Decreased Fgf8 gene dosage on gene expression

Robustness to perturbation, or canalization, is a fundamental and required feature of complex organisms.1 Mutations are the raw material for evolution, but robustness to their effects is required for organisms to tolerate mutational loads.1 Similarly, robustness to environmental perturbations, or limiting environmental responses to an adaptive range, is necessary for organisms to tolerate environmental variation. Robustness is heritable2-4, but the mechanisms that produce it are poorly understood. Explanations range from the role of specific processes such as heat shock proteins to more general embedded features of developmental systems. Nonlinearities are an embedded, ubiquitous feature of development that may modulate how variation in development relates to phenotypic robustness.5,6 Here, we show that variation in Fibroblast Growth Factor 8 (Fgf8) expression across an allelic series has a nonlinear relationship to phenotypic variation, predicting the magnitude of variation within genotypes. These differences in phenotypic variance are independent of genetic variance and so reflect differential robustness to minor environmental variation. Differences in robustness are not related to differences in the variance of gene expression within genotypes. However, mean gene expression levels do vary across genotypes, particularly in genes downstream to FGF8 signaling. Gene expression changes thus explain the genotype-phenotype curve but not the changes in robustness along the curve. This suggests that mechanisms above the gene-regulatory network are important determinants of robustness. Our results show that nonlinearities in developmental mechanisms can persist from genotype to phenotype to produce variation in robustness between genotypes. This suggests that embedded features of development rather than specific canalizing mechanisms explain robustness. How such features vary among individuals in natural populations and relate to genetic variation more generally are key questions for unravelling the origin and evolvability of this fundamental feature of organismal development. Mouse breeding and embryo generation: The Fgf8 neo series is a 5 member series generated from a combination of the neomycin insertion into the intron between exon 2 and 3 of the Fgf8 locus and a null allele generated from loss of exon 2. Fgf8 mice were generated from the Fgf8 flp/ floxed allele originally developed by (Meyers et al., 1998). The neo cassette was maintained in the Fgf8Neo mice. Deletion constructs were developed by crossing with beta-actin cre (FVB/N-Tg(ACTB-cre)2Mrt/J), to delete exons 2 and 3 from all cells. To generate the neo series, crosses were performed between mice that were heterozygous for and mice that were homozygous for the Neo (flp) allele. Genotyping was performed as in (Meyers et al., 1998). For embryos, pregnant dams were sacrificed at embryonic day (E) 10.5 based on visualization of a post-coital plug at E0.5. E10.5 embryos were dissected into PBS on ice and snap frozen at -80C. RNA was extracted in batch preps using Trizol. TapeStation analysis was used to select samples for RNAseq, all samples tested had a RIN number of between 9.0 and 10. Single end reads were performed to a read depth of 40 million reads/ sample on an illumina NextSeq 5000.扰动鲁棒性（亦可称为通道化，canalization）是复杂生物体的一项基础且不可或缺的特征[1]。突变是进化的原材料，但生物体要耐受突变负荷，就需要对突变效应具备鲁棒性[1]。类似地，生物体要耐受环境变异，也需要对环境扰动具备鲁棒性——或将环境响应限制在适应性范围内。鲁棒性具有可遗传性[2-4]，但其产生机制仍鲜为人知。现有解释涵盖了从热休克蛋白等特定过程的作用，到发育系统更为普遍的固有特征等诸多范畴。非线性是发育过程中一种固有且普遍存在的特征，可能会调控发育变异与表型鲁棒性之间的关联[5,6]。 本研究发现，一系列等位基因中成纤维细胞生长因子8（Fgf8）的表达变异与表型变异呈非线性关联，可预测基因型内的变异幅度。这些表型方差的差异与遗传方差无关，因此反映了生物体对微小环境变异的鲁棒性差异。鲁棒性的差异与基因型内基因表达方差的差异并无关联。但不同基因型的平均基因表达水平确实存在差异，尤其是在FGF8信号通路下游的基因中。因此，基因表达的变化可以解释基因型-表型曲线，但无法解释沿该曲线的鲁棒性变化。这表明，基因调控网络之上的机制是鲁棒性的重要决定因素。我们的研究结果显示，发育机制中的非线性可从基因型延续至表型，从而导致不同基因型间的鲁棒性差异。这表明，鲁棒性可由发育的固有特征而非特定的通道化机制来解释。这类特征在自然种群的个体间如何变异，以及其与更广泛的遗传变异的关联，是阐明生物体发育这一基础特征的起源与可进化性的核心问题。 小鼠繁育与胚胎制备： Fgf8 neo系列是由5个成员组成的等位基因系列，其构建方式为：将新霉素抗性基因插入至Fgf8基因座第2外显子与第3外显子之间的内含子中，并结合因第2外显子缺失而产生的无效等位基因。Fgf8 flox/flox小鼠源自Meyers等人于1998年构建的Fgf8 flp/ floxed等位基因。新霉素抗性基因盒（neo cassette）在Fgf8Neo小鼠中得以保留。通过与β-肌动蛋白Cre重组酶（FVB/N-Tg(ACTB-cre)2Mrt/J）小鼠杂交，获得可在所有细胞中删除第2外显子与第3外显子的敲除构建体。为构建neo系列等位基因，将携带Neo（flp）等位基因的杂合子小鼠与纯合子小鼠进行杂交。基因型鉴定方法参照Meyers等人1998年的研究。关于胚胎制备：依据妊娠第0.5天（E0.5）观察到交配栓的情况，在胚胎发育第10.5天（E10.5）处死后取出孕鼠。将E10.5胚胎在冰上的磷酸盐缓冲液（PBS）中解剖分离，随后于-80℃快速冷冻。使用Trizol试剂批量提取RNA。通过TapeStation分析筛选用于RNA测序（RNAseq）的样本，所有待测样本的RNA完整性数（RIN）介于9.0至10.0之间。在Illumina NextSeq 5000测序平台上进行单端测序，测序深度为每样本4000万条读段。