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One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment.

Precursor cells of the B lineage can be enriched from mouse fetal liver by FACS with the aid of the pre-B cell-specific mAb G-5-2. The cells are concomitantly enriched for cells expressing the pre-B cell-specific gene lambda 5, and for cells developing to LPS-reactive mature B cells. The enriched purified precursors are not influenced by rIL-2 through -7, alone or in combination, to develop to mitogen-reactive, sIg+ cells. Marginal proliferation of the precursors is observed in response to IL-3 plus -4, and IL-6 plus -7, and this does not change in the presence of stromal cells. Development to mitogen-reactive, sIg+ cells is dependent on interactions with embryonic stromal cells from fetal liver. Two mAbs raised against the stromal cells inhibit this development. Two phases of precursor cell development can be distinguished in fetal liver. Between days 13 and 15 of gestation, it is dependent on stromal cell interactions, thereafter, from days 16 to 19, it is independent. A sudden increase in the number of mitogen-reactive, sIg+ B lineage cells occurs within 24 h between days 16 and 17. All these results indicate that B cell development occurs in one wave with synchronous steps of changes from a mitogen-insensitive, sIg-, stromal cell dependent to a mitogen-reactive, sIg+, stromal cell-independent B lineage line.

B lymphocyte progenitor cells arise from pluripotent stem cells in a competent environment by a set of critical steps of differentiation . During embryonic development of the mouse, progenitors of B lymphocytes arise in fetal liver at day 13 of gestation (1)(2)(3)(4), then develop into precursor (pre) B cells, which consequently rearrange the gene segments of the sIg V regions of H chains, followed by those of the L chains (5,6). The early stages of progenitor proliferation and induction to Ig gene rearrangements have been found to depend on interactions with cells ofthe environment called stroma (7)(8)(9)(10)(11)(12)(13)(14)(15). At least in part, the effects of stromal cells on B cell development appear to be mediated by soluble cytokines, notably IL-3 or -7 (16)(17)(18)(19)(20)(21). Productive rearrangements of both H and L chain V genes allow the expression of Ig on the surface of pre-B cells, detectable from day 16 of gestation onwards in fetal liver of the mouse (22). 2 d later, these slg+ B cells become reactive to polyclonal activators (1)(2)(3), so that upon stimulation with LPS, they develop into clones of IgMsecreting plasma cells, which can be detected in a plaque assay (23,24).
Tissue culture systems have been established in which the development of B lineage cells in fetal liver from day 13 of gestation can be followed to the final maturation into a clone of IgM-secreting plaque-forming cells (PFC)' (1)(2)(3). The time schedule of development in vitro follows that observed in vivo, so that mitogen-reactive B cells arise at day 19 of gestation and the peak of the IgM PFC response 5 d thereafter, no matter when the cells have been transferred from fetal liver into tissue culture at any time between days 13 and 19 of gestation (1)(2)(3).
In this report, we follow in vitro the development of B lineage cells in fetal liver from day 13 of gestation with time and in dependence ofinteractions with the stromal cell environment and with rIL-2 through rIL-7 (25). B lineage precursors are enriched from fetal liver at different times of gestation by FAGS with the help of a recently developed mAb, G-5-2, specific for pre-B cells (26,27). Enrichment ofpre-B cells is monitored by an in situ hybridization analysis for the expression of the The Basel Institute for Immunology was founded and is supported by F Hoffman-La Roche A . G ., Basel, Switzerland .
