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Figure 1. PACRG expression during early Xenopus development. (A) Expression at the four-cell stage (top view). (A’) Sagittal hemisection of embryo shown in (A). Enlargements (boxes) indicate higher expression levels on the dorsal side. (B) Gastrula embryo. Persistent differences in staining intensities between dorsal and ventral side. (B’) Sagittal hemisection (plane indicated in (B)) revealing mRNA localization in deep mesoderm. Note that the superficial mesoderm (SM) was free of PACRG mRNA (inset in (B’)). Dorsal lip marked by asterisk. (C) Expression in the gastrocoel roof plate (GRP) at stage 17 (dorsal explant shown in ventral view). (C’) Histological section (plane marked in (C)). (D) Expression at stage 23 in ciliated cells of the epidermis and the floor plate (sagittal section shown in (D’)). Enlargements show multiciliated skin cells ((Dâ€); cilia indicated by arrowheads) and GRP cells after intercalation into the notochord ((Dâ€â€™); arrowheads). (E) Staining in the otic vesicle and nephrostomes (arrowheads). (E’,Eâ€) Histological sections (levels indicated in (E)) highlighting expression in the otic vesicle (E’) and nephrostomes ((Eâ€), arrowheads). a = anterior; an = animal; bc = blastocoel; bp = blastopore; d = dorsal; dl = dorsal lip; dm = deep mesoderm; fg = foregut; fp = floor plate; hg = hindgut; l = left; no = notochord; ov = otic vesicle; p = posterior; r = right; s = somite; v = ventral; veg = vegetal.
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pacrg (PARK2 co-regulated) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, posterior view, dorsal up.
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pacrg (PARK2 co-regulated) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
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pacrg (PARK2 co-regulated) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, transverse section, anterior left, dorsal up.
See Xb-IMG-83181 for enlargements indictated by D" and D"'
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pacrg (PARK2 co-regulated) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, head and trunk region only, anterior left, dorsal up.
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Figure 2. PACRG expression in the developing Xenopus brain. (A) Brain anatomy of the 3-day tadpole (stage 45), as highlighted by rhodamine dextran injection into the hindbrain ventricle. (B) Isolated brain following in situ hybridization with an antisense PACRG probe, shown in dorsal (left) and ventral (right) view. (C) Whole-mount of brain in which the hindbrain was removed and a dorsal cut was introduced between forebrain and interbrain to expose the choroid plexus (higher magnification shown in inset). (D-G) Transversal histological sections; levels indicated in (B), dorsal side up. (D) Staining in the thalamic nuclei. (E) Localized expression in roof of cerebral aqueduct (cilia highlighted by arrowheads in the enlargement shown in (E�)). (F) Expression in ventral midline of midbrain region. (G) Staining in roof of hindbrain. (H) Scanning electron micrograph, demonstrating multiciliated cells in roof of hindbrain. ca = cerebral aqueduct; cp = choroid plexus; fb = forebrain; hb = hindbrain; ib = interbrain; mb = midbrain; tn = thalamic nucleus. Note that dark brown spots in whole-mount brain specimens (B) and sections (D-G) represent melanocytes.
doi:10.1186/2046-2530-1-13
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Figure 3. PACRG expression during early rabbit, mouse and zebrafish development. Whole-mount in situ hybridization of staged embryos. (A) Rabbit. Three somite (3S) stage blastodisc revealing PACRG expression in the posterior notochord (PNC) and floor plate. (A’) Sagittal section. (Aâ€) Transversal section (level marked in (A)). Note that the sagittal section revealed absence of PACRG from the node (A’). (B,C) Mouse. (B) E7.5 late headfold mouse embryo displaying PACRG expression in the PNC and floor plate. (B’) Sagittal section. (Bâ€) Transversal histological section. (C) E9.5 embryo. Otic vesicle shown in higher magnification in inset. Box indicates brain region of which a histological section is provided in (C’). (C’) Histological sagittal section, revealing staining in ventral midline of the brain. (D) 6-Somite stage zebrafish embryo. PACRG expression in the Kupffer’s vesicle (KV) and floor plate. a = anterior; d = dorsal; e = eye; fp = floor plate; h = heart; l = left; n = node; no = notochord; ov = otic vesicle; p = posterior; r = right; s = somite; tb = tail bud; v = ventral.
