A DIFFERENTIAL REQUIREMENT FOR THE CILIOPATHY GENE RPGRIP1L IN HUMAN AND MOUSE SPINAL PROGENITOR FATE SPECIFICATION

Studying developmental processes in the context of the human central nervous system is essential to understand neurodevelopmental diseases. In this paper we perform a comparative functional study of the ciliopathy gene RPGRIP1L in human and mouse spinal development using in vitro 3D differentiation of pluripotent stem cells. RGPRIP1L , a causal gene of severe neurodevelopmental ciliopathies such as Joubert and Meckel syndromes, encodes a scaffolding protein of the ciliary transition zone involved in ciliary gating. Previous work has identified a major role for Rpgrip1l in mouse brain and spinal cord development, via controlling the Sonic Hedgehog (SHH)/GLI pathway. We show that spinal organoids derived from Rpgrip1l mutant mouse embryonic stem cells faithfully recapitulate the loss of motoneurons and the strong reduction of SHH signaling observed in the mutant mice. In contrast, human induced pluripotent stem cells mutant for RPGRIP1L produce motoneurons and activate the SHH pathway at levels similar to wild types. Moreover, we show that, in human RPGRIP1L mutant organoids, MNs acquire a more anterior identity, expressing HOX genes and proteins normally present in the hindbrain while motoneurons from wild type organoids strictly display spinal identity. By performing a temporal transcriptome analysis throughout the differentiation process, we find that the anteroposterior specification defect arises in early axial progenitors. Thus, this study uncovers distinct functions in humans and mice for a ciliopathy protein and a novel role for RPGRIP1L in human spinal anteroposterior patterning. These findings have important implications for understanding the role of cilia in human spinal cord development and the pathogenic mechanisms of neurodevelopmental ciliopathies.