The adult human endometrium rapidly cycles through stages of cell proliferation, differentiation and degeneration. Inappropriate endometrial cell differentiation ...
Molecular Human Reproduction vol.4 no.5 pp. 497–501, 1998
Homeobox genes DLX4 and HB24 are expressed in regions of epithelial–mesenchymal cell interaction in the adult human endometrium Leonie M.Quinn, Lynette M.Kilpatrick, Sue E.Latham and Bill Kalionis1 Department of Obstetrics and Gynaecology, Flinders University of South Australia, Flinders Medical Centre, Bedford Park, South Australia, Australia 5042 1To
whom correspondence should be addressed
The adult human endometrium rapidly cycles through stages of cell proliferation, differentiation and degeneration. Inappropriate endometrial cell differentiation is a contributing factor in diseases such as endometrial carcinoma and endometriosis. We have identified two homeobox genes that may play a role in the control of endometrial cell differentiation and development. In-situ mRNA hybridization experiments were used to show differential expression of DLX4 at different phases of the endometrial cycle. Higher levels of DLX4 expression were observed in proliferative phase endometrial epithelium compared with secretory phase endometrial epithelium. The HB24 homeobox gene was shown to be expressed in both the proliferative and secretory phase endometrial epithelium. We predict that DLX4 and HB24 will be required for the transcriptional control of genes important for endometrial cell differentiation. Furthermore, we propose that DLX4 and HB24 are part of a conserved combinatorial code of homeobox genes that are required for controlling epithelial–mesenchymal cell interactions in the endometrium. Key words: endometrial/glandular/homeodomain/luminar/uterus
Introduction The adult human endometrium is a dynamic organ, characterized by rapid cycling of the processes of cell proliferation, differentiation and degeneration. The regenerative events of the menstrual cycle prepare the endometrium for blastocyst implantation. Early in the proliferative phase, the surface epithelium that was lost during menstruation reforms and covers the endometrium. The next stage of the proliferative phase is characterized by repair and proliferation, resulting in a two to three fold increase in the thickness of the endometrium. The primitive stem cell population for regeneration of the postmenstrual endometrium has been localized to the residual epithelium of basal gland stumps of the lamina basalis (Satyaswaroop and Mortel, 1981). During the regenerative phase these stem cells proliferate and as they migrate into the lamina functionalis (Ferenczy, 1976; Ollo, 1991), they differentiate to form precursor cells (Satyaswaroop and Mortel, 1981). At the end of the regenerative phase, the endometrial epithelium is composed of a continuous layer of precursor cells. During the proliferative phase, the precursor cells proliferate to increase the surface area of the endometrial epithelium (Giudice, 1994). In preparation for blastocyst attachment and implantation, precursor cells differentiate to give rise to the secretory cells that comprise the glandular epithelium during the receptive phase of the menstrual cycle (Satyaswaroop and Mortel, 1981; Giudice, 1994). Implantation requires that trophoblast cells surrounding the blastocyst contact and penetrate the endometrial surface epithelium. Successful implantation results from carefully controlled interactions between the receptive © European Society for Human Reproduction and Embryology
endometrium and invasive trophoblast (for review, see Tabibzadeh and Babaknia, 1995). When fertilization does not occur, the entire compact layer and most of the spongy layer of the endometrium are shed. Remnants of the lamina basalis and the entire lamina functionalis remain and undergo regeneration during the beginning of the next proliferative phase (Ferenczy, 1976). The factors required for the genetic control of differentiation within the endometrial cell lineage are poorly understood. There is evidence for a genetic component for endometrial diseases, such as endometrial carcinoma (Lynch et al., 1994; Sumoi et al., 1995) and endometriosis (Moen, 1994; Kennedy et al., 1995). Homeobox genes are good candidates for controlling endometrial differentiation since members of this multigene family encode transcription factors that control embryonic development (for review, see Stein et al., 1996). Preliminary studies suggest that homeobox genes will be required for controlling the earliest stages of reproduction, since their expression has been detected in preimplantation embryos (Verlinsky et al., 1995). Targeted disruption of a number of murine homeobox genes has provided direct evidence that homeobox genes control genito–urinary tract development. Furthermore, similar studies have shown that homeobox genes control specific reproductive events, including embryonic implantation (for review, see Favier and Dolle´, 1997). Despite the importance of endometrial development and function for blastocyst implantation and placentation, very few studies have been carried out on the homeobox genes that are likely to regulate these processes in the adult human endometrium. There is direct evidence that homeobox genes are required for the genetic control of endometrial cell differentiation and 497
