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.
The limb first begins as a ridge; a small outgrowth from the flank of the body. Next, the limb becomes roughly as wide as it is long (bud stage). The limb then flattens into a paddle, and between these stages the digit rays begin to condense. As limb development proceeds, digits become more prominent and tissue between the digits dies, leaving free digits. Some mammals (bats) will retain this tissue, which forms the wing (see figure below). Other mammals (pigs, camels, cows, and many others) undergo a variety of mechanisms to reduce the number of digits.
Studies of candidate genes and transcriptomes of individual species have broadened our understanding of morphological diversity, however to our knowledge no wide-scale analysis of several species at once has been done on the limb. Such a study would permit us to address questions such as: when does gene expression diverge among the limbs of different species? Which pathways and genes differ most in expression in the fore- and hindlimbs of the same and different species?
To test this, we performed an RNA-seq analysis at the ridge, bud, and paddle stages of several representative species. Mouse (Mus musculus) for mammals with a typical 5-digit pattern, pig (Sus scrofa), which have evolved digit reduction, a species of microbat (Carollia perspicillata) – the only mammal capable of powered flight, and the opossum (Monodelphis domestica), a marsupial for which there is a growth disparity at birth in fore- and hindlimb formation.
We found that several genes known in limb development were differentially expressed in the fore- and hindlimbs of all species, including Tbx5, Pax1, and Hand1. 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 the Hoxd and Hoxa families) by whole-mount in situ hybridization (WISH).
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.