AOP ID and Title:

Authors

Vid Modic, Ziva Ramsak, Roman Li, Colette vom Berg, Anze Zupanic

Status

Background

The motivation behind building the AOP was methodological. Our team has recently developed molecular causal networks for developmental cardiotoxicity and neurotoxicity in zebrafish (doi.org/10.1021/acs.chemrestox.0c00095). These networks are highly curated, but rather large, going from adverse outcomes on the organ level upstream to wherever evidence takes us (many times finishing at what would be called MIEs). As there are many causal networks already present on the http://causalbionet.com/ (mostly for humans and for lung conditions), we were wondering how the rich knowledge available in causal pathways could be translated to AOPs. The AOP described in this document is one such example. 

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Overall Assessment of the AOP

References

Appendix 1

List of MIEs in this AOP

Event: 1647: GSK3beta inactivation

Short Name: GSK3beta inactivation

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Evidence for Perturbation by Stressor

CHIR99021

CHIR99021 inhibits GSK3beta (Wu et al., 2015) .

BIO (6-bromoindirubin-3’-oxime)

BIO (6-bromoindirubin-3’-oxime) inhibits GSK3beta (Wu et al., 2015).

Kenpaullone

Kenpaullone inhibits GSK3beta (Yang et al., 2013).

SB216763

SB216763 inhibits GSK3betat (Naujok, Lentes, Diekmann, Davenport, & Lenzen, 2014).

TWS119

TWS119 inhibits GSK3beta (Tang et al., 2018).

CHIR98014

CHIR98014 inhibits GSK3beta (Guerrero et al., 2014; Lian et al., 2014).

Domain of Applicability

Phosphorylation of GSK3beta is induced, which means the inactivation of GSK3beta, in Homo sapiens (Huang et al., 2019). Evidence for this KE is also provided for zebrafish (Anichtchik et al., 2008; Wang et al. 2018)

Key Event Description

The protein encoded by gsk3b gene is a serine-threonine kinase belonging to the glycogen synthase kinase subfamily. It is a negative regulator of glucose homeostasis and is involved in energy metabolism, inflammation, ER-stress, mitochondrial dysfunction, and apoptotic pathways. Defects in this gene have been associated with Parkinson disease and Alzheimer disease (GSK3B Gene - GeneCards). GSK3b has been identified within mitochondria (Hoshi et al., 1996), as well as in the cytoplasm (Anichtchik et al., 2008).

GSK3b kinase is constitutively active in resting cells and undergoes a rapid and transient inhibition in response to a number of external signals. GSK3b activity is regulated by site-specific phosphorylation. Full activity of GSK3b generally requires phosphorylation at tyrosine 216 (Tyr216), and conversely, phosphorylation at serine 9 (Ser9) inhibits GSK3b activity. Phosphorylation of Ser9 is the most common and important regulatory mechanism. Many kinases are capable of phosphorylating Ser9, including p70 S6 kinase, extracellular signal-regulated kinases (ERKs), p90Rsk (also called MAP-KAP kinase-1), protein kinase B (also called Akt), certainisoforms of protein kinase C (PKC) and cyclic AMP-dependent protein kinase (protein kinase A, PKA). In opposition to the inhibitory modulation of GSK3b that occurs by serine phosphorylation, tyrosine phosphorylation of GSK3b increases  the  enzyme’s  activity (Grimes and Jope, 2001; Luo, 2012).

・Glycogen synthase kinase 3beta (GSK3 beta) is inhibited by CHIR99021 (C. H. Li et al., 2017; C. C. Liu et al., 2016; Sineva & Pospelov, 2010).

・Glycogen synthase kinase 3beta (GSK3 beta) is inhibited by BIO (6-bromoindirubin-3’-oxime) (Mohammed et al., 2016; Sineva & Pospelov, 2010).

・Kenpaullone is a dual inhibitor for GSK3 alpha/beta and HPK1/GCK-like kinase (Y. M. Yang et al., 2013; Yao et al., 1999).

・CHIR and BIO treatments lead to a slight upregulation of the primary transcripts of the miR-302-367 cluster and miR-181 family of miRNAs, which activate Wnt/beta-catenin signaling (Y. Wu et al., 2015).

・SB216763 inhibits GSK3beta (Naujok et al., 2014).

・TWS119 inhibits GSK3beta (Tang et al., 2018).

・CHIR98014 inhibits GSK3beta (Guerrero et al., 2014; Lian et al., 2014).

How it is Measured or Detected

Inactivation of GSK3 beta is measured by Wnt/beta-catenin activity assay, in which the vector containing the firefly luciferase gene controlled by TCF/LEF binding sites is transfected in the cells (Naujok et al., 2014). Phosphorylation of GSK3beta at residue Ser9 leads to the inactivation of GSK3beta. Phosphorylation of GSK3 beta is measured by immunoblotting with anti-phospho-GSK3beta (Huang et al., 2019).

References

Anichtchik, O. et al. (2008) ‘Loss of PINK1 function affects development and results in neurodegeneration in zebrafish’, Journal of Neuroscience, 28(33), pp. 8199–8207. doi: 10.1523/JNEUROSCI.0979-08.2008

Grimes, C. A. and Jope, R. S. (2001) ‘The multifaceted roles of glycogen synthase kinase 3β in cellular signaling’, Progress in Neurobiology, 65(4), pp. 391–426. doi: 10.1016/S0301-0082(01)00011-9

GSK3B Gene - GeneCards | GSK3B Protein | GSK3B Antibody (no date). Available at: https://www.genecards.org/cgi-bin/carddisp.pl?gene=GSK3B (Accessed: 3 October 2021)

Guerrero, F., Herencia, C., Almaden, Y., Martinez-Moreno, J. M., Montes de Oca, A., Rodriguez-Ortiz, M. E., . . . Munoz-Castaneda, J. R. (2014). TGF-beta prevents phosphate-induced osteogenesis through inhibition of BMP and Wnt/beta-catenin pathways. PLoS One, 9(2), e89179. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/24586576. doi:10.1371/journal.pone.0089179

Hoshi, M. et al. (1996) Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3p3 in brain, Neurobiology

Huang, J. Q., Wei, F. K., Xu, X. L., Ye, S. X., Song, J. W., Ding, P. K., . . . Gong, L. Y. (2019). SOX9 drives the epithelial-mesenchymal transition in non-small-cell lung cancer through the Wnt/beta-catenin pathway. J Transl Med, 17(1), 143. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/31060551. doi:10.1186/s12967-019-1895-2

