Genetische Kontrolle der Notochordentwicklung: Analyse der truncate Mutation der Maus
Final Report Abstract
The notochord is a rod-like structure, which is located ventral to the neural tube in the midline of chordate embryos, and is an essential signaling center for patterning the surrounding tissues. Truncate (tc) is a spontaneous mutation in mouse that affects development of the caudal notochord. We identified the gene affected by the mutation by positional cloning and identified the long-sought mouse homologue of zebrafish flh as the relevant gene (referred to as Noto). Noto encodes a homeodomain transcription factor specifically expressed in the node and nascent notochord. The tc allele is a missense mutation that changes a conserved phenylalanine residue in helix1 of the homeodomain to cysteine and destabilizes the homeobox. To formally prove that this point mutation causes the tc phenotype we generated homozygous tc ES cells, reverted one Noto allele to wild type by homologous recombination and showed that this reversion restored wild type function in completely ES cell-derived embryos. To further study the role of Noto we generated mice carrying a null allele (NotoGFP). These mice showed notochord defects similar to tc mice. In contrast to tc homozygous NotoGFP mice showed a high rate of post-natal mortality, indicating that tc represents a hypomorphic Noto allele. Analysis of the visceral organs of NotoGFP mice revealed laterality defects ranging from normal situs to heterotaxia, isomerism, and complete situs inversus. Outflow tract malformations were frequently associated with the laterality defects and provide a plausible explanation for the post-natal mortality. The node plays an essential role in generating a symmetry-breaking signal in the early embryo. Here, motile cilia rotate clock-wise and generate a leftward flow which is essential for symmetry breaking. Further analyses of Noto mutants showed that node morphology and ultra structure of nodal cilia were highly abnormal, and nodal cilia were essentially immotile, which are a likely cause for disrupted establishment of left-right asymmetry. To identify Noto targets that might be important for the generation of normal node and functional cilia we performed microarray analyses. In addition to genes with unknown functions known ciliary components and Foxj1, which codes for a transcription factor regulating motile cilia formation in multiple tissues, were identified. The similarity of defects in lung epithelium cells lacking Foxj1, and in node cells lacking Noto, suggested that Noto exerts its function during ciliogenesis at least in part through Foxj1. To address this question, we generated and analyzed a knock-in allele that expresses Foxj1 instead of Noto. Our analyses separated Noto and Foxj1 function in the node: nodal ciliogenesis appears to be controlled by Foxj1 and Rfx3 downstream of Noto, and Noto controls node morphogenesis and potentially planar cell polarity. We showed that Foxj1 expressed from the Noto locus is functional and restores the expression of ciliary genes and the formation of structurally normal motile cilia in the absence of Noto, but is not sufficient for the correct positioning of cilia on the cell surface within the plane of the nodal epithelium, and normal node morphology. To better understand how Noto expression is regulated we have analyzed the Noto promoter and identified an enhancer region of 523 bp approximately 7 kb upstream of exon 1 that is necessary and sufficient for node and notochord specific Noto expression. Further analyses identified three enhancers within this region that contribute to Noto expression in the node and nascent notochord.
Publications
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(2004). The mouse homeobox gene Not is required for caudal notochord development and affected by the truncate mutation. Genes Dev 18, 1725–1736
Abdelkhalek, H. B., Beckers, A., Schuster-Gossler, K., Pavlova, M. N., Burkhardt, H., Lickert, H., Rossant, J., Reinhardt, R., Schalkwyk, L. C., Müller, I., Herrmann, B.G., Ceolin, M., Rivera-Pomar, R., and Gossler A.
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(2007). The mouse homeobox gene Noto regulates node morphogenesis, notochordal ciliogenesis, and left right patterning. PNAS 104, 15765–15770
Beckers, A., Alten, L., Viebahn, C., Andre, P. and Gossler, A.
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(2012). A novel mammal-specific three partite enhancer element regulates node and notochord-specific Noto expression. PLoS ONE 7, e47785
Alten, L., Schuster-Gossler, K., Eichenlaub, M. P., Wittbrodt, B., Wittbrodt, J. and Gossler, A.
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(2012). Differential regulation of node formation, nodal ciliogenesis and cilia positioning by Noto and Foxj1. Development 139, 1276–1284
Alten, L., Schuster-Gossler, K., Beckers, A., Groos, S., Ulmer, B., Hegermann, J., Ochs, M. and Gossler, A.
