Posterior Neural Tissue Research Articles

Early posterior neural tissue is induced by FGF in the chick embryo.

Signals that induce neural cell fate in amniote embryos emanate from a unique cell population found at the anterior end of the primitive streak. Cells in this region express a number of fibroblast growth factors (FGFs), a group of secreted proteins implicated in the induction and patterning of neural tissue in the amphibian embryo. Here we exploit the large size and accessibility of the early chick embryo to analyse the function of FGF signalling specifically during neural induction. Our results demonstrate that extraembryonic epiblast cells previously shown to be responsive to endogenous neural-inducing signals express early posterior neural genes in response to local, physiological levels of FGF signal. This neural tissue does not express anterior neural markers or undergo neuronal differentiation and forms in the absence of axial mesoderm. Prospective mesodermal tissue is, however, induced and we present evidence for both the direct and indirect action of FGFs on prospective posterior neural tissue. These findings suggest that FGF signalling underlies a specific aspect of neural induction, the initiation of the programme that leads to the generation of the posterior central nervous system.

Neural Crest Induction by Xwnt7B in Xenopus

Neural patterning occurs soon after neural induction during early development. In Xenopus, several caudalizing factors transform anterior neural to posterior neural tissue at the open neural plate stages, while other factors are responsible for setting up mediolateral polarity which becomes the dorsoventral (D–V) axis after neural tube closure. Many Wnt ligands are expressed in the neural tube in distinct anteroposterior (A–P) and D–V domains, implying a function in neural patterning. Here we report the cloning of a full-length Xenopus Wnt7B gene. Xwnt7B induces neural crest markers Xslug and Xtwist in ectodermal explants coinjected with neural inducer noggin and in ectodermal cells neuralized by dissociation.In vivo,Xwnt7B expands the Xtwist expression domain when injected in the animal pole. Our results suggest that Wnt members are involved in dorsoventral patterning of the neural tube.

XenopusHindbrain Patterning Requires Retinoid Signaling

We have asked how posterior neural tissue is patterned inXenopusby assaying the involvement of endogenous retinoic acid (RA) in this process and by using the labial Hox gene, HoxD1, as a posterior marker. Although RA is able to inhibit anterior gene expression and activate expression of more posterior genes, the normal role of retinoids in anteroposterior (A/P) patterning is unclear. HoxD1 is an early posterior neurectodermal marker, expressed from midgastrula with a later anterior expression limit in the future hindbrain. We previously showed that HoxD1 was induced as an immediate early response to retinoic acid in naive ectoderm (animal caps). Here, we use a truncated RARα2.2 receptor (RARΔ) to dominantly interfere with retinoid signaling. In embryos injected with RARΔ expression of HoxD1 is eliminated. Conjugates of ectoderm and dorsolateral mesoderm show that retinoid receptors are required in the ectoderm for HoxD1 induction. Further, expression of Krox-20 in r3 and r5 of the presumptive hindbrain is compressed into a single stripe that suggests elimination of r5. RARα2.2 expression almost precisely overlaps that of HoxD1, suggesting that this receptor may normally activate HoxD1. Expression of neither more anterior genes including cement gland, forebrain, and midbrain markers nor a more posterior spinal cord marker is affected by RARΔ. These data suggest that the posterior hindbrain is the region of the nervous system most sensitive to retinoid loss. Finally, we compare the ability of RA and fibroblast growth factor (FGF) to posteriorize isolated anterior neurectoderm and show that both factors can act directly on this substrate. RA acts in a more anterior domain than does FGF; however, neither factor is equivalent to the natural posteriorizing capacity of the posterior mesoderm. We propose that endogenous retinoid and FGF signals pattern largely nonoverlapping regions along the A/P axis and that posterior neural patterning requires multiple inducers.

