HGNC Approved Gene Symbol: TOP2B
SNOMEDCT: 1230295000;
Cytogenetic location: 3p24.2 Genomic coordinates (GRCh38): 3:25,597,905-25,664,907 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 3p24.2 | B-cell immunodeficiency, distal limb anomalies, and urogenital malformations | 609296 | Autosomal dominant | 3 |
The TOP2B gene encodes a type II topoisomerase, an enzyme that generates transient DNA double-strand breaks to relieve topological stress during DNA replication and transcription (summary by Broderick et al., 2019 and Papapietro et al., 2020).
Human cells contain 2 topoisomerase II isozymes: alpha (TOP2A; 126430) and beta (TOP2B). Chung et al. (1989) sequenced human cDNAs that had been isolated by screening a cDNA library derived from a mechlorethamine-resistant Burkitt lymphoma cell line (Raji-HN2) with a Drosophila Topo II cDNA. They identified 2 classes of sequence, which represent the TOP2 isoenzymes TOP2A and TOP2B. Southern blot analysis indicated that the TOP2A and TOP2B cDNAs are derived from distinct genes. Northern blot analysis using a TOP2B-specific probe detected a 6.5-kb transcript in the human cell line U937. Antibodies against a TOP2B peptide recognized a 180-kD protein in U937 cell lysates.
By screening a human B-cell cDNA library with a partial TOP2B cDNA, Jenkins et al. (1992) isolated the complete coding region of the TOP2B gene. The deduced 1,621-amino acid TOP2B protein shares 72% sequence similarity with TOP2A. With the exception of a short region at the extreme N terminus, the sequences of the highly conserved N-terminal ATPase domain and the central breakage/reunion domain of the TOP2A and TOP2B proteins are colinear, with no insertions or deletions required for alignment. In contrast, the C-terminal domains of TOP2A and TOP2B differ markedly both in size and in sequence. The C-terminal domain of TOP2B contains a number of potential phosphorylation sites. Jenkins et al. (1992) examined the expression of TOP2A and TOP2B mRNAs in a panel of human tumor cell lines of different origin using an RNase protection assay. TOP2A and TOP2B were expressed in all cell lines examined, including those of hemopoietic, epithelial, and fibroblast origin, but at levels that differed markedly between different cell types. In particular, TOP2B was expressed at a high level in U937 cells. There was no obvious relationship between the levels of expression of the TOP2A and TOP2B genes.
Using Western blot analysis, Xia et al. (2019) assessed Top2b expression in mouse cochleae and in the HEI-OC1 immortalized auditory cell line, and detected Top2b in the basilar membrane and modiolus of mouse cochleae as well as in the HEI-OC1 cells.
Ju et al. (2006) reported that the signal-dependent activation of gene transcription by nuclear receptors and other classes of DNA binding transcription factors, including AP1 (see 165160), requires TOP2B-dependent, transient, site-specific double-stranded DNA (dsDNA) break formation. Subsequent to the break, poly(adenosine diphosphate-ribose) polymerase-1 (PAP1; 173870) enzymatic activity is induced, which is required for a nucleosome-specific histone H1-high-mobility group B exchange event and for local changes of chromatin architecture. Ju et al. (2006) concluded that their data mechanistically link TOP2B-dependent dsDNA breaks and the components of the DNA damage and repair machinery in regulated gene transcription.
Perillo et al. (2008) analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and showed that a DNA-bound estrogen receptor (see ESRA, 133430) directs transcription by participating in bending chromatin to contact the RNA polymerase II (see 180660) recruited to the promoter. This process is driven by receptor-targeted demethylation of H3K9 (see 602810) at both enhancer and promoter sites and is achieved by activation of resident LSD1 (609132) demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase-1 (601982) and topoisomerase II-beta, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Perillo et al. (2008) concluded that their data showed a strategy that uses controlled DNA damage and repair to guide productive transcription.
