Four-limbed vertebrates, known as tetrapods, have two enlarged areas in their spinal cords. The two enlargements have a correlation with the forelimbs and hind limbs, respectively. These enlargements are thought to be caused by the complex muscular system and the rich sensory networks supplying nerves to the limbs.
Meanwhile, it was long thought that fish had no enlarged areas in their spinal cords due to the absence of limbs. However, a recent study by scientists from Nagoya University in Japan has revealed that zebrafish, in fact, have enlarged areas in their spinal cords, although these areas are not visible to the naked eye.
"We thought that fish also have spinal enlargements because they have paired pectoral and pelvic fins, which correspond to forelimbs and hind limbs in tetrapods, respectively," said Naoyuki Yamamoto , a professor at Nagoya University's Graduate School of Bioagricultural Sciences and the lead author of the study.
In a paper published in the journal Brain, Behavior and Evolution , Professor Yamamoto, along with his colleagues Ryo Takaoka and Assistant Professor Hanako Hagio , investigated whether their hypothesis was valid.
To determine whether certain regions of the spinal cord are enlarged at spinal levels innervating (supplying nerves to) the fins of zebrafish, the researchers first needed to identify which regions of the spinal cord are responsible for innervating each fin: the paired pectoral and pelvic fins, as well as the unpaired dorsal, caudal, and anal fins.
Since the innervation of pectoral, dorsal, and caudal fins had already been reported, the researchers focused on the innervation of pelvic and anal fins. They stained the entire body of the zebrafish specimen using immunohistochemistry, a technique that specifically labels the cell bodies and axons of neurons. The specimen was clarified using a modified version of the CUBIC (clear, unobstructed brain imaging cocktails) method to visualize and identify the deep spinal nerves that connect to the pelvic and anal fins.
They then created serial tissue sections along the entire length of the spinal cord. Using these sections, they examined changes in the cross-sectional areas of both the spinal cord and the gray matter, referring to the levels of the spinal cord that innervate each fin.
These analyses revealed that the spinal cord and gray matter had expanded, innervating not only paired pectoral and pelvic fins but also unpaired dorsal, anal, and caudal fins in zebrafish.
"We showed the presence of spinal enlargements in zebrafish, although they are modest and can only be detected through histological analysis," Yamamoto stated. "Furthermore, we demonstrated that these enlargements are found in all fins—that is, both paired and unpaired fins."
The findings suggest a new evolutionary theory: when tetrapods, which evolved from fish, moved onto land, only the paired fins—adapted for locomotion—transformed into limbs, while the unpaired fins disappeared.
