rabbit polyclonal antibody against h3s10p  (GeneTex)

Journal: Scientific Reports

Article Title: Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris

doi: 10.1038/s41598-024-78278-6

Figure Lengend Snippet: Analysis of epigenetic histone modifications during mitosis and micronuclei formation. Whole-mount immunofluorescent staining of gonads from sexual species ( a , b ) and hybrids (all rest) with antibodies against various epigenetic modification stained in red: H3S10P ( a , c , d ), H3S28P ( b , e ), H3K9me3 ( f , g ) and H3T11P ( h ). Accumulation of all analyzed chromatin modifications indicate normal chromosome condensation during prophase and metaphase. In hybrids, misaligned chromosomes (indicated by white arrows) have similar distribution of the H3S10P ( c ), H3S28P ( e ), H3K9me3 ( f ) and H3T11P ( h ) chromatin modifications as the rest of the chromosomes (showed by white arrowheads) suggesting that misaligned chromosomes condense properly during prophase and metaphase. Micronuclei present during metaphases (indicated by thin white arrows) do not accumulate epigenetic histone modification such as H3S10P ( c ), H3K9me3 ( f ). ( d ) Accumulation of H3S10P epigenetic modification in some micronuclei (indicated by thin red arrows) suggest their formation by chromosomal lagging. Some micronuclei do not show accumulation of H3S10P epigenetic modification (indicated by thin white arrows). ( g ) In contrast to the chromatin in the interphase nucleus of the gonial cell, chromatin in micronuclei accumulated H3K9me3 epigenetic modifications indicating its inactivation and heterochromatinization (micronuclei indicated with thin red arrows). Tubulin (stained in green) visualize cytoskeleton components of the spindle ( a , f ). Chromatin is visualized with DAPI in blue. All pictures represent a single gonadal section of 0.4 μm in thickness after 3D immunofluorescent staining. Scale bars = 10 μm.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antibody against DDX4 (concentration 1:50; C1C3, GeneTex) to detect Vasa protein; mouse monoclonal antibodies against alpha tubulin (concentration 1:100; ab7291, Abcam); rabbit polyclonal antibody against H3S10P (concentration 1:100; GTX128116, GeneTex); rabbit polyclonal antibody against H3S28P (concentration 1:100; #9713, Cell Signaling); rabbit polyclonal antibody against H3T11P (concentration 1:100; #9764, Cell Signaling); rabbit polyclonal antibody against H3K9me3 (concentration 1:100; ab8898, Abcam).

Techniques: Staining, Modification

Journal: Scientific Reports

Article Title: Gradual chromosomal lagging drive programmed genome elimination in hemiclonal fishes from the genus Hypseleotris

doi: 10.1038/s41598-024-78278-6

Figure Lengend Snippet: Schematic overview of selective genome elimination in carp gudgeons. ( a ) Scheme of the reproduction of hybrid individuals and individuals of sexual species, H. bucephala . During gametogenesis in hybrids, H. bucephala genome (red) is eliminated, while H. gymnocephala genome (green) is transmitted to gametes. After fertilization by co-occurring sexual species H. bucephala , hybrid chromosomal composition is restored. ( b ) Suggested simplified scheme of gradual chromosome elimination via micronucleus formation in hybrids. ( c ) During eliminating mitosis all chromosomes accumulate epigenetic chromatin marks H3S10P, H3S28P and H3K9me3. After lagging and inclusion of individual chromosomes in micronuclei, we detected H3S10P modification in at least some micronuclei. In the micronuclei, chromatin accumulates heterochromatin marks.

Article Snippet: We used the following primary antibodies: rabbit polyclonal antibody against DDX4 (concentration 1:50; C1C3, GeneTex) to detect Vasa protein; mouse monoclonal antibodies against alpha tubulin (concentration 1:100; ab7291, Abcam); rabbit polyclonal antibody against H3S10P (concentration 1:100; GTX128116, GeneTex); rabbit polyclonal antibody against H3S28P (concentration 1:100; #9713, Cell Signaling); rabbit polyclonal antibody against H3T11P (concentration 1:100; #9764, Cell Signaling); rabbit polyclonal antibody against H3K9me3 (concentration 1:100; ab8898, Abcam).

