- Research article
- Open Access
Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina
- Sabine Seipold†1,
- Florian C Priller†1,
- Paul Goldsmith2, 3, 4,
- William A Harris2,
- Herwig Baier3 and
- Salim Abdelilah-Seyfried1Email author
© Seipold et al; licensee BioMed Central Ltd. 2009
- Received: 25 March 2009
- Accepted: 08 July 2009
- Published: 08 July 2009
The condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC) and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described.
Here, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the cap-g locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ) whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in cap-g mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes.
Our findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.
- Bacterial Artificial Chromosome
- Retinal Cell
- Neural Retina
- Gastrula Stage
- Chromatid Segregation
SMC family proteins [1, 2] are essential regulators of chromosomal organization in mitotic and meiotic cell cycles and control sister chromatid cohesion and separation, mitotic condensation, recombinational repair, and chromosome-wide gene regulation [3, 4]. Two SMC proteins, SMC2 and SMC4, heterodimerize to form an active ATPase at the core of condensin I and condensin II protein complexes that are essential for the condensation and stability of chromosomes during mitosis in eukaryotes ranging from yeast to humans [5–8]. In addition to the SMC2/SMC4 core proteins, the condensin I complex contains the kleisin subunit CAP-H and the two HEAT domain proteins CAP-D2 and CAP-G. The non-SMC subunits of the condensin complexes have been proposed to activate DNA supercoiling and looping activity of the SMC-ATPases and to play essential roles in directing the association of the condensin holocomplex onto chromosomes at the correct mitotic stage [4, 6]. Components of the condensin I complex are cytoplasmic during interphase and are targeted to chromatin after the breakdown of the nuclear membrane during prometaphase through the A kinase-anchoring protein AKAP95 [9, 10].
In budding and fission yeasts as well as in Xenopus laevis egg extracts, condensin has an important chromosome condensation activity [11–13]. In budding yeast, loss of any component of the condensin complex causes chromosome condensation and segregation defects [2, 13–18]. Similarly, in HeLa cells, depletion of either condensin I or condensin II subunits causes defective chromosome condensation. This effect is enhanced upon simultaneous depletion of subunits from both complexes . In C. elegans and Drosophila, loss of condensin subunits results in the formation of chromosome bridges due to the failure of sister chromatids to separate completely during anaphase [20–23]. In contrast to sister chromatid segregation, the compaction of chromosomes in metazoan organisms is not entirely dependent on condensin complexes. Genetic analyses of different SMC and non-SMC subunits in several metazoan organisms have demonstrated that chromosomal compaction occurs in the absence of condensin [21, 23–30].
In addition to their roles in mitosis, condensin complexes have been shown to regulate transcriptional expression by modulating heterochromatin function during interphase [16, 21, 22, 31–33]. In comparison, the in vivo analysis of condensin function in vertebrates has been scarce. Overexpression of More than blood (MTB), the murine homolog of the condensin II subunit CAP-G2, in murine erythroleukemia cells promotes their erythroid differentiation . A mutation at the murine condensin II kleisin β locus (cap-h2) disrupts T-cell differentiation . To date, vertebrate mutants of condensin I complex components have not been described.
In this study, we report the positional cloning and detailed phenotypic characterization of the zebrafish cap-g mutation. Functional analysis of CAP-G and of its interaction partners CAP-H and CAP-D2 reveals that the condensin I complex ensures the correct segregation of chromosomes during mitosis and maintains the diploid state. Within the retina, proliferative cells in the ciliary marginal zone (CMZ) are particularly sensitive to the loss of the condensin I protein complex, resulting in increased apoptotic cell death whereas postmitotic cells differentiate and are viable. Survival and laminar organization of condensin I complex deficient retinal cells are partially restored upon inhibition of p53-mediated apoptotic cell death, whereas abnormal ploidy levels remain unchanged. These findings imply that differentiation of retinal cells is not impaired upon polyploidization.
