Effects of retinoic acid upon eye field morphogenesis and differentiation.

This study describes a whole embryo and embryonic field analysis of retinoic acid's (RA) effects upon Xenopus laevis forebrain development and differentiation. By using in situ and immunohistochemical analysis of pax6, Xbf1, and tyrosine hydroxylase (TH), gene expression during eye field, telencephalon field, and retinal development was followed with and without RA treatment. These studies indicated that RA has strong effects upon embryonic eye and telencephalon field development with greater effects upon the ventral development of these organ fields. The specification and determination of separate eye primordia occurred at stage-16 when the prechordal plate reaches its most anterior aspect in Xenopus laevis. Differentiation of the dopaminergic cells within the retina was also affected in a distinct dorsoventral pattern by RA treatment, and cell type differentiation in the absence of distinct retinal laminae was also observed. It was concluded that early RA treatments affected organ field patterning by suppression of the upstream elements required for organ field development, and RA's effects upon cellular differentiation occur downstream to these organ determinants' expression within a distinct dorsoventral pattern.

The eyeless mutant gene (e) in the Mexican axolotl (Ambystoma mexicanum) affects pax-6 expression and forebrain axonogenesis.

This study tested the hypothesis that changes in the patterns of pax-6 expression disrupt the anatomy and axonogenesis of the diencephalic areas of the eyeless axolotl. Proper pax-6 expression is necessary for eye and hypothalamus morphogenesis. Since the expression boundaries of pax-6 also provide a permissive environment for axonal outgrowth, an extensive study examining the effects of the eyeless gene (e) in the Mexican axolotl upon pax-6 expression and forebrain axonogenesis was begun. This study used whole embryo in situ hybridization techniques to follow pax-6 expression and whole brain immunocytochemistry to examine axonogenesis and neural differentiation. These studies demonstrated that the mutant gene e in the axolotl alters the response of midanterior neural-plate tissue to signals from the prechordal plate. This response was hypothesized to be a hyper-response to signals (sonic hedgehog?) that suppressed pax-6 expression within the midanterior neural plate and later developmental stages. Alternatively, the affected neuroectoderm of the eyeless embryos may lack competence to express pax-6. Lowered pax-6 expression inhibited eye and forebrain morphogenesis as well as neural axonogenesis and differentiation. Differentiation defects were detected as the suppression of midline dopaminergic neurons within the suprachiasmatic nucleus of eyeless animals. Thus, lowered pax-6 expression by the midanterior neuroectoderm promotes the eyeless condition by inhibiting the role of pax-6 in eye formation. This lowered expression also leads to concurrent alterations in the hypothalamic terrain which disrupt axonogenesis and ultimately promote sterility.

Forebrain differentiation and axonogenesis in amphibians: I. Differentiation of the suprachiasmatic nucleus in relation to background adaptation behavior.

Functional forebrain development is the result of a complex series of early developmental processes which include cell division, cellular rearrangements, tissue-tissue interactions, cellular determinative and differentiation events, and axonogenesis. In these studies, Xenopus laevis embryos were examined for early forebrain neuronal determination, differentiation and axonogenesis with special emphasis on the hypothalamic area known to be involved in regulating pars intermedia function. Whole brain acetylcholine esterase (AChE) histochemistry was used to follow the early pattern of forebrain neuronal differentiation, and whole brain acetylated-tubulin immunocytochemistry was done to follow early forebrain axonogenesis. AChE histochemistry indicated that the source of the tract of the postoptic commissure (stpoc) was the first forebrain area to begin differentiation (stage 22). Whole brain immunocytochemistry for acetylated-tubulin indicated that the tpoc is also the first forebrain tract to develop (at stage 25/26). The main forebrain tracts have developed and become interconnected by stage 35/36. The forebrain undergoes a pronounced extension, with much cellular mixing and rearrangement during stages 37/38 to 43/44. This results in bending and contortions in the already developed tracts. Whole brain immunocytochemistry for tyrosine hydroxylase and extirpation of the stage 14 presumptive suprachiasmatic (SC) area indicated that the dopaminergic cells of the SC are determined by stage 14 and initially undergo differentiation between stages 37/38 and 40. Tadpoles with stage 14 presumptive SC extirpated lacked TH-positive tracts to the pars intermedia, lacked most midline TH-positive forebrain cells, and also failed to background adapt to white background. Thus, the SC tracts to the pars intermedia that inhibit melanotrope secretion probably form during the extension stages of 37/38 and contact the pars intermedia by stage 40 when animals are first capable of background adaptation.

Developmental neurobiology of the anterior areas in amphibians: urodele perspectives.

