Mol. Hum. Reprod. Advance Access originally published online on October 24, 2006
Molecular Human Reproduction 2006 12(12):763-769; doi:10.1093/molehr/gal087
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Homeobox gene DLX4 expression is increased in idiopathic human fetal growth restriction
1Pregnancy Research Centre, Department of Perinatal Medicine, The Royal Women’s Hospital, 2Department of Obstetrics and Gynaecology, The Royal Women’s Hospital and University of Melbourne, Carlton, 3Clinical Epidemiology and Biostatistics Unit, Murdoch Children’s Research Institute and University of Melbourne Department of Paediatrics, The Royal Children’s Hospital, Parkville, Victoria, Australia
4 To whom the correspondence should be addressed at: Pregnancy Research Centre, Department of Perinatal Medicine, The Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. E-mail: padma{at}unimelb.edu.au
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Abstract |
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Idiopathic fetal growth restriction (FGR) is often associated with placental insufficiency. Previously, we isolated and characterized homeobox gene DLX4 from the placenta and provided evidence that DLX4 may regulate placental development. Here, we have investigated whether DLX4 expression levels were altered in idiopathic FGR. FGR-affected placentae were collected based on strict clinical criteria. DLX4 mRNA expression was analysed in placentae obtained from pregnancies complicated by idiopathic FGR and gestation-matched control pregnancies (n = 25 each). Initial RT–PCR results showed a qualitative increase in DLX4 mRNA in both FGR-affected placentae and gestation-matched controls. Real-time PCR showed a 3-fold increase in DLX4 mRNA levels in FGR-affected placentae compared with gestation-matched controls (P < 0.005). Western immunoblotting using a rabbit DLX4 polyclonal antibody revealed significantly increased levels of DLX4 protein in term FGR-affected placentae compared with term controls [5500.1 ± 21.8 (n = 10) versus 3533.2 ± 22.4 (n = 10); P < 0.001]. Qualitative immunohistochemical analyses of term placentae showed moderately increased immunoreactivity for DLX4 antigen in the FGR-affected placentae in syncytiotrophoblasts, residual cytotrophoblast cells and endothelial cells of the fetal capillaries compared with gestation-matched control term placentae. We conclude that the increased expression of homeobox gene DLX4 may be a contributing factor to the developmental abnormalities seen in the FGR-affected placentae.
Key words: DLX4/fetal growth restriction/gene expression/homeobox/placenta
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Introduction |
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Our focus is on the clinically significant pregnancy disorder of idiopathic fetal growth restriction (FGR, also known as intrauterine growth restriction, IUGR). A common definition of FGR is a birthweight at or below the 10th percentile for gestational age and gender, failure of the fetus to grow to its genetically determined potential size and the likely presence of an underlying pathologic process that inhibits the expression of the normal intrinsic growth potential. Not only are there various serious perinatal complications frequently associated with FGR (Illanes and Soothill, 2004
), but also increasing numbers of epidemiological and animal studies provide evidence that the long-term consequences of FGR reach into adulthood (Godfrey and Barker, 2000). Such consequences include an increased risk of chronic somatic disorders such as cardiovascular disease and diabetes (Godfrey and Barker, 2000) as well as asthma (Steffensen et al., 2000) and intellectual impairments such as schizophrenia (Rosso et al., 2000), depression (Gale and Martyn, 2004) and decreased intelligence quotient (Frisk et al., 2002). Therefore, it is becoming increasingly important to understand the molecular mechanism of human FGR.
Only a third of FGR cases can be accounted for by obvious maternal, fetal and placental causes (Brodsky and Christou, 2004), the remainder being idiopathic. Idiopathic FGR pregnancies are distinguished by abnormal umbilical artery diastolic velocities, asymmetric growth of the fetus and reduced liquor volume (Chang et al., 1993). Idiopathic FGR is frequently associated with placental insufficiency (Gagnon, 2003). Uteroplacental ischemia due to failure of placental extravillous cytotrophoblast cells to effectively carry out the critical processes of invasion, transformation and remodelling of the spiral arteries in the maternal decidua (Chaddha et al., 2004) is another significant defect. The effect of abnormal placental function in FGR is reduced transfer of nutrients and growth factors to the fetus, thereby restricting its growth (Mayhew et al., 2004). The morphological changes observed in the idiopathic FGR-affected human placenta are consistent with developmental defects (Chaddha et al., 2004), but the genes that control these processes and their molecular mechanism of action remain largely unknown. A possible causative role for as yet unidentified genetic and familial factors in human FGR has been proposed in some epidemiological studies (Devriendt, 2000; Ghezzi et al., 2003).
