Callithrix jacchus - CSIRO Publishing

CSIRO PUBLISHING

Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD15321

Detection of cross-sex chimerism in the common marmoset monkey (Callithrix jacchus) in interphase cells using fluorescence in situ hybridisation probes specific for the marmoset X and Y chromosomes E. Wedi A,E,H, S. Mu¨ller B, M. Neusser B, P. H. Vogt C, O. Y. Tkachenko A,F, J. Zimmer C, D. Smeets D, H. W. Michelmann G and P. L. Nayudu A A

Department of Reproductive Biology, German Primate Centre, Goettingen, 37077, Germany. Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, 80336 Munich, Germany. C Reproduction Genetics Unit, Department of Gynaecological Endocrinology and Reproductive Medicine, University of Heidelberg, 69047, Germany. D Institute for Anthropology and Human Genetics, Department Biology II, Biocenter, Ludwig-Maximilians-University Munich, 82152 Planegg-Martinsried, Germany. E Departments of Gastroenterology and Endoscopy, Novel Hoˆpital Civil (NHC), University Hospital Strasbourg, 67000, France. F Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center, Oregon Health & Science University, 505 NW 185th Avenue, Beaverton, OR 97006, USA. G Department of Obstetrics and Gynecology, University of Goettingen, Goettingen, 37075, Germany. H Corresponding author. Email: [email protected] B

Abstract. Chimerism associated with placental sharing in marmosets has been traditionally analysed using conventional chromosome staining on metaphase spreads or polymerase chain reaction. However, the former technique requires the presence of proliferating cells, whereas the latter may be associated with possible blood cell contamination. Therefore, we aimed to develop a single-cell analysis technique for sexing marmoset cells. We applied fluorescent in situ hybridisation (FISH) to cell nuclei using differentially labelled X and Y chromosome-specific probes. Herein we present the validation of this method in metaphase cells from a marmoset lymphoblastoid cell line, as well as application of the method for evaluation of cross-sex chimerism in interphase blood lymphocytes and haematopoietic bone marrow cells from marmosets of same- and mixed-sex litters. The results show conclusively that haematopoietic cells of bone marrow and leucocytes from blood are cross-sex chimeric when the litter is mixed sex. In addition, single samples of liver and spleen cell suspensions from one individual were tested. Cross-sex chimerism was observed in the spleen but not in liver cells. We conclude that FISH is the method of choice to identify cross-sex chimerism, especially when combined with morphological identification of nuclei of different cell types, which will allow a targeted tissue-specific analysis. Additional keywords: bone marrow cells, lymphocytes.

Received 6 August 2015, accepted 19 December 2015, published online 15 February 2016 Introduction Twin litters and placental sharing among fetuses are a special characteristic of Callithrix jacchus and other marmosets and tamarins (Benirschke and Brownhill 1962; Gengozian et al. 1964; Ross et al. 2007). The resultant haematopoietic chimerism is a unique natural phenomenon in these multiovulatory species (Sweeney et al. 2012), which contrasts with most other mammals, wherein placental sharing has been described only as Journal compilation Ó CSIRO 2016

an unusual occurrence associated with abnormality (McLaren 1976). In cattle species, chimerism is known to result in freemartinism (Padula 2005). In humans, older reports show blood chimeras with normal fertility (Benirschke and Driscoll 1967), whereas in more recent studies abnormalities have been reported. For example, Souter et al. (2007) reported a case of ambiguous genitalia in a female twin, whereas Choi et al. www.publish.csiro.au/journals/rfd

