Development of bat flight: morphologic and molecular evolution of bat wing digits

Abstract

The earliest fossil bats resemble their modern counterparts in possessing greatly elongated digits to support the wing membrane, which is an anatomical hallmark of powered flight. To quantitatively confirm these similarities, we performed a morphometric analysis of wing bones from fossil and modern bats. We found that the lengths of the third, fourth, and fifth digits (the primary supportive elements of the wing) have remained constant relative to body size over the last 50 million years. This absence of transitional forms in the fossil record led us to look elsewhere to understand bat wing evolution. Investigating embryonic development, we found that the digits in bats (Carollia perspicillata) are initially similar in size to those of mice (Mus musculus) but that, subsequently, bat digits greatly lengthen. The developmental timing of the change in wing digit length points to a change in longitudinal cartilage growth, a process that depends on the relative proliferation and differentiation of chondrocytes. We found that bat forelimb digits exhibit relatively high rates of chondrocyte proliferation and differentiation. We show that bone morphogenetic protein 2 (Bmp2) can stimulate cartilage proliferation and differentiation and increase digit length in the bat embryonic forelimb. Also, we show that Bmp2 expression and Bmp signaling are increased in bat forelimb embryonic digits relative to mouse or bat hind limb digits. Together, our results suggest that an up-regulation of the Bmp pathway is one of the major factors in the developmental elongation of bat forelimb digits, and it is potentially a key mechanism in their evolutionary elongation as well.

Fig. 1.

The relative length of bat forelimb digits has not changed in 50 million years. (a) Icaronycteris index (American Museum of Natural History specimen no. 125000), which is a 50-million-year-old bat fossil. (b) Extant adult bat skeleton. The metacarpals (red arrows) of the first fossil bats are already elongated and closely resemble modern bats. This observation is confirmed by morphometric analysis of bat forelimb skeletal elements. (c) Results of regression analysis of the log of fifth metacarpal length versus a proxy for body size (PC1). (c and d) Extinct bats are indicated by red crosses, and modern bats are indicated by blue circles. Modern and extinct bats fall along the same regression line, indicating that their metacarpals are similar in relative length.

Fig. 2.

Developmental elongation of bat digits occurs after the initial cartilage condensations are formed. (a and b) Cartilage revealed by Alcian blue staining of stage-16 bat forelimb (a; courtesy of C. Cretekos, University of Texas M. D. Anderson Cancer Center) and E12.5 mouse forelimb (b; courtesy of M. Barna, Fox Chase Cancer Center, Philadelphia). (c) Percentage of metacarpal composed of resting (white), proliferative (green), prehypertrophic (yellow), hypertrophic (blue), and ossification (red) zones in mice (Left) and bats (Right) at stages 18–24. The size of the hypertrophic zone is initially comparable in bat and mouse digits, and it then increases in relative size in the bat forelimb at stage 20. (d) Size of the bat hypertrophic zone (blue; measured as a percentage of the total length of the fifth metacarpal) and length of the bat fifth metacarpal (purple; measured in micrometers) against bat developmental stage. At stage 20, the hypertrophic zone greatly increases in size, along with the beginning of a phase of exponential lengthening of the bat digits.

Fig. 3.

Proliferation and Bmp levels are increased in bat forelimb digits relative to mouse forelimb and bat hind limb digits. (a–c) phospho-histone H3 (Ser-10) Ab staining of proliferating cells in a stage-20 bat metacarpal (a), stage-20 bat metatarsal (b), and E14.5 (stage 20) mouse metacarpal (c). Bat forelimb metacarpals show areas of increased proliferation in the growth plate (white arrows). (d–g) Bmp2/4 Ab staining (red; blue is hematoxylin counterstain) of an entire stage-20 bat metacarpal (d) and high-magnification views of the bat metacarpal perichondrium (e), a stage-20 bat metatarsal perichondrium (f) and an E14.5 mouse metacarpal perichondrium of a comparable region (g). (i–k) Phospho-Smad 1/5/8 Ab staining and a high-magnification view of a similar region of the perichondrium as shown in d–g of a stage-20 bat metacarpal (i), stage 20 bat metatarsal (j), and E14.5 mouse metacarpal (k). Bmp2/4 and phospho-Smad 1/5/8 proteins are similarly localized in bat digits as they are in mice (18), but strikingly, these proteins are more intensely expressed in the perichondrium of bats metacarpals than they are in either mouse metacarpals or bat metatarsals. (h) Semiquantitative RT-PCR of Bmp2 RNA from stage-20 bat and stage-20 mouse metacarpals by using 18S rRNA as a control. Bat Bmp2 RNA is expressed at ≈30% higher levels relative to mouse.