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First published online 6 June 2007
doi: 10.1242/dev.003715

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Ftm is a novel basal body protein of cilia involved in Shh signalling

Jeanette Vierkotten¹, Renate Dildrop¹, Thomas Peters^1,*, Baolin Wang² and Ulrich Rüther^1,

¹ Institut für Entwicklungs- und Molekularbiologie der Tiere (EMT), Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.
² Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

View larger version (111K): [in this window] [in a new window]   Fig. 1. The Ftm protein structure and the phenotypes of Ftm-/- embryos. (A) The Ftm protein contains three coiled-coil domains [CC1 amino acids (aa) 43-85, CC2 aa264-375, CC3 aa491-576], one C2 domain (aa792-887) and an RID domain (aa1076-1264). The red arrowhead marks the position where the protein is truncated due to the mutation in Ftm mutant mice. (B) Western blot analysis using an antibody raised against the C-terminus of Ftm. Ftm is absent in Ftm-/- embryos (-/-). The lower lanes show the loading controls with anti-actin antibody. (C-F) Ftm-/- embryos (right side) are shown at different stages in comparison with their wild-type littermates (left side). (C) At E18.5, Ftm mutant embryos reveal polydactyly in fore and hind limbs (white arrowhead), reduction of eye structures and craniofacial malformations. (D) Craniofacial abnormalities in Ftm-/- embryos are shown, including a reduction of mandibular structures and unfused maxilliary and nasal tissues. (E) At E10.5, pericard enlargement (white arrowhead) and exencephaly (black arrowhead) are found in most of the embryos analysed. (F) At E9.5, 19% of analysed mutant embryos displayed dextrocardia (heart looping to the right body side) instead of the normal looping direction to the left. The final position of the ventricular part of the heart tube after looping is marked by the white arrowhead.

View larger version (127K): [in this window] [in a new window]   Fig. 2. Left-right asymmetry specification and limb development are affected in Ftm-/- embryos. (A-D) Hematoxylin and Eosin stainings at E15.5 on transverse sections. (A) In wild-type mouse embryos lungs branch into four right lobes (R1-R4) and into one left lobe (L1), while in mutant embryos (B) only two symmetric lobes are formed (R1 and L1). (C,D) In wild-type embryos the stomach develops in the left body half (C), while in some Ftm mutant embryos the stomach was positioned on the right (heterotaxia) (D). (E,F) Expression pattern of left-right marker gene. Pitx2 is exclusively expressed in the left lateral plate mesoderm in wild-type embryos at the 2- to 5-somite stages (E). In Ftm-/- embryos, Pitx2 shows a bilateral expression pattern (F). (G) Bone-cartilage stainings at E18.5 reveal a preaxial polydactyly in mutant limbs, visible by two extra digits in the fore limb and one extra digit in the hind limb. (H,I) At E11.5, expression of Shh appears to be unaffected in Ftm mutant limb buds (H), whereas Ptc1 expression is reduced (I). fl, fore limb; hl, hind limb; st, stomach; wt, wild type.

View larger version (106K): [in this window] [in a new window]   Fig. 3. Floor plate induction and ventral neural tube patterning is affected in Ftm-/- embryos. (A-H) In situ hybridizations on sections at stage E11.5. In the wild type mouse, Shh is expressed in the notochord (green arrowhead) and in the floor plate (blue arrowhead) (A), and Foxa2 expression is restricted to the floor plate (B). In Ftm-/- embryos, Shh expression in the floor plate is absent (C), and Foxa2 expression is lost (D). In wild-type embryos, Ptc1 is expressed within the neural tube and the mesenchyme sourrounding the notochord (E), and Irx3 is expressed within the whole neural tube except the most ventral region (F). In Ftm-/- embryos, expression of Ptc1 is strongly reduced (G). Irx3-positive cells are expanded into the most ventral part of the neural tube, including the midline (expression borders are marked with red bars) (H). (I-P) Immunohistochemistry at stage E11.5. In wild-type embryos, Shh protein is detectable in the floor plate (white arrowhead) and notochord (green arrowhead) (I), and Nkx2.2-expressing cells are found adjacent to the floor plate (J; white arrowhead). Ftm-/- embryos show a loss of Shh protein in the floor plate (K), and Nkx2.2-expressing cells are greatly reduced (L). In the wild type, MNR2-positive cells are located in two lateral domains (M), and Pax6-expressing cells are restricted to the dorsal neural tube. In Ftm-/- embryos, only a few MNR2-postive cells are found in a spotted fashion (O), and Pax6-expressing cells are expanded into the most ventral part of the neural tube (expression borders are marked with white bars) (P).