Fusions between the TMPRSS2 (602060) and ERG (165080) genes are among the most common oncogenic rearrangements observed in human cancer. Haffner et al. (2010) showed that androgen signaling promotes corecruitment of androgen receptor (AR; 313700) and TOP2B to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated double-strand breaks. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process that required TOP2B and components of the double-strand break repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia cells showed strong coexpression of AR and TOP2B. Haffner et al. (2010) concluded that their findings implicated androgen-induced TOP2B-mediated double-strand breaks in generating TMPRSS2-ERG rearrangements.
King et al. (2013) found that topotecan, a topoisomerase-1 (TOP1; 126420) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kb. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genomewide in neurons, King et al. (2013) found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence autism spectrum disorder (209850) candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. King et al. (2013) concluded that chemicals and genetic mutations that impair topoisomerases could commonly contribute to autism spectrum disorders and other neurodevelopmental disorders.
Lang et al. (1998) reported the complete structures of the TOP2A and TOP2B genes. The TOP2B gene spans at least 49 kb and contains 36 exons.
Crystal Structure
Wu et al. (2011) presented the crystal structure of a large fragment of human TOP2-beta complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations.
By study of somatic cell hybrids with a TOP2B cDNA, Tan et al. (1992) mapped that gene to human chromosome 3. Linkage studies in the CEPH families, performed with a RFLP related to the TOP2B gene, suggested that the gene is located on 3p. By in situ hybridization, Jenkins et al. (1992) mapped the TOP2B gene to 3p24.
In 4 patients from 3 unrelated families with B-cell immunodeficiency, distal limb anomalies, and urogenital malformations (BILU; 609296), Broderick et al. (2019) identified heterozygous mutations in the TOP2B gene (126431.0001-126431.0003). The mutations, which were found by exome sequencing and confirmed by Sanger sequencing, segregated with the disorder in one family and occurred de novo in the other 2 probands. All mutations affected residues in the functional TOPRIM domain. Detailed in vitro studies in yeast showed that the mutations were unable to rescue growth defects and acted in a dominant-negative manner when coexpressed with wildtype TOP2B. The evidence suggested that the mutations caused a partial loss-of-function effect. Mutant mice carrying a heterozygous E587del mutation (126431.0001) recapitulated the BILU phenotype (see ANIMAL MODEL).
In a 13-year-old boy with BILU, Erdos et al. (2021) identified a de novo heterozygous E587del mutation in the TOP2B gene. The mutation was found by whole-exome sequencing and confirmed by Sanger sequencing. Functional studies of the variant were not performed.
In 4 patients from 2 unrelated families (families A and B) with BILU, Papapietro et al. (2020) identified a heterozygous missense mutation in the TOP2B gene (A485P; 126431.0004). The mutation, which was found by whole-exome sequencing and Sanger sequencing, respectively, segregated with the disorder in the families. It was not present in the gnomAD database. Patient-derived cells showed decreased TOP2B levels compared to controls, suggesting that the mutant protein is unstable. In vitro functional expression studies demonstrated that the mutation caused a more than 10-fold reduction in enzymatic activity compared to wildtype, and acted in a dominant-negative manner when coexpressed with wildtype TOP2B and TOP2A (126430). Family A, from Cyprus, was previously reported by Tischkowitz et al. (2004), and family B, from France, was reported by Edery et al. (2001).
Functional Studies of TOP2B Mutations
Broderick et al. (2022) reprogrammed cells from Hoffman syndrome patients with S483L (126431.0002) or G633S (126431.0003) mutations in TOP2B to generate induced pluripotent stem cells (iPSCs). The mutations did not affect maintenance of PSCs, but TOP2B had reduced decatenation activity in iPSCs. Mutant iPSCs could differentiate into natural killer (NK) cells, but they remained functionally impaired with reduced cell-specific cytotoxicity. Based on these findings and studies in mouse models (see ANIMAL MODEL), the authors concluded that Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function.
Associations Pending Confirmation
For a discussion of a possible association between autosomal dominant nonsyndromic hearing loss (see 124900) and variation in the TOP2B gene, see 126431.0005.
