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.

Leadership behaviour similarity as a basis of selection into a management team.

There are contexts where, in order to be effective, a management team needs to display different leadership behaviours as well as possessing a wide range of experiences and skills. But achieving heterogeneity of behaviours is difficult if there are behavioural-similarity biases affecting a selection process. A sample of 126 participants completed a simulation of a selection process for a management team. They compared selection criteria and chose either a task or relations-oriented applicant for an advertised position as well as reporting their perception of their own preferred leadership style. The results of the simulation indicate that leadership behaviour can be a basis for similarity-attraction in the selection of a management team.

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.

Fate of the anterior neural ridge and the morphogenesis of the Xenopus forebrain.

The fate of the anterior neural ridge was studied by following the relative movements of simultaneous spot applications of DiI and DiO from stage 15 through stage 45. These dye movements were mapped onto the neuroepithelium of the developing brain whose shape was gleaned from whole-mount in situs to neural cell adhesion molecule and dissections of the developing nervous system. The result is a model of the cell movements that drive the morphogenesis of the forebrain. The midanterior ridge moves inside and drops down along the most anterior wall of the neural tube. It then pushes forward a bit, rotates ventrally during forebrain flexing, and gives rise to the chiasmatic ridge and anterior hypothalamus. The midanterior plate drops, forming the floor of the forebrain ventricle, and, keeping its place behind the ridge, it gives rise to the posterior hypothalamus or infundibulum. The midlateral anterior ridge slides into the lateral anterior wall of the neural tube and stretches laterally into the optic stalk and retina, and then rotates into a ventral position. The lateral anterior ridge converges to the most anterior part of the dorsal midline during neural tube closure, then rotates anteriorly, and gives rise to telencephalic structures. Whole-mount bromodeoxyuridine labeling at these stages showed that cell division is widespread and relatively uniform throughout the brain during the late neurula and early tailbud stages, but that during late tailbud stages cell division becomes restricted to specific proliferative zones. We conclude that the early morphogenesis of the brain is carried out largely by choreographed cell movements and that later morphogenesis depends on spatially restricted patterns of cell division.

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.

The disposition of albendazole in sheep.

Albendazole (ABZ) was administered intraruminally at 4.75 mg/kg to sheep fitted with a permanent bile-duct cannula to determine if its metabolites might contribute to its flukicidal action. ABZ metabolism was consistent with first-pass clearance by the liver, resulting in ABZ sulphoxide (ABZ-SO) and ABZ sulphone (ABZ-SO2) being present in plasma at maximum concentrations (mean Cmax +/- SD) of 2.0 +/- 0.2 micrograms/ml and 0.4 +/- 0.1 micrograms/ml after 8 +/- 3 h and 24 +/- 5 h, respectively. ABZ-SO, but more particularly ABZ-SO2, appeared to bind to plasma proteins but their clearance rates from plasma were similar. Biliary ABZ metabolites were mainly unconjugated ABZ-SO and 2OH-ABZ-SO (8.0% dose) or conjugated glucuronide and sulphate esters (6.3% dose) mainly of 2OH-ABZ-SO and 2OH-ABZ-SO2. The concentration of the major biliary metabolite, unconjugated ABZ-SO, followed a similar time profile to that of ABZ-SO in plasma except that Cmax was much higher (6.2 +/- 2.2 micrograms/ml). Intraruminal administration of ABZ reduced bile flow rate by 30% which may be attributable to an inhibitory effect of ABZ on microtubule formation in hepatic secretory cells. It is suggested that ABZ is sequestered in the liver. This is unlikely to contribute to its flukicidal action, which is probably attributable to ingestion of ABZ-SO from bile and blood by the fluke.

Significance of Freund's adjuvant/antigen injection granuloma in the maintenance of serum antibody response.

An experimental system involving injections of ovalbumin (OVA) and ferritin (FER) in Freund's incomplete adjuvant (FIA) into the right and left flank skin folds of sheep was used to study the influence of the FIA/antigen depot and the draining lymph node in maintaining an antibody response. Excision of the injection granuloma and draining lymph node from one side 2-3 months after injections resulted in a profound decrease in serum antibody titres. This response was observed in all eight sheep in the experimental group. In five of eight animals in another experiment, excision of the injection sites had no appreciable effect on antigen-specific antibody titres when compared with antibody specific for antigen on the intact side of the sheep. In the remaining three animals, excision of the injection site did cause some fall in titre. Radiotracer studies revealed that about one-third of the original [125I]OVA/FIA injected was present in the granuloma 20 weeks after injection. Lymphatic cannulation approaches were used to study the responsiveness of the lymph node draining an FIA/antigen granuloma established 12 weeks earlier and showed that increments of 1-2 mg OVA in saline administered adjacent to the granuloma at 6-7 day intervals gave rise to strong anti-OVA containing cell (AOCC) responses in lymph. There were 2-6-fold increases in serum antibody titre in response to 3-5 doses of OVA or FER (1-2 mg) in saline injected adjacent to the FIA/antigen injection site (which had been administered 14-16 weeks previously). It is concluded that the release rate of antigen from a FIA/antigen depot is insufficient to sustain maximal antibody levels in blood serum.

