Genetic imprinting in human embryogenesis H19 and IGF2 gene expression

Trophoblast Research 8:285-302, 1994

GENETIC I M P R I N T I N G IN H U M A N E M B R Y O G E N E S I S H19 A N D IGF2 GENE EXPRESSION N a t h a n d e G r o o t l G J. R a c h m i l e w i t z 1, I. Ariel 3, R. G o s h e n 2, O. L u s t i g ~, a n d A. H o c h b e r g ~

1Department of Biological Chemistry The Silberman Institute of Life Sciences The Hebrew University Jerusalem, Israel 2Department of Obstetrics and Gynecology 3Department of Pathology Hadassah University Hospital Mount Scopus Jerusalem, Israel INTRODUCTION Recently, it became evident that genomic imprinting plays a major role in mammalian development (Surani, 1991), pediatric cancers (Sapienza, 1991), and human disease (Sapienza, 1991; Hail, 1990). It is known that both male and female genomes are essential for normal mammalian development, probably because some genes are expressed according to the gamete of their origin (Solter, 1988). As a result of genetic imprinting only one of the two parental alleles which coexist in a cell, is active while the other is repressed. The maternal and the paternal genomes contribute in different ways to the development of embryonic and extraembryonic tissues. This was shown through the construction of androgenetic and gynogenetic zygotes by Solter (1987) and Surani (1986). The results showed that in the androgenetic zygote there was good growth of the placenta and very poor growth of the embryo, whereas in the case of the early gynogenetic zygote the embryo grew relatively well but the placenta developed poorly (Surani et al., 1990). A homologue of an androgenetic zygote in humans is the complete hydatidiform mole. This human placental tumor harbors two sets of chromosomes both derived from the father. Although no embryo is seen, implantation does occur and the placenta is markedly overgrown (Szulman, 1987a). A human disease which is homologous to a gynogenetic zygote is a particular type of ovarian teratoma (Linder et al., 1975), which contains two haploid sets of maternal chromosomes. This tumor contains multiple embryonic tissue types but no extraembryonic tissue (Hall, 1991). The dose effect of maternal and paternal sets of chromosomes on the formation and growth of embryonic and extraembryonic tissues can be observed when compared to the fate of the placenta and the embryos in triploids. Triploids are individuals with 4To Whom Correspondence Should Be Addressed.

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three sets of chromosomes: Theoretically two possibilities exist. One in which the cells contain two sets of paternally derived chromosomes and one set derived from the mother (2p:lm) and in the second two sets of chromosomes are derived from the mother and one from the father (lp:2m). Both possibilities can occur in humans. In the placental tumor, the partial hydatidiform mole with two sets of chromosomes derived from the father and one from the mother, the placenta is overgrown but the embryos or fetus show intrauterine retardation (Szulman, 1987b; McFadden and Kalousek, 1991). On the other hand in a triploid with two sets of maternally derived chromosomes and one paternal set the placenta is undeveloped. The unequal contribution of paternal and maternal chromosomes to the development of extraembryonal and embryonal tissues is also shown by the influence of androgenetic and gynogenetic cells on the phenotype of chimeras. It was found that the presence of these cells in chimeras has a striking phenotypic effect on the growth proliferation of specific cell types and cell-cell interaction and pattern formation (Barton et al., 1991). One of the major effects of androgenetic and gynogenetic cells in chimeras is that of size regulation. The presence of gynogenetic cells is associated with growth retardation and androgenetic cells with growth enhancement up to 50% (Surani et al., 1990). The distribution of androgenetic or gynogenetic cells in chimeras is non uniform and reciprocal. Androgenetic cells contribute to mesodermally derived tissues but less to the brain, the gynogenetic cells contribute relatively more to the brain but very little to skeletal muscle (Barton et al., 1991; Mann et al., 1990). Taking these data together it is possible to conclude that some genes which are expressed only when located on paternally inherited chromosomes are important to placental cell growth and proliferation and genes expressed only when maternally inherited are obligatory for the growth of the early embryo. It was suggested that when the zygote is implanted in the uterus some kind of limitation on its ability to invade the maternal tissue must be imposed if the mother is to survive pregnancy (Moore and Haig, 1991). The differential effect of the parental origin of haploid sets of chromosomes is also well attested in humans in chromosome deletion syndromes (Knoll et al., 1991), in uniparental disomies (Spence et al., 1988), and in the development of tumors in humans (Standbridge, 1992). It is of interest to note that in many cases of the pediatric cancers: Wilms tumor, osteosarcoma, bilateral retinoblastoma, and e m b r y o n a l r h a b d o m y o s c a r c o m a , preferential retention of paternal alleles could be detected (Sapienza, 1991). Genetic studies have demonstrated that some chromosomal regions containing a subset of genes within them are subject to parental imprinting (Cattanach, 1991). So far four imprinted genes in mouse have been identified: IGF2 (De-Chiara et al., 1991), IGF2-R (Barlow et al., 1991), IGF1-R (Rappolee et. al., 1992), and H19 (Bartolomei et al., 1991), by using a number of different approaches. De Chiara mutated the IGF2 gene by homologous recombination and found that in heterozygotes the normal allele was repressed when maternally derived and active when paternally inherited (De-Chiara et al., 1991). Furthermore, Ferguson-Smith et al. (1991) showed that in genetically

