Retinoid signaling is involved in governing the waiting period for axons in chick hindlimb

During embryonic development in chick, axons pause in a plexus region for approximately 1 day prior to invading the limb. We have previously shown that this "waiting period" is governed by maturational changes in the limb. Here we provide a detailed description of the spatiotemporal pattern of Raldh2 expression in lumbosacral motoneurons and in the limb, and show that retinoid signaling in the limb contributes significantly to terminating the waiting period. Raldh2, indicative of retinoid signaling, first appears in hindlimb mesenchyme near the end of the waiting period. Transcripts are more abundant in connective tissue associated with predominantly fast muscles than predominantly slow muscles, but are not expressed in muscle cells themselves. The tips of ingrowing axons are always found in association with domains of Raldh2, but development of Raldh2 expression is not regulated by the axons. Instead, retinoid signaling appears to regulate axon entry into the limb. Supplying exogenous retinoic acid to proximal limb during the waiting period caused both motor and sensory axons to invade the limb prematurely and altered the normal stereotyped pattern of axon ingrowth without obvious effects on limb morphogenesis or motoneuron specification. Conversely, locally decreasing retinoid synthesis reduced axon growth into the limb. Retinoic acid significantly enhanced motor axon growth in vitro, suggesting that retinoic acid may directly promote axon growth into the limb in vivo. In addition, retinoid signaling may indirectly affect the waiting period by regulating the maturation of other gate keeping or guidance molecules in the limb. Together these findings reveal a novel function of retinoid signaling in governing the timing and patterning of axon growth into the limb.