Effects of vitamin A on the behaviour of migratory neural crest cells in vitro

0 CRC Medical Oncology Unit, University of Southampton, Southampton General Hospital , Southampton SO9 $XY, U.K It has been proposed elsewhere that the teratogenic effects of retinoids on craniofacial morphogenesis are caused by a disturbance of the migration of cranial neural crest cells. The effects of 3-5 x io~6 M and 3-5 x io~* M-retinol on the migration of avian neural crest cells in vitro have been investigated by monitoring cell morphology, locomotory behaviour, fibronectin distribution and actin-micronlament organization. Retinol retards migration by affecting cell-to-substratum adhesiveness. Cells exposed to medium containing retinol are less adherent to the substratum, and although the cell surface is very mobile, are unable to extend or maintain lamellipodia. As a consequence the cells do not actively translocate. Fibronectin distribution at the cell surface is sparse, possibly as a result of shedding, and actin distribution remains diffuse. At the retinol molarities used all these effects are reversible. Thus cells allowed to recover in normal medium flatten out, display lamellipodia and commence active translocation. Fibronectin becomes organized into afibrillararray and actin micronlaments become organized into cables. The period needed for this recovery is directly related to the molarity of retinol during the initial exposure; after recovery the retinol-treated cells are virtually indistinguishable from control cells. We propose that in vivo the effects of retinoids might be to impair cell-extracellular matrix interaction, thus impeding a cell's ability to migrate through that matrix. Contrary to previous suggestions, the in vivo effects are probably not in any way ' specific' to neural crest cells but are more accurately considered as ' selective', in that any cell undergoing migration would be similarly affected. EFFECTS OF VITAMIN A ON IN VITRO 1 1 PETER THOROGOOD , LINDA SMITH , ALASTAIR ROSE McGINTY1 AND DAVID GARROD1 Vitamin A and its analogues, collectively termed the retinoids, not only have functional roles in a wide range of normal physiological systems but can also be potent teratogens. Their effects range from changes in the differentiation of integumental structures, such as scale primordia differentiating into feathers (Douailly, Hardy & Sengel, 1980), to congenital limb abnormalities such as phocomelia (Kochar, 1977). Exposure during early development has a dramatic effect on craniofacial morphogenesis ; in the mammalian embryo, for example, exposure, in utero or in vitro, during a critical period of development produces a cleft palate and a deficient facial skeleton arising from a disturbed pattern of skeletogenesis rather than from a failure of skeletal P. Thorogood, L. Smith, A. Nicol. R. McGinty and D. Garrod differentiation per se (e.g. see Morriss & Thorogood, 1978). In work on other vertebrate groups it has been demonstrated that the greater part of the connective and skeletal tissues in the face are ec/omesenchymal, that is to say, derived from cranial neural crest cells migrating into the presumptive facial region during early head development (e.g. see Johnston, 1966; Le Lievre & Le Douarin, 1975). It has been proposed by a number of authors that the craniofacial effect of hypervitaminosis A are due to retarded neural crest migration (Poswillo, 1975; Morriss & Thorogood, 1978). This results in a deficient facial mesenchyme and disturbs the synchrony of morphogenesis, thereby distorting the migration pathways and causing an abnormal distribution of the reduced These proposals have not been tested directly and the precise mode of action of teratogenic retinoid levels has not yet been defined. For the avian embryo, where parameters of migration rate, migration routes and the differentdative fate of cranial neural crest cells have been studied, two brief reports provide circumstantial evidence that migration in vivo might be impeded by retinoids (Hassell, Greenberg & Johnston, 1977; Keith, 1977). In this paper we describe the effects of one particular retinoid, retinol, on the locomotory and social behaviour of avian cranial neural crest cells migrating from primary explants in vitro. The experiments were designed in an attempt to answer the following questions: (i) does retinol affect the locomotory ability of neural crest cells ? (ii) if so, how is this effect implemented ? (iii) is impairment of locomotion specific to neural crest cells as previous reports imply? MATERIALS AND METHODS Blastoderm of the Japanese quail (Coturnix coturnix japonica) at stages 9-9 + (Hamburger & Hamilton, 1951) were washed briefly in Dulbecco's phosphate-buffered saline and placed into 'alpha' Eagle's minimal essential medium (otMEM) containing 10% foetal calf serum (FCS). The tips of the mesencephalic neural folds were dissected free (see fig. 3 of Thorogood, 1981), and placed as primary explants onto heat-sterilized glass coverslips in 30-mm plastic tissueculture dishes (Sterilin) containing z ml of <xMEM supplemented with 10 % FCS, ioo unitg/ml penicillin, 100 mg/ml streptomycin and 0-25 mg/ml Fungizone (GIBCO). Mesenchymal cells migrating from the explants were regarded as migratory neural crest cells; the differentiation of such cells into skeletal tissues, when grown in organ culture, has been described elsewhere (Bee & Thorogood, 1980). Trarei-retinol (Sigma) was dissolved in absolute ethanol and added to experimental cultures to give final concentrations of i-o fig and io-o fig retinol/ml medium, that is3-5Xio~*M and 3-5 x io"5 M, respectively (dose levels of io~* M and above are thought to be cytotoxic to most cell types; for further details, see Lotan, 1981). Control cultures received an amount of ethanol (4 fiX) equal to that added to experimental cultures. Cultures were maintained at 37 C in s % CO, in air, in a humidified incubator. The duration of the culture period and frequency of medium changes varied according to experiment (see below). Analysis of cell locomotion Each culture was assigned a code number and all measurements and photography were carried out ' blind' by a second person. The rate of outgrowth from explants was monitored by measuring the diameter of outgrowth at 24 h intervals; for each culture the average of two measurements made at 900 was recorded. The morphology of living cells was monitored using inverted phase-contrast microscopy and recorded by either 35 mm photography using Ilford Pan F or by time-lapse video recording (see below). Cultures were terminated by washing twice in PBS, and then they were fixed in absolute methanol and stained by the May-Grunwald-Giemsa technique (Paul, 1975). The locomotory and social behaviour of the cells was monitored by time-lapse video recording for periods up to 48 h. An Hitachi low-light intensity camera (model HV-175K) was fitted to an Olympus inverted phase-contrast microscope and coupled to a FOR-A video time generator (model VTG-377) and National time-lapse video (...truncated)