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crossed embryos, with two maternal copies of the distal chromosome 7 and no paternal complement, IGF2 (which maps to the distal region of chromosome 7) was repressed. The IGF2-R which maps in mice to the proximal region of chromosome 17 resides within the only known maternal effect deletion mutant in mice Tme. There was a striking lack of IGF2-R transcripts in embryos inheriting the Tme maternally but not when the deletion was inherited from the father. Hence the maternal allele of IGF2-R appears to be preferentially expressed (Barlow et al., 1991). The third gene H19 was shown to be imprinted by an RNase protection assay in interspecific hybrid F-1 mice. The maternal allele of this gene is preferentially expressed in mouse (Bartolomei et al., 1991). Moreover the human H19 gene is expressed in a monoallelic manner in humans (Zhang and Tycko, 1992). Interestingly, the H19 gene is closely linked (Zemel et al., 1992) both in mice and human to the IGF2 gene.

The IGF2 gene product functions as an embryonic mitogen on a variety of embryonic and extraembryonic tissues during development (Ohlsson et al., 1989b). It also has an important role during embryogenesis (Rappolee et al., 1992) and interacts with high affinity to two different cell surface receptors (Roth, 1988). It is generally accepted that the growth response of IGF2 is mediated primarily via the IGFl-receptor. Recently it was shown that in mice IGF1-R is not expressed when inherited maternally (Rappolee, 1992). The activation of the IGF2 gene in human placenta is a post implantation event that correlates with the growth of the high proliferative trophoblastic shell (Ohlsson et al., 1989b). IGF2 was implied as an important modulator of placenta cell proliferation and maturation. The H19 gene is an unusual gene. It is transcribed by RNA polymerase II, processed by splicing and polyadenylation, yet it does not appear to encode a protein (Pachnis et al., 1988, Brannan et al., 1990). In four mammalian species the H19 sequence has been determined yet no common open reading frame is apparent, despite the fact that the nucleotide sequence is well conserved (Brannan et al., 1990). The transcripts of H19 are not associated with the translational machinery but are located in 28S particles both in mice (Pachnis et al., 1988) and in human placentae. The H19 gene is first expressed in extraembryonic tissues at the time of implantation. By 9.5 dpc (days post coitus) a large number of endoderm and mesoderm embryonic tissues express H19 (Poirier et al., 1991). After birth its expression is repressed in all tissues except muscle (Pachnis et a1.,1988). It was suggested that H19 plays a role in the differentiation of mouse and human embryonic cell lines (Rogers et al., 1990 and Wiles, 1988). Transgenic studies in mice have revealed that extra copies of the intact H19 gene were deleterious to the embryo between day 14 and birth (Brunkow et al., 1991). The mode of action of H19 is not known but the possibility that the H19 product resembles another gene product that functions at the RNA level (XIST) was suggested. H19 and IGF2 genes share more than physical proximity. They are similarly expressed during mouse embryogenesis in terms of their site (tissue) of expression and the time of expression. Both are activated in the extraembryonal tissue at the late blastocyst stage and start to be transcribed in the embryo proper at day 8.5 (Poirier et al., 1991; Rappolee et al., 1992). These data suggest that the two reciprocal imprinted