Yang et al. (2000) disrupted the murine Top2b gene by homologous recombination. Heterozygous mice were phenotypically indistinguishable from their wildtype littermates, but homozygous Top2b -/- embryos from intercrosses of the heterozygotes were dead at birth. Neurogenesis was normal in Top2b mutant mice, but motor axons failed to contact skeletal muscles and sensory axons failed to enter the spinal cord. Despite an absence of innervation, clusters of acetylcholine receptors were concentrated in the central region of skeletal muscles, thereby revealing patterning mechanisms that are autonomous to skeletal muscle. The defects in motor axon growth in Top2b -/- mice resulted in the breathing impairment and death of the pups shortly after birth.
Broderick et al. (2019) found that Top2b-null mice had B-cell developmental defects affecting multiple stages of development likely due to transcriptional defects. These mutant mice had altered splenic follicle structure with reduced marginal zone and follicular B-cell zones; immunophenotyping showed decreased B cells at all stages of development. Mutant mice failed to mount an antibody response to vaccination and B cells failed to proliferate in response to stimulation, indicating deficits in B-cell function. Mice carrying a heterozygous E587del mutation (126431.0001) recapitulated the BILU phenotype. They had impaired antibody response associated with defects in B-cell development and function, decreased expression of B-cell transcription factors Pax5 (167414) and Foxo1 (136533), and increased DNA breaks limited to B cells. T-cell proliferation and function was normal.
Broderick et al. (2022) generated knockin mice heterozygous for the murine equivalent of the human TOP2B EE587E mutation (126431.0001) that causes Hoffman syndrome. Mutant mice displayed NK-cell defects, in addition to B-cell defects. Mutant NK cells exhibited defective progression to the mature NK-cell stage, leading to reduced cytotoxicity. Moreover, expression of several transcription factors associated with generation of mature NK cells was significantly reduced in mutant NK cells.
Xia et al. (2019) generated zebrafish morphants with knockdown of top2b, and observed fewer and smaller lateral line neuromasts at 48 hours postfertilization (hpf) in the morphants compared to wildtype zebrafish. In addition, the morphants showed reduced numbers of hair cells and supporting cells, and cilia on the hair cells were fewer, shorter, and thinner than in wildtype zebrafish. Human TOP2B mRNA rescued the morpholino-induced phenotypes. RNA-seq profiling in morphants with top2b knockdown showed differential expression of the hearing development-associated PI3K (see 171834)-Akt (164730) pathway. RT-qPCR and Western blot analysis showed reduced expression of akt, and akt phosphorylation was also reduced in morphants at 48 hpf. Expression of the downstream cell proliferation-associated genes cyclind1 and myca was also reduced in morphants, and BrdU-positive cells were fewer in morphants than wildtype embryos. The authors suggested that top2b knockdown results in decreased cell proliferation via inhibition of the PI3K-Akt signaling pathway, resulting in hearing loss.
In a mother and daughter (family 1) with B-cell immunodeficiency, distal limb anomalies, and urogenital malformations (BILU; 609296) previously reported by Hugle et al. (2011), Broderick et al. (2019) identified a heterozygous 3-bp in-frame deletion (c.1761_1763delAGA) in the TOP2B gene, resulting in the deletion of a residue (Glu587del, E587del) in the TOPRIM domain. The mutation was found by exome sequencing and confirmed by Sanger sequencing. Yeast complementation studies indicated that the mutation caused a loss of function with a dominant-negative effect. Mice carrying a heterozygous E587del mutation recapitulated the BILU phenotype. They had impaired antibody response associated with defects in B-cell development and function, decreased expression of B-cell transcription factors Pax5 (167414) and Foxo1 (136533), and increased DNA breaks limited to B cells. T-cell proliferation and function was normal. The authors referred to the mutation as EE587E.
In a 13-year-old boy with BILU, Erdos et al. (2021) identified a de novo heterozygous c.1761delAGA mutation in the TOP2B gene. The mutation was found by whole-exome sequencing and confirmed by Sanger sequencing. Functional studies of the variant were not performed.
