Paternal knockout of Slc38a4/SNAT4 causes placental hypoplasia associated with intrauterine growth restriction in mice

The placenta is critical for mammalian embryonic development because the embryo's supply of nutrients, including amino acids, depends solely on mother-to-embryo transport through the placenta. However, the molecular mechanisms underlying this amino acid supply are poorly understood. In this study, we focused on the system A amino acid transporters, Slc38a1/SNAT1, Slc38a2/SNAT2, and Slc38a4/SNAT4, which carry neutral, short-side-chain amino acids, to determine their involvement in placental or embryonic development. A triple-target CRISPR screen identified Slc38a4/SNAT4 as the critical amino acid transporter for placental development in mice. We established mouse lines from the CRISPR founders with large deletions in Slc38a4 and found that, consistent with the imprinted paternal expression of Slc38a4/SNAT4 in the placenta, paternal knockout (KO), but not maternal KO, of Slc38a4/SNAT4 caused placental hypoplasia associated with reduced fetal weight. Immunostaining revealed that SNAT4 was widely expressed in differentiating cytotrophoblasts and maturing trophoblasts at the maternal-fetal interface. A blood metabolome analysis revealed that amino acid concentrations were globally reduced in Slc38a4/SNAT4 mutant embryos. These results indicated that, in mice, SNAT4-mediated amino acid transport plays a major role in both placental and embryonic development. Given that expression of Slc38a4 in placentas is conserved in other species, our Slc38a4/SNAT4 mutant mice could be a promising model for the analysis of placental defects leading to intrauterine growth restriction in mammals. Global gene expression patterns in the placenta of wildtype (WT) and Slc38a4 mutant mice (MKO, PKO, and Null) were analyzed by one-color Mouse Gene Expression 8x60K microarray. RNA was extracted from E13.5 placentas using an RNeasy Mini Kit (Qiagen). Total RNA (200 ng) was amplified and labeled with a Low Input Quick Amp Gene Expression Labeling Kit (#5190-2305, Agilent Technologies). The labeled cRNA was hybridized to a mouse oligo DNA microarray (8x60K, SurePrint G3 Mouse GE v2; #G4852B, Agilent Technologies). After the scan, the signal intensities on the microarray were processed using the Feature extraction software (Agilent Technologies) and analyzed by GeneSpring 14.5 (Agilent Technologies).胎盘对哺乳动物胚胎发育至关重要，因为胚胎的营养供给（包括氨基酸）完全依赖母体通过胎盘向胚胎的转运。然而，这一氨基酸供给的分子机制尚不明晰。本研究聚焦于转运中性短链氨基酸的A型氨基酸转运蛋白Slc38a1/SNAT1、Slc38a2/SNAT2及Slc38a4/SNAT4，以探究其在胎盘或胚胎发育中的作用。通过三靶点CRISPR（成簇规律间隔短回文重复序列）筛选，我们确定Slc38a4/SNAT4是小鼠胎盘发育的关键氨基酸转运蛋白。我们从携带Slc38a4大片段缺失的CRISPR创始小鼠中建立了稳定品系，并发现：与Slc38a4/SNAT4在胎盘中的印记父本表达模式一致，Slc38a4/SNAT4的父本敲除（KO）而非母本敲除，会引发胎盘发育不全，并伴随胎儿体重降低。免疫染色实验显示，SNAT4在母胎界面的分化性细胞滋养层细胞及成熟滋养层细胞中广泛表达。血液代谢组分析表明，Slc38a4/SNAT4突变胚胎的氨基酸浓度整体显著下降。上述结果证实，在小鼠体内，SNAT4介导的氨基酸转运在胎盘及胚胎发育中均发挥核心作用。鉴于其他物种胎盘中Slc38a4的表达具有保守性，本研究构建的Slc38a4/SNAT4突变小鼠有望成为研究哺乳动物宫内生长受限相关胎盘缺陷的理想动物模型。本研究采用单色小鼠基因表达8x60K微阵列，分析了野生型（WT）及Slc38a4突变小鼠（母本敲除型MKO、父本敲除型PKO及纯合缺失型Null）胎盘的全局基因表达模式。实验中，我们使用RNeasy Mini试剂盒（Qiagen）从E13.5胎盘中提取总RNA；取200 ng总RNA，通过Low Input Quick Amp基因表达标记试剂盒（#5190-2305，Agilent Technologies）进行扩增与标记；将标记后的互补RNA（cRNA）与小鼠寡核苷酸DNA微阵列（8x60K，SurePrint G3小鼠基因表达芯片v2；#G4852B，Agilent Technologies）进行杂交。扫描完成后，使用特征提取软件（Agilent Technologies）处理微阵列上的信号强度，并通过GeneSpring 14.5（Agilent Technologies）完成数据分析。