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genes which have a critical role in mouse embryogenesis might share a c o m m o n regulatory element. MATERIALS AND METHODS Tissue Sampling Placental and embryonal specimens were collected from conception products derived from normal first and second trimester terminations. The study protocol was approved by the local ethical committee. Gestational age was determined by means of the last menstrual period and physical examination and was confirmed by ultrasonographic biometry. Partial and complete hydatidiform moles were obtained by vacuum aspiration of the uterus and were diagnosed by ultrasonographic studies, and by histopathological and cytogenetical analysis. Decidual and endometrial tissues were obtained from samples sent for pathological examinations. The tissue type was confirmed by histological observations. The specimens were frozen in liquid nitrogen and then kept at -80~ for further analysis. Cell Cultures Cytotrophoblasts were isolated from h u m a n term placentae as described (Gileadi et al., 1991). JAr choriocarcinoma cells were maintained as described previously (Hochberg et al., 1992). Centrifugal elutriation cytotrophoblast cells were separated by centrifugal elutriation, as described by Rachmilewitz et al. (1993). Cell Size Determination Cells from each of the elutriation fractions were suspended in HBSS (Hank's Balanced Salt Solution), and their number and size distributions in each fraction were determined using a Coulter Counter (Coulter Electronics, Harpenden, Herts, England). Trypsinization Cells grown for 24 and 120 hours were washed with HBSS. Trypsin solution (0.25% trypsin, 0.3% EDTA) was added to the cells and the suspension incubated for 20 minutes. The cell suspension was transferred to a conical tube with 2 volumes of newborn calf serum, collected by centrifugation, and resuspended in Medium 199. Isolation of RNA Total cellular RNA was isolated from the various tissues by the guanidinium thiocyanate method and from the cells by the guanidinium-thiocyanate/cesiumchloride method (McDonald et al., 1987). Northern Blotting For all Northern blots described in this paper equal amounts of RNA (10 ~g) were separated as measured in a Spectrophotometer at both 260 and 280 nm wavelengths by 1% agarose-formaldehyde gel electrophoresis. The RNA was

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transferred to Hybond N Nylon filters. In order to ascertain that the efficiency of RNA transfer from the gel to the filters is equal in all the lanes of the blot, the filters were routinely stained with methylene blue (Sambrook et al., 1989). After the staining, the filters were photographed. Only filters which showed equal amounts of undegraded 18S and 28S ribosomal RNA were hybridized with the probes indicated in the legend of the figures. All other filters were discarded. In Figure 2 a picture of such a methylene blue stained filter is shown. All the other filters mentioned in this paper gave identical pictures. The blots were prehybridized at 42~ in 50% formamide, 5xSSPE, 5xDenhardt's solution, 0.1% SDS and 0.1 m g / m l herring testes DNA, and hybridized with specific cDNA probes. The H19 probe used for hybridization was prepared from the appropriate clone of H19. The probe was labeled according to the Random Primed labeling kit (Boehringer, Mannheim) protocol. The probe for IGF2 was an antisense 40 mer nucleotide purchased from Oncogene Sciences and was labeled according to the manufacturer's instructions. The blots were washed twice in 0.1xSSC, 0.1% SDS at 65~ and exposed to AGFA Curix film at -80~ RESULTS AND DISCUSSION Isolation of a H19 c D N A Clone From Human Placenta