Techniques: Modification

Journal: eLife

Article Title: Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

doi: 10.7554/eLife.21052

Figure Lengend Snippet: ( A – B ) Architectural features of S2 and S3 embryos. ( A ) Expression of the pan embryonic cell marker EF1a-like-1 (blue) in S2 (left) and S3 (right) embryos. Cyan arrowheads: primitive ectoderm cells. Cyan arrows: undifferentiated blastomeres. Red arrowhead: temporary embryonic pharynx. Red arrows: primitive gut cells. ( B ) S3 embryo stained with EF1a-like-1 riboprobes (red) and sytox green nuclear counterstain (green). Cyan arrowheads: primitive ectoderm cells. Cyan arrows: undifferentiated blastomeres. Yellow arrowhead: temporary embryonic pharynx. ( C ) Confocal Z-slice of an ovary from a sexually mature Smed hermaphrodite stained with piwi-1 riboprobes (green) and DAPI (blue). Yellow arrows: oocytes. ( D ) Dispersed cleavage. S2 embryo stained with piwi-1 riboprobes (red, blastomeres) and antibodies raised against the mitotic epitope H3S10p (green). Nuclei stained with DAPI (blue). Yellow arrow: dividing blastomere. ( E ) piwi-1 is expressed in undifferentiated blastomeres of S3 embryos. S3 embryo costained with riboprobes complementary to piwi-1 (red) and EF1a-like-1 (green). 100% piwi-1+ blastomeres coexpressed EF1a-like-1 . n = 159 cells scored, n = 5 S3 embryos. Cyan arrows: undifferentiated blastomeres. Yellow arrowhead: temporary embryonic pharynx. Red arrows: fiduciary beads used for SPIM reconstruction. ( F ) piwi-1 + cells are located in the embryonic wall. Paraffin-embedded cross-section of a S3 sphere stained with piwi-1 riboprobes (blue) and eosin (pink). Cyan arrows: piwi-1+ cells. GC: yolk-filled gut cavity. Inset: magnified view of a piwi-1+ cell. Scale: 25 µm. ( G ) Left: Average RPKM per embryo for piwi-1 (S2–S8). Right: WISH developmental time course with piwi-1 riboprobes (blue) (S2–S8). O, oral hemisphere; V, ventral. ( A – G ) Scale: 100 µm. Left: Observed distribution of piwi-1+ cells in S3–S4 embryos (blue bars) relative to the oral-aboral axis (0–3.14 radians). Maximum likelihood analysis best described distribution by the function ((1-exp(-θ/θ’))*sin(θ), blue line). The optimal calculated θ’ was 0.45 ± 0.045 radians, based on simulations with comparably sized data sets, and was several orders of magnitude more likely to explain the observed distribution than the theoretical normal distribution, sin(θ), (θ’ = 0), red line. S3: n = 32 embryos, n = 1,746 piwi-1+ cells scored. S4: n = 8 embryos, 2,665 piwi-1+ cells scored. Right: observed piwi-1+ cell distributions for individual S3 (top) and S4 (bottom) embryos. ( C – G ) piwi-1+ cells are detected throughout embryogenesis. ( H ) piwi-1 + cell positions are not stereotyped in S3–S4 embryos. DOI: http://dx.doi.org/10.7554/eLife.21052.032

Article Snippet: Colorimetric and fluorescent WISH was performed as described by ) and ), with the following modifications: Immunostaining was performed after fluorescent WISH development with rabbit polyclonal antibodies against H3S10p (1:1000; Millipore # 06–570), mouse monoclonal antibodies against Smith Antigen (Y12) (1:200, ThermoFisher Scientific, PIMA190490), or mouse monoclonal antibodies against Smed PIWI-1 (1:1000, a generous gift from J. Rink).