In a girl (family 2) with B-cell immunodeficiency, distal limb anomalies, and urogenital malformations (BILU; 609296) previously reported by Hoffman et al. (2001), Broderick et al. (2019) identified a de novo heterozygous c.1448C-T transition in the TOP2B gene, resulting in a ser483-to-leu (S483L) substitution in the TOPRIM domain. The mutation was found by exome sequencing and confirmed by Sanger sequencing. Yeast complementation studies indicated that the mutation caused a loss of function with a dominant-negative effect.
In a girl (family 3) with B-cell immunodeficiency, distal limb anomalies, and urogenital malformations (BILU; 609296) previously reported by Kallish et al. (2011), Broderick et al. (2019) identified a de novo heterozygous c.1897G-A transition in the TOP2B gene, resulting in a gly633-to-ser (G633S) substitution in the TOPRIM domain. The mutation was found by exome sequencing and confirmed by Sanger sequencing. Yeast complementation studies indicated that the mutation caused a loss of function with a dominant-negative effect.
In 4 patients from 2 unrelated families (families A and B) B-cell immunodeficiency, distal limb anomalies, and urogenital malformations (BILU; 609296), Papapietro et al. (2020) identified a heterozygous c.1453G-C transversion (c.1453G-C, ENST00000435706) in the TOP2B gene, resulting in an ala485-to-pro (A485P) substitution at a conserved residue in the TOPRIM domain. The mutation, which was found by whole-exome sequencing and Sanger sequencing, respectively, segregated with the disorder in the families. It was not present in the gnomAD database. Patient-derived cells showed decreased TOP2B levels compared to controls, suggesting that the mutant protein is unstable. In vitro functional expression studies demonstrated that the mutation caused a more than 10-fold reduction in enzymatic activity compared to wildtype, and acted in a dominant-negative manner when coexpressed with wildtype TOP2B and TOP2A (126430). Family A, from Cyprus, was previously reported by Tischkowitz et al. (2004), and family B, from France, was reported by Edery et al. (2001).
This variant is classified as a variant of unknown significance because its contribution to autosomal dominant nonsyndromic deafness (see 124900) has not been confirmed.
In affected individuals from a large 5-generation Chinese family (PD-01) segregating autosomal dominant nonsyndromic late-onset hearing loss, who were negative for mutation in common deafness-associated genes, Xia et al. (2019) performed whole-exome sequencing and identified heterozygosity for a c.4837G-C transition (c.4837G-C, NM_001068) in exon 36 of the TOP2B gene, resulting in an asp1613-to-his (D1613H) substitution at a highly conserved residue within the C-terminal domain. Sanger sequencing confirmed the mutation, which segregated fully with disease in 11 affected and 24 unaffected members of the family and was not found in 500 ethnicity-matched controls. The proband (III27) was a 41-year-old woman who had onset of hearing loss at age 33 and whose mother (II14) had noted hearing loss at age 35. The proband's daughter (IV5), who did not yet show hearing loss, was heterozygous for the mutation. Deafness in affected family members was characterized by postlingual low- and mid-frequency hearing loss, with a flat or valley audiogram configuration initially, which then progressed to all frequencies. Most affected individuals also experienced tinnitus. Screening of TOP2B in 66 sporadic Chinese cases of sensorineural hearing loss revealed heterozygosity for the D1613H substitution as well as an L721F variant and a K1435 deletion; the number of mutation-carrying individuals was not reported. The authors injected human TOP2B mRNA with the D1613H mutation into 1-cell zebrafish embryos and observed a reduction in neuromasts, hair cells, and supporting cells in the mutants compared to wildtype fish.
Hamosh (2023) noted that the D1613H variant was present in 16 of 19,412 East Asian alleles in gnomAD (v2.1.1), in heterozygosity only, for an allele frequency of 0.0008242 (March 14, 2023). The variant was not present in any other population in gnomAD.