During placental differentiation the expression level of many genes undergo profound changes. In order to try to identify such genes which may play a role in the regulation of placental differentiation, cDNA libraries were prepared from RNA isolated from term cytotrophoblast cells incubated for 24 and 120 hours. Differential screening of these libraries lead to the isolation of cDNA clones corresponding to genes expressed at greatly different levels at the time points mentioned above. Three of 84 independent clones isolated were subjected to sequencing and their sequences turned out to be identical to the sequence of the human H19 gene which was isolated and sequenced by Brannan et al. (1990). The abundance of the H19 mRNA in total RNA isolated from cultured cytotrophoblast cells was determined at different times during the in vitro differentiation of cytotrophoblast cells (Figure 1). As can be seen from Figure 1, the level of the H19 mRNA remained nearly constant during the first 9 hours of incubation and fell sharply between 9 and 18 hours and rose again continuously after 24 hours till the end of the incubation period (120 hours).

0

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Figure 1. Time-dependent expression of H19 in isolated cytotrophoblast cells in vitro. Autoradiograms of Northern blots containing RNA isolated from cytotrophoblast cells after different incubation times.

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The abundance of the H19 sequence reach very high levels in cytotrophoblast cells. It is probably one of the major RNA polymerase II products in cytotrophoblast cells and in the whole placenta. The reason for the fall in the level of the H19 gene transcript during the first day of the incubation of the cytotrophoblast cells is not known but is very likely due to the "re-equilibration" of the isolated cytotrophoblast cells with their new surroundings. For example, the isolation procedure can damage cell surface receptors and this damage can influence, in an indirect way, the transcription level of specific genes. Certainly, other explanations can be offered and they are presently being investigated in this laboratory. A decrease parallel to that of the H19 transcript was observed for the level of mRNA's for CGc~, IGF2, and FMS during the first day of cytotrophoblast cultivation, followed by an increase in these mRNA levels after 24 hours, similar to that of H19 mRNA (results not shown). Whatever the explanation for the temporary drop in the level of the H19 transcript, it has lead this laboratory to the isolation of a clone derived from a very highly abundant placental transcript, which could be identified as the H19 gene transcript. The Expression of H19 and IGF2 in Complete Hydatidiform Molar Tissue The complete hydatidiform molar tissue is an androgenetic tissue and therefore can be used for the determination of the parental origin of the active allele of embryonal expressed imprinted genes. The normal placenta, the partial hydatidiform molar tissue, and complete hydatidiform molar tissue are three tissues derived from the same cell type of origin, the trophoblastic cell. However, they differ in the ratios between the number of paternal and maternal sets of chromosomes, normal placenta (1P:IM), partial hydatidiform mole (2P:IM) and complete hydatidiform mole (2P). Therefore, the comparison between the extent of the expression of certain genes in those tissues cannot only provide evidence for the parental origin of the imprinted gene, but also enables us to learn about the differential effect of maternal and paternal gene dosage on gene expression. The expression of the H19 and IGF2 genes in these tissues was compared. Cells of the choriocarcinoma cell line JAr as a model for dividing cytotrophoblastic cells were used. First trimester, third trimester placenta, and partial molar tissue express the H19 gene to a similar extent. The highest expression was found in the cells of the choriocarcinoma cell line. In complete hydatidiform molar tissue, the H19 expression was less than 10% of that in the normal placental tissue (Figure 2A). The apparent weak expression of the H19 gene in the complete hydatidiform molar tissue can be due either to a weak expression in the molar tissue or to its expression in contaminating decidual tissue. The molar tissues obtained from the

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operation room were contaminated with decidual tissue. It is relatively easy to distinguish between molar tissue proper and decidual tissue as the molar tissue is avascular (whitish tissue) and the decidual tissue reddish. Great efforts in separating molar tissue from the adherent decidual tissue were invested.

