Splinter was the pet of a ninja by the name of Hamato Yoshi. Yoshi and a woman named Tang Shen loved each other, but a third party, the jealous and malicious Oroku Nagi wanted Shen all to himself. He attacked Shen after she insisted that she loved only Yoshi; in response, Yoshi beat Nagi to death to save his beloved.
As this was a crime of honor, Yoshi was necessitated by the Foot Clan to take his own life. However, he knew in his heart that he had done nothing wrong, so he fled with Shen and Splinter to America. Eventually, Nagi's younger brother Saki tracked down the couple and slew them both. In the scuffle, Splinter's cage broke open, and he leapt on Saki's face, clawing up his cheek. In response, Saki nicked Splinter's ear with his katana and left him among his dead human family.
Splinter wandered the streets of New York City until one day, a truck swerved to avoid a blind man crossing the street. A canister of mutagen ooze bounced out and struck a glass bowl that was held by a boy, dumping his newly-purchased pet turtles into the sewer drain along with the shattered canister. Splinter scuttled after them, and scooped the baby turtles up in an old coffee can. The next morning, he awoke to find that the turtles had doubled in size, and he had, too. Before long, they were all growing and learning. Over the course of time, Splinter taught them the ninjutsu movements he'd often watched his Master Yoshi perform, and named them for European Renaissance artists based on a book he'd found in the sewer.
Fifteen years after this event, Oroku Saki began calling himself The Shredder and ran the Foot Clan like a street gang, taking in teens and using them as petty thieves to work their way up to becoming Foot Ninja. These actions caught the attention of a local newshound, April O'Neil, and her perseverance in uncovering the truth drew attention to her from Shredder. The turtles saved her once before from some hoodlums, but on a off chance, Raphael came across her being jumped by the Foot and took her back to their lair after she was knocked unconscious during the fight. April awoke, startled at those around her, but Splinter calmed her and told her of his and the turtles' origins.
The turtles walked April home and stayed for pizza, but in their absence, the Foot had taken Splinter. Chained up like a test animal, Shredder made every effort to break his spirit. Through a series of events, the turtles, April, and new friend Casey Jones retreated to April's family farmhouse. Eventually, Leonardo caught a communication from Splinter whilst meditating, leading his brothers around a campfire to attempt to speak to Splinter again. Splinter projected his astral form to the fire, imparting potential final words of wisdom to them and telling them that he loves them. With renewed spirit, the turtles head back to New York to rescue their father.
Meanwhile, Danny Pennington, the son of April's boss Charles and a Foot hopeful, had begun holding commune with Splinter, starting to see through the cracks in Shredder's promise of family. He tells the young Pennington of his origins in Japan, and tells him that his family's murderer wears the same symbol as this clan that he has joined. Shredder catches Danny and finds a drawing of Raph that he had taken from April, cluing Shredder in that the turtles are still alive.
Danny ran and collided with Casey, who was about to pound him when he said that they were going to kill Splinter. Casey realized that they needed to work together. Danny stole the key to Splinter's chains, and Casey got him free. Shredder's second-in-command, Tatsu, tried to stop them, but Casey knocked him out cold.
Up on a rooftop, the turtles faced Shredder and were not successful in subduing the ninja master. Splinter appeared and confirmed to Shredder of his identity as the rat who had scratched his face all those years ago, and Shredder vowed to take the remainder of the rat's ear. He charged Splinter with his yari, but Splinter countered with a pair of nunchaku and flipped him overhead, leaving him to dangle over the side of the roof. Splinter tried to warn Shredder about his lack of honor, but Shredder proved his point by trying to throw a tantō at him. Splinter caught the blade before it struck, but loosened his grip on the nunchaku, sending Saki to the ground below to his presumed death.
However, this was not to be the end of Oroku Saki. The Shredder had somehow ended up in a landfill, the same one in which Tatsu and the regrouping Foot had made their new headquarters. Eager on revenge, once he learned of Techno Global Research Industries' attempts at disposing the last of what became later known as mutagen ooze, he sent Tatsu out for it. Meanwhile, after seeing April's interview with T.G.R.I. scientist Jordan Perry, Splinter realized he could no longer hide the truth from his sons. He showed them the canister the ooze came in, with T.G.R.I. written clearly on it.
Later, when the pizza delivery boy the turtles met before, Keno came to April's apartment to find them, Splinter told Keno of their origin story. Keno proposed infiltrating the Foot as a potential recruit, and Raph agreed with the idea. Splinter, however, did not, and forbid the plan from bearing fruit. But, with Splinter having no stewardship over Keno, and Raph being Raph, they went ahead and did it anyway - a maneuver which would get Raphael captured.
Keno ran to tell April and the other turtles of Raph's capture. The others raced to his rescue, but the endeavor was a trap, and they were caught in a net suspended from a crane. Just before they were moved above some rusty spikes, Splinter arrived and severed the net with an arrow from afar.
After the turtles had escaped and brought Professor Perry with them down to the new lair, Donatello bemoaned that he felt disappointed that there wasn't more to the ooze and to their existence, having found out they amounted to nothing more than an accident and now there were two giant, purposeful mutants running amok. Splinter consoled his son by telling them "Do not confuse the specter of your origin with your present worth."
As the turtles went full-on to their final battle with the Foot, Splinter and Keno meditated. Splinter was just trying to keep Keno occupied so he wouldn't rush headstrong into this dangerous fight. The rat finally relented that he had no authority to keep Keno there, and the boy rushed off to help.
After the turtles finally defeated Shredder, perhaps finally once and for all, they returned to the lair. Splinter asked them if they were seen, and they, of course, denied that they were. At this point, Splinter produced a newspaper headline with their photo on the front, reading "Ninja Rap is born!" He "punishes" the turtles by telling them to do twenty flips.
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