Li, C. H., Liu, C. W., Tsai, C. H., Peng, Y. J., Yang, Y. H., Liao, P. L., . . . Kang, J. J. (2017). Cytoplasmic aryl hydrocarbon receptor regulates glycogen synthase kinase 3 beta, accelerates vimentin degradation, and suppresses epithelial-mesenchymal transition in non-small cell lung cancer cells. Arch Toxicol, 91(5), 2165-2178. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/27752740. doi:10.1007/s00204-016-1870-0

Lian, X., Bao, X., Al-Ahmad, A., Liu, J., Wu, Y., Dong, W., . . . Palecek, S. P. (2014). Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of WNT signaling. Stem Cell Reports, 3(5), 804-816. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25418725. doi:10.1016/j.stemcr.2014.09.005

Liu, C. C., Cai, D. L., Sun, F., Wu, Z. H., Yue, B., Zhao, S. L., . . . Yan, D. W. (2016). FERMT1 mediates epithelial–mesenchymal transition to promote colon cancer metastasis via modulation of β-catenin transcriptional activity. Oncogene, 36, 1779. Retrieved from https://doi.org/10.1038/onc.2016.339. doi:10.1038/onc.2016.339 https://www.nature.com/articles/onc2016339 - supplementary-information

Luo, J. (2012) ‘The role of GSK3beta in the development of the central nervous system’, Front. Biol, 7(3), pp. 212–220. doi: 10.1007/s11515-012-1222-2

Mohammed, M. K., Shao, C., Wang, J., Wei, Q., Wang, X., Collier, Z., . . . Lee, M. J. (2016). Wnt/beta-catenin signaling plays an ever-expanding role in stem cell self-renewal, tumorigenesis and cancer chemoresistance. Genes Dis, 3(1), 11-40. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/27077077. doi:10.1016/j.gendis.2015.12.004

Naujok, O., Lentes, J., Diekmann, U., Davenport, C., & Lenzen, S. (2014). Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Res Notes, 7, 273. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/24779365. doi:10.1186/1756-0500-7-273

Sineva, G. S., & Pospelov, V. A. (2010). Inhibition of GSK3beta enhances both adhesive and signalling activities of beta-catenin in mouse embryonic stem cells. Biol Cell, 102(10), 549-560. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/20626347. doi:10.1042/BC20100016

Tang, Y. Y., Sheng, S. Y., Lu, C. G., Zhang, Y. Q., Zou, J. Y., Lei, Y. Y., . . . Hong, H. (2018). Effects of Glycogen Synthase Kinase-3beta Inhibitor TWS119 on Proliferation and Cytokine Production of TILs From Human Lung Cancer. J Immunother, 41(7), 319-328. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/29877972. doi:10.1097/CJI.0000000000000234

Wang, Z. et al. (2018) ‘The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos’, Differentiation, 99(December 2017), pp. 28–40. doi: 10.1016/j.diff.2017.12.005

Wu, Y., Liu, F., Liu, Y., Liu, X., Ai, Z., Guo, Z., & Zhang, Y. (2015). GSK3 inhibitors CHIR99021 and 6-bromoindirubin-3'-oxime inhibit microRNA maturation in mouse embryonic stem cells. Sci Rep, 5, 8666. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25727520. doi:10.1038/srep08666

Yang, Y. M., Gupta, S. K., Kim, K. J., Powers, B. E., Cerqueira, A., Wainger, B. J., . . . Rubin, L. L. (2013). A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell Stem Cell, 12(6), 713-726. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/23602540. doi:10.1016/j.stem.2013.04.003

Yao, Z., Zhou, G., Wang, X. S., Brown, A., Diener, K., Gan, H., & Tan, T. H. (1999). A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway. J Biol Chem, 274(4), 2118-2125. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/9890973.

Event: 1902: Repression of Gbx2 expression

Short Name: Repression of Gbx2 expression

AOPs Including This Key Event

Stressors

Biological Context

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

• Zebrafish embryos were treated with chemical inhibitors or activators of various signaling pathways, such as the Wnt, FGF, retinoic acid (RA), HH, BMP, Nodal, and Notch pathways, and examined gbx2 expression in the telencephalon. First, embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region . In embryos treated with BIO, a selective GSK3 inhibitor that activates Wnt signaling (Sato et al., 2004), gbx2 expression was specifically repressed in the telencephalon, but was unaffected or weakly activated in the isthmus and otic vesicle (OV). In embryos where FGF signaling was inhibited by SU5402, gbx2 was downregulated in the telencephalon and MHB, but its expression in the OV was little affected. Retinoic acid (RA) treatment strongly repressed gbx2 expression in the telencephalon, but not in the MHB and OV. These results suggest that gbx2-dependent telencephalon development is regulated by Wnt, FGF, and RA signaling (Z. Wang et al., 2018).
• To clarify the critical stages of previous study for gbx2 regulation in the telencephalon, chemical treatment started between 14 and 17 hpf and gbx2 expression was examined at 18 hpf. Alternatively, chemical treatment was started at 14 hpf and then embryos were washed between 15 and 18 hpf, cultured in the absence of chemicals, and gbx2 expression was examined at 18 hpf. Resuoults showed that the downregulation of gbx2 by BIO grew less significant as the start time was delayed, and the repression of gbx2 by BIO in the telencephalon became less prominent when the chemicals were removed earlier, suggesting that Wnt signaling remains effective throughout the 4-h period (14–18 hpf) and that the repressive effect of BIO is reversible. Similarly, SU5402  mediated repression of gbx2 expression in the telencephalon and MHB became less significant as the treatment start time was delayed from 14 hpf to 17 hpf, and gbx2 expression was gradually restored with earlier removal of the chemical, showing that FGF signaling is continuously required for gbx2 expression in the telencephalon. Essentially the same results were obtained with RA treatment in terms of gbx2 expression in the telencephalon (Z. Wang et al., 2018).

BIO (6-bromoindirubin-3’-oxime)

Embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region . In embryos treated with BIO, a selective GSK3 inhibitor that activates Wnt signaling (Sato et al., 2004), gbx2 expression was specifically repressed in the telencephalon, but was unaffected or weakly activated in the isthmus and otic vesicle (OV).

Retinoic acid

Zebrafish embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region. Retinoic acid (RA) treatment strongly repressed gbx2 expression in the telencephalon, but not in the MHB and OV.

su5402

Zebrafish embryos were treated with chemicals from 14 hpf to 18 hpf, immediately before the advent of gbx2 expression in the telencephalon, and then gbx2 expression was examined in this brain region. In embryos where FGF signaling was inhibited by SU5402, gbx2 was downregulated in the telencephalon and MHB, but its expression in the OV was little affected (Z. Wang et al., 2018).