ILs. Murine rIL-2, -3, -4, and -5 were obtained as described (25) and used at a concentration of 5%, i.e., at N5-50 U/ml. Human rIL-6, produced by a cell line transfected with the human IL-6 gene (kindly provided by Dr. W. Fiers, Biogent, Gent, Belgium), was used at a dilution of 5% (i.e., at 5-50 U/ml). IL-7 (18,19) (kindly given to us by Drs. S. Gillis and Ch. Henney, Immunex, Seattle) was used at concentrations between 100 and 1,000 U/ml. Tissue Culture. Fetal liver cells were prepared as described earlier (1-3, 26, 27). Total fetal liver cells and G-5-2+ fetal liver cells isolated by cell sorting (see below) were cultured in serum-substituted IMDM (32), in the presence or absence of stromal cells and in the presence or absence of IL-2 through IL-7 (19,20,25), as indicated in Results. Cultures were set up at 0.2 ml in 96-well flat-bottomed plastic culture plates (Costar, Cambridge, MA) . Primary fetal liver stromal cell layers were established from 13-d-old BDF, embryos by overnight culture at a density of 5 x 106 to 10' cells/ml in IMDM containing 5% FCS. Nonadherent cells were thereafter removed by extensive washing with serum-free medium . This stromal layer was subjected to 3,000 rad gamma irradiation before coculture with G-5-2' fetal liver cells.
Limiting dilution experiments were performed in 96-well culture plates in serum-substituted medium in the presence of 3 x 106 rat thymocytes/ml and 50 p.g/ml LPS, (S form ofSalmonella abortus equii; a kind gift of Drs . G. Galanos and O. Luderitz, Max-Planck-Institut fur Immunobiologie, Freiburg, FRG) as described earlier (1-3, 26, 27). Threefold dilutions of fetal liver cells between 5 x 106 and 5 cells/ml were set up with 24 cultures of 200 pl for every cell concentration in rat filler cells and LPS. A positive culture with a clone of IgM-secreting cells arising from one mitogen-reactive B cell contained between 30 and 120 IgM PFC at the day of assay, i.e., at the equivalent time of day 5 after birth in all fetal liver cell experiments . The peak of IgM PFC responses at that day with the various fetal liver cell suspensions cultured in various ways was ascertained in pilot experiments, and reaffirmed earlier results showing that all fetal liver cell responses, regardless from which day ofgestation and regardless oftreatment in tissue culture, showed apeak ofIgM PFC development at the equivalent time ofday 5 after birth (2, 3). Poisson's distribution was used to calculate the frequencies of mitogen-reactive, Ig-expressing cells yielding clones of IgM PFC. Cultures were scored as negative if they contained three IgM PFC or less.
Plaque Assay. The total number ofIgM-secreting PFC in a population of cells was determined by the protein A SRBC plaque assay (24) using a rabbit anti-mouse IgM (MOPC 104E IA/X) antiserum .
Production of Stromal Cell mAbs and Testing their Effects on the Adherence of the Pre-B Lymphoma 40E1 to Stromal Cells. Lewis rats were immunized on day 0 with 2 x 101 stromal cells emulsified in CFA in the hind foot pad . On day 4 and 7 the rats were boosted with 2 x 101 stromal cells in PBS in the same foot pad . On day 8 the regional lymph nodes were removed and fusion was performed as described (33).
Individual hybridoma clones were then tested for the production of antibodies that interfere with the adherence ofthe pre-B lymphoma 40E1 to the fetal liver stromal cell layers (prepared as described above). Stromal cell layers were therefore cocultured with 5 x 104/m1 40E1 cells and 20% final concentration of the different hybridoma clone culture supernatants. Adherence of the 40E1 cells to the stromal cells was read after 24-36 h of coculture using an inverted microscope. Two mAbs called STR4 and STR10 were found that interfered with the adherence of 40E1 to the stromal cells .
Immuno,Jluorescence Analysis and Cell Sorting. Immunofluoreseence staining and analysis, as well as cell sorting, were done as described earlier (26) using biotinylated (Calbiochem-Behring Corp., La Jolla, CA) mAbs and FITC-streptavidin (Amersham International, Amersham, UK) as secondary reagents.