B

Reproduction, Fertility and Development

(2013) published a case report where testicular hypoplasia was observed in a live-born monochorionic diamniotic dizygotic male twin with chimerism. Both reports describe cases of fraternal twins. In addition, there are two reports that suggest possible association between fraternal (Vabres and Bonneau 2005) or maternal (Kowalzick et al. 2005) microchimerism and skin disorders. In marmosets, implantation takes place around Day 12 after ovulation and, at this time, chorionic gonadotropin (CG) is first detected (Hearn 2001). Fusion of the choria begins before the presomite stage around Day 19 and is completed around Day 29 (Benirschke and Layton 1969; Moore et al. 1985). This progresses to placental anastomosis and sharing of placental circulation during the subsequent development. The whole gestation takes 144 2 days (mean s.d.) in the marmoset monkey (Chambers and Hearn 1985). This allows haematopoietic stem cells to circulate to all embryos and a common population to be established in the developing haematopoietic tissues of the liver, spleen and lymph nodes, and subsequently in the bone marrow. However, because all investigations of chimerism to date have been carried out in postnatal animals, it remains unclear where the locations of chimeric cells may be in addition to bone marrow. Furthermore, it is unknown whether chimeric cells located in other organs remain in the stem cell state or whether they integrate into the organ tissue as differentiated cells. Early studies on cross-sex chimerism in Callithrix using conventional cytogenetic methods demonstrated that blood, bone marrow, lymph nodes, spleen and thymus, but not lung and liver, were chimeric (Benirschke and Brownhill 1962; Gengozian et al. 1964, 1969). More recently microsatellite data even argued for germline chimerism, because gonads and sperm cells among the 12 tissues analysed tested positive for chimerism (Ross et al. 2007). However, this conclusion was refuted by an independent study using sex determining region Y (SRY) gene quantitative polymerase chain reaction (qPCR) that suggested that only haematopoietic cell lineages are likely to be chimeric and that chimerism detected in other tissues is presumed to be a result of blood or lymphocyte infiltration (Sweeney et al. 2012). Each of these methods technically limits the questions that can be asked and answered. The issue with conventional cytogenetics (the identification of sex chromosomes in stained metaphase spreads) is the necessity for proliferating cells, which can only be obtained from very few cell types. With PCR, the problem is the inability to differentiate different cell types and, in particular, blood cell contamination in a tissue biopsy is very difficult, or even impossible, to exclude. Highly sensitive molecular methods in general are prone to produce false positives through blood contamination of organ tissue. Therefore, to critically investigate chimerism in marmosets, a single cell-based cytological sexing technique would be the method of choice to determine the specific cell type rate of cross-sex chimerism. We reasoned that fluorescent in situ hybridisation (FISH) using sex chromosome-specific probes would be a suitable method because it can be applied to a large variety of cell types, at any cell cycle phase, and may also allow the identification of cell type by inspection of interphase nucleus morphology.

E. Wedi et al.

Using a FISH technique with appropriate probes, it would also be possible to visualise whole chromosomes or chromosomal subregions (Cremer et al. 1986, 1988; Pinkel et al. 1988) in the interphase or in the metaphase with DNA sequencespecific fluorescent probes (John et al. 1969; Pardue and Gall 1970). In the present validation study, we chose to focus on cross-sex chimerism to minimise the number of chromosomal markers required to obtain accurate first results. We established robust X and Y chromosome FISH probes for marmosets, which can be used in metaphase and in interphase FISH experiments. In the future, additional individually variable chromosome markers could be identified for more detailed studies of chimerism. With the now publicly available C. jacchus genomic resources (Marmoset Genome Sequencing and Analysis Consortium 2014) this becomes an achievable objective. Of interest would be same-sex litters and multiple chimerism (i.e. the presence of more than one foreign genotype) among litters with more than two littermates. This latter phenomenon, common in captivity, has potentially major implications for the immune system and self-recognition. Materials and methods Animals The experimental part of the study was performed in 2008. The animals for the study included female and male common marmosets kept in the German Primate Centre, Goettingen, Germany, according to the standard German Primate Centre practice for this species (Gilchrist et al. 1997; Isachenko et al. 2002). All procedures were performed according to German Animal Experimentation Law (Animal Experiment Permission #33.42502/08-01.03) and were approved by the German Primate Centre animal care committee. The research adhered to the legal requirements and animal care regulations of Germany for the ethical treatment of non-human primates. Sampling, culture and cell fixation were performed in the German Primate Centre, whereas FISH staining, microscopic evaluation and analysis of results were performed in collaboration between the German Primate Centre and the Institute of Human Genetics, Munich, Germany. Marmoset cell samples Common marmoset blood samples (17 from females and four from males) were taken from hand-held, conscious young adult animals. Blood sampling was performed either from the femoral vein or the femoral artery. Bone marrow and organ samples were recovered by surgery from killed animals, which were used in terminal experiments involving only normal physiology. Bone marrow samples were obtained from eight females, and spleen and liver samples were both obtained from one female animal. In addition, the previously described male marmoset lymphoblastoid cell line (LCL) CJA98 (Neusser et al. 2007) was used for validation of the method. For each animal, the composition of the litter it originated from was recovered from the records of the German Primate Centre management data. In general, the present study encompasses three types of litters: (1) with all females (hereafter referred to as 100%f); (2) with two females