Mouse knockout studies of several transcription factors have contributed significantly to our understanding of potentially important regulatory genes in the early stages of human FGR. There is mounting evidence to support a role for a large subfamily of homeobox gene transcription factors in the regulation of murine placental development. Homeobox genes regulate multiple genetic pathways involved in the formation of important placental substructures (Knofler et al., 2000; Hemberger and Cross, 2001; Rossant and Cross, 2001; Sapin et al., 2001; Cross, 2003).
The Distal-less (Dlx) subfamily of homeobox genes includes six identified members referred to as Dlx 1–6 in the mouse and DLX 1–6 in humans (Simeone et al., 1994). Substantial experimental data support the notion that the absence of expression of members of the Dlx/DLX family results in embryonic lethality in mouse and altered expression of these genes may play a role in human tumorigenesis (Merlo et al., 2000; Shimamoto et al., 2000; Ferrari et al., 2003b; Morasso and Radoja, 2005). Expression patterns of members of the Dlx genes have been well characterized in the developing embryo (Quint et al., 2000) and specifically in organ substructures that are dependent on epithelial mesenchymal cell interactions for their formation (Zhao et al., 1994; Morasso et al., 1995; Morasso and Radoja, 2005).
A Dlx homeobox gene, Dlx3, has been shown to play an important role in the development of the murine placenta, an organ dependent on epithelial mesenchymal cell interactions. Dlx3 null mutant mice die at mid-gestation due to placental defects (Morasso et al., 1999). These data reveal that Dlx3 has a unique role in the placenta that cannot be substituted by other related family members including Dlx4 which is closely linked to Dlx3 (within 10 kb) on the chromosome (Nakamura et al., 1996). Although the Dlx3 mutant shows substantial structural defects, subsequent studies of Dlx3 and its human homologue DLX3 show the gene also regulates very specific functions of differentiated trophoblast cells (Roberson et al., 2001; Peng and Payne, 2002).
Our interest is in the role of DLX4, a homeobox gene closely related to DLX3, in the human placenta. In previous studies, the isolation, chromosome mapping and characterization of DLX4 in the human placenta were reported (Quinn et al., 1997a). As in the mouse, the human DLX3 and DLX4 genes are physically very close (Nakamura et al., 1996; Quinn et al., 1997a), their homeodomain show 80% identity (Nakamura et al., 1996; Quinn et al., 1997a), but outside the homeodomain sequence, the amino acid sequences are not significantly related. Furthermore, as in the mouse (Beanan and Sargent, 2000), DLX3 and DLX4 expression patterns overlap but are not identical (Quinn et al., 1998b; Roberson et al., 2001). Therefore, the functions of DLX3 and DLX4 are expected to differ in the human placenta as they do in the mouse.
We postulated a regulatory role for DLX4 in the development of human placenta (Quinn et al., 1998b) based on the observation that DLX4 mRNA expression is in regions where epithelial and mesenchymal cell layers contact. Most important to this current work is that the cell types in which DLX4 is expressed are those that are abnormal in the FGR-affected placenta i.e. syncytiotrophoblast, villous and extravillous cytotrophoblast and placental endothelial cells.
Two isoforms of DLX4 have been identified; DLX7 and ß-protein 1 (BP1) (Fu et al., 2001; Chase et al., 2002). DLX4, DLX7 and BP1 contain identical homeodomain sequences and strong homology between mRNA and protein sequences in the 3' regions, but all the three isoforms exhibit unique mRNA and protein sequences in the 5' regions (Chase et al., 2002). The isoforms are not functionally equivalent as shown by the observation that DLX7 and BP1 bind identical DNA sequences found in ß-globin silencer elements but differ in their ability to repress ß-globin transcription (Berg et al., 1989; Fu et al., 2001; Chase et al., 2002). Therefore, for quantitative measurements of DLX4, it is essential to use assays that measure only DLX4 and not its other isoforms.
In this study, we have determined DLX4 expression in placentae obtained from a clinically well-defined group of placentae from idiopathic FGR-affected pregnancies compared with gestation-matched controls.
