Distal-less 3 haploinsufficiency results in elevated placental oxidative stress and altered fetal growth kinetics in the mouse

Abstract

Distal-less 3 (Dlx3)^−/− mice die at E9.5 presumably due to an abnormal placental phenotype including reduced placental vasculature and secretion of placental growth factor. To examine the role of Dlx3 specifically within the epiblast, Dlx3 conditional knockout mice were generated using an epiblast-specific Meox2^CreSor allele. Dlx3^−/fl, Meox2^CreSor animals were born at expected frequencies and survived to weaning providing indirect evidence that loss of Dlx3 within the trophoectoderm plays a critical role in fetal survival in the Dlx3^−/− mouse. We next examined the hypothesis that loss of a single Dlx3 allele would have a negative impact on placental and fetal fitness. Dlx3^+/− mice displayed reduced fetal growth beginning at E12.5 compared with Dlx3^+/+ controls. Altered fetal growth trajectory occurred coincident with elevated oxidative stress and apoptosis within Dlx3^+/− placentas. Oral supplementation with the superoxide dismutase mimetic, Tempol, rescued the fetal growth and placental cell death phenotypes in Dlx3^+/− mice. To determine the potential mechanisms associated with elevated oxidative stress on the Dlx3^+/− placentas, we next examined vascular characteristics within the feto-placental unit. Studies revealed reduced maternal spiral artery luminal area in the Dlx3^+/− mice receiving water; Dlx3^+/− mice receiving Tempol displayed maternal spiral artery luminal area similar to control Dlx3^+/+ mice. We conclude that reduced Dlx3 gene dose results in diminished fetal fitness associated with elevated placental cell oxidative stress and apoptosis coincident with altered vascular remodeling. Administration of antioxidant therapy ameliorated this feto-placental phenotype, suggesting that Dlx3 may be required for adaptation to oxidative stresses within the intrauterine environment.

Introduction

Distal-less 3 (Dlx3) is a homeodomain-containing transcription factor that is involved in key developmental events in several tissues including placental morphogenesis in the mouse as well as expression of key endocrine products of the human and mouse placentas [1], [2], [3], [4], [5]. In general, Dlx3 plays a role in the differentiation of epithelial compartments such as skin. For example, ectopic expression of Dlx3 in the basal layer of mouse epidermis results in cessation of proliferation of basal cells and enhanced differentiation of keratinocytes [6]. Complete genetic loss of Dlx3 in mice results in mid-gestation lethality presumably due to inadequate placental development, suggesting an important role for this transcription factor at the maternal fetal interface [2]; however, clearly delineated roles for Dlx3 within the trophoectoderm and epiblast in the Dlx3^−/− mouse model have not been established. In trophoblast cell culture models, Dlx3 is a required transcriptional regulator of the α subunit of chorionic gonadotropin (CG) [7]. Immunohistochemical studies of human placental sections obtained at 8 weeks gestation suggest that Dlx3 is expressed in villous cyto- and syncytial trophoblasts at a time when CG biosynthesis is high [4], [7]. Recently, Murthi and colleagues reported that Dlx3 transcript levels increased coincident with in vitro differentiation of human villous cytotrophoblasts where differentiation was measured by increased expression of the β subunit of CG as well as 3β-hydroxysteroid dehydrogenase [8]. These studies support the conclusion that Dlx3 may be playing a role in the regulation of expression of α and β subunits of CG as well as steroid hormone biosynthesis in the human placenta.

Pathological conditions such as intrauterine growth restriction (IUGR) and pre-eclampsia (PE) have been linked to failure in placental morphogenesis correlated with aberrant gene expression, abnormalities in proangiogenic growth factor production and secretion, endothelial cell dysfunction and improper placental vascularization; all of which can lead to reduced placental perfusion and elevated levels of reactive oxygen species (ROS) within the placenta [9], [10], [11], [12]. Interestingly, the Dlx3 null mice display dysregulated vascularization within the placental labyrinth [2], [4] coincident with reduced expression and secretion of the proangiogenic placental growth factor (PGF), a direct target of Dlx3 action [13]. Notably in women, reduced PGF levels in maternal serum serve as a key biomarker of PE [10], [14]; in addition, the BPH/5 mouse model of PE also displays altered labyrinth morphogenesis, reduced PGF expression and maternal vascular remodeling [15], [16]. Taken together, these studies suggest that reduced expression of Dlx3 may be associated with placental insufficiency due to inappropriate expansion of the labyrinth within an environment of a reduced angiogenic potential; however, since the Dlx3 null animal is embryonic lethal, little is known regarding the role of Dlx3 governing the feto-placental unit past E9.5.

The present studies provide evidence that conditional loss of Dlx3 specifically within the epiblast (rather than within the trophectoderm and epiblast in the Dlx3^−/− model) results in viable albeit phenotypically abnormal mice. These data provide indirect support for the conclusion that loss of Dlx3 within the placenta in the Dlx3^−/− model is likely central to embryonic lethality. Loss of a single Dlx3 allele has marked effects on intrauterine fetal growth patterns coincident with elevated oxidative stress and vascular dysfunction in murine placentas. These Dlx3-dependent placental defects are rescued with oral administration of an antioxidant given prior to conception and throughout gestation providing evidence that Dlx3 may be playing a role in the adaptation of the murine placenta to oxidative stresses during gestation.