Molecular Human Reproduction Vol.12, No.12 pp. 763–769, 2006 Advance Access publication October 24, 2006
doi:10.1093/molehr/gal087
Homeobox gene DLX4 expression is increased in idiopathic human fetal growth restriction P.Murthi1,2,4, J.M.Said1,2, V.L.Doherty1, S.Donath2,3, C.J.Nowell1,2, S.P.Brennecke1,2 and B.Kalionis1,2 1
Pregnancy 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:
[email protected] 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
Introduction 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.,
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P.Murthi 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 β-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 β-globin silencer elements but differ in their ability to repress β-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.
Materials and methods 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 gestationmatched 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) 1.2). The exclusion criteria for the study for both control and FGR-affected pregnancies were multiple pregnancies, chemical
Table I. Clinical characteristics of samples included in the study Characteristics Gestation age weeks (mean ± SD) Maternal age years (mean ± SD) Placental weight (g) Parity Primaparous Multiparous Mode of delivery Vaginal delivery Caesar in labour Caesar not in labour New born characteristics Male Female Birthweight (mean ± SD) 10–90% 5–10%