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Figure 4. PACRG protein localization. (A-D) Immunohistochemistry. PACRG shown in green and acetylated tubulin in red. (A) Ventral view of gastrocoel roof plate (GRP) (dashed white line) at stage 17. (A�) Enlargement of GRP cilia. (B) Epidermis. Overview and schematic depiction of multiciliated epidermal cell. (C,D) Enlargements of cilia (C) and cytoplasmic PACRG staining (D). Note that within the cell PACRG localizes to vesicle-like structures and the perinuclear region. (E,F) Expression of a PACRG-enhanced green fluorescent protein (PACRG-eGFP) fusion protein. (E) Maximum intensity projection of timelapse movie of stage 17 GRP cilia, demonstrating elliptic shape of ciliary beating. (F) Squash preparation and (F�) maximum intensity projection of timelapse movie of stage 25 multiciliated skin cell. a = anterior; ac = acetylated; l = left; p = posterior; r = right.
doi:10.1186/2046-2530-1-13
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Additional file 2. Additional Figure 2 Localization of PACRG to primary cilia of the pronephric duct. Tadpoles at stage 37/38 were fixed and processed for immunohistochemistry with antibodies specific for PACRG (green) and acetylated tubulin (red). Specimens were sectioned on a vibratome (30μm) and viewed in the confocal laser scanning microscope. The lumen of the pronephric duct is outlined by a white dashed line. White rectangles indicate regions shown in higher magnification in the lower left corner.
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Figure 5. Impaired laterality in PACRG morphants in the absence of superficial mesoderm (SM) defects. (A)Pitx2c gene expression in the lateral plate mesoderm (LPM). Note that the morpholino oligonucleotide (MO) dose chosen (0.4 pmol/embryo) did not affect dorsoanterior development. (B,C) Unaltered Foxj1 expression levels in the SM of morphants. Assessment of expression levels was as described previously [36]. Embryos in (C) are shown in vegetal view, dorsal side up. Numbers in brackets represent number of analyzed specimens. *** = very highly significant (P <0.001); n.s., not significant (P = 0.891).
doi:10.1186/2046-2530-1-13
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Figure 6. Absence of leftward flow and altered gastrocoel roof plate (GRP) morphology in PACRG morphants. (A,B) Loss of leftward flow in PACRG morphant GRPs. Gradient time trail (GTT) projection of directed bead trajectories (25 s, indicated in color bar). (A',B') Circular histograms of mean angles of trajectories. (C) Quality of flow, as depicted by the dimensionless number rho. (D,E) SEM analysis of wild-type (D) and PACRG morphant (E) dorsal explants. GRP area indicated by dashed line. Note the loss of posterior polarization of cilia in enlargements of characteristic cells in (D',E'), and the marked increase in non-ciliated cells (D'',E''). (F) Altered ciliation rates, mean cilia lengths, polarization of residual cilia and mean apical cell surface areas, as assessed in standardized central GRP areas of 86 μm2 each [21].
doi:10.1186/2046-2530-1-13
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Figure 7. Gastrulation and neural tube closure defects in high dose PACRG morphant embryos. (A) Phenotypes. Note that with increasing morpholino oligonucleotide (MO) doses the anterior-posterior axis shortened, head structures were lost and the blastopore (arrowhead) failed to close. At 2 pmol/embryo (bottom) gastrulation was arrested on the injected side. (B,C) Still frames taken from timelapse movies (Additional file 5: 3) at the stages indicated. (B) Uninjected control specimen. (C) Embryo injected with 2 pmol of PACRG-MO into the dorsal marginal zone at the four cell stage. Note that the dorsal lip did not form in morphant, while the uninjected ventral side gastrulated normally and in time. (D) Immunohistochemistry with an antibody against acetylated tubulin (red), demonstrating non-dynamic microtubules in the deep mesoderm of the marginal zone of the stage 11 gastrula embryo. (E,F) Rescue of neural tube closure defect (NTD) in morphants by parallel injection of a mutated PACRG-eGFP construct such that MO binding was abolished (rescue construct; see also Methods). (E) Unilateral right-sided MO injection (0.5 pmol) into the dorsal marginal zone resulted in NTD and loss of cement gland (white arrowhead) on the injected side. (F) Rescue of NTD and partial rescue of the cement gland. (G) Wild-type development upon overexpression of PACRG-eGFP mRNA. Yellow dashed line = lateral border of neural folds; white dashed line = dorsal midline; blue arrowheads = dorsal lip; green arrowheads = ventral lip; red asterisk = lack of dorsal lip formation. a = anterior; an = animal; d = dorsal; l = left; r = right; veg = vegetal.
doi:10.1186/2046-2530-1-13
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