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

ABSTRACT: UNLABELLED: BACKGROUND: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. 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. METHODS: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 ?2 tests. RESULTS: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. CONCLUSIONS:The PACRG protein is a novel essential factor of cilia in Xenopus.

SUBMITTER: Thumberger T

PROVIDER: S-EPMC3555705 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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

Thumberger Thomas T Hagenlocher Cathrin C Tisler Matthias M Beyer Tina T Tietze Nina N Schweickert Axel A Feistel Kerstin K Blum Martin M

Cilia 20120801 1

<h4>Unlabelled</h4><h4>Background</h4>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. 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 lef ...[more]

PMID: 23351225

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Project description:BackgroundRenin-angiotensin system (RAS) components have been identified in human ciliary body and aqueous humour, pointing to a role for the RAS in the regulation of aqueous humour dynamics. Here, the authors examine the functional expression of a RAS and the effects of angiotensin II (AII) on a signal transduction pathway and ion secretion mechanism in cultured human ciliary body non-pigmented epithelium (HNPE).MethodsRAS expression was examined in cultured HNPE cells using polymerase chain reaction (PCR) analysis. Secretory function was determined using spectrofluorescence imaging microscopy to measure cell calcium (Ca(2+)(I)) and volume responses. Single channel patch clamp techniques were employed to investigate ion channel activity.ResultsPCR analysis demonstrated the expression of angiotensinogen and the AT(1b) receptor in HNPE cells. A large conductance potassium (BK) channel (mean 190 (SEM 5.6) pS, n = 22 cells), was observed in plasma membrane patches. This channel was calcium sensitive with channel open probability (Po) increasing with increasing Ca(2+)(I) (K(0.5) 10.79 (0.44) microM Ca(2+), Hill coefficient of 1.04 (0.04)). AII (100 nM) increased the number (N) of active BK channels in HNPE cells and also the probability of channel opening (Po). N.P(o) increased from 0.008 (0.002) to 1.38 (0.4) following the addition of AII (p=0.0064). AII also induced a rapid rise in Ca(2+)(I) from resting values of 112 (14) nM to a peak of 992 (106) nM (p<10(-4)). A simultaneous cell volume reduction of 24.70% (3.34%) (p<10(-4)) occurred during this calcium signal. Losartan (1 microM) significantly blocked the AII induced BK channel activation (p=0.0131), the Ca(2+)(I) response (p<10(-4)), and the AII induced volume effect (p=0.0046).ConclusionIt was demonstrated that AII activates a Ca(2+)(I) signalling system which subsequently increases potassium ion channel activity. These effects are accompanied simultaneously by cell volume loss, indicating that AII acts as receptor operated secretagogue in HNPE cells. The ability of an AT(1) receptor antagonist to inhibit these processes may thus offer a new family of pharmaceutical agents to the current armamentarium in the treatment of glaucoma.

Dataset Information

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

Publications

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

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