An adaptable chromosome preparation methodology for use in invertebrate research organisms
- Longhua Guo†1,
- Alice Accorsi†2, 3,
- Shuonan He2,
- Carlos Guerrero-Hernández2,
- Shamilene Sivagnanam4,
- Sean McKinney2,
- Matthew Gibson2 and
- Alejandro Sánchez Alvarado2, 3Email authorView ORCID ID profile
© Sánchez-Alvarado. 2018
Received: 1 December 2017
Accepted: 7 February 2018
Published: 26 February 2018
The ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed.
We optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization.
Our simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.
Chromosome preparation is critical to our understanding of animal genetics and genomics. As the price for genome sequencing has dropped, we have witnessed in the last decade an explosive number of research organisms being sequenced and studied. Identifying the basic chromosome composition of an organism sets a foundation to query its unique biological attributes [1, 2]. Behaviors of cells and chromosomes can be fundamentally different in animals that are stable diploids or stable polyploids or those that possess mixed ploidy. Hence, experimental observations at genetic/genomic, tissue, and organismal levels need to be interpreted accordingly.
Isolated chromosomes can be used directly to study multiple questions in genetics and epigenetics. For example, they can be used to collapse scaffolds into chromosomal-level genome assembly , to detect insertion or deletion of large fragments of DNA , to visualize physical positions of multiple genes [5, 6], to localize histone modifications or to identify DNA regions interacting with chromatin regulators [7, 8]. Moreover, genomes assembled into chromosomes can be used for comparative mapping to study chromosome and genome evolution [9–12].
For communities interested in evolutionary genetics and evolutionary developmental biology, it is favorable to learn about chromosome compositions of multiple species of interest. In these situations, the main challenges come when classical protocols do not work on new research organisms. The development of freshwater planarians as a popular invertebrate for studying regeneration and adult stem cells illustrates many of these challenges and opportunities [13–17]. The existing literature since the early twentieth century on chromosome compositions of freshwater planarians showcased the dynamic changes of ploidy within a species and the wide range of chromosome numbers across closely related species . While these phenomena provided opportunities to study genetics and evolution , they also demonstrated the necessity of a standardized chromosome preparation protocol to identify animal ploidy .
An optimized chromosomal preparation protocol that can be easily adapted to multiple species would be advantageous. Conventional karyotyping protocols in mammalian cells involve tissue disassociation and cell culture , techniques which are not applicable to most invertebrate organisms. Furthermore, solution osmolarity, incubation temperature and time, and centrifugation speed are all complex factors to optimize for karyotyping an invertebrate species of interest. The variety of protocols used in the past for chromosome preparation in planarians and the difficulties in comparing the obtained results  clearly demonstrate the necessity to develop a universal protocol that can be easily adapted to as many species as possible.
Here, we optimized a chromosome preparation protocol for chromosome visualization and manipulation in freshwater planarians (phylum Platyhelminthes) and two other research organisms, Pomacea canaliculata (phylum Mollusca) and Nematostella vectensis (phylum Cnidaria). We demonstrate that (1) both adult and embryonic tissues, treated with colchicine, can provide an adequate number of mitotic cells, (2) deionized (DI) water is a convenient hypotonic reagent, and (3) there is no need for tissue dissociation, centrifugation, or cell culture. We further demonstrate that the chromosomes prepared with this protocol are suitable for downstream applications, including fluorescent in situ hybridization (FISH) in all the tested species. Our protocol is simple and inexpensive, and it can be potentially adapted to a wide variety of invertebrate organisms.
Both sexual and asexual strains of Schmidtea mediterranea, in addition to asexual strains of Dugesia japonica, Phagocata velata, and wild planarians collected in the field (Sardinia, Italy) were maintained in the dark at 21–22 °C in 1× Montjuïc water (1.6 mM NaCl, 1.0 mM CaCl2, 1.0 mM MgSO4, 0.1 mM MgCl2, 0.1 mM KCl, and 1.2 mM NaHCO3 in DI H2O, pH 6.9–8.1) . The above-mentioned species were historically defined by their karyotype, anatomy, and physiological attributes (e.g., asexual reproduction by fission). All animals were fed organic beef liver paste twice per week, and the culture water was exchanged after each feeding. The animals were starved for 1 week before the experiments.
