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. 2018 Feb 1;102(2):249–265. doi: 10.1016/j.ajhg.2017.12.017

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© 2017 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Cells Derived from Sall1^+/Δ Mouse Embryos Exhibit Cilia Defects

(A) PCR genotyping of MEFs derived from WT homozygous (Sall1^+/+) or Sall1-ΔZn^2–10 heterozygous (Sall1^+/Δ) embryos.

(B) Micrographs of Sall1^+/+ and Sall1^+/Δ MEFs analyzed during cilia assembly. Cilia were visualized by acetylated alpha-tubulin and gamma-tubulin (AcTub and γTub, respectively; purple), and nuclei were visualized by DAPI. Scale bar, 1 μm.

(C and D) Graphical representation of cilia length (C) and cilia frequency (D) measured in Sall1^+/+ (n = 38 cilia and 24 micrographs for cilia length and frequency, respectively; blue circles) and Sall1^+/Δ MEFs in (B) (n = 37–45 cilia and n = 16–38 micrographs for cilia length and frequency, respectively; orange triangles).

(E) Immunofluorescence micrographs of Sall1^+/+ and Sall1^+/Δ MEFs showing the centrosomes (AcTub, purple), CCP110 foci (green), and nuclei (DAPI). Scale bars, 0.5 μm. Cells that underwent 48 hr of starvation were compared with non-starved cells. Pictures were taken with a Deltavision fluorescence microscope (GE Healthcare Life Sciences) with a 60× objective.

(F) Graphical representation of the percentage of cells showing CCP110 in two foci in Sall1^+/+ and Sall1^+/Δ MEFs in (E) (n > 10). The graphs represent the mean and SEM. p values were calculated by one-way ANOVA: ^∗∗p < 0.01, ^∗∗∗p < 0.001.

(G) The presence of a truncated SALL1 underlies cilia malformations in TBS. In control cells (left), SALL1^FL is mainly nuclear, and CCP110 and CEP97 localize in the mother and daughter centrioles, inhibiting cilia formation. Upon starvation, CCP110 and CEP97 are depleted from the MC, which will allow the formation of the primary cilia. By contrast, in TBS cells (right) a truncated form of SALL1, together with sequestered SALL1^FL, localizes in the cytoplasm, where it interacts with centrosomal proteins such as CCP110 and CEP97, displacing them from the MC. As a result, the frequency of cilia formation increases, and cilia are longer than in control cells. Problems in cilia assembly are accompanied by altered SHH signaling in TBS cells.