Discussion

This investigation has demonstrated that mouse trunk neural crest cells in culture constitutively synthesize gelatinase-A and -B. Immunolocalization detected gelatinase- AB within cells o f the neural tube and in cells that had emigrated from neural tube explants during 24-48 hours in culture. SDS-gelatin gel zymography detected both active gelatinase-A and -B in serum-free culture supernatants from trunk neural crest cell cultures.

Previous attempts to localize MMPs and TIMP-1 in neural tubes and migrating neural crest cells using monensin treated whole embryos have been unsuccessfrd. Using an established technique o f neural tube explant culture it has been possible to monensin treat migrating neural crest cell cultures in vitro. Unfortunately, no reliable cellular markers exist for characterization o f mammalian neural crest cells. It has been assumed cells capable o f migrating from 9.5 day p.c. neural tube explants during 24-48 hours in culture are neural crest cells. It has also been assumed that, in the absence o f an inducting epithelium and complex extracellular matrix components, the neural crest cell phenotype was stable in the short term (24 - 48 hours) and cells have not undergone significant differentiation during culture.

Immunolocalization in monensin-treated neural crest cell cultures was o f low intensity and rarely showed the bright perinuclear staining seen in differentiated connective tissue cells, which is characteristic o f enzyme accumulation within the Golgi apparatus. This suggests gelatinase-AB synthesis is at a low level. Fluorescence for gelatinase-AB, both within cells at the periphery o f the neural tube e?q)lant and in cells at the periphery o f neural crest cell cultures, shows gelatinase synthesis is not uniform It is interesting to speculate that cells of the neural tube synthesize gelatinase in order to degrade adjacent basement membrane. This is consistent with the supposed preconditions for neural crest emigration (Erickson and Perris, 1993) and the observed micro structure o f neural tube basement membranes by electron microscopy (Tosney, 1978; Erickson and Weston, 1983; Martins-Green and Erickson, 1987). However, gelatinase positive cells within the neural tube may not necessarily be presumptive neural crest cells; degradation may be acconiphshed by a separate population. Interestingly, neural tube explants cultured with ortho-phenanthroline (an MMP inhibitor) showed a marked reduction in emigrated cells, suggesting that MMPs may play a role in neural crest emigration and, or migration (Erickson and Isserofl^ 1989).

Zymography has revealed that neural crest cell cultures constitutively synthesize both gelatinase-A and -B. In addition, the technique has demonstrated that gelatinase synthesis is upregulated by recombinant mouse IE -la , suggesting that mouse neural crest cells in culture express IE -la receptors. Synthesis o f gelatiuases by neural crest cell cultures also responds to the culture substrate. Although fibronectin appears to support a more pronounced cell emigration, the effect on gelatinase synthesis was less than type I collagen. By contrast, fibronectin has been shown to upregulate coUagenase and stromelysin expression by human fibroblasts (Tremble et a l, 1992).

Gelatin-gel zymography was able to detect both active- and pro- forms of gelatmases because SDS within the gel autoactivates enzymes after separation by electrophoresis. The majority o f gelatinase detected was active gelatmase-A and -B. However, significant levels o f pro- gelatinase-A and -B were detected. Concentrated culture supernatants, particularly from explants grown on Matrigel, also contain gelatinolytic activity at other molecular weights. The absence o f bands on duphcate ortho-phenanthroline treated gels suggests that degradation was by MMPs and not plasmin or PAs. It is possible that the multiple bands are an artefact arising from concentration o f the medium Gelatinases -A and -B may conçlex with other proteins in the medium thereby increasing their perceived molecular weight. However, the molecular weights o f additional bands correlate with previously reported con^lexes of gelatinases with either TIMP-1 or TIMP-2 (Ward et a l, 1991b; see Table 6.2). TIMP-1 can complex with gelatinase-B, either pro- or active form, and progelatinase-A to give bands at 115-, 108- and 85-kDa respectively. TIMP-2 can complex with gelatinase-A, either pro- or active form, and active gelatinase-B to give bands at 77-, 61- and 86-kDa respectively. It has been suggested that most TIMP-2 and gelatinase produced may exist as a complex (Murphy and Reynolds, 1993). In addition, TIMP-2 conplexed with gelatinase-A shows continued MMP inhibitory activity, the ‘high molecular mass TIMP activity’. The physiological significance o f both these phenomena is unknown.

Neural tube explants cultured on Matrigel failed to produce substantial emigration o f cells except for a few elongated neuron-like cells. MatrigeF^ is a basement membrane matrix prepared from an extract from the Engelbreth-Hohn-Swarm (EHS) mouse sarcoma, a tumour rich in extracellular matrix proteins. Its major conponent is laminin, but it also contains type IV collagen, heparan sulphate proteoglycan, entactin

Table 6-2 Molecular masses of metalloproteinase complexes with either TIMP-1 or TIMP-2

determined by gel filtration on Sephacryl S-200 (from Ward et al, 1991).

MMP TIMP-1 C28-kDa) TIMP-2 C224cDa)

pro-gelatinase-A (66-kDa) no conq)lex 77

active gelatinase-A (64-kDa) 85 61

pro-gelatinase-B (97-kDa) 115 no canq)lex

active gelatinase-B (92-kDa) 108 86

and nidogen. Laminin and type IV collagen are known to promote neural crest cell migration; therefore it is surprising to find emigration is not promoted by Matrigel. Culture supernatants fi'om neural crest cell cultures grown on Matrigel show a high level of both pro- and active gelatinase-B:TIMP-1 complex, as well as pro- and active gelatinase-B, active gelatinase-B:TIMP-2 con^lex, pro- and active gelatinase-A, active gelatinase-A: TIMP-1 conq)lex, and pro- and active gelatinase-A: TIMP-2 complexes. The high level o f pro- and active forms o f both gelatinase-A and gelatinase-B may not be due to neural crest cell synthesis as Matrigel is known to contain gelatinases (MacKay et a l, 1993). I have reproduced these results (data not presented) but found gelatinolytic bands produced under similar conditions were faint compared with my test samples. Matrigel also contains TGF-P, FGF and t-PA. It is interesting to speculate that TGF-P and FGF in combination, which are known to upregulate gelatinase and TIMP synthesis by fibroblasts (Overall et a l, 1989), may be responsible for the multiple additional bands. The TIMP content o f Matrigel was not detected by reverse zymography and is thought to be low (Overall et a l, 1989); although if it were present it may reduce emigration similar to ortho-phenanthroline treatment (Erickson and Isserofl^ 1989).

Gelatinase synthesis by cranial neural crest cell cultures was disappointing. Only with the explant lefl: in situ could gelatinolytic activity be demonstrated by zymography.

Cranial fold expiants are much smaller than neural tube e?q)lants and these diBferences may reflect much smaller cell numbers.

The present results suggest that the concentrated supernatants flrom trunk neural crest cell cultures contain pro- and active forms o f both gelatinase-A and -B. Additional bands representing gelatinase: TIMP conçlexes may be an in vitro artefact resulting fi'om concentration o f the medium or may reflect the con^lex nature o f gelatiuase and TIMP synthesis in vitro.

7. General Discussion and Suggestions for Future