Specimens of the apple snail P. canaliculata were initially obtained from Prof. Davide Malagoli from a population stably maintained at the University of Modena and Reggio Emilia (Italy) . The species was defined based on both morphological features and transcriptome and genome sequencing. P. canaliculata specimens were maintained in the lab at 26–27 °C with a 14:10 h light:dark cycle. The animals were housed in tanks filled with artificial freshwater (2.7 mM CaCl2, 0.8 mM MgSO4, 1.8 mM NaHCO3, 1:5000 Remineraliz Balanced Minerals in Liquid Form [Brightwell Aquatics, Fort Payne, AL, USA]). The water was changed twice per week, followed by cleansing of the tanks. The snails were fed ad libitum with lettuce leaves and kale. Deposited egg clutches were collected daily and stored in dry conditions.
Specimens of starlet sea anemone N. vectensis Stephenson were initially obtained from Prof. Mark Martindale and Prof. Craig Magie (University of Hawaii, USA). This particular strain was originally collected from the Rhode River (Maryland, USA) and kindly distributed by Dr. Cadet Hand and Prof. Kevin Uhlinger [24, 25]. Species identification was performed based on both morphological features, such as the presence of nematosomes in the gastric cavity, as well as transcriptome and genome sequencing. Colonies of the sea anemone N. vectensis were maintained at 18–20 °C in a 1:3 dilution of artificial seawater (12 parts per thousand [ppt] of Instant Ocean Sea Salt) and were fed the larvae of the brine shrimp Artemia salina two to five times per week. Spawning induction of sexually mature individuals, egg de-jellying treatment, and fertilization were carried out as previously described .
Planarian sample preparation for chromosome spreads
The posterior or tail region of planarians (~ 2.5 mm) was amputated using a blade, and the regenerating tail fragments were incubated in 0.25% colchicine (Sigma Aldrich) in 1× Montjuïc water for 0 or 6 h in the dark. To test the efficiency of the colchicine treatment, some tail fragments were incubated for 0 or 6 h in 1× Montjuïc water without colchicine.
P. canaliculata sample preparation for chromosome spreads
P. canaliculata embryos 5, 6, 8, or 10 days post-fertilization (dpf) were collected, and both the external egg shell and the bright pink perivitelline fluid were removed with forceps. The embryos were incubated in 0.25% colchicine in Pc-embryonic salt solution (33.3 mM NaCl, 6 mM KCl, 6.7 mM CaCl2, 3.3 mM MgCl2, 1.67 mM hydroxyethyl piperazineethanesulfonic acid [HEPES]) for 6 or 24 h in the dark. To test the efficiency of colchicine treatment, some embryos were incubated in Pc-embryonic salt solution without colchicine.
Adult snails (2.8–3.2 cm shell length) were incubated in 0.25% colchicine in artificial freshwater for 6, 24, 48, or 72 h. After the incubation, small fragments of the organs of interest (gonads, gills, gut, anterior and posterior kidney, and digestive gland) were dissected with scissors.
N. vectensis sample preparation for chromosome spreads
Fertilized N. vectensis embryos were cultured for approximately 5 h post-fertilization (hpf). At the 64- or 128-cell stage, around 200 embryos were transferred to a sterile 50-mm petri dish. They were rinsed twice with 12 ppt artificial seawater to remove extra sperm and incubated in freshly made 0.04% colchicine in 12 ppt artificial seawater for 45 min with gentle rotation in the dark.
Preparation of siliconized coverslips
Siliconized coverslips (22 × 22 mm) were submerged in Sigmacote® (Sigma Aldrich) for 2–3 s in a fume hood. The coverslips were then propped up vertically for 10 min to dry, rinsed in DI H2O for 2–3 s, and propped up vertically to dry.
