Abstract
BackgroundPark2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals. In Chlamydomonas and trypanosomes, the PACRG protein associates with flagella. Loss of PACRG results in shortened or absent flagella. In mouse the PACRG protein is required for spermatogenesis. The purpose of the present study was to analyze (1) the expression patterns of PACRG during vertebrate embryogenesis, and (2) whether the PACRG protein was required for left-right (LR) axis specification through cilia-driven leftward flow in Xenopus laevis.MethodsPACRG 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.ResultsPACRG 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.ConclusionsThe PACRG protein is a novel essential factor of cilia in Xenopus.
Highlights
Park2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals
PACRG was further detected in Lewy bodies: these are neuronal inclusions frequently found in the brain of Parkinson’s disease (PD) patients that are positive for Parkin, the protein encoded by Park2 [2]
Onset of PACRG expression correlated with the outgrowth of cilia, unlike other ciliary genes such as Foxj1 or dnah9, which are already induced in the superficial mesoderm (SM) and persist to be expressed in the gastrocoel roof plate (GRP) proper [18,26]
Summary
Park2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals. In Chlamydomonas and trypanosomes, the PACRG protein associates with flagella. Loss of PACRG results in shortened or absent flagella. In mouse the PACRG protein is required for spermatogenesis. In mammals PACRG shares a bidirectional promoter with Park, 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]. A precise function has yet to be ascribed, the available evidence suggests that the PACRG protein is cerebrospinal fluid flow [6]. Structural investigations suggested that PACRG associated with nexin interdoublet links in trypanosomes [4]. PACRG was further detected in Lewy bodies: these are neuronal inclusions frequently found in the brain of PD patients that are positive for Parkin, the protein encoded by Park2 [2]
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