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Immunofluorescent techniques were used t o examine several aspects of B cell ontogeny in humans. Large lymphoid cells containing intracytoplasmic IgM (pre-B cells) were present in fetal liver as early as 7 weeks of gestation, approximately 2 weeks prior t o the appearance of surface IgM positive (sIgM+) B lymphocytes. Pre-B cells outnumbered sIgM+ B lymphocytes in fetal liver u p until the 13th week of gestation. In fetuses older than 13 weeks, pre-B cells and sIgM+ B lymphocytes were present in approximately equal proportions in liver and bone marrow. Pre-B cells in fetal liver, and fetal and adult marrow, were large and small (indicating a heterogeneous population of cytoplasmic IgM+. sIg-cells in these sites), while only the small pre-B cells were present i n fetal spleen, blood and lymph node. Lymphocytes bearing sIgG were detected earlier than those bearing sIgD o r sIgA, which were present by the 12th gestational week. Using double-staining techniques, we determined that during fetal life, (a) the proportion of B lymphocytes bearing only sIgM, as opposed t o those bearing both sIgM and sIgD, was much higher in liver and bone marrow than in spleen, blood and lymph node, and ( b ) sIgG, sIgA and sIgD appear independently on lymphocytes bearing sIgM. Studies of the frequency of doublestained cells for each combination of the four sIg isotypes indicated that B lymphocytes from neonatal humans may simultaneously bear three o r more sIg isotypes, whereas sIgG+ and sIgA+ B lymphocytes in adult blood usually express only the single isotype. Lower concentrations of anti+ antibodies were required for modulation of sIgM from B lymphocytes of fetal liver and adult bone marrow than for equivalent removal of sIgM from circulating B cells of mature individuals.In conjunction with data obtained in mice, our observations indicate that (a) the presence of large and small pre-B cells, (b) a high ratio of sIgM single t o sIgM.sIgD double B lymphocytes, and (c) increased sensitivity t o modulation of B cell sIgM by divalent anti-p antibodies define the fetal liver and adult bone marrow as bursa-equivalent sites in humans. Our results are consistent with a model o f isotype diversification in which immature sIgM+ cells give rise to B cell sublines devoted t o synthesis of each of the Ig classes, and sIgD is secondarily expressed on unstimulated B cells of all of these sublines.

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SummaryMyoD and Myf5 are fundamental regulators of skeletal muscle lineage determination in the embryo, and their expression is induced in satellite cells following muscle injury. MyoD and Myf5 are also expressed by satellite cell precursors developmentally, although the relative contribution of historical and injury-induced expression to satellite cell function is unknown. We show that satellite cells lacking both MyoD and Myf5 (double knockout [dKO]) are maintained with aging in uninjured muscle. However, injured muscle fails to regenerate and dKO satellite cell progeny accumulate in damaged muscle but do not undergo muscle differentiation. dKO satellite cell progeny continue to express markers of myoblast identity, although their myogenic programming is labile, as demonstrated by dramatic morphological changes and increased propensity for non-myogenic differentiation. These data demonstrate an absolute requirement for either MyoD or Myf5 in muscle regeneration and indicate that their expression after injury stabilizes myogenic identity and confers the capacity for muscle differentiation.

Abstract. Development of heterogeneity of immunoglobulin classes has been investigated in the chicken by studying the effects of antibody-mediated suppression of IgM synthesis. Treatment of 13-day embryos with purified goat antibodies to IgM resulted in the elimination of IgM-containing The chicken has been extremely useful for studying differentiation of immunoglobulin-producing cells, primarily because development of this line of cells depends on the functional integrity of the bursa of Fabricius.1 Effective bursectomy prior to or at the time of hatching, combined with procedures to destroy cells already seeded from the bursa, results in agammaglobulinemia.2-5 The ability to form germinal centers, plasma cells, and immunoglobulins6 and to respond to antigen! may be conferred upon bursectomized recipients by bursal lymphocytes. Immunoglobulin synthesis first occurs in the bursa89. Impetus for the experiments described in this report stemmed from recent observations made in this laboratory on the ontogeny of immunoglobulin synthesis in chickens.9 With the aid of sensitive fluorescent-antibody techniques, it was found that IgM-and IgG-containing cells appear first in the bursa of Fabricius of 14-and 21-day embryos, respectively. The time of appearance of each class of immunoglobulin is unrelated to exogenous antigenic stimulation. In the mature bursa, contiguous localization of IgM and IgG was observed in the medullary areas of most follicles. IgG-containing cells were found only in the medullas of follicles that contained IgM, whereas some follicles 1918

Purified goat antibodies against mouse mu-chains and rabbit antibodies against mouse Ig determinants, and their Fab fragments, inhibited the development of IgM-bearing B cells in explant cultures of 14-day mouse fetal liver, and caused the disappearance of cell surface IgM in explant and dissociated cell cultures of more developed lymphoid tissues. While treatment of cultures of fetal or newborn liver, or adult bone marrow, with low concentrations (less than or equal to 10 mug/ml) of anti-Ig for less than or equal to 24 h caused the complete, but reversible, disappearance (modulation) of cell surface IgM, treatment for greater than or less than 48 h produced irreversible IgM suppression. In contrast, anti-Ig-induced suppression of cell surface IgM in cultures of adult spleen or lymph nodes required much higher concentrations of antibody (greater than or equal to 100 mug/ml) and was always reversible. These differences between immature and mature IgM-bearing cells could not be related to differences in the amount of surface IgM on the cells. The remarkable sensitivity of newly formed B cells to IgM modulation and irreversible IgM suppression when ligands bind to their Ig receptors, may have important implications for B-cell tolerance to self antigens.

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