Because of its evolutionary grade and its relative simplicity, the Urodele brain provides an excellent archetype for the study of forebrain development. Early experiments on Primary Induction took advantage of the Urodele's manipulatability and ease of use, but due to the fact that its ectoderm was very readily neuralized Anurans (especially Xenopus) became the vertebrate of choice for early developmental neurobiology. Recent advances in the molecular biology of neuralization in Xenopus may rejuvenate Urodele use in solving the complicated sequence of events during this process of neural induction and to ascertain if separate or a combination of events (de fault and inductive) are involved. In the future, the combined use of Urodeles and Anurans will provide much information with regard to the evolutionary conservation of the mechanisms of regional specification, gene expression events, neurulation, neuroblast migration, and axonogenesis during the development of the nervous system. The present review provides some recent examples of this approach of using Urodeles and Anurans in a combinatorial fashion to decipher specific aspects of developmental neurobiology.

Mapping of the presumptive brain regions in the neural plate of Xenopus laevis.

Two cell autonomous fluorescent labels (DiI and Hoechst) were used as vital markers in a fate map study of the Xenopus neural plate and ridge. Most areas of the brain derive from the neural plate in a fate map that is consistent with the topology of a sheet rolling into a tube, i.e., neighboring areas are maintained as neighbors. This has enabled us not only to plot the fates of larval brain structures, but also to suggest their primordial orientation in the neural plate. Since overlapping areas of the plate gave rise to overlapping regions of the central nervous system (CNS), we have been able to construct a space-filling model of the neural plate, whereby the number of founder cells for each brain region fate-mapped may be estimated roughly. Much of the telencephalon, ventral forebrain, and dorsal brain stem derives from the neural ridge and not the neural plate in the stage 15 Xenopus embryo. The structures of the forebrain were examined in detail because there were indications of substantial cell movements in this region. The anterior pituitary arises from the mid-anterior ridge, while hypothalamic structures arise from the midline regions of the anterior neural plate. Consistent groups of ventral hypothalamic structures were labeled when fluorescent markers were applied to these parts of the neural plate, indicating stereotyped cell movements. Detailed comparisons were made between the fate map of the Ambystoma neural plate (Jacobson, 1959) and that of Xenopus.

Paraboloid of axolotl retinal photoreceptor and bovine pituitary thyrotropin fraction share an antigen.

Lenses of newts (genera Notophthalmus, Triturus, Cynops) regenerate from irises in the presence of retinae of larval frogs (Rana) or adult salamanders (Hynobius, Ambystoma), species which are themselves incapable of lens regeneration from the iris. In newts, bovine pituitary thyrotropin preparation NIH-TSH-B8 can also stimulate lens regeneration from the iris. An antiserum against NIH-TSH-B7 (purified as is NIH-TSH-B8), absorbed with bovine lutropin preparation NIH-LH-B9, cross-reacts with bovine retinal glycoprotein extracts in immunodiffusion tests, and with retinal photoreceptor cells of the axolotl (Ambystoma mexicanum), as evidenced by immunofluorescence. In normal adult eyes and in eyes 21 days after lens removal, the paraboloid portion of the photoreceptor inner segments, and in some cases the perinuclear cytoplasm of the photoreceptor cells, contained the antigen. The cross-reacting antigen appears to be different from thyrotropin, and also different from the basic and acidic retinal fibroblast growth factors. However, immunodiffusion reveals a precipitation arc with retina-derived growth factor fraction III (EDGF III). If bovine pituitary thyrotropin preparations produce lens regeneration, and if these preparations cross-react with an antigen in the retinal photoreceptors, the retinal antigen may be involved in the stimulation of lens regeneration as well.

A scanning electron microscopy and histological study on the effects of the mutant eyeless (e/e) gene upon the hypothalamus in the Mexican axolotl Ambystoma mexicanum Shaw.

A scanning electron microscopy, histological, and immunochemical investigation examined the effects of the mutant gene (e) upon hypothalamic development in the Mexican axolotl. The adult eyeless mutant is sterile. Previous studies indicated that this reproductive defect was due to the mutation's effect upon the hypothalamus. The present study demonstrated the pleiotropic effects of the eyeless gene upon development of the hypothalamus. Scanning electron microscopy studies looked at the early ontogeny of the hypothalamohypophyseal system. The major morphological difference observed in the hypothalamus of normals compared to eyeless mutants was the reduced nature or complete lack of a preoptic recess in eyeless mutants. Early embryonic tissue movements also differed when normal siblings were compared to eyeless mutant axolotls. Histological examination looking for paraldehyde-fuchsin-positive secretory neurons revealed a paired nucleus preopticus in both normals and eyeless mutants, but this region lacked the emanating paraldehyde-fuchsin-positive fiber tracts in eyeless mutants. The neurohypophysis of the eyeless mutants was atrophied and contained far less paraldehyde-fuchsin-positive material when compared to normal axolotls. Immunochemical studies were done to look at the distribution of immunoreactive luteinizing-hormone-releasing hormone (ir-LHRH) in brains of eyed and eyeless mutant axolotls of different stages. This study detected deficiencies in ir-LHRH in the anterior hypothalamus of eyeless mutants. In general in the eyeless mutant axolotl, the observed anterior hypothalamic deficiencies are comparable to those observed in anurans which have had their optic vesicles removed. These observations suggest a possible utility of the eyeless mutant axolotl for studies concerned with endocrine development in the absence of hypothalamic modulation.