The zebrafish creature from the black lagoon (cbl)locus encodes the condensin I protein CAP-G
Additional file 3: Early lethality of MO cap-g injected embryos. Timelapse movie of wild-type (left side) and cap-g morphants (right side) between the 50%-epiboly and 6-somite stages. cap-g morphants display a high rate of death as evidenced by rupture of the yolk ball between the tailbud and 4-somite stages. The same phenotype was observed for MOcap-g+p53 co-injected embryos (not shown). (AVI 1 MB)
Loss of CAP-G causes a severe reduction in retinal cell numbers, which is partly caused by p53-mediated apoptosis
The severe reduction of eye and head regions in cap-gs105 mutants led us to examine whether proliferation was affected. To this end, we stained mitotic cells with an antibody against phosphorylated histone H3 (PH3) to label mitotic nuclei. At 30 hours post fertilization (hpf), there was no significant difference in the number of mitotic cells throughout the entire embryo between wild-type (278 ± 43 mitotic cells) and mutants (229 ± 33 mitotic cells; p > 0.05). In contrast, apoptotic events were much more frequent in cap-gs105 mutants compared to wild-type, as assayed by staining with acridine orange. Analysis of 24 hpf retinae revealed that many PH3-positive mitotic cells displayed TUNEL positive apoptotic nuclei (Figure 3B). At later stages, we detected cell death using an antibody against activated caspase 3 and found that apoptosis was largely restricted to the proliferative CMZ of the neural retina which suggests that proliferative retinal progenitor cells are primarily affected by loss of CAP-G, whereas postmitotic and terminally differentiated cells of the retina are viable (Figure 3C). This observation prompted us to test whether suppression of apoptotic cell death via inactivation of p53 would rescue the cap-gs105 mutant phenotype . Indeed, injection of 150 μM MOp53 led to a significant increase in cell numbers throughout all retinal cell layers within cap-gs105 embryos at 3 dpf, which were identified by PCR genotyping (Figure 3D, E). Only regions with a clear separation of the INL, GCL and PRL were assessed in this analysis. Taken together, these results demonstrate that CAP-G is required in proliferative rather than in postmitotic differentiated cells. Moreover, the severe reduction in retinal cell numbers is, in part, caused by p53-mediated apoptosis.
Loss of CAP-G causes increased adhesion of sister chromatids during anaphase and aberrant nuclear sizes and shapes
To solidify our findings from morphants, we next tested whether similar mitotic defects could be observed in cap-gs105 mutant embryos. To this end, we analyzed nuclear divisions by high-resolution live imaging at 32 hpf in Tg [H2A::GFP] transgenic embryos that were PCR genotyped (n = 3 cap-gs105 mutant embryos analyzed). We focused on the ventricular highly proliferative zone of the CNS at the hindbrain level. Analysis of mitotic stages revealed that in wild-type embryos, the progression from prometa- to anaphase takes between 7–12 minutes (0% of mitoses delayed, n = 39 mitoses analyzed) (Figure 4B) [see Additional file 4]. In cap-gs105 mutants, the progression from prometa- to anaphase was frequently delayed to 12–18 minutes (58.3% of mitoses delayed to >12 minutes, n = 24 mitoses analyzed) (Figure 4B) [see Additional file 5]. Taken together, live imaging demonstrates that the zebrafish CAP-G protein is an essential player in chromatid segregation during mitosis. Moreover, loss of CAP-G affects the timely progression of mitosis from prometa- to telophase.
CAP-G is required for the maintenance of correct nuclear sizes and shapes
Quantification of nuclear sizes in wild-type and cap-gs105 retinal cross sections.
nuclear cross section area
cross section area
17.3 ± 5.9 μm2
19.0 ± 10.8 μm2
p > 0.01
cap-gs105 + MOp53
20.0 ± 9.9 μm2
p > 0.00001
Loss of other non-SMC condensin I genes phenocopies cap-gs105
Loss of the condensin I complex causes polyploidization
Accumulation of genomic material in nuclei of cap-gs105 mutants, cap-h, and cap-d2 morphants.