Studies on the Role of Fibroblast Growth Factor Signaling in Neurogenesis Using Conjugated/Aged Animal Caps and Dorsal Ectoderm-Grafted Embryos

Basic fibroblast growth factor (bFGF) has been shown to induce neural fate in dissociated animal cap (AC) cells or in AC explants cultured in low calcium and magnesium concentrations. However, long-term disclosure of the cap may cause diffusion of the secreted molecule bone morphogenetic protein 4 (BMP-4), a neural inhibitor present in the AC. This may contribute to the subsequent neurogenesis induced by bFGF. Here we used conjugated and aged blastula AC to avoid diffusion of endogenous molecules from the AC. Unlike noggin, bFGF failed to induce neural tissue in this system. However, it enhanced neuralization elicited by a dominant negative BMP receptor (DN-BR) that inhibits the BMP-4 signaling. Posterior neural markers were turned on by bFGF in AC expressing DN-BR or chordin. Blocking the endogenous FGF signal with a dominant negative FGF receptor (XFD) mainly inhibited development of posterior neural tissue in neuralized ACs. These in vitro studies were confirmed in vivo in embryos grafted with XFD-expressing ACs in the place of neuroectoderm. Expression of some regional neural markers was inhibited, although markers for muscle and posterior notochord were still detectable in the grafted embryos, suggesting that XFD specifically affected neurogenesis but not the dorsal mesoderm. The use of these in vitro and in vivo model systems provides new evidence that FGF, although unable to initiate neurogenesis on its own, is required for neural induction as well as for posteriorization.

Epithelial Cell Wedging and Neural Trough Formation Are Induced Planarly inXenopus,without Persistent Vertical Interactions with Mesoderm

In this study we investigate the induction of the cell behaviors underlying neurulation in the frog,Xenopus laevis.Although planar signals from the organizer can induce convergent extension movements of the posterior neural tissue in explants, the remaining morphogenic processes of neurulation do not appear to occur in absence of vertical interactions with the organizer (R. Kelleret al.,1992,Dev. Dyn.193, 218–234). These processes include: (1) cell elongation perpendicular to the plane of the epithelium, forming the neural plate; (2) cell wedging, which rolls the neural plate into a trough; (3) intercalation of two layers of neural plate cells to form one layer; and (4) fusion of the neural folds. To allow planar signaling between all the inducing tissues of the involuting marginal zone and the responding prospective ectoderm, we have designed a “giant sandwich” explant. In these explants, cell elongation and wedging are induced in the superficial neural layer by planar signals without persistent vertical interactions with underlying, involuted mesoderm. A neural trough forms, and neural folds form and approach one another. However, the neural folds do not fuse with one another, and the deep cells of these explants do not undergo their normal behaviors of elongation, wedging, and intercalation between the superficial neural cells, even when planar signals are supplemented with vertical signaling until the late midgastrula (stage 11.5). Vertical interactions with mesoderm during and beyond the late gastrula stage were required for expression of these deep cell behaviors and for neural fold fusion. These explants offer a way to regulate deep and superficial cell behaviors and thus make possible the analysis of the relative roles of these behaviors in closing the neural tube.

Graded amounts of Xenopus dishevelled specify discrete anteroposterior cell fates in prospective ectoderm

Signals emitted from the prospective dorsal marginal zone (the organizer) are thought to specify neuroectodermal cell fates along the anteroposterior (AP) axis, but the mechanisms underlying this signaling event remain to be elucidated. To assess the effect of Xenopus Dishevelled (Xdsh), a proposed component of the Wnt, Notch and Frizzled signal transduction pathways, on AP axis determination, it was supplied in varying doses to presumptive ectodermal cells. Two-fold increments in levels of microinjected Xdsh mRNA revealed a gradual shift in cell fates along the AP axis. Lower doses of Xdsh mRNA activated anterior neuroectodermal markers, XAG1 and Xotx2, whereas the higher doses induced more posterior neural tissue markers such as En2, Krox20 and HoxB9. At the highest dose of Xdsh mRNA, explants contained maximal amount of HoxB9 transcripts and developed notochord and somites. When compared with Xdsh, Xwnt8 mRNA also activated anterior neuroectodermal markers, but failed to elicit mesoderm formation. Analysis of explants overexpressing Xdsh at the gastrula stage revealed activation of several organizer-specific genes which have been implicated in determination of neural tissue (Xotx2, noggin, chordin and follistatin). Whereas Goosecoid, Xlim1 and Xwnt8 were not induced in these explants, another early marginal zone marker, Xbra, was activated at the highest level of Xdsh mRNA. These observations suggest that the effects of Xdsh on AP axis specification may be mediated by combinatorial action of several early patterning genes. Increasing levels of Xdsh mRNA activate posterior markers, whereas increasing amounts of the organizer stimulate the extent of anterior development (Stewart, R.M. and Gerhart, J.C. (1990) Development 109, 363–372). These findings argue against induction of the entire organizer by Xdsh in ectodermal cells and implicate signal transduction pathways involving Xdsh in AP axis determination. Thus, different levels of a single molecule, Xdsh, can specify distinct cell states along the AP axis.

