Loss of Apela peptide in mice causes low penetrance embryonic lethality and defects in early mesodermal derivatives

Apela (also referred to as Elabela, Ende and Toddler) is a small signaling peptide that activates the G protein-coupled receptor Aplnr. We used CRISPR/Cas9 to generate a null, reporter-expressing allele, in order to study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low penetrance cardiovascular defects that manifest after the onset of circulation. Targeted Apela null alleles exhibited different transcriptional activity depending on the presence or absence of a Neomycin selection cassette. These are referred to as Apela KO NEO-IN and Apela KO NEO-OUT strains, respectively. Despite subtle phenotypic characteristics that were unique to the NEO-OUT mutants, both Apela null strains shared the same variable expressivity of cardiovascular defects and the same penetrance of embryonic lethality. To investigate the earliest regulatory events leading to physical abnormalities in Apela mutants, we performed RNA-Seq on whole stage-matched and morphologically normal E7.5 embryos (3 wild-type, 6 Apela KO NEO-IN, and 6 Apela KO NEO-OUT individuals). We chose this stage because Apela is initially expressed in the embryo at late gastrulation, shortly after the emergence of extraembryonic mesoderm progenitors. Since modification of the Apela locus may influence the expression of neighboring genes, we examined the expression of upstream and downstream sequences and found no significant difference in their expression. Downregulated genes of interest included several mitochondrial genes, Ceacam2, Ulk4, and Mov10l1. Upregulated genes included the vascular endothelial growth factor Vegfc. Principal component analysis identified outliers (KO1 and KO9), both of which expressed lower levels of mesoderm markers. KO9 was further characterized by aberrant upregulation of erythroid and myeloid markers. This finding was confirmed in our study by qRT-PCR analysis of additional Apela null individuals. 15 individual embryos were analyzed at E7.5. Embryos were stage-matched according to morphological landmarks. Control samples were wild-type (n=3), and Apela KO samples were null embryos from the NEO-IN (n=6, "KO1-6") and NEO-OUT (n=6, "KO7-12") mutant strains. Whole embryos (including embryonic and extraembryonic tissues) were used for the analysis. Apela KO samples were isolated from homozygous KO intercrosses and therefore did not require genotyping.Apela（亦称为Elabela、Ende及Toddler）是一种小型信号肽，可激活G蛋白偶联受体Aplnr。我们利用CRISPR/Cas9技术构建了可表达报告基因的无效等位基因，以期探究Apela在发育中小鼠胚胎中的生物学功能。研究发现，Apela缺失会导致低外显率的心血管缺陷，该缺陷会在血液循环启动后显现。靶向构建的Apela无效等位基因的转录活性会因新霉素筛选盒（Neomycin selection cassette）的存在与否产生差异，两类品系分别命名为Apela KO NEO-IN与Apela KO NEO-OUT。尽管NEO-OUT突变体存在独特的细微表型特征，但两种Apela无效等位基因敲除品系均表现出相同的心血管缺陷可变外显率，以及一致的胚胎致死外显率。为探究Apela突变体出现形态异常的最早调控事件，我们对发育阶段匹配、形态正常的E7.5期全胚胎进行了RNA测序（RNA-Seq），样本包括3例野生型个体、6例Apela KO NEO-IN个体与6例Apela KO NEO-OUT个体。我们选择该时期的原因在于，Apela最早于原肠胚晚期在胚胎中表达，此时恰为胚外中胚层祖细胞出现后不久。由于Apela基因座的修饰可能会影响邻近基因的表达，我们对其上下游序列的表达水平进行了检测，未发现显著差异。显著下调的目的基因包括多个线粒体基因、Ceacam2、Ulk4及Mov10l1；显著上调的基因则包括血管内皮生长因子Vegfc。主成分分析（PCA）识别出两个异常样本（KO1与KO9），二者的中胚层标志物表达水平均较低。KO9的特征进一步表现为红系与髓系标志物的异常上调。我们通过对额外的Apela无效等位基因敲除个体进行实时定量反转录PCR（qRT-PCR）分析，验证了这一发现。本研究共分析了15枚E7.5期胚胎，所有胚胎均根据形态学标志匹配发育阶段。对照样本为野生型胚胎（n=3），Apela敲除样本则分别来自NEO-IN突变品系（样本量6，即KO1至KO6）与NEO-OUT突变品系（样本量6，即KO7至KO12）的无效等位基因敲除胚胎。本研究采用全胚胎（包含胚胎组织与胚外组织）进行分析。Apela敲除样本均来自纯合敲除小鼠的互交后代，因此无需进行基因分型。