Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid generating enzyme in the early embryo. Here we report the immunolocalization of this enzyme (RALDH-2-IR) in stage 6-29 chicken embryos; we also show that tissues that exhibit strong RALDH-2-IR in the embryo contain RALDH-2 and synthesize retinoic acid. RALDH-2-IR indicates dynamic and discrete patterns of retinoic acid synthesis in the embryo, particularly within the somitic mesoderm, lateral mesoderm, kidney, heart, and spinal motor neurons. Prior to somitogenesis, RALDH-2-IR is present in the paraxial mesoderm with a rostral boundary at the level of the presumptive first somite; as the somites form, they exhibit strong RALDH-2-IR. Cervical presomitic mesoderm exhibits RALDH-2-IR but thoracic presomitic mesoderm does not. Neural crest cells do not express detectable levels of RALDH-2, but migrating crest cells are associated with RALDH-2 expressing mesoderm. The developing limb mesoderm expresses little RALDH-2-IR; however, RALDH-2-IR is strongly expressed in tissues adjacent to the limb. The most lateral, earliest-projecting motor neurons at all levels of the spinal cord exhibit RALDH-2-IR. Subsequently, many additional motor neurons in the brachial and lumbar cord regions express RALDH-2-IR. Motor neuronal expression of RALDH-2-IR is present in the growing axons as they extend to the periphery, indicating a potential role of retinoic acid in nerve influences on peripheral differentiation. With the exception of a transient expression in the facial/vestibulocochlear nucleus, cranial motor neurons do not express detectable levels of RALDH-2-IR.

These results indicate that at least one mechanism for the teratogenicity of VPA involves a direct effect on developing tissue. The nature of the abnormalities observed implies that this effect may be mediated by disruption of genes that regulate pattern formation.

The spinal cord of the bullfrog (Rana catesbeiana) tadpole contains primary neurons, born during embryonic stages, and secondary neurons born for the most part during larval stages. Electrophysiological and anatomical characteristics of these two categories of neurons were examined during larval development to trace the development of secondary neurons and to determine whether primary neurons persist into adult life or are replaced by secondary neurons. Five classes of primary neuron were identified on the basis of their distinctive locations, morphologies, cytoplasmic melanin content, and presence at the earliest larval stages examined: primary motoneurons, Rohon-Beard cells, commissural cells, dorsal marginal cells, and anterolateral marginal cells. Secondary neurons of the lateral motor column and dorsal root ganglia underwent extensive developmental changes during larval life manifested both in anatomical studies with horseradish peroxidase and electrophysiological experiments on the isolated spinal cord. Primary motoneurons that innervate the tadpole tail were not found in the adult, although those innervating thoracic musculature persisted, as did at least some primary neurons projecting to other spinal segments or brainstem. Primary neurons are thus replaced or maintained through metamorphosis depending on their class and location.

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Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid (RA) generating enzyme in the embryo. Here, we report immunolocalization of this enzyme (RALDH-2-IR) in the developing wings of stage 17-30 chicken embryos. RALDH-2-IR is located in the area of the presumptive muscle masses, although it is not colocalized with developing muscle cells. RALDH-2-IR is located in tendon precursor cells and may be present in muscular connective tissue. We show that motor neurons and blood vessels, tissues showing RALDH-2-IR as they enter the limb, are capable of synthesizing and releasing RA in culture. RALDH-2-IR in the limb mesenchyme is under the control of both the vasculature and the motor innervation; it is decreased with denervation and increased with hypervascularization. RALDH-2-IR is present in the motor neuron pool of the brachial spinal cord, but this expression pattern is apparently not under the control of limb target tissues, RA in the periphery, or somitic factors. RA is known to be a potent inducer of cellular differentiation; we propose that locally synthesized RA may be involved in aspects of wing tissue specification, including cartilage condensation and outgrowth, skeletal muscle differentiation, and recruitment of smooth muscle cells to the vasculature.