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An in vitro microculture system of early gastrula cells of Xenopus laevis has been developed; deep layer cells from the lateralmarginal zone (prospective somite region) or ventralectoderm (prospective epidermis region) were fully dissociated, and the desired number of each (1-100) was distributed into a microculture well and cultured under appropriate conditions. When examined with the tissue-specific Mabs (Mu1 for muscle and E3 for epidermis, respectively), a substantial portion of the deep layer cells from the two regions followed their respective normal embryonic fates. It was found that reproducible cellular differentiation was dependent on the intimate reaggregation of dissociated cells and on the size of the resultant aggregate. About 20 lateralmarginal zone cells were found to be sufficient, when put into a culture well, for supporting successful muscle differentiation, whereas about 100 ventralectoderm cells were necessary for epidermal differentiation.
Fig. 1. Tissue specificities of monoclonal antibodies, E3 and Mul. (A) Schematic drawing of gross anatomy in larval cross- section used (stage 37/38). (B) Binding of E3 to epidermis. (C) Binding of Mul to myotomemuscle. Abbreviations: epi, epidermis; sc, spinal cord; n, notochord; m, myotome; en, endodermal mass. Bars = 100um
Fig. 2. Development of deep layer cells from the lateralmarginal zone of early gastrula in microculture wells. A suspension of dissociated deep layer cells was prepared from the lateralmarginal zone from which about 100 cells (A) or 12 cells (B) were initially added into culture wells; each referred to as 100-cell or 12-cell culture etc., in the text and following figure legends. These cultures were started with (al-a3) or without (bl-b3) the centrifugation step for reaggregation. Volume of culture medium was 24 fA for each well. Cultures were fixed after incubation of 36 h and examined for the presence of Mul antigen, (al, bl); phase-contrast appearance. (a2, b2); nuclear staining with DAPI. (a3, b3); Mul labelling. Bar= 100 um
Fig. 3. Development of deep-layer cells from the ventralectoderm of early gastrula in microculture wells. In A, 100- cell cultures were started with (al-a3) or without (bl-b3) the centrifugation step. In B and C, 50-cell cultures (B) or 25-cell cultures (C) were started with the centrifugation. After 36 h of incubation, cultures were fixed and examined for the presence of E3 antigen. Typical examples of positive (al-a3 in B and C) and negative (bl-b3 in B and C) wells for E3 antigen are shown. Volume of culture medium was 24/il for each well in A to C. (al, bl); phase-contrast appearance, (a.2, b2); nuclear staining. (a3, b3); E3 labelling. Bar= 100 um
Fig. 4. Dependence of epidermal (A) and muscular (B,C) cell differentiation on reaggregation and the initial number of cells in the culture well. About 10 to 100 deep layer cells from the ventralectoderm (A) or lateral marginal zone (B) of early gastrula were initially added to culture wells. These cultures were started with ( [black triangle], [black circle] in A and B) or without ([open circle] in A and B) the centrifugation step for reaggregation. Volume of culture medium was adjusted to 12 uI per well ([black triangle] in A and B) or 24 ul pe rwell ([black circle] [open circle] in A and B ). In C, initial number of marginal zone cells in each well (1-12) were directly counted under microscope just after centrifugation. After 36h of incubation, cultures were fixed and examined for the presence of E3 (A) or Mul (B,C) antigen. In A and B, the proportion of positive wells for
the respective antigens is plotted against initial number of cells in well. The total number of culture wells examined was 16 to 18 for each point. In C, mean number of Mul- positive cells per well is plotted against initial number of cells in well.
