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In order to more accurately determine the mechanism by which retinoic acid causes embryonic defects, we have developed a simple method of locally applying retinoic acid rather than immersing the whole embryo in retinoic acid solutions. Retinoic acid was suspended in corn oil and then injected between the surface and the deep ectodermal layers of an early gastrula Xenopus embryo. When droplets containing retinoic acid were injected into the presumptive head region, the embryos exhibited inhibited development of anterior structures near the injection site. Development of the eye, cement gland, hatching gland, olfactory pits, and expression of engrailed protein were all disrupted near the injection site. Inhibited development of anterior structures was far greater on the injected side of the embryo than on the uninjected side. The retinoic acid droplet did not cause an anterior shift of structures on the injected side relative to the uninjected side. These experiments suggest that retinoic acid does not cause global respecification of axial level in the head, but rather suppresses development of anterior structures. Retinoic acid injected into presumptive trunk regions had no discernible effect.
FIG. 1. Possible patter ns of development following retinoic acid microinjection.
injection of tetinoic acid between the deep and the sul'face
ectodermal layers of a stage 10.5gastrulaembryo (top drawings)
might have one of two effects upon subsequent head development.
Control injections of carrier medium alone (open circle) should have
no effect upon endogenous concentrations of retinoic acid. The resulting
neural plate would have anterior-posterior specification and a
normal tadpole would develop. Injection of retinoic acid (closed circles)
might alter pattern formation by locally inhibiting differentiation
of anterior structures so that they fail to develop on the injected
side (represented by faded lines). Alternatively, if the axial level is
specified by a gradient of retinoic acid, on the injected side the injected
retinoic acid would supplement the endogenous retinoic acid
gradient. thereby respecifying (represented by the upturned lines) the
axial coordinates in the neural plate. In subsequent embryos, structures
woold then appear more rostral than contralateral ones.
FIG. 2. Effects of droplet position and retinoic acid concentration on subsequent development. Embryos were categorized according to retinoic
acid concentration (pg/ml) and droplet placement. The effects were scored according to the degree of inhibition from normal (O) to complete
inhibition (3). The means of the scores are graphed. Cement gland was inhibited only when the injected droplet was near the front of the
embryo. The injected side showed hi11her levels of hatching gland and eye inhibition than the contralateral side. indicating that the effects of
the injection were graded and localized to the droplet region. Hatching gland was most sensitive to the retinoic acid, and both sides of the
hatching gland pattern were often affected. Eyes were les.s sensitive. The eye on the injected side was usually only reduced in size with no
apparent effect on the contralateral one. Occasionally, the eye on the injected aide was absent, with the contralateral eye appearing normal.
FIG. 3. Local inhibition of cement and hatching gland by retinoic acid. (A) A retinoic acid droplet (arrow) lying posterior to the cement gland
causing local inhibition of the cement gland. (B) Two examples of embryos that had the retinoic acid droplet (arrow) injected anterior to the
cement gland. In both cases the entire middle of the cement gland was inhibited, resulting in two patches of cement gland on either side of the
droplet. Scale bar, 200 ,..m. (C) Hatching gland, visualized under epifluorescence, was strongly inhibited when in close proximity to the retinoic
acid droplet. The droplet, not visible in this view, was on the left side, where there are fewer, more dispersed cells in the Y pattern. Note that the
overall pattern is still symmetric. Anterior is up. (D} The injected droplet(i) can be visuali-z.ed on the left side of the embryo ~cause of the Oil
fluorescence). The hatching gland (h) is completely absent on the left side of the embryo. Anterior is up. Scale bar, 100 um.
FIG. 5. Engrailed expression is inhibited by retinoic acid injection. (A) The band of engrailed staining (e) is normal and symmetric when a
control droplet (d) is injected. When a retinoic add droplet (d) is injected either anterior (B) or posterior (C) to the normal engrailed position (e),
the ~engrailed band is inhibited on the injected side. No anterior shifting of the engrailed band was seen. Scale har, 100 um.
Fie. 6. (A) Microinjection of control and retinoic acid-containing droplets appeared to have no effect upon ectoderm integrity. This specimen,
fixed 3 hrs following retinoic acid injection, appears normal, with the exception that the surface ectoderm has bulged slightly. No cytotoxicity
was visible either 1.5, 3.0, or 14 hr postinjection. Scale bar = 100 ~tm. (B) This photomicrograph is of a 8-h postinjection specimen, the injection
site of which was broken open just prior to sputter coating. Such specimens demonstrated that injections were being successfully placed
between deep and surface ectoderm layers, and furthermore, that cells in both layers appear to suffer no obvious ill effects from the administration
of retinoic acid. The compound seems unl ikely to inhibit anterior structures by cytotoxic effects. Scale bar, 30 um.
