Hedgehog Signaling Controls Astral Microtubules and Mitotic Spindle Orientation in Neural Progenitors and iPSCs

Fengming Liu¹Anna Medyukhina¹Kris M Olesen¹Abbas Shirinifard¹Hongjian Jin²Lei Li²Marina Mapelli³Khaled Khairy¹Young-Goo Han^1*

• ¹Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States
• ²Center for Applied Bioinformatics, St. Jude Children's Research Hospital, Memphis, United States
• ³European Institute of Oncology (IEO), Milan, Lombardy, Italy

Mitotic spindle orientation is crucial for cell fate determination and tissue organization. Although the intracellular machinery governing spindle orientation is well characterized, whether and how secreted factors, such as morphogens, regulate this process remains poorly understood. This study investigated the role of Hedgehog (HH) signaling in modulating mitotic spindle orientation in neural progenitor cells and in induced pluripotent stem cells (iPSCs). Time-lapse microscopy of cerebral organoids and iPSCs revealed that HH signaling increases the angle of the mitotic spindle relative to the apical surface, prolongs mitosis, and enhances spindle rotation. Mechanistically, HH signaling reduces both the number and the length of astral microtubules, key regulators of spindle orientation. This reduction correlates with increased spindle angle in iPSCs. Furthermore, we show that canonical HH signaling, involving GLI-dependent transcriptional regulation, contributes to these effects. RNA sequencing and gene set enrichment analysis (GSEA) revealed that HH signaling upregulates genes associated with microtubule depolymerization, suggesting a transcriptional mechanism by which HH signaling influences astral microtubule dynamics and, consequently, mitotic spindle orientation. These findings highlight a novel link between a morphogen, transcriptional regulation, and the control of mitotic spindle orientation, with implications for development and tissue homeostasis.