Transcriptome comparison of bat (Miniopterus natalensis) forelimb and hindlimb autopods at developmental stages CS16 and CS17 and E13.5 mouse forelimb autopods

The bat offers an alternative paradigm to the standard mouse and chick model of limb development as it has extremely divergent forelimbs (long digits supporting a wing) and hindlimbs (short digits and claws) due the distinct requirements of both aerial and terrestrial locomotion. We used a cross-species microarray approach to identify differentially expressed (DE) genes between the bat (Minniopterus natalensis) forelimb and hindlimb autopods at Carollia developmental stages (CS) 16 and CS17, and between the bat (CS17) and mouse (E13.5) forelimb autopods. Several DE genes were identified, including two homeobox genes, Meis2, a proximal limb-patterning gene, and Hoxd11, a gene involved in digit elongation. Both genes are significantly over-expressed in the developing bat forelimb as compared to the hindlimb and equivalently staged mouse forelimbs. A reference design was used in this microarray experiment. A pool of left and right mouse forelimb autopods from 24 embryos was used as the reference sample. This sample was directly compared to individual CS16 and CS17 bat fore- and hindlimbs (left and right of one individual pooled) that were classified as the test conditions. Four experimental sessions were performed using an independently amplified mouse reference pool and 4 biological repeats for the bat limbs. These samples were co-hybridised to OPERON Mouse OpArray (ver. 4.0) spotted oligonucleotide slides to perform a competitive Cross-Species Hybridisation experiment. The bat aRNA (test) samples were labelled with Cy3 dye (green signal), the mouse aRNA (reference) sample was labelled with Cy5 dye (red signal).蝙蝠为经典的小鼠与鸡肢发育模型提供了替代性研究范式。由于空中飞行与陆地行走的运动需求存在显著差异，蝙蝠的前肢（支撑翼膜的修长指骨）与后肢（短指带爪）结构差异极大。本研究采用跨物种微阵列技术，针对卡罗利亚发育分期（Carollia developmental stages, CS）16期与CS17期的纳塔尔长翼蝠（Minniopterus natalensis）前肢、后肢肢端，以及CS17期蝙蝠与E13.5期小鼠的前肢肢端，鉴定差异表达（differentially expressed, DE）基因。本次研究共鉴定出多个差异表达基因，其中包含2个同源框（homeobox）基因：作为肢体近端模式形成调控基因的Meis2，以及参与指骨延长的Hoxd11。相较于蝙蝠后肢以及同期发育的小鼠前肢，这两个基因在发育中的蝙蝠前肢中均显著高表达。本微阵列实验采用参考设计方案：将24枚小鼠胚胎的左右前肢肢端混合制备为参考样本，将该样本与合并为单一样本的单只蝙蝠CS16、CS17期前、后肢肢端（左右肢合并）直接进行比较，后者作为测试组样本。实验采用独立扩增的小鼠参考混合样本，针对蝙蝠肢体设置4次生物学重复，共开展4轮实验。将所有样本与印有OPERON小鼠OpArray（版本4.0）寡核苷酸探针的玻片进行共杂交，以完成竞争性跨物种杂交实验：蝙蝠的反义RNA（antisense RNA, aRNA）测试样本以Cy3染料标记（呈现绿色信号），小鼠的反义RNA（aRNA）参考样本以Cy5染料标记（呈现红色信号）。