First indication of segmentation seen with:
Morphological segmentation first becomes molecularly evident when pair-rule products accumulate in stripes at late syncytial blastoderm after gap domains refined. Unlike gap genes in broad domains, pair-rule proteins exhibit seven regularly spaced bands detectable by in situ hybridization, marking future parasegment boundaries before cellularization. Homeotic genes confer identity later but do not create periodic pattern. Gap genes precede pair-rule but lack periodicity. Observation of eve stripes represents earliest indication of segmentation converting continuous maternal gradients into discrete repeating units, linking nonperiodic information to repetitive body plan architecture characteristic of insect embryos and evolutionary patterning.
Ref: Gilbert, Developmental Biology, 12th ed., Chapter 9: First segmentation indication - pair-rule stripe patterning.