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Materials and methods |
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Patient details and tissue sampling
Informed patient consent and approval from the Research and Ethics Committees of The Royal Women’s Hospital, Melbourne, were obtained. Placentae from pregnancies complicated by idiopathic FGR (n = 25) and gestation-matched control pregnancies (n = 25) were used. Growth-restricted fetuses were identified prospectively using ultrasound. The clinical features of the FGR-affected pregnancies as well as the gestation-matched controls employed in this study are summarized in Table I. There was no significant difference in the gestational age, maternal age, parity or mode of delivery between the two groups. As expected, the mean birthweight and mean placental weight were significantly lower in FGR-affected patients compared with the controls (P < 0.025, n = 25, t-test). Table II summarizes the inclusion criteria for this study, and these were a birthweight less than the 10th centile for gestation age using Australian growth charts (Guaran et al., 1994
) and any two of the following criteria diagnosed on antenatal ultrasound; abnormal umbilical artery Doppler flow velocimetry, oligohydramnios as determined by amniotic fluid index (AFI) <7 on antenatal ultrasound performed before delivery or asymmetric growth of the fetus as quantified from the head circumference (HC) to abdominal circumference (AC) ratio (>1.2). The exclusion criteria for the study for both control and FGR-affected pregnancies were multiple pregnancies, chemical dependency, maternal smoking, pre-eclampsia, prolonged rupture of the membranes, placental abruption, suspicion of intrauterine viral infection or fetal congenital anomalies. In this study, we also excluded cases with pre-eclampsia and FGR and included only normotensive patients with idiopathic FGR. Last menstrual period (LMP) dates were used to calculate the gestation times for both FGR and control patients, and these were confirmed by early pregnancy ultrasound. Control patients were selected to match FGR cases according to gestation. Control patients were included if they required elective delivery by the induction of labour/Cesarean section or presented with spontaneous labour. Pre-term control patients presented in spontaneous idiopathic pre-term labour or underwent elective delivery for conditions not associated with placental dysfunction (e.g. one patient included in this study delivered pre-term electively by Cesarean section for maternal breast cancer). Control patients had no clinical evidence of pre-eclampsia, FGR, placental abruption, ascending infection or prolonged rupture of the membranes. Control patients gave birth to normally formed babies with birthweights appropriate for gestation, and control placentae from these patients were grossly normal with no obvious infarcts. Placental tissue samples were excised from randomly selected areas of central placental cotyledons. Any attached decidua was carefully removed by dissection. All samples were processed within 10 min of delivery of the placenta. Tissues were divided into small pieces and thoroughly washed in phosphate-buffered 0.9% saline (PBS) to minimize blood contamination and then snap frozen and stored at –80°C for RNA analyses. Initial experiments (n = 3 for FGR-affected placentae and control group) to determine the DLX4 mRNA expression were carried out in at least three replicate samples excised from different locations of the placenta but avoiding the peripheral cotyledons. No significant change in DLX4 mRNA expression was observed between replicate samples taken from the different location of a placenta (data not shown). Subsequently, DLX4 mRNA expression was analysed just in one sample from each placenta, and the samples were not pooled.
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RT–PCR
The modified acid guanidinium isothiocyanate-phenol-chloroform extraction and lithium chloride precipitation method (Chomczynski and Sacchi, 1987; Puissant and Houdebine, 1990) or Qiagen RNeasy midi-kits (Qiagen, Australia) were used to isolate total RNA. The yield, purity, integrity of the placental RNA and lack of any high molecular weight genomic DNA contamination were determined by both spectrophotometric analysis and gel elctrophoresis. Total RNA was used for first strand synthesis as described previously (Murthi et al., 2006a,b). Approximately 50 ng of cDNA was amplified in an ABI PRISM 9700 thermocycler (Applied Biosystems) using PCR-Platinum Supermix (Invitrogen, Australia). Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as the housekeeping gene (Murthi et al., 2006a,b). A term control was used to normalize the GAPDH levels for both control and FGR-affected placentae, and the cDNA volumes were adjusted using scanning densitometry. Oligonucleotide primers for the DLX4 gene were forward primer, 5'-CCGCCGTGGCTGAACTCCGACC-3' and reverse primer, 5'-CCCCCACGTTCACCGCGCC AGGTG-3' (Hollington et al., 2004). The primers detect DLX4 and the closely related isoform DLX7 but not isoform BP1 (Chase et al., 2002). Adjusted volumes of the control and FGR-affected placental cDNA were used for DLX4 PCR amplification, and the conditions were 94°C for 10 min followed by 40 cycles of 94°C for 60 s, 72°C for 45 s and primer extension at 72°C for 60 s. Amplified products of GAPDH (452 bp) and DLX4 (160 bp) were fractionated on a 2% agarose gel. The PCR reaction for each sample was performed on both cDNA and corresponding RNA that was not reverse transcribed, to control for contaminating genomic DNA.