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Figure 2. Expression of H19 (A), IGF2 (B), in placental, hydatidiform mole tissues and in choriocarcinoma cells (JAr). Northern blots containing RNA isolated from first trimester placentae (n=4; 12 weeks; lane 1). Term placental tissues (n=5; lane 2). Partial hydatidiform mole (n=3; 18 weeks; lane 3). Complete hydatidiform mole (n=3; 12 weeks; lane 4). JAr cells (lane 5). The RNA on the Nylon filters was stained with methylene blue and photographed. The photo is shown in the lower panel of the figure.

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Figure 3. Expression of H19 in different decidual tissues. Autoradiograms of Northern blots with RNA isolated from the following tissues, lane 1) term decidua, lane 2) complete hydatidiform mole decidua, 12 weeks, lane 3) normal term placenta, lane 4) myometrium, non-pregnant, lane 5) endometrium, non pregnant, secretory phase.

The H19 expression was lowest in molar tissue which had the smallest contamination with decidual tissue. After the separation of the two tissues each from the other, the level of CG~ mRNA in the two tissues was investigated. It was high in the molar tissue and negligible in the decidual tissue (results not shown). The expression of the H19 gene in human decidual tissue obtained from normal pregnancy, from patients with complete hydatidiform mole and from endometrium in the secretory phase was also determined. The results are summarized in Figure 3 and they clearly show that the endometrial tissue under different physiological conditions strongly expresses the H19 gene. Recently in-situ hybridization experiments with tissue samples consisting of complete hydatidiform molar tissue with adjacent decidual tissues were carried out in this laboratory. These experiments showed a strong expression of the H19 gene in the decidual cells, but no or nearly no H19 expression in the molar tissue (results to be published). It has to be stressed that the decidua is a non-embryonal adult tissue and that in all other adult tissues (except skeletal muscle) the H19 is not expressed or hardly expressed. H19 expression in the myometrium was much lower than in the decidua (Figure 3). It is worthwhile mentioning here that human decidual tissue also expresses the IGF-II gene at a high level (Glaser et al., 1992). Zhang and Tycko (1992) reported the monoaUelic expression of the H19 gene in several human fetal tissues, including liver, kidney, lung, and heart. The only example of biallelic expression was found in placentae. According to Zhang and Tycko (1992), "the apparent biallelic expression in the placenta containing maternal tissues might

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represent a variable contribution from an expressed allele in a population of maternal cells". Only the decidua can be the source of these maternal cells in the placental samples investigated. One has also to take into consideration that Zhang and Tycko used PCR in their experiments and therefore the slightest contamination of placental tissue with decidual cells can explain the biallelic expression of H19 in placenta, as described by them. The results from this laboratory strongly support Zhang and Tycko's explanation of their results. These findings, together with Zhang and Tycko's findings, serve as a proof for the parental imprinting of the H19 gene in humans, its active allele derived form the mother. The IGF2 gene is expressed in all the tissues examined (Figure 2B). Several mRNAs of the single copy IGF2 gene are formed as a result of the use of different promoters, differential splicing and polyadenylation (de Pagter-Holthuizen et al., 1987; Daimon et al., 1992). The ratio between the concentrations of the different transcripts is not identical in the tissues examined. In JAr cells only one minor transcript could be detected. Gene imprinting is considered to be a property of certain chromosomal areas (Hall, 1990). The human IGF2 gene is tightly linked to the imprinted H19 gene on chromosome 11p 15.5, an area syntenic with the distal part of mouse chromosome 7 (Zemel et al., 1992), which carries the imprinted genes IGF2 (Lalley and Chirgwin, 1984) and H19 (Pachnis, 1984). IGF2 is parentally imprinted in mice with the paternally inherited allele the active one (de Chiara et a1.,1991). The finding that IGF2 is expressed in complete molar tissue, shows explicitly that the paternal allele of the IGF2 gene can be expressed. All these observations together make a strong argument for the IGF2 gene being also imprinted in humans with the active allele paternally derived. It could be expected that in complete hydatidiform molar tissue endowed with two sets of paternal genomes and lacking a maternal chromosome set, the expression of IGF2 should be higher than in placentae of comparable age. This is, not the case, being actually, somewhat lower. It is possible, however, that in the absence of the maternal genome one of the factors involved in the regulation of IGF2 expression is missing or less active. Therefore, the above mentioned finding is not contradictory to the proposed parental imprinting of the IGF2 gene. In contrast, the finding that IGF2 is expressed from the paternally inherited allele, does not prove that the IGF2 gene is imprinted in humans, as long as its monoallelic expression in humans has not been established. In several tumors the IGF2 gene was found to be highly expressed, making its low expression in the cells of the JAr cell line remarkable. JAr cells have a wide variety of chromosomal abnormalities with a modal chromosome number of 72. Several chromosomes are presented by more than two copies (Surti and Habibian, 1989). Also, several chromosomal breakpoints have been observed, including in chromosome ] 1, where both the H19 and the IGF2 genes are located. These chromosomal aberrations m a y be partly or wholly responsible for the high H19 expression and low IGF2 expression in JAr cells and may account for the expression of only one single IGF2 transcript.