Chromosome spread preparation for planarians
Tail fragments were placed in a petri dish after colchicine treatment and rinsed with DI H2O. The tissue was punctured using forceps or needles to increase its permeability and then incubated in DI H2O for 20 min at room temperature (RT). The samples were fixed with freshly made Carnoy’s fixative (3:1 dilution of methanol:acetic acid) for 30 min on ice. A small portion of the tail fragment was then placed onto a slide using a pair of forceps or a pipette. The sample was soaked in a drop of 60% acetic acid (10–20 μl) and incubated for 5 min. A siliconized coverslip was placed on top of the tissue. The tissue was then squashed by applying constant pressure to the coverslip for 2–3 s to create a single layer of nuclei. Care was taken to keep the coverslip from shifting laterally during its application. After an overnight (ON) incubation at 4 °C, the slides were chilled on dry ice for 10–20 min. While the slides were still sitting on the dry ice, the coverslips were quickly removed using a blade. The blade was inserted between the slide and one corner of the coverslip and used as a lever for removing the coverslip, with care taken not to scratch the slide where the sample was sitting. The slides were subsequently returned to RT, rinsed 3 times with 1× phosphate-buffered saline (PBS), and stained with a 1:5000 dilution of 4′,6-diamidino-2-phenylindole (DAPI) in 1× PBS for 10 min at RT. The slides were then rinsed twice with 1× PBS for 5 min each and mounted with either Prolong Diamond (Molecular Probes) or Vectashield (Vector Laboratories) mounting media. The slides were stored at 4 °C.
Chromosome spread preparation for P. canaliculata
The adult tissues were punctured using a needle, but not the embryos.
Before tissues were placed on the slides, the older embryos (10 dpf) were cut in half and only the anterior portion was squashed.
Chromosome spread preparation for N. vectensis
Because Nematostella embryos are extremely fragile during early cleavage stages, significant caution was taken when changing solutions. Specifically, solutions were added slowly dropwise and enough solution was left in the petri dish to keep the embryos fully submerged to avoid blastomeres being burst as a result of the surface tension exerted by the solutions.
The embryos were not punctured with a needle and the initial DI H2O incubation was performed for only 5 min due to the fragility of the blastomeres.
After 5 min in 60% acetic acid, the weight of the siliconized coverslip was sufficient to flatten the cells, and no additional pressure was applied.
Image acquisition and processing
The spreads were imaged on a Zeiss LSM 780 or Zeiss LSM 700 confocal microscope using a 63× or 100× magnification lens. The acquired images were processed and karyograms were generated using Photoshop and Fiji softwares.
If the organism has regenerative potential and adult tissues are to be used, the temporal kinetics of the mitotic peak(s) following amputation may differ from that for the planarians.
Head/trunk fragments with mitotic cells and/or embryos can be used for planarian species with different regenerative capacities.
Puncturing the tissue with needles to break the external epithelium aids the penetration of DI H2O into the tissues and cells. The duration of DI H2O incubation, the size of the tissue fragment, the number of perforations made in the tissue, and the colchicine concentration and incubation time are all critical steps to optimize.
Colchicine treatment is usually recommended since it both increases the yield of metaphase chromosomes and produces more reliable chromosome morphologies .
The pressure applied to the coverslip during the squash is correlated to both size and texture of the tissue fragment. If the applied pressure is too great, the chromosomes may not remain grouped together. However, if the pressure is too minimal, nuclei or chromosomes may overlap.
Imaging with a confocal microscope is not required, and a common compound epifluorescence microscope could be used as well.
By varying the length of the colchicine treatment, the level of chromosome condensation can be regulated.
Chromosome spreads can be used immediately for DNA staining or within a few days for in situ hybridization as reported here. Otherwise, they can be aged for a longer time (if required) for different downstream applications, such as G-banding .