DNA content fraction
2C – 4C
2.2% ± 0.3%
87.9% ± 0.1%
4.5% ± 0.5%
3.5% ± 0.4%
1.3% ± 0.1%
6.8% ± 1.7%
65.8% ± 4.0%
6.1% ± 0.5%
15.5% ± 1.3%
4.2% ± 0.7%
4.3% ± 1.9%
70.1% ± 3.7%
8.2% ± 0.8%
13.1% ± 0.3%
2.6% ± 1.0%
3.7% ± 1.2%
74.2% ± 8.7%
6.6% ± 2.1%
11.2% ± 3.8%
3.0% ± 1.2%
9.8% ± 5.4%
61.4% ± 10.7%
11.3% ± 3.0%
10.0% ± 1.5%
4.7% ± 1.5%
7.6% ± 3.5%
64.4% ± 14.2%
9.3% ± 3.2%
12.2% ± 4.2%
4.8% ± 2.9%
WT vs. cap-gs105
p < 0.05
p < 0.01
p < 0.01
p < 0.001
p < 0.01
WT vs. MO cap-h
p > 0.05
p < 0.01
p < 0.01
p < 0.001
p > 0.05
WT vs. MOcap-d2
p > 0.05
p < 0.05
p < 0.05
p < 0.005
p < 0.05
MO cap-h vs. MOcap-h+p53
p > 0.05
p > 0.05
p > 0.05
p > 0.05
p > 0.05
MOcap-d2 vs. MOcap-d2+p53
p > 0.05
p > 0.05
p > 0.05
p > 0.05
p > 0.05
We have reported the first functional analysis of the condensin I complex in a vertebrate organism. The lack of other homologs of cap-g, cap-h and cap-d2 within the zebrafish genome and phenotypic similarities between the appropriate mutants and morphants suggests that a single condensin I complex is present in zebrafish. High-resolution live imaging of mitoses in cap-gs105 mutants and cap-g morphants revealed that progression through mitosis is delayed and that chromatid segregation defects occur during anaphase. Together these findings confirm a role of the condensin I complex that is conserved between zebrafish and other eukaryotic organisms. Our analysis has extended previous studies from other eukaryotic model systems in demonstrating that the loss of CAP-G, CAP-H or CAP-D2 causes aberrant sizes and shapes of retinal cell nuclei most likely caused by tetraploidization. This is rather a direct effect of chromosomal non-disjunction during anaphase than G2/M arrest, as levels of mitotic cells do not increase upon loss of condensin I subunits, as would be expected in case of the latter .
In our analysis of the condensin I complex we have focused on retinal development since retinal patterning and proliferation have been well described . The neural retina is a tissue which is largely derived from a small pool of highly proliferative progenitor cells that are located within the CMZ region . We could show that expression of cap-g, cap-h and cap-d2 correlates with the expression of pcna which marks regions of cell proliferation within the retina. In comparison, there was no overlap with the expression of elavl3, a late stage marker of neurogenesis, which indicates that cap-g, cap-h and cap-d2 expression is down-regulated upon differentiation. We also showed that loss of CAP-G had no effect on the transcriptional expression of pcna or elavl3, indicating that the severe loss of cell number within the neural retina is not caused by transcriptional silencing of genes involved in proliferation or neurogenesis. Moreover, since several neuronal differentiation markers are correctly expressed, we could not detect any obvious developmental consequences caused by tetraploidization.
Our observation that some mitoses are delayed until their elimination by the apoptotic machinery suggests that this probably is a major cause of death among retinal cells. In a recent study, Plaster and colleagues showed that loss of the DNA polymerase delta catalytic subunit 1 compromises DNA replication, which is followed by apoptosis. Similar to our study, they reported that knockdown of p53 led to a phenotypic rescue of mutants which suggests that p53 eliminates cells that are stalled within the cell cycle but that otherwise can finish their developmental program . Our observation that loss of p53 does not completely prevent the massive reduction of retinal cells in cap-gs105 mutants indicates that other forms of cell death or a generally slowed proliferation rate affect the retina.