Domain of Applicability

The gastrulation brain homebox (Gbx) group of transcription factor genes, composed of two genes, gbx1 and gbx2, in vertebrates, is also present in invertebrates (Chiang et al., 1995), and can be regarded as widely conserved among animals (Wang et al., 2018). Gbx2 functions in a variety of developmental processes after midbrain-hindbrain boundary (MHB) establishment. (Burroughs-Garcia et al., 2011) data demonstrate that the role of gbx2 in anterior hindbrain development is functionally conserved between zebrafish and mice. This gene was shown to be required for neural crest (NC) formation in mice (B. Li et al., 2009; Roeseler et al., 2012). In Xenopus gbx2 is the earliest factor for specifying neural crest (NC) cells, and that gbx2 is directly regulated by NC inducing signaling pathways, such as Wnt/β-catenin signaling (Li et al., 2009).

Key Event Description

During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain (Z. Wang et al., 2018). The genes encoding the Gbx-type homeodomain transcription factors have been identified in a variety of vertebrates, and are primarily implicated in the regulation of various aspects of vertebrate brain development (Nakayama et al., 2017). Gbx2 exhibits DNA-binding transcription factor activity, RNA polymerase II-specific. Involved in cerebellum development; iridophore differentiation; and telencephalon regionalization. Predicted to localize to nucleus. Is expressed in several structures, including midbrain hindbrain boundary neural keel; midbrain hindbrain boundary neural rod; midbrain neural rod; nervous system; and presumptive rhombomere 1. Orthologous to human GBX2 (gastrulation brain homeobox 2) (ZFIN Gene: Gbx2, n.d.)

Retinoids such as retinoic acid (RA) are chemopreventive and chemotherapeutic agents. One source of RA is vitamin A, derived from dietary β-carotene. RA regulates cell proliferation, differentiation, and morphogenesis (X. J. Wang et al., 2007). It inhibits tumorigenesis through suppression of cell growth and stimulation of cellular differentiation (Soprano et al., 2004). Also, RA promotes apoptosis (Atencia et al., 1997; Herget et al., 2000), and this property may contribute to its antitumor properties. The effects of retinoids are mediated by specific nuclear receptors, namely, retinoic acid receptors (RAR-α, -β, and -γ) and retinoid X receptors (RXR- α, - β, and - γ) (Rochette-Egly & Chambon, 2001). RXRs form heterodimers with RARs or other nuclear hormone receptors and function as transcriptional regulators. Retinoids can either activate or repress gene expression through RAR/RXR heterodimers interacting with other transcription factors, such as AP-1, estrogen receptor α, and NF-κB activities (Shaulian & Karin, 2002). Retinoic acid has been shown to repress Gbx2 expression in talencephalon in Zebrafish and other vertebrate models in early stages of development.

References

Atencia, R., García-Sanz, M., Pérez-Yarza, G., Asumendi, A., Hilario, E., & Aréchaga, J. (1997). A structural analysis of cytoskeleton components during the execution phase of apoptosis. Protoplasma, 198(3–4), 163–169. https://doi.org/10.1007/BF01287565

Chiang, C., Young, K. E., & Beachy, P. A. (1995). Control of Drosophila tracheal branching by the novel homeodomain gene unplugged, a regulatory target for genes of the bithorax complex. Development, 121(11), 3901–3912.

Herget, T., Esdar, C., Oehrlein, S. A., Heinrich, M., Schützei, S., Maelicke, A., & Van Echten-Deckert, G. (2000). Production of ceramides causes apoptosis during early neural differentiation in vitro. Journal of Biological Chemistry, 275(39), 30344–30354. https://doi.org/10.1074/jbc.M000714200

Li, B., Kuriyama, S., Moreno, M., & Mayor, R. (2009). The posteriorizing gene Gbx2 is a direct target of Wnt signalling and the earliest factor in neural crest induction. Development, 136(19), 3267–3278. https://doi.org/10.1242/dev.036954

Luu, B., Ellisor, D., & Zervas, M. (2011). The Lineage Contribution and Role of Gbx2 in Spinal Cord Development. PLoS ONE, 6. https://doi.org/10.1371/journal.pone.0020940

Nakayama, Y., Inomata, C., Yuikawa, T., Tsuda, S., & Yamasu, K. (2017). Comprehensive analysis of target genes in zebrafish embryos reveals gbx2 involvement in neurogenesis. Developmental Biology, 430(1), 237–248. https://doi.org/10.1016/j.ydbio.2017.07.015

Rochette-Egly, C., & Chambon, P. (2001). F9 embryocarcinoma cells: A cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling. Histology and Histopathology, 16(3), 909–922. https://doi.org/10.14670/HH-16.909

Roeseler, D. A., Sachdev, S., Buckley, D. M., Joshi, T., & Wu, D. K. (2012). Elongation Factor 1 alpha1 and Genes Associated with Usher Syndromes Are Downstream Targets of GBX2. PLoS ONE, 7(11), 47366. https://doi.org/10.1371/journal.pone.0047366

Shaulian, E., & Karin, M. (2002). AP-1 as a regulator of cell life and death. Nature Cell Biology, 4(5), E131–E136. https://doi.org/10.1038/ncb0502-e131

Soprano, D. R., Qin, P., & Soprano, K. J. (2004). Retinoic acid receptors and cancers. Annual Review of Nutrition, 24, 201–221. https://doi.org/10.1146/annurev.nutr.24.012003.132407

Wang, X. J., Hayes, J. D., Henderson, C. J., & Roland Wolf, C. (2007). Identification of retinoic acid as an inhibitor of transcription factor Nrf2 through activation of retinoic acid receptor alpha. Proc Natl Acad Sci U S A, 104(49), 19589–19594. www.pnas.org/cgi/content/full/

Wang, Z., Nakayama, Y., Tsuda, S., & Yamasu, K. (2018). The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos. Differentiation, 99(December 2017), 28–40. https://doi.org/10.1016/j.diff.2017.12.005