Real-time PCR
The quantification of DLX4 mRNA expression in placental samples was performed in an ABI Prism 7700 (Perkin-Elmer-Applied Biosystems) using pre-validated Assays on DemandTM that consisted of a mix of unlabelled DLX4 PCR primers and TaqMan® FAMTM labelled MGB probe (DLX4 Assays on Demand catalogue number Hs00231080_m1, Applied Biosystems). The DLX4 real-time assay only detects DLX4 and not the isoforms DLX7 or BP1 (Chase et al., 2002). Gene expression quantification was performed as the second step in a two-step RT–PCR protocol as described previously (Murthi et al., 2006a,b). The relative quantification of DLX4 expression relative to GAPDH (Amu et al., 2006; Murthi et al., 2006a,b) and 18S rRNA (Hs 99999901_s1, Applied Biosystems; Meyer et al., 2005) was calculated according to the 2–
CT method (Livak and Schmittgen, 2001) with a term control used as a calibrator. A standard curve ranging from 100 to 0.01 ng, generated using Human Universal Total RNA standard (BD Biosciences/Clontech), was used for primer efficiency as described by Meller et al. (2005). The amplification efficiencies of DLX4, GAPDH and 18S rRNA were 95, 90 and 92%, respectively (ABI Prism 7700 Sequence Detection System, User Bulletin number 2, 2001).
Western immunoblotting
Randomly selected term FGR-affected placentae (n = 10 of 15) and gestation-matched term controls (n = 10 of 12) were used to determine the level of DLX4 protein. Total protein was extracted, and immunoblotting was performed as described (Murthi et al., 2006b). Briefly, 25 µg of total protein per lane was loaded and fractionated using 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Mini Protean II System, Bio-Rad, Australia). Proteins were transferred to nitrocellulose membranes and blocked with 5% non-fat milk in Tris-buffered saline (TBS, pH 7.4). A rabbit polyclonal antibody to DLX4 which was raised to the N-terminal region of the DLX4 protein and verified by Hollington et al. (2004) was used as primary antibody. To visualize the signal, we used peroxidase-conjugated goat anti-rabbit secondary antibody and an enhanced chemiluminescence system (Amersham, Australia). Coomassie blue staining of total protein in each well was used to ensure constant protein load. Semi-quantitative determination of the levels of expression of DLX4 protein relative to the protein load was carried out by scanning densitometry (Image Quant, Australia).
Immunohistochemistry
Randomly selected term FGR-affected placentae (n = 6 of 15) and gestation-matched term control placentae (n = 6 of 12) were fixed in 4% paraformaldehyde/PBS pH 7.4, paraffin embedded and sections cut to 5 µm as described elsewhere (Murthi et al., 2006b). Immunohistochemistry was performed as described previously (Murthi et al., 2006b) using a Histostain-Plus Broad spectrum kit (Zymed, Australia). Briefly, the sections were deparaffinized in Xylene for 10 min after which the paraffin sections were rehydrated in graded ethanol for 6 min (100, 90, 70 and 50%). After digestion of the tissue sections in Proteinase K enzyme (100 µg/ml; Roche, Australia), the slides were rinsed in PBS and blocked with the blocking agent provided in the Histostain-Plus Broad Spectrum kit (Zymed). Rabbit DLX4 polyclonal antibody was applied to the sections and incubated overnight at 4°C. The incubations of the biotinylated secondary antibody and streptavidin-conjugated enzyme from the Histostain-Plus Broad Spectrum kit were carried out as instructed by the manufacturer (Zymed). Colour detection was performed using 3-amino-9-ethylcarbazole (AEC; Zymed). Sections were mounted with 80% glycerol.
Data analysis
All parameters for the FGR-affected pregnancies and controls were described as mean ± SEM. Either the Chi-squared test or Student’s t-test was used where appropriate to analyse the significance of differences between the clinical characteristics of the FGR-affected pregnancies and the control patients. Multiple linear regression was used to model the relationship between mRNA expression of DLX4 and gestation for FGR-affected and control placentae. The likelihood ratio test was used to assess whether the relationship between mRNA expression of DLX4 and gestation differed significantly between FGR-affected and control placentae. The difference in protein expression of DLX4 between FGR-affected and control pregnancies was assessed by t-test and a P value of <0.05 was considered significant.
