Deficiency of the bZIP transcription factors Mafg and Mafk causes misexpression of genes in distinct pathways and results in lens embryonic developmental defects

Deficiency of the small Maf proteins Mafg and Mafk cause multiple defects, namely, progressive neuronal degeneration, cataract, thrombocytopenia and mid-gestational/perinatal lethality. Previous data shows Mafg-/-:Mafk+/- compound knockout (KO) mice exhibit cataracts age 4-months onward. Strikingly, Mafg-/-:Mafk-/- double KO mice develop lens defects significantly early in life, during embryogenesis, but the pathobiology of these defects is unknown, and is addressed here. At embryonic day (E)16.5, the epithelium of lens in Mafg-/-:Mafk-/- animals appears abnormally multilayered as demonstrated by E-cadherin and nuclear staining. Additionally, Mafg-/-:Mafk-/- lenses exhibit abnormal distribution of F-actin near the “fulcrum” region where epithelial cells undergo apical constriction prior to elongation and reorientation as early differentiating fiber cells. To identify the underlying molecular changes, we performed high-throughput RNA-sequencing of E16.5 Mafg-/-:Mafk-/- lenses and identified a cohort of differentially expressed genes that were further prioritized using stringent filtering criteria and validated by RT-qPCR. Several key factors associated with the cytoskeleton, cell cycle or extracellular matrix (e.g. Cdk1, Cdkn1c, Camsap1, Col3A1, Map3k12, Sipa1l1) were mis-expressed in Mafg-/-:Mafk-/- lenses. Further, the congenital cataract-linked extracellular matrix peroxidase Pxdn was significantly overexpressed in Mafg-/-:Mafk-/- lenses, which may cause abnormal cell morphology. These data also identified the ephrin signaling receptor Epha5 to be reduced in Mafg-/-:Mafk-/- lenses. This likely contributes to the Mafg-/-:Mafk-/- multilayered lens epithelium pathology, as loss of an ephrin ligand, Efna5 (ephrin-A5), causes similar lens defects. Together, these 35 findings uncover a novel early function of Mafg and Mafk in lens development and identify their new downstream regulatory relationships with key cellular factors. For RNA-sequencing (RNA-Seq) experiments, mouse embryonic lenses at stage E16.5 (n = 8 lenses per biological replicate) were collected from collected from control (Mafg+/-:Mafk+/-), compound (Mafg-/-:Mafk+/-) and double KO (Mafg-/-:Mafk-/-). RNA isolation was performed using the mirVanaTM RNA isolation kit (Life Technologies, Grand Island, NY). Total RNA isolation, followed by removal of small molecular weight RNA was performed according to manufacturer's instructions. Sequencing was performed on a 2x101 paired end run using standard protocols on an NovaSeq 6000 sequencing system and FASTQ sequence files were obtained小Maf家族蛋白Mafg与Mafk的缺失可引发多种异常表型，具体包括进行性神经元变性、白内障、血小板减少症，以及妊娠中期/围产期致死。既往研究表明，Mafg基因敲除纯合、Mafk基因敲除杂合（Mafg-/-:Mafk+/-）的复合基因敲除（compound knockout, KO）小鼠，会在4月龄及之后出现白内障症状。值得注意的是，Mafg-/-:Mafk-/-双基因敲除（double knockout, double KO）小鼠会在生命早期即胚胎发育阶段出现晶状体缺陷，但此类缺陷的病理发生机制尚未明确，本研究即针对该问题展开探究。在胚胎第16.5天（embryonic day 16.5, E16.5）时，通过E-钙粘蛋白（E-cadherin）染色与细胞核染色可观察到，Mafg-/-:Mafk-/-小鼠的晶状体上皮层出现异常多层化现象。此外，Mafg-/-:Mafk-/-小鼠的晶状体在“支点（fulcrum）”区域附近的丝状肌动蛋白（F-actin）分布异常——该区域是上皮细胞在伸长、重定向为早期分化晶状体纤维细胞前发生顶端收缩的部位。为明确该缺陷背后的分子层面变化，我们对E16.5期的Mafg-/-:Mafk-/-小鼠晶状体开展了高通量RNA测序（RNA-sequencing, RNA-Seq），筛选得到一组差异表达基因；随后通过严格的过滤标准对其进行优先级排序，并利用实时定量聚合酶链反应（RT-qPCR）完成验证。多项与细胞骨架、细胞周期或细胞外基质相关的关键因子（如Cdk1、Cdkn1c、Camsap1、Col3A1、Map3k12、Sipa1l1）在Mafg-/-:Mafk-/-小鼠的晶状体中出现表达异常。此外，与先天性白内障相关的细胞外基质过氧化物酶Pxdn在Mafg-/-:Mafk-/-小鼠的晶状体中显著高表达，这可能会导致细胞形态异常。本研究数据还显示，Ephrin信号通路受体Epha5在Mafg-/-:Mafk-/-小鼠的晶状体中表达下调。由于Ephrin配体Efna5（ephrin-A5）的缺失会引发类似的晶状体缺陷，因此Epha5表达下调可能是导致Mafg-/-:Mafk-/-小鼠晶状体上皮层多层化病理表型的原因之一。综上，本研究的35项发现揭示了Mafg与Mafk在晶状体发育过程中一种全新的早期功能，并明确了二者与关键细胞因子间的新型下游调控关联。在RNA测序（RNA-sequencing, RNA-Seq）实验中，我们从对照组（Mafg+/-:Mafk+/-）、复合敲除组（Mafg-/-:Mafk+/-）与双基因敲除组（Mafg-/-:Mafk-/-）小鼠中采集了E16.5期的小鼠胚胎晶状体（每个生物学重复包含8个晶状体）。总RNA提取采用mirVana™ RNA提取试剂盒（Life Technologies公司，纽约州格兰德岛）完成。总RNA提取及小分子RNA去除步骤均严格按照试剂盒制造商的说明书进行操作。测序采用NovaSeq 6000测序系统，按照标准流程完成2×101双端测序，最终得到FASTQ格式的测序文件。