The Joubert gene TMEM67 is required for the correct establishment of spinal dorsal identities in human organoids.
Primary cilia are essential signaling organelles that mediate key developmental pathways, including Sonic Hedgehog (SHH) and WNT, and are crucial for tissue patterning and homeostasis. Ciliary dysfunction underlies a spectrum of human ciliopathies--such as Joubert syndrome (JBTS) and Meckel syndrome (MKS)--which present with profound neurodevelopmental abnormalities. Although the role of cilia in SHH-dependent ventral spinal cord patterning is well established, their contribution to dorsal spinal cord development, particularly in human systems, remains poorly defined. To address this gap, we utilized human spinal organoids to investigate the function of a ciliopathy-associated protein in dorsal neural tube patterning downstream of BMP4, independent of exogenous SHH. Using TMEM67 knockout human iPSC-derived dorsal spinal organoids, we demonstrate that loss of TMEM67 disrupts the specification of dorsal interneurons, most notably within the dI1 lineage, while concomitantly expanding intermediate dorsal progenitor populations (dI4-dI6). These patterning defects are associated with defective roof plate induction and attenuated BMP4 signaling. Mechanistically, TMEM67 deficiency alters ciliary morphology, decreases cilia number, and impairs recruitment of BMPR2 to the ciliary base, suggesting a direct role for cilia in modulating BMP-induced dorsal spinal patterning. Together, these findings provide new mechanistic insights into the pathogenesis of ciliopathies and underscore the value of human organoid models for elucidating human-specific aspects of neurodevelopmental disorders.