Loss of Sip1 leads to migration defects and retention of ectodermal markers during lens development

We report the application of RNA-sequencing technology for the high-throughput profiling of mammalian lens gene expression at embryonic day 15.5. The lens has a particularly biased transcriptome, with the top 50 genes encoding approximately 90% of the protein content. Using RNA-Seq, we have shown that there are over 7,700 genes being expressed in the lens at embryonic day 15.5. As expected, the crystallins and many structural genes were amoung the most highly expressed; however, numerous genes expressed at lower transcript abundance were also identified. This study provides a framework for the application of RNA-Seq technology towards characterization of the mammalian lens transcriptome during development. Using high-throughput RNA-sequencing, gene expression in the mammalian lens was compared between an inbred C56Bl/6<har> strain and a mix background strain at E15.5. This analysis identifies almost 2,000 genes being differentially expressed between the inbred and mixed background lenses, ranging from 6.5 fold upregulated to 5.2 fold downregulated in the mixed background compared to the inbred strain. This list does not include unknown/predicted genes or pseudogenes which are known to change between strains. Further, it appears that approximately 98% of these genes are altered at levels less than 2.5 fold. This study therefore provides a fold change threshold cutoff (2.5 fold) to use in the analysis of differentially expressed lens genes at E15.5 using RNA-Seq technology as it takes into account genetic variation due to background strain differences. SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases where it functionally links TGFb signaling to the loss of epithelial preferred gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes involved in EMT/cancer. Furthermore, 34% of the upregulated genes in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes downregulated in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts. RNA-Seq of inbred background wild type lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and inbred wild type (C57Bl/6<har>) lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and Sip1 conditional knockout lenses at E15.5本研究报道了RNA测序（RNA-sequencing）技术在胚胎第15.5天对哺乳动物晶状体基因表达进行高通量谱分析的应用。晶状体的转录组具有显著的偏倚性，排名前50的基因编码的蛋白质约占总蛋白含量的90%。通过RNA测序技术，我们证实胚胎第15.5天的晶状体中存在超过7700个表达基因。正如预期，晶状体蛋白（crystallins）与众多结构基因位列高表达基因之列；不过研究同时鉴定出了大量转录本丰度较低的基因。本研究为RNA测序技术应用于发育过程中哺乳动物晶状体转录组的表征提供了研究框架。 本研究借助高通量RNA测序，对比了胚胎第15.5天（E15.5）时近交系C56Bl/6<har>与混合背景品系小鼠晶状体的基因表达差异。该分析鉴定出近交系与混合背景品系晶状体间存在近2000个差异表达基因，与近交系相比，混合背景品系中这些基因的表达上调幅度最高达6.5倍，下调幅度最高达5.2倍。该差异基因列表未包含品系间已知存在表达差异的未知/预测基因及假基因。此外，近98%的差异表达基因的表达变化幅度小于2.5倍。鉴于背景品系差异带来的遗传变异，本研究确立了2.5倍的表达变化阈值，可用于基于RNA测序技术的胚胎第15.5天晶状体差异表达基因分析。 SIP1编码一种DNA结合转录因子，可调控多种发育过程，这一点可由该基因突变引发的莫瓦特-威尔森综合征（Mowat-Wilson Syndrome）所表现出的多效性缺陷得到佐证。此外，在成体中，SIP1表达失调已被证实与癌症及纤维化疾病相关，其可通过功能层面将TGFb信号通路与上皮偏好性基因表达的缺失联系起来。在对视觉至关重要的上皮组织——眼晶状体中，Sip1与E-钙粘蛋白（E-cadherin）等上皮标志物共表达，且在早期眼形态发生过程中，它对于晶状体泡与头部外胚层的完全分离不可或缺。然而，晶状体泡早期形态发生完成后，Sip1的功能仍未明确。 本研究在小鼠晶状体泡闭合后不久，对发育中的小鼠晶状体条件性敲除Sip1，导致晶状体纤维细胞尖端协同迁移异常、缝线形成缺陷及白内障发生。值得注意的是，对Sip1敲除晶状体的RNA测序分析鉴定出190个差异表达基因，所有这些基因均与此前报道的参与上皮间质转化（Epithelial-Mesenchymal Transition，EMT）/癌症的Sip1靶基因有所不同。此外，Sip1敲除晶状体中上调的基因有34%在晶状体从晶状体泡向早期晶状体发育的过程中通常是下调的；而Sip1敲除晶状体中下调的基因有49%在早期晶状体发育过程中通常是上调的。综上，这些数据表明，Sip1在重编程晶状体泡的细胞命运中发挥关键作用，使其从表面外胚层细胞命运转向发育透明晶状体所需的细胞命运；同时证实Sip1在不同细胞环境中调控的基因集存在显著差异。 胚胎第15.5天近交系背景野生型晶状体的RNA测序；胚胎第15.5天混合背景野生型对照与近交系野生型（C57Bl/6<har>）晶状体的RNA测序对比；胚胎第15.5天混合背景野生型对照与Sip1条件性敲除晶状体的RNA测序对比。