Identifying domains of EFHC1 involved in ciliary localization, ciliogenesis, and the regulation of Wnt signaling

Abstract

EFHC1 encodes a ciliary protein that has been linked to Juvenile Myoclonic Epilepsy. In ectodermal explants, derived from Xenopus laevis embryos, the morpholino-mediated down-regulation of EFHC1b inhibited multiciliated cell formation. In those ciliated cells that did form, axoneme but not basal body formation was inhibited. EFHC1b morphant embryos displayed defects in central nervous system (CNS) and neural crest patterning that were rescued by a EFHC1b-GFP chimera. EFHC1b-GFP localized to ciliary axonemes in epidermal, gastrocoele roof plate, and neural tube cells. In X. laevis there is a link between Wnt signaling and multiciliated cell formation. While down-regulation of EFHC1b led to a ~2-fold increase in the activity of the β-catenin/Wnt-responsive TOPFLASH reporter, EFHC1b-GFP did not inhibit β-catenin activation of TOPFLASH. Wnt8a RNA levels were increased in EFHC1b morphant ectodermal explants and intact embryos, analyzed prior to the on-set of ciliogenesis. Rescue of the EFHC1b MO’s ciliary axonemal phenotypes required the entire protein; in contrast, the EFHC1b morpholino’s Wnt8a, CNS, and neural crest phenotypes were rescued by a truncated form of EFHC1b. The EFHC1b morpholino’s Wnt8a phenotype was also rescued by the injection of RNAs encoding secreted Wnt inhibitors, suggesting that these phenotypes are due to effects on Wnt signaling, rather than the loss of cilia, an observation of potential relevance to understanding EFHC1’s role in human neural development.

Microtubule-associated defects caused by EFHC1 mutations in juvenile myoclonic epilepsy

“…Therefore, structural modeling of EFHC1 mutants is a useful step in understanding the importance of structure–function relationships in DM10 domains. Interestingly, recent functional evidence suggests that DM10 domains of EFHC1 are involved in ciliogenesis and regulate aspects of Wnt signaling during development in Xenopus leavis (Zhao, Shi, Winey, & Klymkowsky, ).…”

Using Xenopus to understand human disease and developmental disorders

“…Numerous studies in Xenopus elucidate mechanisms of morphogenesis and organogenesis ( Agricola et al, ; Bestman, Huang, Lee‐Osbourne, Cheung, & Cline, ; Metikala, Neuhaus, & Hollemann, ; Mimoto, Kwon, Green, Goldman, & Christian, ; Nie & Bronner, ; Okada, Wen, Miller, Su, & Shi, ; Ossipova et al, ; Tanizaki, Ishida‐Iwata, Obuchi‐Shimoji, & Kato, ). Many of the components of the well‐known signaling pathways were discovered in Xenopus and fly, and new discoveries in this field continue to rely on Xenopus (e.g., Lee et al, ; Lee, Shi, & Zheng, ; Park et al, ; Zhang et al, ; Zhao, Shi, Winey, & Klymkowsky, ). Nuclear reprogramming and stem cell research began in amphibians, particularly frog (Gurdon, ), and Xenopus continues to be used for seminal discoveries (e.g., Ali et al, ; Buitrago‐Delgado, Nordin, Rao, Geary, & LaBonne, ; Jullien et al, ; Kole, Ambady, Page, & Dominko, ; Kurmann et al, ; Maza & Hanna, ; Paranjpe & Veenstra, ).…”

TetrahymenaRIB72A and RIB72B are Microtubule Inner Proteins in the ciliary doublet microtubules

“…Mutations in the human RIB72 homolog, hsEFHC1, cause juvenile myoclonic epilepsy (Suzuki et al, 2004). A common feature of RIB72 homologs are three DM10 domains that are important for ciliary localization (Zhao et al, 2016) and in many cases a Cterminal, Ca 2+ -binding EF-hand motif (Zhao, 2015) (Fig. 1C).…”