Fig. 2

Morphometric analyses of the hindlimb in WT and HKM at different developmental stages. A Dissecting the hindlimb buds of WT and HKM embryos using Leica Microsystems (E10.5–E12.5). Scale bar, 1:2500 μm. B–D ImageJ software was used to measure the area of hindlimb bud area for t-test analysis, B E10.5, C E11.5, D E12.5, Scale bar, 1:2500 μm. E Double staining of the hindlimb skeleton elements with alcian blue (blue; cartilage) and alizarin red (violet; bone), top: E18.5 embryos (n = 14), bottom: P1 embryos (n = 12). Pg: pelvis girdle; Fe: femur; Ti: tibia; Fi: fibula, D1-D5: digit1, digit2, digit3, digit4, digit5. Scale bar, 1:2500 μm. F, G Proportional results of hindlimb skeletal element lengths relative to body length (from the cervical to the caudal spine) at E18.5 and P1. The length of individual elements of the hindlimb skeleton was quantified using LAS X software (https://www.leica-microsystems.com.cn/cn/products/microscope-software/p/leica-las-x-ls/), while body length was measured with vernier calipers for t-test analysis, F E18.5, G P1. Scale bar, 1:2500 μm. All the above measurements were performed in three independent replicates. The results were shown as Mean ± SD (standard deviation), and asterisks indicate significant differences (*: p < 0.05, **: p < 0.01, ns: Not significant)