Development of Precursors of LPS-reactive B Cells in Cultures of Unseparated Fetal Liver
Rpulations. Tissue culture conditions have been developed in which resting mature B cells from a variety of lymphoid organs of the mouse can be stimulated by polyclonal activators such as LPS under conditions of limiting dilution to develop in high frequencies to clones ofPFC (1-3, 26, 27). These culture conditions, in which single B cells are plated in serum-substituted medium (32), in the presence of rat thymus cells known as "fillers," are also suitable to stimulate in vitro those B cells that have developed, or will develop in this foreign environment, to the stage of a mitogen-reactive, Ig+ B cell. When cells are taken from fetal liver at different days of gestation between days 13 and 19 of embryonic development of the mouse and directly cultured with LPS and rat thymus cells under limiting dilutions, the peak of the PFC response occurs always at the same time, i.e., at the day equivalent to day 5 after birth . Therefore,, fetal liver cells of day 13 ( Fig. 1, circles) and of day 14 ( Fig. 1, triangles) of gestation were cultured in high density suspensions (see legend to filter cells and LPS were done in the presence of IL-6 and -7 . As can be seen from the data in Fig. 1, filled and dotted triangles, IL-6 and -7 did not show any significant effects on the frequencies of pre-B cells capable of development to mitogen-reactive B cells and to clones of IgM PFC. The frequencies of precursors that can develop in this environment increases from N1 in 107 at day 13 to N1 in 30 at day 19 of gestation (1-3). The change in precursor frequency is particularly high, i.e., -100-fold, between days 16 and 17 ( Fig. 1).
When fetal liver cells of early times of gestation (days [13][14][15]) are cultured at high density, e.g., when the progenitors and precursors are not diluted in rat thymus cells but kept in contact with each other until the time equivalent to day 19 and then plated in limiting dilution with LPS and rat thymus cells, the frequency ofprecursors of LPS-reactive cells increases. Over 100-fold more LPS-reactive B cells have developed in high density cultures in contact with each other (Figs. 1, open circles and triangles, at day 19). This indicates that their own environment is favorable for the development of precursors to mature, sIg+ B cells, while that of rat thymus cells is not. The change in precursor frequencies capable of development to IgM PFC between days 13 and 19 is, in fact, as high in the equivalent time in vitro as it is in vivo (compare Figs. 1 and 2) and reaches frequencies comparable with those in vivo, i.e., ti 1 in 30 cells. The culture conditions of fetal liver cell suspensions at high densities, thus, appear as good as those of cells in vivo in fetal liver. They make it further unlikely that exogenously added IL-6 and -7 could show any stimulatory effects.
When fetal liver cells of either day 13 ( Fig. 1, circles) or 14 ( Fig. 1, triangles) of gestation are removed at different times of tissue culture from their own environment into that ofrat thymus cells, the 100-fold change in LPS-reactive cells developing in the second environment (observed above) occurs within 24 h, between the time equivalent to days 16 and 17 ofembryonic development (Fig. 1) . This sudden change indicates that pre-B cells in fetal liver change from dependence to independence on their own environment to develop to LPS-reactive cells at that time. Failure of Purified Fetal Liver B Cell Precursors to Develop Outside their Own Environment. Pre-B cells express a lineage-and developmental stage-specific glycoprotein, PB76, on their surface that is recognized by mAb G-5-2 (26,27). B cell precursors of fetal liver can be enriched by FAGS using this mAb, as shown by the concomitant enrichment of cells expressing the pre-B cell-specific gene X5 (27,28). Thus, mAb G-5-2' fetal liver cells of day 14 were -30%, of day 16 -60%, and of day 18 -85% X5' . In the same G-5-2' cells, w H chain mRNA-positive B cell precursors were present at 1 % (at day 13), 6 % (at day 14), 50 % (at day 16), and 85 % (at day 18) . When plated in limiting dilutions with rat thymus cells immediately after FACS, these cell populations proved to be enriched for precursors of LPS-reactive B cells (26,27). Although between 30 and 85% of all G-5-2' cells expressed X5 at days 14-18 and could, therefore, be regarded as pre-B cells, only a very small fraction of all G-5-2' cells before days 15 and 16 of gestation could develop into clones of IgM PFC in the foreign environment of rat thymus cells . The frequencies of these precursors in the enriched populations increased from N1 in 3 x 103 at day 14 to ti 1 in 10 at day 19 of gestation . The highest increase in frequency was detected within 24 h of gestation, between days 15 and 16 ( Fig . 2) (see also Discussion).
