Within the limb bud, expression of specific Hox genes varies as a function of the position along the anterior-posterior axis. The Hox genes are linked in four chromosomal clusters: Hoxa, Hoxb, Hoxc, and Hoxd. Their physical position on the chromosome correlates with the time and place of expression. This statement is supported by the knowledge that Hox gene expression is initiated during gastrulation in primitive somitic mesoderm by FGF signaling which effects the primitive somitic mesoderm cells at different times depending on their axial location during organism development—and is even further specified with other anterior-posterior axis signals (such as retinoic acid). Additional evidence for the role that Hox genes play in limb development was found when researchers effected Hox gene expressions in zebrafish by adding retinoic acid during gastrulation; This experiment resulted in a duplication of limbs. Although excess retinoic acid can alter limb patterning by ectopically activating Shh expression, genetic studies in mouse that eliminate retinoic acid synthesis have shown that RA is not required for limb patterning.