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Anatomical integration of the sacral–hindlimb unit coordinated by GDF11 underlies variation in hindlimb positioning in tetrapods


Elucidating how body parts from different primordia are integrated during development is essential for understanding the nature of morphological evolution. In tetrapod evolution, while the position of the hindlimb has diversified along with the vertebral formula, the mechanism responsible for this coordination has not been well understood. However, this synchronization suggests the presence of an evolutionarily conserved developmental mechanism that coordinates the positioning of the hindlimb skeleton derived from the lateral plate mesoderm with that of the sacral vertebrae derived from the somites. Here we show that GDF11 secreted from the posterior axial mesoderm is a key factor in the integration of sacral vertebrae and hindlimb positioning by inducing Hox gene expression in two different primordia. Manipulating the onset of GDF11 activity altered the position of the hindlimb in chicken embryos, indicating that the onset of Gdf11 expression is responsible for the coordinated positioning of the sacral vertebrae and hindlimbs. Through comparative analysis with other vertebrate embryos, we also show that each tetrapod species has a unique onset timing of Gdf11 expression, which is tightly correlated with the anteroposterior levels of the hindlimb bud. We conclude that the evolutionary diversity of hindlimb positioning resulted from heterochronic shifts in Gdf11 expression, which led to coordinated shifts in the sacral–hindlimb unit along the anteroposterior axis.

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Fig. 1: Topographical relationship between the limbs and vertebral formula is conserved in tetrapods.
Fig. 2: When Gdf11 expression is initiated, the pAM and the adjacent LPM correspond to the prospective sacral vertebrae and hindlimb regions.
Fig. 3: GDF11 integrates hindlimb positioning with the sacral vertebrae by coordination of 5′ Hox gene expression in both the pAM and the LPM.
Fig. 4: The onset of GDF11 activity in the LPM is essential for the determination of hindlimb positioning in chick embryos.
Fig. 5: Species with more posterior hindlimb primordia in the LPM show later onset of Gdf11 expression.


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We thank Y. Yamamoto-Shiraishi for discussions; G. Sheng for help with collecting the emu embryos; and A. Sakai for help with collecting the snake embryos. This work was supported by JST PRESTO, Grants-in-Aid for Scientific Research—KAKENHI grant numbers 2529150, 15KT0150, 16H01444 and 17H05764.

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Y.M., A.K. and T.Suz. conceived the project and designed the experiments. Y.M. performed most experiments; T.H. performed the statistical analyses; S.E. collected ocelot gecko embryos; A.H. performed implantation experiments. T.Sug. and Y.K. performed the cell-tracking study for African clawed frog embryos; T.N. performed gene-expression studies; K.T. contributed ocelot gecko embryos; and Y.M., T.H., S.K., A.K and T.Suz. wrote the paper, with comments from co-authors.

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Correspondence to Atsushi Kuroiwa or Takayuki Suzuki.

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Animated model regarding anatomical integration of the sacral–hindlimb unit and the mechanism that brought about variation in hindlimb positioning in tetrapod evolution.

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Matsubara, Y., Hirasawa, T., Egawa, S. et al. Anatomical integration of the sacral–hindlimb unit coordinated by GDF11 underlies variation in hindlimb positioning in tetrapods. Nat Ecol Evol 1, 1392–1399 (2017).

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