Sex chromosome FISH in Callithrix jacchus

and one male (67%f); and (3) with an equal proportion of males and females, either twin or quadruple litters (50%f). Marmoset cell culture and cell preparation for FISH Seventy-two hour blood and bone marrow cell cultures were set up immediately on the day of sampling. Cell culture and fixation of blood and bone marrow cells was done as described previously (Delimitreva et al. 2013). The marmoset LCL CJA98 was cultured and mitotic cells for cytogenetic analysis were harvested and fixed according to standard procedures. (Neusser et al. 2001, 2007). Sixty minutes before the end of the culture, 10 mg mL1 colcemid was added to the culture medium. Liver and spleen single cell suspensions were prepared by mechanically dissociating the respective fresh tissue samples, followed by fixation in Carnoy’s fixative at room temperature for 30 min and two further washes in Carnoy’s fixative. The fixed cell suspension was then dropped onto glass slides. All slides with interphase or metaphase cell preparations obtained as described above were permeabilised by pepsin treatment for 2 min when using metaphase preparations from cultured cells, or for 30 min in the case of liver and spleen cell suspensions, respectively, then dehydrated in ascending ethanol series (70%, 90%, 100%) for 3 min each, before finally being air dried. Background and detailed description of FISH using marmoset X and Y chromosome-specific probes DNA probes for interphase FISH analysis are required to meet the highest standards in terms of specificity and sensitivity. Hence, for human interphase FISH diagnostics, chromosomespecific centromere probes are preferred. However, it is well known that (peri-)centromeric satellite repeat motifs show rapid evolutionary sequence divergence (Plohl et al. 2012), which prevents the use of human centromeric probes in non-human species and, to date, marmoset centromere probes are not available. Initial tests using probes from human bacterial artificial chromosome (BAC) cloned genomic sequences were found unsuitable for interphase FISH copy number profiling in marmosets because they would not show a sufficiently high signal intensity combined with a low signal : noise ratio (data not shown). Because this project was initiated before publication of the C. jacchus genome (Marmoset Genome Sequencing and Analysis Consortium 2014) and the release of the corresponding genome-wide BAC map (http://genome.ucsc.edu, accessed 15 September 2014), there were only very few resources available, for example from genomic regions sequenced during the ENCODE pilot project (The ENCODE Project Consortium 2007). Therefore, we used the C. jacchus BAC clone CH259274F24, from ENCODE region 324 (X: 122.8 Mb; The ENCODE Project Consortium 2007), as a FISH marker, which was purchased from the BACPAC Resources Centre at the Children’s Hospital Oakland Research Institute (CHORI; Oakland, CA, USA). The Y chromosome-specific cosmid probe MPMGc160D0581Q2 was identified by PCR assays with Y chromosome-specific sequence primers on the primary pools and subsequently secondary pools of the genomic C. jacchus


C

library #160 of the RZPD (Deutsches Ressourcenzentrum Prima¨rdatenbank; http://www.rzpd-ia.de/, accessed 13 October 2005; P. H. Vogt, M. Vogel and J. Zimmer, unpubl. obs.). The X-BAC was labelled with biotin–dUTP and the Y-cosmid was labelled with Cy3–dUTP by nick translation according to standard protocols (Cremer et al. 1986). For FISH experiments, 200–400 ng mL1 of labelled DNA per FISH probe was mixed with 3 mg mL1 sheared C. jacchus genomic DNA to suppress non-specific cross-hybridisation. Metaphase FISH and interphase FISH were performed using standard procedures (Neusser et al. 2007; Solovei et al. 2007). Briefly, the XY-FISH probe mixture was denatured for 7 min at 728C and then incubated for 30–60 min at 378C in a water bath to prehybridise repetitive sequences. Further, the glass slides with the fixed and permeabilised cell preparations were incubated at 728C for 90 s in 70% formamide–2 standard saline citrate (SSC), pH 7. Subsequently, the slides were dehydrated in an ascending ethanol series (70%, 90%, 100%; 3 min each at room temperature). The XY-FISH probe mixture was added to the slide, covered with a coverslip and sealed with rubber cement. The hybridisation took place overnight at 378C in a dark chamber. After hybridisation, the slides were washed at 608C for three time for 5 min each time in 0.1 SSC, followed by an incubation step in Avidin-Alexa 488 (Life Technologies, Carlsbad, CA, USA). Finally, the slides were mounted in Vectashield embedding medium containing 40 ,60 -diamidino-2phenylindole (DAPI; Vector Laboratories, Peterborough, UK). Microscopic evaluation For all cell types, the number of X- and Y-FISH signals per cell was analysed by visual inspection under an epifluorescence microscope (Axioplan 2; Zeiss, Jena, Germany). The number of FISH signals in each nucleus or metaphase was determined according to standardised criteria (Wolff et al. 2007). The specificity of the XY-FISH probe set was determined by metaphase FISH to the C. jacchus LCL CJA98. C. jacchus have a submetacentric X chromosome and a very small acrocentric Y chromosome (Sherlock et al. 1996). Because of their individual morphology, the sex chromosomes can be easily distinguished from the other chromosomes in metaphase. Next, the sensitivity of the interphase FISH assay was validated in two independent evaluations of the same cell line, LCL CJA98. In this cell line, more than 300 nuclei were analysed per evaluation. In the remaining interphase FISH analyses, where body tissues were analysed, at least 100 nuclei were scored per experiment, except for lymphocytes from one female animal (100%f litter), for which sufficient material was only available to score at least 50 nuclei. Statistical analysis The results are presented as the mean s.d. for n animals from which the cells were derived. Data regarding the proportion of cells with XX/XY chromosome sets in animals originating from litters with different female to male ratios (100%f, 67%f or 50%f) were analysed using analysis of variance (ANOVA). In case of a significant difference between groups, the data were subjected to further analysis using t-tests to determine the level

