Comparison of regenerating and non regenerating transgenic stage 52 Xenopus hind limbs

Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus in which the BMP inhibitor Noggin can be over-expressed at any time during development. We have previously shown that activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles. In the current study, we have taken advantage of this transgenic line to directly compare gene expression in same stage regenerating vs. non-regenerating hind limb buds. Using Affymetrix gene chip analysis, we have identified genes whose expression levels are linked to regenerative success. These include the BMP inhibitor Gremlin and the stress protein Hsp60 (no blastema in zebrafish). Analysis of overrepresented Gene Ontology functional groupings suggests that successful regeneration in the Xenopus hind limb depends on induction of stress response pathways. Furthermore, as expected, genes involved in embryonic development and growth are also significantly over-represented in regenerating early hind limb buds. Keywords: Differential expression, regeneration This series was used to identify differentially expressed genes between regenerating wild type stage 52 hind limb buds and those of their transgenic siblings which are blocked in BMP signalling and fail to regenerate. Two biological replicates of each type were examined. Each replicate is made up of dissected distal stump tissue from 20 hindlimbs 3 days after amputation. The single timepoint of 3 days was chosen to fall after wound healing had occurred but before re-differentiation and corresponds to the cone shaped blastema stage of successfull regenerates. BMP signalling is blocked using a single insertion transgene (N1) expressing noggin under the control of an inducible heat shock promoter, and all tadpoles are siblings derived from a single backcross to a wild type animal to minimise genetic variation.

形态发生性再生（Epimorphic regeneration）是指通过增殖性芽基（blastema）的形成，实现肢体等复杂结构完全再生的过程。这类再生在脊椎动物中极为罕见，但可在非洲爪蟾（Xenopus laevis）中实现——后者是早期发育研究的经典模式生物。非洲爪蟾蝌蚪可再生尾部、肢芽（limb bud）以及眼晶状体，不过后两种器官的再生能力会随发育阶段推进而逐渐减弱。本研究借助一种仅在激活状态下才会表达的热激诱导型转基因（heat shock-inducible transgene），构建了可在发育任意时段过表达骨形态发生蛋白（Bone Morphogenetic Protein, BMP）抑制剂Noggin的稳定转基因非洲爪蟾品系。我们此前已证实，激活该转基因可阻断非洲爪蟾蝌蚪尾部与肢体的再生过程。在本研究中，我们利用该转基因品系，直接对比了同一发育阶段的再生型与非再生型后肢芽的基因表达差异。
通过Affymetrix基因芯片分析，我们鉴定出了表达水平与再生成功率相关的基因，其中包括BMP抑制剂Gremlin以及应激蛋白Hsp60（斑马鱼中无再生芽基形成）。对富集的基因本体（Gene Ontology, GO）功能聚类的分析显示，非洲爪蟾后肢的成功再生依赖于应激应答通路的激活。此外，正如预期，参与胚胎发育与生长过程的基因在再生型早期后肢芽中同样显著富集。
关键词：差异表达、再生
本数据集系列用于鉴定再生型野生型52期后肢芽，与BMP信号通路受阻且无法完成再生的转基因同系幼体的后肢芽之间的差异表达基因。每组设置2次生物学重复，每个生物学重复由截肢3天后采集的20条后肢的远端残端组织构成。本研究选取截肢后3天这一单一时间点，该时间点处于伤口愈合完成后、重分化开始前，对应成功再生个体的锥状芽基阶段。本研究通过单插入位点转基因品系（N1）阻断BMP信号通路：该品系在热激诱导启动子的调控下表达noggin，且所有实验蝌蚪均来自与野生型个体单次回交产生的同系幼体，以最大限度降低遗传变异带来的影响。