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development in the mouse. In females homozygous for the loss of Hoxa10 and Hoxa11 genes, blastocysts die by 3.5 days post-coitum, but when transplanted into surrogate wildtype females blastocysts develop to term normally (Hsieh-Li et al., 1995; Satokata et al., 1995). Hoxa10 and Hoxa11 are therefore required to generate a uterine environment conducive to embryonic implantation and development in the mouse (HsiehLi et al., 1995; Satokata et al., 1995). Other murine homeobox genes reported to be expressed in the uterus include Hox2.2 (Shen et al., 1991), MHox (Cserjesi et al., 1992) and Msx-1 (Pavlova et al., 1994), but their role in endometrial development is unknown. Studies on the role, if any, of the human homologues of the murine homeobox genes described above have not been carried out in the human endometrium. Northern analysis suggests that the human homeobox gene, HOX4E, is expressed in the uterus (Redline et al., 1992). However, the mRNA expression pattern for HOX4E in the uterus has yet to be reported. The role of homeobox genes in the development of the adult human endometrium is poorly understood. We have studied expression of two homeobox genes called DLX4 and HB24 in the human endometrium based on the prediction that a common molecular mechanism will be required for controlling the development of tissues dependent on epithelial–mesenchymal cell interactions for morphogenesis. There is evidence for a conserved combinatorial code of homeobox genes, that includes DLX4 (also called DLX7) and HB24, for the control of epithelial–mesenchymal cell interactions in the human (Quinn et al., 1997b, 1998). Expression of the murine homologues of these genes has not been reported in the murine endometrium, and although targeted gene disruption experiments have been carried out for Hlx (the murine homologue of HB24) the effects, if any, on endometrial or placental development have not been reported (Hentsch et al., 1996). However, homologues of these homeobox genes are likely to regulate epithelial–mesenchymal interactions in the developing mouse embryo (Weiss et al., 1994, 1995; Hentsch et al., 1996). Since endometrial development requires controlled interaction between the mesenchymal cells that form the stroma and the epithelial cells that line the glands and lumen, we expected that this combinatorial code of homeobox genes would also be expressed in the endometrium. Based on previous evidence that suggests HB24 and DLX4 are required for differentiation of the haematopoietic cell lineage and the trophoblast lineage, we predict that DLX4 and HB24 will be important for controlling endometrial cell differentiation. For example, in the haematopoietic cell lineage, DLX4 and HB24 have been shown to be required for proliferation, differentiation and lineage commitment (Deguchi et al., 1992; Shimamoto et al., 1997). Expression of HB24 is detected in haematopoietic progenitor cells, but when differentiation to granulocytes or monocytes occurs, expression is not detected. Down-regulation of HB24 expression appears to be required for normal differentiation since transient expression of HB24 in haematopoietic progenitor cell lines inhibits differentiation (Deguchi et al., 1992). Furthermore, elevated levels of HB24 expression have been detected in haematopoietic cells from 498
patients with acute myeloid leukaemia, compared with normal patients (Deguchi et al., 1993). Inhibition of DLX4 expression in haematopoietic cell lines and leukaemic cell lines results in decreased levels of c-myc expression and induces apoptosis (Shimamoto et al., 1997). DLX4 and HB24 were isolated from a human placental cDNA library (Quinn et al., 1997a) and are predominantly expressed in the villous cytotrophoblast stem cell population of the developing placenta (Quinn et al., 1997b, 1998). These expression patterns suggest a role for DLX4 and HB24 in the control of cytotrophoblast differentiation. Support for this notion has been provided by the differential expression of DLX4 and HB24 in cell lines derived from choriocarcinoma, an invasive cytotrophoblast cancer (Quinn et al., 1997b, 1998). Here we have shown by mRNA in-situ hybridization that DLX4 and HB24 are expressed in the adult human endometrium. These are the first reported human endometrial homeobox gene expression patterns. Expression of DLX4 and HB24 has been detected in the immature, proliferative phase glandular epithelial cells. This expression pattern would be consistent with a role for these homeobox genes in endometrial cell differentiation.