When high concentrations (5 x 104 to 5 x 105 cells/ml) of mAb G-5-2' day 14 fetal liver cells were cultured in rat thymus filler cells (5 x 106/ml) plus LPS, no significant cell death was observed between day 14 and the time equivalent to day 19 of gestation, indicating that rat thymus cells had no adverse effect on the viability of precursor cells. Lower concentrations of the fetal liver cells, as those used in the limiting dilution analyses, cannot be monitored for survival, due to the vast excess of rat thymus cells in culture .
The Influence ofIL-2, -3, -4, -5, -6, and -7. The inability of early fetal liver cells (day 14) to develop in high frequencies to LPS-reactive B cells did not change when IL-2, -3, -4, -5, -6, and -7, either alone or in all possible combinations of one, two, three, four, five, or all six ILs, i.e., 37 different sets of ILs (data not shown), were added to the cultures of G-5-2' cells (shown only for IL-6 and -7 in Fig. 1 and for IL-3 and IL-7 in Fig. 4). All ILs were used in concentrations (5-50 U/ml or more) that would be expected to suffice for occupancy of high avidity receptors for these ILs. We conclude that neither rat thymus cells with LPS nor these six ILs, by themselves or in combinations, induce the development to mitogen-reactive B cells under conditions of high avidity IL-R occupancy.
The Influence of Embryonic Stromal Cells. Since the own environment of fetal liver precursors of the B lineage appeared beneficial for their development to mature cells, i.e ., since high density suspensions of fetal liver cells allowed the development of mitogen-reactive B cells, while limiting numbers of fetal liver cells in the foreign environment of high density rat thymus filler cells and LPS did not, the influence of adherent "stromal" cells on this development was investigated .
Cultures of primary embryonic stromal cells were established from day 13 fetal liver cells. Cells that adhered to the plastic tissue culture wells in overnight culture were either trypsinized and replated for various periods of time up to 2 wk, or were used directly after removal of the nonadherent cells in the primary culture as sources of embryonic stromal cells. They were irradiated before coculture with FACS-sorted G-5-2' B cell precursors .
In the presence of such irradiated embryonic stromal cells, G-5-2' precursors were now induced to the development of mitogen-reactive B cells in high frequencies . The frequencies increased from ti 1 in 3 x 10 3 at day 14 to N1 in 30 when precursors of day 14 and stromal cells were cocultured until the time equivalent to day 19, before they were replated in LPS and rat thymus cells (Fig. 3) . Addition of the various ILs led to no significant increase in the frequencies of mitogen-reactive B cells, although addition of IL-6 plus IL-7 tended to increase the size of a single LPS-reactive B cell clone by one quarter to one half (data not shown) . For all subsequent experiments these two ILs were included in the cultures, unless specified otherwise . We conclude from these results that irradiated embryonic stromal cells provide an environment in culture that is inductive for B cell differentiation to mitogenreactive B cells. Exogenously added IL-2 through IL-7 are not needed .
A Stromal Cell-dependent, Followed by a Stromal Cell-independent Period ofB Cell Development. Experiments with unseparated and FACS-sorted G-5-2 + fetal liver cells described above had indicated that the environment of stromal cells in fetal liver pro-  vides the conditions for development to mitogen-reactive B cells at high frequencies.