D


E. Wedi et al.

of significance between the groups. Two-tailed P , 0.05 was considered significant. Statistical analyses were performed using Statistica Version 10 (StatSoft GmBH, Hamburg, Germany). Results Validation of the marmoset XY-FISH probe set The specificity of the XY-FISH probe set was determined by metaphase FISH analysis of the LCL CJA98, established from immortalised lymphocytes of a male C. jacchus. Both X and Y probes showed FISH signals of high intensity and low unspecific background (Fig. 1). Furthermore, both probes were shown to be highly specific for their respective chromosomes, because the X probe exclusively hybridised to the human chromosome Xq28 homologous region on the long arm of the C. jacchus X chromosome and the Y probe FISH signal was confined to the q-arm pericentromeric region of the minuscule C. jacchus Y chromosome. In all, 113 metaphases were analysed to determine the frequency of male XY and female XX cells for the cell line CJA98, as well as to delineate other potential gonosomal aneuploidies and the percentage of polyploid cells in this cell line. No XX metaphases were observed, 97.3% (110/113) of analysed cells were XY (Fig. 1a, b), and 2.7% (3/113) of cells were tetraploid with an XXYY sex chromosome set (Fig. 1c, d ). Using the metaphase FISH results on cell line CJA98 as a reference, the sensitivity of the interphase FISH assay was determined using the same cell line. In all, 1051 interphase cells were evaluated by two independent experienced cytogeneticists (S. M. and M. N.; 689 cells and 362 cells, respectively. The mean ( s.d.) proportion of normal XY cells for both analyses was 96.1 0.6%, and again there was no evidence for the presence of XX cells. After validation of the XY-FISH probe set using the cell line, we evaluated short-term cultured blood and bone marrow samples by interphase FISH. In addition, single cell suspensions from liver and spleen were evaluated to determine whether the method could be used with different types of tissue and preparations, and further to determine whether

(a)

lymphocytes could be correctly identified and left out of the evaluation. Cross-sex chimerism in interphase blood lymphocytes and bone marrow cells after culture Interphase nuclei from 21 samples of cultured blood cells, each from a different individual, were labelled with the FISH method after culture and fixation. In addition, cells from cultured bone marrow samples from eight of these individuals (all females) were treated in the same way. Representative FISH images of nuclei from blood and bone marrow cells, with clear signals for both X and Y chromosomes, are shown in Fig. 2. Fig. 3 shows the relationship between the proportion of females in the litters and the degree of cross-sex chimerism in female individuals. Only females are shown because the number of males tested was too small to make a meaningful analysis. The lymphocytes of females in the 100%f group had only 1 1% XY cells (n ¼ 6), whereas the proportion of male cells was significantly higher in both the 67%f group (45 21%; n ¼ 6) and the 50%f group (52 26%; n ¼ 5). The results for bone marrow followed a similar pattern, although because of lower numbers the difference was significant only between the 100%f and 50%f groups. There were 4 3% of XY cells in females from the 100%f group (n ¼ 4), and 17 16% (n ¼ 2) and 46 8% (n ¼ 2) in the 67%f and 50%f groups, respectively. It also should be noted that the degree of cross-sex chimerism varied significantly in animals from mixed-sex litters (e.g. from 22% to 70% among females from the 67%f group, and from 20% to as high as 90% among females from the 50%f group). The same variability was observed in males from the 67%f litters, where the proportion of XX lymphocytes in the blood was in the range 4%–44% (mean 36 30%; n ¼ 3). Preliminary testing of disassociated tissue mixed with blood cells: liver and spleen cells As part of our method optimisation studies we performed preliminary tests aimed at evaluating the potential of XY

(b)