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Figure 4. Expression of H-19 in fetal tissue throughout pregnancy. Autoradiogram of Northern blots containing RNA isolated from whole embryos, 7-13 weeks of pregnancy. The positions of 28S and 18S rRNAs are indicated.

H19 and IGF2 Genes During Human Embryogenesis and Placental Development This laboratory has studied the expression of the H19 and IGF2 genes during human placental development and in human embryos. The abundance of H19 and IGF2 gene transcripts in RNA extracted from placenta at different stages of pregnancy was more or less constant, notwithstanding great changes in the level of the transcripts coding for placental specific proteins, such as CG~ and hPL mRNAs during the course of pregnancy (results not shown). The results describing the expression of the IGF2 gene are in agreement with those described by Ohlsson et al., 1989b. Since murine H19 and IGF2 genes are expressed in a broad array of tissues in the developing murine embryo and the IGF2 gene is expressed in human embryos (Rechler and Nissley, 1990), the expression of the two genes in human embryos in greater detail was investigated in this laboratory. RNA was isolated from whole human embryos (between 7 and 13 weeks old) and the abundance of the H19 transcripts in these RNAs was determined (Figure 4). There was a definite increase in the level of the transcripts between the eighth and tenth week. In order to investigate the abundance of the H19 and IGF2 transcripts in the different organs of the human fetus, RNA was isolated from the organs of fetuses aborted during the second trimester (Figure 5). The fetal adrenal gland showed the highest level of H19 transcripts followed by muscle, liver, and placenta (Figure 5A). Expression is lower in lung and kidney and much lower in spleen and heart. In thymus the expression was still lower and no expression was detected in brain cortex. The fact that H19 is not expressed in fetal brain tissue is well attested in the mouse (Pachnis et al., 1988). Hybridization of the H19 probe with RNA isolated from fetal adrenal, muscle, and liver showed the existence of a second and smaller transcript. As the H19 gene is a single copy gene (Brannan et al., 1990) the above mentioned finding may provide evidence for the alternative splicing of the primary H19 transcript in these tissues (Bartolomei and Tighiman, 1992).

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Figure 5. Expression of H19 (A) and IGF2 (B) in various fetal organs. Autoradiogram of Northern blots containing isolated RNA from fetal organs derived from second trimester abortions. The positions of 28S and 18S rRNAs are indicated.

