Newsletter No 4 December 2010

Lysyl oxidases in zebrafish development and teratogenesis

Antonius L. van Boxtel, PhD Dissertation Defended October 8, 2010, VU University Amsterdam

cbOn October 8, 2010, Antonius (Thijs) van Boxtel brilliantly defended his PhD thesis entitled “Lysyl oxidases in zebrafish development and teratogenesis.” This research revealed a new mechanism of developmental toxicity of dithiocarbamates, a widely used group of pesticides. This has led to the discovery of novel roles for proteins called lysyl oxidases in vertebrate development.

Summary PhD dissertation

Dithiocarbamates (DTCs) are widely used chemicals that are teratogenic to vertebrates through mechanisms that are poorly understood (Robens, 1969,  Simsa et al. 2007; Tilton et al. 2006). This study has utilized the zebrafish as a vertebrate model to study DTC induced teratogenic effects and the molecular mechanisms underlying these effects. Novel teratogenic endpoints of DTCs in zebrafish were investigated by exposing developing embryos to three model DTCs: disulfiram, metam and thiram. It was found that all three compounds lead to abnormal development of cartilage and bone elements that make up the zebrafish craniofacial skeleton (van Boxtel et al, 2010a). These malformations were characterized by twisted, reduced and missing cartilages and a lack of ossification of endochondral bone. To gain insight into the molecular mechanisms underlying these defects, DTC induced changes in gene expression in embryonic zebrafish (PAC2) cells were measured using microarray analysis. Both thiram and disulfiram caused differential expression of genes that are related to signalling of transforming growth factor β1 (TGF-β1), a crucial growth factor in endochondral bone formation (Janssens et al. 2005). Importantly, the identified TGF-β1 signalling related network contained an essential transcription factor for craniofacial development, sox9a (Yan et al. 2002). Subsequent in situ hybridization experiments revealed that expression of this differentiation marker and its direct downstream target col2a1 is perturbed in the developing ceratobranchial arches of DTC exposed embryo. This suggested that abnormal expression of sox9a is an important event in the development of zebrafish craniofacial abnormalities induced by these compounds. Though a causal relationship between DTC exposure, loss of sox9a expression and abnormal development of the ceratobranchial arches has not yet been established, these findings provide a starting point for further investigation of the molecular mechanisms of DTC induced bone and cartilage malformations in vertebrates.

cb2In contrast to the craniofacial defects identified, the effects of DTCs on notochord formation are well described (Tilton et al. 2006). However, underlying mechanisms are unclear and understanding these mechanisms will contribute to unravelling DTC induced teratogenesis. Therefore, the hypothesis was investigated that DTC induced notochord malformations in zebrafish are in part mediated by inhibition of lysyl oxidase (Loxl) proteins. Chemical inhibition of lysyl oxidase activity with β-aminoproprionitrile (βAPN) or exposure to DTCs resulted in an identical notochord phenotype (van Boxtel et al. 2010b). Electron microscopy revealed abnormal crosslinking of collagens in the notochord sheath which was indicative of impaired lysyl oxidase function. In addition, morpholino mediated loss of function of three individual Loxl proteins, Lox, Loxl1 and Loxl5b, caused similar notochord defects as observed after exposure to DTCs. Moreover, partial knock down of Lox, Loxl1 and Loxl5b sensitized developing zebrafish embryos to DTCs, suggesting that lysyl oxidase activity is inhibited by DTCs in vivo and that this can contribute to the development of notochord abnormalities. Finally, a LOXL protein inhibition assay revealed that the activity of zebrafish lysyl oxidases was greatly reduced when incubated with DTCs while this was not observed after incorporation of the copper chelator NCu. Together, these data demonstrated that DTCs inhibit zebrafish lysyl oxidase activity ex vivo and in vivo (van Boxtel et al. 2010b).