Caudalization by the amphibian organizer: brachyury, convergent extension and retinoic acid.

Caudalization, which is proposed to be one of two functions of the amphibian organizer, initiates posterior pathways of neural development in the dorsalized ectoderm. In the absence of caudalization, dorsalized ectoderm only expresses the most anterior (archencephalic) differentiation. In the presence of caudalization, dorsalized ectorderm develops various levels of posterior neural tissues, depending on the extent of caudalization. A series of induction experiments have shown that caudalization is mediated by convergent extension: cell motility that is based on directed cell intercalation, and is essential for the morphogenesis of posterior axial tissues. During amphibian development, convergent extension is first expressed all-over the mesoderm and, after mesoderm involution, it becomes localized to the posterior mid-dorsal mesoderm, which produces notochord. This expression pattern of specific down regulation of convergent extension is also followed by the expression of the brachyury homolog. Furthermore, mouse brachyury has been implicated in the regulation of tissue elongation on the one hand, and in the control of posterior differentiation on the other. These observations suggest that protein encoded by the brachyury homolog controls the expression of convergent extension in the mesoderm. The idea is fully corroborated by a genetic study of mouse brachyury, which demonstrates that the gene product produces elongation of the posterior embryonic axis. However, there exists evidence for the induction of posterior dorsal mesodermal tissues, if brachyury homolog protein is expressed in the ectoderm. In both cases the brachyury homolog contributes to caudalization. A number of other genes appear to be involved in caudalization. The most important of these is pintavallis, which contains a fork-head DNA binding domain. It is first expressed in the marginal zone. After mesoderm involution, it is present not only in the presumptive notochord, but also in the floor plate. This is in contrast to the brachyury homolog, whose expression is restricted to mesoderm. The morphogenetic effects of exogenous RA on anteroposterior specification during amphibian embryogenesis are reviewed. The agent inhibits archencephalic differentiation and enhances differentiation of deuterencephalic and trunk levels. Thus the effect of exogenous RA on morphogenesis of CNS is very similar to that of caudalization, which is proposed to occur through the normal action of the organizer. According to a detailed analysis of the effect of lithium on morphogenesis induced by the Cynops organizer, lithium has a caudalizing effect closely comparable with that of RA. Furthermore, lithium induces convergent extension in the prechordal plate, which normally does not show cell motility.(ABSTRACT TRUNCATED AT 400 WORDS)

Bilateral eye formation in the eyeless mutant Mexican salamander following unilateral, partial excision of neural fold tissues: A quantitative study

AbstractPrevious results showed that ectopic neural fold grafts, performed unilaterally during neurulation on eyeless mutant axolotl embryos, frequently stimulated eye formation bilaterally. The analysis of the eyeless mutant was expanded in order to obtain quantitative data on the effect of various types of neural fold grafts. While performing these experiments, significant eye formation occurred in controls. Larvae in which neural fold tissues were not grafted but only partially removed at neurula stages formed eyes in some experiments, but not in others. In those cases that eyes did form, partial removal of the posterior neural fold of the prospective ear region stimulated eyes to differentiate more frequently than partial removal of the anterior neural fold of the prospective nose area. In both cases, unilateral neural fold ectomy frequently resulted in bilateral eye formation. There was a positive correlation between the amount of posterior neural fold tissues excised and the frequency of cases in which eyes formed. This increase was roughly 10% if the posterior neural fold excision was performed bilaterally instead of unilaterally.A comparison of eye formation between neural fold excision experiments and ectopic neural fold grafts was performed. There was a statistically significant different between anterior and posterior neural fold operations, but not between grafting and excision experiments. I interpret these results to suggest that there is a signal in the posterior neural fold of the eyeless mutant that prevents eye formation. This signal can be neutralized most efficiently by wounding of the posterior neural folds. In addition, the results provide strong evidence that the effect of wounding propagates from posterior to anterior regions, as well as from one side of the developing neuroepithelium to the other. © 1993 Wiley‐Liss, Inc.

Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay.

Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.

Planar and vertical signals in the induction and patterning of the Xenopus nervous system.

The cellular mechanisms responsible for the formation of the Xenopus nervous system have been examined in total exogastrula embryos in which the axial mesoderm appears to remain segregated from prospective neural ectoderm and in recombinates of ectoderm and mesoderm. Posterior neural tissue displaying anteroposterior pattern develops in exogastrula ectoderm. This effect may be mediated by planar signals that occur in the absence of underlying mesoderm. The formation of a posterior neural tube may depend on the notoplate, a midline ectodermal cell group which extends along the anteroposterior axis. The induction of neural structures characteristic of the forebrain and of cell types normally found in the ventral region of the posterior neural tube requires additional vertical signals from underlying axial mesoderm. Thus, the formation of the embryonic Xenopus nervous system appears to involve the cooperation of distinct planar and vertical signals derived from midline cell groups.

In the Mexican salamander (Ambystoma mexicanum) homozygous for the geneeyeless, unilateral neural fold rearrangements stimulate bilateral eye formation

The objective of the present investigation was to test the hypothesis that the absence of eyes in the eyeless mutant Mexican salamander (Ambystoma mexicanum) might be caused by an antero-posterior morphogenetic system out of balance in the head of the mutant. To test this hypothesis the antero-posterior sequence of the neural fold of eyeless mutant neurulae was rearranged in two ways: unilateral grafting of posterior neural fold tissues from mutant donors into the anterior nasal area of mutant hosts (and vice-versa) was performed; and in addition, the eye region of the mutant was moved into posterior temporal positions as well as shifted forward into the nasal area. The results of these experiments demonstrate that larvae homozygous for the eyeless mutant gene are capable of forming eyes in vivo following such antero-posterior rearrangements of their neural folds. In addition, operations performed unilaterally had a bilateral effect: eye structures differentiated on the operated as well as on the unoperated side of the larvae. These results suggest that the eyeless mutant fails to form eyes because of an inhibitory effect on the optic vesicles emanating from neighboring tissues, not because of an inherent defect in the neural plate to form retina.

Neuropathology with spinal instrumentation.

Neurohistologic examination of the spinal cord and cauda equina were compared for 28 beagles undergoing anterior and posterior spinal destabilization procedures--Group I (n = 7), destabilized operative controls; Group II (n = 7), posterolateral bone grafting; Group III (n = 7), Harrington distraction instrumentation and posterolateral fusion; and Group IV (n = 7), Luque rectangular instrumentation and posterolateral fusion. All dogs had appeared neurologically intact upon repeated examinations prior to death. Neurohistological abnormalities (Wallerian degeneration of the dorsal columns, corticospinal tracts, and nerve roots, focal cystic degeneration, and intraspinal central cavitation) occurred in only 1 of the 14 animals (7%) in Groups I and II (noninstrumented) and in 9 of the 14 animals (64%) in Groups III and IV (instrumented). This result is statistically significant (p less than 0.001). Transient sensory disturbances and radicular paresthesias have been described in clinical reports of spinal instrumentation. It is probable that subclinical neurologic injuries, such as intraspinal and nerve root infarction in posterior neural tissue, can occur with the use of sublaminar hooks or wires. The chondrodystrophic beagle spinal model in this study should be considered a "worst case situation," and the clinical incidence of neurohistologic changes is expected to be lower.