good polarized positioning of buy inhibitor BBs and establishment of ?uid performedintraperitonealtamoxifeninjectionsintomousepups
?ow is finished by day 9 of postnatal growth . To usingtwodifferentschemes(SupplementaryFig.2a):(1)inducing
assess the role of Pten throughout ependyma development, we Pten knockout with tamoxifen at P0�CP2 (when multicilia are
NATURE COMMUNICATIONS |6:8388|DOI: 10.1038/ncomms9388 |www.nature.com/naturecommunications
forming) and analysing the defects at P9, or (2) inducing the explorethephysiologicalrelevanceofPTEN�CDVLinteraction,we
knockout at P3�C5 (after multicilia are already formed) Dasatinib and co-injected PTEN morpholino together with either full-length
analysing the defects at P10. Similar to our observations in GFP-Dsh or lower doses of a dominant-negative version Xdd1
tracheal cells, decreased Pten expression drastically affected the (ref. 33) and analysed the morphological changes in Xenopus
number of ependymal cilia (Fig. 2b), which was more apparent embryos, speci?cally focusing on defects in ciliogenesis and CE
To establish ?uid ?ow, ependymal MCCs (EMCCs) require polarizedmotilitywithindesignatedgroupsofcellsthatleadsto
First,BBsmigratetowardstheanterior their ordered intercalation with each apply for it other, resulting in tissue
side of a cell, creating so-called translational polarity. Next, elongation all through gastrulation and neural tube closure .
similar to Xenopus MCCs, basal rootlets of cilia within one cell Overexpression of either lower doses of a dominant-negative
are aligned, creating rotational polarity . Analysis of EMCCs DVL (Xdd1) , or full-length GFP-tagged DVL combined with
stained with gamma-tubulin, showed that in cells lacking Pten, PTEN loss of function, markedly increased the number of
BBpatcheslocalizeabnormallyandoftenmigrateinadirection embryos bearing CE defects (Fig. 3c; Supplementary Fig. 3e).
different from the neighbouring cell (Fig. 2c; Supplementary GiventheimportanceofDVLdosageandlocalizationduringCE
2b). In addition, a small portion of BB patches retained movements, similar effects from Xdd1 and Dsh-GFP can be
centrallocalizationwithjustextensiontowardsonesideofacell. expected. Moreover, when the embryos co-injected with Xdd1
This misalignment in BB localization was found signi?cant and PTEN morpholino were analysed for cilia formation, we
upon quanti?cation, as shown in Fig. 2d, suggesting that observedenhancedcilialoss,aswellasmoresevereBBdocking
PTEN regulates translational polarity of ependymal cells. Taken defects, in comparison with either treatment alone (Fig. 3d,e).