Ciliary and non-ciliary expression and function of PACRG during vertebrate development

PACRG cDNAs were cloned and expression was analyzed during early embryonic development of Xenopus, mouse, rabbit and zebrafish. Antisense morpholino oligonucleotide (MO) mediated gene knockdown was applied in Xenopus to investigate LR development at the level of tissue morphology, leftward flow and asymmetric marker gene expression, using timelapse videography, scanning electron microscopy (SEM) and whole-mount in situ hybridization. Results were statistically evaluated using Wilcoxon paired and χ2 tests.PACRG mRNA expression was found in cells and tissues harboring cilia throughout the vertebrates. Highly localized expression was also detected in the brain. During early development, PACRG was specifically localized to epithelia where leftward flow arises, that is, the gastrocoel roof plate (GRP) in Xenopus, the posterior notochord (PNC) in mammals and Kupffer’s vesicle (KV) in zebrafish. Besides its association with ciliary axonemes, subcellular localization of PACRG protein was found around the nucleus and in a spotty pattern in the cytoplasm. A green fluorescent protein (GFP) fusion construct preferentially labeled cilia, rendering PACRG a versatile marker for live imaging. Loss-of-function in the frog resulted dose dependently in LR, neural tube closure and gastrulation defects, representing ciliary and non-ciliary functions of PACRG.The PACRG protein is a novel essential factor of cilia in Xenopus.PACRG was originally identified as a gene related to Parkinson’s disease (PD) in humans [1,2]. In mammals PACRG shares a bidirectional promoter with Park2, the target gene for early onset juvenile PD. PACRG represents an evolutionarily very highly conserved gene, which is present from green algae to mammals [1,3,4]. Although a precise function has yet to be ascribed, the available evidence suggests that the PACRG protein is associated with the ciliary axoneme: antibodies or green fluorescent protein (GFP) fusion proteins detected PACRG in flagellae of Chlamydomonas re