Eosinophil responses in sheep selected for high and low responsiveness to Trichostrongylus colubriformis.

A breeding programme, based on selection for faecal egg counts, has produced lines of sheep which demonstrate either increased resistance (high responder) or susceptibility (low responder) to challenge infection with T. colubriformis after vaccination with irradiated larvae. Circulating blood eosinophilia, a hallmark of helminth infections, was examined in third generation lambs from two separate selective matings and random bred control lambs. Numbers of eosinophils were higher in high responder lambs when compared to low responders after vaccination and challenge infections. Analysis of eosinophil counts confirmed a strong line effect and there was no evidence of a sex effect. Random bred lambs showed wide individual variations in eosinophil numbers and their response to infection. It was concluded that peripheral eosinophilia was more a measure of host responsiveness to infection than an indicator of helminthiasis. As such the eosinophil may serve as an indicator of the hosts ability to respond to T. colubriformis vaccination and infection.

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.

Prolonged exposure to an odor or deodorized air alters the size of mitral cells in the olfactory bulb.

The size and distribution of mitral cells in the olfactory bulbs of rats were determined using a computer-assisted morphometric technique. Rats were reared from 2 weeks of age for 10 weeks in an animal colony, or in cages through which a stream of deodorized air or cyclohexanone vapor passed. Statistical analyses indicated that each treatment produced a different distribution of cell sizes in the coronal but not along the rostrocaudal plane of the bulb. The mean size of mitral cells in rats reared in deodorized air was substantially smaller than that of normal rats, and the mean length of their mitral cell layer in the coronal plane was less than in the other groups. Overall, these findings indicate that mitral cells which were of normal size in rats exposed to cyclohexanone had been stimulated by this odor. Cells which had not been stimulated by an odor in either of the experimental groups were smaller than normal and were degenerating or underdeveloped. The results also suggest that there are bands of mitral cells aligned along the rostrocaudal axis of the olfactory bulb which are responsive to specific odors and other evidence is presented which supports this view. The functional significance of exposure effects and their implications for the spatial coding of odor quality are discussed.

The distribution of the size and number of mitral cells in the olfactory bulb of the rat.

A computer assisted method was used to measure the distribution of the size and number of cells in the mitral cell layer of ten olfactory bulbs from six Sprague-Dawley rats. The size of mitral cells was found to vary around the coronal but not along the rostrocaudal extent of the bulb. Cells were smaller in the ventral and dorsal aspects of the bulb, and larger in the medial and lateral aspects; possible causes of these differences are discussed. The pattern of cell size variation in the coronal plane was consistent both within and between animals, although the average size of mitral cells varied from rat to rat. These changes in mean cell size in the coronal plane were due to overall shifts in the size of cells and not to altered proportions of large and small cells. There was no consistent pattern however for cell density as measured by inter-cell distances of mitral cells, either in the coronal or the rostrocaudal planes. Estimates of the number of cells per bulb and the size of cells were observer dependent, whereas the pattern of cell size variation was observer independent. This characteristic pattern of size variation of mitral cells in rats reared in a normal rat colony environment may be due to a functional differentiation of mitral cells, as size has been shown to be associated with the amount of information processed by a cell. That mitral cells possess such a distinct pattern of size variation should therefore be considered if representative data are to be obtained from anatomical, electrophysiological and other studies of the olfactory bulb.

Evaluation of a rosette inhibition test for pregnancy diagnosis in pigs.

A rosette inhibition test was developed using pig lymphocytes and sheep red blood cells. Antilymphocyte serum (ALS) in the presence of complement inhibited rosette formation by greater than 95% at 1/250 declining to no inhibition at 1/8000. Sera obtained from a total of 14 pregnant sows before and 1, 2, 3 and 4 wk after mating were tested for their ability to augment the rosette depression caused by ALS. In one experiment in which the responses of 4 pregnant sows were compared to 4 non-pregnant sows by discriminant analysis, sera were classified correctly in 83% of the samples taken from either pregnant or non-pregnant sows. When the more usual method of calculating the rosette inhibition titre was used, the responses of sera from pregnant pigs were classified with 31% accuracy and those from non-pregnant pigs with 80% accuracy. In a second experiment, sera from 10 pregnant sows were classified with 25% accuracy using the rosette inhibition titre. Thus 4 of these pigs were classified as non-pregnant by this method. Data from the second experiment were not suitable for discriminant analysis. It was concluded that there was some factor present in the sera of pregnant pigs, particularly by 3 or 4 wk post-mating, which could be detected by the rosette inhibition test. However, the test is not sensitive enough to allow specific diagnosis of early pregnancy in pigs.

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.