Our study of the zebrafish cap-g mutant has revealed an essential function of the condensin I complex in rapidly proliferating progenitor cells of the retina and for the maintenance of the diploid state of cell types throughout the entire embryo. Our work has extended previous studies performed in invertebrate models or tissue culture systems to the vertebrate organismal level and has enabled us to characterize the effects of polyploidization on differentiation processes within the entire embryo. A recent report has demonstrated that the Drosophila Retinoblastoma family protein 1 Rbf1 physically interacts with dCap-D3 and is required for efficient localization of dCap-D3 with chromatin . This interaction has uncovered a potentially important mechanism by which the inactivation of Rbf members contributes to genome instability which is a hallmark of many tumors. Consistent with this finding, mutations in SMC2 and SMC4 subunits were found in several cases of pyothorax-associated lymphoma  and loss of heterozygosity at the Cap-D3 locus is frequently associated with breast cancer . As we have now reported similar genomic instability defects in cap-g mutants, it is tempting to speculate that the loss of non-SMC condensin I components will find a similar correlate with tumorigenesis.
Fish maintenance and stocks
Zebrafish were maintained at standard conditions. Embryos were staged by hpf at 28.5°C and fixed at desired timepoints using 4% paraformaldehyde (PFA) in PBS. The following fish strains were used: wild type AB, wild-type WIK, Tg [H2A::GFP] . The cbls105 mutation (Tüpfel longfin background) was isolated during an ethyl-nitrosourea mutagenesis screen performed in the laboratory of Herwig Baier [36, 37].
Mapping, cloning and genotyping of cap-gs105 mutants
To map the cap-gs105 locus, F2 embryos from a mapping cross between heterozygous carriers of the cap-gs105 allele (Tüpfel longfin) and wild-type (WIK) homozygotes were genotyped using a panel of 2982 F2 homozygous mutant embryos. The ZFIN gene names of the condensin I complex genes are as follows: cap-g, [ZFIN:si:dkeyp-26a9.1]; cap-h, [ZFIN:zgc:158618]; cap-d2, [ZFIN:si:dkey-175g20.1].
The following markers were utilized for mapping:
For genotyping of cap-gs105 mutant embryos, we used the following primer pair:
Generation of the cap-g-mcherryfusion construct
We designed PCR primers to amplify full-length cap-g cDNA which was inserted into the pCS2+ vector. Next, we PCR amplified monomeric cherry from Gateway construct p3E mcherry poly A and subcloned the insert into the pCS2+ cap-g expression vector at the 3' of the coding sequence.
Injections of mRNAs and antisense oligonucleotide morpholinos
Constructs were transcribed using the SP6 MessageMachine kit (Ambion). For overexpression (to determine the subcellular localization patterns), 75–100 pg of mRNA were used. For injections of the ATG-directed MOs, the following concentrations were used: 200 μM (MO cap-g ; MO cap-h ; MOcap-d2) and 100 μM (MOp53).
