Ciliary and non-ciliary expression and function of PACRGduring vertebrate development
© Thumberger et al.; licensee BioMed Central Ltd. 2012
Received: 16 March 2012
Accepted: 30 May 2012
Published: 1 August 2012
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.
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 reinhardtii and of Trypanosoma brucei as well as in mouse spermatocytes . Parallel RNAi-mediated knockdown of two paralogous genes in trypanosomes resulted in motility-impaired specimens with flagella of apparently normal length but outer microtubule doublet defects . In the viable mutant mouse quaking (qk v ) male fertility was lost due to a deletion of PACRG, which resulted in failure to complete spermatogenesis . Mutant mice were also affected by acquired hydrocephalus due to a defect in ependymal cilia function, resulting in reduced cerebrospinal fluid flow . Structural investigations suggested that PACRG associated with nexin interdoublet links in trypanosomes . In contrast, a localization between A and B tubules was proposed in the axoneme of Chlamydomonas flagellae . Non-ciliary localizations were reported as well. PACRG was found in a large molecular chaperone complex containing heat shock proteins 70 and 90 as well as chaperonin components . PACRG was further detected in Lewy bodies: these are neuronal inclusions frequently found in the brain of PD patients that are also positive for Parkin, the protein encoded by Park2.
Cilia play a pivotal role during early vertebrate embryogenesis [7–10], with the establishment of the LR body axis as the first event where cilia are required [11–14]. During gastrulation a ciliated epithelium forms at the posterior pole of the emerging notochord . This epithelium harbors rotating monocilia, which due to their posterior polarization produce a leftward flow of extracellular fluid. Epithelia vary in shape and size but are structurally and functionally homologous . They comprise the Kupffer’s vesicle (KV) in bony fish, the gastrocoel roof plate (GRP) of amphibian embryos and the PNC in mammals. Experimental or genetic inhibition of flow, ablation or mispolarization of cilia or impairment of ciliary motility in all cases results in LR axis defects [11, 13, 17, 18]. Downstream of leftward flow, the asymmetric Nodal gene cascade, consisting of the growth factor Nodal, its feedback inhibitor Lefty and the homeobox transcription factor Pitx2, is initiated in the left lateral plate mesoderm (LPM) and governs asymmetric organ morphogenesis and placement at later stages of development .
Here, we asked whether PACRG plays a role during embryogenesis as well, specifically during LR axis formation. PACRG expression was predominantly found in tissues harboring cilia in early frog, mouse, rabbit and zebrafish embryos. A GFP fusion protein labeled cilia in the frog GRP and epidermis. Gene knockdown in the frog demonstrated an embryonic role of PACRG in gastrulation, LR development and neural tube closure.
Cloning of constructs
Total RNA was isolated from embryos of various stages (frog, mouse, rabbit and zebrafish) and cDNAs were prepared using standard protocols. Primers for PCR amplification of X. laevis PACRG (accession number JQ771622) were designed based on X. tropicalis expressed sequence tags (accession numbers CX959700.1, CU025070.1): untranslated region (UTR) forward 5′-TAGGCAACCGAACGTAAACAACAG-3′; forward 5′-ATGGTGTTTGAGACAAGCAAAGCAACA-3′; reverse 5′-GTTCAGCAAGCAGGATTCAT-3′. In order to clone the enhanced GFP (eGFP) fusion construct, BamHI and XhoI restriction sites were introduced into forward and reverse primers, respectively. eGFP was cloned using primers forward 5′-CTCGAGATGGTGAGCAAGGGCGAGGAGC-3′ (including XhoI site); reverse 5′-TCTAGATTACTTGTACAGCTCGTCCATG-3′ (including XbaI site).
Xenopus PACRG and eGFP were ligated into the BamHI/XbaI linearized CS2+ vector. The Xenopus rescue-eGFP construct was cloned using mutated PACRG forward primer 5′-ATGGTCTTCGAAACTAGTAAGGCAACA-3′ to prevent morpholino oligonucleotide (MO) binding. Mouse PACRG (accession number BC120740.1) was cloned using primers forward 5′-CCCTCTCCTCCCCTAAACTC-3′; reverse 5′-GGTCAGTTCAGCAAGCACG-3′. A rabbit PACRG fragment was cloned using primers designed to match regions conserved between human (accession number BC044227.1) and mouse PACRG (see above): forward 5′- ATGCCGAAGAGGACTAAACTGCTG-3′; reverse 5′-ACCTACGAGTCTTGCTTGCT-3′ (accession number JQ771623). Zebrafish PACRG (accession number ENSDARG00000004736) was cloned using primers forward 5′-ATGAGAACCTTTGAACCTTTGGCTA-3′; reverse 5′-GTTGAGAAGGCAGGACTCGTAGGTGGG-3′.