The filters used in these hybridization experiments were stained with methylene blue as in all previous hybridization experiments. The 18S and 28S ribosomal RNAs produced well defined and sharp bands indicating the integrity of the RNA. In the human fetus the highest expression of IGF2 was detected in the adrenal gland followed by muscle and placenta (Figure 5B). The expression was lower in liver and kidney and considerably lower in all other organs tested, brain cortex, thymus, lung, heart, and spleen. The observation that for both H19 and IGF2 the highest

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expression occurs in the fetal adrenal gland followed by muscle and placenta and the lowest expression occurs in brain and thymus deserves special attention. During the course of gestation, the fetal adrenal gland not only undergoes extensive anatomical and biochemical changes but also exhibits a very high rate of growth which is due to the rapid growth of the fetal cortical zone, Which comprises between 80-90% of the weight of the gland during the second half of gestation (Pepe and Albrecht, 1990). In situ hybridization experiments revealed that H19 expression occurred throughout the cortex but was significantly higher in the definite cortical zone than in the fetal zone. Products of the fetal adrenal gland appear to play important roles in the growth regulation of various fetal organs. In all fetuses examined, the levels of the H19 and IGF2 transcripts in the adrenal were at least three times higher than in the corresponding placentae. Relatively high expression of the two genes was also detected in muscle and liver. The fetal adrenal, the liver, and the placenta together constitute the feto-placental unit which is responsible for the high level of estrogen synthesis during pregnancy. H19 and IGF2 genes are involved in embryogenesis in mice and are reciprocally imprinted in mice and probably also in humans. The two genes are closely physically linked both in mice and humans (Zemel et al., 1992). Both genes are highly expressed during placental and embryonal development in mice and humans and the expression of both genes is postnatally d o w n regulated. These observations have lead to the proposal that the two genes, in-spite of the reciprocally of their imprinting, have one or more regulatory element(s) in common, responsible for their expression in embryonic tissues and the down regulation of their expression in adult tissues (Bartolomei and Tighlman, 1992). H19 and IGF2 in Differentiating Cytotrophoblast Cells In Vitro

As shown in Figure 1, the level of H19 mRNA in the cells of a cytotrophoblast culture continues to rise up until 120 hours like the mRNA levels of placental specific proteins which are markers of placental differentiation, CG[~, hPL (Gileadi et al., 1991). To ascertain whether the rise in the expression of H19 is linked to the f o r m a t i o n of s y n c y t i o t r o p h o b l a s t s , a n o t h e r h a l l m a r k of c y t o t r o p h o b l a s t differentiation, a method to isolate mononuclear cells from a cell culture containing both m o n o and m u l t i n u c l e a r cells, based upon the high sensitivity of syncytiotrophoblasts as compared to cytotrophoblast towards trypsin treatment was applied. This was done by trypsinizing a cell culture after 72 hours of incubation. The surviving cells were reisolated and consisted nearly exclusively of mononuclear cytotrophoblast cells (Rachmilewitz et al., 1993). From the results shown in Figure 6, it can be seen that there was no difference between the abundances of the H19 RNAs in mononuclear cells and that in cells of a culture containing predominantly multinuclear cells. As the increased expression of H19 was not dependent on the formation of syncytial elements, the linkage between H19 expression and cytotrophoblast differentiation was further investigated.

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Figure 6. Expression of H19 in mono and multinuclear cytotrophoblast cells. Cells were incubated for either 24 or 120 hours. After trypsinization, 20% of the original cells were recovered, all were mononuclear cells. Autoradiogram of Northern blots with RNA isolated from cells after trypsinization (T) or from untreated cells (C).

Cells can be separated according to their size with high resolution by centrifugal elutriation (Sharpe, 1988). This method was applied to fractionate cytotrophoblasts, isolated from term placentae. Fractions of cells which differed not only in their size but also in their biochemical and morphological characteristics (Rachmilewitz et al., 1993; N e ' e m a n et al., 1994) were obtained. The numerical distribution of cells in each elutriation fraction and their average diameter are shown in Figure 7. 40 ~ I

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Figure 7. Percentage distribution of cytotrophoblast cells and their average diameter in the fractions separated by centrifugal elutriation. Eleven fractions were collected, and the cells in each fraction were counted and their average size was determined using a Coulter Counter.