         ZFIN Gene: gbx2. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-020509-2

List of Key Events in the AOP

Event: 1903: foxi1 expression, increased

Short Name: foxi1 expression, increased

AOPs Including This Key Event

Biological Context

Domain of Applicability

Foxi I class genes have been described in zebrafish (Hans et al., 2004; Solomon et al., 2003),  humans (Larsson et al., 1995; Pierrou et al., 1994), mouse (Hulander et al., 1998; Overdier et al., 1997), rat (Clevidence et al., 1993) and Xenopus (Lef et al., 1994, 1996). However, it is unclear whether zebrafish foxi1 is orthologous to any one of these genes. The Xenopus FoxI1c (Lef et al., 1996), FoxI1a and FoxI1b genes (Lef et al., 1994) share the highest degree of sequence conservation with the zebrafish gene. The expression pattern of the two Xenopus pseudoallelic variants FoxI1a/b does not suggest functional similarity to zebrafish foxi1. Of the three Xenopus FoxI genes, FoxI1c (XFD-10) is most similar to foxi1 in sequence. However, Xenopus FoxI1c was reported to be expressed in the neuroectoderm and somites but not in the otic placode, unlike the pattern for foxi1 reported in (Lef et al., 1996). (Pohl et al., 2002) report provides a more detailed description of Xenopus FoxI1c, which suggests that this gene is expressed in preplacodal tissue and the branchial arches, similar to observations for zebrafish foxi1. Thus, it appears probable that Xenopus FoxI1c represents the ortholog of zebrafish foxi1 (Solomon et al., 2003).

Key Event Description

Foxi1 exhibits DNA-binding transcription factor activity. Involved in several processes, including animal organ development; epidermal cell fate specification; and neuron development. Predicted to localize to nucleus. Is expressed in several structures, including ectoderm; epibranchial ganglion; head; neural crest; and neurogenic field. Human ortholog(s) of this gene implicated in autosomal recessive nonsyndromic deafness 4. Orthologous to human FOXI1 (forkhead box I1) (ZFIN Gene: Foxi1, n.d.). The zebrafish Foxi1 protein shares 52% identity with Xenopus FoxI1c and 40% with human FOXI1; the forkhead domains are 95% and 94% identical, respectively (Solomon et al., 2003).

Zebrafish Foxi1 is expressed in nonneural ectoderm. Based on double in situ labeling with otx2, the anterior-most region of foxi1 expression lies just posterior to the midbrain hindbrain boundary. At the three-somite stage, the two domains of foxi1 expression become more compact, but are still located in approximately the same position lateral to the hindbrain (Solomon et al., 2003).

How it is Measured or Detected

Inhibition of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) (Wong & Medrano, 2005). Combined RT-PCR and qPCR are routinely used for analysis of gene expression.

References

Clevidence, D. E., Overdier, D. G., Taot, W., Qian, X., Pani, L., Lait, E., & Costa, R. H. (1993). Identification of nine tissue-specific transcription factors of the hepatocyte nuclear factor 3/forkhead DNA-binding-domain family (tissue-specific transcription factors/gene family/differentiation). In Proc. Natl. Acad. Sci. USA (Vol. 90).

Hulander, M., Wurst, W., Carlsson, P., & Enerbäck, S. (1998). The winged helix transcription factor FKh10 is required for normal development of the inner ear. Nature Genetics, 20(4), 374–376. https://doi.org/10.1038/3850

Larsson, C., Hellqvist, M., Pierrou, S., White, I., Enerback, S. and, & Carlsson, P. (1995). Chromosomal Localization of Six Human Forkhead Genes, freac-1 (FKHL5), -3 (FKHL7), -4 (FKHL8), -5 (FKHL9), -6 (FKHL10), and -8 (FKHL12). Genomics, 30, 464–469.

Lef, J., Clement, J. H., Oschwald, R., Köster, M., & Knöchel, W. (1994). Spatial and temporal transcription patterns of the forkhead related XFD-2/XFD-2′ genes in Xenopus laevis embryos. Mechanisms of Development, 45(2), 117–126. https://doi.org/10.1016/0925-4773(94)90025-6

Lef, J., Dege, P., Scheucher, M., Forsbach-Birk, V., Clement, J. H., & Knöchel, W. (1996). A fork head related multigene family is transcribed in Xenopus laevis embryos. International Journal of Developmental Biology, 40(1), 245–253. https://doi.org/10.1387/ijdb.8735935

Overdier, D. G., Ye, H., Peterson, R. S., Clevidence, D. E., & Costa, R. H. (1997). The Winged Helix Transcriptional Activator HFH-3 Is Expressed in the Distal Tubules of Embryonic and Adult Mouse Kidney*. In THE JOURNAL OF BIOLOGICAL CHEMISTRY (Vol. 272, Issue 21). https://doi.org/10.1074/jbc.272.21.13725

Pierrou, S., Hellqvist, M., Samuelsson, L., Enerbäck, S., & Carlsson, P. (1994). Cloning and characterization of seven human forkhead proteins: Binding site specificity and DNA bending. EMBO Journal, 13(20), 5002–5012. https://doi.org/10.1002/j.1460-2075.1994.tb06827.x

Pohl, B. S., Knöchel, S., Dillinger, K., & Knöchel, W. (2002). Sequence and expression of FoxB2 (XFD-5) and FoxI1c (XFD-10) in Xenopus embryogenesis. Mechanisms of Development, 117(1–2), 283–287. https://doi.org/10.1016/S0925-4773(02)00184-3

Solomon, K. S., Kudoh, T., Dawid, I. B., & Fritz, A. (2003). Zebrafish foxi1 mediates otic placode formation and jaw development. Development, 130(5), 929–940. https://doi.org/10.1242/dev.00308

Wong, M. L., & Medrano, J. F. (2005). Real-time PCR for mRNA quantitation. 39(1), 75–85. https://doi.org/10.2144/05391RV01

         ZFIN Gene: foxi1. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-030505-1

Event: 1904: six1b expression, increased

Short Name: six1b expression, increased

AOPs Including This Key Event

Biological Context

Domain of Applicability

Evidence was provided for vertebrates ((Brodbeck & Englert, 2004; Heanue et al., 1999; Li et al., 2003; Wawersik & Maas, 2000) and Drosophila (Bui et al., 2000).

Key Event Description

Six1b is predicted to have DNA-binding transcription factor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity. Involved in several processes, including muscle organ development; nervous system development; and regulation of skeletal muscle cell proliferation. Human ortholog(s) of this gene implicated in autosomal dominant nonsyndromic deafness; branchiootorenal syndrome; and nephroblastoma. Orthologous to human SIX1 (SIX homeobox 1) (ZFIN Gene: Six1b, n.d.).

Six1b is a Member of the Pax–Six1b–Eya–Dach ( paired box–sine oculis homeobox–eyes absent– dachshund) gene regulatory network, involved in the development of numerous organs and tissues (Bessarab et al., 2004; Bricaud et al., 2006). It has been proposed to play an important role in inner ear development (Baker & Bronner-Fraser, 2001; Whitfield et al., 2002). Six1b expression appears to be regulated by pax2b and also by foxi1 (forkhead box I1) as expected for an early inducer of the otic placode (Bricaud et al., 2006).