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Results |
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RT–PCR for DLX4 mRNA expression was performed on FGR-affected placentae (n = 25) and in gestation-matched control (n = 25). Figure 1 shows a representative RT–PCR for DLX4 mRNA expression in term FGR-affected placentae (n = 5, 37–39 weeks) and gestation-matched term controls (n = 5, 37–41 weeks). A single band at the predicted size of 160 bp was observed in both FGR-affected placentae (tracks 1–5 in upper panel) and gestation-matched term controls (tracks 6–10 in upper panel). Constant levels of the housekeeping gene GAPDH was observed in both term FGR-affected placentae and term controls (tracks 1–10 in lower panel). A qualitative increase was observed in DLX4 levels in the FGR-affected placental samples compared with the gestation-matched controls.
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The effect of gestation on DLX4 mRNA expression was analysed in control placentae with gestation ranging from 27 to 41 weeks. Figure 2A shows a representative placental DLX4 mRNA expression in 27–37 weeks of gestation in control placentae. Although there appears to be a discernible increase in the level of expression of DLX4 mRNA with advancing gestation, semi-quantification using scanning densitometry revealed no significant change in the level of DLX4 mRNA normalized to GAPDH [0.93 ± 0.03 of densitometric units for DLX4 mRNA relative to GAPDH in pre-term (n = 13) versus 0.98 ± 0.00 of densitometric units for DLX4 mRNA relative to GAPDH in term (n = 12), P = 0.16, t-test].
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The relative quantification of DLX4 mRNA to GAPDH using real-time PCR on all pre-term (27–36 weeks, n = 13) and term (37–41 weeks, n = 12) control placentae and in pre-term (28–36 weeks, n = 10) and term (37–39 weeks, n = 15) FGR-affected placentae is shown in Figure 2B. The level of expression of DLX4 mRNA was not significantly altered between the pre-term and the term FGR placentae nor was it significantly altered between pre-term and term control samples. However, a significant increase in DLX4 mRNA expression was observed [1.2 ± 0.4 (n = 25) versus 3.3 ± 0.2 (n = 25), P < 0.01] when comparing grouped pre-term and term controls with pre-term and term FGR samples.
Multiple linear regression analysis as illustrated in Figure 3A showed that in control placentae, for each additional week of gestation, DLX4 mRNA expression levels relative to GAPDH reduced by 0.014-fold of the calibrator for each additional week of gestation [95% confidence interval (CI) –0.033–0.006, P < 0.16]. This analysis further confirmed that there was no evidence of significant difference in DLX4 mRNA expression levels relative to GAPDH in control placentae as the length of gestation increased. In FGR-affected placentae, regression analysis showed that DLX4 mRNA expression relative to GAPDH increased by 0.078-fold of the calibrator for each additional week of gestation; however, the apparent increase was not significantly different with advancing gestation (95% CI –0.050–0.206, P < 0.22).
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DLX4 mRNA expression relative to 18S RNA is illustrated in Figure 3B. The difference between the FGR-affected placentae and controls indicated strong evidence for a difference between the two groups (95% CI 2.02–3.96 P < 0.0005).
DLX4 expression in randomly selected term control (n = 10 of 12, 37–41 weeks), and term FGR-affected placentae (n = 10 of 15, 37–41 weeks) was investigated at the protein level. A representative immunoblot for DLX4 protein in term FGR-affected placentae (n = 5) and in gestation-matched term control placentae (n = 5) is shown in Figure 4A. Coomassie staining of proteins in the gel showed a constant amount of protein load in each well. As shown, the level of DLX4 protein increased in FGR-affected term placentae compared with that in the gestation-matched term control placentae. Semi-quantitative analyses using scanning densitometry showed that the levels of DLX4 protein relative to the protein load were significantly increased in FGR-affected term placentae compared with the term controls [5500.1 ± 21.8 (n = 10) versus 3533.2 ± 22.4, (n = 10), P < 0.001] (Figure 4B).
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Immunohistochemical localization for immunoreactive DLX4 protein provided qualitative evidence that DLX4 was seen in the syncytiotrophoblasts, residual cytotrophoblasts and in the endothelial cells of the fetal capillaries in both FGR-affected term placentae compared with the term control placentae (Figure 5; n = 6, 37–41 weeks).