Since DTCs caused craniofacial abnormalities in developing zebrafish embryos and were found to inhibit of lysyl oxidase activity, the hypothesis was investigated that Loxl proteins are involved in zebrafish craniofacial development. Inhibition of lysyl oxidase activity with βAPN in developing zebrafish embryos from 3 hpf until 5 dpf and subsequent analysis of craniofacial development indeed revealed abnormal bone and cartilage development which were highly similar to the effects found after exposure to DTCs (van Boxtel et al., submitted for publication). By varying the βAPN exposure window, a critical window for LOXL protein activity was identified from 24 to 72 hpf. Together, these findings suggested a functional role for lysyl oxidases in a window during development that is important for ceratobranchial arch condensation, differentiation and cartilage maturation. To investigate the involvement of Loxl proteins in craniofacial development in detail, the zebrafish Loxl family was first completed by identifying two previously unknown Loxl proteins that are highly homologous to LOXL4; loxl4a and loxl4b (van Boxtel et al., submitted for publication). Analysis of spatiotemporal expression of all ten loxl genes implicated the involvement of four loxl genes, loxl1, loxl2a, loxl2b and loxl3b, in zebrafish craniofacial development. LOF studies using antisense morpholino oligonucleotides demonstrated that loxl1 and loxl3b indeed caused abnormal craniofacial development while loss of loxl2a or loxl2b was lethal before full development of the craniofacial cartilages. To gain understanding in Loxl protein function during zebrafish craniofacial development, the functional roles of loxl3a and loxl3b were investigated in more detail (van Boxtel et al., 2010c). Two previously unidentified full length loxl3a and loxl3b cDNAs were cloned and detailed in situ hybridisation experiments revealed that loxl3b but not loxl3a is abundantly expressed in and around the maturing craniofacial cartilage elements between 30 and 96 hpf. LOF of loxl3b led to a severe reduction of craniofacial cartilage elements which was due to a lack of maturation of cartilage elements and not to increased p53 mediated cell death. Analysis of expression of crestin, sox10, dlx2a, hand2, sox9a and col2a1 in loxl3b morphant embryos, revealed that migration of neural crest cells (NCC) and initial pharyngeal arch patterning occurred normally but chondrogenic progenitor cells within the mesenchymal arch condensations failed to express the differentiation markers sox9a and col2a1. These findings were in conjunction with aberrant expression of both markers in the ceratobranchial arches of DTC exposed developing embryos. Subsequent analysis of cellular proliferation within pharyngeal arch condensations revealed that chondrogenic progenitor cell proliferation was severely reduced. Together, these findings indicated important and novel roles for individual loxl genes during zebrafish craniofacial development.

In summary, this research has contributed to understanding DTC induced teratogenesis through the identification of a novel teratogenic mechanism that may contribute to cartilage and bone abnormalities in vertebrates. Importantly, these studies have also revealed novel roles for Loxl proteins during vertebrate development which provides insight into the genetic and environmental factors that may contribute to craniofacial pathogenesis.

- Janssens, K., ten, D. P., Janssens, S., and Van, H. W. (2005). Transforming growth factor-beta1 to the bone. Endocr. Rev. 26(6), 743-774.
- Robens, J. F. (1969). Teratologic studies of carbaryl, diazinon, norea, disulfiram, and thiram in small laboratory animals. Toxicol. Appl. Pharmacol. 15, 152-163.
- Simsa, S., Hasdai, A., Dan, H., and Ornan, E. M. (2007). Induction of tibial dyschondroplasia in turkeys by tetramethylthiuram disulfide (thiram). Poult. Sci. 86(8), 1766-1771.
- Tilton, F., La Du, J. K., Vue, M., Alzarban, N., and Tanguay, R. L. (2006). Dithiocarbamates have a common toxic effect on zebrafish body axis formation. Toxicol. Appl. Pharmacol. 216(1), 55-68.
- Van Boxtel, A.L., Pieterse, B., Cenijn, P., Kamstra, J., de Boer, J. Brouwer, A., van Wieringen, W. And Legler, J. (2010a) Dithiocarbamates induce craniofacial abnormalities and downregulate sox9a during zebrafish development. Tox. Sciences, 117(1):209-17.
- Van Boxtel, A.L., Kamstra, J.H., Fluitsma, D.M. and Legler, J. (2010b) Dithiocarbamates are teratogenic to developing zebrafish through inhibition of  lysyl oxidase activity, Toxicol. Appl. Pharm. 244(2):156-61.
- Van Boxtel, A.L., Gansner, J.M., Hakvoort, H., Snell, H., Legler, J. and Gitlin, J.D. (2010c) Lysyl oxidase-like 3b is critical for proliferation of chondrogenic progenitor cells during zebrafish craniofacial development. Matrix Biology, accepted.
- Yan, Y. L., Miller, C. T., Nissen, R. M., Singer, A., Liu, D., Kirn, A., Draper, B., Willoughby, J., Morcos, P. A., Amsterdam, A., Chung, B. C., Westerfield, M., Haffter, P., Hopkins, N., Kimmel, C., and Postlethwait, J. H. (2002). A zebrafish sox9 gene required for cartilage morphogenesis. Development 129(21), 5065-5079.

For more information contact Dr Juliette Legler.