Thus, we investigated how long this environment had to be present for the precursors to reach the mitogen-reactive stage as assayed in the presence of rat thymus cells and LPS in limiting dilution analysis . Previous experiments have shown that the LPS-reactive stage is reached in culture at the time equivalent to day 19 of gestation (1-3, 26, 27). G-5-2+ precursors of day 14 fetal liver were cocultured with irradiated stromal cells for either 1, 2, 3, 4, or 5 d, and then replated in limiting dilutions with LPS and rat thymus cells. The results in Fig. 3 show that the frequencies of LPS-reactive B cells remained low (between 1 in 3,000 and 1 in 1,000) until the time equivalent to day 16 when it changed to between 1 in 100 and 1 in 30 within 24 h. This indicates that B cell development is stroma cell dependent until day 16 of gestation, and becomes independent thereafter. This change occurs at the time when adherence of precursors to stromal cells is lost and when pre-B cells become sIg+ in these cultures (see below) . The rapid change in frequencies of precursors indicates that this development is synchronous for the majority of the precursors .
The same rapid change from stromal cell dependence to independence was observed in experiments where G-5-2' cells at various stages of development in vivo in fetal liver were cultured in limiting dilutions with LPS and rat thymus cells immediately after FACS. Results in Fig. 2 show that G-5-2 + fetal liver cells from days 14 and 15 of gestation did not develop in high frequencies to mitogen-reactive B cells, while from day 16 onwards they did, indicating a change in their independence of the fetal liver cell environment, e.g., as shown above in vitro on stromal cells.
Limited Proliferation ofPrecursors in the Presence of Stromal Cells and/or ILs, but Change in Adherence. When G-5-2+ B cell precursors from day 14 fetal liver were plated on layers of adherent irradiated embryonic stromal cells, they adhered in clusters to the stromal cells. They remained adherent for 2 d, then became enlarged and detached from the stromal cell layer. At best, only a twofold increase in the number of cells cultured in the absence or presence of either IL-3 or IL-6 plus IL-7, with or without stromal cells, was observed over a 5-d culture period (Fig . 4, top) . These results provide evidence that G-5-2+ B cell precursors do not proliferate polyclonally at a high rate . They suggest that the precursors change their interactions with stromal cells at a time equivalent to day 16 of gestation from adherent to nonadherent.
Inhibition of the Influence ofStromal Cells by Specific mAbs. Adherence to embryonic stromal cell layers could also be observed with Abelson murine leukemia virus-transformed B cell lymphomas, but not with mature B or T cells (data not shown) . This indicated that transformed pre-B cell lines could be used to assay inhibition of adherence by specific ligands such as mAbs. mAbs were raised against embryonic stromal cells from day 13 fetal liver expanded for 2 wk. Two of them, STR4 and STR10, interfered with the binding of the Abelson murine leukemia virus-transformed pre-B cell line 40E1 to stromal cells and stained stromal cells as well as pre-B lymphoma cells, as detected by FACS. These mAbs were then tested in cocultures of G-5-2* precursors of day 14 fetal liver with embryonic stromal cells for their capacity to influence the development to mitogen-reactive B cells. Both mAbs inhibited the adherence of the precursor to the stromal cell layer. Both mAbs also inhibited the development to mitogen-reactive B cell, but did not influence the number of cells in culture. As controls, the isotype-matched progenitor, pre-B cell-, and B cell-specific  Fig. 4 (bottom) show that within 24 h, in the time between the equivalent of days 16 and 17 of gestation, 5-10% of all cultured cells became sIg' and remained in that state for the next 2 d. Both mAbs STR4 and STR10 inhibited the development of sIg' cells (data not shown) . These results indicate that the change to sIg' cells occurs in a majority of G-5-2 +-sorted cells, and does so synchronously for the majority of them . This change occurs at the same time that precursors become independent of stromal cells in their development to mitogenreactive B cells. Based on these results, a model of B lymphocyte lineage development (Fig . 5) is discussed below.

