The Polycomb Group Protein Pcgf1 Is Dispensable in Zebrafish but Involved in Early Growth and Aging - Fig 2

<p><b>Expression of the Pcgf family member during caudal fin regeneration</b> (<b>A</b>) Schematic representation of the regenerating caudal fin structures. (<b>B</b>) <i>In situ</i> hybridization with <i>pcgf1</i>, <i>bmi1a</i>, <i>bmi1b</i>, <i>pcgf5a</i>, <i>pcgf5b</i> and <i>pcgf6</i> RNA anti-sense probes on sections of regenerating caudal fins from 6 month-old zebrafish at 4 days post-amputation (dpa). Dashed lines indicate the amputation plane.</p

Cranial musculature in <i>pcgf1</i>^<i>-/-</i> mutants.

<p>Cranial musculature revealed by immunocytochemistry using the anti-myosin MF20 antibody shows that muscle development is delayed at 3 dpf but normal at 6 dpf in <i>pcgf1</i>^<i>-/-</i> larvae. adm: adductor mandibulae; ah: adductor hyomandibulae; ao: adductor operculi; bm: branchial musculatur; do: dilatator operculi; h: heart; hh:hyohyoideus; ih: interhyoideus; ima: intermandibularis anterior; imp: intermandibularis posterior; lap: levator arcus palatini; lo: levator operculi; om: occular muscle; pf: pectoral fin; sh: sternohyoideus.</p

Expression of Psc orthologs during zebrafish development.

<p>Whole-mount <i>in situ</i> analyses of <i>pcgf1</i>, <i>bmi1a</i>, <i>bmi1b</i>, <i>pcgf5a</i>, <i>pcgf5b</i> and <i>pcgf6</i> zebrafish genes at 0.75, 2.25, 4, 8, 12, 16 and 24 hpf were representatively shown.</p

Growth of <i>pcgf1</i>^<i>-/-</i> mutants at early developmental stages.

<p>(<b>A</b>) Comparison of <i>pcgf1</i>^<i>+/+</i> and <i>pcgf1</i>^<i>-/-</i> embryo development during the cleavage period. Embryos from wild-type and mutant lines were fixed at 0.5, 1, 1.5 and 2 hpf, then 1-, 2-, 4-, 8-, 16-, 32-, 64-, 128- and 256-cell embryos were then counted, and results are expressed as the number of cells per embryo; n > 100 embryos per time point for each genotype. (<b>B</b>) Comparison of <i>pcgf1</i>^<i>+/+</i> and <i>pcgf1</i>^<i>-/-</i> embryo development at blastula and gastrula periods. Embryos were fixed at 4.5 and 6 hpf and representative embryos are shown. At 6 hpf embryos can be classified into 2 classes and the proportion of embryos of each group is indicated; n > 100 embryos. (<b>C</b>) Comparison of the size from head to tail, of fixed <i>pcgf1</i>^<i>+/+</i> and <i>pcgf1</i>^<i>-/-</i> larvae at 2, 3, 4, 5 and 6 dpf; Error bars indicate ± SD. Statistical significance was assessed by Student t-test analysis and significance expressed as the value of p (*, p < 0.1; **, p < 0.01, ***, p < 0.001). Measurements on embryos and larvae were done on 3 independent experiments using materials from different (3 to 6) layings.</p

Generation of a pcgf1 mutant line using the TALEN technology.

<p>(<b>A</b>) Schematic representation of the genomic structure of the <i>pcgf1</i> gene, with coding and untranslated exon depicted as solid and open boxes, respectively. The exonic regions coding for the conserved RING finger motif are shown in orange. The location of the <i>pcgf1</i> TALEN in exon 2 is indicated. The <i>pcgf1</i> TALEN target sequence with Left and Right TALEN binding sites in red is shown. The ClaI restriction site is indicated in green. (<b>B</b>) Identification of mutant embryos using a diagnostic restriction. Genomic DNA was prepared from an uninjected (Control) and a <i>pcgf1</i> TALEN injected (TAL-pcgf1) embryo. The TALEN targeted DNA region is amplified by PCR and subjected to ClaI digestion. The TAL-pcgf1 injected embryo contains undigested material (arrow at 507 bp), indicating that the ClaI diagnostic restriction site has been disrupted. (<b>C</b>) Sequence of the mutant allele compared to its wild-type counterpart. Dashes indicate deleted nucleotides. For the peptide sequence, the gray line indicates residues read out of frame prior to encountering a premature stop codon and the red triangle corresponds to the RING finger motif in the wild-type protein. Size of the predicted proteins is indicated. (<b>D</b>) Pictures of siblings from a <i>pcgf1</i>^<i>+/-</i> x <i>pcgf1</i>^<i>+/-</i> cross at 7 months. The genotype of the fish was established using the ClaI diagnostic restriction on genomic DNA obtained by fin clipping. (<b>E</b>) Global monoubiquitinylation of histone H2A is not affected in <i>pcgf1</i>^<i>-/-</i> mutants. Total histone from pools of 5 embryos at 7 dpf were extracted and analyzed by western blotting. The efficiency of the transfer on the membrane is verified by Ponceau staining (Left) before incubation with anti-H2AK119ub1 and anti-H4 antibodies (Right).</p

Cell proliferation measured by whole-mount immunohistochemistry using an anti-phosphohistone H3 antibody.

<p>(<b>A</b>) Antibody staining against phosphorylated histone H3 (anti-H3S10p) in <i>pcgf1</i>^<i>+/+</i> and <i>pcgf1</i>^<i>-/-</i> 24 hpf embryos. The caudal fin fold region of representative embryos is shown. (<b>B</b>) Quantification of H3S10p spots in the caudal fin fold expressed per unit of surface since <i>pcgf1</i>^<i>+/+</i> and <i>pcgf1</i>^<i>-/-</i> embryos have different sizes at this developmental stage. Error bars indicate ± SD. Statistical significance was assessed by Student t-test analysis and significance expressed as the value of p and * indicates p < 0.01.</p

Development of cartilages and bones in <i>pcgf1</i>^<i>-/-</i> mutants.

<p>Alcian blue stained head cartilages and Alizarin red stained head bones of <i>pcgf1</i>^<i>+/+</i> (Left) and <i>pcgf1</i>^<i>-/-</i> mutant lines (Right) at the indicated developmental points. Note that ossification is visible at 8 dpf in the wild-type but not in the mutant. cb: ceratobranchials; ch: ceratohyal; ep: ethmoid plate; m: Meckel’s cartilage; n: notochord; ot: otoliths; pch: parachordal plate; pq: palatoquadrate.</p