Digital gene expression tag profiling of bat digits provides robust candidates contributing to wing formation

Abstract

Background: As the only truly flying mammals, bats use their unique wing - consisting of four elongated digits (digits II-V) connected by membranes - to power their flight. In addition to the elongated digits II-V, the forelimb contains one shorter digit (digit I) that is morphologically similar to the hindlimb digits. Here, we capitalized on the morphological variation among the bat forelimb digits to investigate the molecular mechanisms underlying digit elongation and wing formation. Using next generation sequencing technology, we performed digital gene expression tag profiling (DGE-tag profiling) of developing digits in a pooled sample of two Myotis ricketti and validated our sequencing results using real-time quantitative PCR (RT-qPCR) of gene expression in the developing digits of two Hipposideros armiger.

Results: Among hundreds of genes exhibiting significant differences in expression between the short and long digits, we highlight 14 genes most related to digit elongation. These genes include two Tbx genes (Tbx3 and Tbx15), five BMP pathway genes (Bmp3, RGMB, Smad1, Smad4 and Nog), four Homeobox genes (Hoxd8, Hoxd9, Hoxa1 and Satb1), and three other genes (Twist1, Tmeff2 and Enpp2) related to digit malformations or cell proliferation. In addition, our results suggest that Tbx4 and Pitx2 contribute to the morphological similarity and five genes (Acta1, Tnnc2, Atp2a1, Hrc and Myoz1) contribute to the functional similarity between the thumb and hindlimb digits.

Conclusions: Results of this study not only implicate many developmental genes as robust candidates underlying digit elongation and wing formation in bats, but also provide a better understanding of the genes involved in autopodial development in general.

Figure 1

Limbs of the adult and fetal bats (modified from our previous study [18]). (A) Left limbs of adult Myotis ricketti. DI, DII, DIII, DIV and DV represent digits I-V of the forelimb; (B, C) Left limbs of Miniopterus schreibersii fuliginosus in the Fetal Stage as an example of samples used for the Myotis ricketti libraries. Libraries Hand DI and Hand DII-V are constructed from forelimb digit I and digits II-V, respectively. Library Foot is constructed from hindlimb digits I-V. Bar = 1 cm in A; bar = 1 mm in B and C.

Figure 2

Saturation measured as the number of new distinct tags added with increasing sequencing depth in each library Hand DI, Hand DII-V and Foot. FRQ indicates the frequency of distinct tags in the library.

Figure 3

Pairwise comparison of distinct tag expression between all three libraries (Hand DI, Hand DII-V and Foot) using DEGseq software. Genes exhibiting significant differences in expression are provided in red (p < 0.001), genes with similar expression in the compared libraries are shown in black.

Figure 4

The number of genes differentially expressed between distinct digit morphologies and digit limb association (p < 0.001). (A) Comparison between the elongated wing digits (Library Hand DII-V) and the short digits (Libraries Hand DI and Foot). (B) Comparison between the forelimb digits (Libraries Hand DII-V and Hand DI) and the hindlimb digits (Library Foot).

Figure 5

Patterns of gene expression in hand digit I (HDI), hand digits II-V (HDII-V), and all five foot digits (Foot). To validate DGE-tag profiling, expression of 14 genes, exhibiting differences in expression among the M. ricketti libraries, were examined in two independent H. armiger embryos using RT-qPCR. (A) Meis2; (B) Hoxd10; (C) Tbx3; (D) Tbx15; (E) Bmp3; (F) Rgmb; (G) Twist1; (H) Hoxa1; (I) Tmeff2; (J) Enpp2; (K) Krt17; (L) Tbx4; (M) Acta1; (N) Tnnc2. The overall pattern and significance of gene expression differences among the samples were concordant using the distinct methods and taxa. The fold changes in expression among samples were reduced in the RT-qPCR in several cases; however this difference is not surprising given the difference in sensitivity of gene detection in the two methods. The y-axis indicates fold change in expression between the samples - HDI, HDII-V, and Foot - using the results from RT-qPCR (dotted bars) and DGE-tag profiling (grey bars) independently. Significance of pairwise comparisons (HDI vs. HDII-V and Foot vs. HDII-V) are indicated by the asterisks between the bars. *, p < 0.05; **, p < 0.01; ***, p < 0.001.