Normal vertebral and intervertebral micro-anatomy of the guppy P. reticulata. (A) Whole-mount skeletal preparation of tail vertebrae shown in lateral view. Vertebral centra in P. reticulata are amphicoelous and bear paired arches dorsally and ventrally. (B-G) Micrographs of guppy vertebral sections stained by either Mallory's trichrome for collagen (B,C) or with a combination of Picrosirius Red and Alcian Blue (D-G), which stain for collagen and sulfated proteoglycans, respectively. (B) Sagittal section through vertebral column, showing three successive intervertebral ligaments (visible in whole-mount in boxed area in panel A). (C) Magnified view of boxed area in B. The intervertebral ligament of P. reticulata comprises two structures: an outer ring-like structure and a spongy, notochord-derived tissue that forms the core of the ligament and runs between adjacent centra. For clarity, these structures have been digitally colorized in panel C'. The spongy layer stains strongly for collagen, while the outer ring-like structure is enriched for sulfated proteoglycans (arrowhead in inset). (D-G) Consecutive transverse sections through a single vertebra, spanning the intervertebral region to the tapered middle of the centrum (see vertical lines in panel B for approximate axial level). Remnants of the embryonic notochord line the inside of the vertebral centrum and, at intervertebral levels, form a wall between vertebrae. Inset in panel D depicts a schematized intervertebral ligament in transverse section; colors correspond to those shown in panel C'. Abbreviations: ao, aorta; ha, hemal arch, m, axial muscle; na, neural arch; sc, spinal cord, v, vacuole; vc, vertebral centrum. Scale bar equals 200 μm.