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Discussion |
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The cohort of FGR-affected pregnancies that was employed was carefully defined in clinical terms and represents the severe end of spectrum of idiopathic FGR. Fetuses showed reduced growth by the late-second and early-third trimester. Reduced villous tree elaboration, diminished surface area of the placenta and abnormal end-diastolic blood flow in the umbilical artery are characteristic of pregnancies with severely growth-restricted infants (Kingdom et al., 2000
; Chen et al., 2002). Well-defined clinical groups with gestation age-matched controls reveal reproducible gene expression changes (Murthi et al., 2006a,b). RT–PCR analysis showed a qualitative increase in the intensity of DLX4 mRNA expression at the expected product size of 160 bp in FGR-affected term placentae compared with the gestation-matched controls. Therefore, more quantitative and sensitive assays of real-time PCR and western analysis were employed to confirm the elevated levels of DLX4 expression in FGR-affected placentae compared with gestation-matched controls. Both assays detected only DLX4 and not two other isoforms (DLX7 and BP1, see Materials and methods for details).
Our study is the first to demonstrate that a member of the Distal-less family of homeobox genes is significantly increased in the placenta in a human pregnancy disorder. The real-time PCR and immunoblotting analyses revealed quantitative differences in DLX4 expression in FGR-affected placentae compared with controls. However, these methods do not provide information on the spatial distribution of DLX4 expression and on whether changes in DLX4 expression between FGR-affected and control placentae are restricted to specific cell types. Therefore, immunohistochemical analysis was performed to detect the spatial distribution of DLX4 immunoreactive protein. As described, DLX4 was detected in syncytiotrophoblasts, residual cytotrophoblast cells and in the endothelial cells of term control and term FGR-affected placentae. These results are consistent with previous in situ mRNA hybridization studies of DLX4 (Quinn et al., 1997a). Furthermore, immunohistochemical analysis suggests that the quantitative differences in DLX4 expression observed between FGR and control placentae reflect changes in DLX4 expressing cell types in both groups.
Targeted mutation of the DLX4 gene in the mouse has not yet been carried out, and therefore, the importance and functional role of DLX4 in the embryo and placenta have yet to be definitively determined. In the human endometrium, significantly higher levels of DLX4 mRNA expression in proliferative phase glandular epithelia are seen compared with secretory phase epithelia, suggesting that the down-regulation of DLX4 may be important in the differentiation of epithelial cells (Quinn et al., 1998a). DLX4 down-regulation is also observed with hematopoietic stem cell differentiation in culture, and up-regulated DLX4 expression is seen in leukaemias (Haga et al., 2000; Ferrari et al., 2003a; Li et al., 2004). Thus, increased levels of DLX4 in FGR may reflect aberrant cell differentiation, proliferation or cell death.
Increased apoptosis is frequently associated with idiopathic FGR (Endo et al., 2005; Murthi et al., 2005; Straszewski-Chavez et al., 2005). Several studies have described a possible functional role for DLX4 expression in the regulation of apoptosis. DLX4 is a positive regulator of apoptosis in several tumour cell lines (Ferrari et al., 2003a). However, in other cell types, DLX4 acts as a negative regulator of apoptosis (Shimamoto et al., 1997; Shimamoto et al., 2000). The RNAi inactivation of DLX4 expression in placental choriocarcinoma cell lines also suggests that DLX4 is a negative regulator of apoptosis (Sun et al., 2006). The reasons for these conflicting results may reflect differences in cell lines but also different contributions of the isoforms DLX7 and BP1 in various cell types. Thus, although DLX4 expression has been correlated to the regulation of apoptosis in various malignant cell types, at this stage, the precise role of DLX4 in apoptosis in the normal or FGR-affected placenta has yet to be defined.
From the above studies, it can be seen that DLX4 may have several potentially important roles in the normal development of human placenta and that there may be significant deleterious consequences of increased DLX4 expression in the FGR-affected placenta.
In recent studies, we provided evidence that homeobox genes HLX1 (Murthi et al., 2006b) and ESX1L (Murthi et al., 2006a) are decreased in the placentae from FGR-affected pregnancies. Together with the current study on DLX4, it appears that changes in homeobox gene transcription factor levels are features of FGR-affected placentae, and they may form an important part of the molecular mechanisms of FGR.
Further investigations on the functional role of homeobox genes such as DLX4 in various normal placental cell types will contribute substantially to our understanding of the molecular mechanisms of idiopathic human FGR.
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Acknowledgements |
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The authors would like to thank the Lynne Quayle Charitable Trust Fund (Equity Trustees), Marian and EH Flack Trust Funds, Sunshine Foundation for funding support for this project and also the University of Melbourne for the award of a Melbourne Research Fellowship and Early Career Researcher Grant to Dr P. Murthi.
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Submitted on October 1, 2006; accepted on October 22, 2006.
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