Notochord grafts do not suppress formation of neural crest cells or commissural neurons

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia

Cranial and trunk neural crest cells use different mechanisms for attachment to extracellular matrices

We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the β_1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to α_1 intergrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and Bronner-Fraser, M. (1991) Development 113, 1069–1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, α_1 integrin and β_1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties

Delayed Formation of the Floor Plate after Ablation of the Avian Notochord

We have examined the long-term effects of notochord ablation at chick stages 9–10 on formation of the floor plate and motor neurons. Although missing or reduced 2 days postablation, the floor plate and motor neurons were morphologically normal by 4 postoperative days. When isolated whole or ventral, but not lateral, neural plate fragments from stage 9 embryos were cultured for 4 days in collagen gels, floor plate and neural markers were observed. Our results suggest that floor plate and motor neurons can form in a delayed fashion in vivo after notochord ablation and in vitro from isolated neural plates. This suggests that either there is an early induction of floor plate by the chordamesoderm of Hensen's node, or only limited interactions between the neural plate and notochord immediately after neurulation are required for floor plate determination

Partial Restriction in the Developmental Potential of Late Emigrating Avian Neural Crest Cells

Trunk neural crest cells migrate along two major pathways: a ventral pathway through the somites whose cells form neuronal derivatives and a dorsolateral pathway underneath the ectoderm whose cells become pigmented. In avian embryos, the latest emigrating neural crest cells move only along the dorsolateral pathway. To test whether late emigrating neural crest cells are more restricted in developmental potential than early migrating cells, cultures were prepared from the neural tubes of embryos at various stages of neural crest cell migration. “Early” and “middle” aged neural crest cells differentiated into many derivatives including pigmented cells, neurofilament-immunoreactive cells, and adrenergic cells. In contrast, “late” neural crest cells differentiated into pigment cells and neurofilament-immunoreactive cells, but not into adrenergic cells even after 10–14 days. To further challenge the developmental potential of early and late emigrating neural crest cells, they were transplanted into embryos during the early phases of neural crest cell migration, known to be permissive for adrenergic neuronal differentiation. The cells were labeled with the vital dye, DiI, and injected onto the ventral pathway at stages 14–17. Two and three days after injection, some early neural crest cells were found to express catecholamines, suggesting they were adrenergic neuroblasts. In contrast, DiI-labeled late neural crest cells never became catecholamine-positive. These results suggest that the late emigrating neural crest cell population has a more restricted developmental potential than the early migrating neural crest cell population

Early Migrating Neural Crest Cells Can Form Ventral Neural Tube Derivatives When Challenged by Transplantation

Once neural crest cells undergo an epithelial–mesenchymal transition to leave the neural tube, it has been classically assumed that they are fated to differentiate within the neural crest lineage. To test this idea, we challenged the developmental potential of recently emigrated neural crest cells by transplanting them into the ventral portion of the neural tube at the open neural plate stage. Newly migrating neural crest cells were isolated in tissue culture, labeled with the lipophilic dye DiI, and microinjected into the ventral portion of the neural plate. After 2 days, some neural crest cells became incorporated into the neuroepithelium in positions characteristic of floor plate cells and motor neurons. Some of the labeled cells within the ventral neural tube expressed FP-1, characteristic of floor plate cells. A few labeled cells were found in positions characteristic of motor neurons and expressed islet-1. In contrast, neural crest cells transplanted onto neural crest pathways expressed the HNK-1 epitope, but no ventral neural tube markers. Injection of neural crest cells into the mesenchyme adjacent to the notochord or culturing them in the presence of Sonic hedgehog failed to elicit FP-1 expression. These results suggest that migrating neural crest cells are flexible in their fate and retain the ability to form neural tube derivatives even after emigrating from the neural tube