RNA in situhybridization, immunohistochemistry and histological analysis
Embryos or explanted larval brains were fixed in MEMFA (1 part of (1M MOPS (pH 7.4, Roth), 20 mM EGTA (Applichem), and 10 mM MgSO4,(Applichem)), 8 parts H2O and 1 part formaldehyde (37%, Roth)) or 4% paraformaldehyde (PFA, Roth) for 2 h and processed following standard protocols . Digoxigenin-labeled (Roche) RNA probes were prepared from linearized plasmids using SP6 or T7 RNA polymerase (Promega). In situ hybridization was according to . Immunohistochemistry was performed on whole-mount embryos fixed in 4% PFA for 1 h at room temperature. Embryos were processed according to standard procedures . Antibodies used include mouse monoclonal antibody directed against acetylated alpha tubulin (1:700; Sigma), rabbit polyclonal antibody directed against PACRG (1:100, Rockland Immunochemicals, Inc.) and Cy2-conjugated or Cy3-conjugated secondary polyclonal rabbit or sheep anti mouse antibodies (Jackson Immunoresearch or Sigma; both 1:250). RNA encoding membrane red fluorescent protein (mRFP) (50 to 100 ng/μl) or rhodamine-B dextran (0.5 to 1.0 μg/μl; Molecular Probes) were used as lineage tracers. For histological analysis embryos were embedded in gelatin-albumin and sectioned on a vibratome at 30 μm (standard) to 40 μm (brain sections). Statistical calculations of marker gene expression patterns were performed using Pearson’s χ2 test (statistical R; http://cran.r-project.org/). SEM analysis was performed as described . GRP cilia and cell parameters were determined in a square of 1,000 × 1,000 pixels (magnification 500-fold, corresponding to 86 μm2) at the center of GRP in SEM pictures . Cilia lengths, polarization (posterior, central, other) and cell surface areas were determined manually in ImageJ . Ciliation rates were calculated as the ratio of cilia over cells (separately in each GRP SEM photograph). Posterior polarization was quantified for each GRP and statistical significances were calculated by Student’s t test in statistical R (http://cran.r-project.org/). The whiskers of the box plots extend to maximal 1.5 × interquartile range (IQR), outliers are displayed as dots.
Microinjections and MO-mediated knockdown of frog PACRG
Embryos were injected at the four to eight cell stage using a Harvard Apparatus set-up. Drop size was calibrated to about 7–8 nl/injection. Morpholinos (Gene Tools, Philomath, OR, USA) were used at 0.4-2 pmol/embryo as indicated. Lineage tracer RNAs were prepared using the Ambion message machine kit (Ambion) and diluted to a concentration of about 50–100 ng/μl. In all experiments care was taken to exclusively use four to eight cell embryos with a clear dorsoventral segregation of pigment [23, 24], and only correctly targeted specimens (controlled by coinjected lineage tracer) were processed for further analysis. The AUG blocking MO for frog PACRG (PACRG-MO) comprised 5′-TGCTTGTCTCAAACACCATATTCAC-3′.
Video analysis of cilia, blastopore closure and leftward flow
Fluorescent in vivo imaging of epidermal and GRP cilia was performed following injection of 80 ng/μl PACRG-eGFP mRNA. Timelapse sequences were recorded on a Zeiss Axioskop equipped with a CCD camera (AxioCam Hsm, Zeiss) using AxioVision 4.6 (Zeiss) at 62 fps (beating cilia) or 2 fps (leftward flow). For blastopore closure timelapse acquisition, specimens were mounted onto an inverse microscope in a glass-bottom Petri dish onto a nitex mesh and cultivated in 0.1 × MBSH (Modified Barth‘s Saline). Timelapse movies were acquired at one frame every 2 minutes. Preparation of dorsal explants, recording of timelapse movies, processing and analysis of leftward flow were according to . Significances were calculated by Student’s t test in statistical R (http://cran.r-project.org/). The whiskers of the box plot extend to maximal 1.5 × IQR.
