Limb proportions show developmental plasticity in response to embryo movement

A S Pollard,I M Mcgonnell,A A Pitsillides,T Gustafsson,J A Timmons,B G Charlton,J R Hutchinson

doi:10.1038/srep41926

Abstract

Animals have evolved limb proportions adapted to different environments, but it is not yet clear to what extent these proportions are directly influenced by the environment during prenatal development. The developing skeleton experiences mechanical loading resulting from embryo movement. We tested the hypothesis that environmentally-induced changes in prenatal movement influence embryonic limb growth to alter proportions. We show that incubation temperature influences motility and limb bone growth in West African Dwarf crocodiles, producing altered limb proportions which may, influence post-hatching performance. Pharmacological immobilisation of embryonic chickens revealed that altered motility, independent of temperature, may underpin this growth regulation. Use of the chick also allowed us to merge histological, immunochemical and cell proliferation labelling studies to evaluate changes in growth plate organisation, and unbiased array profiling to identify specific cellular and transcriptional targets of embryo movement. This disclosed that movement alters limb proportions and regulates chondrocyte proliferation in only specific growth plates. This selective targeting is related to intrinsic mTOR (mechanistic target of rapamycin) pathway activity in individual growth plates. Our findings provide new insights into how environmental factors can be integrated to influence cellular activity in growing bones and ultimately gross limb morphology, to generate phenotypic variation during prenatal development.

Highlights

Been observed in lizards but has yet to be explained[8]
We tested whether West African Dwarf crocodile embryos incubated at the extremes of the normal range for this species (32 °C and 28 °C) exhibited alterations in motility and limb proportions; we made measurements of body size, limb length and stylopod, zeugopod and autopod element length
We tested whether pharmacological immobilization alone alters limb proportions, when temperature is maintained constant to remove any influence it may have on embryo movement as a variable

Summary

Introduction

Been observed in lizards but has yet to be explained[8]. Phenotypic differences seen in the North American lizard Sceloporus occidentalis include longer limbs relative to body size in individuals from warmer locations, and a reduction in relative limb length in cooler, more northern populations. We explore the hypothesis that developmental plasticity during prenatal stages of ontogeny allows for variation in limb proportions to emerge via environmentally-triggered alterations in movement This hypothesis was tested in experiments using embryonic crocodiles (Osteolaemus tetraspis Cope 1861) and chickens (Gallus gallus). Limb element lengths were measured at selected time-points during development and their growth monitored in some experiments by repeat MRI imaging of individual control and immobilised chicken embryos to examine whether pharmacologically-induced alterations in movement alone produced modifications in limb proportions. Use of the embryonic chick as a model allowed us to identify the mechanisms which underpin regulation of limb proportions by embryonic movement by examining the impact of altered embryo movement on both cellular behaviour and gene expression in individual growth plates. Our findings provide evidence that environmental factors, and genetic pre-specification may differentially influence limb element growth during prenatal development and thereby introduce phenotypic variation, which provides a novel insight into the potential evolutionary importance of developmental plasticity

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Conclusion