MO cap-g : 5'-CAGATCCGCGTCTCCAGGCATGATG-3'
MO cap-h : 5'-ACTAAATGCGCTCATAACGAAACTG-3'
Antibodies, immunohistochemistry and sections
Antibody stainings were performed as previously described . The following antibodies were used: rabbit anti-activated caspase 3 (1:200, BD Pharmingen), rabbit anti-phosphorylated histone 3 (1:1000, Upstate Biotechnology), zn5 to label ganglion cells (1:1000, Oregon Monoclonal Bank), zpr1 to label red/green double cones (1:200, Oregon Monoclonal Bank). Nuclei were counter-stained with propidium iodide or DAPI (both 1:1000). Phalloidin labeled with Alexa Fluor 647 was used to mark actin (1:100, Molecular Probes). For sectioning, stained embryos were postfixed over night at 4°C in 2%PFA, 0.3 M sucrose. Embryos were embedded in 4% low melting agarose and sectioned on a Leica VT1000 Vibratome. Confocal images were obtained using the Zeiss LSM 510 Meta confocal microscope with a 63× oil lens and zoom 1–3×. Whole embryos were documented under a Leica MZFLIII stereomicroscope using the 1× and 4× objectives with 5–10× zoom and Leica IM50 software package. Photos were processed using Photoshop (Adobe).
In situ hybridization
Whole mount in situ hybridization was carried out as described . Stained embryos were mounted in glycerol and images taken with an Axioplan2 microscope (Zeiss) equipped with a SPOT CCD-camera (Diagnostic Instruments). Templates for probe synthesis were generated from 72 hpf wild-type cDNA by PCR:
DNA content analysis and apoptosis detection
For a single preparation, approx. 50 embryos were collected in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4), deyolked in 1/2 Fish Ringer without Calcium (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3), and washed with Hanks. Retinae dissociated from the embryo proper after 20 min incubation with 0.25% trypsine at 4°C. Single cell suspension was achieved after 20–30 min trypsine treatment and nuclei were stained with propidium iodide according to Shepard et al. . Profiles were recorded on a FACS Canto II flow cytometer (Becton Dickinson). Analysis was carried out with CellQuest Pro for n = 4 independent experiments for each condition. Standard deviations and probabilities associated with Student's t-test (2-tailed, paired) were calculated using Microsoft Exel software.
The TUNEL assay was performed on cryosections using the "Terminal deoxynucleotidyl transferase mediated dUTP Nick End labelling" and the "In situ cell death detection kit, TMR red or Fluorescein" according to the manufacturer's instructions (Roche).
Time-lapse analysis and live imaging
For time-lapse analysis, embryos were embedded in 1.5% low melting agarose (NuSieve GFT agarose; Cambrex). Spontaneous movements of embryos at 32 hpf were reduced with 3-aminobenzoic acid ethyl ester (Tricaine) (Sigma). Tg[H2A::GFP] transgenic embryos were imaged on the Zeiss LSM 510 Meta confocal microscope using 40× magnification and a capture rate of 1 frame per either 30 seconds or 60 seconds. Data were collected and analyzed using Zeiss LSM software. Individual image files were cropped and processed using Photoshop (Adobe) and the movies were assembled using ImageJ software.
Nuclear circularity and cross section area measurements
Confocal sections of retinae taken at 63× magnification, 0.7× zoom at a resolution of 1024 × 1024 pixels with a Zeiss LSM 510 Meta were first processed with Photoshop software (Adobe) and subsequently imported into Image J software http://rsbweb.nih.gov/ij/ for further analysis of particle measurements for circularity and area. Area sizes were converted from pixels to μm2 by recalculating measurements taken from the original LSM images. In total, properties of 100–200 nuclei per cell layer and condition were analyzed with Microsoft Excel software. Significance of differences in between nuclei of different conditions was assessed by Student's t-test (2-tailed, unpaired, unequal variance). Circularity values for nuclei range between 1.0 (perfectly round) and 0.0 (stretched line).
Mitotic cell count and Immunohistochemistry with α-Phospho-Histone H3
Analysis was performed according to Shepard et al. . Mitotic cells were counted for only one side of the embryo. Probabilities were calculated in Excel using t-test (2-tailed, unpaired).
We are indebted to C.B. Chien and N.D. Lawson for sharing reagents and tools and to Robby Fechner for expert technical assistance with the fish facility. Moreover, we are grateful to S. Kreher for help with the FACS analysis. We would like to thank Manfred Gossen for comments on the manuscript. We would like to apologize to colleagues whose work may not have been cited.
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