Mar. 15, 1999 Boston, MA--March 8, 1999--The structural differences between an arm and a leg are crucial to their proper function: digits that flex and curl are needed in a hand for grasping while strong muscles in the legs allow for walking and running. Now researchers at Harvard Medical School have unveiled the molecular instructions that command these differences, and have identified a gene that can partially transform the upper limb of a vertebrate into a structure that resembles its lower limb. Their findings, published in the March 12 Science, open new doors to understanding how vertebrate limbs acquire their identity. Postdoctoral fellow Malcolm Logan and Clifford Tabin, professor of genetics at Harvard Medical School, took a gene that is normally only active in legs and transferred it to the forming wings of chick embryos. The resulting structures lost many of their wing characteristics and gained those of a leg: feathers were gone, claws appeared at the end on the digits, and leg-specific muscles were clearly identifiable.
"This is the first time that a gene has been shown to direct a transformation of forelimb (arm or wing) to hindlimb (leg) structures," said Logan, principal author of the study.
The gene, called Pitx1, is one of three genes thought to play a role in giving upper and lower extremities their identity, and the first one whose function has been confirmed. The other two, Tbx4 and Tbx5, are members of the same gene family and are active only in the leg or in the wing, respectively. Scientists had long speculated that genes like these would be needed to set up forelimb and hindlimb structural differences. However, as researchers began to isolate genes involved in guiding the growth and development of limbs, it became obvious that the crucial signals for limb patterning were present in all four "limb buds"--the outgrowths on the sides of an embryo's trunk that represent the beginnings of the future limbs. If the same signals supervise the development of all extremities, how do the differences between a wing and a leg arise? According to Tabin, the wing- and leg-specific genes discovered within the last three years allowed them to postulate an explanation that has been corroborated with the present study.
"Our current work shows that the limb identity genes of the Pitx and Tbx families provide a context for the limb bud to make the correct forelimb and hindlimb structures in response to the common signals," he said. In their analysis of the limb-identity genes, Logan and Tabin focused on Pitx1 for a simple reason. "We knew that Pitx1 and Tbx4 were only present in the leg, but we started the analysis of Pitx1 because it comes up earlier than Tbx4," said Logan. The logic was that Pitx1 could be an earlier signal that controls activation of Tbx4 in the leg.
In fact, when Logan used a virus to deliver the Pitx1 gene into the future wings of chick embryos, Tbx4 was activated in the wing tissue even though it is normally never present in the wing. Pitx1 also caused activation of two other genes--HoxC10 and HoxC11--normally found only in legs later in development. But delivering Pitx1 to the wing did not affect Tbx5, the gene that is normally present only in the wing. Therefore, wings infected with the virus carrying Pitx1 had not only the normal presence of Tbx5, but also the induced activation of Tbx4. According to the authors, this provides an explanation for the partial transformation of the wings, since both wing-specific and leg-specific genes were present within the same appendage.
While the infected wings did not undergo a full transformation into legs, many individual structures unequivocally became leg elements. Changes in the digits were in some cases dramatic: while wings normally have three digits of different size, Pitx1 infected wings had digits of approximately equal length--sometimes four instead of three--as is the case in the leg. Also, claws at the ends of the digits were apparent in many of the wings, even thought these structures are only ever present in legs. Muscle patterns were also transformed.
All three limb-identity genes are present in mice, and are similarly restricted to the forelimbs or hindlimbs. In humans, mutations in Tbx5 are associated with Holt-Oram syndrome, which results in forelimb truncations among other symptoms. Thus, the roles of Pitx1 and Tbx genes in limb identity seem to be conserved throughout evolution. In the near future, Logan and Tabin hope to figure out how these limb-identity genes are working.
"For example, what cellular properties are changed by Pitx1 such that the limb tissue condenses to form four digits instead of the three in the absence of Pitx1?" Tabin said. "This is the ultimate goal of the field, to understand how developmental control genes actually regulate morphogenesis."
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