Regulatory modulation of the T-box gene Tbx5 links development, evolution, and adaptation of the sternum

Abstract

The sternum bone lies at the ventral midline of the thorax where it provides a critical attachment for the pectoral muscles that allow the forelimbs to raise the body from the ground. Among tetrapods, sternum morphology is correlated with the mode of locomotion: Avians that fly have a ventral extension, or keel, on their sterna, which provides an increased area for flight muscle attachment. The sternum is fused with the ribs attaching on either side; however, unlike the ribs, the sternal precursors do not originate from the somites. Despite the crucial role of the sternum in tetrapod locomotion, little attention has been given to its acquisition, evolution, and embryological development. We demonstrate an essential role for the T-box transcription factor gene Tbx5 in sternum and forelimb formation and show that both structures share an embryological origin within the lateral plate mesoderm. Consistent with this shared origin and role of Tbx5, sternum defects are a characteristic feature of Holt-Oram Syndrome (OMIM 142900) caused by mutations in TBX5. We demonstrate a link between sternum size and forelimb use across avians and provide evidence that modulation of Tbx5 expression underlies the reduction in sternum and wing size in a flightless bird, the emu. We demonstrate that Tbx5 is a common node in the genetic pathways regulating forelimb and sternum development, enabling specific adaptations of these features without affecting other skeletal elements and can also explain the linked adaptation of sternum and forelimb morphology correlated with mode of locomotion.

Keywords: Tbx5; sternum adaptation; sternum defects; sternum development.

Fig. 1.

Sternum length and keel height are correlated with mode of locomotion in avians. (A) Scatterplot of measurements for sternum length and keel height, normalized for bird size by dividing by thorax length, for a range of bird groups. Each point on the graph represents one species. When possible, multiple specimens were measured per species. Error bars show SE between multiple specimen measurements. Flying species are represented as diamonds; flightless species as triangles. Thoracic skeletons of volant cormorant (Phalacrocorax carbo) with the keel indicated (white arrow) (B) and flightless cormorant (Phalacrocorax harissi) with the reduced keel indicated (black arrow) (C).

Fig. 2.

The sternum precursor cells reside in the LPM, ventral to the forelimb bud. (A) Schematic of transverse section through HH20 chick showing LPM subdivision into somatic and splanchnic domains. (B) Schematic of DiI injection sites and adjacent somites with sternum precursor population highlighted (blue). Ventral whole-mount view (C) and transverse section of HH20 embryos showing DiI-labeling (arrows) (D) following injection into site 4. Limb bud is labeled LB; dorsal aorta is labeled DA. (E–G) Ventral whole-mount view of harvested, skinned HH36 embryos showing DiI-labeled cells at the midline (boundaries of population shown by white arrowheads) following injection into HH20 embryos at sites 2, 4, and 6, respectively. (H) Transverse section through a harvested embryo showing DiI-labeling in the sternum (arrow). S, sternum.

Fig. 3.

The sternal bands and forelimbs fail to form in the absence of Tbx5. Ventral views of control (A and D), Tbx5 conditional mutant (Tbx5^lox/lox;Prx1Cre) (B and E), and Fgf10 mutant (Fgf10^−/−) (C and F) embryos at E17.5. In whole-mount (A–C) and Alcian Blue/Alizarin Red (D–F) skeletal preparations. The most distal forelimb structures have been cropped in the control image. (G–L) Ventral views of Runx1 expression in the sternal precursors (arrows) at E12.5 and E13.5 in control, Tbx5^lox/lox;Prx1Cre, and Fgf10^−/− mouse embryos. Herniation of the internal organs following the failure of body wall closure present in H and K. (M–O) Ventrolateral view showing Tbx5 expression in the forelimb and ventral body wall detected by in situ hybridization in E10.5, 11.5, and 12.5 mouse embryos..(P) Ventrolateral view of HH22 chick showing Tbx5 expression in the forelimb bud (FL) and LPM ventral to the limb bud (black arrow). (Q and R) Ventrolateral views of Emu embryos at eqHH22 and eqHH23, respectively, showing expression of Tbx5 in the small forelimb bud (FL) and LPM ventral to the limb bud (black arrows).

Fig. 4.

Modulation of Tbx5 expression accompanies forelimb and sternum adaptation in the emu. (A–I) In situ hybridization showing Tbx5 expression in the forelimb-forming region (bracket), forelimb (FL), and heart (*) of chick (A–D) and emu (F–I) embryos. (K–N) In situ hybridization showing Pitx1 expression in the emu hindlimb forming region (bracket) and hindlimb (HL). (E and J) Lateral view of Alcian Blue/Alizarin Red-stained chick and emu skeletons at days 10 and 27, respectively, highlighting forelimbs (yellow) and sterna (orange). (A–M) Lateral views. (N and Inset) Dorsal views.