Limb proportions show developmental plasticity in response to embryo movement

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.

Figure 1. Motility and growth of West African Dwarf crocodile (Osteolaemus tetraspis) embryos incubated at 28 °C or 32 °C (n = 3 per group) and euthanised at E70, approximately hatching age.

(a) Individuals from the 28 °C (left) and 32 °C (right) groups, demonstrating large differences in growth. Scale bar = 20 mm. (b) Number of embryo movements per 3 minute monitoring period during mid- to late incubation. (c) Body (snout to vent) length upon euthanasia. (d) Limb length. (e) Relative limb length (% snout-cloaca length). (f) Limb element lengths (% total limb length). Values are shown as mean ± SEM and were analysed using Welch’s t-test. *Indicates P < 0.05, ***indicates P < 0.001.

Figure 2. Total hindlimb length is reduced by removal of embryo movement at selected time-points during development.

Mean ± SEM total limb length in control (n = 5) and immobilized (n = 4) chicken embryos is unchanged by (a) immobilization between E10–14. Total limb length is reduced by immobilization between (b) E13–16 and (c) E15–18 when compared by Mann-Whitney U-tests (P < 0.05 and 0.001 respectively). (d) MRI monitoring of mean ± SEM limb length daily in control (n = 6) and immobilized (n = 4) chicks. *Indicates P < 0.05.

Figure 3. Limb proportions are altered by immobilization of embryonic chicks at selected time-points during development.

Mean ± SEM lengths of femur, tibiotarsus (TBT) and tarsometatarsus (TMT) in control (n = 5) and immobilized (n = 4) chicken embryos is unchanged by (a) immobilization between E10–14. (b) Mean length of the femur and TMT but not TBT is reduced by immobilization between E13–16. (c) The length of all elements is reduced by immobilization between E15–18 when analysed by Mann Whitney U-test. MRI monitoring of mean ± SEM limb growth daily in control (n = 6) and immobilised (n = 4) chickens: (d) femur, (e) TBT and (f) TMT. *Indicates P < 0.5 and **indicates P < 0.01.

Figure 4. Altered embryo motility results in targeted changes in growth plate dynamics in the embryonic chick.

(a) Growth plate of an embryonic day 18 (E18) control and (b) immobilized embryos (treated from E10 onwards) stained with toluidine blue. The proliferative/maturing zone (P/M), prehypertrophic (PH) and hypertrophic (H) zones are indicated by dotted lines. Quantification of average width ± SEM of (c) proliferative/maturing, (d) prehypertrophic and (e) hypertrophic zones as % total growth plate width in E18 control and immobilised limbs (n = 5 in each group). These were analysed by Mann-Whitney U-test. (f) Average width ± SEM of proliferative, prehypertrophic and hypertrophic zones in the distal femur of control & immobilised chick limbs (n = 4) at E14 shows a lack of modified growth plate dynamics at earlier stages. Values for control limbs are shown in black and immobilised in grey. Scale bar represents 200 μm. *Indicates P < 0.05, **indicates P < 0.01.

Figure 5. Proliferation in the embryonic chicken growth plate is disrupted by the removal of mechanical stimuli.

PCNA expression in (a) control and (b) immobilized distal femur at E14. The mean ± SEM % total cells which express PCNA in the 1) articular cartilage, 2) “resting zone” cartilage, 3) proliferative zone and 4) prehypertrophic/hypertrophic zone was quantified in (c) control and (d) immobilised (n = 5 for each group) limbs at E14, after 4 days of treatment. (e) Mean ± SEM % total cells in each zone. Phosphohistone H3 expression in the (f) control and (g) immobilized distal femur at E14. The mean ± SEM % total cells that are phosphohistone H3 positive was quantified from a representative view of the epiphysis of (h) control and (i) immobilized limbs at E14, after 4 days of treatment, and E18, after 8 days of treatment (n = 5 in each group). (j) Mean ± SEM % total cells phosphohistone H3 positive. BrdU incorporation in (k) control and (l) immobilized E16 chick limbs sacrificed 4 hours after BrdU administration, after 6 days of treatment. The mean ± SEM % total cells which are BrdU positive in the proliferative zone (shown by dotted lines) or the prehypertrophic/hypertrophic zones of (m) control and (n) immobilised limbs (n = 5 in each group; indicated by yellow squares) 4 hours after BrdU administration was quantified. (o) Mean ± SEM % total cells phosphohistone positive in these regions. Mean values from the two treatment groups were analysed by Mann-Whitney U test. *Indicates P < 0.05. Scale bars represent 200 μm.

Figure 6. Differential expression of mTOR associated genes characterise femur and TBT elements at E15.

(a) Upon analysis of E15 femur versus TBT (SAMR analysis), it was noted that 59 genes were differentially regulated between these limbs. These included ‘structural constituent of ribosome’ (p < 1E-08) and metabolic genes. Ingenuity up-stream analysis of this gene-list noted that less mTOR activity (p < 1E-09) was predicted in the femur, compared with the TBT (i.e. rapamycin being the inhibitor of mTOR) based on analysis of 54/59 genes in the database. mTOR regulated genes, shown in blue, are expressed at a lower level in E15 femur than E15 TBT. (b) The expression of these mTOR regulated genes was not significantly different between limbs at E12/13. Note that SLC38A3 expression is omitted from this graph due to its disproportionately higher expression level in the femur obscuring the other gene expression values. (c) Expression of the mTOR associated genes in the femur and TBT in growth cartilage in control and immobilised chicks. Expression patterns of these mTOR associated genes were unaltered by immobilisation. Femur data are in red and TBT in blue. Horizontal lines indicate mean expression values for all genes (n = 2–3 at each time point).

Figure 7. Gene expression in limb element growth cartilage in response to development and immobilisation.

Analysis of changes in gene expression from E12/13 to E15 in both femur and TBT identified a robust expression of skeletal muscle genes at E12/13 followed by a strong down-regulation. Up-stream analysis of these genes identified that this included inhibition of MEF2c (p < 1E-10) (a) Plotting the 17 ‘myogenic’ genes directly associated with MEF2c finding, we noted these were further down-regulated during immobilisation in both the femur and TBT at E15 (plotting all muscle genes would have yielded a similar pattern). (b) Analysis of gene expression differences between DMB treated E15 femur and DMB treated E15 TBT (SAMR analysis) revealed that genes regulated by greater Wnt3a activity (Z = 2.1, p < 1E-10) and greater CTNNB1 (Z = 2.1, p < 1E-9) activity, were expressed at a greater level in the femur than TBT. Genes expressed at a higher level in DMB treated E15 femur are shown in red and lower-level are shown in blue. (c) The genes down-stream of Wnt3a are plotted using data from femur and TBT in growth cartilage from control and immobilised (DMB treated) chicks. Horizontal black lines indicate mean expression values for each gene.