(c)

XY (d)

XY

XXYY

XXYY

Fig. 1. Validation of the Y chromosome cosmid 2 and X chromosome bacterial artificial chromosome (BAC)specific probes using (a, b) metaphase and (c, d ) interphase cells from an XY Callithrix jacchus lymphoblastoid cell line (CJA98). Red, Y chromosome; green, X chromosome. The lymphocytes in (b) and (d ) are polyploid and have two X chromosomes and two Y chromosomes. All X and Y chromosome signals are indicated by the arrows.



interphase FISH to determine the degree of cross-sex chimerism in non-cultured fixed single cell suspensions from two different organs. Representative FISH images of nuclei from liver and spleen cells are shown in Fig. 2. First, and most importantly, fixed single cell suspensions from liver and spleen analysed were obtained from the same

Lymphocytes

female individual originating from a mixed-sex triplet litter (67%f). Cross-sex chimerism was present in 48% of splenic cells, but not in liver cells, where 99% of cells were XX. This result is important because it demonstrates a clear difference between a lymphatic and non-lymphatic organ within one individual. Further, both organs contain many blood cells,

Liver

Spleen XY

XX

XY XY

XX

Bone marrow XX

XX XY

XY

XX

XX

Fig. 2. Representative interphase nuclei from cell suspensions from Callithrix jacchus after fluorescence in situ hybridisation (FISH) labelling with our C. jacchus X (green) and Y (red) chromosome-specific probes. Nuclei of peripheral blood lymphocytes, bone marrow, spleen and liver cells from female individuals from mixed-sex litters are shown, with both XX- and XY-labelled cells. All cell types, with the exception of the liver, show cross-sex chimerism. The respective type of cell of origin and the genotype assessed based on FISH signals are indicated.

80%

Percentage of XY-cells

[6]

[2]

60%

[5]

[2]

b

c

a 40%

20%

[6]

[4]

ab

c

0% 100%f

E

67%f

50%f

Animal groups depending on original litters Fig. 3. Percentage of XY cells in lymphocytes (grey columns) and bone marrow (black columns) derived from Callithrix jacchus females from litters with different proportions of females to males. 100%f, litters with all females; 67%f, litters with two females and one male; 50%f, litters with an equal proportion of males and females. The number of animals analysed in each group is shown in square brackets above each columns. Data are the mean s.d. Values with different letters differ significantly (P , 0.05).

F


which could invalidate the result if they are not correctly identified. Blood cells were excluded based on nuclear morphology. Discussion A FISH method specific for X and Y chromosomes has been developed and validated for the investigation of cross-sex chimerism in haematopoietic and organ tissues of common marmosets. In the present study, the indirect labelling method using fluorescent dyes was chosen. The reporter molecules biotin and dinitrophenol and the two antibody-bound fluorochromes Alexa 488 (green; marks the X chromosome) and Cy3 (red; marks the Y chromosome) were used. The primary aims of the study were to first validate the specificity of the method on metaphase chromosomes of a marmoset cell line and thereafter to evaluate XY chimerism in interphase leucocytes and bone marrow. Additional preliminary tests were also performed on other tissues to determine whether the method was functional for other cells and preparation methods. The method validation was performed on XY metaphase cells from a C. jacchus LCL (CJA98) to validate the specificity of the labelling and then confirmed on interphase cells from the same line. The advantages of the FISH method are its use on interphase cells, its high degree of accuracy and that it maintains the cell organisation and allows cell types to be identified so that one can distinguish in which cell type chimerism occurs. Previously, this technique has never been used to investigate chimerism in any species, and no probes for C. jacchus chromosomes were available. Therefore, we have generated the probes, established a procedure for their use and have performed a preliminary study using the validated probes. We evaluated the chimerism status of interphase bone marrow cells and interphase lymphocytes isolated from blood. The results of the evaluation of blood and bone marrow showed that in individuals from all-female litters, only minimal or no XY cells were found either in bone marrow or circulating lymphocytes. This result provided confirmation of the stability of the method and was evidence that the main source of the XY cells was the male littermate(s). In mixed-sex litters, virtually all animals were cross-sex chimeric to a variable degree. The degree of chimerism in both bone marrow and lymphocytes obtained from plasma appeared to reflect the proportion of males to females in the litter. One more interesting aspect is the not infrequent occurrence of a dominance of the opposite sex haemopoietic cells. It would seem unlikely that this could have an effect on the reproductive and hormonal systems, because it involves haemopoietic stem cells and cells derived from this population. However, it is possible that chimerism involving more than two individuals, regardless of which sex, could broaden the immune tolerance or, in extreme cases, interfere with the accuracy of self-recognition and thereby promote the occurrence of autoimmune diseases. This issue remains to be investigated. It is also unknown at present whether other tissues may be involved in the chimerism. Logically, chimerism may be predicted to only involve myeloid or lymphoid tissues, but the migration of stem cells to other organ sites during early

