90021-0 [DOI] [PubMed] [Google Scholar]
- 62.Bakrania P, Efthymiou M, Klein JC, et al. : Mutations in BMP4 cause eye brain, and digit developmental anomalies: overlap between the BMP4 and hedgehog signaling pathways. Am J Hum Genet. 2008;82(2):304–319 10.1016/j.ajhg.2007.09.023 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63.Suzuki S, Marazita ML, Cooper ME, et al. : Mutations in BMP4 are associated with subepithelial, microform, and overt cleft lip. Am J Hum Genet. 2009;84(3):406–411 10.1016/j.ajhg.2009.02.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.Lane KB, Machado RD, Pauciulo MW, et al. : Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. Nat Genet. 2000;26(1):81–84 10.1038/79226+J2 [DOI] [PubMed] [Google Scholar]
- 65.Panizzi JR, Becker-Heck A, Castleman VH, et al. : CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms. Nat Genet. 2012;44(6):714–719 10.1038/ng.2277 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 66.Blanchon S, Legendre M, Copin B, et al. : Delineation of CCDC39/CCDC40 mutation spectrum and associated phenotypes in primary ciliary dyskinesia. J Med Genet. 2012;49(6):410–416 10.1136/jmedgenet-2012-100867 [DOI] [PubMed] [Google Scholar]
- 67.Becker-Heck A, Zohn IE, Okabe N, et al. : The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation. Nat Genet. 2011;43(1):79–84 10.1038/ng.727 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 68.Duquesnoy P, Escudier E, Vincensini L, et al. : Loss-of-function mutations in the human ortholog of Chlamydomonas reinhardtii ODA7 disrupt dynein arm assembly and cause primary ciliary dyskinesia. Am J Hum Genet. 2009;85(6):890–896 10.1016/j.ajhg.2009.11.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 69.Loges NT, Olbrich H, Becker-Heck A, et al. : Deletions and point mutations of LRRC50 cause primary ciliary dyskinesia due to dynein arm defects. Am J Hum Genet. 2009;85(6):883–889 10.1016/j.ajhg.2009.10.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 70.Reardon W, Winter RM, Rutland P, et al. : Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet. 1994;8(1):98–103 10.1038/ng0994-98 [DOI] [PubMed] [Google Scholar]
- 71.Jabs EW, Li X, Scott AF, et al. : Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat Genet. 1994;8(3):275–279 10.1038/ng1194-275 [DOI] [PubMed] [Google Scholar]
- 72.Wang B, Yan J, Mi R, et al. : Forkhead box H1 (FOXH1) sequence variants in ventricular septal defect. Int J Cardiol. 2010;145(1):83–85 10.1016/j.ijcard.2009.05.030 [DOI] [PubMed] [Google Scholar]
- 73.Kott E, Duquesnoy P, Copin B, et al. : Loss-of-function mutations in LRRC6, a gene essential for proper axonemal assembly of inner and outer dynein arms, cause primary ciliary dyskinesia. Am J Hum Genet. 2012;91(5):958–964 10.1016/j.ajhg.2012.10.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 74.Gillis LA, McCallum J, Kaur M, et al. : NIPBL mutational analysis in 120 individuals with Cornelia de Lange syndrome and evaluation of genotype-phenotype correlations. Am J Hum Genet. 2004;75(4):610–623 10.1086/424698 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 75.Krantz ID, McCallum J, DeScipio C, et al. : Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat Genet. 2004;36(6):631–635 10.1038/ng1364 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 76.Guo J, Cagatay T, Zhou G, et al. : Mutations in the human naked cuticle homolog NKD1 found in colorectal cancer alter Wnt/Dvl/beta-catenin signaling. PLoS One. 2009;4(11):e7982 10.1371/journal.pone.0007982 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 77.Bergmann C, Fliegauf M, Bruchle NO, et al. : Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia. Am J Hum Genet. 2008;82(4):959–970 10.1016/j.ajhg.2008.02.017 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 78.Olbrich H, Fliegauf M, Hoefele J, et al. : Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis. Nat Genet. 2003;34(4):455–459 10.1038/ng1216 [DOI] [PubMed] [Google Scholar]
- 79.Fiskerstrand T, Houge G, Sund S, et al. : Identification of a gene for renal-hepatic-pancreatic dysplasia by microarray-based homozygosity mapping. J Mol Diagn. 2010;12(1):125–131 10.2353/jmoldx.2010.090033 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 80.Mochizuki T, Wu G, Hayashi T, et al. : PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science. 1996;272(5266):1339–1342 10.1126/science.272.5266.1339 [DOI] [PubMed] [Google Scholar]
- 81.Digilio MC, Conti E, Sarkozy A, et al. : Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene. Am J Hum Genet. 2002;71(2):389–394 10.1086/341528 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 82.Tartaglia M, Niemeyer CM, Fragale A, et al. : Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet. 2003;34(2):148–150 10.1038/ng1156 [DOI] [PubMed] [Google Scholar]
- 83.Tartaglia M, Mehler EL, Goldberg R, et al. : Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet. 2001;29(4):465–468 10.1038/ng772 [DOI] [PubMed] [Google Scholar]
- 84.Renfro DP, McIntosh BK, Venkatraman A, et al. : GONUTS: the Gene Ontology Normal Usage Tracking System. Nucleic Acids Res. 2012;40(Database issue):D1262–D1269 10.1093/nar/gkr907 [DOI] [PMC free article] [PubMed] [Google Scholar]