Discussion
Development of B lineage cells from pluripotent stem cells and committed progenitors has been found to be dependent on interactions with so called stromal cells in organs where B cells are generated, e.g ., mainly in fetal liver and bone marrow of the mouse. Tissue culture conditions and stromal cells lines have been developed with which the contributions of stromal cells to B lineage development can be studied in vitro (7)(8)(9)(10)(11)(12)(13)(14)(15) . Once B lineage development has become stromal cell independent, single cells can be followed in limiting dilutions with rat thymus filler cells (1)(2)(3) or other feeder cells (4), and under the stimulating influence of LPS in their development to clones of IgM-secreting PFC (24). In fetal liver this later development is programmed in time, so that the majority of all cells in vivo or in vitro gain mitogen reactivity at the same time, i.e., at day 19 of gestation (1-3, 26, 27). It then takes another 5 d to stimulate these mature B cells to the peak of clonal proliferation and maturation to IgM PFC (24). The results presented in this paper confirm the observations that two phases are distinguishable, one stromal cell dependent, the other independent (36). Our results specify the time ofembryonic development when this change occurs, i.e., between days 15 and 16 of gestation. They show that the change occurs in the majority of all cells at the same time. The two mAbs able to interfere with the interactions of B cell precursors and stromal cells that we have developed should be useful in studies ofthe molecular and cellular requirements of such interactions necessary to induce the precursor to become sIg`, and then mitogenreactive. Our results further support the idea that specific cellular contacts between the progenitors/precursors and the stromal cells mediate the induction to the development of mature B cells.
Whenever G-5-2+ precursor cells of day 13, 14, or 15 fetal liver were plated immediately in rat filler cells, the change from stromal cell dependence to independence was observed to occur between days 15 and 16. On the other hand, when these purified precursor cells were first plated on stromal cells and then replated in the time equivalent until day 19 of gestation in rat filler cells, the change occurred almost 24 h later (compare Figs. 1-3). This suggests that a second handling of the precursors (washing, cooling down, etc.) might delay the normal development. Therefore, we think that the time of change to stromal cell independence in vivo is between days 15 and 16 of gestation in fetal liver.
None of the exogenously added ILs had any influence on the purified precursor cells that would replace the requirement for stromal cells in the development towards mature B cells. Similar findings have been reported for adult bone marrow precursors (21). It, however, does not exclude that ILs, notably IL-7 (18)(19)(20) and IL-1 (21), could play a role in this development. The stromal cells that we use could well synthesize these ILs and thereby provide, endogenously and locally, the necessary growth and differentiation signals.
The contacts with stromal cells, and any IL that may be produced endogenously in this interaction, do, however, not induce extensive pre-B cell proliferation (Fig.  4, top) . This is in line with many observations (37,38) that point to a very limited proliferation of precursors between days 14 and 19 of gestation in fetal liver. In this time of gestation, all rearrangements of V gene segments of H and L chain genes take place in a time-ordered fashion (5,6). If rearrangements occur when cells divide, then it is tempting to speculate that every division at this time of development in fetal liver is critical. The first division might rearrange DH to J., the second V to DJ , the third VK to J,,, and a fourth VX to JX. A very well ordered machinery for these rearrangement processes should exist, controlled in its activity by the interactions with stromal cells (Fig. 5).
At the end of the rearrangement processes, sIg+ cells appear, provided that these rearrangements were functional, and provided that the Ig could be transported to and inserted into the surface membrane . The rearrangement process is known to lead not only to productive, but also to nonproductive Ig genes, due to incomplete, out-of-frame, and pseudogene rearrangements in either the H and/or L chain genes. Consequently, no sIg can be made . It is difficult to estimate how high the percentage of nonproductive rearrangements should be, since we do not know whether the rearrangement processes are purely stochastic, or guided by molecular interactions that favor productive rearrangements . We also do not know how many pseudogenes participate in these rearrangement processes. Our findings that between 5 and 10% of all G-5-2+ precursors develop to sIg' cells ( Fig . 4, bottom) suggests that such nonproductive rearrangements could happen in 90% or more of all precursors . This number is, at least, within the range of frequencies ofproductive vs. nonproductive rearrangements expected from a stochastic process of rearrangements . The appearance of Ig on the surface of pre-B cells at day 16 of gestation is obviously dependent on successful rearrangements of V gene segments, and on transcription and translation of the genes. The deposition of Ig in the surface membrane, however, could also be dependent on interactions with neighboring molecules, like mb-1 (39), which could have functions similar to the CD3 complex associated with TCRa and -S, respectively, and y and 6 chains (reviewed in reference 40). mb-1 is expressed from the earliest stages of pre-B cell development, i .e., when D segments rearrange to JH segments, and continues to be expressed to the stage of the mature B cell . While this is true for mRNA expression, a protein with CD3 line functions might only be expressed at a given, later stage of precursor development. This could, then, also control expression of surface Ig on pre-B cells and its transition from a stromal cell-dependent to an independent stage.