Six1b promotes hair cell fate and, conversely, inhibits neuronal fate by differentially affecting cell proliferation and cell death in these lineages. Gain/loss-of-function experiment results indicate that, when six1 is overexpressed, not only are fewer neural progenitors formed but many of these progenitors do not go on to differentiate into neurons (Bricaud et al., 2006).

How it is Measured or Detected

Inhibition of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) (Wong & Medrano, 2005). Combined RT-PCR and qPCR are routinely used for analysis of gene expression.

References

Baker, C. V. H., & Bronner-Fraser, M. (2001). Vertebrate cranial placodes. I. Embryonic induction. Developmental Biology, 232(1), 1–61. https://doi.org/10.1006/dbio.2001.0156

Bessarab, D. A., Chong, S., & Korzh, V. (2004). Expression of Zebrafish six1 During Sensory Organ Development and Myogenesis. June, 781–786. https://doi.org/10.1002/dvdy.20093

Bricaud, O., Leslie, A. C., & Gonda, S. (2006). Development/Plasticity/Repair The Transcription Factor six1 Inhibits Neuronal and Promotes Hair Cell Fate in the Developing Zebrafish (Danio rerio) Inner Ear. Journal of Neuroscience, 26(41), 10438–10451. https://doi.org/10.1523/JNEUROSCI.1025-06.2006

Brodbeck, S., & Englert, C. (2004). Genetic determination of nephrogenesis: The Pax/Eya/Six gene network. Pediatric Nephrology, 19(3), 249–255. https://doi.org/10.1007/s00467-003-1374-z

Heanue, T. A., Reshef, R., Davis, R. J., Mardon, G., Oliver, G., Tomarev, S., Lassar, A. B., & Tabin, C. J. (1999). Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation. www.genesdev.org

Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., & Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature, 426(6964), 247–254. https://doi.org/10.1038/nature02083

Wawersik, S., & Maas, R. L. (2000). Vertebrate eye development as modeled in Drosophila. In Human Molecular Genetics (Vol. 9, Issue 6). http://hgu.mrc.ac.uk/Softdata/PAX6/

Whitfield, T. T., Riley, B. B., Chiang, M. Y., & Phillips, B. (2002). Development of the zebrafish inner ear. Developmental Dynamics, 223(4), 427–458. https://doi.org/10.1002/dvdy.10073

Wong, M. L., & Medrano, J. F. (2005). Real-time PCR for mRNA quantitation. 39(1), 75–85. https://doi.org/10.2144/05391RV01

         ZFIN Gene: six1b. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-040426-230

Event: 1905: eya1 expression, inhibited

Short Name: eya1 expression, inhibited

AOPs Including This Key Event

Biological Context

Domain of Applicability

Evidence was provided zebrafish (Kozlowski et al., 2005), Drosophila and vertebrates (Li et al., 2003; Zimmerman et al., 1997), and human (Abdelhak et al., 1997)

Key Event Description

Eya1 is predicted to have protein tyrosine phosphatase activity. Involved in adenohypophysis development; otic vesicle morphogenesis; and otolith development. Predicted to localize to nucleus. Is expressed in several structures, including adenohypophyseal placode; brain; ectoderm; head; and lateral line system. Orthologous to human EYA1 (EYA transcriptional coactivator and phosphatase 1) (ZFIN Gene: Eya1, n.d.).

Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome and hereditary syndromic deafness, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system (Kozlowski et al., 2005; Xu et al., 2002).

In zebrafish, the eya1 gene is widely expressed in placode-derived sensory organs during embryogenesis. Eya1 function appears to be primarily required for survival of sensory hair cells in the developing ear and lateral line neuromasts (Kozlowski et al., 2005).

How it is Measured or Detected

Inhibition of expression can be measured with reverse transcription polymerase chain reaction (RT-PCR). This technique is primarily used to measure the amount of specific RNA which is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR) (Wong & Medrano, 2005). Combined RT-PCR and qPCR are routinely used for analysis of gene expression

References

Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samoson, D., Vincent, C., Weil, D., Cruaud, C., Sahly, I., Leibovici, M., Bitner-Glindzicz, M., & Francis, M. (1997). A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nature Genetics, 15, 157–167. https://doi.org/10.1038/ng0297-157

Kozlowski, D. J., Whitfield, T. T., Hukriede, N. A., Lam, W. K., & Weinberg, E. S. (2005). The zebrafish dog-eared mutation disrupts eya1, a gene required for cell survival and differentiation in the inner ear and lateral line. Developmental Biology, 277(1), 27–41. https://doi.org/10.1016/j.ydbio.2004.08.033

Li, X., Oghi, K. A., Zhang, J., Krones, A., Bush, K. T., Glass, C. K., Nigam, S. K., Aggarwal, A. K., Maas, R., Rose, D. W., & Rosenfeld, M. G. (2003). Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature, 426(6964), 247–254. https://doi.org/10.1038/nature02083

Wong, M. L., & Medrano, J. F. (2005). Real-time PCR for mRNA quantitation. 39(1), 75–85. https://doi.org/10.2144/05391RV01

Xu, P.-X., Weiming, Z., Laclef, C., Maire, P., Maas L., R., Peters, H., & Xin, X. (2002). Eya1is required for the morphogenesis of mammalian thymus, parathyroid and thyroid. Development, 129, 3033–3044.

ZFIN Gene: eya1. (n.d.). Retrieved April 12, 2021, from https://zfin.org/ZDB-GENE-990712-18

Zimmerman, J. E., Bui, Q. T., Kur Steingrimsson, E. [, Nagle, D. L., Fu, W., Genin, A., Spinner, N. B., Copeland, N. G., Jenkins, N. A., Bucan, M., & Bonini, N. M. (1997). Cloning and Characterization of Two Vertebrate Homologs of the Drosophila eyes absent Gene. Development, 124(23), 4819–4826.

Event: 1825: Increase, Cell death

Short Name: Increase, Cell death

AOPs Including This Key Event

Stressors

Biological Context

Cell term

Organ term

Evidence for Perturbation by Stressor

Food deprivation

Autophagy can be initiated by a variety of stressors, most notably by nutrient deprivation (caloric restriction) or can result from signals present during cellular differentiation and embryogenesis and on the surface of damaged organelles (Mizushima et al., 2008).

Gentamicin

Gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish (Uribe et al., 2013).

Domain of Applicability

The process of cell death is highly conserved within multi‐cellular organisms. (Lockshin & Zakeri, 2004).