Immunohistochemical staining on planarians
The tail fragments were incubated in 5% N-acetyl cysteine (NAC) (Sigma Aldrich) in 1× PBS for 10 min at RT to remove mucous from the epithelium. The tissues were then fixed in pre-chilled Carnoy’s fixative solution (6:3:1 dilution of methanol:chloroform:glacial acetic acid) for 2 h at 4 °C. The samples were then rinsed in 100% methanol. The rehydrated tissues were bleached in 3% formamide and 6% H2O2 in 0.5% Triton X-100 in 1× PBS for 1 h under direct light. The blocking step in 5% horse serum in 0.3% Triton X-100 in 1× PBS for 1–2 h at RT was followed by the incubation ON in 1:500 Rabbit anti-phospho(Ser10)-Histone H3 (H3P)Ab (Cat. ab-32107, Batch GR37459-28, RRID AB_732930; Abcam) at RT. The samples were rinsed and then incubated in 1:500 Goat anti-Rabbit IgG (H + L) Ab conjugated with Alexa Fluor® 488 (Cat. ab-150081, Batch GR297619-1, RRID N/A; Abcam) ON at RT. The samples were rinsed and mounted with ScaleA2 (4 M urea, 0.1% Triton X-100, 20% glycerol, 2.5% 1,4-diazabicyclo-[2,2,2]-octane [DABCO] in double-distilled water [ddH2O]) mounting media.
Z-stack images were acquired with a Nikon Eclipse Ti microscope equipped with a Yokogawa W1 spinning disk head and robotic plate loader. Slides were loaded automatically and imaged with a low magnification objective. Objects were identified using custom software and imaged again with a Plan Apo 20× 0.75 NA air objective. Images were batch stitched and processed using custom plugins and macros in Fiji similar to the procedure in previous reports .
Chromosome spreads were prepared according to the species-specific protocols described previously through the coverslip removal step. Once the slides were warmed to RT, they were rinsed 3 times with 1× PBS for 5 min each. The slides were then dehydrated in an ice-cold ethanol series (70%, 80%, and 100% ethanol) for 2 min each. Slides were then stored at RT for at least 2 days and as long as 1 month.
The telomere DNA probe (sequence [TTAGGG]×7) was obtained from Integrated DNA Technologies (USA) and labeled with digoxigenin (DIG)-deoxyuridine triphosphate (dUTP) using the recombinant Terminal Transferase (Roche) according to the manufacturer’s protocol. For each slide, 20 ng of labeled telomere DNA probe was mixed with 10 volumes of Master Hybridization Mix (4× saline-sodium citrate buffer [SSC], 20% dextran sulfate, 2 mg/ml nuclease-free bovine serum albumin [BSA], 50% deionized formamide in ddH2O). The total volume (~ 22 μl) was placed on the area containing the chromosome spreads, covered with a 22 × 22 mm coverslip, and sealed with mineral oil. The slides were incubated for 5 min at 70 °C to denature the DNA, followed by 24–36 h at RT for hybridization. Next, the slides were submerged in 2× SSC in a slide staining jar to remove the coverslips and then rinsed successively with 2× SSC, 0.5× SSC, and TNT (100 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) for 15 min each at RT. The areas containing the chromosome spreads were covered with 1:200 anti-DIGAb conjugated with Rhodamine (Cat. 11207750910, Batch N/A, RRID AB_514501; Roche) and 1:1000 DAPI in TNB (5% fetal bovine serum [FBS] in TNT) in the dark for either 1–4 h at RT or ON at 4 °C. The slides were rinsed with TNT for 15 min at RT and then mounted, imaged, and stored at 4 °C.
An unpaired two-sample t test was performed for calculation of the statistical difference between the number of H3P-positive cells in tail fragments at 0 h post-amputation (hpa) incubated with (N = 20) and without colchicine (N = 19) and at 6 hpa incubated with (N = 18) and without colchicine (N = 17). The differences were considered statistically significant with P < 0.05.
