Transcriptomic insights into the genetic basis of mammalian limb diversity

Abstract

Background: From bat wings to whale flippers, limb diversification has been crucial to the evolutionary success of mammals. We performed the first transcriptome-wide study of limb development in multiple species to explore the hypothesis that mammalian limb diversification has proceeded through the differential expression of conserved shared genes, rather than by major changes to limb patterning. Specifically, we investigated the manner in which the expression of shared genes has evolved within and among mammalian species.

Results: We assembled and compared transcriptomes of bat, mouse, opossum, and pig fore- and hind limbs at the ridge, bud, and paddle stages of development. Results suggest that gene expression patterns exhibit larger variation among species during later than earlier stages of limb development, while within species results are more mixed. Consistent with the former, results also suggest that genes expressed at later developmental stages tend to have a younger evolutionary age than genes expressed at earlier stages. A suite of key limb-patterning genes was identified as being differentially expressed among the homologous limbs of all species. However, only a small subset of shared genes is differentially expressed in the fore- and hind limbs of all examined species. Similarly, a small subset of shared genes is differentially expressed within the fore- and hind limb of a single species and among the forelimbs of different species.

Conclusions: Taken together, results of this study do not support the existence of a phylotypic period of limb development ending at chondrogenesis, but do support the hypothesis that the hierarchical nature of development translates into increasing variation among species as development progresses.

Keywords: Bat; Differential expression; Diversification; Limb; Mammalian; Mouse; Opossum; Pig; Transcriptome.

Fig. 1

Representative stages of limb development used in this study. This study assembled the transcriptomes of four species (bat, mouse, opossum, and pig) from three stages of limb development, namely the ridge (a), bud (b), and paddle (c) stages. The embryos shown here are from Erophylla sezekorni (Buffy Flower Bat)

Fig. 2

Similarity of gene expression profiles within species. Heat-maps of the gene expression profiles for the fore- and hind limbs of all developmental stages for bat (a), mouse (b), opossum (c), and pig (d). FL = forelimb, HL = hind limb. The stages for each species are as follows: b_St13 = bat ridge, b_St14 = bat bud, b_St15 = bat paddle, m_StW2 = mouse ridge, m_StW3_4 = mouse bud, mStW6 = mouse paddle, o_St27 = opossum ridge, o_St28 = opossum bud, o_St29 = opossum paddle, o_St30 = opossum ridge, o_St31 = opossum bud, o_St32 = opossum paddle, p_St20 = pig ridge, p_St22 = pig bud, p_St26 = pig paddle. Branches with statistically significant clustered sub-branches (p-value < =0.05) are indicated with asterisks (*)

Fig. 3

Similarity of gene expression profiles among species. Pairwise Spearman coefficient values for forelimbs at the (a), bud (b), paddle (c) stages for all species, and for the hind limbs of the ridge (d), bud (e), paddle (f) stages for all species. M. = mouse, O. = opossum. Abbreviations for stages and species are as in the legend for Fig. 2. In each label for the heat map, Stage 2 corresponds to ridge, Stage 3–4 to bud, and Stage 6 to paddle

Fig. 4

Conservation of gene expression profiles. The conservation of the gene expression profiles of all study species (bat, mouse, opossum, and pig) across embryonic development of the fore- (a) and hind limb (b), as determined by the mean of all species pairwise Spearman coefficients (X axis). Results for the ridge stage are shown in red, for the bud stage in green, and for the paddle stage in blue. To test the robustness of differences in Spearman coefficients with respect to the gene selection, we randomly sub-sampled gene sets at intensities ranging from 50 to 100% of all genes (Y axis). In the box plots, the bottom and top of the boxes represent the first and third quartiles of the data, and the line through the middle of the box the second quartile (median). The whiskers range from the 2^nd to 98^th percentiles, and the open circles depict the outliers to these percentiles

Fig. 5

Evolutionary Age. The evolutionary ages of the genes of each species in this study are shown in (a). Genes were assigned to phylostratums (ps) based on their degree of conservation. At one end of the spectrum, genes that are specific to a single species (i.e., they do not have homologues in other species) were assigned to ps 1, and at the other end genes that are common to all study species were assigned to ps 4. Ps 2 and ps 3 correspond to the branching points of the phylogenetic tree between ps 1 and ps 4. Genes in ps 1, ps 2, ps 3, and ps 4 were assigned evolutionary ages of 1, 2, 3, and 4, respectively. A hierarchical clustering analysis based on the pair-wise number of homologue genes (b) exactly mirrors the phylogeny of the study species. Results shown in (a) were used to calculate the transcriptome age indices (TAIs) of each species across development (c) for the fore- (left) and hind (right) limbs. Smaller TAIs correspond to younger ages, and larger TAIs older ages. Bat is shown in blue, pig in purple, mouse in red, and opossum in green

Fig. 6

Hoxd13 Expression in Limb Stages of Three Species. a-c: Mouse forelimb Hoxd13 in ridge, bud, and paddle, respectively. a’-c’: Mouse hindlimb Hoxd13 in ridge, bud, and paddle. d-f and d’-f’: Opossum Hoxd13 in forelimb and hindlimb ridge, bud, and paddle, respectively. g-i and g’-i’: Bat Hoxd13 in forelimb and hindlimb ridge, bud, and paddle. Purple staining denotes Hoxd13 expression boundary. In bat forelimb ridge (g) the posterior limb was damaged in processing, though the area of Hoxd13 expression is intact. The inset in a is an earlier bud stage between that of ridge and image shown in b. Scale bars = 0.2 mm

Fig. 7

Hoxa13 Expression in Stages of Mouse, Opossum, and Bat Paddle Stages. a, a’: Fore- and hindlimb of mouse, respectively. b, b’: Opossum, c, c’: Bat

Fig. 8

Evx2 Expression in Limb Stages of Mice and Opossums. a-b, a’-b’: Mouse Evx2 in forelimb and hindlimb of the bud and paddle, respectively. c-d, c’-d’: Opossum Evx2 in forelimb and hindlimb of bud and paddle, respectively. The insets in a and a’ are mouse Evx2 in earlier fore- and hindlimb buds, respectively. Scale bars = 0.2 mm