Key Event Description

Cell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e.. micro organisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (Kanduc et al., 2002). Many physiological processes require cell death for their function (e.g.., embryonal development and immune selection of B and T cells) (Bertheloot et al., 2021). Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer (Crowley et al., 2016).

Cell death can be divided into: programmed cell death (cell death as a normal component of development) and non-programmed cell death (uncontrolled death of the cell). Although this simplistic view has blurred the intricate mechanisms separating these forms of cell death, studies have and will uncover new effectors, cell death pathways and reveal a more complex and intertwined landscape of processes involving cell death (Bertheloot et al., 2021).

Programmed cell death: is a form of cell death in which the dying cell plays an active part in its own demise (Cotter & Al-Rubeai, 1995).

Apoptosis At a morphological level, it is characterized by cell shrinkage rather than the swelling seen in necrotic cell death. It is characterized by a number of characteristic morphological changes in the structure of the cell, together with a number of enzyme‐dependent biochemical processes. The result of it being the clearance of cells from the body, with minimal damage to surrounding tissues. An essential feature of apoptosis is the release of cytochrome c from mitochondria, regulated by a balance between proapoptotic and antiapoptotic proteins of the BCL-2 family, initiator caspases (caspase-8, -9 and -10) and effector caspases (caspase-3, -6 and -7). Apoptosis culminates in the breakdown of the nuclear membrane by caspase-6, the cleavage of many intracellular proteins (e.g., PARP and lamin), membrane blebbing, and the breakdown of genomic DNA into nucleosomal structures (Bertheloot et al., 2021). Mechanistically, two main pathways contribute to the caspase activation cascade downstream of mitochondrial cytochrome c release:

• Intrinsic pathway is triggered by dysregulation of or imbalance in intracellular homeostasis by toxic agents or DNA damage. It is characterized by mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome c into the cytosol.
• Extrinsic pathway is initiated by activation of cell surface death receptors. Proapoptotic death receptors include TNFR1/2, Fas and the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5.

Other pathways of programmed cell death are called »non-apoptotic programmed cell-death« or »caspase-independent programmed cell-death« (Blank & Shiloh, 2007).

Necroptosis: This type of regulated cell death, occurs following the activation of the tumor necrosis receptor (TNFR1) by TNFα. Activation of other cellular receptors triggers necroptosis. These receptors include death receptors (i.e., Fas/FasL), Toll-like receptors (TLR4 and TLR3) and cytosolic nucleic acid sensors such as RIG-I and STING, which induce type I interferon (IFN-I) and TNFα production and thus promote necroptosis in an autocrine feedback loop. Most of these pathways trigger NFκB- dependent proinflammatory and prosurvival signals.

Pyroptosis is a type of cell death culminating in the loss of plasma membrane integrity and induced by activation of so-called inflammasome sensors. These include the Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor.

Autophagy: is a process where cellular components such as macro proteins or even whole organelles are sequestered into lysosomes for degradation (Mizushima et al., 2008; Shintani & Klionsky, 2004). The lysosomes are then able to digest these substrates, the components of which can either be recycled to create new cellular structures and/or organelles or alternatively can be further processed and used as a source of energy (D’Arcy, 2019).

Anoikis is apoptosis induced by loss of attachment to substrate or to other cells (anoikis). Anoikis overlaps with apoptosis in molecular terms, but is classified as a separate entity because of its specific form od induction (Blank & Shiloh, 2007). Induction of anoikis occurs when cells lose attachment to ECM, or adhere to an inappropriate type of ECM, the latter being the more relevant in vivo (Gilmore, 2005).

Cornification: is programmed cell death of keratinocytes. Cell death in the context of cornification involves distinct enzyme classes such as transglutaminases, proteases, DNases and others (Eckhart et al., 2013).

Non-programmed cell death: occurs accidentally in an unplanned manner.

Necrosis is generally characterized to be the uncontrolled death of the cell, usually following a severe insult, resulting in spillage of the contents of the cell into surrounding tissues and subsequent damage thereof (D’Arcy, 2019).

How it is Measured or Detected

Assays for Quantitating Cell Death:

• Cell death can be measured by staining a sample of cells with trypan blue, assay is described in protocol: Measuring Cell Death by Trypan Blue Uptake and Light Microscopy (Crowley, Marfell, Christensen, et al., 2015d). Or with propidium Iodide, assay is described in protocol: Measuring Cell Death by Propidium Iodide (PI) Uptake and Flow Cytometry (Crowley & Waterhouse, 2015a)
• TUNEL technique: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Uribe et al., 2013).

Assays for Quantitating Cell Survival          

Colony-forming assay can be used to define the number of cells in a population that are capable of proliferating and forming large groups of cells. Described in Protocol: Measuring Survival of Adherent Cells with the Colony-Forming Assay (Crowley, Christensen, & Waterhouse, 2015c); Measuring Survival of Hematopoietic Cancer Cells with the Colony-Forming Assay in Soft Agar (Crowley & Waterhouse, 2015b).

ASSAYS TO DISTINGUISH APOPTOSIS FROM NECROSIS AND OTHER DEATH MODALITIES

Detecting Nuclear Condensation: The nucleus is generally round in healthy cells but fragmented in apoptotic cells. Dyes such as Giemsa or hematoxylin, which are purple in color and therefore easily viewed using light microscopy, are commonly used to stain the nucleus. Other features of apoptosis and necrosis, such as plasma membrane blebbing or rupture, can be identified by staining the cytoplasm with eosin. Eosin is pinkish in color and can also be viewed using light microscopy. Hematoxylin and eosin are, therefore, commonly used together to stain cells. Assay is described in Protocol: Morphological Analysis of Cell Death by Cytospinning Followed by Rapid Staining (Crowley, Marfell, & Waterhouse, 2015c); Analyzing Cell Death by Nuclear Staining with Hoechst 33342 (Crowley, Marfell, & Waterhouse, 2015a).

Detection of DNA Fragmentation: Apoptotic cells with fragmented DNA can be identified and distinguished from live cells by staining with Propidium Iodide (PI) and measuring DNA content by flow cytometry. This assay is described in Protocol: Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry (Crowley, Chojnowski, & Waterhouse, 2015a). TUNEL technique can also be used: in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling can be used to detect apoptotic cells (Bever & Fekete, 1999; Crowley, Marfell, & Waterhouse, 2015b; Uribe et al., 2013).