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Figure 8. Relative abundance of H19 (A) and IGF2 (B) mRNA in elutriation fractions of cytotrophoblast cells. Autoradiogram of Northern blots containing RNA isolated from elutriated cytotrophobtasts. 1-11, Cell elutriation fractions; U, unfractionated cells.

The pattern of expression of the H19 and the 1GF2 genes among the elutriation fractions is shown in Figure 8A and B. The figures show unequivocally that the H19 and IGF2 transcripts are unevenly distributed among the cell populations obtained by centrifugal elutriation. Cells infraction one express the H19 gene at a much lower level than cells in all the other fractions. In general, H19 expression increases parallel with the cell size and the highest expression was found in Fraction 11. The expression of IGF2 is even more unevenly distributed among the different cell fractions. No or only low expression could be detected in fractions 1-6. The expression of IGF2 in fraction 7 is low, but increases gradually until fraction 11 in which the IGF2 expression was comparatively very high. The exact function of the two genes in cytotrophoblasts is not known, but the results show that there are great differences in the level of their expression among cytotrophoblast cells separated by centrifugal elutriation. Their highest expression occurs in a relatively small portion of the total cell population, which differS from the

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majority of the cytotrophoblast cells by their larger size, their biochemical differentiation, such as their higher hPL synthesis (Rachmilewitz et al., 1993), and in their structural differentiation, such as their highly developed endoplasmic reticulum (Ne'eman et al., 1994). These observations may indicate specific roles of their gene products in placental development. The exact role of these genes in placental development is at present under investigation. REFERENCES

Barlow, D.P., Stoger, R., Herrmann, B.G., Saito, K., and Schweifer, N. (1991) The mouse insulin like growth factor type 2 receptor is imprinted and closely linked to the tme locus. Nature 349, 84-87. Bartoiomei, M.S., Zemel, S., and Tilghman, S.M. (1991) Parental imprinting of the mouse H19 gene. Nature 351,153-155. Bartolomei, M.S. and Tighlman, S.M. (1992) Parental imprinting of mouse chromosome 7. Develop. Biol. 3, 107-117. Barton, S.C., Ferguson-Smith, A.C., Fundele, R., and Surani, M.A. (1991) Influence of paternally imprinted genes on development. Development 113, 679-688. Brannan, C.I., Dees, E.C., Ingram, R.S., and Tilghman S.M. (1990). The product of the H19 gene may function as an RNA. Mol. Cell. Biol. 10, 28-36. Brunkow, M.E. and Tilghman, S.M. (1991) Ectopic expression of the H19 gene in mice causes prenatal lethality. Genes Dev. 5, 1092-1101. Cattanach, B.M. (1988) Chromosome imprinting in the mouse. Mouse Newsletter 82, 93. Daimon, M. Johnson, T.R., Ilan, J. and Ilan, J. (1992) The third IGF2 promoter specifies transcription of three transcripts out of the five in human placenta. Mol. Rep. Dev. 33, 413-417. De Chiara, T.M., Efstratiadis, A., and Robertson, E.J. (1990) A growth deficiency phenotype in heterozygous mice carrying an insulin like growth factor II gene disrupted by targeting. Nature 345, 78-80. De Chiara, T.M., Robertson, E.J., and Efstratiadis, A. (1991) Parental imprinting of the mouse insulin like growth factor 2 gene. Cell 64, 849-859. de Pagter-Holthuizen, P., Jansen, M., van Schaik, F.M.A., van der Kammen, R., Oosterwijk, C., van den Brande, J.L., and Sussenbach, J.S. (1987) The human insulin like growth factor II gene contains two development-specific promoters. FEBS Letters 214, 259-264. Ferguson-Smith, A.C., Cattanach, B.M., Barton, S.C., Beechey, C.V., and Surani, M.A. (1991) Embryological and molecular investigations of parental imprinting on mouse chromosome 7. Nature 351,667-670.

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