E. Wedi et al.

stages of organogenesis may also make organ chimerism a possibility, but this is most likely to occur in organs containing lymphoid tissue. Therefore, we performed a small preliminary experiment to determine whether FISH would be useful to evaluate chimerism in organ tissues and to compare isolated cells from an organ containing lymphoid tissue (spleen) and one non-lymphoid organ (liver) from one female from a mixed-sex litter. Both organs are haemopoietic during embryonic development (Rohen and Lu¨tjen-Drecoll 2002). The results showed the spleen to be strongly chimeric and liver not chimeric. Important for an accurate result was that only the organ cells were evaluated, not the blood within the organ. This result contrasted with that published by Ross et al. (2007) in Callithrix kuhlii, namely that spleen and liver are both chimeric, as determined using qPCR. Sweeney et al. (2012) performed a similar study in Saguinus oedipus and C. jacchus and suggested that only haematopoietic cell lineages are chimeric and that chimerism detected in other tissues is the result of blood or lymphocyte infiltration. However, because both studies used qPCR, a method that does not allow differentiation between tissue and blood cells (Ross et al. 2007; Sweeney et al. 2012), the conclusions using this method remain speculative. Chimerism in Callithrichidae is a natural phenomenon and stems from the normal sharing of placental circulation between or among littermates. However, because of the previous lack of a technique that allowed the identification of specific cells types as well as highly specific chromosomal markers for this species, this phenomenon has not yet been explored in depth. The FISH technique presented herein specifically marks marmoset X and Y chromosomes and can be used on metaphase spreads and interphase cells identifiable by their morphology, thus providing a powerful tool to investigate this phenomenon. Further, by using additional markers for differentiation status (Aeckerle et al. 2015), it could also be possible in future to determine whether chimeric cells in organs, for example, are differentiated or have remained as stem cells within the organ. The results using this method for the first time suggest a proportionally variable but mixed-sex litter-specific XX/XY chimerism in mature lymphocytes and bone marrow, which appears to proportionally reflect the mixed sex litter composition. In addition, preliminary tests on spleen and liver cells from a single female from a mixed-sex litter showed a high level of chimerism in spleen in contrast with the liver, which showed no chimerism. This tentatively suggests that, as may have been predicted, lymphoid-containing tissues may preferentially develop chimerism, even though the liver is haemopoietic before the development of the bone marrow. This requires a much larger study to confirm or disprove the hypothesis, in both pre- and postnatal animals. The suggestions that haemopoietic chimerism may extend to the gametes we find unlikely, because primordial germ cells have a different embryological origin and a different mode of transport through the embryo to the genital ridge (Ross et al. 2007). However, it warrants careful investigation, perhaps with newborn ovaries and testes, which still contain a large number of primordial germ cells (Mitchell et al. 2008; Fereydouni et al. 2014). However, it could be feasible that gametic stem cells may be exchanged among the embryos and a separately developed


chimerism could theoretically occur. This issue needs detailed investigation to resolve (Mitchell et al. 2008). A further question that needs to be asked is what benefit chimerism may have for the embryo or fetus, and later the adult. One possibility is that it may be a mechanism to facilitate immune tolerance between or among the embryos or fetuses within the uterus. This would appear to be a necessary feature for the proper functioning of the more efficient fused placental circulation. However, abnormally large litters (more than two) commonly found in captive marmosets may, in fact, reduce any benefit through the increased complexity of the placental sharing, and thereby also a more generalised immune tolerance. In summary, we have developed a marmoset XY-specific FISH method that we expect to be a powerful tool to clarify developmental or evolutionary benefits of cross-sex chimerism in marmosets and tamarins by providing accurate cell typespecific results in any cell cycle stage, and in any tissue. We also have shown that these probes can reveal the patterns of cross-sex chimerism extant in our test group. These results allow us to build more focused hypotheses using this new and more powerful tool for the next stage of the investigations. Acknowledgements The authors thank the laboratory assistants K. Fuhrmann, N. Umland and A. Berenson for blood sampling and technical help during operations and laboratory work. The authors also thank Tanja Koppe and Anja Kellermann for their generous support with the screening assays for Y-specific cosmids using their primary and secondary pools of the Callithix jacchus cosmid library (#160) from the RZPD. The authors acknowledge T. Becker and J. Lademann for surgery. OYT was supported, in part, by The German Academic Exchange Service (DAAD) and a Young Scientists grant from the German Primate Centre.

