The mammalian limb displays a breadth of morphological diversity: from the single toe of the horse, to the wings of the bat, to the flippers of the dolphin, to our own five digits. Yet, decades of studies on classical vertebrate models (e.g. chickens, mice) have shown that a similar suite of genes drives limb patterning across species. With this, how is such variety of form achieved?

It is now becoming more widely accepted that regulatory changes are a main driver of evolution and diversity. Many excellent studies on this topic have been done with invertebrates and, more recently, mammals.

肢体首先以山脊开始。从身体的侧面出现小出现。接下来，肢体变得大致宽尽如人意（芽阶段）。然后，肢体会变平成桨，在这些阶段之间，数字射线开始凝结。随着肢体发育的进行，数字变得更加突出，并且数字之间的组织死亡，留下了自由数字。一些哺乳动物（蝙蝠）将保留该组织，该组织形成机翼（请参见下图）。其他哺乳动物（pigs, camels,cows, and many others) undergo a variety of mechanisms to reduce the number of digits.

Studies ofcandidate genes和transcriptomes单个物种扩大了我们对形态多样性的理解，但是据我们所知，在肢体上没有对几种物种进行广泛的分析。这样的研究将使我们能够解决以下问题：基因表达何时在不同物种的四肢之间发散？哪些途径和基因在同一和不同物种的前肢和后肢的表达方面有最大的不同？

To test this, we performed an RNA-seq analysis at the ridge, bud, and paddle stages of several representative species. Mouse (mus musculus）对于具有典型5位模式的哺乳动物，猪（Sus scrofa), which have evolved digit reduction, a species of microbat (Carollia perspicillata) – the only mammal capable of powered flight, and the opossum (单层家族), a marsupial for which there is a growth disparity at birth in fore- and hindlimb formation.

我们发现，肢体发育中已知的几个基因在所有物种的前端和后肢中差异表达，包括TBX5,Pax1, andHand1. In addition, some other known limb genes (Wnt5a,TBX4,Hoxa13, others) were greatly divergently expressed in the fore- and hindlimbs of most species and among limbs of all species. We confirmed a few select genes (of theHoxd和Hoxafamilies) by whole-mountin situ杂交（愿望）。

In general, we found that the fore- and hindlimbs of all species are most similar at the ridge stage of formation, and they begin to diverge at the onset of cartilage condensation (between the bud and paddle stages). We also calculated the evolutionary age of genes expressed at these stages across species and found that in general, oldest genes were from the ridge stages, while younger genes were at the paddle stage. Thus, our results suggest there is more diversity of gene expression as limbs become more morphologically diverse between species.

This work is a first step to examining regulatory changes that lead to differences in gene expression between diverse species. We were able to determine across stages and species a large number of divergently expressed genes. We are currently examining other genes by WISH to confirm their divergent expression, and investigating the enhancers surrounding our genes of interest.