Optimization of a chromosome preparation protocol
Treatment with the mitosis-inhibiting drug colchicine significantly increased the number of mitotic cells in planarians (Fig. 2m, n), similarly as in other systems . When colchicine binds tubulin, it prevents spindle formation and halts dividing cells in metaphase. An antibody detecting phospho(Ser10)-Histone H3 (H3P) was used to assess the number of mitotic cells in a planarian tail with or without 6 h of colchicine treatment. In the absence of colchicine, the number of dividing cells approximately tripled at 6 hpa compared to baseline (0 hpa). Hence, with colchicine treatment, the number of mitotic cells increased about seven times by 6 hpa compared to baseline, yielding a significantly higher number of chromosome spreads in the tissue (Fig. 2n).
Applicability of the protocol to multiple planarian species
Second, we applied the protocol to Phagocata velata, a fissiparous species that reproduces asexually by fission  (Fig. 4e). The protocol worked successfully on its tail fragments, and we observed that this species has the largest number of chromosomes in planarians reported thus far (Fig. 4f). The specific number of chromosomes seems to vary from cell to cell, but on average there are about 84 chromosomes per nucleus (Fig. 4g). Altogether, the method reported here was successful in rapidly generating unambiguous karyotypes for a diverse cohort of planarian species.
The protocol is broadly applicable to species belonging to different phyla
To further test the adaptability of this protocol on different tissues, we used it on various adult organs of P. canaliculata. After an incubation for 72 h in colchicine, the snails were dissected to collect organs known to undergo rapid cell turnover including gonads, gills, gut, anterior and posterior kidney, and digestive gland. We obtained good chromosome spreads from the anterior and posterior kidney and the digestive gland (representative karyotype and karyogram are shown in Fig. 5d, e). The tissue producing the most chromosome spreads was the gonads, but they contain many dividing polyploid cells (unpublished data), which could result in a misleading karyotype analysis. Shorter colchicine incubation (6, 24, and 48 h) provided no or extremely rare mitotic cells in the analyzed adult tissues.
Chromosome preparation protocol is compatible with downstream applications
Techniques providing easy and inexpensive access to chromosome composition (i.e., number and morphology) are invaluable for our understanding of chromosome behaviors and genome architecture in any organism of interest. Here, we reported an optimized protocol to prepare chromosomes for karyotyping and FISH in species belonging to three different phyla: Platyhelminthes, Mollusca, and Cnidaria. We expect that the protocol reported here will be highly adaptable to the study of multiple invertebrate species.
In planarians, the analysis of karyotypes across multiple species was pioneered by Benazzi and his coworkers [18, 36] and spanned from the early to late twentieth century. This body of work identified a large cohort of sexual and asexual planarian species and provided information on both the karyotypes and anatomy of sexual reproductive systems. However, few detailed karyotyping methodologies are available. Traditional methods employed regenerating blastemas 3 dpa for the preparation of chromosome spreads , which provided very little material to work with, as the blastema are usually rather small. Furthermore, the traditional methods required a long processing time. Another issue associated with this literature is that the conditions for chromosome preparation are highly variable from researcher to researcher. Here, we showed that a fragment of tissue amputated from adult worm and treated with colchicine for 6 h and successively with DI water as hypotonic reagent is sufficient to yield well-spread chromosomes in multiple planarian species. This can potentially function as a standardized methodology for planarian chromosome preparation.
Recently, interest in the apple snail P. canaliculata (originally from South America) is significantly rising among researchers. This snail is listed among the 100 most invasive species worldwide (Global Invasive Species Database). Nowadays, many studies are focused on both the characterization of its immune system and a search for methods to limit its diffusion [38–40] but also on establishing this animal as a molluscan research organism for laboratory research [41–43]. We confirmed that the P. canaliculata genome is diploid with 2n = 28 chromosomes [44, 45], applying the protocol optimized on planarian to both embryos at different developmental stages and adult tissues. It is important to highlight that only a small number of optimizations were required to successfully isolate a significant number of good-quality chromosome spreads.