Detecting Phosphatidylserine Exposure: Apoptosis is also characterized by exposure of phosphatidylserine (PS) on the outside of apoptotic cells, which acts as a signal that triggers removal of the dying cell by phagocytosis. Annexin V, can selectively bind to PS to label apoptotic cells in which PS is exposed. Purified annexin V can be conjugated to various fluorochromes, which can then be visualized by fluorescence microscopy or detected by flow cytometry. This assay is described in protocol: Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry (Crowley, Marfell, Scott, et al., 2015e).

Detecting Caspase Activity: antibodies that specifically recognize the cleaved fragments of caspases and their substrates can be used to specifically detect caspase activity in apoptotic cells by immunocytochemistry. Flow cytometry (using primary antibodies conjugated to fluorescent molecules, or by counter staining with fluorescently labeled antibodies against the primary antibody) can then be used to quantitate the number of apoptotic cells. This assay is described in protocol: Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry (Crowley & Waterhouse, 2015a).

Detecting Mitochondrial Damage: flow cytometry can be used to quantitate the number of cells that have reduced mitochondrial transmembrane potential, which is commonly associated with cytochrome c release during apoptosis. For this assay see protocol: Measuring Mitochondrial Transmembrane Potential by TMRE Staining (Crowley, Christensen, & Waterhouse, 2015b).

References

Bertheloot, D., Latz, E., & Franklin, B. S. (2021). Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cellular & Molecular Immunology, 18, 1106–1121. https://doi.org/10.1038/s41423-020-00630-3

Bever, M. M., & Fekete, D. M. (1999). Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears. Journal of Neurocytology, 28(10–11), 781–793. https://doi.org/10.1023/a:1007005702187

Blank, M., & Shiloh, Y. (2007). Cell Cycle Programs for Cell Death: Apoptosis is Only One Way to Go. Cell Cycle, 6(6), 686–695. https://doi.org/10.4161/cc.6.6.3990

Cotter, T. G., & Al-Rubeai, M. (1995). Cell death (apoptosis) in cell culture systems. Trends in Biotechnology, 13(4), 150–155. https://doi.org/10.1016/S0167-7799(00)88926-X

Crowley, L. C., Chojnowski, G., & Waterhouse, N. J. (2015a). Measuring the DNA content of cells in apoptosis and at different cell-cycle stages by propidium iodide staining and flow cytometry. Cold Spring Harbor Protocols, 10, 905–910. https://doi.org/10.1101/pdb.prot087247

Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015b). Measuring mitochondrial transmembrane potential by TMRE staining. Cold Spring Harbor Protocols, 12, 1092–1096. https://doi.org/10.1101/pdb.prot087361

Crowley, L. C., Christensen, M. E., & Waterhouse, N. J. (2015c). Measuring survival of adherent cells with the Colony-forming assay. Cold Spring Harbor Protocols, 8, 721–724. https://doi.org/10.1101/pdb.prot087171

Crowley, L. C., Marfell, B. J., Christensen, M. E., & Waterhouse, N. J. (2015d). Measuring cell death by trypan blue uptake and light microscopy. Cold Spring Harbor Protocols, 7, 643–646. https://doi.org/10.1101/pdb.prot087155

Crowley, L. C., Marfell, B. J., Scott, A. P., Boughaba, J. A., Chojnowski, G., Christensen, M. E., & Waterhouse, N. J. (2016). Dead cert: Measuring cell death. Cold Spring Harbor Protocols, 2016(12), 1064–1072. https://doi.org/10.1101/pdb.top070318

Crowley, L. C., Marfell, B. J., Scott, A. P., & Waterhouse, N. J. (2015e). Quantitation of apoptosis and necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harbor Protocols, 11, 953–957. https://doi.org/10.1101/pdb.prot087288

Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015a). Analyzing cell death by nuclear staining with Hoechst 33342. Cold Spring Harbor Protocols, 9, 778–781. https://doi.org/10.1101/pdb.prot087205

Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015b). Detection of DNA fragmentation in apoptotic cells by TUNEL. Cold Spring Harbor Protocols, 10, 900–905. https://doi.org/10.1101/pdb.prot087221

Crowley, L. C., Marfell, B. J., & Waterhouse, N. J. (2015c). Morphological analysis of cell death by cytospinning followed by rapid staining. Cold Spring Harbor Protocols, 9, 773–777. https://doi.org/10.1101/pdb.prot087197

Crowley, L. C., & Waterhouse, N. J. (2015a). Detecting cleaved caspase-3 in apoptotic cells by flow cytometry. Cold Spring Harbor Protocols, 11, 958–962. https://doi.org/10.1101/pdb.prot087312

Crowley, L. C., & Waterhouse, N. J. (2015b). Measuring survival of hematopoietic cancer cells with the Colony-forming assay in soft agar. Cold Spring Harbor Protocols, 8, 725. https://doi.org/10.1101/pdb.prot087189

D’Arcy, M. S. (2019). Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biology International, 43(6), 582–592. https://doi.org/10.1002/cbin.11137

Eckhart, L., Lippens, S., Tschachler, E., & Declercq, W. (2013). Cell death by cornification. Biochimica et Biophysica Acta - Molecular Cell Research, 1833(12), 3471–3480. https://doi.org/10.1016/j.bbamcr.2013.06.010

Gilmore, A. P. (2005). Anoikis. Cell Death and Differentiation, 12, 1473–1477. https://doi.org/10.1038/sj.cdd.4401723

Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A. A., Natale, C., Santacroce, R., Di Corcia, M. G., Lucchese, A., Dini, L., Pani, P., Santacroce, S., Simone, S., Bucci, R., & Farber, E. (2002). Cell death: apoptosis versus necrosis (review). International Journal of Oncology, 21(1), 165–170. https://doi.org/10.3892/ijo.21.1.165

Lockshin, R. A., & Zakeri, Z. (2004). Apoptosis, autophagy, and more. International Journal of Biochemistry and Cell Biology, 36(12), 2405–2419. https://doi.org/10.1016/j.biocel.2004.04.011

Mizushima, N., Levine, B., Cuervo, A. M., & Klionsky, D. J. (2008). Autophagy fights disease through cellular self-digestion. Nature, 451(7182), 1069–1075. https://doi.org/10.1038/nature06639

Shintani, T., & Klionsky, D. J. (2004). Autophagy in health and disease: A double-edged sword. Science, 306(5698), 990–995. https://doi.org/10.1126/science.1099993

Uribe, P. M., Sun, H., Wang, K., Asuncion, J. D., & Wang, Q. (2013). Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio). PLoS ONE, 8(3), 58755. https://doi.org/10.1371/journal.pone.0058755

Event: 1930: altered, inner ear development

Short Name: Altered, inner ear development

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Evidence for Perturbation by Stressor

Gentamicin

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls (Uribe et al., 2013).

