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Abstract
Microtubules are structural components of the cell cytoskeleton and key factors for mitosis and ciliogenesis in eukaryotes. The regulation of MT dynamics requires non-motor MAPs. We previously showed that, in human cells in culture, MAP9 (also named ASAP) is involved in MT dynamics and is essential for mitotic spindle formation and mitosis progression. Indeed, misexpression of MAP9 leads to severe mitotic defects and cell death. Here, we investigated the in vivo role of map9 during zebrafish development. Map9 is expressed mainly as a maternal gene. Within cells, Map9 is associated with the MT network of the mitotic spindle and with centrosomes. Morpholino-mediated depletion of map9 leads to early development arrest before completion of epiboly. Map9 localizes to the MT array of the YSL. This MT network is destroyed in Map9-depleted embryos, and injection of anti-map9 morpholinos directly in the nascent YSL leads to arrest of epiboly/gastrulation. Finally, map9 knockdown deregulates the expression of genes involved in endodermal differentiation, dorso–ventral and left–right patterning, and other MT-based functions. At low morpholino doses, the surviving embryos show dramatic developmental defects, spindle and mitotic defects, and increased apoptosis. Our findings suggest that map9 is a crucial factor in early zebrafish development by regulating different MT-based processes.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors are grateful to Drs A Lander, A Muto, A Bruce, M Halpern, T Lepage, and M Poulain for providing the sox17, sox32, oep, and cyc cDNA clones to C Gonzalez, Drs M N’Guyen and K Kissa for advice and help in manipulating zebrafish embryos, to Dr Chris Jopling for help in map9 ISH experiments, and to Dr A Renucci for helpful discussions. We also would like to thank Montpellier Rio Imaging facility for microscopy support. We thank Drs E Andermarcher and R Kiernan for the careful reading of the manuscript and the English language corrections. This work was supported by the CNRS and grants from Association de la Recherche contre le Cancer (ARC N°4027), Ligue Nationale contre le Cancer (Comité Languedoc-Roussillon 2009), and the European Community’s Seventh Framework Programme [FP7-PEOPLE-2011-ITN] under grant agreement no. PITN-GA-2011–289209 for the Marie-Curie Initial Training Network FishForPharma. L.F. was a recipient of a MERT fellowship.