Nematostella has gained attention over the last decade due to both its unique phylogenic position as an early branching metazoan and its amenability to experimental manipulation. The sequencing of the Nematostella genome in 2007 provided crucial insights into the ancestral eumetazoan gene repertoire and evolutionarily conserved signaling pathways . In contrast to its apparent morphological simplicity, Nematostella possesses a complex genome [46, 47], whose sequence and high-quality annotation have provided a robust platform for applying modern molecular biological tools such as genome editing to study gene function in the context of evolution [48, 49]. Yet, despite the rapid expansion of Nematostella as a research organism, little attention has been paid to its chromosomal structure and organization. Previous scattered experimental evidence showed that Nematostella is a diploid animal with chromosome number 2n = 30 . Interestingly, this number is identical to those of several other hydrozoans and it is also comparable to those of several species of Acropora corals (2n = 28) [50, 51]. Utilizing the karyotyping method discussed above on early stage embryos (64- and 128-cell stage) and optimizing a few steps, we easily obtained highly synchronized metaphase spreads.
Many times, karyograms have been used as a powerful tool for species identification, among both planarians and mollusks [21, 52]. We showed that when planarians are collected in the wild, it can be difficult to identify the species only on the basis of morphological features. The high regenerative potential of these animals allows the researcher to karyotype each individual without sacrificing it. Historically, planarian species identification relies on the anatomy of sexual reproductive organs [18, 53, 54], which leaves the cohort of asexual planarian species largely uncatalogued. A proper characterization of the karyotypes of both sexual and asexual species would facilitate the identification and cataloging of planarian species, while also providing a rich resource for the understanding of karyotype evolution . Similarly, over the years, the Gastropoda class has been reorganized multiple times in subclasses and families, and new species have been integrated, but the lack of a unique and established method for species identification has created multiple incongruences [55, 56]. In particular, Hayes and colleagues faced this problem within the Ampullariidae family, suggesting a combination of morphological and molecular features for species identification [57, 58]. The karyogram is potentially a very useful feature that can be added to the plethora of characteristics required for species identification [44, 52, 59, 60]. The possibility to easily obtain unambiguous karyotypes from material collected in the field irrespective of embryonic developmental stages or from adult tissue without extensive optimization stands to be extremely useful in comparative analyses and population genetic studies.
In conclusion, we optimized a chromosome preparation protocol in invertebrate species belonging to three phyla. The protocol is simple, inexpensive, and highly adaptable to multiple species. Prepared chromosomes can also be used for multiple downstream applications including FISH for localizing telomeres and potentially any other gene of interest.
We thank S.A. Elliott and S. Nowotarski for helpful comments on the manuscript; P. Newmark and O. Guedelhoefer for earlier work on the planarian karyotyping protocol; M. Miller for illustration assistance; the Stowers Aquatics Facility for maintaining all animals used in this study. We also thank members of Sánchez Alvarado and Gibson Laboratories for helpful comments and discussions and Sebastian Sánchez-Piotrowski for his assistance in obtaining wild cultures of planarians.
Funding for this project was provided by the Howard Hughes Medical Institute (A. Sánchez Alvarado), the Stowers Institute for Medical Research (A. Sánchez Alvarado), the Society for Developmental Biology (SDB) Emerging Models grant (A. Accorsi), and the American Association of Anatomists postdoctoral fellowship (A. Accorsi).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and are also available at the Stowers Original Data Repository, http://www.stowers.org/research/publications/libpb-1241.
LG established the chromosome preparation protocol on planarians and was a major contributor in writing the manuscript. AA established the chromosome preparation protocol on apple snails and was a major contributor in writing the manuscript. SH established the chromosome preparation protocol on Nematostella and contributed in writing the manuscript. CGH collected part of the data on planarians. SS contributed in screening already-published protocols for karyotyping and testing different variables on both planarians and Nematostella. SM acquired images after H3P Ab staining and quantified the number of H3P-positive cells. MG and ASA funded the research and helped write the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The studies with planarians, snails, and sea anemones in this research abide to ethical regulations.
Consent for publication
The authors declare that they have no competing interests.
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