Materials and methods Probe preparation for in-situ hybridization DLX4 riboprobes were prepared from a pBluescriptSK1 clone (DLX470) containing a 470bp DLX4 cDNA fragment which did not contain a homeobox sequence, as described by Quinn et al. (1997b). Templates for transcription of the anti-sense and sense riboprobes were prepared after first linearizing DLX470 with EcoRI and KpnI respectively. The anti-sense transcription product generated from the EcoRI linearized template using the T7 polymerase was 484 bp and the sense transcription product generated from the KpnI linearized template using the T3 polymerase was 543 bp. HB24 riboprobes were prepared from a pBluescriptSK1 clone containing a 1.2 kb HB24 cDNA fragment. Templates for transcription of the anti-sense and sense riboprobes were prepared after first linearizing plasmid DNA with AvaII and DdeI respectively. The anti-sense transcription product generated from the AvaII linearized template using the T7 polymerase was 405 bp and the sense transcription product generated from the DdeI linearized template using the T3 polymerase was 424 bp, as described by Quinn et al. (1998). Transcription reactions were carried out using the T3 and T7 RNA polymerases and digoxigenin riboprobe kit according to the instructions of the manufacturer (Boehringer Mannheim, Germany). Tissue collection and ethics approval Endometrial tissue was collected from women undergoing laparoscopy/hysteroscopy operations for either infertility investigation or tubal ligation. The tissue was collected by cervical dilation and curettage of the endometrium. Informed consent was obtained in accordance with the Committee on Clinical Investigation, Flinders Medical Centre, South Australia. This project was approved by the Flinders Medical Centre Committee on Clinical Investigation/Drug and Therapeutics Advisory Committee; approval number 1994030. In-situ hybridization to human endometrial sections Fresh tissue was fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS), embedded in paraffin wax and 4 µm sections were cut.
Homeobox genes in human endometrium Sections were treated with protease K (125 µg/ml) at 37°C for 10 min and rinsed in PBS before post-fixing in 4% paraformaldehyde/ PBS for 10 min. Slides were rinsed twice for 5 min in PBS, dehydrated using ethanol and dried in a dessicator under vacuum. Hybridization was carried out overnight at 50°C using the digoxigenin-labelled riboprobes (150 ng/µl) in the following hybridization buffer: 50% deionized formamide, 0.5% TritonX-100, 2.5% (w/v) dextran sulphate, 700 µg/ml Escherichia coli tRNA, 0.3 M NaCl, 10 mM Na2HPO4, pH 6.8, 10 mM Tris–HCl, pH 7.5, 5 mM EDTA pH 8.0, 13 Denhart’s reagent. The slides were washed three times for 45 min at 50°C in hybridization buffer without E.coli tRNA or dextran sulphate. Sections were treated with RNaseA (125 µg/ml) for 1 h at 37°C and then with 10% BSA in 50 mM Tris–HCl/150 mM NaCl, pH 7.5 for 1 h. Hybridized probe was detected using a 1/750 dilution of anti-digoxigenin alkaline phosphatase fragments (Boehringer Mannheim) in 50 mM Tris–HCl/150 mM NaCl, pH 7.5 by incubating at room temperature for 1 h. Colour detection was carried out overnight using nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, as described by the manufacturer (Boehringer Mannheim) and 0.04% levamisole was added to inhibit endogenous alkaline phosphatase activity. The sections were counterstained with the nuclear dye Methyl Green.
Results In-situ hybridization to endometrial sections with the DLX4 riboprobe On the basis of histological analysis, patient samples were assigned to either the proliferative or secretory phase of the endometrial cycle. The mRNA expression pattern for DLX4 in proliferative phase endometrium is shown in Figure 1A and C and can be compared with the background generated using a sense riboprobe in Figure 1B and D. DLX4 expression was detected in the glandular epithelium (ge) and luminal epithelium (le). Expression was also detected in mesenchymal cells that comprise the endometrial stroma(s). The mRNA expression pattern for DLX4 in secretory phase endometrium is shown in Figure 1E. The signal in the glandular epithelium and luminal epithelium was not above the background signal shown in Figure 1F. Expression of DLX4 in the endometrial stroma during the secretory phase was not above background (Figure 1E). Reproducible results were obtained by in-situ hybridization to a second patient sample (data not shown). In-situ hybridization to endometrial sections with the HB24 riboprobe The expression pattern for HB24 in proliferative phase endometrium is shown in Figure 2A and can be compared with the background in Figure 2B. HB24 expression was detected in the glandular epithelium and the luminal epithelium. HB24 expression was also detected in mesenchymal cells that comprise the endometrial stroma. The HB24 expression pattern in secretory phase endometrium is shown in Figure 2C and can be compared with the background in Figure 2D. Expression was detected in the glandular epithelium and in the stroma.
Discussion Expression patterns for HB24 and DLX4 We report the first in-situ expression patterns for any homeobox genes in the human endometrium. The DLX4 homeobox gene
Figure 1. In-situ mRNA hybridization to endometrial sections with digoxigenin-labelled DLX4 riboprobes. (A, C) In-situ hybridization to proliferative phase endometrial sections with the digoxigenin labelled anti-sense DLX4 riboprobe. ge 5 glandular epithelium; s 5 stroma; le 5 luminal epithelium. (B, D) Negative control; insitu hybridization to proliferative phase sections using the sense DLX4 probe shows the level of non-specific background signal. (E) In-situ hybridization with the anti-sense DLX4 probe to secretory phase endometrial sections through the glandular epithelium, luminal epithelium and stromal cells. (F) Negative control, in-situ hybridization to secretory phase sections with sense DLX4.
is differentially expressed in the adult endometrial epithelium. During the proliferative phase DLX4 expression was detected in the undifferentiated precursor cells, whilst expression was found to be downregulated in differentiated secretory phase epithelial cells. In contrast, HB24 expression was detected in precursor endometrial epithelial cells and secretory phase epithelial cells. Unlike DLX4, HB24 mRNA expression may be required for both phases of endometrial epithelial differentiation. DLX4 or HB24 expression in the glandular epithelial stem cells that remain at the base of the glands post-menstruation has not yet been investigated.
