The metabolic pathway map showing changes in each metabolite levels per individual yolk sac (open column) or embryo (filled column) during development. The horizontal axes represent developmental stages {stages 4, 24 (glucose and glycogen only), 27, 31, 32} and the vertical axes represent nmol/sample. Data are presented as mean ± standard error (N = 6). Different letters indicate significant differences (p < 0.05) between groups. Tests for significant differences were performed by one‐way ANOVA and Tukey's post‐hoc test separately for yolk sac or embryo samples after log transformation. Fructose‐1,6BP, fructose 1,6‐bisphosphate; ‐P, ‐phosphate. Genes responsible for respective pathways were shown beside the arrows. For the complete map with all metabolites measured in this study, please refer to Figure S2.

LC–MS‐based isotope tracking of yolk sac membrane (YSM) in stage 31. Abundance of M + 3 isotopologues of glucose, glucose‐6‐phosphate (‐P), fructose‐6‐P, glucose‐1‐P, and sedoheptulose‐7‐P in the YSM after 3‐h incubation with ¹³C‐labeled substrates. Horizontal axes show each ¹³C‐labeled substrate and the control without them, and vertical axes show the levels of M + 3 (as % M + 0). Data are presented as mean ± standard error (N = 9), and asterisks (*) indicate significant differences (p < 0.05) between the control and each group. Tests for significant differences were conducted with one‐way ANOVA followed by Dunnett's test after log‐transformation of the values. N/A, not available.

Changes in expression levels of the selected genes during development. The expression levels of gluconeogenesis‐related genes and glucose transporter (GLUT) gene slc2a2 were measured. Horizontal and vertical axes indicate developmental stages and mRNA levels (× 10⁹ copies/g RNA), respectively. Open and filled columns indicate the mRNA levels in the yolk sac membrane (YSM) and embryos, respectively. Data are presented as mean ± standard error (N = 6), and different letters indicate significant differences (p < 0.05) between groups. Tests for significance were performed by two‐way ANOVA and Tukey's post‐hoc test. *I indicates a significant interaction between the two factors (developmental stage and site), and *S and *P indicate significant main effects of developmental stage and the position (yolk sac or embryo), respectively. For other genes assessed in this study, please refer to Figure S3.

In situ hybridization analysis for g6pc1, fbp1, pck2, gpd1, ldha, and slc2a2 at yolk sac membrane (YSM) at stage 27. For all transcripts, sense probes (right) were used as negative controls for antisense probes (left), which showed true signals. Positive signals for transcripts are indicated by arrowheads. Hematoxylin and eosin (HE) staining shows general morphology of the YSM in stage 27. se, squamous epithelium; yp, yolk platelet; YSN, yolk syncytial nuclei. Scale bars = 50 μm.

In situ hybridization analysis for g6pc1, fbp1, pck1, pck2, gpd1, gpd1l, ldha, pc, and slc2a2 at yolk sac membrane (YSM) at stage 31. Black arrowheads indicate signals in YSL‐like tissue (YSL‐L) and their nuclei (YSN), while white arrowheads indicate the signals in yolk‐sac endoderm (YSE). Hematoxylin and eosin (HE) staining shows general morphology of YSM in stage 31. fct, fibrillar connective tissue; se, squamous epithelium; yp, yolk platelet. Scale bars = 50 μm.

In situ hybridization for g6pc1, fbp1, pck1, pck2, gpd1, gpd1l, ldha, pc, and slc2a2 at yolk sac membrane (YSM) in stage 32. Black arrowheads indicate signals in YSL‐like tissue (YSL‐L) and their nuclei (YSN), while white arrowheads indicate the signals in yolk‐sac endoderm (YSE). Hematoxylin and eosin (HE) staining shows general morphology of YSM in stage 32. fct, fibrillar connective tissue; se, squamous epithelium; yp, yolk platelet. Scale bars = 50 μm.

Model of functional changes in the yolk sac over the near‐prehatching stages. (a) Until stage 31, where pre‐hatching takes place, yolk degradation, absorption, and gluconeogenesis occur mainly in the yolk sac; (b) At stage 32 or later, yolk degradation and absorption become more active in both embryonic gut and yolk sac, and glucogenesis and other processes occur actively in the embryo.