Results and discussion
Cloning and expression analysis of PACRGmRNA during vertebrate embryonic development
PACRG protein localizes to cilia and intracellular compartments
In order to confirm the immunohistochemistry data, we injected a PACRG-eGFP fusion construct into four-cell Xenopus embryos and targeted the mRNA to the GRP or epidermal cells. As shown in Figure 4E,F, the fusion protein marked both monocilia on the GRP and cilia of multiciliated skin cells. Vesicle-like structures and the perinuclear region were positive for the fusion protein as well (not shown), demonstrating that the fusion protein localized in an identical manner as the endogenous PACRG.
The ciliary localization of PACRG-eGFP afforded the opportunity of testing whether this fusion protein enabled live imaging of motile cilia in the frog Xenopus. The thickness of dorsal explants and the high yolk content of cells resulted in scattering of polarized light, which prevented in vivo imaging of GRP cilia in top view in the past . Additional file 3, movie 1 shows a field of GRP cilia, confirming their rotational and, due to the posterior tilt, elliptical beat pattern (see also Figure 4E). The whip-like wave form of epidermal cilia bundles could likewise be recorded (Additional file 3: Movie 1). These data demonstrated that PACRG-eGFP could be used as a cilia marker for live imaging in frog, and perhaps in other vertebrate model organisms as well.
LR axis defects in PACRGmorphants
In order to investigate the function of PACRG during Xenopus LR development, an antisense morpholino oligonucleotide (MO) was designed which targeted the translational start site. PACRG-MO or a random control MO (co-MO) were injected into the GRP lineage by targeting the dorsal marginal zone at the four-cell stage as described . Embryos were cultured until control uninjected specimens reached stage 34. Dose-dependently a series of axial defects were recorded (see below). Following injections of 0.4 pmol PACRG-MO per embryo specimens developed with wild-type dorsoanterior index (DAI; ) of 5 (n = 89/91). Alterations of dorsoanterior development (DAI ≠ 5) frequently indicate midline defects, which inevitably cause altered LR marker gene expression and organ situs [34, 35]. Therefore LR parameters were only evaluated in DAI5 PACRG morphants.
Gastrulation and neural tube closure defects in morphants
Neural tube closure depends on apical constriction of neural plate cells as well, and as with bottle cell formation this process has been shown to require intact microtubules . Neural tube closure defects (NTD) seen at intermediate PACRG-MO doses might therefore be related to altered assembly of parallel microtubule arrays as well. NTD represented a specific PACRG phenotype, as we were able to rescue closure by coinjection of PACRG-eGFP mRNA using a construct in which the MO binding site was mutated (Figure 7E-G). In summary, intermediate and high doses of MO caused phenotypes unrelated to cilia but associated with intracellular arrangement of ordered microtubule bundles.
Our study of PACRG in four vertebrate model organisms revealed a pronounced degree of conservation at the level of amino acid sequences and embryonic expression. In particular, PACRG was highly correlated with motile cilia during development, an aspect that we confirmed in depth by our functional analysis of LR development in PACRG morphants in Xenopus. The remarkable non-ciliary PACRG functions are worth being analyzed in greater depth in future studies. The preliminary evidence presented here points to a more general role related to non-dynamic microtubules in their recently shown involvement in apical cell constriction. In addition, the expression in the embryonic brain at sites where lesions in populations of non-dopaminergic neurons occur in PD patients may deserve further attention. Lastly, PACRG may serve as a versatile marker of motile cilia in live imaging.
We would like to thank Ray Keller, in whose lab the blastopore closure timelapse videos were recorded with the help of Dave Shook, Philipp Vick for providing the gastrula-stage tubulin immunohistochemistry and for critical reading of the manuscript, Susanne Bogusch for expert help with some of the experiments and Jochen Wittbrodt, in whose lab TT performed the zebrafish experiments. TT and TB were recipients of a PhD fellowship from the Landesgraduiertenförderung Baden-Württemberg, CH and KF are indebted to the Baden-Württemberg Stiftung for the financial support of their research by the Eliteprogramme for Postdocs; KF was supported by a Margarete-von-Wrangell fellowship, funded by the European Social Fund in Baden-Württemberg, and work in the Blum lab was funded through DFG grants Bl285/9-2 and Bl285/10-1.
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