The pituitary adrenocorticotropes originate from neural ridge tissue in Xenopus laevis.

A series of grafting experiments was conducted to determine pituitary origins prior to brain tube closure in Xenopus laevis. Extirpation experiments indicated that the ventral neural ridge (VNR) tissue of stage-18+ embryos was essential for pituitary development. Bolton-Hunter reagent was used to label stage-18+ VNR tissue with 125I, and this tissue was then returned to the donor and its subsequent ontogenesis followed. Labelled tissue was ultimately found in the ventral hypothalamus, the ventral retina, and the anterior pituitary. Using immunocytochemical techniques with antisera to adrenocorticotropin (ACTH), it was found that some of the VNR-derived cells were corticotropes. A region of the nucleus infundibularis which was radioactive labelled also gave ACTH-positive immunoreaction. This might indicate that some ACTH-containing neurones of the hypothalamus are VNR in origin. We suggest that stage-18+ VNR is the site of attachment of brain and anterior pituitary ectoderm. Part of this adherence point is eventually incorporated into the anterior pituitary and will form corticotropes. It is concluded that the ventral retina, the preoptic region of the hypothalamus, some hypothalamic ACTH-immunoreactive cells, and the most anterior portion of the adenohypophysis are all ventral neural ridge in origin.

T4-induced metamorphosis in Ambystoma gracile (Baird) from two populations: the effects of aging and temperature.

In order to determine the factors affecting the maturation of the functional hypothalamo-pituitary-thyroid axis, Ambystoma gracile (Baird) larvae of different ages and acclimated to different temperature regimes were exposed to a concentration of thyroxine (T4) which activates the pituitary-thyroid axis. A. gracile from a montane and low-altitude population were utilized. These studies, plus observations concerning spontaneous metamorphosis, indicated that populations of Ambystoma gracile are highly polymorphic as suggested by Sprules (1974 b). A. gracile populations consist of 'obligate transformers', 'obligate neotenes', and 'facultative transformers'--which will metamorphose depending on environmental conditions. The colder high-altitude conditions tend to select against facultative individuals, whereas the less certain low-altitude conditions permit a much higher proportion of animals that exhibit a facultative response with respect to metamorphosis. Interrelationships between environment and physiological parameters that determine morphology in salamanders are discussed with regard to these findings.

Localization of the pituitary lactotropes and thyrotropes within Ambystoma gracile by histochemical and immunochemical methods. A developmental study of two populations.

The prolactin-producing cells are the first hormone-producing cells of the pars distalis to be differentiated within Ambystoma gracile. They first appear when the larvae attain a length of approximately 3.0 cm snout to vent length (SVL). Thyrotropes are observed as the next chromophilic cells to appear, and they occur when the larvae are approximately 4.5 cm SVL. Both thyrotropes and lactotropes increase in numbers until metamorphosis. Gonadotropes begin to appear when larvae attain a size of 5.0 cm SVL and become extremely abundant when larvae are in excess of 7.0 cm SVL. Animals, generally, exhibit the greatest number of thyrotropes just prior to the mean size for metamorphosis, and metamorphosing animals exhibit a dramatic reduction in the number of thyrotropes. Neotenous larvae have an abundant number of thyrotropes which are mainly located along the caudal periphery of the pituitary.

Factors affecting the larval growth and development of laboratory-reared Ambystoma gracile (Baird) from natural populations of different temperature regimes.

Temperature-transfer of Ambystoma gracile larvae from 11 degrees C to 21 degrees C does not significantly alter growth rates at the higher temperature when compared to larvae continuously grown at 21 degrees C. Temperature-transfer does not alter the incidence of neoteny within populations, even though interpopulation neotenic tendencies differ. Salamander larvae from the high altitude population exhibit larvae from the high altitude population exhibit faster growth rates than the larvae reared from eggs from the low altitude-population. These high altitude larvae exhibit faster growth rates both at the lower and higher temperatures suggesting a greater sensitivity to growth-promoting factors and/or higher rates of synthesis or secretion of such factors. Experiments utilizing ovine prolactin indicated that prolactin-injected experimentals were significantly longer (P less than 0.025) after 60 days of treatment than were placebo-injected controls. A prolactin-like hormone is suggested as thepossible growth-promoting factor in larval Ambystoma gracile.