References Aeckerle, N., Drummer, C., Debowski, K., Viebahn, C., and Behr, R. (2015). Primordial germ cell development in the marmoset monkey as revealed by pluripotency factor expression: suggestion of a novel model of embryonic germ cell translocation. Mol. Hum. Reprod. 21, 66–80. doi:10.1093/MOLEHR/GAU088 Benirschke, K., and Brownhill, L. E. (1962). Further observations on marrow chimerism in marmosets. Cytogenetics 1, 245–257. doi:10.1159/ 000129734 Benirschke, K., and Driscoll, S. G. (1967). ‘Pathology of the Human Placenta.’ 1st edn. (Springer-Verlag: New York.) Benirschke, K., and Layton, W. (1969). An early twin blastocyst of the golden lion marmoset, Leontocebus rosalia L. Folia Primatol. (Basel) 10, 131–138. doi:10.1159/000155191 Chambers, P. L., and Hearn, J. P. (1985). Embryonic, foetal and placental development in the common marmoset monkey (Callithrix jacchus). J. Zool. 207, 545–561. doi:10.1111/J.1469-7998.1985.TB04951.X Choi, D. H., Kwon, H., Lee, S. D., Moon, M. J., Yoo, E. G., Lee, K. H., Hong, Y. K., and Kim, G. (2013). Testicular hypoplasia in monochorionic dizygous twin with confined blood chimerism. J. Assist. Reprod. Genet. 30, 1487–1491. doi:10.1007/S10815-013-0109-8 Cremer, T., Landegent, J., Bruckner, A., Scholl, H. P., Schardin, M., Hager, H. D., Devilee, P., Pearson, P., and van der Ploeg, M. (1986). Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84. Hum. Genet. 74, 346–352. doi:10.1007/BF00280484


G

Cremer, T., Lichter, P., Borden, J., Ward, D. C., and Manuelidis, L. (1988). Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum. Genet. 80, 235–246. doi:10.1007/BF01790091 Delimitreva, S., Wedi, E., Bakker, J., Tkachenko, O. Y., Nikolova, V., and Nayudu, P. L. (2013). Numerical chromosome disorders in the common marmoset (Callithrix jacchus): comparison between two captive colonies. J. Med. Primatol. 42, 177–185. doi:10.1111/JMP.12050 Fereydouni, B., Drummer, C., Aeckerle, N., Schlatt, S., and Behr, R. (2014). The neonatal marmoset monkey ovary is very primitive exhibiting many oogonia. Reproduction 148, 237–247. doi:10.1530/REP-14-0068 Gengozian, N., Batson, J. S., and Eide, P. (1964). Hematologic and cytogenic evidence for chimerism in the marmoset, Tamarinus nigricollis. Sam-Tdr-64-61. AMD TR Rep. Nov, 1–10. Gengozian, N., Batson, J. S., Greene, C. T., and Gosslee, D. G. (1969). Hemopoietic chimerism in imported and laboratory-bred marmosets. Transplantation 8, 633–652. doi:10.1097/00007890-196911000-00009 Gilchrist, R. B., Nayudu, P. L., and Hodges, J. K. (1997). Maturation, fertilization, and development of marmoset monkey oocytes in vitro. Biol. Reprod. 56, 238–246. doi:10.1095/BIOLREPROD56.1.238 Hearn, J. P. (2001). Embryo implantation and embryonic stem cell development in primates. Reprod. Fertil. Dev. 13, 517–522. doi:10.1071/ RD01068 Isachenko, E. F., Nayudu, P. L., Isachenko, V. V., Nawroth, F., and Michelmann, H. W. (2002). Congenitally caused fused labia in the common marmoset (Callithrix jacchus). J. Med. Primatol. 31, 350–355. doi:10.1034/J.1600-0684.2002.T01-1-02002.X John, H. A., Birnstiel, M. L., and Jones, K. W. (1969). RNA–DNA hybrids at the cytological level. Nature 223, 582–587. doi:10.1038/223582A0 Kowalzick, L., Artlett, C. M., Thoss, K., Baum, H. P., Ziegler, H., Mischke, D., Blum, R., Ponnighaus, J. M., and Quietzsch, J. (2005). Chronic graft-versus-host-disease-like dermopathy in a child with CD4þ cell microchimerism. Dermatology 210, 68–71. doi:10.1159/000081489 Marmoset Genome Sequencing and Analysis Consortium (2014). The Marmoset Genome Sequencing and Analysis Consortium. The common marmoset genome provides insight into primate biology and evolution. Nat. Genet. 46, 850–857. doi:10.1038/NG.3042 McLaren, A. (1976). ‘Mammalian Chimaeras.’ (Cambridge University Press: Cambridge.) Mitchell, R. T., Cowan, G., Morris, K. D., Anderson, R. A., Fraser, H. M., McKenzie, K. J., Wallace, W. H., Kelnar, C. J., Saunders, P. T., and Sharpe, R. M. (2008). Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human. Hum. Reprod. 23, 2755–2765. doi:10.1093/HUMREP/ DEN295 Moore, H. D., Gems, S., and Hearn, J. P. (1985). Early implantation stages in the marmoset monkey (Callithrix jacchus). Am. J. Anat. 172, 265–278. doi:10.1002/AJA.1001720402 Neusser, M., Stanyon, R., Bigoni, F., Wienberg, J., and Muller, S. (2001). Molecular cytotaxonomy of New World monkeys (Platyrrhini): comparative analysis of five species by multi-color chromosome painting gives evidence for a classification of Callimico goeldii within the family of Callitrichidae. Cytogenet. Cell Genet. 94, 206–215. doi:10.1159/000048818 Neusser, M., Schubel, V., Koch, A., Cremer, T., and Muller, S. (2007). Evolutionarily conserved, cell type and species-specific higher order chromatin arrangements in interphase nuclei of primates. Chromosoma 116, 307–320. doi:10.1007/S00412-007-0099-3 Padula, A. M. (2005). The freemartin syndrome: an update. Anim. Reprod. Sci. 87, 93–109. doi:10.1016/J.ANIREPROSCI.2004.09.008 Pardue, M. L., and Gall, J. G. (1970). Chromosomal localization of mouse satellite DNA. Science 168, 1356–1358. doi:10.1126/SCIENCE.168. 3937.1356