Techniques: Expressing, Marker, Staining

Journal: eLife

Article Title: Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

doi: 10.7554/eLife.21052

Figure Lengend Snippet: ( A – B ) Left: Colorimetric WISH depicting expression of PCNA ( A ) or RRM2-2 ( B ) during stages S2–S8. Right: Average RPKM values per embryo for PCNA ( A ) or RRM2-2 ( B ) in Y (yolk) and S2–S8. V, ventral. Scale: 100 µm. ( C – D ) S3 (top), S4 (middle) and S5 (bottom) embryos costained with piwi-1 (red) and PCNA (green [ C ]) or RRM2-2 (green [ D ]) riboprobes. The percentage of piwi-1+ cells coexpressing the indicated cell cycle marker (red) and the percentage of PCNA+ or RRM2-2+ cells coexpressing piwi-1 (green) appear in the lower left corner of merged images. Scale bars: 100 µm. ( C ) S3: n = 273 cells, n = 6 embryos. S4: n = 1,267 cells, n = 4 embryos. S5: n = 734 cells, n = 3 embryos. ( D ) S3: n = 130 cells, n = 4 embryos. S4: n = 1,295 cells, n = 5 embryos. S5: n = 350 cells, n = 3 embryos. ( E ) Mitotic activity is restricted to the piwi-1+ cell compartment in S3–S5 embryos. Left: S4 embryo costained with piwi-1 and the embryonic pharynx marker LYAG-like (both in green) and antibodies against the mitotic epitope H3S10p (red). White arrows: dividing blastomeres. White arrowhead: temporary embryonic pharynx. Scale bar: 100 µm. Right: Bar graph depicting the percentage of mitotic cells scored that expressed piwi-1 in S3–S5 embryos. ( F ) The mitotic index for the piwi-1+ cell compartment did not vary significantly during S3–S5. Average percentage of piwi-1+ cells in mitosis during S3–S5. Error bars represent the standard deviation of the mean. Observed distribution of mitotic ( piwi-1+ , H3S10p+) cells in S3-–S4 embryos (blue bars) along the oral-aboral axis (0–3.14 radians). Using the function derived with maximum likelihood estimation for the piwi-1+ cell distribution, (1-exp(-θ/θ’))*sin(θ) (blue line), and simulations using equivalent sample sizes, the optimal θ’ was calculated to be 0.58 ± 0.33, and was 50-fold more likely to explain the observed trend than a simple normal distribution, sin(θ), where θ’=0 (red line). S3: n = 82 mitotic cells, n = 18 embryos. S4: n = 110 mitotic cells, n = 8 embryos. ( G ) Mitotic cell positions are not stereotyped in early embryos. DOI: http://dx.doi.org/10.7554/eLife.21052.035

Article Snippet: Colorimetric and fluorescent WISH was performed as described by ) and ), with the following modifications: Immunostaining was performed after fluorescent WISH development with rabbit polyclonal antibodies against H3S10p (1:1000; Millipore # 06–570), mouse monoclonal antibodies against Smith Antigen (Y12) (1:200, ThermoFisher Scientific, PIMA190490), or mouse monoclonal antibodies against Smed PIWI-1 (1:1000, a generous gift from J. Rink).

Techniques: Expressing, Marker, Activity Assay, Standard Deviation, Derivative Assay