Uribe, P. M. et al. (2013) ‘Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)’, PLoS ONE, 8(3), p. 58755. doi: 10.1371/journal.pone.0058755.

Domain of Applicability

Evidence was provided for  Zebrafish, Chick and Mouse (Whitfield, 2015)

Key Event Description

Zebrafish:            

The zebrafish (Danio rerio), a genetically tractable vertebrate, lends itself particularly well as a model system in which to study the ear. Zebrafish do not possess outer or middle ears, but have a fairly typical vertebrate inner ear, the normal development and anatomy of which has been described in a series of atlas-type papers (Haddon and Lewis, 1996; Bang, Sewell and Malicki, 2001). Although the zebrafish ear does not contain a specialized hearing organ—there is no equivalent of the mammalian cochlea—many features are conserved with other vertebrate species (Whitfield, 2002).

Inner ear develops from an ectodermal thickening, the otic placode, visible on either side of the hindbrain from mid-somite stages. In the zebrafish, this placode cavitates to form a hollow ball of epithelium, the otic vesicle, from which all structures of the membranous labyrinth and the neurons of the statoacoustic (VIIIth) ganglion arise (Haddon and Lewis, 1996; Whitfield et al., 2002).

The mature organ, found in all jawed vertebrates, has two functions: it serves as an auditory system, which detects sound waves, and as a vestibular system, which detects linear and angular accelerations, enabling the organism to maintain balance (Whitfield et al., 1996).

How it is Measured or Detected

Zebrafish:

• Direct observation of internal anatomic structures of zebrafish embryos. Defects visible under the dissecting microscope (Whitfield, 2002)
• Comparison of swimming patterns with wild-type fish. Dog-eared embryos are less responsive to vibrational stimuli, fail to maintain balance when swimming, and may circle when disturbed, a behavior characteristic of fish with vestibular defects  (Nicolson et al., 1998)
• High-throughput behavioral screening method for detecting auditory response defects in zebrafish. Assay monitors a rapid escape reflex in response to a loud sound (Bang et al., 2002).

References

Bang, P. I. et al. (2002) ‘High-throughput behavioral screening method for detecting auditory response defects in zebrafish’, Journal of Neuroscience Methods, 118(2), pp. 177–187. doi: 10.1016/S0165-0270(02)00118-8.

Bang, P. I., Sewell, W. F. and Malicki, J. J. (2001) ‘Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio)’, Journal of Comparative Neurology, 438(2), pp. 173–190. doi: 10.1002/cne.1308.

Haddon, C. and Lewis, J. (1996) ‘Early ear development in the embryo of the zebrafish, Danio rerio’, Journal of Comparative Neurology, 365(1), pp. 113–128. doi: 10.1002/(SICI)1096-9861(19960129)365:1<113::AID-CNE9>3.0.CO;2-6.

Nicolson, T. et al. (1998) ‘Genetic analysis of vertebrate sensory hair cell mechanosensation: The zebrafish circler mutants’, Neuron, 20(2), pp. 271–283. doi: 10.1016/S0896-6273(00)80455-9.

Uribe, P. M. et al. (2013) ‘Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)’, PLoS ONE, 8(3), p. 58755. doi: 10.1371/journal.pone.0058755.

Whitfield, T. T. et al. (1996) ‘Mutations affecting development of the zebrafish inner ear and lateral line’, Development, 123, pp. 241–254. doi: 10.1242/dev.123.1.241.

Whitfield, T. T. et al. (2002) ‘Development of the zebrafish inner ear’, Developmental Dynamics, 223(4), pp. 427–458. doi: 10.1002/dvdy.10073.

Whitfield, T. T. (2002) ‘Zebrafish as a Model for Hearing and Deafness’, J Neurobiol, 53, pp. 157–171. doi: 10.1002/neu.10123.

Whitfield, T. T. (2015) ‘Development of the inner ear’, Current Opinion in Genetics and Development, 32, pp. 112–118. doi: 10.1016/j.gde.2015.02.006.

Event: 1008: Reduced, Hearing

Short Name: Reduced, Hearing

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

• A sense of hearing is known to exist in a wide range of vertebrates and invertebrates, although the organs and structures involved vary widely.

Key Event Description

Hearing refers to the ability to perceive sound vibrations propagated as pressure changes through a medium such as air or water. Reduced hearing in the context of this key event can refer to reduction in the perceived volume of a sound relative to the amplitude of sound waves. Reduced hearing may also refer to a reduced range of frequencies that can be perceived.

How it is Measured or Detected

Hearing is generally measured behaviorally or electrophysiologically.

• Common behavioral tests involve transmission of pure tones of defined amplitude and frequency using and audiometer or PC and using a behavioral response (e.g., clicking a button; startle response) to determine whether the tone is perceived.

Electrophysiological tests:

• Auditory brainstem response (ABR): Uses electrodes placed on the head to detect auditory evoked potentials from background electrical activity in the brain.

Hearing tests in Fish:

• Through the mid-late 1980s conditioning and behavioral tests were most commonly employed in testing fish hearing. Methods reviewed by Fay (1988)
• A high throughput behavioral test for detecting auditory response in fish has been described (Bang et al. 2002).
• Invasive electrophysiological methods involving surgical insertion of electrodes into the auditory nerves have been employed.
• Non-invasive recording of Auditory Evoked Potentials (AEPs; synonymous with ABRs) are now the most common approach for measuring hearing in fish. AEPs can be recorded via electrodes attached cutaneously to the head (see review by Ladich and Fay, 2013).

References

• Fay RR (1988) Hearing in vertebrates: a psychophysics databook. Hill-Fay Associates, Winnetka, Ill
• Ladich F, Fay RR. Auditory evoked potential audiometry in fish. Reviews in Fish Biology and Fisheries. 2013;23(3):317-364. doi:10.1007/s11160-012-9297-z.
• Bang PI, Yelick PC, Malicki JJ, Sewell WF. High-throughput behavioral screening method for detecting auditory response defects in zebrafish. J Neurosci Methods. 2002 Aug 30;118(2):177-87. PubMed PMID: 12204308.

Event: 351: Increased Mortality

Short Name: Increased Mortality

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

All living things are susceptible to mortality.

Key Event Description

Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.

How it is Measured or Detected

Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.

Depending on the species and the study setup, mortality can be measured:

• in the lab by recording mortality during exposure experiments
• in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species
• in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.

Regulatory Significance of the AO

Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.

Appendix 2

List of Key Event Relationships in the AOP