H


E. Wedi et al.

Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. (1988). Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc. Natl Acad. Sci. USA 85, 9138–9142. doi:10.1073/PNAS.85.23.9138 Plohl, M., Mestrovic, N., and Mravinac, B. (2012). Satellite DNA evolution. Genome Dyn. 7, 126–152. doi:10.1159/000337122 Rohen, J. W., and Lu¨tjen-Drecoll, E. (2002). ‘Funktionelle Embryologie: Die Entwicklung der Funktionssysteme des menschlichen Organismus.’ (Schattauer: Stuttgart.) Ross, C. N., French, J. A., and Orti, G. (2007). Germ-line chimerism and paternal care in marmosets (Callithrix kuhlii). Proc. Natl. Acad. Sci. USA 104, 6278–6282. doi:10.1073/PNAS.0607426104 Sherlock, J. K., Griffin, D. K., Delhanty, J. D., and Parrington, J. M. (1996). Homologies between human and marmoset (Callithrix jacchus) chromosomes revealed by comparative chromosome painting. Genomics 33, 214–219. doi:10.1006/GENO.1996.0186 Solovei, I., Grasser, F., and Lanctot, C. (2007). FISH on histological sections. CSH Protoc. 2007, pdb.prot4729. doi:10.1101/PDB. PROT4729

Souter, V. L., Parisi, M. A., Nyholt, D. R., Kapur, R. P., Henders, A. K., Opheim, K. E., Gunther, D. F., Mitchell, M. E., Glass, I. A., and Montgomery, G. W. (2007). A case of true hermaphroditism reveals an unusual mechanism of twinning. Hum. Genet. 121, 179–185. doi:10.1007/S00439-006-0279-X Sweeney, C. G., Curran, E., Westmoreland, S. V., Mansfield, K. G., and Vallender, E. J. (2012). Quantitative molecular assessment of chimerism across tissues in marmosets and tamarins. BMC Genomics 13, 98. doi:10.1186/1471-2164-13-98 The ENCODE Project Consortium (2007). Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816. doi:10.1038/NATURE05874 Vabres, P., and Bonneau, D. (2005). Childhood dermatosis due to microchimerism. Dermatology 211, 388–389. doi:10.1159/000088521 Wolff, D. J., Bagg, A., Cooley, L. D., Dewald, G. W., Hirsch, B. A., Jacky, P. B., Rao, K. W., and Rao, P. N. Association for Molecular Pathology Clinical Practice Committee, and American College of Medical Genetics Laboratory Quality Assurance Committee (2007). Guidance for fluorescence in situ hybridization testing in hematologic disorders. J. Mol. Diagn. 9, 134–143. doi:10.2353/JMOLDX.2007.060128

www.publish.csiro.au/journals/rfd