Journal: eLife

Article Title: Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

doi: 10.7554/eLife.21052

Figure Lengend Snippet: ( A ) Schematic depicting the workflow for heterochronic transplantation experiments. S4, S5, S6, S7 or S8 embryonic cell suspensions were injected into the tails of lethally irradiated sexual adult hosts at 3 days post-irradiation (dpi). Cohorts of transplanted animals were fixed at 1 hr and 5 days post-transplantation (1 hpt and 5 dpt, respectively) for staining with piwi-1 riboprobes and H3S10p antibodies. Lethally irradiated, uninjected host controls were fixed and stained at 5 dpt. ( B ) Percentage of transplanted animals fixed at 1 hpt (blue bars) or 5 dpt (red bars) containing one or more donor-derived piwi-1+ cell(s). X-axis: stage (S) of donor cells. ( C ) Number of donor-derived piwi-1+ cell(s) per transplant at 1 hpt and 5 dpt. Each point represents one transplanted animal. Mean ± standard deviation (black bars) are shown. Statistical tests were performed using a generalized linear model, assuming that the counts followed a Poisson distribution. S4 transplants contained significantly fewer piwi-1+ cells at 1 hpt than S5, S6, S7 or S8 transplants (Tukey post-hoc comparisons, S4 vs S5, S4 vs S6 and S4 vs S7, S4 vs S8: p<0.001). Group differences in the number of piwi-1+ cells at 1 hpt for S5 and S6 transplants were not statistically significant (p=0.21). Significantly fewer S4 and S5 donor-derived piwi-1+ cells persisted at 5 dpt than were observed for later stages (Tukey post-hoc comparisons: S4 vs S5, S4 vs S6, S4 vs S7 and S4 vs S8: p<0.001. S5 vs S6, S5 vs S7, S5 vs S8: p<0.001). ( D ) Percentage of transplants with mitotic piwi-1+ cell(s) at 5 dpt (green bars). X-axis: Donor cell stage. ( E ) Mitotic index for donor-derived piwi-1+ cells at 5 dpt. Stage-specific differences were not observed for S4–S8 embryonic cell populations using a generalized linear model, assuming counts followed a Poisson distribution and the number of piwi-1+ cells as a covariate. ( B – E ) Numbers of transplants scored: S4: n = 36 (1 hpt), n = 43 (5 dpt), four independent experiments. S5: n = 15 (1 hpt), n = 16 (5 dpt), two independent experiments. S6: n = 31 (1 hpt), n = 29 (5 dpt), four independent experiments. S7: n = 31 (1 hpt), n = 30 (5 dpt), four independent experiments. S8: n = 19 (1 hpt), n = 20 (5 dpt), three independent experiments. ( F ) Confocal maximal projections of S4, S5, S6, S7 and S8 embryonic cell transplants fixed at 1 hpt and 5 dpt. Animals were stained with piwi-1 riboprobes (green), antibodies against the mitotic marker H3S10p (red, 5 dpt only) and DAPI nuclear counterstain (blue). S6, S7 and S8 insets: mitotic piwi-1+ cells. Red arrows indicate mitotic cells magnified in insets. Yellow arrows: mitotic piwi-1+ cells. Scale bar (inset): 20 µm. Scale bar (panel): 100 µm. ( B – C ) S4–S8 embryonic piwi-1+ cells were reliably introduced into hosts. S6–S8 embryonic piwi-1+ cells persisted in an adult microenvironment. ( D - E ) S6–S8 embryonic piwi-1+ cells proliferated in an adult microenvironment. DOI: http://dx.doi.org/10.7554/eLife.21052.059

Article Snippet: Colorimetric and fluorescent WISH was performed as described by ) and ), with the following modifications: Immunostaining was performed after fluorescent WISH development with rabbit polyclonal antibodies against H3S10p (1:1000; Millipore # 06–570), mouse monoclonal antibodies against Smith Antigen (Y12) (1:200, ThermoFisher Scientific, PIMA190490), or mouse monoclonal antibodies against Smed PIWI-1 (1:1000, a generous gift from J. Rink).

Techniques: Transplantation Assay, Injection, Irradiation, Staining, Derivative Assay, Standard Deviation, Marker