Evidence for a combinatorial code of homeobox genes in the placenta and endometrium In the rodent, members of the Dlx family are expressed in those structures including the branchial arch, bone, ear, whisker and tooth, that require epithelial–mesenchymal cell interactions for development (Porteous et al., 1991; Price et al., 1991; 499
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Figure 2. In-situ mRNA hybridization to endometrial sections with digoxigenin-labelled HB24 riboprobes. (A) In-situ hybridization with the digoxigenin-labelled anti-sense HB24 riboprobe to proliferative phase endometrial sections complete with the glandular epithelium (ge), luminal epithelium (le) and stroma (s). (B) Negative control, in-situ hybridization to proliferative phase sections using the sense HB24 riboprobe. (C) In-situ hybridization with the anti-sense HB24 probe to secretory phase endometrial sections through the glandular epithelium and the stroma. (D) Negative control, in-situ hybridization to secretory phase sections with the sense HB24 probe.
Simeone et al., 1994; Robinson and Mahon, 1994; Zhao et al., 1994). A combinatorial code of homeobox genes, that includes members of the Dlx, Msx and Mox families, has been predicted to regulate epithelial–mesenchymal interactions important for embryogenesis (Weiss et al., 1994). The murine homologue of HB24 (Hlx) is expressed in mesenchymal cell types during organogenesis (Lints et al., 1996) and targeted gene disruption experiments suggest that Hlx is important for controlling epithelial–mesenchymal cell interactions (Hentsch et al., 1996). Hlx may be part of the combinatorial code of homeobox genes required for regulating epithelial–mesenchymal interactions during embryogenesis. Studies carried out in the placenta with HB24 and DLX4 suggest a role for these homeobox genes in controlling epithelial–mesenchymal cell interactions (Quinn et al., 1997b, 1998). DLX4 (Quinn et al., 1998) and HB24 (Quinn et al., 1997b) are expressed predominantly in epithelial trophoblast cell types throughout placental development. As yet there is no direct evidence for epithelial–mesenchymal cell interactions 500
between epithelial trophoblast cells and the stromal cells that comprise the mesenchyme of the placenta. However, there is evidence that epithelial cell differentiation requires interaction with mesenchymal cells in the endometrium. The stromal cells, that provide structural support for the glands and blood vessels, are also important regulators of endometrial growth and differentiation (Cunha et al., 1983). Furthermore, many hormone responses that occur in epithelial cells may not be elicited directly in these target cells, but instead may be mediated by factors produced by stromal cells in response to hormonal stimulation. Hormonal effects may not require the presence of hormone receptors within the responding epithelial cell provided they are associated with hormonally-responsive mesenchymal cells. For example, macrophage-derived growth factor and platelet-derived growth factor effects on endometrial tissue are mediated by the stromal component rather than by direct interaction with the endometrial epithelium (Whitworth et al., 1994). More specifically, expression of the Msx1 homeobox gene in the murine uterine epithelium is dependent on interaction with the underlying mesenchyme (Pavlova et al., 1994). Therefore homeobox gene expression is induced as a result of epithelial– mesenchymal interactions in the murine endometrium. The combinatorial expression of homeobox genes may also be important for controlling epithelial–mesenchymal interactions in human reproductive tissues. HB24, DLX4, MSX2 and MOX2 are co-expressed in regions where epithelial and mesenchymal cell layers contact in the placenta (Quinn et al., 1997b, 1998). Expression of HB24 and DLX4 (this work) and MSX2 (C.Briton-Jones and B.Kalionis, unpublished data) has also been detected in endometrial epithelial and mesenchymal cell types. Co-expression of these homeobox genes in the endometrium suggests that this combinatorial code has been conserved in reproductive tissues.
Acknowledgements We thank Dr Enzo Lombardi, the surgeon responsible for obtaining the endometrial tissue, and Chris Briton-Jones, for collection and preparation of tissue for sectioning. This work was carried out with the support of NH and MRC grant number 940191. L.Quinn was supported by an Australian Postgraduate Award.
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