Broderick, L., Clay, G. M., Blum, R. H., Liu, Y., McVicar, R., Papes, F., Booshehri, L. M., Cowell, I. G., Austin, C. A., Putnam, C. D., Kaufman, D. S. Disease-associated mutations in topoisomerase II-beta result in defective NK cells. J. Allergy Clin. Immun. 149: 2171-2176, 2022. [PubMed: 35063500] [Full Text: https://doi.org/10.1016/j.jaci.2021.12.792]
Broderick, L., Yost, S., Li, D., McGeough, M. D., Booshehri, L. M., Guaderrama, M., Brydges, S. D., Kucharova, K., Patel, N. C., Harr, M., Hakonarson, H., Zackai, E., and 11 others. Mutations in topoisomerase II-beta result in a B cell immunodeficiency. Nature Commun. 10: 3644, 2019. [PubMed: 31409799] [Full Text: https://doi.org/10.1038/s41467-019-11570-6]
Chung, T. D. Y., Drake, F. H., Tan, K. B., Per, S. R., Crooke, S. T., Mirabelli, C. K. Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. Proc. Nat. Acad. Sci. 86: 9431-9435, 1989. [PubMed: 2556712] [Full Text: https://doi.org/10.1073/pnas.86.23.9431]
Edery, P., Le Deist, F., Briard, M.-L., Debre, M., Munnich, A., Griscelli, C., Fischer, A., Lyonnet, S. B cell immunodeficiency, distal limb abnormalities, and urogenital malformations in a three generation family: a novel autosomal dominant syndrome? J. Med. Genet. 38: 488-492, 2001. [PubMed: 11476068] [Full Text: https://doi.org/10.1136/jmg.38.7.488]
Erdos, M., Lanyi, A., Balazs, G., Casanova, J.-L., Boisson, B., Marodi, L. Inherited TOP2B mutation: possible confirmation of mutational hotspots in the TOPRIM domain. J. Clin. Immun. 41: 817-819, 2021. [PubMed: 33459963] [Full Text: https://doi.org/10.1007/s10875-020-00963-8]
Haffner, M. C., Aryee, M. J., Toubaji, A., Esopi, D. M., Albadine, R., Gurel, B., Isaacs, W. B., Bova, G. S., Liu, W., Xu, J., Meeker, A. K., Netto, G., De Marzo, A. M., Nelson, W. G., Yegnasubramanian, S. Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Nature Genet. 42: 668-675, 2010. [PubMed: 20601956] [Full Text: https://doi.org/10.1038/ng.613]
Hamosh, A. Personal Communication. Baltimore, Md. 03/14/2023.
Hoffman, H. M., Bastian, J. F., Bird, L. M. Humoral immunodeficiency with facial dysmorphology and limb anomalies: a new syndrome. Clin. Dysmorph. 10: 1-8, 2001. [PubMed: 11152140] [Full Text: https://doi.org/10.1097/00019605-200101000-00001]
Hugle, B., Hoffman, H., Bird, L. M., Gebauer, C., Suchowerskyj, P., Sack, U., Kohlhase, J., Schuster, V. Hoffman syndrome: new patients, new insights. Am. J. Med. Genet. 155A: 149-153, 2011. [PubMed: 21204224] [Full Text: https://doi.org/10.1002/ajmg.a.33678]
Jenkins, J. R., Ayton, P., Jones, T., Davies, S. L., Simmons, D. L., Harris, A. L., Sheer, D., Hickson, I. D. Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Nucleic Acids Res. 20: 5587-5592, 1992. [PubMed: 1333583] [Full Text: https://doi.org/10.1093/nar/20.21.5587]
Ju, B.-G., Lunyak, V. V., Perissi, V., Garcia-Bassets, I., Rose, D. W., Glass, C. K., Rosenfeld, M. G. A topoisomerase II-beta-mediated dsDNA break required for regulated transcription. Science 312: 1798-1802, 2006. Note: Erratum: Science 332: 664 only, 2011. Erratum: Science 343: 839 only, 2014. [PubMed: 16794079] [Full Text: https://doi.org/10.1126/science.1127196]
