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Fig. 3. Knockdown of xNubp1 leads Ciliogenesis defects. ((A)�(C)) Immunofluorescence data showing surface views of ciliated epidermis from intact embryos. (A) xNubp1 SpMO (13 ng) morphant embryo, showing mainly internal acetylated α-tubulin positive structures. Very few cilia project outward from the apical surface. (B) Co-injection of xNubp1 SpMO with xNubp1 mRNA (90pg) rescues the phenotype and confirms the specificity of the MO. (C) Control epidermis showing normal ciliated cells. (D) Quantification of ciliated cells which lack cilia and have internal acetylated tubulin positive structures. Co-Injection of xNubp1 mRNA with either xNubp1MO1 or SpMO partially rescues cilia phenotype (E) TEM of cross section from stage 31 tailbud injected with xNubp1�SpMO, showing an internal ciliary axoneme right below the apical surface (black arrow). (F) Ventral view of X. laevis tadpoles. Unlike control embryos, xNubp1 morphants have various gut looping defects. (G) Maximum intensity projection (MIP) of optical sections from immunostaining showing cilia projecting out from posterior apical surface of a normal GRP (G′) and shorter cilia projecting for the apical surface of a SpMO morphant GRP (G′). In some morphant cells the cilia remain centrally localized and have not migrated to the posterior side of the cell. (H) Average length of GRP cilia is significantly reduced in xNubp1 morphants (2.4� 0.06 μm) compared to controls (6.7�0.1 μm). Horizontal lines indicate the mean, Vertical line SEM, p<0.0001 Mann�Whitney test. ((I)�(J)) Pitx2c and Xnr1expression at stage 24. (I) In wild type embryos Pitx2c is expressed in left lateral plate mesoderm (LPM) (black arrow). In xNubp1 morphants Pitx2c is absent. (J) Xnr1 expression is also lost from in xNubp1 morphants (black arrow, bottom panel). |
