Asymmetric repression of the Nodal inhibitor Coco by cilia-driven leftward flow in Xenopus
Final Report Abstract
Motile cilia are of central importance for symmetry breakage in fish, amphibian and mammalian embryos. At the left-right organizer (LRO), an epithelium localized in the dorsal midline of neurula stage embryos, polarized monocilia rotate in a clockwise fashion to produce a vectorial flow of extracellular fluids from the right to the left side. Lack of cilia or defects in ciliary motility invariably result in laterality phenotypes, as exemplified by misplaced organs in the chest and abdomen or, at earlier stages of development, inaccurate or absent activation of the Nodal signaling cascade. During normal embryogenesis the genes of this cascade get activated exclusively in the left lateral plate mesoderm (LPM). Deviations of these patterns include absence of any LPM expression, bilateral induction of the cascade and inverted expression on the right side. Therefore, both sides are equally competent for Nodal cascade induction. Central questions of laterality research concern the nature of the original left-sided signals, which are generated by leftward flow, and the mechanisms of Nodal cascade induction in response to flow. We have previously pinpointed the Nodal inhibitor dand5 (previously known as Coco) as a central target of leftward flow in the frog Xenopus. Nodal and dand5 are co-expressed in cells bordering the LRO, originally in a bilaterally symmetrical manner. As a consequence of leftward flow, dand5 mRNA becomes down-regulated on the left side of the LRO, which in Xenopus localizes to the dorsal roof of the archenteron and is addressed as gastrocoel roof plate (GRP). Importantly, Nodal cascade expression can be manipulated by gain- and loss-of-function of dand5 to adopt every possible pattern in a predictable manner. Absence of Nodal induction as a consequence of impaired ciliary motility, for example, can be rescued to the normal left-sided pattern by dand5 knockdown on the left side of the GRP. On the other hand, right-sided injection of a morpholino oligonucleotide (MO), which prevented dand5 translation, resulted in inverted, right-asymmetric Nodal expression in flow-impaired embryos. From these experiments the view emerged that leftward flow inhibits dand5 on the left to release repression of Nodal. Nodal in turn signals and/or transfers from the GRP to the left LPM to induce the asymmetric signaling cascade. This basic scenario has been confirmed in zebrafish and mouse. The mechanism by which leftward fluid flow represses dand5, however, has remained elusive. During our previous studies we noted that left-sided dand5 mRNA was only reduced in 70-80% of embryos at post flow stages. While statistically highly significant, the remaining 20-30% of embryos retained a symmetrical pattern, however, or even displayed higher levels on the left as compared to the right side of the GRP. This result is somewhat in conflict with the about 95% of tadpoles with wildtype organ placement (situs solitus). In the light of the unequivocal potential of dand5 to direct the induction of the Nodal cascade, this observation suggests a post-transcriptional mode of dand5 repression. We thus wondered whether microRNAs (miRNAs, miRs) were involved in the flow-dependent regulation of dand5. We therefore investigated the role of miRNAs in this process in the frog Xenopus. dicer was expressed at the LRO and required for dand5 inhibition. Potential miRNA binding sites were identified in the dand5 3‘-UTR and functionally characterized. miR-15a was expressed at the frog LRO and required for symmetry breakage. Clusters miR-15a/16a and miR-17~92 rescued laterality in flow-impaired embryos, following delivery of pri-miRNAs to the left but not the right side of the LRO. Our results demonstrate that miRNAs act downstream of flow and upstream of dand5 inhibition to regulate biased symmetry breakage in the frog Xenopus.
Publications
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(2013). Acquisition of leftward flow in Xenopus laevis. Bioprotocols
Thumberger, T. and Blum, M.
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(2013). Embryonic exposure to propylthiouracil disrupts left.right patterning in Xenopus embryos. FASEB J. 27, 684-691
Van Veenendaal, N., Ulmer, B, Boskovski, M.T., Fang, X., Khoka, M.K., Wendler, C.C., Blum, M., and Rivkees, S.A.
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(2013). Wnt11b is involved in cilia-mediated symmetry breakage during Xenopus left-right development. PLOS One 8, e73646
Walentek, P., Schneider, I., Schweickert, A., and Blum, M.
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(2014). Symmetry breakage in the frog Xenopus: Role of Rab11 and the ventral-right blastomere. Genesis
Tingler, M., Ott, T., Tözser, J., Getwan, M., Kurz, S., Tisler, M., Schweickert, A. andBlum M.
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(2014). Symmetry breakage in the vertebrate embryo: when does it happen and how does it work? Dev. Biol. 393, 109-123
Blum, M., Schweickert, A., Vick, P., Wright, C.V.E and Danilchik, M.
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(2014). The evolution and conservation of left-right patterning mechanisms. Development141, 1603-1613
Blum, M., Feistel, K., Thumberger, T. and Schweickert, A.
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(2015). ATP4a is required for development and function of the Xenopus mucociliary epidermis - a potential model to study proton pump inhibitor-associated pneumonia. Dev. Biol. 408, 292-304
Walentek, P., Hagenlocher, C., Beyer, T., Hagenlocher, C., Müller, C., Feistel, K., Schweickert, A., Harland, R. and Blum, M.
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(2015). TGF-β signaling regulates the differentiation of motile cilia. Cell Reports, 11, 1000-1007
Tözser, J., Earwood, R., Kato, A., Brown, J., Tanaka, K., Didier, R., Megraw, T., Blum, M. and Kato, Y.
