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The origin story in the television series differs greatly from that of the original Mirage Studios comics. In this version, Splinter was formerly a human being, an honorable ninja master named Hamato Yoshi who had studied art history as a hobby. Yoshi was banished from the Foot Clan in Japan after being deceived by the seditious Oroku Saki, who pinned Hamato Yoshi's dogi to the wall with a knife, preventing him from kneeling before their sensei which was seen as an insult. When Yoshi removed the knife, the sensei was again insulted believing Yoshi was drawing the blade in opposition to him. Exiled from the ninja clan, the disgraced Yoshi moved to the United States, specifically New York City. Being penniless, Yoshi is forced to live in the sewers. Back in Japan, Saki has been given command of the Foot Clan, which he corrupts by teaching his students how to commit robberies and other crimes.
While living in the sewers with the rats as his friends, Yoshi one day found four turtles, recently bought from a pet store by an unnamed boy who accidentally dropped them in the sewer. Yoshi returned one day from his explorations around New York to find the turtles covered with a strange glowing ooze. The substance caused the turtles, most recently exposed to Yoshi, to become humanoid, while Yoshi, most recently exposed to sewer rats, became a humanoid rat, and started going by the pseudonym "Splinter". This, and the following Archie TMNT Adventures Comics, is the only origin story in the TMNT franchise where the Turtles come to Yoshi before being exposed to mutagen. Also, Yoshi becomes a rat, whereas in most other versions, he is Yoshi's pet rat that becomes humanoid. This is also the only version in which the Turtles become fully grown immediately after exposure to the mutagen, whereas Splinter raises them from infancy in other versions. Yoshi adopts the four turtles as his sons and trains them in the art of ninjitsu. He names them after his favorite Italian renaissance artists: Leonardo da Vinci (Leonardo), Donato di Niccolò di Betto Bardi (Donatello), Raffaello Sanzio da Urbino (Raphael), and Michelangelo di Lodovico Buonarroti Simoni (Michelangelo). In most versions, the Turtles tend to go by nicknames Leo, Donnie, Raph, and Mikey, but in this version they are always addressed by their full names. Each Ninja Turtle wears a mask over his eyes with a distinctive color, and is trained in the art of a distinct weapon.
Meanwhile, Oroku Saki has left Japan and tracked Yoshi to New York City, where he intends to destroy him once and for all. He has become associated with Krang, a disembodied alien brain who has been banished from his home, Dimension X, where he was a great warlord. Saki has taken on a new persona, donning a suit covered with razor spikes, complemented by a long cape, and a metal mask over his mouth. He has also taken on the pseudonym "The Shredder".
It becomes clear early in the series that the mutagen that transformed the Turtles and Splinter into their new forms was dumped into the sewer by Shredder in an effort to murder Yoshi. Shredder, not knowing the full effect of mutagen at the time, thought he had concocted a deadly poison. The Turtles vow to take revenge on the Shredder for dishonoring their master, as well as turning him into a rat. The Turtles' vendetta evolves into stopping Shredder's ongoing criminal career with the aid of Channel 6 reporter April O'Neil. Another goal of the Turtles is to force Shredder to restore Hamato Yoshi's human form, but this is also dangerous as it could also result in the Turtles reverting to ordinary turtles and losing their humanoid abilities. The Turtles begin to take on the role of vigilante crime-fighters operating outside of the jurisdiction of law enforcement against any criminals, much like Casey Jones in the third season. For the first couple of seasons, it seems as if the Turtles are constantly preoccupied with hiding their existence. This seems to be slowly relaxed and, by the last few seasons, most citizens seem to be well aware of them. They also frequently have to deal with citizens misunderstanding them, thanks to the efforts of Burne Thompson, April's employer, and Vernon Fenwick, a Channel 6 cameraman, who distrust the Turtles and frequently blame them for the trouble that the Shredder and Krang cause. Eventually the Turtles manage to build up a small amount of trust with New Yorkers whom they have saved from Shredder or other villains.
Shredder, Krang, Bebop and Rocksteady (two street thugs morphed into animal form by exposure to the Shredder's mutagen), and a small army of robotic Foot Soldiers try to destroy the Turtles and take over the world. Much of their quest for world domination hinges on bringing the Technodrome (Krang's mobile fortress, and their base of operation) to the surface as it was either stuck in the Earth's core, Dimension X, the Arctic, or on the sea bottom of the Arctic Ocean.
In the last three seasons, the show, which had already lasted well past the average lifespan of most Saturday morning cartoon series, went through dramatic changes. The animation became darker and closer to the movies' style, the color of the sky in each episode changed from the traditional blue to a continuous and ominous dark-red sky (which was commonplace with newer action-oriented children's programming at that time), the theme song was changed, the introduction sequence added in clips from the first live-action film, and the show took on a darker, more action-oriented atmosphere.
The Turtles finally banish The Shredder and Krang to Dimension X at the end of the eighth season. They destroy the engines and the "trans-dimensional portal" of the Technodrome preventing them from returning to Earth, though they later return for a few episodes in season ten. A new villain, Lord Dregg, an evil alien warlord, appeared as their new chief nemesis for the final two seasons. Lord Dregg begins a propaganda campaign against the Turtles, turning the general population against them and in favor of him and his forces. The Turtles also suffered from subsequent mutations that would temporarily metamorphosize them into hulks with diminished intelligence. Also the TMNT gained a close new ally, Carter, a black male with an incurable mutation disease before he left to look for a cure in the future. Dregg is eventually outed as a villain, but the Turtles are never hailed as heroes within the city. In the last episode of the series, the Turtles trap Dregg in Dimension X.
In 2009, the Turtles, Shredder, Krang, and various other characters from the 1987 series returned for the 25th anniversary crossover movie Turtles Forever, in which they meet up with their counterparts from the 2003 series. Due to financial reasons, none of the original voice actors were able to reprise their roles, and replacement actors were used instead. In relation to this particular series, the plot suggests the film is most likely set sometime before Shredder and Krang are banished to Dimension X, during the time the Technodrome is stuck near the Earth's core.
In April 2013, Ciro Nieli, the executive producer of the 2012 Turtles series, confirmed in an interview that the 1987 Turtles would cameo in a one-hour special in Season 2. Cam Clarke, Townsend Coleman, Barry Gordon and Rob Paulsen (who voiced Donatello in the 2012 series) reprise their roles as Leonardo, Michelangelo, Donatello, and Raphael, respectively, in the closing of the episode "Wormquake!".