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Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement

Abstract

Embryonic cell movement is essential for morphogenesis and the establishment of body shapes1,2, but little is known about its mechanism. Here we report that pregnenolone, which is produced from cholesterol by the steroidogenic enzyme Cyp11a1 (cholesterol side-chain cleavage enzyme, P450scc)3, functions in promoting cell migration during epiboly. Epiboly is a process in which embryonic cells spread from the animal pole to cover the underlying yolk. During epiboly, cyp11a1 is expressed in an extra-embryonic yolk syncytial layer4. Reducing cyp11a1 expression in zebrafish using antisense morpholino oligonucleotides did not perturb cell fates, but caused epibolic delay. This epibolic defect was partially rescued by the injection of cyp11a1 RNA or the supplementation of pregnenolone. We show that the epibolic delay is accompanied by a decrease in the level of polymerized microtubules, and that pregnenolone can rescue this microtubule defect. Our results indicate that pregnenolone preserves microtubule abundance and promotes cell movement during epiboly.

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Figure 1: Two steroidogenic genes, cyp11a1 and hsd3b , are expressed in blastomeres and the yolk syncytial layer during early zebrafish embryogenesis.
Figure 2: Reduced cyp11a1 expression results in epibolic delay.
Figure 3: Pregnenolone rescues epiboly defect and increases microtubule content.
Figure 4: Fluorescein-conjugated pregnenolone co-localizes with microtubules.

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Acknowledgements

We would like to thank V. Korzh and C.-H. Hu for the plasmids for in situ hybridization, and K. Deen and M. Wyatt for editing the manuscript. This work was supported by grants from the National Science Council and Academia Sinica, Taiwan. Author Contributions H.-J.H. performed all the experiments; M.-R.L. synthesized F–P5; C.-T.C. devised and supervised the F–P5 synthesis scheme; B.-c.C. oversaw the execution of the entire project.

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Correspondence to Bon-chu Chung.

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Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1–9 (PPT 7800 kb)

Supplementary Figure Legends

This file contains text to accompany the above Supplementary Figures. (DOC 37 kb)

Supplementary Table

Reducing cyp11a1 expression resulted in epibolic defect. (DOC 38 kb)

Supplementary Methods

This file details the methods for fish maintenance, morpholinos and analysis of genomic structures, plasmids, microinjection and detection of gene expression, steroid detection, RT-PCR, and synthesis of fluorescein conjugated pregnenolone. (DOC 215 kb)

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Hsu, HJ., Liang, MR., Chen, CT. et al. Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement. Nature 439, 480–483 (2006). https://doi.org/10.1038/nature04436

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