Skeletal ciliopathies result from defects in primary cilia, which are crucial for embryonic development because they transduce extracellular signals, including Hedgehog. Selective transport of ciliary proteins is mediated by the intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes and the kinesin-2 and dynein-2 motors. Biallelic loss-of-function variants in genes encoding dynein-2-specific subunits, including DYNC2LI1, cause skeletal ciliopathies. As mesenchymal stem cells (MSCs) differentiate into osteoblasts, we investigated the effects of pathogenic variants of DYNC2LI1 on osteogenic differentiation of the MSC-like line C3H10T1/2. Dync2li1-knockout cells expressing disease-causing DYNC2LI1 variants demonstrated defects in the retrograde ciliary protein trafficking, including Hedgehog pathway GPCRs, Smoothened and GPR161. Furthermore, Dync2li1-knockout cells expressing the pathogenic variants demonstrated impaired Hedgehog signaling, in particular, a reduced ratio of the GLI3 repressor form to total GLI3, resulting in impaired osteogenic differentiation of MSCs. By contrast, osteogenic differentiation via BMP signaling was derepressed in Dync2li1-knockout cells. This suggests that skeletal ciliopathies caused by DYNC2LI1 variants could be attributable in part to impaired osteogenic differentiation due to defects in Hedgehog signaling, resulting from defects in retrograde ciliary protein trafficking.

Author contributions

Conceptualization: Y.I., H.-W.S., Y.K., K.N.; Formal analysis: Y.I., H.H., K.K.; Investigation: Y.I., H.H., K.K.; Project administration: Y.K., K.N.; Supervision: H.-W.S., Y.K., K.N.; Validation: Y.I.; Writing – original draft: K.N.; Writing – review & editing: Y.I., H.-W.S., Y.K., K.N.

Funding

This work was supported in part by grants from the Japan Society for the Promotion of Science (JSPS; grant numbers 20H04904 and 24K02022 to K.N., and 21H02427 and 22H05539 to Y.K.). Y.I. was supported by a JSPS Research Fellowship (grant number 22J20116).

Data and resource availability

All relevant data can be found within the article and its supplementary information.

Special Issue

This article is part of the Special Issue ‘Cilia and Flagella: from Basic Biology to Disease’, guest edited by Pleasantine Mill and Lotte Pedersen. See related articles at https://journals.biologists.com/jcs/issue/138/20.

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