Journal: eLife

Article Title: Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

doi: 10.7554/eLife.21052

Figure Lengend Snippet: ( A ) Schematic for heterochronic transplantation experiments. S5, S6, S7 or S8 embryonic cell suspensions were injected into the tail parenchyma of lethally irradiated sexual adult hosts at 1 day post irradiation (dpi). Cohorts of transplanted animals and uninjected host controls were fixed at 5 days post-transplantation (dpt) for staining with piwi-1 riboprobes and H3S10p antibodies. The remaining animals were monitored for 70 dpt for survival and rescue. ( B ) Percentage of transplants with persistent, donor-derived piwi-1+ cell(s) (blue) or donor-derived mitotic ( piwi-1+ , H3S10p+) cell(s) (red) at 5 dpt. X-axis: Embryonic donor cell stage. ( C ) Number of piwi-1+ cells per transplanted host at 5 dpt for S5–S8 embryonic cell transplants. Each point represents one transplanted animal. Means ± standard deviation (SD) are shown (black bars). Statistically significant differences in the number of persistent piwi-1+ cells per transplant at 5 dpt were observed using a generalized linear model, assuming that count data followed a Poisson distribution. S5 transplants contained fewer persistent piwi-1+ cells than S6 or S7 transplants (Tukey post-hoc comparisons, S5 vs S6: p<0.0001, S5 vs S7: p<0.0001, S5 vs S8: p<0.0001). ( D ) Mitotic index for donor-derived piwi-1+ cells at 5 dpt for S5–S8 embryonic cell transplants. Each point represents one transplanted animal. Means ± standard deviation (SD) are shown (black bars). Statistically significant differences in the piwi-1+ cell mitotic index were observed using a generalized linear model with piwi-1+ cell counts as a covariate, assuming that count data followed a Poisson distribution. S5 transplants contained significantly fewer cycling cells than S6, S7 or S8 transplants (Tukey post-hoc comparisons, S5 vs S6: p<0.01, S5 vs S7: p<0.01, S5 vs S8: p<0.001). ( E ) Confocal maximal projections for S5, S6, S7 and S8 embryonic cell transplants fixed at 5 dpt and stained with piwi-1 riboprobes (green), H3S10p antibodies (red) and DAPI (blue). S6, S7 and S8 insets show mitotic piwi-1+ cells. Red arrows indicate mitotic cells magnified in the insets. Yellow arrows: mitotic piwi-1+ cells. Scale bar (inset): 20 µm. Scale bar (panel): 100 µm. ( B – E ) Numbers of transplants scored in four independent experiments: S5 n = 22; S6 n = 24; S7 n = 21; S8 n = 27 in ( C ), n = 21 in ( D ). ( F ) Survival curves for S5, S6, S7 and S8 embryonic cell transplants and uninjected 6,000-Rad-irradiated host controls as a function of time (days) post-transplant. ( G ) Live images of regenerating S6, S7 and S8 rescue animals. Left: Tail fragment after self-amputation of head and trunk tissue. Middle: Tail fragment with unpigmented anterior blastema (yellow arrowheads). Right: Animal with new head tissue and developing eyes (yellow arrows) and a regenerated pharynx (yellow asterisk). Animals from different experiments are shown in the S7 panels; the same animals are shown in the S6 and S8 panels. Dorsal view. Anterior: top. Scale: 100 µm. ( F – G ) Numbers of transplants scored in four independent experiments: host controls n = 89; S5 n = 105; S6 n = 90; S7 n = 92; S8 n = 85. Rescue animals were obtained in two experiments for S6 and S7 transplants, and four experiments for S8 transplants. ( B – E ) S6, S7 and S8 embryonic donor cells persist and divide in the adult parenchyma. ( F – G ) S6, S7 and S8 embryonic cells can rescue lethally irradiated adult hosts. DOI: http://dx.doi.org/10.7554/eLife.21052.060

Article Snippet: Colorimetric and fluorescent WISH was performed as described by ) and ), with the following modifications: Immunostaining was performed after fluorescent WISH development with rabbit polyclonal antibodies against H3S10p (1:1000; Millipore # 06–570), mouse monoclonal antibodies against Smith Antigen (Y12) (1:200, ThermoFisher Scientific, PIMA190490), or mouse monoclonal antibodies against Smed PIWI-1 (1:1000, a generous gift from J. Rink).

Techniques: Transplantation Assay, Injection, Irradiation, Staining, Derivative Assay, Standard Deviation