Objective:

Although primary cilia are widely distributed in the developing CNS, their dysfunction results in region-specific defects in neurodevelopmental ciliopathies like Joubert Syndrome. The pathogenetic mechanisms connecting ciliary dysfunction to these spatially distinct developmental defects remain poorly understood. Advances in human induced pluripotent stem cell (hiPSC)-derived organoid models provide new avenues to investigate the context-dependent roles of primary cilia in human neural development.

Methods:

We employed hiPSC-derived ventral and dorsal spinal organoids, combined with time-resolved transcriptomic profiling and high-resolution imaging, to uncover previously unrecognized roles of the ciliopathy-associated proteins RPGRIP1L and TMEM67 in human neural patterning.

Results:

Utilizing two distinct organoid models -mimicking ventral and dorsal spinal cord development- enabled us to dissect the contributions of ciliary proteins to SHH and BMP signaling in spatial patterning and highlighted the differential dependency of these signaling pathways on cilia integrity in human development.

Neural progenitors deficient for RPGRIP1L or TMEM67 in ventral spinal organoids retained cilia, responded to cilia-mediated Sonic Hedgehog (SHH) signaling, and were capable of terminal motor neuron (MN) differentiation. However, RPGRIP1L-deficient MNs exhibited anterior-posterior patterning abnormalities pointing towards a SHH independent role of cilia in early spinal patterning (Wiegering et al., 2025).

Here, we show that RPGRIP1L- or TMEM67-mutant progenitors differentiated under dorsalizing conditions also maintained cilia but displayed patterning defects resembling those of impaired BMP signaling; and this, in contrast to in vivo data, uncoupled from ventral SHH cues. Analysis of BMP pathway components in conjunction with ciliary phenotypes will provide insight into how distinct patterning abnormalities emerge in ciliopathies.

Conclusion:

By uncoupling ventral and dorsal differentiation cues, our studies reveal distinct functions for cilia and ciliopathy proteins in regional human spinal patterning. Further exploration of context-specific cilia functions will enhance our understanding of the regional patterning defects and pathogenic mechanisms observed in neurodevelopmental ciliopathies.