Kallish, S., McDonald-McGinn, D. M., van Haelst, M. M., Bartlett, S. P., Katowitz, J. A., Zackai, E. H. Ablepharon-macrostomia syndrome--extension of the phenotype. Am. J. Med. Genet. 155A: 3060-3062, 2011. [PubMed: 22002929] [Full Text: https://doi.org/10.1002/ajmg.a.34287]
King, I. F., Yandava, C. N., Mabb, A. M., Hsiao, J. S., Huang, H.-S., Pearson, B. L., Calabrese, J. M., Starmer, J., Parker, J. S., Magnuson, T., Chamberlain, S. J., Philpot, B. D., Zylka, M. J. Topoisomerases facilitate transcription of long genes linked to autism. Nature 501: 58-62, 2013. [PubMed: 23995680] [Full Text: https://doi.org/10.1038/nature12504]
Lang, A. J., Mirski, S. E. L., Cummings, H. J., Yu, Q., Gerlach, J. H., Cole, S. P. C. Structural organization of the human TOP2A and TOP2B genes. Gene 221: 255-266, 1998. [PubMed: 9795238] [Full Text: https://doi.org/10.1016/s0378-1119(98)00468-5]
Papapietro, O., Chandra, A., Eletto, D., Inglott, S., Plagnol, V., Curtis, J., Maes, M., Alisaac, A., Albuquerque, A. S., Basseres, E., Hermine, O., Picard, C., Fischer, A., Durandy, A., Kracker, S., Burns, S. O., Cuchet-Lourenco, D., Okkenhaug, K., Nejentsev, S. Topoisomerase 2-beta mutation impairs early B-cell development. Blood 135: 1497-1501, 2020. [PubMed: 32128574] [Full Text: https://doi.org/10.1182/blood.2019003299]
Perillo, B., Ombra, M. N., Bertoni, A., Cuozzo, C., Sacchetti, S., Sasso, A., Chiariotti, L., Malorni, A., Abbondanza, C., Avvedimento, E. V. DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression. Science 319: 202-206, 2008. [PubMed: 18187655] [Full Text: https://doi.org/10.1126/science.1147674]
Tan, K. B., Dorman, T. E., Falls, K. M., Chung, T. D. Y., Mirabelli, C. K., Crooke, S. T., Mao, J. Topoisomerase II-alpha and topoisomerase II-beta genes: characterization and mapping to human chromosomes 17 and 3, respectively. Cancer Res. 52: 231-234, 1992. [PubMed: 1309226]
Tischkowitz, M., Goodman, F., Koliou, M., Webster, D., Edery, P., Jones, A., Wilson, L. C. Autosomal dominant B-cell immunodeficiency, distal limb anomalies and urogenital malformations (BILU syndrome)--report of a second family. Clin. Genet. 66: 550-555, 2004. [PubMed: 15521984] [Full Text: https://doi.org/10.1111/j.1399-0004.2004.00349.x]
Wu, C.-C., Li, T.-K., Farh, L., Lin, L.-Y., Lin, T.-S., Yu, Y.-J., Yen, T.-J., Chiang, C.-W., Chan, N.-L. Structural basis of type II topoisomerase inhibition by the anticancer drug etoposide. Science 333: 459-462, 2011. [PubMed: 21778401] [Full Text: https://doi.org/10.1126/science.1204117]
Xia, W., Hu, J., Ma, J., Huang, J., Jing, T., Deng, L., Zhang, J., Jiang, N., Ma, D., Ma, Z. Mutations in TOP2B cause autosomal-dominant hereditary hearing loss via inhibition of the PI3K-Akt signalling pathway. FEBS Lett. 593: 2008-2018, 2019. [PubMed: 31198993] [Full Text: https://doi.org/10.1002/1873-3468.13482]
Yang, X., Li, W., Prescott, E. D., Burden, S. J., Wang, J. C. DNA topoisomerase II-beta and neural development. Science 287: 131-134, 2000. [PubMed: 10615047] [Full Text: https://doi.org/10.1126/science.287.5450.131]