It was previously shown (Roberts, C., Platt, N., Streit, A., Schachner, M. and Stern, C. D. (1991) Development 112, 959-970) that grafts of Hensen's node into chick embryos enhanced and maintain expression of the L5 carbohydrate in neighbouring epiblast cells, and that antibodies against L5 inhibit neural induction by such a graft. We now show that L5 is initially widely expressed in the epiblast, but as neural induction proceeds it gradually becomes confined to and up-regulated in the early neural plate. L5 can therefore be considered as a marker for cells that are competent to respond to neural induction. We also show that Hepatocyte Growth Factor/Scatter Factor (HGF/SF) promotes the expression of L5 by extraembryonic epiblast in collagen gels after overnight culture. Explants cultured for several days in the presence of HGF/SF, as well as explants of prospective neural plate, can differentiate into cells with neuronal morphology expressing neuronal markers. To investigate whether HGF/SF is expressed in the chick embryo at appropriate stages of development, we produced specific cDNA probes and used them for in situ hybridization. We find that at the primitive streak stage, HGF/SF is expressed specifically in Hensen's node. We therefore propose that HGF/SF plays a role during the early steps of neural induction, perhaps by inducing or maintaining the competence of the epiblast to respond to neural inducing signals.

A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation

Modulation of neural commitment by changes in target cell contacts in Pleurodeles waltl.

In amphibia, noggin, one of the neural inducers expressed in the Spemann organizer, acts by neutralizing the effects of bone morphogenetic protein-4 (BMP-4). It is shown that noggin is able to activate L-type calcium channels. The fos proto-oncogene is known to be induced within minutes by calcium signaling. Here it is reported that in animal cap explants of the amphibian Pleurodeles waltl, noggin can induce upregulation of a FOS-related protein in a calcium-dependent manner. Specific inhibition of the dihydropyridine sensitive L-type calcium channels blocked both calcium influx and the induction of FOS-related protein. When animal cap explants were treated with caffeine in order to release calcium from an internal store or with a specific agonist of the L-type calcium channels, FOS-related protein could be detected in cell nuclei by 5 or 15 min, respectively. Additionally, the calcium calmodulin kinase inhibitor, KN62, could block the upregulation of FOS-related protein induced by agents that increased intracellular calcium ([Ca2+]i). The present results suggest that transcription factors from the FOS family are downstream targets of neural inducer noggin.

Noggin upregulates Fos expression by a calcium-mediated pathway in amphibian embryos.

Lectins (SBA and PSA) were used to provoke crowding and structural modifications of the presumptive ectoderm cell surface in order to investigate the role of the membrane organization of the competent target cells in neural induction. Are specific characteristics of the cell surface essential for this phenomenon to occur? From amphibian gastrulae, it is possible to obtain neural induction in vitro by association of presumptive ectoderm (target cells) with chordamesoderm (inductor tissue): 4 h of contact is sufficient in Pleurodeles waltl for transmission of the inductive signal. Very quickly, the treatment of the normal ectoderm by lectins (SBA-FITC or PSA-FITC) provoked surface modifications. Lectin-treatment (50 µg ml 1− , 30 min) of presumptive ectoderm did not result in any neural induction. Lectin-treatment (50 µg ml 1− , 30 min) of presumptive ectoderm previous to its association with the natural inductor for 4 h, disturbed the phenomenon: no induction. Similar treatment followed by association with the inductor for 24 h: induction. Treatment of SBA or PSA with their respective hapten inhibitors prior to addition to ectodermal cells completely blocked the suppressive effects on induction. The structural integrity of the membrane of competent target cells is necessary for neural induction to occur. The cell membrane could thus play, directly or indirectly, an active role in the specificity of this process

Neural induction and the structure of the target cell surface

SUMMARY The competent ectoderm of Pleurodeles waltl comprises two cell layers with characteristic differences in their morphology, their composition and the molecular arrangement of the various constituents. The use of labelled lectin probes for observations of ectoderm tissue in vitro with u.v. microscopy (epi-illumination) and the quantification of the results show the following:1) Differences in labelling according to the nature of the lectins (SB A, PSA, LCA and Con A). These differences provide information on the nature of the carbohydrates which are present at this stage and on the number of receptors. 2) Differences in fluorescence intensity of the surfaces studied. The internal surface of the ectoderm is labelled more densely than the external surface. 3) Rearrangement of the lectin receptors with a new molecular configuration, stressing the fluidity of the membrane (by the mobility of the receptors throughout the membrane) and its importance for the occurrence of neural induction. 4) Existence of membrane glycoconjugate turnover. 5) A difference in behavioural characteristics between the internal and the external surfaces with respect to the lectins and the formation of an extracellular matrix on the internal surface alone. The extracellular matrix seems to have a role in morphogenetic movements.

L. Gualandris

Papers

A rapid experimental method to study primary embryonic induction.