Particularly striking is the synchrony of B cell development in one wave in fetal liver. This indicates that stem cells and progenitors may populate the fetal liver at any time in development, but are induced only within a time window of 24 h, around day 13 of gestation, to enter the pathway of B cell development by Ig gene rearrangements and other processes connected with this program of differentiation. Before and after this time, progenitors may exist but they are not drawn. It is reasonable to assume as a working hypothesis that the environment of stromal cells is competent only for this short period of time to convey the necessary interactions with progenitors . Synchrony of B cell development in this wave is high for a remarkably long period of time in embryonic development, i.e., for 6 d between days 13 and 19 of gestation, pointing to an ordered process all along the way. It is, therefore, not very likely that normal cells at any one of these stages of B cell development would, without transformation, be capable of extensive cell proliferation.
Other waves of lymphocyte development are known. In mice, the wave of B cell development in fetal liver is preceded by one in fetal placenta and embryonic blood (41) . In sheep (and maybe in other species), a late wave has been observed in Peyer's patches (42) . In chicken, colonization of the bursa occurs at a specific time before hatching (43). Development of T cells in the thymus of birds occurs in three waves (44) . Initially, these waves are initiated by a similar short 24-h period of competence of the organs for immigration and induction to development of the precursors . Whether these waves of lymphocyte development use the total repertoire of V gene segments or prefer, as in fetal liver, only part of it (V 7183), and whether different environments actually select the expressed V genes according to their binding specificities for self determinants in the special environments, remains to be seen .
In bone marrow, throughout life, the generation of B cells is continuous . At the same time, turnover of the mature, primary B cell is high (43,44). Therefore, if pre-B cells in bone marrow are equally limited in their capacity to proliferate as they rearrange their V segmcnts, become sIg' and, finally, mitogen and antigen reactive, a pool of progenitor cells before rearrangements must proliferate very actively. This progenitor pool, possibly reactive to another stromal cell and to IL-7 (45), should continuously generate by stem cell-like proliferation those cells that can then be drawn into the pathway of B cell development at rates that keep up the pool of primary mature B cells (Fig . 5) .

Summary
Precursor cells of the B lineage can be enriched from mouse fetal liver by FAGS with the aid of the pre-B cell-specific mAb G-5-2. The cells are concomitantly enriched for cells expressing the pre-B cell-specific gene X5, and for cells developing to LPS-reactive mature B cells. The enriched purified precursors are not influenced by rIL-2 through -7, alone or in combination, to develop to mitogen-reactive, sIg+ cells. Marginal proliferation of-the precursors is observed in response to IL-3 plus -4, and IL-6 plus -7, and this does not change in the presence of stromal cells. Development to mitogen-reactive, sIg+ cells is dependent on interactions with embryonic stromal cells from fetal liver. Two mAbs raised against the stromal cells inhibit this development. Two phases of precursor cell development can be distinguished in fetal liver. Between days 13 and 15 of gestation, it is dependent on stromal cell interactions, thereafter, from days 16 to 19, it is independent. A sudden increase in the number of mitogen-reactive, sIg+ B lineage cells occurs within 24 h between days 16 and 17 . All these results indicate that B cell development occurs in one wave with synchronous steps of changes from a mitogen-insensitive, sIg -, stromal cell dependent to a mitogen-reactive, sIg+, stromal cell-independent B lineage line.