View larger version (102K): [in this window] [in a new window]   Fig. 4. Rescue of neural tube patterning in Ftm-/- embryos by abrogating Gli3 function. Shown are immunohistochemistry data at stage E11.5 mouse embryos from four different genotypes. (A-C) In wild-type embryos, MNR2 (A) is expressed lateral to Nkx2.2 (B), which is expressed adjacent to the floor plate. Furthermore, Pax2 is expressed in the dorsal region of the neural tube (C). (D-F) By contrast, Ftm-/- embryos show a strong reduction of MNR2-positive cells (D) and Nkx2.2-expressing cells (E). Expression of Pax2 is expanded into the most ventral part of the neural tube (F). (G-I) In Ftm-/-; Xt/+ embryos, the number of MNR2-positive (G) and Nkx2.2-positive (H) cells is increased, but these cells still cross the midline. Pax2-expressing cells are expanded into more ventral parts within the neural tube, but there are fewer cells crossing the midline (I). (J-L) In Ftm-/-; Xt/Xt embryos, MNR2-expressing (J) and Nkx2.2-expressing (K) cells are absent from the midline and located in two lateral distinct domains. Pax2-expressing cells (L) seem to be completely restored.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Ftm is necessary for the establishment of a proper Gli3-190/Gli3-83 ratio in mice. (A) Immunoblots show the amounts of Gli3-190 and Gli3-83 in E11.5 embryos. In wild-type and Ftm+/- embryos, the majority of Gli3 protein was processed to form Gli3-83, and only small amounts of Gli3-190 are detectable. In Ftm-/- embryos, the amount of Gli-83 is equal to wild-type levels, but the amount of Gli3-190 is increased. Note that there is an increase in total Gli3 protein in Ftm-/- embryos. The lower part shows the loading control with anti-tubulin antibody. (B) Graphical evaluation of the Gli3-190/Gli3-83 ratio in wild-type, Ftm+/- and Ftm-/- embryos. At least three embryos of each genotype have been analysed. (C) RT-PCR analysis of RNA from E11.5 embryos. The amount of Gli3 transcripts is increased in Ftm-/- embryos in comparison with the wild type. The lower part shows the Hprt normalization. (D) Comparison of relative Gli3 transcription levels in wild-type and Ftm-/- embryos (n=3). In Ftm-/- embryos, the number of Gli3 transcripts is increased nearly twofold.

View larger version (76K): [in this window] [in a new window]   Fig. 6. Ftm is located at the basal body and is necessary for normal cilia numbers in mice. (A) Scanning electron microscopy images at E7.5. In the wild-type node (left), each cell possesses a single cilium on its surface, whereas in Ftm-/- embryos (right), the number of cilia is reduced and the cilia are malformed. (B) Immunohistochemistry on sections of limb mesenchyme at E11.5. In the wild type (left), each mesenchymal cell exhibits cilia stained by acetylated -tubulin antibody. By contrast, in Ftm-/- embryos (right), the number of cilia is reduced. (C) Immunostainings on MEFs. (i) Ftm (red) is located at cilia, but does not overlap with acetylated -tubulin (green). (ii) Ftm (red) is co-localized with -tubulin (green) in the basal body of the cilia. The blue staining marks the nuclei by DAPI.

View larger version (69K): [in this window] [in a new window]   Fig. 7. Ciliogenesis and Shh signalling in Ftm-/- cells in mice. (A) Upper part: immunohistochemistry on G0 phase (left) and proliferating (right) MLCs. (Left panels) In G0 phase, each wild-type and Ftm-/- cell exhibits a single cilium, detected by acetylated -tubulin antibody (green staining). Ftm (stained in red) is present only in wild-type MLCs. (Right panels) In proliferating wild-type and Ftm-/- MLCs, cilia are absent and the cytoplasm is enriched with cytoskeletal tubulins, marked by acetylated -tubulin antibody (green staining). Note that Ftm antibodies were not used in this staining. Lower part: Shh response in G0-phase (left) and proliferating (right) wild-type and Ftm-/- MLCs. G0-phase wild-type MLCs show a strong induction of Gli1 and Ptc1 after incubation with recombinant Shh protein for 24 hours. In G0-phase Ftm-/- MLCs, the induction of Gli1 and Ptc1 is strongly reduced. Both, proliferating (non-ciliated) wild-type and Ftm-/- MLCs show no induction of Shh target genes after stimulation. (B) Quantification of Ptc1-expression levels in G0-phase (left panels) and proliferating (right panels) wild-type and Ftm-/- MLCs after Shh stimulation. The quantification of Ptc1 induction reveals that G0-phase wild-type MLCs show an increase of Ptc1 transcripts by a factor of 9.4, whereas G0-phase Ftm-/- MLCs show an increase of only a factor of 3.1. Proliferating (non-ciliated) wild-type and Ftm-/- MLCs show no changes in the relative expression levels of Ptc1 upon treatment with Shh (wild-type MLCs 1.1; Ftm-/- MLCs 0.8). Data represent the quantification of three independent experiments. wt, wild type.