Endogenous retinoids in the zebrafish embryo and adult.

Retinoic acid and its isoforms are considered to be endogenous compounds which regulate embryonic development. In the work reported here we have determined which retinoids are present in zebrafish embryos and how their levels change throughout development and into adulthood. All-trans-RA is present and its level does not change significantly during embryogenesis. We failed to detect other retinoic acid isomers such as 9-cis-RA and 4-oxo-RA, but we did observe a rapid rise in the level of didehydroretinol after gastrulation. The most striking result is that the zebrafish embryo, like Xenopus and tunicates, contains a vast excess of t-retinal whereas the embryos of higher vertebrates have an excess of t-retinol. However, as the zebrafish grows, the levels of t-retinol rise so that by adulthood t-retinol and t-retinal concentrations are more equivalent, indicating a changing pattern of retinoid metabolism with growth. To examine the significance of the use of t-retinal as a precursor of t-RA we treated embryos with disulphiram, an inhibitor of retinaldehyde dehydrogenase. This resulted in embryos with an undulating notochord and correspondingly abnormal somites and ventral floor plate. In contrast to this effect, 4-methylpyrazole, which inhibits alcohol dehydrogenases, had no effect on development. This effect of disulphiram suggests that t-RA may be involved in the establishment of the anteroposterior axis of the embryo.

Types of supernumerary outgrowths produced after inverting the dorsoventral limb axis of the anuranBufo bufo.

Contralateral grafts were performed on the larval limb buds of the anuranBufo bufo. The dorsoventral axis of 80 buds at stages IV or V was inverted. Ten tadpoles were used as controls. Fifty-two supernumerary structures developed, all of them in dorsal or ventral locations on the host stump. The majority (32 out of the 44 outgrowths with more than 3 toes) were normal limbs of stump handedness. However, the following abnormal structures were also observed: 2 double-posterior, 3 mixed-symmetric, and 7 undetermined cases. These results are in agreement with the predictions of a hierarchical polar coordinate model for epimorphic regeneration.

Diverse supernumerary structures develop after inverting the anteroposterior limb axis of the anuran.

Contralateral limb bud graftings were performed on tadpoles of the anuran Bufo bufo. The anteroposterior axis was inverted while the larvae were at stage IV or V (e.g., between 22-30 days after fertilization). Eighty-four tadpoles were operated on, 10 of which were used as controls. At anterior or posterior location 104 supernumerary structures developed in toto. They were collected and whole-mount examined after being stained with Alcian blue. They were further prepared for serial sectioning, mounting, and staining with hematoxylin and eosin. The majority of these supernumerary structures were found to be normal limbs of the stump handedness in agreement with all models and experiments on the urodeles axolotl and newt. However, some of the structures were clearly abnormal: double symmetric or of mixed handedness. This result is consistent with a prediction of a hierarchical polar coordinate model. The fact that no such structures have been found in the experiments on the urodeles may be due to the expected low probability for their appearance and the fact that only few such limbs have been sectioned and analyzed as yet.